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11th International Symposium on Rice Functional Genomics Program

Conference Chair and Co-‐Chair Akhilesh K Tyagi (NIPGR, New Delhi) Jitendra P Khurana (Univ. of Delhi South Campus, New Delhi)

Patrons S Ayyappan, DG, ICAR SK Brahmachari, DG, CSIR Gurdev Khush, UC Davis

T Ramasami, Secretary DST AK Sharma, Calcutta Univ. M Sharma, Fmr. Secretary, DBT

D Singh, VC, Univ of Delhi RB Singh, President, NAAS SK Sopory, VC, JNU

MS Swaminathan, MSSRF K Vijayraghavan, Secretary, DBT RS Zeigler, DG, IRRI

International Organizing Committee Gynheung An, Korea Antonio Costa de Oliveira, Brazil Emmanuel Guiderdoni, France

Bin Han, China Yue-‐ie Caroline Hsing, Taiwan Ju-‐Kon Kim, Korea

Hei Leung, Philippines Jiayang Li, China David Mackill, USA

Makoto Matsuoka, Japan Susan Rutherford McCouch, USA Oliveir Panaud, France

Pamela C. Ronald, USA Blanca San Segundo, Spain Ko Shimamoto, Japan

Akhilesh K. Tyagi, India Narayana M. Upadhyaya, Australia Apichart Vanavichit, Thailand

Rod A. Wing, USA Masahiro Yano, Japan Qifa Zhang, China

National Organizing Committee Kailash C. Bansal, NBPGR Niranjan Chakraborty, NIPGR KS Charak, DBT,

B. B. Chattoo, Univ of Baroda Asis Datta, NIPGR Swapan Datta, ICAR

HS Gupta, IARI Sanjay Kapoor, Univ of Delhi Paramjit Khurana, Univ of Delhi

Arun L Majumder, Bose Institute Trilochan Mohapatra, CRRI KK Narayanan, Metahelix

Ashwani Pareek, JNU Saurabh Raghuvanshi, Univ of Delhi Umesh Rai, Univ of Delhi

MK Reddy, ICGEB SK Sen, IIT Kharagpur Tilak R. Sharma, NRCPB

EA Siddiq, ANGRAU, Hyderabad Ashok K. Singh, IARI NK Singh, NRCPB

Alok K. Sinha, NIPGR Ramesh V. Sonti, CCMB Renu Swarup, BIRAC

Rakesh Tuli, NABI, Mohali M Udayakumar, UAS, Bangalore K. Veluthambi, MKU, Madurai

Usha Vijayraghavan, IISc BC Viraktamath, DRR, Hyderabad Usha B. Zehr, MAHYCO

11th International Symposium on Rice Functional Genomics Program

Scientific Programme Committee Anil Grover, Univ of Delhi Mukesh Jain, NIPGR, New Delhi Sanjay Kapoor, Univ of Delhi

P Ananda Kumar, NRCPB Madan Mohan, Univ of Delhi AK Nandi, JNU

Ajay Parida, MSSRF MK Reddy, ICGEB VS Reddy, ICGEB,

Ashok K Singh, IARI NK Singh, NRCPB Akhilesh K Tyagi, NIPGR-‐Chair

Finance & Resource Committee Kailash C Bansal, NBPGR Sandeep Datta, NIPGR Jitendra Giri, NIPGR

Jitendra P Khurana, Univ of Delhi – Chair Manoj Majee, NIPGR

Ashwani Pareek, JNU Tilak R. Sharma, NRCPB Alok K. Sinha, NIPGR

Logistics Committee V Chinnusamy, IARI Indranil Dasgupta, Univ of Delhi Paramjit Khurana, UDSC – Chair

Sneh Lata Pareek, ICGEB Saurabh Raghuvanshi, Univ of Delhi Arun K Sharma, Univ of Delhi

R Srinivasan, NRCPB Jitender Thakur, NIPGR DP Wankhede, NBPGR

Program

GBR – GRAND BALLROOM i

Day 1: November 20, 2013; Wednesday 8:00

Onwards REGISTRATION: Grand Ballroom (GBR) Prereception area

09:30 -‐ 11:00 OPENING CEREMONY & KEYNOTE ADDRESS (GBR)

Chair Person: Manju Sharma Chief Guest: MS Swaminathan

Welcome: Akhilesh K Tyagi Guests of Honour: Qifa Zhang, Dinesh Singh, S Ayyapan

Introduction of the keynote speaker and remarks: Manju Sharma Keynote address: MS Swaminathan Vote of Thanks: Jitendra P Khurana

11:00 -‐ 11:30 TEA/COFFEE BREAK (GBR Lawns)

11:30 -‐ 13:30

PLENARY LECTURES (GBR) Chair Persons: Sudhir Sopory, Rod Wing

Rod Wing, USA Qifa Zhang, China

Masahiro Yano, Japan Usha Vijayraghavan, India

13:30 -‐ 14:30 LUNCH (GBR Lawns)

14:30 -‐ 16:00

Concurrent Session I (GBR 1) Abiotic Stress I Chair: Anil Grover, San Segundo Blanca

Concurrent Session II (GBR 2) Architecture & Development Chair: Dabing Zhang, R Srinivasan

Concurrent Session III (GBR 3) Comparative and Evolutionary Genomics Chair: Antonio Costa de Oliveira, RP Sharma

Anil Grover, India Dibyendu N Sengupta, India Tiago Lourenço, Portugal R Venkategowda, USA

Yongzhong Xing, China Ajay Kohli, Philippines Sanjay Kapoor, India Yang-‐Seok Lee, Korea

N K Singh, India Pankaj Jaiswal, USA Jitendra K Thakur, India Ramanjulu Sunkar, USA


16:30 -‐ 18:00

Ajay Parida, India Niranjan Chakraborty, India Sneh-‐Lata Singla-‐Pareek, India Girdhar K Pandey, India

Changyin Wu, China Gauravi Deshpande, India Letian Chen, China

Yesheng Zhang, China Rachel S Meyer, USA Masahiko Kumagai, Japan Jorge Duitama, USA

18:00 -‐ 19:00 Discussion & Personal Interaction

19:00 WELCOME DINNER (GBR Lawns)

Program

GBR – GRAND BALLROOM ii

Day 2: November 21, 2013; Thursday

09:00 -‐ 10:30

PLENARY LECTURES (GBR) Chair Persons: Masahiro Yano, Jitendra P Khurana

Lizhong Xiong, China Christophe Perin, France Naoka Nishizawa, Japan


11:00 -‐ 12:30

PLENARY LECTURES (GBR)

Peter Westhoff, Germany Jie Luo, China

Gynheung An, Korea

12:30 -‐ 15:30 LUNCH (GBR Lawns) & POSTER SESSIONS I & II (Brix) (Co-‐ordinators: Sanjay Kapoor, Alok Sinha, Saurabh Raghuvanshi, Mukesh Jain)

15:30 -‐ 16:30

Concurrent Session IV (GBR 1) Abiotic Stress 2 Chair:Naoko Nishizawa, Arun Lahiri Majumder

Concurrent session V (GBR 2) Small RNA and Epigenomics Chair:Blake Meyers, M Udaykumar

Concurrent session VI (GBR 3) Informatics and Systems Chair: Bin Han, Paramjit Khurana

Arun L Majumder, India Nelson Saibo, Portugal

Yoshiki Habu, Japan Dao-‐Xiu Zhou, France

Antonio C de Oliveira, Brazil Sushma Naithani, USA


17:00 -‐ 18:20

M K Reddy, India Alok K Sinha, India Ashwani Pareek, India Ming-‐Der Shih, Taiwan

Meenu Kapoor, India Saurabh Raghuvanshi, India Sarah Anderson, USA

Bijayalaxmi Mohanty, Singapore Olivia Wilkins, USA Sunil Archak, India

19:00 BUFFET DINNER (GBR Lawns)

Program

GBR – GRAND BALLROOM iii

Day 3: November 22, 2013; Friday

09:00 -‐ 10:30

PLENARY LECTURES (GBR) Chair Persons: Gynheung An, RB Singh

Blake Meyers, USA Dabing Zhang, China

Venkatesan Sundaresan, USA


11:00 -‐ 13:00

PLENARY LECTURES (GBR)

Trilochan Mohapatra, India Bin Han, China

Olivier Panaud, France Yue-‐le Caroline Hsing, Taiwan

13:00 -‐ 14:00 LUNCH (GBR Lawns)

14:00 -‐ 15:30

Concurrent session VII (GBR 1) SHIMAMOTO session

(Biotic Stress): Chair: Hiroshi Takatsuji,

Bharat B Chattoo

Concurrent session VIII (GBR 2)

Molecular Breeding Chair: HS Gupta, Yue-‐Ie Hsing

Concurrent session IX (GBR 3) Translational Genomics

Chair: Venkatesan Sundaresan, Rakesh Tuli

Shiping Wang, China Tilak R Sharma, India Sampa Das, India Yoji Kawano, Japan

Yaoguang Liu, China Arvind Kumar, Philippines Darshan S Brar, India Endang M Septiningsih, Philippines

Karuppannan Veluthambi, India Ashok K Singh, India Hiroaki Saika, Japan Karabi Datta, India


16:00 -‐ 17:30 Masaki Mori, Japan Indranil Dasgupta, India RM Sundaram, India

Shunsuke Adachi, Japan Kshirod Jena, Philippines Dak Deborah, India Vandna Rai, India

Bharat B Chattoo, India Amol Samant, India Paras Yadav, ILS, India AK Bhattacharya, Leica

17:30 -‐ 18:30 Meeting of the International Organizing Committee (Board room)

18:30 -‐ 19:30 CULTURAL EVENING (GBR)

19:30 BANQUET DINNER (GBR Lawns)

Program

GBR – GRAND BALLROOM iv

Day 4: November 23, 2013; Saturday

09:00 -‐ 10:30

PLENARY LECTURES: (GBR) Chair Persons: Ebrahimali A Siddiq, Peter Westhoff

Blanca San Segundo, Spain Hiroshi Takatsuji, Japan Ramesh Sonti, India


11:00 -‐ 12:30

PLENARY LECTURES: (GBR)

Daniel Zilberman, USA Akhilesh Tyagi, India Andy Pereira, USA

12:30 -‐ 15:00 LUNCH (GBR Lawns) & POSTER SESSIONS III—VIII (Brix)

(Co-‐ordinators: Sanjay Kapoor, Alok Sinha, Saurabh Raghuvanshi, Mukesh Jain)

15:00 -‐ 16:30

PANEL DISCUSSION on "Rice Functional Genomics in Sustaining Food & Nutritional Security" Chair Persons: K Vijayraghavan, Swapan K Datta

(GBR)

K Vijayraghavan, Swapan K Datta, Rod Wing, Qifa Zhang, Kailash C Bansal, KK Narayanan, Usha Zehr

16:30 -‐ 17:00 Announcement of ISRFG 2014

Closing ceremony (GBR)

17:00 -‐ 17:30 High Tea (GBR Lawns)

ISRFG2013 New Delhi Table of Contents

Page Keynote Lecture 1

Plenary Lectures 3

Concurrent Session I – Abiotic Stress 1 16

Concurrent Session II – Architechture & Development 21

Concurrent Session III – Comparative and Evolutionary Genomics 25

Concurrent Session IV – Abiotic Stress 2 29

Concurrent session V – Small RNA and Epigenomics 32

Concurrent session VI – Informatics and Systems 35

Concurrent session VII – Biotic Stress 38

Concurrent session VIII – Molecular Breeding 42

Concurrent session IX – Translational Genomics 46

Poster Session I – Abiotic Stress 50

Poster Session II – Architechture & Development 84

Poster Session III – Comparative and Evolutionary Genomics 106

Poster Session IV – Small RNA and Epigenomics 114

Poster Session V – Informatics and Systems 121

Poster Session VI – Biotic Stress 124

Poster Session VII – Molecular Breeding 133

Poster Session VIII – Translational Genomics 152

Author Index … … … … … … 155

ISRFG2013 New Delhi Keynote Address

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Science and Sustainable Food and Nutritional Security MS Swaminathan UNESCO, Chair in Ecotechnology, MSSRF, Chennai At the close of the 2012 London Olympics, the United Kingdom announced a programme designed to rescue 25 million children worldwide from malnutrition by the time of the next Olympics in Brazil. In May 2012, the World Health Assembly agreed to a target of reducing the number of stunted children by 40 per cent (a reduction of about 70 million children) by 2025. To meet these targets and nourish the 870 million hungry people in the world, the world's leaders will need to prioritise an innovative science-based marriage of nutrition and agriculture.

The problem of hunger is not simply a lack of sufficient quantities of food. The chronic hunger caused by protein and calorie undernutrition is exacerbated by malnutrition (the “hidden” hunger caused by the deficiency of micronutrients, which include iron, iodine, zinc, vitamin A, and vitamin B12), and sometimes by human diseases that disable the body's ability to absorb the micronutrients it receives. To address such intertwined problems, there must be synergy among national programmes dealing with the availability, access to, and absorption of food. These nutrition security programmes should be based on a life-cycle approach that starts with the “first 1000 days” from pregnancy to 2-years old, the critical period when stunting can cause irreversible brain damage.

The United Nations (UN) High Level Panel on Food Security and Nutrition which I chair, released in 2012, a comprehensive report on Social Protection for Food Security, with recommendations for combating chronic childhood hunger. One of its recommendations — the concept of a “food security floor” is particularly worthy of mention. The food security floor recognizes that freedom from hunger is a fundamental human right, defining the minimal steps needed for hunger elimination. These include nutrition literacy, clean drinking water, sanitation and primary health care.

In some “hunger hotspots” of the world where agriculture is the backbone of survival, as in sub-Saharan Africa and South Asia, mainstreaming nutrition in agriculture programmes is the most effective and low-cost method of eliminating malnutrition. This requires greater attention to the net income of smallholder farmers, whose women food producers have particular needs that require specific policies and support. As an example, the M. S. Swaminathan Research Foundation in Chennai, India, has designed a Farming System for Nutrition initiative, comprising specific steps. They include carrying out a nutritional survey of the area and identifying the major causes of chronic and hidden hunger, and redesigning the farming system so that specific agricultural remedies are introduced for each nutritional malady, such as the cultivation of bio-fortified crops and crop-livestock integration. Thanks to the work carried out since 2004 under the Harvest Plus programme of the Consultative Group on International Agricultural Research (CGIAR), micronutrient-enriched varieties are becoming available in several crops, such as iron- and zinc-rich rice; iron-rich beans and pearl millet; zinc-rich wheat; and vitamin A–rich cassava, sweet potato and maize. BRRI dhan 62, the world’s first zinc – rich rice variety was released in Bangladesh this year. Iron-rich Pearl Millet hybrid is getting popular among farmers and consumers in India.

The year 2014 has been designated the International Year of Family Farming by the UN, and every effort should be made by developing countries to make each family farm a bio-fortified farm. If synergy can be created among scientific know-how, political do-how, and farmers' participation, it should be possible to achieve the goal of overcoming chronic and hidden hunger in large sections of the population of developing countries during this decade.

Climate Change and price volatility pose ecological and economic threats to sustainable food security. IRRI estimates that by 2035, the world will need more than 100 million tonnes of additional rice to meet the requirement of a population of 8.5 billion. In India, it has been estimated that inspite of production advances, there is a deterioration in the prevalence of undernourished during the period 1987-88 to 2004-05 based on energy intake. About 40% children under the age of 5 years are underweight. To overcome this Indian enigma of adequate food production being accompanied by the persistence of widespread chronic and hidden hunger, the Government of India has introduced the world’s largest social protection measure against hunger, in the form of the National Food Security Act, 2013.

Science is the prime mover of change. Fortunately, the new genetics represented by molecular biology provides new opportunities to achieve a balance between human numbers and the human capacity to produce food. For example, the International C4 Rice Consortium, coordinated by the International Rice Research Institute, is

ISRFG2013 New Delhi Keynote Address

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developing a new kind of rice capable of C4 phytosynthesis. C4 rice will not only be high yielding, but also tolerant to high temperature caused by global warming. It will also need less water. Recent studies by Consortium Members have shown that the yield gain of C4 rice could be more than 40% in Kerala, Odisha, Andhra Pradesh and Karnataka. This is just one example of the uncommon opportunities opened by advances in science in improving the productivity and profitability of small farms. Appropriate regulatory structures should be established to measure risks and benefits in a transparent and professionally credible manner. The Biosafety Regulatory Mechanism should be so structured that it inspires public, political, professional and media confidence. This will enable us to derive benefit from the uncommon opportunities for sustainable food and nutrition security opened up by functional genomics.

ISRFG2013 New Delhi Plenary Lectures

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1. 15 Million Years of Oryza Evolution as Seen Through the Analysis of 12 High-Quality Genome Assemblies

Wing RA, Chen M, Han B, Henry R, Hsing YC, Kurata N, de Oliveira A, Panaud O, Wang W

The genus Oryza contains 23 species (2 cultivated and 21 wild), encompasses ~15 million years of diverse evolutionary history, and contains a virtually untapped reservoir of genes that can be used to improve cultivated rice. The International Oryza Map Alignment Project formed in 2007 at the ISRFG meeting in Tsukuba, Japan and set 3 major goals: 1) Generate RefSeqs & Transcriptome data sets for all eight AA genome species, and a representative species of the nine other genome types; 2) Generate, map, and phenotype advanced ABC, CSSL, RIL populations for the AA genome species for functional and breeding studies; and 3) Identify collections of naturally occurring populations of the wild Oryza species for diversity, conservation, population and evolutionary analyses. Recently, IOMAP completed the sequencing, assembly (genomic and transcriptomic), and annotation of 9 Oryza genomes and the outgroup Leersia perrieri. When this data set is combined with the previously published O. sativa ssp. japonica and indica genomes, we now a total 11 high-quality Oryza genomes available for interrogaton. My talk will discuss the generation and analysis of this 12 genome data package and how we can used this resource to help solve the 9-billion people question - i.e. How can grow enough food to feed 2 billion additional human inhabitants in less than 40 years.

2. Understanding reproductive isolation based on the rice model Zhang Q

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China

Reproductive isolation plays important roles in speciation and maintaining species identity, thus has remained a key issue in evolutionary biology for many decades in a wide range of organisms. We have been using rice as the model to illustrate the biological mechanism of the reproductive barriers. Hybrid sterility is a major form of post-zygotic reproductive isolation. The Asian cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. Hybrids between indica and japonica varieties are usually highly sterile thus are reproductively isolated. A special group of rice germplasm, referred to as wide-compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. Cytological studies showed that both male and female gamete abortion, and reduced affinity between the uniting gametes all occur in the hybrids, resulting in greatly reduced fertility. Genetic analyses have identified a large number of loci controlling indica-japonica hybrid sterility and wide compatibility. Several loci have been cloned and molecularly characterized in recent years. Thus rice has provided an excellent model for studying the mechanisms and evolutionary processes of reproductive isolation in plants. In my presentation, I will use the results of cloning and molecular characterization of S5, a major locus for indica-japonica hybrid sterility and wide compatibility, to exemplify the genetic and molecular mechanisms of reproductive isolation and to demonstrate the molecular nature of indica-japonica differentiation. The results may have direct implications for understanding the mechanisms of reproductive isolation in other organisms, and for overcoming reproductive isolation in rice genetic improvement.


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3. Genomics-based allele mining and improvement of drought avoidance in rice Yano M, Uga Y

National Institute of Agrobiological Sciences, Tsukuba, Japan. [email protected]

Allele mining for traits of biological and economic interest in rice is enhanced by a large amount of sequence information, diverse accessions and mapping populations. We have been continuously developing mapping populations such as recombinant inbred lines and chromosome segment substitution lines (CSSLs) for detecting useful alleles in rice breeding. In particular, we have used CSSLs to evaluate the effects of QTLs / small chromosomal regions on various agronomic traits. Upon detection of an allele or chromosomal region of interest, we can straightforwardly perform integration of such information to breeding applications. Recently, we have been focusing on root architecture associated with deeper rooting to facilitate drought avoidance. Deeper rooting 1 (Dro1), a rice quantitative trait locus controlling deep rooting has been successfully cloned using mapping populations derived from a cross between IR64, a lowland rice with shallow roots, and Kinandang Patong, an upland rice with deep roots. Dro1 encodes an unknown protein expressed particularly in the cell division zone of the root tips. A putative auxin response element in its promoter region interacted specifically with AUXIN RESPONSE FACTOR (ARF), suggesting that ARF regulates Dro1 directly in the auxin-signaling pathway. For breeding applications, we developed a near-isogenic line for Dro1 (Dro1-NIL) in the IR64 genetic background. The effect of Dro1 in improving drought avoidance was clarified by exposing Dro1-NIL and IR64 to drought stress under upland conditions. As a result, Dro1-NIL showed significantly higher yield capacity than IR64, demonstrating that Dro1 contributes to drought avoidance and improves the productivity of rice under soil drought stress. To provide more opportunities for improving drought avoidance, we are currently analyzing other genes associated with root architecture and several candidate QTLs have been identified so far. A series of NILs carrying target traits will be promising materials for molecular breeding of root system architecture in the future.

4. Functional genomic studies of regulatory transcription factor networks during inflorescence and floret development

Vijayraghavan U, Khanday I, Yadav SR, Deshpande G, Chongloi GL, Ramakrishna K, Rao NN

Department of MCB, Indian Institute of Science, Bangalore 560012, India [email protected]

We use various functional genomics approaches to investigate regulatory networks controlling rice panicle branching, spikelet specification and floret development. RFL, the homolog of Arabidopsis LFY a floral meristem specification transcription factor, is expressed in a distinct profile in the developing rice panicles. Phenotypic analyses of loss-of-function mutant plants attribute a role for RFL in panicle branching and in certain ecotypes also for tiller formation from vegetative axillary meristems. We have probed the expression status of candidate genes and pathways implicated in vegetative axillary meristem development. We find that RFL negatively regulates the strigolactone biogenesis and perception while activating LAX1- a bHLH transcription factor for axillary meristem formation and OsPIN3- an auxin efflux pump. In branching inflorescence meristems RFL modulates the expression of other transcription factors (with MADS domain, bHLH domain, AP2-ERF and homeodomain) and affects signaling pathways (GA and auxin). The data suggest RFL regulates meristem development, at least in part, by regulating transcription factors and hormone signaling and that some of these downstream targets are tissue-dependent. MADS box transcription factors have many regulatory functions in floral development. The Class E factors encode co-activators and in rice these are five SEP-like genes. These genes perform both redundant and non-redundant roles during panicle and floret development. OsLHS1/OsMADS1 regulates floret meristem identity, its determinacy and organ development. To understand the molecular mechanisms of OsMADS1 action, transcript levels for candidate target genes, deduced from global transcript profiling, were validated after induced expression of OsMADS1. Further, immunoprecipitation analyses of OsMADS1 bound chromatin in panicles with spikelets and young florets was adopted probe direct DNA-protein interactions at target loci. Together these data show that genes encoding different classes of transcription regulators (homeodomain, YABBY, GARP, MADS domain) and genes for factors controlling multiple events during auxin and cytokinin signaling are regulated by OsMADS1. We discuss these regulatory relationships with respect to floret meristem specification and its determinate development.


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5. Control of drought resistance through cuticular wax deposition in rice Zhu X, Xiong L

National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. [email protected].

Drought resistance is a very complex trait controlled by distinct molecular and physiological mechanisms. Irrigated rice has been domesticated in full irrigation ecosystem and it is extreme sensitive to drought. With a long-term goal of improving drought resistance in irrigated rice, we have adopted a strategy by integrating the approaches to identify loci/genes effective for drought resistance improvement of rice. We will present a case study of a gene DWA1 (Drought induced Wax Accumulation 1) that controls drought-induced cuticular wax deposition and thus contributes to drought resistance. DWA1 is a very large protein containing multiple enzymatic structures including including an AMP-binding domain. This previously unreported putative mega-enzyme is conserved in vascular plants. A dwa1 knockout mutant was highly sensitive to drought stress relative to the wild type. DWA1 was strongly induced by drought stress. The dwa1 mutant was impaired in cuticular wax accumulation under drought stress, which significantly altered cuticular wax composition of the plant resulting in increased drought sensitivity. The mutant had reduced levels of very long chain fatty acids (VLCFAs), and plants overexpressing DWA1 showed elevated levels of VLCFAs relative to the wild type. The expression of many wax-related genes was significantly suppressed in dwa1 under drought conditions. The AMP-binding domain exhibited in vitro enzymatic activity in activating long chain fatty acids to form Acyl-CoA. Our results suggest that DWA1 controls drought resistance by regulating drought-induced cuticular wax deposition in rice.

6. Root anatomy and architecture as determinants of abiotic stress tolerance in rice: future is underground

Périn C, Divol F, Henry S, Puig J, Diévart A, Ahmadi N, Courtois B, Guiderdoni E

CIRAD, UMR AGAP, 34398 Montpellier cedex 5, France. [email protected]

Development of crops tolerant to adverse conditions is urgently needed but remains difficult due to the complex genetic determinism of these traits. Nowadays, root traits become major breeding targets towards the release of rice cultivars with enhanced tolerance to environmental stresses. Root architecture, at a macroscopic level and internal root structure, at a cellular level are indeed two key determinants of stress tolerance for rice as well as for many other species. Root depth, root biomass and root diameter represent three important root architectural traits that significantly impact drought tolerance in rice. The internal structure of the rice roots includes several tissues that play important role in the tolerance to various abiotic stresses. Endodermis and exodermis tissues are for instance major selective barriers limiting soil sodium and aluminum toxicities. We seek to identify the gene networks controlling root architecture and root tissue formation and differentiation by combining forward and reverse genetics. For instance (meta) QTL analysis and association mapping and fine mapping of a root depth QTL is underway. In a reverse genetics approach to isolate genes underlying the development and differentiation of root tissues, we are analyzing the function of the rice orthologs of A. thaliana SCR and SHR, two key genes involved in root ground tissue formation. A non-targeted approach is also used to identify among members of the large LRR-RLK receptor family those specifically involved in the development of ground tissue structure in the rice root. We anticipate that the combination of the favorable genes identified through the two approaches will fasten the development of rice cultivars with enhanced tolerance to different environmental constraints.


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7. Sensing iron and regulation of iron homeostasis in rice Kobayashi T1,2, Nakanishi-‐Itai R3, Senoura T2, Nakanishi H3 and Nishizawa NK2,3

1 Japan Science and Technology Agency (JST), PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. 2 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836, Japan. 3Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. [email protected]

Most living organisms require iron for their growth and proliferation. Since humans rely on plants as the primary source of both energy and iron, clarification of iron homeostasis in plants is of particular importance. Although abundant in mineral soils, iron is sparingly soluble in the physiological pH range under aerobic conditions. To avoid a deficit of iron, higher plants induce various genes involved in iron uptake and translocation in response to low iron availability. The rice transcription factor IDEF1 (Iron Deficiency-responsive Element-binding Factor 1), which specifically binds to the IDE1 sequence, confers iron deficiency response and tolerance in rice. IDEF1 expression was constitutively observed in various organs including roots, leaves, flowers and seeds throughout rice life, and was not affected by iron nutritional status. IDEF1 positively regulated the majority of known iron uptake and/or translocation-related genes during iron sufficiency and the early stages of iron deficiency. In subsequent stages of iron deficency, however, IDEF1 partially changed the species of its downstream genes. IDEF1 possessed characteristic histidine–asparagine repeat and proline-rich regions, which bound to various divalent metals, including iron and zinc. Deletion of these metal-binding regions impaired the IDEF1-mediated gene activation when IDEF1 was overexpressed in rice, primarily at an early stage but not at subsequent stages of iron deficiency. These results suggest that the metal-binding regions in IDEF1 might sense the cellular metal ion balance caused by changes in iron availability to shift the regulation pattern from the ‘early’ to ‘subsequent’ deficiency mode.

8. Evolution of C4 Photosynthesis in the Genus Flaveria – Lessons for C4 Engineering

Westhoff P

Department of Biology, Heinrich Heine University, D-40225 Duesseldorf, Germany. [email protected]

C4 photosynthesis is characterised by a division of labour between two different leaf cell types, mesophyll and bundle sheath cells. The photosynthetic, but also other metabolic activities of these two cells are highly integrated and based on differential gene expression. The cell-type specific expression of genes is mostly controlled by transcription; however, post-transcriptional regulation has been reported, too. The C4 photosynthetic process concentrates CO2 at the site of Rubisco in the bundle-sheath cells and due to this CO2 pumping device photorespiration is drastically reduced. It follows that C4 plants achieve high rates of photosynthesis and are among the most effective producers of plant biomass. The C4 photosynthetic pathway is of polyphyletic origin indicating that in genetic terms is must have been relatively easy to evolve a C4 from a C3 ancestral species. To understand the evolutionary genomics of C4 photosynthesis and to develop strategies for the introduction of the C4 photosynthetic pathway into C3 species, we are using the Asteracean genus Flaveria as a model system. Flaveria contains C3 and C4 species and, in addition, a large number of C3-C4 intermediates. This genus is therefore a playground of C4 evolution. To gain insight into C3-C4 associated changes in gene expression, the leaf transcriptomes from C3, C3-C4 and C4 Flaveria species were compared by using RNA seq technology. To identify the molecular changes in the cis-regulatory modules for mesophyll and bundle-sheath specific gene expression, respectively, the gene families encoding phosphoenolpyruvate carboxylase and the glycine decarboxylase P protein, respectively, are being investigated. To understand the specific kinetic and regulatory properties the C4 enzymes had to acquire for their functioning in the metabolic context of C4 photosynthesis phosphoenolpyruvate carboxylase is used as a case example. The implications of these findings for engineering C4 photosynthesis will be discussed.


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9. Genetical genomics studies of the rice metabolome Luo J, Chen W1, Gong L1, Gao Y1, Xie W1, Lyu K, Liu X, Yu S, Lian X, Zhang Q 1National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. 2College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. [email protected]

Plants produce far more metabolites than any other organisms that are crucial for both plant and human. Despite recent advances in metabolomics and functional genomics, the processes of identification/annotation of metabolites and their underlying genes are still tedious and time-consuming, and the genetics of most metabolites remain largely unknown. Application of a newly developed widely-targeted metabolomics strategy simultaneously detected hundreds of both primary and secondary metabolites in rice and disclosed a number of subspecies-specific metabolites that may reflect, as well as affect the subspecies differentiation of rice. Distinct and overlapped accumulation was observed and complex genetic regulation of metabolism was revealed in two different tissues. Further genetical genomics studies have uncovered hundreds of loci with high resolution and large effects. Interactive gene/metabolite identification/annotation was facilitated for both functional genomics and metabolomics. Data mining revealed over 30 candidate genes underlying metabolites that are of physiological and agronomical importance, with seven of them experimentally verified. Subsequently, a comprehensive forward-genetics-based metabolic network was constructed. Our study not only reveals novel biochemical and genetic insights of important aspects of plant and human such as development, stress resistance, and nutrition/health-promoting, but also provides vast amount of high-quality data for further understanding plant metabolome which may help bridge the gap between the genome and phenome. The strategy describes here is a powerful tool for large-scale gene identification, pathway elucidation, and for knowledge-based crop genetic improvement.

10. Usage of T-DNA tagging lines An G

Kyung Hee University, Yongin, Republic of Korea [email protected]

We have established over 100,000 T-DNA insertional mutant lines in japonica rice and their insertion positions have been determined. The information is open to public and more than 3,000 lines are distributed yearly to rice community. Because the vectors used for the insertional lines contain the promoterless GUS gene and the enhancer elements of the 35S promoter, the lines can be used for various objectives. I will present examples how we and others are using the mutant lines. For example, activation tagging of OsVIL2, which controls chromatin remodeling by binding to a polycomb group complex, resulted in increasing biomass and grain yield. Some knockout mutants showed beneficial phenotypes toward yield or stress. Although we and several others generated various insertional mutant resources, the number is not enough to cover entire genes. I will discuss the limitations of the current resources and need for enhancing rice research.

11. The biogenesis and diversity of small RNAs in grass inflorescences Meyers B

The University of Delaware, USA. [email protected]

Non-coding RNAs, especially small RNAs, play important roles in many biological processes. Plant small RNA types including microRNAs (miRNAs) and small interfering RNAs (siRNAs), as well as secondary siRNAs that include trans-acting siRNAs (tasiRNAs). Many small RNAs along with their targets have been characterized with deep sequencing technologies, but much remains to be learned about their function. My lab has developed a suite of computational tools for the analysis and visualization of data for small RNAs and cleaved mRNAs (miRNA targets). We have applied these tools to a range of plant species and their variants, such as mutants in small RNA biogenesis or downstream pathways. This has led to the characterization of a number of new miRNAs and their targets, as well as the identification of diverse populations of phased, secondary siRNAs and their miRNA triggers. Most recently, we have focused on phased, secondary siRNAs, like tasiRNAs, and their diverse


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roles in plant gene and genome regulation. I will discuss a novel class of small RNAs (sRNAs) in grass anthers, produced by dispersed loci and synthesized either (1) pre-meiotically during cell fate specification and cell proliferation, or (2) coordinate with germinal cell maturation for meiosis, peak during meiosis, and persisting into mature pollen. Analysis of male sterile mutants indicates there are distinct regulatory features of these small RNAs, both spatially and temporally.

12. Molecular control of tapetal cell death during rice male reproductive development Zhang D

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240. [email protected]

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. We use the tapetal cells as the model to investigate the mechanism underlying programmed cell death in monocot rice (Oryza sativa). We identified several key regulators such as Tapetum Degeneration Retardation (TDR), PERSISTENT TAPETAL CELL 1 (PTC1), MADS3 and MICROSPORE AND TAPETUM REGULATOR 1 (MTR1), ETERNAL TAPETUM 1 (EAT1) in controlling tapetal cell death. TDR positively triggers tapetal PCD by activating the expression of a cysteine protease gene OsCP1. MTR1 encoding a fasciclin domain protein and is specifically expressed in the male reproductive cells, but it is able to affect both tapetal cell death and microspore development, suggesting it’s critical role in coordinating the development of reproductive cells and their adjacent somatic cells. EAT1 encodes a bHLH transcription factor conserved in land plants, positively regulates PCD in tapetal cells in rice. EAT1 directly regulates the expression of two aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that EAT1 acts downstream of TDR, and EAT1 can also interact with the TDR protein. Our study demonstrates that a dynamic regulatory cascade and effective proteases triggering plant PCD, in regulation male reproductive development in rice.

13. Genomics of the zygotic transition in rice: From gametes to embryos Anderson S1 , Johnson C1, Jones D2, Conrad L1, Gou X3, Russell S2, Sundaresan V1 1University of California-Davis, USA; 2Univesity of Oklahoma, USA; 3Lanzhou University, China. [email protected]

Plant reproduction involves the fusion of two highly differentiated gametic cells, an egg cell and a sperm cell, to generate the zygote, a single totipotent cell that will regenerate a new plant. The transition from fertilized egg to autonomous embryo requires extensive changes at the genomic level, including reprogramming of the chromatin, activation of zygotic genes, and removal of maternally-supplied RNA. We have investigated the dynamics of this transition in rice by transcriptome characterization of individual male and female gametes, and isolated time-staged zygotes. The transcriptomes of the gametes, i.e. the egg and the sperm cell, are highly divergent from each other, as well as from that of the pollen vegetative cells that contain and support the sperm cells. The egg cell and sperm cell transcriptomes reveal major differences in expression of genes regulating epigenetic modifications and in the RNAi pathways, suggesting differential silencing of genes and transposons in the two germlines. Although studies in Arabidopsis have proposed delayed activation of the paternal genome as a feature of plant embryogenesis, high expression of genes promoting active chromatin configurations in the rice sperm cell suggests that the paternal genome is ready for activation. Preliminary analysis of time-staged zygotes indicates that partial activation of the zygotic genome and loss of maternal transcripts takes place prior to the division of the unicellular zygote. We conclude that flowering plants, in contrast to animals, initiate the zygotic transition during the earliest cell cycle after fertilization. Further characterization of isolated zygotes, currently in progress, should help elucidate the nature of the genetic and epigenetic factors promoting this fundamental transition in the plant life cycle.


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14. Functional Genomic Analysis of Genetic Variation in Plant Height and Seed Size Induced by EMS in Upland Rice Variety Nagina22

Mohapatra T1,2, Ngangkham U1,2, Kulkarni K1, Robin S3, Sarla N4, Seshashayee M5, Singh AK6, Singh K7, Singh NK1

and Sharma RP1 1National Research Centre on Plant Biotechnology, New Delhi, India; 2Present Address: Central Rice Research Institute, Cuttack; 3Tamilnadu Agricultural University, Coimbatore; 4Directorate of Rice Research, Hyderabad; 5University Agricultural Sciences, Bangalore; 6Indian Agricultural Research Institute, New Delhi; 7Punjab Agricultural University, Ludhiana, India

One of the approaches for understanding the functions of the genes predicted in the rice genome requires use of mutants. Mutants facilitate unveiling the causal relationships between coding/regulatory sequences and plant performance, and also cloning of the corresponding genes. Induced mutations can be efficiently integrated with functional genomics approaches to understand the phenome. In an indigenous effort funded by the Department of Biotechnology (DBT), Government of India, more than 20,000 EMS mutagenised lines have been generated in the background of an upland variety Nagina-22. The uniqueness of this national effort is phenotyping that has led to identification of mutants for a range of traits including plant growth and architecture, flowering, maturity, grain number, shape and size, yield, resistance to blast and bacterial leaf blight diseases, phosphorus use efficiency, and tolerance to drought and salinity. Inheritance of selected mutants has been carried out using F2 populations developed from the cross of the mutants with wild type Nagina22. Mutant loci for seed size and plant height have been mapped using SSR markers on chromosomes 5 and 4, respectively. Transcriptome profiling using rice microarrays has revealed altered expression of only a limited set of genes in these mutants. Two of the mutants, one for plant height and the other for seed size were characterized in detail. The dwarf mutant showed proportionate reduction in each of the internodes as compared to wild type revealing that it belonged to the category of dn-type of dwarf mutants. Besides, exogenous application of GA3 and 24-epibrassinolide did not have any effect on the phenotype of the mutant. The gene was mapped on the long arm of Chromosome 4, identified through positional candidate approach and verified by co-segregation analysis. It was found to encode Carotenoid Cleavage Dioxygenase7 (CCD7) and identified as an allele of htd1. The mutant carried substitution of two nucleotides CC to AA in the sixth exon of the gene that resulted in substitution of serine by a stop codon in the mutant, and thus formation of a truncated protein, unlike amino acid substitution event in htd1. A short grain mutant was similarly characterized. Exogenous application of auxin, cytokinin, gibberellin and brassinosteroid in this mutant did not have significant effect on grain size in the mutant. The cells were shorter, narrower, and lesser in number in the mutant as compared to the wild type. The candidate genomic region was identified on short arm of chromosome no. 5 to an interval of 250kb. Among the 60 protein coding genes present in 250kb genomic region, one (srs3) was already reported to control grain size in rice. It encodes kinesin 13 family proteins with a major role in mediation of microtubule organisation during mitosis. Sequencing of 11kb srs3 gene region revealed a substitution of C to T in the coding region of the mutant leading to creation of a stop codon. This resulted in synthesis of a truncated protein of 459 amino acid residues as against the 1100 long amino acid in the wild type Nagina22. The relationship between the mutation and the phenotype was validated by sequencing the segregants from mutant x IR64 population having contrasting parental phenotypes. Sequencing of 96 germplasm lines including short and long grains in the kinesin motor domain region of the gene did not reveal presence of this mutation. Functional analysis of the two mutants revealed new alleles generated by EMS mutagenesis.


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15. Rice genome variation and domestication studies Han B

National Center for Gene Research & Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China. [email protected]

Rice is one of the most important crops in the world. The cultivated rice (Oryza sativa), which is grown worldwide, is considered to be domesticated from wild rice (Oryza rufipogon) thousands of years ago. The differences are reflected in a wide range of morphological and physiological traits. The current rice germplasm reserved in the world will be the most important resource for rice genetic improvement for humanity. So it is imminent to profile the genetic diversity of both wild and cultivated rice species for breeding elite variety. We performed large-scale genome sequencing for analyzing genome-wide genetic variations to further understanding of rice gene functions in agronomic traits and for bridging the knowledge gap between genotype and phenotype in rice. To construct a comprehensive map of rice genome variation, we collected wild rice and cultivated rice germplasm throughout the entire geographic range of rice in the world. We sequenced and analyzed the genomes of the collected rice germplasm. The constructed map of rice genome variation thus enabled investigation of the domestication processes and identification of domestication associated genes. This study provides an important resource for rice breeding and demonstrates an effective genomics approach for crop domestication research. The results and discussion will be presented.

16. Rice as a model species to study the impact of Transposable Elements on plant genome structure and evolution.

Panaud O

Laboratoire Génome et Développement des Plantes, UMR 5096 CNRS/UPVD. Université de Perpignan Via Domitia

Transposable Elements (TEs) are ubiquitous components of eukaryotic genomes and thus strongly impact their structure, function and evolution. Rice, with a high quality genome sequence, has been a model of choice to study TEs at the whole genome scale. Pioneering work on this species allowed us to define the « increase/decrease » model for TE-driven genome evolution. Recent comparative genomics surveys across plant kingdom showed that this model holds true for both monocots and dicots as well as large and small genome species : plant genomes recurrently increase in size through catastrophic retrotranpositional bursts, while these TE-related sequences are quickly eliminated through recombination and/or deletions, therefore leading to a very rapid turn-over of the intergenic sequences. The availability of several genome sequences of wild rice relatives (iOMAP initiative) opens new perspective to study genome evolution at a shorter time scale (less than one Million years) and therefore to refine our model, in particular for what concerns the dynamics of TE elimination. Finally, we will present some recent results on horizontal transfers of TEs across plant kingdom and propose a new hypothesis for their origin as well as their evolutionary success among higher eukaryotes.


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17. Introgression occurs in the rice field Hsing Y-‐I, Wei F-‐J, Lai M-‐H, Huang L-‐T, Chen Y-‐A

Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan. Department of Agronomy, National Taiwan University, Taipei, Taiwan. Crop Division, Taiwan Agriculture Research Institute, Taichung, Taiwan. [email protected]

Being a self-pollinated crop, the outcrossing rate of the cultivated rice is in general low. Nevertheless, pollen-mediated gene flow from cultivated rice to weedy rice, wild rice, and wild relative barnyard grass under field conditions has been reported. The gene flow was estimated in the open field using two japonica rice varieties, with one non-waxy variety Tainung 67 (TNG67) and another waxy variety TNG73. The mean frequency of outcrossed seeds was 1.68%, 0.74% and 0.61% at 1, 2 and 3 meters, respectively (Tseng et al., 2012). By T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing promoter trap and gene activation/knockout lines using a japonica rice cultivar TNG67, with the website at http://trim.sinica.edu.tw. We are currently estimating the sequence changes of the TRIM mutant line using NGS. We noticed one specific line had very high SNP rates, compared with the published data. We found out that more than 10% of the chromosome regions were indica sequences, that is, introgression occurred in the field seasons ago before this specific seed was used for transformation. In addition, we also found out Taichung 65, a japonica variety with the parental lines Shinriki and Kameji, must also have a major introgression from other land race. We’ll present the detail results at the meeting.

18. MicroRNAs (miRNAs) in plant innate immunity: miR7695, a novel miRNA from rice involved in pathogen resistance

Baldrich Patricia, Peris-‐Peris Cristina, Campo Sonia, Wu Ming-‐Tsung, Hsing Yue-‐Ie and San Segundo Blanca

Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB. Campus Autonomous University of Barcelona (UAB). Bellaterra (Cerdanyola del Vallés). 08193 Barcelona, Spain. [email protected]

Plants have evolved an innate immune system to counteract pathogen infection. Increasing evidence is emerging to support the notion that host endogenous small RNAs represent a fundamental layer of post-transcriptional gene regulation for fine-tuning of gene expression in host adaptative responses to pathogen infection. Our interest is to understand the regulatory roles of miRNAs in plant immunity. Towards this end, we approach the identification and functional characterization of pathogen-regulated miRNAs from rice (rice/Magnaporthe oryzae interaction). High-throughput sequencing of small RNA libraries prepared from elicitor-treated rice tissues (leaves, roots) revealed dynamic alterations on the accumulation of a diverse set of known miRNAs, both conserved and non conserved miRNAs. The identification of novel miRNAs from rice is also pursued in our group. Transcriptome analysis in combination with degradome sequencing is being used to investigate target gene expression for known miRNAs while allowing the identification of target genes for novel miRNAs. A new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6), has been recently identified. This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers resistance to infection by the rice blast fungus M. oryzae. Finally, recent progress on the identification of rice mutants (Taiwan Rice Insertional Mutants, TRIM) affected in small RNA biogenesis will be presented. The discovery of novel pathogen-regulated miRNAs and their mode of functioning offers new opportunities to develop novel strategies for rice protection against the rice blast disease.


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19. Induced disease resistance in rice through salicylic pathway and its modification by genetic engineering

Takatsuji H, Goto S, Shimoda FS, Suetsugu M, Ueno Y, Yoshida R

National Institute of Agrobiological Sciences, Disease Resistant Crops Research Unit, and Tsukuba University, Graduate School of Life and Environmental Sciences. Tsukuba Japan. [email protected]

Rice blast, caused by the fungus Magnaporthe oryzae, and bacterial leaf-blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), are among the most devastating diseases of cultivated rice worldwide. Chemical defense inducers such as benzothiadiazole (BTH) and probenazole protect rice plants from these diseases by acting on the salicylic acid (SA) signaling pathway. A transcription factor WRKY45 plays a central role in this defense-signaling pathway and its overexpression in rice (Oryzae sativa cv. Nipponbare) conferred extremely strong resistance against the two diseases, as well as brown spot disease due to Cochliobolus miyabeanus. However, substantial levels of adverse effects on the growth and yield, which were exacerbated by environmental factors such as low temperature and high salinity, were observed in WRKY45-overexpression rice. To address this problem due to the tradeoff between WRKY45-dependent disease resistance and rice yield, we attempted to optimize WRKY45 expression by the use of a constitutive promoter with moderate activity or a pathogen-inducible promoter in combination with a translational enhancer from rice alcohol dehydrogenase gene. In both the cases, the transformants exhibited favorable growth and yield traits in the field, which were nearly comparable with untransformed rice, even under the abiotic stresses, while retaining strong disease resistance. Thus, we introduced the optimized constructs to a forage rice cultivar to develop disease resistant transgenic forage rice lines that are amenable to low-cost cultivation. Low temperature suppresses the induction of SA-pathway-dependent disease resistance by chemical defense inducers, and the suppression is mediated by activation of ABA signaling. We showed that a MAP kinase, which phosphorylates WRKY45, is a node of convergence of the antagonistic crosstalk of ABA signaling to the SA pathway, leading to suppression of WRKY45-dependent disease resistance.

20. Induction and suppression of DAMP induced innate immunity in rice-Xanthomonas interactions

Sonti RV

CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad

Plants have powerful inducible innate immune responses that protect them against the vast majority of potential pathogens. These immune responses are induced following the detection of either MAMPs (microbe associated molecular patterns) or DAMPs (damage associated molecular patterns). We are studying the mechanisms by which plant innate immune responses are induced and suppressed using the interaction between rice and the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo) as a model. We had previously shown that secreted cell wall degrading enzymes such as two Cellulases (ClsA and CbsA), Lipase/esterase (LipA), and Xylanase (XynB) are important virulence factors of Xoo. Conversely, these enzymes are potent inducers of rice immune responses as their activity in degrading the cell wall releases DAMPs that serve as a mark of infection. Xoo is able to cause disease only because it has the capacity to suppress rice innate immunity, a task which it accomplishes using proteins that are secreted into plant cells using the Type 3 secretion system (T3S). We are exploring the role of Xoo T3S secreted proteins in suppression of innate immunity and the role of specific rice genes in induction of innate immunity. Our results obtained using Agrobacterium mediated transient transfection assays indicate that four Xoo T3S secreted proteins, called XopN (Xanthomonas Outer protein N), XopQ, XopX and XopZ are involved in suppression of cell wall damage induced rice innate immunity. Recent results on characterization of the XopQ protein will be presented. The characterization of certain rice functions whose expression is upregulated at early stages after cell wall damage, and which might be involved in signal transduction, will also be discussed.


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21. Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm

Zilberman D

Department of Plant and Microbial Biology, University of California, 211, Koshland Hall, Berkeley, CA 94720, USA. [email protected]

Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally-inherited chromosomes. Demethylation mediates parent-of-origin-specific (imprinted) gene expression, but is apparently unnecessary for the extensive accumulation of maternally-biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin-specific was unknown. Imprinted expression of sRNA was also uninvestigated in monocot seeds. We found that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini, and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small Class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.

22. Identification of genes/alleles regulating grain development in rice Tyagi AK1, Parida SK1, Bajaj D1, Das S1, Malick N1, Anand D 2, Kapoor S 3, Singh AK 2, Agarwal P1 1National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India, 2 Division of Genetics, Indian Agricultural Research Institute, New Delhi 110012, India, 3 Department of Plant Molecular Biology, Delhi University South Campus, New Delhi 110021, India. [email protected]

We are working to define major genes related to seed development by forward and reverse genetics approaches. Specific gene targets from HAP and MED gene families have been taken-up for functional analysis based on their expression characteristics. More recently, SNP marker-based genome-wide association (GWAS) mapping has been utilized for dissection of complex quantitative grain size traits in rice. Genotyping-by-sequencing (GBS) of 96 phenotypically and genotypically diverse rice genotypes contrasting for three major grain size traits identified 199,085 SNPs, including 105,517 SNPs in 31,267 genes. The reliability of SNPs identified through GBS analysis has been ascertained by their large-scale validation in a selected set of genes using Illumina GoldenGate genotyping assay and cloned amplicon sequencing. Genome-wide linkage disequilibrium (LD) decay and GWAS analysis identified 82 target genomic loci showing strong association with grain length, grain width and grain weight in rice. A selected set of these grain size-associated genes has been validated through SNP haplotyping and traditional bi-parental linkage mapping. The identified genetic loci and haplotypes regulating grain development have potential for genetic improvement in rice.


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23. Systems level analysis of photosynthetic carbon metabolism and grain yield in rice

Pereira A, Ambavaram MM, Krishnan A, Venkategowda R, Basu S, Batlang U, Baisakh N

University of Arkansas, Fayetteville, AR, USA, 6AG2286WW, [email protected]

To dissect the complex traits of grain yield and stress tolerance in rice, and understand the network of genes and biological processes involved, we used publicly available rice gene expression data to develop a rice gene regulatory network that represents the association of biological processes/pathways to all rice transcription factors (TFs). We identified a TF gene HYR that is positively associated with photosynthetic carbohydrate metabolism (PCM), including key biological processes affected by environmental stresses. HYR is upregulated by drought in vegetative and reproductive stages, and was used in overexpression constructs to transform rice genotypes for functional analysis. Morpho-physiological analysis of the rice HYR lines showed an increase in biomass, water use efficiency, root growth, photosynthesis and sugars under normal and drought stress conditions at vegetative stage. HYR lines exhibited higher yield when treated to drought or high nighttime temperature at critical reproductive stages. Gene expression profiles of HYR lines revealed that genes involved in PCM, other metabolic and regulatory processes were significantly up-regulated. Affinity-tagged HYR expressing rice lines were used to show binding of HYR protein in vivo to promoters of genes involved in photosynthesis namely LHC2, PSII and electron transport, and also to promoters of TFs regulating carbon metabolism. Direct binding and regulation was further validated using estrogen-inducible expression of HYR and other downstream TFs in rice protoplast assays showing induction/repression of specific rice promoters. The results suggest a model of HYR as a master regulator of PCM and stress responsive processes, maintaining the expression of genes involved in plant growth and grain yield under environmental stresses.

ABIOTIC STRESS 1 Chair: Anil Grover, San Segundo Blanca

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Concurrent Session I

1. Hot topic: molecular biology of ‘heated’ rice seedlings Anil Grover

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India [email protected]

Rice (Oryza sativa L.) is the most important food crop. For future breeding of heat-resistant rice cultivars, there is a need to identify the molecular components that underlie the adaptation of rice to sub-lethal heat stress levels. While literature is replete with information on heat stress response of Arabidopsis, account of heat stress response in rice is far from complete. Our research aims to fill up this gap. We note that the global transcriptome/ proteome profiles of rice tissues are rapidly altered upon heat stress. The rice transcriptional profile is significantly modulated within 10 min of heat stress: the enrichment of GO terms protein kinase activity/ protein serine kinase activity, response to heat and reactive oxygen species in up-regulated genes after 10 min signifies the role of signal transduction events and reactive oxygen species during early heat stress. Overall, the response of rice to heat stress involves signal perception and transduction, activation/ synthesis of heat shock factors, genomic and proteomic alterations, ROS metabolism and a host of other proteins associated with biochemical, cellular and physiological processes and ‘unknown’ functions. We note that proteins associated with chaperones and protein degradation machineries of the cell are stimulated as one of the early steps in minimizing damages to other proteins. The synthesis of heat shock proteins appears an important response to heat stress: high and rapid up-regulation of heat shock protein genes is actually a hallmark of high temperature stress response. Most heat shock proteins reportedly function in protecting cells against damage due to high temperature by stabilization and/or refolding of denatured proteins. Genome-wide biology of rice Hsp20, Hsp40, Hsp70 and Hsp100 as well as heat shock factors has been unearthed from our studies. Hsp100 is a major heat-regulated protein family in diverse organisms. Our group identified that yeast Hsp104 equivalent Hsp100 protein is synthesized in rice against heat stress. We have shown that rice ClpB/Hsp100 family proteins is constituted of three isoforms which are localized in three different cell compartments namely cytoplasm (ClpB-c), chloroplast (ClpB-p) and mitochondria (ClpB-m). Extensive work carried out on the structure, function and regulation biology of rice ClpB-c/Hsp100 gene will be presented.

2. Expression of DNA pol lambda enzyme in rice (Oryza sativa L) seed germination and enhancement after high salinity or dehydration stress

Sengupta DN1*, Sihi S1, Bakshi S2 1 Division of Plant Biology, Bose Institute, 93/1 A.P.C. Road, Kolkata-700009, West Bengal, India. 2 Vidyasagar College For Women, 39 Sankar Ghosh Lane Kolkata-700006, West Bengal, India. [email protected]

Replication and repairing of genomic DNA are necessary for stable maintenance during growth and differentiation so that successful transfer of genetic material is possible. DNA polymerase enzymes are known to synthesize DNA using a template DNA strand and the 3’OH end of a nucleic acid (DNA/RNA) act as a primer. X-family DNA Pol λ and β are known to be involved in DNA repairing. While both are present in human system, in plant system only DNA Polλ has been detected. While the cDNA for DNA Polλ has been cloned from Arabidopsis and japonica rice and characterized from E.coli expressed protein, very little is known about its activity at protein level in plants. Here we report on the DNA Pol λ enzyme activity after partial purification and compared it during the progress of IR-8 indica rice seed germination. By in vitro DNA Polλ activity assay, in-gel activity assay and western blot analysis the enzyme was detectable after imbibition and found to be enhanced from low level to high level upto 3 days/72 h during the germination steps. On the upstream sequence of its gene presence of several cis-acting elements including LREs, DRE, Myb binding sites, etc. have been detected. Therefore 4-day-old germinated seedlings of IR-29, a salt sensitive but high yielding and Nonabokra, a salt tolerant but low yielding cultivar, were treated with water (control) or 250 mM NaCl or 20% Polyethyleneglycol-6000 for 4 and 8 hours. Both salinity and dehydration stress were found to enhance the activity and the protein level, whereas in control tissue the activity was very low and the protein was undetectable in both the cultivars. This was the first evidence of the salinity or dehydration stress induced DNA Pol λ activity in the plumules of rice cultivars.


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3. The role of OsHOS1, an E3-ubiquitin ligase involved in abiotic stress response and root mechano-sensing

Lourenço T, Serra T, Sapeta H, Abreu I, Gilroy S, Saibo N, Oliveira M

ITQB - Universidade Nova de Lisboa and iBET, Av. da República, 2780-157 Oeiras, Portugal. [email protected]

The proteosome-ubiquitin system controls the abundance of key proteins involved in plant stress responses. This control mediates a rapid and precise transcription regulation and physiological changes that, eventually, lead to plant stress adaptation. To better understand the involvement of the proteasome in abiotic stress responses in rice, we have investigated the role of an E3-ubiquitin ligase (OsHOS1) in the modulation of the response. Using a RNA interference (RNAi) transgenic approach, we found that, under cold conditions, the RNAi::OsHOS1 plants showed a higher expression level of OsDREB1a (1). This was correlated with an increased amount of OsICE1, a master transcription factor (TF) of the DREB1 regulon. In addition, we showed that OsHOS1 interacts with OsICE1, confirming the involvement of the proteasome in this response mechanism. Interestingly, when grown in flasks, the RNAi::OsHOS1 plants showed a straight root phenotype as compared to WT. We showed that this phenotype is due to the sensing of a mechanical barrier and that it could be reverted by exogenous application of jasmonic acid. The straight root phenotype of the OsHOS1 silencing plants was correlated with a higher expression of OsRMC (ROOT MEANDER CURLING), a gene involved in salt stress response and root curling. In order to better understand the modulation of OsRMC gene expression, we used the Yeast-One hybrid system and identified two transcription factors binding to its promoter (2). In addition, we showed that OsHOS1 interacts with these two TFs. These results suggest that OsHOS1 regulates OsRMC expression through interaction with the identified TFs. Further evidences of the involvement of OsHOS1 in the modulation of abiotic stress responses and root mechano-sensing will be present and discussed.

4. Dissection of genetic regulatory networks of drought stress response in rice using knockout mutants

Venkategowda R, Basu S, Ambavaram MRM, Krishnan A, Pereira A

Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA. [email protected]

Drought is one of the major environmental stress factors faced by crops. In the wake of global climate change the incidence of drought is likely to increase in the future resulting in substantial yield loss in major crops like rice. Therefore, understanding the molecular response of rice plants to drought stress is necessary to provide opportunities to develop cultivars tolerant to stress. In this study, using multiple publicly available rice gene expression datasets, a Rice Environment Coexpression Network (RECoN) was constructed. Rice drought transcriptome data of seedling, vegetative and reproductive stages was then used to mine RECoN and derive sub-networks of drought transcriptional clusters enriched for drought stress responsive genes. 1347 regulatory genes comprising of transcription factors, kinases, phosphatases and other signaling genes which are up- or down-regulated under drought stress were selected from drought sub-networks for perturbation analysis using knockout mutants to unravel the underlying biological processes involved in drought resistance. T-DNA or Tos17 retrotransposon insertion lines were identified in cultivar Nipponbare from publicly available rice knockout mutant resources for 126 genes and homozygous mutant lines were identified for 74 genes by genotyping. Testing for the ABA response of the insertion mutant lines in a plate based assay showed altered growth of 20 mutant lines. Drought stress response analysis of ABA responsive mutant lines under controlled drought stress treatment showed significant reduction in biomass, photosynthetic capacity and instantaneous water use efficiency in 10 mutant lines which include four transcription factors and five kinases. To unravel the signal transduction events controlled by these transcription factors and kinases, RNA-seq and phosphoproteome analysis is being carried out and the results will be presented.


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5. Mangroves as a novel source for developing abiotic stress tolerant crop varieties

Parida A

M. S. Swaminathan Research Foundation, III Cross Street, Institutional Area, Taramani, Chennai, India. [email protected]

Agricultural productivity in the coastal region is severely affected due to the impact of salinization of soil and water. Sustaining agriculture in these regions would require employing new and advanced technologies. In this context, we have used halophytes and mangroves for developing abiotic stress tolerant genotypes. Mangroves and halophytes have evolved over million years to cope with extreme conditions of abiotic stress, which is evident by their unique structural adaptations. Thus, mangroves serve as a valuable source of stress tolerance genes. Food crops particularly Rice is grown extensively in coastal lands, which face the danger of sea water intrusion due to climate change and its effects. MSSRF initiated an anticipatory research programme to identify and isolate genes for salt tolerance from mangroves and their associates that are naturally tolerant to salinity, and later transfer them to salt sensitive locale-specific cultivars of crops to render them salt tolerant. As a first step towards the identification of genes that contribute to combating salinity stress, cDNA libraries were prepared from A. marina, P. coarctata and other mangrove associates. Random expressed sequence tag (EST) sequencing of a cDNA library prepared from salt treated seedlings of A. marina suggested that several genes were specific or unique for the plant and were not previously characterized. Differential expression analysis using RNA gel blot analysis at different time points after salt treatment revealed that twenty six genes were up regulated and five were down-regulated after salt treatment. Thirty six full length genes have been isolated from A. marina representing genes related and were showing homology to Reactive Oxygen Species scavengers, and genes involved in Ion homoestasis Some of these characterised genes were introduced into rice for developing location specific crop varieties in locally adapted cultivars of Rice. These studies have provided information on how these genes help in enhancing their abiotic stress tolerance. The studies revealed that mangroves could be potential sources for isolating novel genes for abiotic stress tolerance and are capable of tolerating a high degree of salinity and abiotic stress.

6. Quest for stress-responsive proteins in the nucleus: a comparative proteomic approach

Chakraborty N

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India. [email protected]

National Institute of Plant Genome Research, Nucleus is the regulatory hub and a dynamic repository of various molecules that dictate cellular signaling. It not only hosts the genome but also administers its transcription and the regulated expression of proteins, thereby playing a critical role as a modulator of cellular phenotype. Nuclear proteins constitute a highly organized but complex network that play diverse roles during plant development and stress response. In an attempt to dissect the molecular circuitry of stress tolerance in plants, we examined the nuclear proteome of rice (Oryza sativa L.) under dehydration. A critical screening of the proteome led to identification of an Alba-family protein, designated OsAlba1, distantly related to the archaeal DNA/RNA-binding Alba protein. Here we describe, for the first time, the complete sequence of OsAlba1, its genomic organization, and its possible function. Phylogenetic analysis shows its close proximity with Alba proteins from other monocots and suggests a separate lineage from dicots. DNA interaction site prediction indicates a possible DNA-binding property for OsAlba1. The OsAlba1-GFP fusion protein revealed that OsAlba1 localizes to the nucleus and also sparsely to the cytoplasm. The OsAlba1 transcripts are upregulated by dehydration, cold and high salinity, along with abscisic acid (ABA) treatment, suggesting that its stress-responsive function might be associated with ABA-dependent network. The preferential expression of OsAlba1 in the flag leaves indicates its possible role in panicle development, grain filling. Our findings suggest that the Alba-family proteins, especially from plants where there is no evidence for a major chromosomal role, might play important function in stress adaptation.


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7. Multigene engineering bridges the yield gap in rice under abiotic stress conditions

Sahoo KK1, Gupta B1, Tripathi AK1, Pareek A2, Sopory SK1 and Singla-‐Pareek SL1 1Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi- 110 067, India. 2Stress Physiology and Molecular Biology, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110 067, India. [email protected]

Considering the multigenic nature of the stress response in plants, gene pyramiding or multigene engineering is a promising approach to develop stress tolerant plants. Glyoxalase pathway enzymes, viz. glyoxalase I (Gly I) and glyoxalase II (Gly II), are the two enzymes required for glutathione-based detoxification of a metabolically produced cytotoxin methylglyoxal. Due to the presence of sodium proton antiporter (NHX) in membrane, vacuole functions to sequester excess of Na+ ions thus contributing to cellular ion homeostasis. In the present study, we have shown that gene pyramiding using GlyI, GlyII and NHX significantly improves tolerance to multiple abiotic stresses in both tobacco and rice. The photosynthetic efficiency of triple (GlyI+GlyII+NHX) transgenic tobacco and rice lines was significantly higher than double (GlyI+GlyII) or single (GlyI) gene transgenic plants and they were able to flower and set more seeds under salinity and drought conditions. Further, as compared to wild type, single or double gene transgenic plants, the yield penalty was significantly lower in the plants expressing all the three genes (GlyI, GlyII and NHX) under such stress conditions. These results suggest that strategies of pyramiding genes to remove the cytotoxic metabolites like methylglyoxal (by engineering glyoxalase system) and sequestration of sodium ions (by engineering ion transporters) may significantly improve stress tolerance limits. Future studies may utilize a similar strategy to generate stress tolerant varieties in other agronomically important crops as well.

8. Expression and functional analysis of calcium transport elements in rice during abiotic stress

Pandey GK, Singh A, Yadav AK, Mishra M, Baranwal V, Pandey V, Kapoor S, Tyagi AK

Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi-110021, India. [email protected]

Changes in the cytosolic Ca++ are one of the primary response to external stimuli such as biotic and abiotic stresses. In response to various stimuli, generation of specific “Ca2+ signature” takes place, which encompasses differences in Ca2+ oscillation frequency, amplitude and location. The specific changes in the Ca2+ signature provide vital clue about the nature and intensity of the stimuli to the cell. Specific Ca2+ signatures are generated due to spatial and temporal Ca2+ fluxes in response to stimuli, which are mediated by regulated activity of membrane localized calcium channels and other calcium transporting elements. In plants, there are three major classes of calcium transporters; channels, pumps (ATPases) and exchangers. Apart from these, some groups of protein have been identified in plants, which non-specifically transport Ca2+ and include cyclic nucleotide gated channels (CNGC), glutamate receptor homologs (GLR) and annexins. Various calcium transporters have been implicated in a number of cellular processes such as hormone responses, biotic and abiotic stress responses, light signaling and development in plants. Majority of these findings have been established in Arabidopsis and other plant species but knowledge is minuscule about the role of the Ca2+ transporting elements in crop plants especially rice. My group is focusing on functional characterization of calcium transport element in rice by undertaking a detail genomic, expressional, and functional characterization of several representative members of this calcium transport elements. Exhaustive in-silico data mining and analysis resulted in the identification of 81 Ca2+ transport element genes, which belongs to various groups such as Ca2+-ATPases (pumps), exchangers, channels, glutamate receptor homologs (GLRs) and annexins. Phylogenetic analysis revealed that different Ca2+ transporters are evolutionary conserved in rice and Arabidopsis. Comprehensive expression analysis by gene chip microarray and quantitative RT-PCR revealed that a substantial proportion of Ca2+ transporters genes were expressed differentially under abiotic stresses (salt, cold and drought) in rice. These findings suggest possible role of rice Ca2+ transporters in abiotic stress triggered signaling pathways. Subcellular localization of Ca2+ transporters from different groups in Nicotiana benthamiana and onion epidermal peel cells revealed their variable localization to different compartments, which could be their possible site of action. A detail


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Concurrent Session I complementation of rice Ca2+ transporter in different mutants of yeast defective in various calcium transport related gene lead to identify their functional role during abiotic stress such as salinity and heavy metal tolerance.

ARCHITECTURE & DEVELOPMENT Chair: Narayan Upadhyaya, R Srinivasan

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Concurrent Session II

9. The heading date genes Ghd7, Ghd8 and Ghd7.1 enhanced rice grain productivity under long-day conditions

Zhang J, Zhou X, Liu H, Yan W, Xing Y

National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan, 430070 China. [email protected]

Heading date is a key factor to determine the growing seasons and regional adaptability of rice cultivars. Natural selections under seasonal changes in day length make rice flowering photoperiod sensitive. Several photoperiod flowering genes have been cloned in rice and Arabidopsis. Comparative analysis indicated that they shared a similar CO/Hd1 mediated photoperiod flowering pathway. Besides, rice has a unique Ehd1 mediated flowering pathway. Via map based cloning, we isolated three flowering genes Ghd7, Ghd8 and Ghd7.1, which simultaneously and dramatically affected the performance of grain number, plant height and heading date. Photoperiod treatment of the near isogenic lines (NILs) for Ghd7, Ghd8 and Ghd7.1 showed that difference in the three targeted traits between paired NILs sharply decreased under short-day conditions, while greatly increased under long-day conditions (LD). These results indicated that the three genes are photoperiod sensitivity. Ghd7 and Ghd7.1 belong to CCT family, respectively. Ghd8 was classified into HAP2 family. They exhibited diurnal expression patterns and peaked in the morning. Under LD, all the three genes promoted the expression of Ehd1 and suppressed the expression of Hd3a, which resulted in a delayed flowering and caused a high grain yield under LD. Sequencing of a rice germplasm with broad genetic diversity indicated that there were plenty of natural variations of the three genes. Ghd7 alleles showed a distinct geographic distribution, that is, the cultivars carried nonfunctional or weak alleles with short life cycle were grown in the temperate regions or as early rice grown in double-cropping season regions. The single-cropping rice in tropic and sub-tropic regions frequently carried strong alleles. Comparative sequencing of Ghd7.1 between cultivars and wild rice indicated that retention of its pre-exsiting genetic variants in ancestral species and the acquisition of mutations after domestication together made great contributions to rice adaptation. Reasonable allele combinations among the three genes provide the flexibility to establish an ideal balance between vegetative growth and reproductive growth duration for breeding rice cultivars, which can fully take advantage of light and solar energy resources in specific eco-geographic regions.

10. A trifunctional rice germin like protein plays a critical role in grain development Tsakispaloglou N, Raorane M, Chuba T, Kohli A

Plant Molecular Biology Laboratory, PBGB, IRRI, DAPO 7777, Manila, Philippines. [email protected]

Germin-like proteins (GLPs) are a class of plant specific proteins. GLPs contain the conserved cupin domain but could otherwise be active in various processes and perform different enzymatic functions. Most GLPs that have been characterized have a role in pathogen response. A cluster of GLPs on chromosome 8 is also responsive to pathogens except one among them. We have characterized this GLP (OsGLP8960) in detail. It is a trifunctional protein. Like most known mono- or bi-functional GLPs it acts as a superoxide dismutase (SOD) and oxalate oxidase (OxOx). Another important activity identified in one GLP of barley in leaf tissue was the nucleotide sugar pyrophosphatase (NSPPase) activity. It has proven elusive to find this activity in any cereal endosperm where it can make a difference to the process and/or rate of starch synthesis. We have been able to show that OsGLP8960 possesses NSPPase activity. The three activities of OsGLP8960 depend on differential oligomerization. Such oligomerization state changes at different stages of rice grain development leading to more or less activity of the three-enzymatic activities associated with it. The OsGLP8960 is developmentally upregulated in the panicle tissue but more in spikelets and in the developing grain. Various lines of evidence have been accumulated to confirm that it has a critical role in grain filling and may have a role in grain quality. Its SOD and OxOx activities may be involved in plant cell death (PCD) during endosperm development while its NSPPase activity may affect starch synthesis. This is the first time that the elusive NSPPase activity has been localized for a protein expressed in the endosperm thus supporting the hypothesis that cereal grain starch synthesis is a balance between its synthesis and breakdown. The results also underscore the value of homo-oligomer-mediated capacity of a protein to perform enzymatic activities for which there are This is the first time that the elusive NSPPase activity has been localized for a protein expressed in the endosperm thus supporting the hypothesis that cereal grain starch synthesis is a balance between its synthesis and breakdown. The results also underscore the value of homo-oligomer-mediated capacity of a protein to perform enzymatic activities for which there are no discernible domains in the monomer.


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11. Rice MADS29: Its role in embryo and endosperm development and regulation of its function

Nayar S1, Sharma R1*, Kapoor M2, Tyagi AK3, Kapoor S1 1Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. 2University School of Biotechnology, Guru Gobind Singh Indraprastha University, Dwarka, New Delhi New Delhi-110078, India. 3National Institute for Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India. *Present address: Department of Plant Pathology, University of California, Davis, CA 95616, USA. [email protected]

The analyses involving transcript and protein profiles and molecular characterization of the gain-of-function and knockdown phenotypes have indicated the role of OsMADS29 in embryo and endosperm development by affecting hormone homeostasis. The MADS29 transcripts accumulated to high levels soon after fertilization; however, protein accumulation was found to be delayed by at least 4 days. Immunolocalization studies revealed that the protein accumulated initially in the dorsal-vascular trace and the outer layers of endosperm, and subsequently in the embryo and aleurone and subaleurone layers of the endosperm. Ectopic expression of MADS29 resulted in a severely dwarfed phenotype, exhibiting elevated levels of cytokinin, thereby suggesting that cytokinin biosynthesis pathway could be one of the major targets of OsMADS29. Overexpression of OsMADS29 in heterologous BY2 cells was found to mimic the effects of exogenous application of cytokinins that causes differentiation of proplastids to starch-containing amyloplasts and activation of genes involved in the starch biosynthesis pathway. Suppression of MADS29 expression by RNAi severely affected seed set. The surviving seeds were smaller in size, with developmental abnormalities in the embryo and reduced size of endosperm cells, which also contained loosely packed starch granules. Microarray analysis of overexpression and knockdown lines exhibited altered expression of genes involved in plastid biogenesis, starch biosynthesis, cytokinin signaling and biosynthesis. We have also found that MADS29 interacts with several seed-expressed transcription factors and its capability to localize in the nucleus is affected by these interactions. Taken together, these data suggest that MADS29 is monocot-specific transcription factor that affects multiple aspects of seed development in rice and probably in other cereals as well.

12. OsGI controls flowering time via balancing various floral regulators and is modulated by phytochromes

Lee Y-‐S, Yi J, Lee D-‐Y, An G

Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. Department of Plant Systems Biotech, Kyung Hee University, Yongin 446-701, Korea. [email protected]

Day length is one of the most important factors determining flowering time in a facultative short day plant, rice. It was previously reported that OsGI and SE5 are concerned with photoperiodic sensitivity. In this study, we observed that osgi mutants lost the photoperiod sensitivity, flowering at about 90 days after germination regardless of the photoperiod. We also observed that osphyA osphyB double mutants flowered 60 days after germination under both SD and LD. Transcript levels of most photoperiodic flowering regulators were significantly decreased in the osgi mutants, but chromatin remodeling factors were not significantly changed. We observed that transcript levels of OsGI were decreased in the osphyA osphyB mutants. Transgene analysis supported that Phy genes control OsGI expression. However, flowering activators such as Ehd1, Hd3a and RFT1 were dramatically increased in the double mutants. These results indicate that there should be another pathway to control Ehd1 expression by phytochromes. In addition to, diurnal patterns of OsGI were changed and decreased at daytime in the double mutants. For this reason, those of other flowering regulators such as Ehd1, Hd3a and RFT1 were also changed in the mutants. These observations indicate that phytochromes are needed to maintain OsGI expression under light and that OsGI controls flowering time via balancing various regulators.


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13. OsRPA2c Coupled with OsRPA1c Regulates Crossover Formation during Meiosis in Rice

Wu C, Li X, Chang Y, Xin X, Zhu C

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, Hubei Province, China [email protected]

Replication protein A (RPA) is a conserved heterotrimeric protein complex, comprising RPA1, RPA2, and RPA3 subunits, and is involved in multiple DNA metabolism pathways due to its single- stranded DNA-binding property. Arabidopsis and rice have multiple genes for most RPA subunits. Our previous investigation revealed that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice. However, the precise functions of the other RPA subunits in DNA metabolism have never been elucidated in rice. Recently, we have identified OsRPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (osrpa2c) exhibited reduced bivalent formation, leading to chromosome non-disjunction. In osrpa2c, chiasma frequency is reduced by ~78% compared to the wild type and accompanied by loss of the obligate chiasma. The residual ~22% chiasmata fit a Poisson distribution suggesting loss of crossover control. OsRPA2c showed a dynamic chromosome localization pattern from pre-meiotic S-phase to pachytene. At S/G2 phase, OsRPA2c co-localized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of OsRPA2c to the chromosomes. In addition, OsRPA2c partially co-localized with OsMER3 during late-leptotene, thus indicating that OsRPA2c is required for a late stage in class I crossover formation. Furthermore, we identified OsRPA1c, an RPA1 subunit with nearly overlapping distribution to OsRPA2c, required for ~79% chiasmata. Our results demonstrate that an RPA complex comprising of OsRPA2c and OsRPA1c is required to promote meiotic crossovers in rice.

14. Functional characterization of RFL as a regulator of rice inflorescence architecture

Deshpande G, Ramakrishna K, Vijayraghavan U

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangaluru, India. [email protected]

LEAFY (LFY) encodes a plant specific transcriptional factor, necessary and sufficient to specify meristem as floral meristem in Arabidopsis. The temporal and spatial expression profile of RFL, the rice LFY homolog, differs from its other plant homologs suggesting distinct functions. RFL is expressed throughout the apical inflorescence meristem from the earliest stage of its specification and its expression continues in the primary and secondary rachis branch primordia. Knockdown of RFL activity or loss-of-function mutants cause significantly reduced panicle branching and in few instances, reduction in vegetative axillary branching. These phenotypes attribute unique functions for RFL as a regulator of rice plant architecture. Here we examined mechanistic aspects of RFL as a transcriptional regulator of downstream targets in different meristems. To obtain a global profile of genes and pathways downstream to RFL, genome-wide transcript profiling was done using branching panicle tissues and axillary meristems using RFL knockdown and wild type plants. The data on de-regulation of gene expression was validated by qRT-PCR analyses for several transcripts. This includes genes with functions implicated in panicle development, axillary meristem development and also several novel genes. In an independent approach to identify downstream genes whose expression status can be modulated by RFL we have created transgenic plants that express a dominant repressive form of RFL in panicle tissues. The expression status of candidate RFL downstream targets was examined in branching panicle tissues and axillary meristems of these transgenics. These analyses implicate that RFL functions to regulate in a tissue-specific context other transcription factors (e.g., YABBY and homeodomain), hormone signaling (GA, strigolactone), signaling factors (F box factors; Ubiquitin conjugating enzymes) and metabolic enzymes. The direct regulation of several of these genes by RFL was ascertained by determining occupancy of RFL at cis-elements of some of these deregulated genes. Overall, we delineate mechanisms by which RFL contributes to panicle branching and axillary meristem pathway.


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15. Characterization and fine-mapping of the rice premature senescence mutant ospse1

Chen L, Wu HB, Wang B, Chen Y, Liu YG

South China Agricultural University, [email protected]

Premature senescence can limit crop productivity by limiting the growth phase. In the present study, a spontaneous premature senescence mutant was identified in rice (Oryza sativa L.). Genetic analysis revealed that the early senescence phenotype was controlled by a recessive mutation, which we named ospse1 (Oryza sativa premature senescence1). The ospse1 mutants showed premature leaf senescence from the booting stage and exhibited more severe symptoms during reproductive and ripening stages. Key yield-related agronomic traits such as 1000-grain weight and seed-setting rate, but not panicle grain number, were significantly reduced in ospse1 mutants. Chlorophyll content, net photosynthetic rate, and transpiration rate of ospse1 flag leaves were similar to the wild-type plants in vegetative stages, but these parameters decreased steeply in the mutants after the heading stage. Consistent with this, the senescence-associated genes OsNYC1 and OsSgr were up-regulated in ospse1 mutants during leaf senescence. The ospse1 locus was mapped to a 38-kb region on chromosome 1 and sequence analysis of this region identified a single-nucleotide deletion, leading to a frame shift and a novel stop codon in the 3’ UTR of an open reading frame encoding a putative pectate lyase.

COMPARATIVE & EVOLUTIONARY GENOMICS Chair: Antonio Costa De Oliveira, RP Sharma

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Concurrent Session III

16. Integrating Genetic, Transcriptomics and Proteomics Approaches for Identifying Genes and QTLs for Salt Stress tolerance in Rice

Singh NK1, Tiwari S1, Pandit A1, Bhowmick R1, Mishra V1, Singh B1, Sinha S1, Rai V1, Bhowmik PK2, Singh AK2 , Gautam RK3, Sharma SK3, Krishnamurthy SL3 1National Research Centre for Plant Biotechnology, IARI, New Delhi. 2Division of Genetics, IARI, New Delhi, India. 3Central Soil Salinity Research Institute, Karnal, India

About 77 mha of the cultivated land area of the world is covered by saline and sodic soils where productivity is severely depressed due adverse effect of the salt ions. Salinity is measured in terms of electrical conductivity (EC) of the soil extract (EC > 4 dS/m indicating saline soil), whereas sodic soils are characterized by high concentration of exchangeable Na+ percentage (ESP), which raises the pH of a saturated soil extract to more than 8.5 with an excess of carbonates and bicarbonates of Na+ and low hydraulic conductivity. Plants adapt to salt stress in different ways and land races of rice have been identified possessing different mechanisms of salt tolerance. Salt tolerance in rice is a quantitative trait controlled by multiple genes. We have identified several QTLs for salt tolerance in the Indian rice varieties derived from Indian land races Nona Bokra and Damodar using conventional QTL mapping and bulk segregant analysis (BSA) approaches. The two parents and two bulks of RILs with extreme phenotype were analyzed for marker genotype as well as RNA and protein expression profiles for identification of candidate genes for the trait. Phenotyping for salt tolerance was done in controlled microplots under moderate (pH 9.5) and high sodicity (pH 9.9) conditions. Using this approach we have identified 5 QTL regions on chromosomes 1, 2, 8, 9 and 10 showing tight linkage with the trait. Two of these locations match with QTLs previously mapped on chromosomes 1 and 8. Genetic map of the three novel QTLs on chromosomes 2, 9 and 10 has been prepared to identify their precise location and effect. Functional complementation is being carried out for the candidate genes located in the QTL intervals for further validation of function and use in molecular breeding of rice.

17. Transcriptome characterization and expression analyses in salt-tolerant and salt-sensitive cultivars of Oryza sativa after exposure to salinity stress

Jaiswal P, Christie SFM, Hanumappa M, Sullivan C, Sage A, Kimbrel J

Oregon State University, USA. [email protected]

Salinity stress causes serious global crop losses and rice is particularly sensitive to salt stress. Significant progress has been made over recent years at identifying individual biological mechanisms and genes that are required for plant tolerance to salt stress. However, the present challenges are to elucidate how these complex genetic, physiological, and biochemical interactions are coordinated within rice and how gene expression differences and SNPs may contribute to tolerance to salinity stress. We are most interested in how these genetic differences differ in salt-tolerant plants compared to salt-sensitive plants. In this study, we used Illumina RNA-Seq to analyze the transcriptomes of salt-tolerant and salt-sensitive cultivars of Oryza sativa after exposure to salinity stress. We conducted genome-scale de novo transcriptome assemblies of the two rice cultivars and functionally annotated the assembled contigs. We also analyzed the SNPs and gene expression differences among the two cultivars. De novo assembly and clustering of the sequence data resulted in ~55,000 contigs for each transcriptome. We also identified >26,000 SNPs unique to the salt-tolerant rice cultivar. A number of genes were identified based on their differential regulation in response to stress and comparisons were made between the salt-tolerant and salt-sensitive cultivars leading to the identification of genes associated with salt tolerance response. This data will aid in understanding the genetic elements that confer salt-tolerance in rice that occurs in rice. Additionally, this de novo assembly and characterization of the two transcriptomes will be helpful in future transcriptome studies in non-reference rice cultivars.


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18. Understanding the importance of KIX domain proteins in plants Thakur JK, Dwivedi N, Kumar V, Kumar A, Agarwal P, Parida SK

National Institute of Plant Genome Research, New Delhi, India. [email protected]

The KIX domain, required for protein-protein interaction, was first discovered as a part of the large multidomain transcriptional activator histone acetyltransferase p300/CBP. Later on, this domain was identified in Mediator subunit MED15. In both of these proteins, the KIX domain has been shown to be a target of activation domains of diverse transcription activators and found to be essential in several specific gene-activation pathways in fungi and metazoans. However, not much is known about KIX domain proteins in plants. We made an attempt to characterize all the KIX domain proteins coded by Arabidopsis and rice genomes. Interestingly, KIX domain was found not only in p300/CBP- and MED15-like plant proteins as known earlier, but also in F-box containing proteins in rice and DNA helicase in Arabidopsis. These findings suggest new roles of KIX domain in ubiquitin mediated proteasomal degradation of protein and genome stability. In Arabidopsis, we have found more than twenty proteins interacting with the KIX domain of MED15. In rice, expression analysis revealed overlapping expression of OsKIX_3, OsKIX_5 and OsKIX_7 in seeds of different stages of development, suggesting their individual or combined role during rice seed development. Moreover, the association analysis using the genotyping data of 136 in silico mined SNP loci in 23 contrasting rice genotypes and their grain length-specific phenotypic information identified three non-synonymous SNP loci in these three rice genes showing strong association with long- and short-grain differentiation. Interestingly, these SNPs are located within KIX domain encoding genomic regions. It, thus, indicates the additional importance of novel SNP loci/alleles identified in KIX domain containing genes for determining seed size in rice.

19. Comparative integrative “Omics” approaches to decipher salt tolerance in rice Sunkar R

Oklahoma State University, USA. [email protected]

Soil salinity is one of the primary causes of crop losses including rice production. Naturally adapted salt-tolerant relatives are potential sources for elucidating molecular networks important for salinity tolerance. Comparative analysis of closely related genotypes with contrasting salt tolerance may lead to better understanding of salinity tolerance at the molecular level. We have been studying molecular mechanisms important for salt tolerance in rice utilizing two well-characterized rice genotypes with contrasting salt sensitivities, i.e., salt-tolerant “Pokkali” and salt-sensitive “IR29.” We have used a comprehensive and comparative integrative “Omics” (transcriptome, translatome, miRNome and metabolome) to elucidate gene regulations and metabolite accumulations that are different in a tolerant genotype compared to sensitive genotype. These recent results will be discussed in my presentation.


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20. A draft genome assembly and comparative transcriptomics of the African wild rice species Oryza longistaminata

Zhang Y, Li X, Cai J, Li L, Bian C, Dong Y, Fu B

CAS-Max Planck Junior Research Group, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China [email protected]

Oryza longistaminata, an African wild AA genome (2n=24) relative of Asian cultivated rice (Oryza sativa L.), is characterized by a number of valuable traits that could be used for improvement of cultivars. Here we report the draft genome assembly of O. longistaminata and transcriptomes of four pairs of relative traits’ tissues.Although high heterozygosity from obligate outcrossing obstructed our ability to generate a high-quality reference genome, we were able to produce a relatively good draft genome assembly that can be used to annotate 32,485 protein-coding genes. The total length of the assembled draft genome is 341 Mb with a scaffold N50 size of 89.6 kb. The average sequence divergence between O. longistaminata and O. sativa is 4.92% in aligned regions. Consistent with the fact that O. longistaminata is highly resistant to many plant pathogens, our genome annotation revealed more disease resistant genes than in cultivated rice. Detailed analysis of four pairs of transcriptomes permitted the identification of some rhizome- and self-incompatibility-related genes. This study provides one more reference gnome for AA genome rice, and offers new insights into the evolution of the genus Oryza and genic basis of rhizome and self-incompatibility.

21. Evolution of salinity tolerance in African rice Meyer RS, Barretto A, Amas J, Gregorio G, Purugganan M

NYU Center for Genomics and Systems Biology, 12 Waverly Place, New York, NY 10003. USA. [email protected]

High salinity affects 50% of global irrigated land area. Farmers in the Atlantic Coast region stretching from Senegal to Liberia plant indigenous crops that are tolerant to saline conditions, and this project focuses on one such crop, African rice (Oryza glaberrima Steud.). African rice is irrigated by rivers with a conspicuous salinity gradient. Thus, the agricultural system developed for African rice implies that varieties have been arranged with matched fitness to local saline levels. However, farmers also practice agriculture in unique ways including dyking of mangrove swamps, soil overturning, and sometimes desalination, which spurs questions about the nature of selection on African rice. How has the O. glaberrima genome been modified to tolerate salinity? Do tolerant varieties more frequently occur in coastal saline areas? We selected 100 African rice landraces from upland and coastal West African environments and screened landraces for salinity tolerant phenotypes. Phenotypes were correlated with each other and with location. We then performed whole genome sequencing on these 100 landraces using Illumina HiSeq aiming for 10x coverage. Twenty percent of landraces were highly tolerant to saline conditions, although leaf relative Na+/K+ concentrations suggest there are multiple strategies for tolerance. We built a SNP map for O. glaberrima and identified DNA regions that distinguish landraces from different environment types and with different levels of salinity tolerance. Herein we discuss different approaches taken to perform mapping and to impute SNPs. There was no conspicuous correlation between location and genotype or phenotype, suggesting trade and soft selection pressures have influenced geographic patterns. Nonetheless, preliminary candidate regions that confer tolerance have been found.


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Concurrent Session III 22. Significant contribution of gene introgression for formation of varietal groups of

Asian cultivated rice Kumagai M, Kawahara Y, Wu J, Itoh T

National Institute of Agrobiological Sciences, Japan. [email protected]

This study aims to elucidate the pattern and extent of introgression events between varietal groups of Asian rice, Oryza sativa, which led to generation of the group-specific genetic background. Previous studies have indicated that two major groups of Asian rice, japonica and indica, originated from different wild progenitor populations of O. rufipogon and subsequently underwent hybridizations that resulted in introgressions of key domestication genes. Here we conducted genome-wide phylogenetic analysis using recently published genome resequencing data of 40 domesticated and 10 wild rice accessions as well as two newly sequenced genomes of wild rice of India and Malaysia. Our NGS read mapping could cover 38% of the Nipponbare reference genome with uniquely placed reads of all 52 accessions. As a result, more than 3 million SNPs were detected. We conducted genome-wide molecular phylogenetic analysis, and found that 6% of the genomic regions have experienced introgression between temperate japonica and indica. Although the direction of introgression between temperate japonica and indica was largely unidirectional from japonica to indica, it was indicated that other japonica varietal groups, tropical and aromatic japonica, possess a significant amount of introgressed regions derived from indica. In addition, the aus subgroup was found to have greater amount of japonica-derived genomic regions than the pure indica subgroup, which made the aus subgroup unique among indica accessions. In several subgroups, reduced genetic diversity was observed with significantly high frequencies around introgressed regions. This observation suggests that introgressed regions were artificially selected in recipient varietal groups during the cultivation. We discuss the importance of introgression between cultivars that generated the genetic makeup of today’s rice varietal groups in both japonica and indica.

23. Whole genome sequencing of elite rice cultivars reveals recurrent copy number variation, diversity patterns, and variety specific introgression events in Oryza Sativa spp. japonica and indica

Duitama J, Cruz D, Scheffler B, Farmer A, Torres E, Tohme J, Oard J 1School of Plant, Environmental, and Soil Sciences, 104 Sturgis Hall, Louisiana State University Agricultural Center, Baton Rouge, LA 70803. USA 2Agrobiodiversity research area, International Center for Tropical Agriculture (CIAT), Cali, Colombia. 3USDA-ARS Genomics and Bioinformatics Research Unit, 141 Experiment Station Road, JWDSRC, Stoneville, MS 38776; National Center for Genome Resources, 2935 Rodeo Park Dr. East, Santa Fe, NM 87505. USA. [email protected]

Current advances in sequencing technologies and bioinformatics allow to determine a nearly complete genomic background of rice, a staple food for the poor people. Consequently, comprehensive databases of variation among hundreds and even thousands of varieties will be released within the next years. Proper analysis of this massive resource is expected to give novel insights into the structure, function, and evolution of the rice genome, and to produce significant improvements for development of rice varieties through marker assisted selection or genomic selection. In this work we present sequencing and bioinformatics analyses of 60 rice varieties belonging to the major subspecies of Oryza sativa (indica and japonica). We built a database of repetitive elements and recurrent copy number variation covering about 200 Mbp of the rice genome. We identified close to 1 million SNPs in unique and copy number neutral regions, from which about 100 thousand allow to differentiate indica and japonica cultivars. Over 20,000 SNPs show high minor allele frequency (MAF) within indica and over 10,000 SNPs showed high MAF within japonica. Window-based analysis of the genome revealed regions of high diversity and high conservation, and also variety specific indica-japonica introgressions. Combining gene ontologies, predicted sites of conservation, and observed non-synonymous variation, we identified several candidate genes with a high percentage of allele sharing associated with grain yield and quality, heading date, biotic and abiotic stress, and gene regulation. Selected non-synonymous SNPs on different genes of interest are currently under testing for marker assisted selection. We expect that this database of genomic variation will be of great use to understand the genetic basis of observed differences between indica and japonica cultivars and to aid the development of useful markers for genomic selection.

ABIOTIC STRESS 2 Chair: Naoko Nishizawa, Arun Lahiri Majumder

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Concurrent Session IV

24. Engineering Inositol Metabolic Network as a potential approach for amelioration of salt stress in rice

Majumder AL, Sengupta S, Mukherjee S, Mukherjee R, Mukherjee A, Basak P, Bandopadhyay SA

Division of Plant Biology, Bose Institute (Centenary Building), Kolkata, India. [email protected]

With emerging knowledge of the diverse metabolic fate of inositol and the growing amount of data about their multiple roles in stress signaling indicates the importance of engineering inositol metabolism in plants. A significant number of attempts have been reported from the present laboratory in the last decade towards the characterization of the genes involved in myo-inositol metabolic network in plants. Majority of these genes and their regulatory elements have been isolated from the wild rice, Porteresia coarctata. The resulting data connects inositol experimentally to a large number of life processes, majority of which are regulated by abiotic stress induced by salinity and related stresses. Overexpressing native genes related to the inositol module, or by overexpressing natural salt-tolerant variant or recombinant designer proteins that may ensure increased inositol production under several stresses have been shown to give a multiple stress tolerant phenotype. Such applications in rice plants are underway; especially in the arena of generating single-gene crop transgenics in the inositol metabolic pathway, or manipulating one of the downstream metabolic fates by introducing all genes in a branch. The exploration of such pathways indicates that inositol itself and many of its derivatives can impart abiotic stress tolerance (against salinity, dehydration, chilling or oxidative stress, known to date) to plants when overexpressed. It is also necessary to understand how an alteration of myo-inositol pathway can change the channeling profile of the molecule in different pathways. The growing field of systems biology presents the scope to manipulate the inositol metabolic pathway specifically in the form of pathway engineering or network engineering. We propose that engineering inositol metabolic network is a potential approach towards stress tolerant transgenic rice plant generation and thus its exploitation in agricultural biotechnology is the call of time.

25. Novel regulators involved in rice response to abiotic stress Santos T, Serra T, Figueiredo D, Almeida D, Cordeiro A, Chander S, Gorska A, Barros P, Ferreira L, Costa M, Sapeta H, Santos AP, Abreu I, Oliveira MM, Saibo N

Instituto de Tecnologia Química e Biológica - Universidade Nova de Lisboa, Av da Republica, 2780-157 Oeiras, Portugal, Instituto de Biologia Experimental e Tecnológica, Av da Republica, 2780-157 Oeiras, Portugal [email protected]

Rice is the staple food for more than half of the world population and it is also a very important crop for Portugal, which is the largest rice consumer (per capita) in Europe. Rice productivity is highly affected by different abiotic stresses, such as high salinity, cold and drought. In order to identify and characterize the function of novel stress response regulators, which may be useful in future crop improvement, we have followed an integrative and complementary approach. Many novel transcription factors (TF) binding to the promoters of stress responsive genes (e.g OsDREB1A, OsDREB1B, OsNHX1, OsRMC, OsHOS1) have been identified (yeast-one-hybrid) and functionally characterized. This characterization includes trans-activation assays, protein-DNA and protein-protein studies, gene expression, post-translational modifications (e.g. SUMOylation, phosphorylation), and stress tolerance of transgenics/mutants. We have already shown that some of these novel TFs play important roles in the abiotic stress response (1,2), while others are currently being studied. In addition to the TF functional characterization, we have investigated how epigenetics is involved in the regulation of the stress responsive genes. The function of other players in the abiotic stress-signalling pathway in rice, such as OsICE1, OsHOS1 (3) and OsRMC, has also been investigated. These proteins have been shown to play key roles in abiotic stress responses and some of them have unveiled unexpected functions in either abiotic stress response or plant development. Our studies will help to better understand the signalling pathways regulating abiotic stress response in rice and provide new breeding tools.


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26. Genetic Engineering of rice for environmental stresses tolerance Reddy MK

International Centre for Genetic Engineering and Biotechnology, New Delhi-110067, India.

Plants are exposed to all kinds of environmental stresses because of their sessile nature that effect their growth and productivity. Under adverse environmental stresses the carbon assimilation through photosynthesis is restricted and excess photochemical energy trapped by the chloroplasts are transferred to molecular oxygen (O2) and induce oxidative stress. The enhanced production of Reactive Oxygen Species (ROS) can pose a threat to plants. We successfully transformed entire ascorbate-glutathione pathway encoding genes into Indica rice cultivar “Swarna” for effectively scavenge reactive oxygen intermediates in multiple redox reactions and protect the transgenic rice cultivar from environmental stress-induced oxidative stress damage. In another approach we developed a strategy to prevent the generation of ‘Reactive Oxygen Species (ROs) by engineering enhanced photosynthetic efficiency for effectively using the photochemical energy trapped by chloroplasts for Carbon reduction rather than generation of ROS production and subsequently to minimize the environmental stress induced cellular damage. Currently we are evaluating the integration and expression of these transgene(s) in putative transgenic swarna rice cultivar.

27. Understanding the role of mitogen activated protein kinase (MAPK) cascade in rice

Sinha AK

National Institute of Plant Genome Research [email protected]

Mitogen activated protein kinase (MAPK) cascade is a ubiquitously conserved cascade among eukaryotes. The basic functions of MAPK cascades involve the stimulus-triggered activation of a MAPK kinase kinase (MAPKKK or MEKK), which phosphorylates a MAPK kinase (MAPKK or MKK or MEK) that in turn phosphorylates a MAP kinase (MAPK or MPK). Phosphorylation and activation of MAPK can lead to changes in its subcellular localization and its interaction with various cytoplasmic and nuclear proteins, thereby controlling several cellular processes. It is reported that rice encodes 75 MAPKKKs, 8 MAPKKs and 16 MAPKs. We report cloning of entire members of MAPKs and MAPKKs from Oryza sativa indica cultivar group cv Pusa Basmati 1. Using in-silico approach we have predicted existence of 75 members of MAPKKK in rice. The cloned MAPKs and MAPKKs from rice were classified into four different groups and their expression and activity under different abiotic stress were analyzed. Specific MAPKs getting activated by heavy metal (arsenic) stress has been biochemically characterized. A stable transgenic rice plant harboring constitutive active OsMKK6 was generated. The transgenic plant showed tolerance towards cold and UV stress. The transgenic line also revealed that OsMKK6 and OsMPK3 network transduce the upregulation of phyotalexin biosynthesis genes by UV stress signal. With protein modeling and docking approach we tried to generate an interactome map between rice MAPKKs and MAPKs and validated it using yeast-2-hybrid analysis. We also report a unique protein-protein interaction between two rice MAP kinases and found that this interaction has a role in plant defense mechanism. In nutshell, an overview of complexity of MAPK cascade in rice will be presented.


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28. Raising rice plants for saline and dry areas: hunting for new ‘candidate genes’ Pareek A, Soni P, Soda N, Saran A, Nongpiur R, Singla-‐Pareek SL

School of Life Sciences, Jawaharlal Nehru University, New Delhi -110067, India. [email protected]

To increase crop production under saline and dry areas, it is necessary to identify pathways and novel members in crops that control tolerance to environmental stresses. We have made an attempt to dissect out the osmosensing pathway in rice. For this purpose, we performed transcript profiling of members of two-component system in rice which distinguished OsHk3 as ABA inducible multi-stress responsive receptor having high expression in all tissues. OsHk3b acts as homo-dimer and extra-cellular domain of OsHk3b is important for homo-dimerization. Yeast two-hybrid analysis showed that OsHk3b interacts with OsPhp3 which in turn interacts with type-B response regulators- OsRr22, OsRr23, OsRr24, and OsRr26. These type- B response regulators also interacts with type-A response regulators; OsRr22 with OsRr10; OsRr24 with OsRr12 and OsRr26 with OsRr4. Altogether, these results lead us to hypothesize that OsHk3b and downstream interacting members could be partners in a multi-step phosphorelay system involved in the osmosensing in rice. In another approach, we explored salinity related QTL Saltol, present on chromosome I of rice accounting for >40% of salinity tolerance. A total of 783 loci were retrieved from in-silico analysis of Saltol QTL. Among them 2% and 24% genes encode signaling related proteins (SRPs) and protein of unknown functions (PUFs), respectively. We have analysed the expression pattern of these genes in contrasting rice genotypes and interesting leads have been obtained based on the temporal transcript accumulation to help us comment on their precise role in salt physiology of plants. These results suggested that gene encoding Intermediate filament (OsIF) protein may have important role in stress tolerance. OsIF is differentially regulated under stress in contrasting cultivars and provides survival advantage to diverse organisms. This study might serve as first evidence of involvement of cytoskeletal genes in stress tolerance.

29. The expression profiles of drought-tolerant NaN3-induced rice (Oryza sativa L. spp. indica) mutant

Shih M-‐D, Wu J-‐J, Wei F-‐J, Wu Y-‐P, Hsing YC,

Distinguished Research Fellow, Institute of Plant and Microbial Biology, Academia Sinica. Department of Agronomy, National Taiwan University, Taipei, Taiwan. Department of Agronomy, Taiwan Agricultural Research Institute, Chiayi, Taiwan. [email protected]

Rice (Oryza sativa L.) plant is highly drought sensitive. In most of cases, drought stress is the key factor to limit yield in the rice reproductive area of Asia. Hence, to improve varieties through breeding process, chemical mutagenesis was used to generate variations and to select drought-tolerant mutants. Chemical mutagenesis has been widely used to generate genetic variation for basic research and breeding programs. Besides, the development of high-throughput genotyping also provides the efficient detection of point mutations generated by chemical agents. In this study, IR64 mutated population generated by sodium azide (NaN3) treatment was used to screen for drought-tolerant individuals by treatment with 20% PEG solution. The total RNA were then extracted from both type to establish the high-throughput RNA-seq profiles. Our results suggest that two drought-tolerant individuals have totally different expression profiles. Functional analysis also suggests that both individuals have diverse GO profiles. We will discuss the possible drought-droved pathway of two drought-tolerant individuals and wild-type.

SMALL RNA & EPIGENOMICS Chair: Blake Meyers, M Udaykumar

31

Concurrent Session V

30. Genome-wide methylation profile of DECREASE IN DNA METHYLATION1 (DDM1) knockdown in rice

Habu Y, Numa H, Yamaguchi K, Shigenobu S 1Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2National Institute for Basic Biology, [email protected]

Distribution of cytosine methylation in the genome of higher plants is not uniform throughout the chromosome but biased to heterochromatin regions surrounding centromeres where remnants of transposons accumulate. DECREASE IN DNA METHYLATION1 (DDM1) in Arabidopsis thaliana is a chromatin remodeling factor that maintains silent chromatin modifications of heterochromatin and keeps various types of transposons silent. DDM1 is thought to act on repetitive sequences in heterochromatin rather than single copy euchromatin regions, however, the mechanism that directs DDM1 to heterochromatin regions and the mode of action for the maintenance of silent chromatin modifications by DDM1 is unclear. Towards understanding the function of DDM1 in maintenance of silent chromatin, we analyzed genome-wide changes in cytosine methylation by bisulfite-sequencing using a rice line deficient in the DDM1 function. CG and CHG methylation was strongly affected in the pericentromeric heterochromatin regions but decrease in CHH methylation was not evident, suggesting that independency between de novo RNA-directed DNA methylation (RdDM) and the DDM1 function in the pericentromeric region of rice. However, characteristic changes in CHH methylation were observed in limited regions of the chromosomes of the DDM1-deficient line, indicating that local, but not global, interaction between RdDM and DDM1 on specific regions of rice chromosomes.

31. Dynamics, regulation and function of epigenomic modifications in rice gene expression and plant growth

Zhou DX1, Zhao Y2 1Institut de Biologie des plantes, Université Paris sud, Orsay France. 2National Key laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan China University Paris-sud 11. [email protected]

Epigenomic information such as DNA methylation, histone modifications and nucleosome positioning are important for genome activity, gene expression and plant development and growth. Studying the establishment, the maintenance, and the recognition mechanisms of epigenomic modifications is essential to understand the epigenetic regulation of gene regulation and plant growth. We have been studying the function of genes involved in rice histone modification and recognition mechanisms. Our study led to the identification of a number of rice chromatin regulators involved in histone modification (e.g. acetylation and emthylation) homeostasis and gene expression of specific developmental and stress-responsive pathways. We will present our recent data on the interplay between a few rice chromatin regulators in epigenomic modifications and recognition that affect rice gene expression and phenotypic variation. We will discuss their possible implication in epigenetic transmission of gene expression states.


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32. Conserved Interaction Between the De-novo Methyltransferase, OsDRM2, and the ATP-dependent RNA helicase, eIF4A, in Rice and Arabidopsis

Dangwal M1, Malik G1, Kapoor S2, Kapoor M1 1University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector 16C, Dwarka, New Delhi-110078, India. 2 Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. [email protected]

Domains Rearranged Methyltransferases (DRM) are the DNA methyltransferases that catalyze the establishment of new cytosine methylation patterns. This de novo methylation process is similar to the methylation activity carried out by the animal de novo methyltransferases, DNMT3a and DNMT3b. The de novo methyltransferases are known to be guided to the target sites by small RNAs through the process of RNA-directed DNA Methylation (RdDM). In this process, interactions among transcription factors and/or chromatin modifying proteins and the large subunits of plant specific polymerases, Pol IV and Pol V, are known to regulate de novo methylation of cytosines by the 24nt siRNAs. The RNA-induced Silencing Complex (RISC) finally facilitates recruitment of the methyltransferase to chromatin sites by unknown mechanism/protein interactions. In the present study, using the yeast two-hybrid method, Bimolecular Fluorescence Complementation (BiFC) and histidine-pull down assays we describe specific interaction between the de novo methyltransferase, OsDRM2 and the ATP-dependent RNA helicase, eIF4A in young rice panicles. Deletion analysis of conserved domains was carried out to characterize the motifs/domains essential for the observed interaction between the two proteins in vivo. We also describe the possible conservation of role of this interaction between DNA methyltransferases and the translation initiation factor, eIF4A, in Arabidopsis and the early land plant, Physcomitrella patens, in the process of RdDM across land plants.

33. Drought regulated miRNAs in indica rice: a comparative profile Raghuvanshi S

Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. [email protected]

MicroRNAs define a complete dimension of the multifaceted and intricate molecular regulatory mechanism operative in eukaryotic systems. They have been reported to regulate several key developmental and biochemical pathways in plants as well. In our current study we endeavor to investigate the role of miRNA in regulating the drought response in rice. Drought is a significant deterrent to global rice production as major economically viable cultivars are sensitive to drought. Nevertheless, several rice varieties, although not economically viable, are naturally tolerant to drought. We have exploited such contrasting characteristics to perform a comparative analysis of the global miRNA population to identify miRNAs that play key role in regulation of drought response. We performed NGS (Next Generation Sequencing) based sequencing of small RNA population from different tissues of control and drought treated (field grown conditions) plants of drought sensitive (Pusa Basmati 1) and tolerant (Nagina 22) indica rice varieties. Subsequent analysis revealed that miRNA population behaved very distinctly in the tolerant variety as compared to the sensitive one. We could identify several miRNAs that had an opposite expression response under similar stress conditions in both the varieties. Characterization of these miRNAs revealed that they regulate several key processes such as free-radicle scavenging, copper homeostasis, starch metabolism etc. In addition we were also able to identify several novel drought responsive miRNAs. Generation and sequencing of the degradome libraries led to identification of several of these novel miRNAs.


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34. Expression of epigenetic regulators in rice gametes suggests dynamic changes during plant fertilization

Anderson S, Johnson C, Jones D, Conrad L, Gou X, Russell S, Sundaresan V

University of California, Davis, USA. [email protected]

The formation of a zygote by the fusion of egg and sperm relies heavily on the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, while the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells, and pollen vegetative cells from rice, and identified transcripts for ~36,000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three quarters of those genes differentially expressed in the different cell types. Interestingly, the egg cell and sperm cell transcriptomes reveal major differences in expression of genes regulating epigenetic modifications that must be resolved in the zygote. We found histone variants indicative of active chromatin in both gametes, a somewhat unexpected result considering the highly-condensed confirmation of the sperm cell chromatin. In addition, we observed high expression in the sperm cell of genes promoting active chromatin configurations, and low expression in both sperm and vegetative cell of genes required for siRNA-mediated silencing. Taken together, these results suggest that the male germline is being prepared for activation after fertilization, and that both gametes will contribute to chromatin reorganization and gene expression in the fertilized zygote.

INFORMATICS & SYSTEMS Chair: Bin Han, Paramjit Khurana

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Concurrent Session VI

35. ERF gene regulation and expression profiles under different stresses in rice Costa de Oliveira A, Santos RS, Pegoraro C, Kruger MM, Farias DR, Maia LC, Manica-‐Berto R, Mertz LM, Madabula FP, Rombaldi CV

Federal University of Pelotas, Brazil, [email protected]

The plant response to stresses occurs via a plethora of signaling pathways, where a large number of genes is induced or repressed. Among the genes responsive to environmental stresses are the ERFs (Ethylene Responsive Transcription Factors) which bind to conserved domains found in promoters of many genes involved in responses to ethylene. Our laboratory has been working aiming to identify transcriptional response of ERFs to different environmental biotic and abiotic stresses such as anoxia, salinity, iron overload and Magnaporthe grisea, as well as to compare the expression profile of different cultivars under some of these stresses. Moreover, we have also been working, through the use of computational tools, on the identification of cis regulatory elements, as well as potentially methylated regions in the promoter region of these genes, and trying to establish an association between them and the expression profiles found. Results indicate a range of transcriptional expression profiles when comparing members of the ERF family under different stresses. It is also possible to notice that some of the analyzed genes have complex regulation, with many cis elements acting in its promoters, while others do not, having simple control. When comparing the expression profile of ERFs on the different tested genotypes, it is difficult to establish a connection between susceptibility to stress and expression levels of these ERFs in response to stresses by hypoxia and anoxia, however methylation of promoters seems to be an interesting research line when dealing with oxygen deficiency in rice and maybe other plants. When comparing cultivars in response to iron overload stress it is possible to observe an association between the presence of some cis elements and the transcriptional expression of some of the tested genes. Our group is pursuing further studies about the transcriptional and epigenetic regulation of ERFs.

36. Comparative plant genomics resources at Gramene Naithani S1, Monaco MK2, Stein J2, Dharmawardhana P1, Amarasinghe V1, Preece J1, Wei S2, Youens-‐Clark2, Ware D2,5 , Jaiswal P1 1OSU, Botany and Plant Pathology, Corvallis, OR, 97331, 2CSHL, Ware Lab, Cold Spring Harbor, NY, 11724, 3OICR, Bioinformatics, Toronto, M5G 1L7, Canada, [email protected]

Gramene (www.gramene.org), a resource for comparative genomics of plants, currently hosts 27 complete reference genomes accessible through Ensembl Genome Browser, ten species-specific plant Pathways Databases, a beta version of the recently developed Plant Reactome, and genetic diversity datasets for rice, maize, sorghum, Brachypodium, barley, grape, and Arabidopsis. Gramene derives its strength from the application of phylogenetics and integration of genome annotation and functional data using ontologies. The Genome Browser allows users to upload and visualize genomic datasets (i.e. genome-wide SNP associations, QTLs, linkage studies, ESTs, Microarrays, RNA-Seq and proteomics) mapped onto the genome. Several sets of genome-wide data can be displayed simultaneously; users can define a threshold value for their data, and thus choose to view only regions that cross the threshold. A plant Pathways database based on the BioCyc platform provides a cellular overview of the metabolic networks, supports queries for metabolites, genes, enzymes, and pathways and allows uploading, visualization and analysis of genomic expression datasets. We are developing the Plant Reactome (http://plantreactome.oicr.on.ca), a platform for the comparative analysis of plant metabolic and regulatory networks, currently featuring 133 curated rice pathways. Gramene collaborates with EBI-EMBL, OICR, and ASPB. Gramene is supported by NSF grant (IOS-1127112). Financial Support for attending the 11th International Symposium on Rice Functional Genomics to SN was provided by USDA.


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37. In silico model-driven alternate pathway design for controlling photorespiration in rice

Mohanty B1, Lakshmanan M1, Cotton C2, Lee D-‐Y1,3 1Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore. 2Department of Life Sciences, Imperial College, London, UK. 3Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01, Centros, Singapore 138668, Singapore. [email protected]

Most of the C3 plants, including rice, have limited CO2 assimilatory capacity due to the occurrence of energetically wasteful process, photorespiration. This process involves the salvage of two molecules of unwanted 2-phosphoglycolate (2-PG) to one molecule of 3-phosphoglycerate (3-PGA) which requires a significant amount of cellular energy, i.e. ATP, and releases a part of the fixed CO2 in the mitochondria. Therefore, in order to control photorespiration and to improve crop productivity, several efforts have been initiated either through engineering RuBisCO such that it can be more specific to CO2 or by minimizing the flux through photorespiratory pathways by implementing alternate 2-PG salvage pathways in the chloroplast itself. While comparing these two techniques, the latter route has shown better success in improving plant productivity. Following such an approach, in this work, we systematically assessed all the possible metabolic routes available for the salvage of 2-PG into 3-PGA through in silico modeling and analysis. To do so, we first identified and compiled all the possible metabolic routes for the conversion of 2-PG into 3-PGA from KEGG database. The resulting designs of 14 potential pathways, having a length of 3-9 enzymatic steps, were then incorporated into the plastid compartment of our recently published rice central metabolic network model and photorespiration was simulated. From these simulations, the transgenic rice cells with the alternative 2-PG salvage pathway designs showed better CO2-to-biomass conversion than that of wild-type, indicating that introduction of such alternative pathways is an attractive option for improving rice productivity. Furthermore, we also rank the suggested designs based on several factors such as biomass yield, pathway length, cofactor regeneration, possible difficulties in gene cloning and others.

38. Polyomic interrogation of rice environmental gene regulatory interaction networks

Wilkins O1, Hafemeister C1, Plessis A1, Holloway-‐Phillips M-‐M2, Pham G1, Nicotra AN2, Septiningsih E3, Bonneau R1,4, Purugganan M1 1Center for Genomics and Systems Biology, New York University; 2Research School of Biology, Australian National University; 3Plant Genetics and Biotechnology Division, International Rice Research Institute, Los Baños, the Philippines, 4Courant Institute, New York University, New York. [email protected]

Plants activate flexible networks of co-expressed and co-regulated genes to respond to the fluctuating environmental conditions they experience in nature. These networks evolve as distinct plant populations experience divergent ecological regimes. Our research aims to infer the environmental gene regulatory interaction networks (EGRINs) in the leaves of rice plants (Oryza sativa) associated with changes in ambient air temperature and water availability. Our study includes four rice cultivars, encompassing the two major genetic rice subgroups – indica and japonica – and the diverse ecological niches occupied by cultivated rice – flooded paddies and drier, rain-fed fields. By including multiple cultivars in this analysis, we are able to identify EGRINs that are conserved across all tested varieties as well as elaborations to these networks that are cultivar specific. To learn these networks, we performed a common garden experiment wherein all varieties were germinated and grown in identical environmental conditions and then were shifted into novel conditions, either a 10°C increase in air temperature or a complete removal of root-accessible water. We harvested leaves from each genotype and treatment at 15-minute intervals for four hours. This experiment generated 480 whole transcriptome profiles, measured by RNA-sequencing, and 100 metabolome profiles, determined by LC-MS/MS and GC-MS. We are adapting computational methods for network inference (Inferelator) for use with high sample-number transcriptome, metabolome and environmental datasets to learn these networks. The integration of polyomic and time series data makes the identification of functionally and biologically significant heat- and drought-induced changes in transcript abundance possible. The tools we have developed for inferring networks are open-source


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and will be made available to the plant science community; they will allow users to explore inferred EGRINs using a web-based platform. This research lays the foundation for a broad dissection of plant response to environmental fluctuations and advances a systems biology approach to understanding plant-environment interactions.

39. National Rice Resource Database: Information source on 15K Indian rice germplasm

Archak S, Srinivasan K, Singh R, Patra BC, Singh SP, Sarawgi AK, Sharma SK, Viraktamath BC, Subbarao LV, Kumar V, Shaheen R, Singh A, Rai A, Das S, Mohapatra S, Ojha GC, Pachauri A, Chauhan MS, Singh V, Bansal KC

NRRD Coordinating Centre, National Bureau of Plant Genetic Resources (NBPGR), New Delhi 110 012 India [email protected]

Advancements in high-throughput genomic technologies allow plant breeders to exploit agronomically useful alleles that were hitherto inaccessible due to limitations of the conventional breeding strategies as well as domestication bottlenecks. Success of this approach depends upon the access to the diversity conserved in genebanks. The systematic characterization of naturally occurring genetic variation is a prerequisite to genomics-facilitated germplasm enhancement. In rice, all possible genomic resources and integrated genetic maps are available. Their employment— in trait mapping and allele mining for resistance to biotic and abiotic stresses and for agronomic traits— is however subject to availability of well phenotyped genebank accessions and designation of specialized germplasm sub sets such as core, mini core and reference sets. National Bureau of Plant Genetic Resources (NBPGR), New Delhi hosts the National Genebank with about 0.1 millions rice germplasm collections. Utilization of this valuable rice genetic diversity for crop improvement begins with characterization and trait-specific evaluation. NRRD Project, coordinated by NBPGR, is an initiative to generate characterization data of about 15,000 selected rice germplasm accessions and to develop a user friendly interface to access information on 19 qualitative and 11 quantitative descriptors. The rice accessions were characterized at five different centres (BHU, Varanasi; CRRI, Cuttack; IGKV, Raipur; CSSRI, Karnal; and DRR, Hyderabad) over a three years period (Aug 2010-Aug 2013). The NRRD intends to provide opportunities for both breeders and genomics researchers to select trait-specific accessions based on multi-location multi-season characterization data. NRRD uses SQL Server 2012 (database), Visual Studio 2010 (applications), ASP.NET (interface) and C# (coding) and is hosted on a high-end server. The database has a provision for passport data, characterization data (1.35 million data points), evaluation data (10 traits: 7 biotic and 3 abiotic; 2 seasons), hvSSR marker data and core set. NRRD has built-in applications for data analysis, visualization and reporting. A CMS based web portal is developed and hosted on NBPGR web server. Portal can be accessed at www.nbpgr.ernet.in/nrrd.

SHIMAMOTO SESSION: BIOTIC STRESS Chair: Hiroshi Takatsuji, Bharat B Chattoo

37

Concurrent Session VII

40. A signaling network for broad-spectrum disease resistance in rice Wang S, Cheng H, Deng Y, Xiao J

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China [email protected]

Breeding crops with the quality of broad-spectrum resistance to different pathogen species is one of the principal goals of crop improvement. However, the molecular mechanisms of broad-spectrum resistance are largely unknown. We have identified a defense signaling network which is involved in the regulation of rice resistance to Xanthomonas oryzae pv. oryzae (Xoo) causing bacterial blight disease, Xanthomonas oryzae pv. oryzicola causing bacterial streak, and Magnaporthe grisea causing fungal blast disease. This defense network is downstream of the major disease resistance (MR) gene Xa3/Xa26, which confers race-specific resistance to Xoo, and the MR gene Rbr2, which confers race-specific resistance to M. oryzae. At least three WRKY genes function in this network.

41. Molecular Analysis of Allelic Variations in Rice Blast Resistance Gene Pi54 and their Utilization in Resistance Breeding

Sharma TR

National Research Centre on Plant Biotechnology, IARI, New Delhi-110012, India. [email protected]

Rice blast is a major production constraints in different parts of the world because of highly variable nature of the pathogen Magnaporthe oryzae . More than 100 major blast resistance genes and many QTLs have been identified in different rice cultivars during the past many years and are important components of rice breeding. We cloned and characterized a major blast resistance gene Pi54 (Pikh) from the rice line Tetep using positional cloning approach and functionally validated it in the susceptible rice line. We showed that Pi54 is the smallest and unique gene among all the disease resistance genes cloned from different plant species. This gene has unique Zn finger domain which plays an important role in its expression. The single Pi54 gene regulates resistance mechanism in rice lines in a typical incompatible interaction. The orthologues of Pi54 gene cloned from wild species of rice showed extensive sequence variations in both coding and non-coding regions. The position of these genes on different rice chromosomes in the transgenic rice lines does not affect its phenotype. The orthologues cloned from the blast resistant wild species has a typical Suppressor of cytokine signalling (SOCS) domain which is associated with its resistance phenotype. In the alleles of Pi54 genes cloned from different aromatic and non-aromatic rice lines and Indian land races showed the presence of many single nucleotide polymorphisms (SNPs) and InDels of different sizes which results in the identification of many unique haplotypes. We developed allele specific markers which are now being used in the introgression of Pi54 gene and its alleles in combination with other blast resistance genes in the mega varieties and hybrids of rice being grown in different parts of India.


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42. Xanthomonas oryzae pv oryzae infection induces very early transcriptomic modulations in rice

Grewal RK, Das S,

Division of Plant Biology, Bose Institute, Kolkata 700054, India. [email protected]

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice (Oryza sativa L.), a major disease that constrains production of this staple crop in many parts of the globe including India where it causes huge economic loss. The variability of pathogenic races make the control of disease more difficult. However, the plant adaptive strategies are unique, versatile and still grossly unknown which have attracted the researchers since decades towards looking into the responses and diversification of plant adaptation at cell and molecular level when challenged with pathogen. In this particular rice–Xoo interaction process, DNA Microarray was used to study the transcriptional modulations in eighteen days old rice seedling leaves of both susceptible and resistant genotypes one hour after pathogen inoculation. Hundreds of genes were found to be differentially expressed. Annotations could be assigned to a number of up- and down-regulated transcripts and gene interaction maps of some transcripts were generated. Expressional data and interaction maps were used to develop a hypothetical scheme of the molecular events taking place during defense response as early as one hour of pathogen infection. Network analysis with the differential transcripts showed up-regulation of major clusters of cell signaling proteins and transcription factors while growth and basal metabolic components were largely found to be down-regulated. This study provides an understanding of the early defense signaling in rice cells. Components of the calcium and lipid signaling as well as MAPK cascade were modulated by signals from surface receptors and cytosolic R-proteins, to arouse jasmonic acid and ethylene signaling and suppress auxin signaling through various transcription factors. Abscisic acid modulation was also evident through the expression regulation of transcription factors involved with its functions. Moreover adjustments in expression levels of components of primary as well as secondary metabolism, protein trafficking and turnout were apparent, highlighting the complexity of defense response of rice against the important pathogen, Xanthomonas oryzae pv. Oryzae. The pattern of gene modulations that undertaken by rice cells at such early a stage, highlighting the ability of plant cells to rapidly mount a complex defense response has been documented for the first time.

43. Study of Defensome in rice immunity with Dr. Shimamoto Kawano Y, Akamatsu A, Hamada S, Shimamoto K

Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Japan. [email protected]

We have been studying molecular signaling in rice innate immunity by studying the small GTPase OsRac1 and its interacting proteins by using a variety of methods. We have identified a number of OsRac1-interacting proteins and studied their functions and interactions with other proteins. We found that OsRac1 interacts with two types of receptors; membrane-bound receptor-like kinases and NB-LRR type receptors. OsRac1 forms a protein network with several cheperones and co-chaperones, SGT1, RAR1, Hsp90, Hsp70, and Hop/Sti1. A scaffloding protein, RACK1, also interacts with OsRac1. The OsRac1 network includes enzymes such as NADPH oxidase and CCR1 which are important for immune responses. Based on genetic, protein-protein interaction, and biochemical studies we propose that OsRac1 is a hub of rice innate immunity where PTI and ETI pathways merge. We also propose that these proteins form complex termed defensome. Based on the recent biochemical analysis we found PTI and ETI receptors form separate defensomes but contain the same chaperones in each defensome. Our results suggest that the defensome complex is a key regulatory unit for rice innate immunity.


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44. Broad-spectrum disease resistance by overexpression of rice BSR1 and its application to crop improvement

Mori M, Maeda S, Sugano S, Naoki Y, Hayashi N, Goto S, Jiang C-‐J, Oda K, Hirochika H, Takatsuji H

National Institute of Agrobiological Sciences [email protected]

Broad-spectrum disease resistance against two or more types of pathogen species is desirable for crop improvement. In rice, Xanthomonas oryzae pv. oryzae (Xoo), the causal bacteria of rice leaf blight, and Magnaporthe oryzae, the fungal pathogen causing rice blast, are two of the most devastating pathogens. We have identified the rice BROAD-SPECTRUM RESISTANCE 1 (BSR1) gene for a BIK1-like receptor-like cytoplasmic kinase that confers resistance to a bacterial pathogen, Pseudomonas syringae pv tomato DC3000 (PstDC3000) and a fungal pathogen, Colletotrichum higginsianum in Arabidposis by using FOX hunting system. BSR1-overexpressing (OX) rice showed strong resistance to at least three different races of Xoo. In addition, it showed resistance to M. oryzae and Cochliobolus miyabeanus, the fungal pathogen causing brown spot. Disease resistance of BSR1-knockdown rice was weaker than that of WT, which supports the involvement of BSR1 in innate immunity of rice. Microarray analyses revealed that ~20% of the genes upregulated in BSR1-OX rice overlapped with BTH (SA analog)-inducible genes. To investigate SA dependency of the BSR1-dependent disease resistance, we crossed BSR1-OX rice with transgenic rice lines overexpressing bacterial NahG gene, in which endogenous SA levels are undetectably low, and examined the blast resistance of resulting hybrid transformants. Results indicated that the disease resistance in BSR1-OX rice is largely independent of the SA pathway. Because rice plants overexpressing BSR1 under maize Ubiquitin promoter displayed a decreased germination rate, we attempted BSR1 expression using weaker or pathogen-inducible promoters. Resulting transgenic rice lines showed germination rates comparable to WT while retaining disease resistance. We have been introducing BSR1 to several crops. Overexpression of BSR1 conferred PstDC3000 resistance to tomato (Micro-Tom) without any notable growth defect or morphological changes.

45. Development and optimization of gene silencing using RTBV-MVIGS, a virus-derived tool for transient gene silencing in rice

Dasgupta I, Kant R, Purkayastha A

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India, [email protected]

Virus-induced gene silencing (VIGS) is an established transient gene silencing technology available for a number of dicot plants. However, for monocots, the VIGS technology is much less robust. Realizing this gap in the technology, a VIGS system, based on the rice DNA virus Rice tungro bacilliform virus (RTBV) has been recently developed for rice. The conversion of the DNA of RTBV to RTBV-MVIGS involved cloning of a truncated viral DNA, to remove genes unnecessary for viral replication and spread, as a partial dimer in a binary plasmid; replacing the tissue-specific RTBV promoter with the constitutive maize ubi promoter; modification of the translation start sequences and introduction of multi-cloning sites. These modifications allow RTBV-MVIGS to replicate in 20-day old rice plants, when inoculated at the crown region of the plant with Agrobacterium cells containing the vector, with the help of a syringe and needle, a process termed agroinoculation. Although RTBV-MVIGS itself causes no symptoms in the plant, rice phytoene desaturase (pds) cDNA, when cloned in RTBV-MVIGS and agroinoculated, causes leaf streaks to appear in emerging leaves within 14 days and reduces the accumulation of pds transcripts up to 70%, as assessed by Real-time PCR. The RTBV-MVIGS has also been used to silence rice Magnesium chelatase H subunit, with comparable levels of silencing. Current efforts to improve RTBV-MVIGS include the determination of the ideal orientation of the insert for optimal silencing and to enhance the silencing levels by inactivating potential RNAi-suppressors in RTBV.


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46. Towards durable bacterial blight resistance in rice Sundaram RM, Laha GS, Viraktamath BC, Hajira Sk, Yugander A, Madhav MS, Balachandran SM, Shobha Rani N, Priya MRV, Sonti RV

Directorate of Rice Research-ICAR, Rajendranagar, Hyderabad, Andhra Pradesh, 500030, India. [email protected]

Bacterial blight (BB) is a major disease of rice caused by Xanthomonas oryzae p.v. oryzae (Xoo) and deployment of resistant varieties is the only option for its management. More than 38 genes conferring resistance to BB have been identified in rice and some of them have been deployed. However, the emergence of virulent pathotypes of the bacterium is a threat to the strategy of host-plant resistance and pyramiding of two or more effective BB resistance genes in a single genetic background can enhance the magnitude and spectrum of resistance. Keeping this in focus, we have introgressed three major resistance genes- Xa21, xa13 and xa5 into the genetic background of the elite rice cultivar Samba Mahsuri marker-assisted backcross breeding (MABB) in collaboration with CCMB, Hyderabad. Four three-gene pyramid lines of Samba Mahsuri were tested under the All India Coordinated Rice Improvement Project (AICRIP) trials at multiple locations and were observed to display a high level of resistance to multiple races of Xoo and were identical to Samba Mahsuri in terms of grain and cooking quality features and yield. After rigorous testing under AICRIP for multiple seasons, one of the three-gene pyramid lines, RPBio-226 was found to be consistently resistant to BB and identical to Samba Mahsuri in terms of agro-morphological parameters and yield. RPBio-226 was released and notified by the Central sub-Committee on Crop Standards and Notification of Varieties (CCS & NV) in the year 2008 as a new variety, Improved Samba Mahsuri (ISM). The new variety is being enthusiastically cultivated in an area of ~ 70,000 ha in the states of Andhra Pradesh, Karnataka, Tamilnadu, Chattisgarh and Maharashtra. In order to increase of the durability and spectrum of resistance, we have introgressed a newly identified novel resistance gene, Xa33 into ISM and developed breeding lines of ISM, Sampada and Akshayadhan possessing Xa33 and other novel resistance genes. With a focus on monitoring the virulence of Xoo strains across the country, we have deployed molecular tools to characterize Xoo pathotypes, identified rice susceptibility gene targets of the virulent isolates of Xoo and identified rice resistance gene combinations for durable resistance.

MOLECULAR BREEDING Chair: HS Gupta, Yue-le Hsing

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Concurrent Session VIII

47. The Origin and Molecular Mechanism of the Wild-abortive Cytoplasmic Male Sterility in Rice

Liu Y-‐G

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources; South China Agricultural University, China. [email protected]

Cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes in plants and is crucial for hybrid crop breeding. The Wild Abortive CMS (CMS-WA) has been widely exploited in the “three-line” hybrid rice production. By whole mitochondrial transcriptom analysis among CMS-WA line, maintainer line, and fertility-restored line, and transgenic study, we have identified a mitochondrial gene, WA352, conferring CMS-WA. WA352 is a chimeric gene generated from multiple recombination events involving three mitochondrial fragments (but not from essential known genes) and one of unknown origin. We traced the evolutionary rout of WA352 and found a number of intermediate structures related to completing the WA352 origination, as well as several variants of WA352, in different wild rice species. We also found that other types (GA, ID, Dissi, DA, K, Y, X) of rice sporophytic CMS lines used for hybrid rice breeding, which were bred using different female parents of indica rice or O. rufipogon, also contained WA352. Although WA352 mRNA species are transcribed constitutively, WA352 protein accumulates specifically in the anther tapetum of microspore mother cell stage. WA352-induced sterility can be suppressed by two restorer genes, Rf4 and Rf3, with different different mechanisms, probably Rf4 functioning post-transcriptionally and Rf3 post-translationally. WA352 protein interacts with the nuclear-encoded mitochondrial protein COX11, which is highly conserved in eukaryotes and functions as an assembly factor of the cytochrome c oxidase complex and has a role in degrading reactive oxygen species (ROS). This specific interaction inhibits the COX11 function in peroxide metabolism, leading to ROS burst and release of cytochrome c from mitochondria to the cytosol. These signals trigger premature programmed cell death in the tapetum thereby causing sporophytic CMS. Thus, the CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental interaction between a newly-evolved mitochondrial gene and a conserved, essential nuclear gene.

48. Major QTLs for grain yield under drought: is rice different from other cereals? Kumar A

International Rice Research Institute, South Asia Hub, Patancheru, Hyderabad 502324, [email protected]

Extensive QTL mapping studies on QTLs for grain yield under drought undertaken at IRRI from 2003-2013 led to the identification of fourteen large effect QTLs showing large effect in the background of popular high yielding varieties Swarna, IR64, MTU1010, Vandna, Sabitri and TDK1. Among the identified QTLs, nine were effective across multiple genetic backgrounds and six among the nine were effective in both lowland and upland ecosystem. Pyramiding of two to three QTLs was an appropriate strategy to achieve yield gain of 1.0 t/ha or more under drought. Accordingly, qDTY1.1, qDTY3.1 and qDTY12.1 combination for lowland and qDTY12.1, qDTY2.3, qDTY3.2 and qDTY6.1 combination for upland were identified as effective for high yield gains under moderate to severe drought. Identified QTLs/QTLs combinations have been introgressed in Vandna, Anjali, IR64, Swarna, MRQ74, and MR219 and predicted yield gains of 1.0 t/ha or more under moderate to severe drought was achieved without any yield penalty under irrigated situation. Drought grain yield QTLs have been successfully combined with submergence tolerance in Swarna sub 1 background to develop rice variety providing tolerance to both submergence and drought in same or different seasons. Presence of multiple sub QTLs within some of these QTLs and their digenic interactions leading to high grain yield under drought were also revealed. Many QTLs were linked with days to early flowering and/or tall plant height. The undesirable linkage drags removed and dwarf near isogenic lines with single/QTLs combinations developed at IRRI for use in regular breeding program to develop dwarf, high yielding drought tolerant rice varieties. Physiological and molecular studies revealed that multiple, often complementary mechanistic routes to tolerance may exist and that even known tolerant cultivars could be improved further. Large-effect QTLs thus are a practical alternative to the elusive single gene master-regulators. Existence of such large effect QTLs for grain yield under drought have not been reported in other major cereals.


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Concurrent Session VIII 49. Genomic approaches to accelerate rice breeding programs for increasing

productivity and sustainability Brar DS, Singh K

School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana (Punjab), India. [email protected]

During the last few decades, major advances have been made in increasing rice productivity and world rice production has more than doubled. This was achieved primarily through the application of principles of Mendelian genetics and conventional plant breeding. However, we need 25 % more rice to meet the growing demand in 2030. This is even more challenging given the continual threat by several diseases, insects and abiotic stresses like drought, floods, salinity, cold, heat, etc., particularly in the context of global climate change. The major challenge at present is how to overcome these constraints and produce more rice in an eco-friendly way using less land, water, labor and chemicals. Advances in molecular biology and genomics have opened new avenues to clone and understand the function of genes governing agronomic traits, accelerate breeding programs and to develop improved rice germplasm with new genetic properties. Some such developments include: (i) dense molecular maps and new generation of markers such as SNPs, facilitating mapping of genes/ QTL and precise characterization of genetic resources, (ii) availability of whole genome sequence from a number of diverse lines for gene annotation, SNP development and allele mining, (iii) high-throughput genotyping for MAS and gene chips for gene discovery and expression analysis. (iv) tagging of many important genes/QTLs for tolerance to biotic and abiotic stresses and improved quality traits, which have been pyramided and used in MAS to accelerate rice breeding programs (v) allele mining for identifying alleles suited to a particular environment, iv) characterization and precise transfer of novel genes from wild relatives (s) into elite breeding lines. Several successful examples have been published in each of the above mentioned developments in rice and other crops. In addition, genome wide selection and sequence based MAS offer opportunities to accelerate rice breeding, especially for the difficult traits. Integration of genomic approaches with conventional breeding is emphasized to accelerate rice improvement and to further increase rice productivity and sustainability.

50. Identification of QTLs and genes for varietal improvement of tolerance to flooding during germination in rice

Septiningsih EM, Kretzschmar T, Ignacio JC, Pelayo MA, Casal C, T hein SZM, Mackill DJ, Ismail AM

International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines [email protected]

Tolerance of flooding or anaerobic conditions during germination, referred to as anaerobic germination (AG), is an essential trait in direct-seeded rice ecosystems. Most rice varieties fail to germinate or sustain further growth under anaerobic conditions when water is standing in the fields leading to poor seedling establishment. However, a few rice landraces having an exceptional capability to overcome this stress have been recently identified from the IRRI genebank. Since then, studies to unravel the molecular and physiological mechanisms of AG stress tolerance and varietal improvement of AG tolerant varieties have been ongoing and considerable progress has been made. Exploring novel QTLs for tolerance of anaerobic conditions during germination from diverse germplasm is essential to capture the complexity of the trait and to provide additional targets for marker assisted breeding. Thus far, several germplasm accessions have been utilized in QTL studies using SSR and SNP markers, and significant QTLs for AG tolerance have been identified from a number of donor landraces. Selected major QTLs have been fine-mapped and are being pursued for cloning and for use in molecular breeding to transfer them into popular varieties as well as varieties carrying the SUB1 QTL. Pyramiding multiple QTLs in the right genetic backgrounds will be critical to successfully develop more robust AG-tolerant varieties, both to combine multiple AG QTLs as well as AG plus SUB1. Ultimately, this trait can also be combined with other abiotic and biotic stress tolerance QTLs to provide more resilient rice varieties for both irrigated and rainfed conditions and to cope with the challenges of future rice production under various global climate change scenarios.


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51. CARBON ASSIMILATION RATE 8, a gene identical to DTH8/Ghd8 responsible for heading date, pleiotropically increases the rate of leaf photosynthesis in rice

Adachi S, Yamanouchi U, Tanabata T, Sun J, Hirasawa T, Yamamoto T, Yonemaru J 1Natl. Inst. Agrobiol. Sci. 2RIKEN CSRS 3Graduate. School. of Agriculture, Tokyo Univ. of Agriculture and Technology.. [email protected]

Increases in rates of individual leaf photosynthesis (Pn) are critical for future increases in rice yields. The use of natural genetic variation among cultivars can be an effective strategy for the genetic improvement of rice photosynthesis. The high-yielding indica variety Habataki has one of the highest recorded Pn among Asian rice cultivars. In previous reports, we have detected four QTLs for Pn of the flag leaves at the full heading stage by using progeny derived from a cross between Habataki and Koshihikari, a leading variety in Japan. Among these QTLs, we aimed to identify the causal gene for CAR8 (CARBON ASSIMILATION RATE 8), detected on chromosome 8, and to evaluate its effects on photosynthesis-related processes. Near-isogenic line NIL(CAR8), which has a Habataki chromosome segment of less than 1 Mb in the Koshihikari background, showed higher stomatal conductance but similar stomatal density in comparison with Koshihikari. NIL(CAR8) showed a maximum rate of carboxylation (estimated from A/Ci curves) similar to that of Koshihikari. These results indicate that the Habataki allele at CAR8 increases Pn by increasing stomatal aperture. We narrowed the CAR8 region to 11.0 kb by using newly developed mapping populations. The RAP database predicts only one annotated gene, Os08g07740, in the region. This gene encodes a protein of 297 amino acids in Koshihikari, but only 125 amino acids in Habataki because of a frameshift mutation. Os08g07740 has been reported as DTH8/Ghd8, a gene affecting heading date (Wei et al., 2010; Dai et al., 2012); in line with these reports, NIL(CAR8) flowered 7 days earlier than Koshihikari. These results suggest that CAR8 and DTH8/Ghd8 are identical, and are pleiotropically involved in Pn.

52. Transfer of value-added genes from wild Oryza species for rice improvement Jena KK

Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, Los Baños, Laguna, Philippines. [email protected]

The Oryza genus has two cultivated species (O. sativa and O. glaberrima) and 22 wild species with 10 different genomes. The wild species have enormous untapped economically important genes/traits to increase rice production and productivity. It is hard to transfer genes from secondary and tertiary genepools compared to the species of primary gene pool. Novel resistance genes for several traits could be transferred into cultivated rice across crossability and recombination barriers. The rarest interspecific hybrids involving the wild species O. punctata (BB genome) and O. granulata (GG genome) possessing yellow motle virus and sheath blight resistance, water-use efficiency and low light intensity tolerance were produced through bridge crosses. Multi-parent Advanced Generation Inter Cross (MAGIC) populations involving six AA genome wild species and two cultivated species were produced to isolate transgressive variants for many traits unavailable in cultivated rice germplasm. Several value-added genes for broad-spectrum resistance to major diseases and insects were transferred into cultivated rice through production of sesquidiploids and monosomic alien addition lines. The results of these studies will be presented.


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53. Association mapping for grain size traits in rice Deborah DAK, Patil S, Veeraghattapu Roja, Anuradha G, Siddiq E A, Vemireddy LR

Institute of Biotechnology, Acharya N. G. Ranga Agricutural University, Hyderabad 500030, India. [email protected]

Introduction Grain size, an important quality trait of rice is affected by length, breadth and length-breadth ratio influenced by multiple genes. Association mapping is a powerful tool for genetically dissecting complex traits controlled by multiple quantitative trait loci (QTLs). In the present study, an attempt was made to identify markers associated with kernel size, kernel weight, and grain size and grain weight by candidate gene based association mapping. Methodology In all, 96 rice genotypes were used for recording grain size traits viz., length (L), breadth (B), L/B ratio and grain weight for both kernel and polished rice and for screening with molecular markers associated with the grain size QTLs/genes from previous reports. Association mapping was done using GLM and MLM models of TASSEL 2.1. Results Of the markers used, the locus RM18616 explained maximum phenotypic variance (14.6%) followed by RM430 (10.42%). The locus RM18582 linked to the major QTL for grain size on chromosome 5 was found to be associated with all the grain size traits and grain weight both in kernel and polished form of rice with a phenotypic variance range of 2-10%. The markers linked to the known genes viz., GW5, qSS7, qGW8 and SRS5 also showed association with the majority of the grain size traits but with low phenotypic variance ranging from 2.0-8.44%. Discussion and Conclusion From the present study, RM18582 and RM18616 loci found to be strongly associated with grain size and have the potential to be used in marker assisted breeding of grain size traits.

54. A comprehensive study for salt tolerance in high yielding rice introgression lines derived from Oryza rufipogon and Oryza nivara

Rai V, Pushpalatha G, Sukumar M, Sreenu K, Brajendra P, Subrahmanyam D, Subbarao LV, Ram T, Srala N

National Research Centre on Plant Biotechnology, IARI, Pusa, New Delhi, 110012, India. [email protected]

High yielding introgression lines (ILs) were evaluated at different developmental stages for their salinity-stress-mediated morphophysiological, biochemical and molecular responses. Based on seed germination and seedling growth responses at 0-200 mM NaCl, ILs were categorized into sensitive (SSIL) and tolerant (STIL) and the STILs have higher yield as compared to SSILs at 100 mM NaCl. A differential accumulation pattern of Na+ across different ILs ranging from no accumulation in STILs (K467, K463, K381, K478) to higher accumulation in both STIL K458 and SSIL K40 was observed. Tolerance to salinity could be mediated either through Na+ exclusion in STILs (K467, K463, K381, K478) or its compartmentalization of STIL K458. SSIL K40 accumulated Na+ to toxic level due to its inability to maintain Na+. To study gene expression 64 differentially expressed genes (DEGs) were short listed based on their involvement in stress mediated responses through transcriptomics of K478 and K198. Among all, 55 genes were differentially expressed and only 3 were expressed in shoots and 52 in roots and SSIL line has higher DEGs than the STIL. The study highlights the prevalence of wide spectrum genetic variability across SSILs and STILs that helps in dissecting the mechanism of salinity stress responses.

TRANSLATIONAL GENOMICS Chair: Venkatesan Sundaresan, Rakesh Tuli

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Concurrent Session IX

55. T-DNA tagging and functional analysis of an NEP-MEF2B domain protein gene which controls stamen development in rice

Veluthambi K, Majhi BB, Shah JM, Basu D, Sarwade R

Department of Plant Biotechnology, School of Biotechnology, Madurai Kamarraj University, Madurai-625021, India. [email protected]

The T-DNA-tagged rice mutant TC-19, which harbored a single copy of the T-DNA, displayed male sterile phenotype in the homozygous condition. Analysis of the junctions between the T-DNA ends and the rice genome revealed a complex integration in which the T-DNA, together with a 6.55-kb region of chromosome 3 and a 29.8-kb region of chromosome 9, was translocated into chromosome 8. The translocated segments of chromosomes 3 and 9 were found to be restored in the native chromosomes by duplication. Four genes present at the chromosome 3 and 9 breakpoints expressed in both control and TC-19 homozygous mutant plants. Thus, male sterility is not due to the disruption of these four genes. The final insertion site of the complex T-DNA was in the first intron of a ‘hypothetical gene’ Os08g0152500 on chromosome 8. Expression of the Os08g0152500 gene, which was predominant in stamens, was disrupted in the TC-19 homozygous mutant. This gene encodes a hypothetical protein of 268 amino acids. The protein contained an uncharacterized NEP (N-terminal eighty residues of a family of proteins) domain and a MEF2B (MADS-box transcription enhancer factor 2) domain. Blast search revealed that the NEP domain is conserved in many plant species but its function is not known. MEF2B is a member of the MADS domain family and has potential DNA binding properties. The promoter of the ‘hypothetical’ gene showed the presence of various cis-acting elements (GTGA, AGAAA and E-box) known to confer anther- and pollen-specific gene expression. Promoter-GUS fusion analysis showed a low level of GUS expression in leaf but a very high level of GUS expression in P1, P2, P3 and P4 stages of panicle development. In a genetic ablation study, the promoter fused to the cytotoxic diphtheria toxin A-chain gene caused male sterility in the transgenic rice plants. Silencing of Os08g0152500 gene expression severely affected pollen viability. These results demonstrate a crucial role of the tagged Os08g0152500 gene in stamen development in rice. The promoter of this gene holds promise to engineer ‘heterosis’ in rice.

56. Integrating genomics with conventional breeding for the improvement of rice Singh AK1, Ellur RK1, Gopala-‐Krishnan S1, 1Division of Genetics, Indian Agricultural Research Institute, New Delhi, India. [email protected]

Basmati rice from Indian subcontinent is acclaimed internationally for its unique cooking quality and pleasant aroma. The genetic improvement of Basmati rice at IARI has not only helped in improving the productivity but also in realization of better profits by farmers’, industry as well as consumers and the earnings from Basmati rice export has increased to ~ US $ 3.2 billion in 2012-13, out of which 75% is contributed by IARI Basmati rice varieties. Advances in rice genomics have provided an unprecedented opportunity for dissection of genes/ QTLs underlying major grain quality traits, mining alleles for important grain quality traits and marker assisted selection for precise transfer of genes/ QTLs for biotic and abiotic stress tolerance into various popular Basmati rice varieties. MAS have been successfully utilized in development and release of one of the first MAS derived rice variety Improved Pusa Basmati 1 with resistance to bacterial blight (BB), which have been followed by recent release of blast resistant variety Pusa 6 (Pusa1612). Further, a huge array of isogenic lines in Basmati background have been developed utilizing diverse germplasm for combating multiple biotic and abiotic stresses such as BB, blast, sheath blight, Bakanae, BPH, salt tolerance; for development of provitamin A rich golden rice through effective integration of genomic tools in rice breeding. These genotypes have also been registered as genetic stocks for utilization in Basmati rice improvement. Genomic resources complimented by suitable genetic resources have also been used in mapping genes, discovering novel QTLs for yield and yield related traits, grain and cooking quality traits, allele mining, development of heterotic pools and in assessing genetic purity of hybrid seeds.


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57. Gene-targeting-mediated mutagenesis in rice Saika H, Osakabe K, Yokoi-‐Nishizawa A, Kwon Y-‐I, Ohtsuki N, Mori A, Endo M, Toki S

National Institute of Agrobiological Sciences, Tsukuba, Japan. [email protected]

Gene targeting (GT) allows the introduction of desired mutations into a target gene via homologous recombination (HR). GT can be used in the fields of both molecular breeding and functional genomics as a powerful tool for reverse genetics. In 2002, Terada et al. reported the first successful disruption of the waxy gene in rice via GT. To date, target modification via GT has been reported in ~ 10 genes of rice. Using GT, we have produced novel rice plants with improved agronomical traits, e.g., herbicide-tolerance, tryptophan fortification and reduction of potential allergen content. Currently, the most serious obstacle to GT in plants, including rice, is its low frequency; GT occurs at a rate of 10-3 to 10-6 compared with random integration of T-DNA. We have succeeded in drastically improving the frequency of HR and GT by induction of DNA double-strand breaks (DSBs) at the target locus in combination with enhanced resection of DSBs. In a general and universal GT system, positive and negative selection markers should be used for efficient selection of cells in which GT has occurred successfully. A positive selection marker to select cells in which stable transformation has succeeded is inserted into the target sequence in the GT vector, whereas negative selection markers that will kill cells in which T-DNA is inserted at random loci are added at the ends of the target sequence. Thus, exogenous sequences including the positive selection marker remain inside or adjacent to the target gene, resulting in the possibility of altered gene expression levels and patterns. We have succeeded in the complete elimination of the positive selection marker leaving no footprint using piggyBac transposon, showing that the combination of target modification via GT and subsequent marker elimination results allows introducing only the desired point mutations into the target gene.

58. Improvement of Iron in Rice: Accumulation and Bioavailability Datta K, Ali N, Paul S, Datta SK

Department of Botany, University of Calcutta, Kolkata, India. [email protected]

Iron deficiency anaemia (IDA) is recognised as major form of micronutrient malnutrition in human population followed by other micronutrients like zinc, folic acid, essential amino acids. Rice, as a staple cereal food provides nutrition for majority of people in developing country. Therefore iron improvement of rice will have a positive impact for human health specially for people suffering from IDA. Genetic Engineering approach have been employed to increase the iron component in rice endosperm, the edible portion of grains. Iron storage protein gene ferritin, cloned from soybean/rice driven by rice glutelin promoter has been overexpressed for storing more iron molecules in polished grains. More than two fold iron content in polished seeds of transgenic ferritin rice comparing with that of non-transgenic rice seeds has been achieved. Phytic acid, inositol-hexakisphosphate is a phosphorus containing compound that strongly binds mineral cations to form phytate. Phytic acid in rice seed embryo and aleurone layer plays a negative role in bioavailability of iron and other bivalent minerals together with phosphorus. Bioavailability of iron from rice can be increased by reducing the level of phytic acid in the rice embryo and aleurone layer. Rice embryo and aleurone specific promoter (ole18) and aleurone layer specific promoter (lpt2) has been cloned from rice to down regulate phytic acid biosynthetic enzymes. myoinositol- phosphate synthase (mips), for the first enzyme for phytic acid biosynthesis from glucose-6-phosphate and inositol phosphate kinase (ipk1) for the final step in phytic acid biosynthesis has been cloned from rice. To achieve reduction of phytic acid level in rice embryo and aleurone layer, down regulation of the two key enzymes has been carried out by RNAi mediated silencing of the two genes driven by ole18 as well as lpt2 independently. Down regulated transgenic rice lines exhibited higher level of inorganic phosphorus with increment of iron level in the polished seeds.


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59. Analysis of pathogenicity in the rice blast fungus Mohanan VC, Chandarana P, Chattoo BB

Genome Research Centre, Department of Microbiology and Biotechnology Centre, M.S.University of Baroda, Baroda -390002, India

Rice blast disease is a major limiting rice productivity. Current methods of management involve the use of chemicals or deployment of resistant varieties bred for resistance against specific strains of the fungus. The pathogen offers challenges since it is highly variable and is able to overcome host resistance within a few years. We have been exploring the possibility of identifying potential novel targets in the fungus for improved management of the disease. We have used bioinformatics analysis to identify possible targets and have used gene disruption and gene silencing to assess the effects on growth and development of the fungus. Several interesting phenotypes have been obtained and are being evaluated for their effects on pathogenicity. Transcriptome of selected mutants is being analysed to obtain a comprehensive view of the effects of specific mutations on cell growth and metabolism which may lead us towards a better understanding of pathogenicity.

60. Investigation of the mechanism of salt tolerance among rice genotypes: Existence of different mechanisms for ion homeostasis

Samant A, Saini A, Jawali N

Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai-400085. [email protected]

An increased Na+ level in salinity disrupts ion homeostasis in plant cells and affects metabolism. In plant mechanisms involved in ion homeostasis are a) Na+ exclusion and recirculation (especially in lower shoot), b) minimizing Na+ influx into photo synthetically active cells and c) minimizing the cytosolic Na+ concentration by vacuolar compartmentation. Among Oshkt family Na+ selective transporters, Oshkt1;4 and Oshkt1;5 are involved in Na+ exclusion from active transpiration stream in xylem and protects leaves from salinity induced damage. Whereas Osnhx family (Na+/H+ and K+/H+) antiporter and Osvp (Vacuolar H+-translocating inorganic pyrophosphatase) are involved in sequestration of Na+ in vacuoles. Early seedling stage is most salt sensitive development stage in rice and hence, mechanisms involved in ion exclusion from xylem stream and recirculation into phloem has paramount importance in overcoming ion toxicity. We are investigating the mechanism of salt tolerance among 11 rice genotypes having different salt tolerance/sensitivity by analyzing biochemical and transcript changes in response to salinity stress. All genotypes increased Proline levels under salinity stress. However, the salt tolerance and Proline level are negatively correlated. Analysis of ions in shoot tissue showed a wide range of change in the Na+, K+, and Ca2+ content under salinity stress and did not correlate with the salt tolerance. An interesting observation is that genotypes with similar salt tolerance showed large differences in Na+ content; suggesting different mechanisms of ion homeostasis was operational. We analyzed relative transcript level of genes that are involved in the ion homeostasis mechanism viz. Oshkt1; 5, Osnhx family genes and Osvp1. The results of the transcript profiling of these genes in response to salinity stress and their relationship with the mechanisms operational in the rice genotypes will be presented.


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61. Innovative approaches for rice genotyping to meet the increasing global demand for rice

Yadav P

Imperial Life Sciences Pvt Ltd, Gurgaon, Haryana 122001, India

Rice is one of the most important crops for mankind. It feeds virtually half the world’s population and accounts for more than 50% of their daily calorie intake. Even though in the last few years, world’s rice production have doubled but it is still insufficient to cope up with the ever-increasing global demands. To feed the world population expected to be 8 billion by 2030 require’s 22% increase in rice production. Advancement in technology and utilization is the best possible approach to meet the future demand. Among the accessible molecular methods for breeding and selection, SNP genotyping is the most promising functional approach. Since SNPs are most abundant, stable, amenable to automation, informative, efficient cost-effective method. The availability of high throughput SNP genotyping platforms and chemistry provides the opportunity to simultaneously screen hundreds to thousands of markers per individual. The method can be effectively used in marker-assisted and genomic selection, association, QTL mapping, variety identification seed purity testing etc. Low cost and high throughput assay makes it possible to implement the genotyping strategy on large populations and shorten the time for the development of new varieties in diverse crop type. Currently available technologies offer an economical approach to type a large set of SNP markers, without SNP dropout from lot-to-lot. Thus, it maintain the power of panel and is high throughput, performing automated analysis for an assorted study type. Next generation genotyping by hybridization (GBH) approach on Axiom technology from Affymetrix fits in the above mentioned necessities; the readily available arrays and myDesign genotyping customized and semi-customized assays offer panel for genome-wide association, replication, fine mapping and candidate gene studies. Axiom agrigenomics genotyping solutions offer breeders and researchers cost-effective genotyping tools to identify, validate, and screen complex genetic traits for leveraging the highest economic value of the plant. The Axiom genotyping by hybridization technology is high throughput, automated, robust, flexible, automatically assigns genotypes for haploid, diploid and polyploid genomes for speedy and successful genomic selection programs. The available catalog arrays for gene expression and miRNA on Affyemtrix technology complement the genotyping studies. Axiom Rice Genotyping arrays are used globally by leading rice researchers including many Key Opinion leaders from India.

62. Current Trends in Multi Colour Confocal Imaging Bhattacharya AK

Life Science Research, Leica Microsystems, A Division of DHR Holding India Pvt Ltd, New Delhi [email protected]

Confocal microscopy is becoming more demanding with investigations aiming to visualize multiple cellular and sub-cellular targets in the same specimen. This therefore poses challenges both for the excitation of the fluorescent probes for these targets and as well as for their detection. On the excitation side all large number of excitation wavelengths need to be available so as to make optimal excitation of the probes as effective as possible. On the detection side the ability to detect multiple signals needs to be realized. Besides, multi colour confocal imaging acquires an additional dimension for the observation of living cells as ideally excitation and detection of multiple dyes needs to be simultaneously executed in real time. Also, on the detection side fluorescence signals will be present in varying intensities and therefore there is a need to ensure that they are adequately represented in the image. These issues will be elaborated and discussed with reference to the Leica confocal microscope, TCS SP8.

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49

Poster Session I

PI-01. Constitutive activation of abscisic acid responsive transcription factor OsbZIP23 improves drought tolerance in rice

Dey A, Sen SK

Advanced Laboratory for Plant Genetic Engineering1, Advanced Technology Development Center, Indian Institute of Technology, Kharagpur, India. [email protected]

Drought occurs when there is insufficient soil moisture to meet the need of crop plants. Rice (Oryza sativa L.) is a moisture-loving plant and thus its productivity is adversely affected by soil moisture stress (SMS). The phytohormone,abscisic acid (ABA), the central regulator of drought resistance in plants,is produced under SMS condition. Plants under adverse condition of SMS triggers ABA signal transduction pathway which involves several transcription factors (TFs) that regulates the expression of many target genes.OsbZIP23 is one such TF which is SMS responsive and is involved in ABA signaling pathway.The present study about natural allelic variations in genomic resources suggested us to find variation in allelic forms of OsbZIP23 gene in related rice genotypes(RGs)and to transform a better allelic form to sensitive RG through transgenic approach to make it relatively tolerant to drought. Keeping this in view,we searched for presence of variation in the allelic forms of OsbZIP23 gene and their regulatory elements.Allelic polymorphism in different RGs was checked through molecular cloning and sequencing of OsbZIP23 gene, which showed presence of deletions and SNPs.Variation in regulatory elements was checked through molecular cloning of 1.6kb upstream of OsbZIP23 gene from different RGs. By searching the promoter sequence against PLACE and plantCARE database many putative stress response-related cis-elements were predicted. To check the activity,a OsbZIP23 promoter-GUS assay construct was transformed to a tolerant cultivar. A polymorphic form of OsbZIP23 gene was overexpressed under a constitutive promoter in a sensitive cultivar. Transgenic analysis of overexpression lines were done through PCR and Southern blot analysis. Since OsbZIP23 is strongly induced by ABA, so we tested ABA-sensitivity of independent overexpression lines at the germination stage. The relative expression level of OsbZIP23 in the overexpression lines was checked through Real Time PCR analysis. Relative grain yield per plant was checked at the reproductive stage. The results so far shows that overexpression of OsbZIP23 in a sensitive elite cultivar improve drought tolerance and grain yield in rice.

PI-02. Molecular and biochemical characterization of protein L-isoaspartyl methyl transferases from rice

Petla BP, Kamble NU, Majee M

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi - 110067, India. [email protected]

Protein L-Isoaspartyl (D-aspartyl) methyltransferases (PIMT or PCMT EC 2.1.1.77) recognize and repair the isomerized and racemized products from the spontaneous damage of L-aspartyl and L-asparaginyl residues. This widespread enzyme maintains a high degree of sequence conservation in organisms such as bacteria, nematodes, flies, plants and mammals, including humans. In animals, the enzyme is reported to be abundant in brain and in plants the enzyme is concentrated in seeds.The unusual property of this methyltransferase to recognize damaged aspartyl residues was first shown for the human and bovine enzyme. Interestingly the over expression of the chickpea (Cicer arietinum) PIMT in E. coli mitigated the oxidative stress induced growth inhibition. Attention was paid to plant PIMTs when the sacred lotus aged nearly thousand years showed remarkable germination efficiency which was found to contain good amounts of active PIMT. In chickpea CaPIMT1 repairs damaged proteins in the membrane and cytoplasm, whereas CaPIMT2 is mainly involved in the repair of nuclear proteins enhancing seed vigor and longevity. In the present study, an attempt was made to characterize PIMTs from monocot model plant rice. In rice PIMT activity was found to be localized mostly in seeds followed by stem and leaves. PIMT activity was also found to be enhanced during environmental stresses. Genes encoding PIMT (OsPIMT1 and OsPIMT2) from rice were isolated and cloned for molecular and biochemical characterization. Both OsPIMT1 and OsPIMT2 were bacterially expressed, purified to homogeneity and biochemically characterized. Both enzymes showed different temperature optima although they shared same pH optima and fairly similar Km, Vmax values. Stress survivability test with E. coli expressing OsPIMT1 and OsPIMT2 showed reduced growth inhibition compared to control, suggesting that PIMTs play an important role in mitigating oxidative stress-induced growth inhibition.

PI-03. Identification of local rice germplasm with multiple abiotic stress tolerance

ABIOTIC STRESS

50

Poster Session I Kumar BS, Jena PP, Kumar J, Singh ON, Reddy JN, Mohapatra T

Central Rice Research Institute (Indian Council of Agricultural Research). [email protected]

Rice is the staple food for more than half of the world’s population, especially those living in developing countries such as India, China, Pakistan, Bangladesh, Laos, Vietnam and Indonesia. However, the growth and productivity of rice are severely affected by various abiotic stresses, such as submergence, drought, high salinity and high/low temperature under changing climate. In the present study, through phenotypic as well as genotypic screening using SSR markers linked with major QTLs associated with submergence, salinity and drought tolerance, we have identified local rice gernplasm associated with multiple abiotic stress tolerance. In phenotypic screening, out of 122 genotypes, 19, 23 and 46 were found as highly tolerant to submergence, salinity and drought, respectively. In PCR screening, 19, 46 and 44 genotypes showed presence of FR13A alleles with IYT1 (located in promoter region of SUB1A gene), Sub1BC2 (in region between SUB1B and SUB1C genes) and ART5 (located in promoter region of SUB1C gene) markers, respectively for submergence tolerance. For salinity tolerance, 31, 31, 24 and 53 genotypes showed alleles similar to Pokkali genotype (salinity tolerant) with RM3412, AP3206, RM8094 and RM493, respectively. In case of drought tolerance, 79 and 45 genotypes showed alleles similar to that of drought tolerant line IR81896-BB-195 with RM324 and RM520 markers linked with DTY2.1 and DTY3.1 QTLs, respectively. Rice genotypes which were classified as tolerant or highly tolerant in the phenotypic screening were corroborated with results of genotyping using PCR markers, IYT1 (submergence tolerance), RM8094 (salinity tolerance) and RM520 (drought tolerance). Moreover, in this study, we have found that some of the rice genotypes showed tolerance to multiple stresses. For example, Mugei and Bodala Champa genotypes had submergence and salinity tolerances; Kalaketaki and Khoda genotypes showed submergence and drought tolerance; Gangasiuli, Hatimala and Pohasali genotypes gave tolerance to submergence, salinity and drought. The rice genotypes having multiple abiotic stress tolerance will be highly useful in rice breeding for improving rice varieties to survive under changing climate.

PI-04. Roles of rice protein phosphatase 2C subclass A in response to abiotic stresses revealed by phylogenetic analysis and expression profiling

Bhatnagar N1,2, Jung K-‐H1, Kim B-‐G2 1Department of Plant Molecular Systems Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea, 2Molecular breeding division, National academy of agricultural science, RDA, Suwon 441-707 Korea. [email protected]

Plants encounter various abiotic and biotic stresses due to several climatic changes resulting into adverse impact on their growth and yield. PP2C is a major class of protein phosphatase gene family that plays a vital role to overcome these stresses. A total of 78 PP2C genes have been reported in rice and is further divided into 11 subclasses. Of them, Arabidopsis orthologs of subclass A have been observed showing elevated expression in abiotic stress. In this study, the protein sequences of all the members of subclass A of rice were retrieved from Rice Genome Annotation Project (http://rice.plantbiology.msu.edu) and were then subjected for multiple sequence alignment with ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2). The collective result of phylogenetic analysis and meta-expression profiling analysis of OsPP2C subclass A gave a prominent result showcasing significant differential expression in drought, salt and cold from most of members: Under drought and salt stresses 7 genes were up-regulated, and under cold stress 5 genes were down-regulated. Our result enlightened towards the importance of these genes when subjected to various stress tolerance mechanisms. The current data and further research avenues will ultimately widen the knowledge of the role OsPP2C subclass A in rice, forming path for better yield and improved quality of the crop.

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51

Poster Session I PI-05. Exploring the role of rice homeobox transcription factors in abiotic stress

responses Bhattacharjee A, Jain M

Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067. [email protected]

Homeobox transcription factors are well-known regulators of plant growth and development. They contain 180 bp long conserved DNA sequence encoding a 60 amino acid long, DNA binding homeodomain. They have been categorized into 14 classes based on their domain composition and phylogenetic relationship. A total of 110 homeobox transcription factors have been identified in rice, which is a crop of great agronomic importance. Its productivity is greatly compromised due to several abiotic stress factors. Hence, there is a need to identify most promising candidate genes to engineer stress tolerance in rice. A few of the homeobox transcription factors have been implicated in abiotic stress responses in different plants. In this study, we aim to investigate the role of two selected rice homeobox transcription factors, belonging to HD-ZIP I class, in abiotic stress responses. Previous microarray studies have shown them to be highly upregulated under abiotic stress conditions. We also found highly upregulated gene expression of these transcription factors, under different abiotic stress conditions during various developmental stages of rice. Additionally, it was seen that exogenous hormonal treatments led to altered gene expression. Moreover, these genes have been found to be highly upregulated under dehydration and salinity stress in different rice varieties with contrasting stress response. Promoter analysis of these genes revealed that they are highly enriched in stress-responsive cis-regulatory elements. The transient expression of full-length GFP fusion proteins in onion epidermal cells showed that these transcription factors are nuclear-localized. In addition, autoactivating property of these proteins was established by transactivation studies in yeast. Arabidopsis transgenics have shown increased susceptibility to abiotic stress conditions at the seedling stages as compared to the wild-type. These preliminary evidences give an indication that the selected transcription factors are involved in abiotic stress responses. Further, investigations to establish their role in abiotic stress responses are in progress.

PI-06. Multiple stress inducible ETHE1-like protein from rice is highly expressed in roots and is regulated by calcium

Kaur C, Mustafiz A, Sarkar AK, Ariyadasa TU, Singla-‐Pareek SL, Sopory SK

Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg New Delhi, India. [email protected]

ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) encodes sulfur dioxygenase activity regulating sulfide levels in living organisms. It is an essential gene and mutations in ETHE1 leads to ethylmalonic encephalopathy (EE) in humans and embryo lethality in Arabidopsis. Presently, very little is known regarding the role of ETHE1 beyond the context of EE and almost nothing is known about factors affecting its regulation. In the present study we have identified, cloned and characterized OsETHE1, a gene encoding ETHE1-like protein from Oryza sativa. ETHE1 proteins in general are most similar to glyoxalase II (GLYII) and hence OsETHE1 has been earlier annotated as OsGLYII1, a putative GLYII gene. Here we show that OsETHE1 lacks GLYII activity and is instead an ETHE1 homolog being localized in mitochondria like its human and Arabidopsis counterparts. We have isolated and analyzed 1,618 bp OsETHE1 promoter (pOsETHE1) to examine the factors affecting OsETHE1 expression. For this, transcriptional promoter pOsETHE1: β-glucuronidase (GUS) fusion construct was made and stably transformed into rice. GUS expression pattern of transgenic pOsETHE1:GUS plants reveal a high root specific expression of OsETHE1. The pOsETHE1 activity was found to be stimulated by Ca(II) and required light for induction. Moreover, pOsETHE1 activity is highly induced under multiple abiotic stresses specifically, heat and oxidative stress, suggesting a potential role of OsETHE1 in stress response.

ABIOTIC STRESS

52

Poster Session I

PI-07. Molecular characterization and functional analysis of rice transcription factor OsbHLH035 under salinity stress

Chen H-‐C, Yang Y-‐W, Chen Y-‐J, Tsai C-‐N, Chang M-‐C

Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. [email protected]

The basic/helix-loop-helix (bHLH) transcription factors play important roles in the regulation of many developmental and physiological processes in plants. So far, at least 167 bHLH genes have been identified in rice. It is noteworthy that expression levels of several OsbHLH genes were significantly up-regulated under abiotic stresses, such as OsbHLH035 gene. However, whether the OsbHLH035 actually involved in the regulation of rice abiotic-stress responses and the corresponding molecular mechanism are still unknown. In this study, the role of OsbHLH035 under salinity stress was investigated by using reverse-genetic approaches. The germination rate of Osbhlh035 seeds was lower than that of wild-type (Nipponbare), especially in 250 mM NaCl conditions. The photosynthetic efficiency of Osbhlh035 seedlings was lower than that of wild-type, whereas the malondialdehyde (MDA) content was higher. Histochemical staining of promoter::GUS activity in transgenic rice indicated that OsbHLH035 was expressed in root tips and lateral root initiation sites. Interestingly, the GUS signal was significantly enhanced in 250 mM NaCl conditions. Both OsbHLH035-GFP and GFP-OsbHLH035 fusion proteins are localized in the nucleus and cytoplasm. Taken together, our preliminary data suggests that OsbHLH035 plays a positive regulatory role in salinity responses.

PI-08. Development of drought tolerant transgenic indica rice (cv. IR64 and Pusa Sugandh2) carrying AtDREB1A gene

Aggarwal D1, Latha GM1, Raman KV1, Rao SR1, Dhandapani G1, Solanke AU1, Sreevathsa R1, Kumar PA1, Mohapatra T2, Pattanayak D1 1National Research Centre on Plant Biotechnology, New Delhi-10012, 2Central Rice Research Institute, Cuttack, Odisha-753006. [email protected]

Rice is one of the most important crops and is the staple food for nearly half of the world’s population. The rice grain production and quality are dramatically affected by drought which is a major abiotic stress factor. The increasing population and global climate change make the situation worse. It has become imperative to focus research on developing drought tolerant rice varieties to achieve food security. Recent progress in genomics and genetic manipulation of plants opens up opportunities to utilize cellular and molecular techniques to overcome the effect of several abiotic factors including drought. In the present research a cis-acting dehydration responsive element (DRE) binding transcription factor gene DREB1A from Arabidopsis thaliana is used for the development of drought tolerant rice. Drought-sensitive indica rice varieties, IR64 and Pusa Sugandh2, were transformed with DREB1A gene, under the transcriptional control of stress inducible promoter At: rd29A, via biolistic and Agrobacterium mediated genetic transformation using binary vector pCAMBIA1200:: Prd29A:AtDREB1A:NOS-T. Putative transformants were screened and confirmed for the gene integration and copy number evaluation by PCR and Southern blotting. The selected transgenic lines were evaluated for their responses under water deficit conditions after seven days of withholding irrigation. High level of expression of DREB gene in stressed transgenic plants as compared to non-stressed transgenic plants was observed indicating up-regulated expression pattern of this gene under stress. The qRT-PCR data, phenotypic and physiological parameters revealed significant level of tolerance of the transgenics under stress condition compared to the untransformed wild type.

ABIOTIC STRESS

53

Poster Session I

PI-09. Promoter of rice glyoxalaseI gene shows appreciable degree of constitutive expression

Sudhakar D, Arul L, Balaji S, Rastogi K, Mageswari L, Kumar KK, Kokiladevi E, Balasubramanian P

Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore - 641 003. [email protected]

Promoter is an important component of the gene expression cassette and directly controls the expression of transgene(s). The most commonly used promoters in transgenic rice are CaMV35S, maize ubiquitin and rice actin1 which drive constitutive expression of transgenes. Given the limited choice of promoters, the present study aimed at cloning and characterization of a novel promoter of glyoxalaseI (glyI) gene from rice. The glyoxalase pathway consists of two enzymes, glyoxalaseI and glyoxalaseII which convert methylglyoxal into 2-hydroxy acids using reduced glutathione as cofactor. An increased glyoxalaseI activity was reported in metabolically active cells/tissues including cells undergoing stress, thus signifying the importance of glyI expression in plants under stress. In the present study a 1545 bp upstream region of glyI sequence was amplified and cloned upstream of uidA gene in a promoter-less plant transformation vector (pCAMBIA1391z). Stable transformants of Pusa Basmati 1 with the above construct were generated. T3 progenies of three events were raised on MS medium containing 100 mg/l hygromycin and analyzed for transgene homozygosity. Towards understanding the glyI promoter performance, MUG assay was carried out at the seedling stage. Three rice events transformed with uidA under the control of pCAMV35S were used as control. The expression of uidA driven by glyI promoter was comparable to that of CAMV35S promoter.

PI-10. The promoter region of Aeluropus littoralis A20/AN1 Stress Associated Protein exhibits contrasted activities in rice and tobacco

Emmanuel G, Rania BS, Walid BR, Donaldo M, Delphine M, Jean-‐Luc V, Abdullah AD, Afif H


Constitutive expression of the Stress Associated Protein (AlSAP) gene of the C4 halophyte grass Aeluropuslittoralis, encoding an A20/AN1 zinc-finger protein, confers enhanced tolerance to multiple abiotic stresses in tobacco, durum wheat and rice plants. In Aeluropus, AlSAP transcripts accumulate in response to a range of abiotic stresses and preferentially in roots. To determine whether rice and tobacco possess factors allowing a correct regulation of the AlSAP promoter, we isolated a 586 bp genomic fragment, containing a 5’UTR, upstream the AlSAP translated sequence designated as the “PrAlSAP” promoter. This region was fused to the GUSA reporter gene and introduced into the C3 monocot rice and dicot tobacco. GUSA transcripts exhibited similar accumulation patterns in PrAlSAP:GUSA rice plants than AlSAP transcripts in Aeluropus: the accumulation of GUSA transcripts was more important in roots than in leaves and up-regulated by salt, drought, cold and heat treatments. Histochemical staining confirmed GUS activity in roots, coleoptile, leaves and glumes. GUS activity was detected neither in the root cell elongation zone nor in dry seeds while a wound treatment strongly induced GUS accumulation in leaves and imbibed seeds. Histological sections revealed that GUSA activity is located in all leaf and root tissues except in xylem and leaf epidermal cells. Contrastingly, while GUSA transcripts correctly accumulated in tobacco roots, GUSA was not detected both by western and histochemical assays. This indicates that a factor is either missing or existing in tobacco that interferes with the correct translation of the transcribed sequences driven by the AlSAP promoter region. These results also demonstrate that the 586bp PrAlSAP promoter possesses sufficient essential cis-acting elements to drive root-preferential and stress-inducible gene expression in rice.

ABIOTIC STRESS

54

Poster Session I

PI-11. Overexpression of SOS1 in rice enhances salinity stress tolerance and grain yield with reduced cytosolic Na+ and protection from the oxidative damage

Gupta BK1, Sahoo K1, Pareek A2, Sopory SK1, Singla-‐Pareek SL1 1Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, 2Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. [email protected]

Soil salinity is one of the major factors for crop’s stunted growth, loss in productivity and in extreme conditions, causes plant death. To cope up with these extreme conditions, plant cell adapt different mechanisms and one such mechanism is to throw Na+ out of cytosol spaces. Salt-Overly Sensitive (SOS) pathway, which comprises of SOS1 (Na+/H+ antiporter) gene, has been suggested to mediate cellular signaling and to maintain ion homeostasis under salinity stress. In this study, we overexpressed Oryza sativa SOS1 (OsSOS1) gene in rice (cv. IR64) to functionally validate its role under salinity stress. OsSOS1 transgenics showed enhanced tolerance level with high seed germination percentage and seedling survival capacity under NaCl stress in comparison to untransformed control. Further, at mature plant stages, OsSOS1 transgenics showed better growth with substantial protection of vigour under stress. Additionally, confocal microscopic studies in root tips of transgenics using fluorescent dyes specific to Na+ ion (CoroNa Green), oxidative damage (DCFDA) and cell death (Propidium Iodide) showed reduced accumulation of Na+ ion leading to less oxidative damage and decreased cell death under salt stress. Transgenics also showed better photosynthetic efficiency with high chlorophyll content under salt stress and also maintained crop yield under stress condition as evident by more tiller formation, average number of flower, panicles and fertile seeds. These studies dissect the functional role of OsSOS1 gene which confers salinity tolerance in rice.

PI-12. Genome wide analysis and expression profiling of GATA gene family of rice discerns their role in abiotic stress

Gupta P1, Nutan KK1, Singla-‐Pareek SL2, Pareek A1 1Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India, 2Plant Molecular Biology, International Center for Genetic Engineering and Biotechnology, New Delhi 110067, India. [email protected]

Major constrains limiting crop production includes adverse environmental conditions such as high/low temperature, salinity and drought. Among these, salinity stress has a major impact on plant growth and development. GATA family of transcription factors are one of the most conserved transcription factors playing significant role in different aspects of cellular processes in organisms ranging from fungi to angiosperms. These are DNA-binding proteins which bind to a consensus sequence ‘WGATAR’ and contain the class IV zinc finger motif CX2CX17–20CX2C followed by a highly basic region. Varied roles of the members of GATA family have been shown such as photomorphogenesis and regulation of nitrogen metabolism in fungi. In plant system, members of this family are not very well explored but they are reported to be involved in light dependent gene regulation, nitrate assimilation, abiotic stress response etc. Rice (Oryza sativa L.) is an important cereal crop feeding around 50% of total world population and is also a powerful model plant for genomic explorations. In this study, we have analyzed the rice genome for the genes encoding GATA transcription factors. Our analysis reveals presence of 28 genes encoding 35 putative GATA transcription factors. In addition to this, we have also carried out their expression analysis which indicate their differential expression in response to various abiotic stress such as salinity, drought, high and low temperature in contrasting genotype - IR64 (salt sensitive) and Pokkali (salt tolerant). Differential expression of the GATA members was also noticed in response to exogenous application of ABA indicating the ABA mediated regulation of these genes. Developmental regulation of these genes was also noticed based on publically available database (https://www.genevestigator.com/gv).

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Poster Session I

PI-13. Structural analysis of hybrid type HK protein OsHK3b from rice with an ortholog AtHK1 from Arabidopsis

Kushwaha HR, Singla-‐Pareek SL, Pareek A

Synthetic Biology and Biofuel Group, International Center for Genetic Engineering and Biotechnology, New Delhi-110067, India. [email protected]

Various prokaryotes and eukaryotes regulate their cellular responses towards their environment using two-component system (TCS). The TCS now has emerged as a major signaling system in plants regulating their response towards various abiotic stresses. In Arabidopsis, hybrid type sensory histidine kinase - AtHK1 has been shown to be a putative ‘osmosensor’, which strongly interacts with AtHPt1. However, nothing is known about the osmosensing cascade in rice. A sensory type histidine kinase from rice, OsHK3b was found homologus to AtHK1, making it a strong candidate member of TCS machinery possibly involved in osmosensing. The protein-protein interaction database, PRIN strongly suggest towards the interaction of OsHK3b with OsHPt2. With an objective of dissecting out their possible interactions and identifying conserved residues among the interacting orthologs of TCS signaling machinery involved in osmosensing, we have modeled various domains present these proteins from both the genera. The analysis of amino acid sequence of OsHK3b showed the presence of three conserved domains namely CHASE (signaling domain), Transmitter domain (TD) and Receiver Domain (RD) while the protein sequence of AtHK1 showed the presence of Transmitter domain (TD) and Receiver Domain (RD). The binding analysis of the RD domain of these sensory proteins and HPt protein in Arabidopsis and O. sativa shed light on the role of various residues in HPt protein which are essential for the interaction, such as histidine. The analysis preformed in the current work would help in characterizing the structural features of various functional domains present in orthologous osmosensing HKs and their interacting HPt proteins.

PI-14. Analysis of alternative splicing in drought regulated genes for stress tolerance in rice

Thomas J, Venkategowda R, Srivastava S, Reddy AN, Pereira A

University of Arkansas, Fayetteville, AR, USA, Colorado State University, Fort Collins, CO, USA. [email protected]

Alternative splicing (AS) can expand the transcriptome and proteome diversity in plants, and its occurrence in response to environmental stresses suggest a role in the regulation of stress response and tolerance. The overall goal of this research is to characterize AS in the response of rice to drought stress and evaluate the role in stress tolerance. AS will be characterized in drought stressed and well-watered rice genotypes using RNA-Seq, to identify differentially expressed AS transcripts under drought stress, and evaluate stress responsive AS isoforms for association to drought stress response phenotypes or stress parameters in rice genotypes differing in drought tolerance/sensitivity. To achieve the objectives, we will catalog the AS changes in rice genotypes differing in drought response phenotypes to drought treatments at vegetative and reproductive stages: Nipponbare, drought sensitive mutant lines in Nipponbare representing a ‘drought sensitive physiological state’, and drought tolerant cultivars representing the ‘drought tolerant physiological state’. The genotypes will be used for RNA-Seq experiments, and the data analyzed to identify and quantify differentially expressed genes and splice junctions by Tophat/Cufflinks, Splicegrapher and the other bioinformatics tools. The RNA-Seq based predictions of AS isoforms will be validated by quantification of drought responsive AS transcripts (using isoform specific primers for qPCR), followed by characterization of the isoform structure of AS genes by amplification, cloning and sequencing of individual isoforms of specific genes of interest. The drought specific AS splice junctions will be assayed by screening a set of genotypes differing in drought sensitivity from a rice diversity panel, using RNA for qRT-PCR analysis with drought responsive isoform specific primers. The results will provide new information on post-transcriptional regulation of drought response and tolerance, and potentially molecular markers for drought tolerance in rice. Ongoing results of the project will be presented.

ABIOTIC STRESS

56

Poster Session I

PI-15. Over expression of rice (Oryza sativa L.) Saltol QTL localized GATA transcription factor improves salt tolerance in transgenic rice

Nutan KK, Singla-‐Pareek SL, Pareek A

Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India, Plant Molecular Biology, International Center for Genetic Engineering and Biotechnology, New Delhi 110067, India, Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. [email protected]

One-third of the world’s population consumes rice as a staple food, but this crop is highly sensitive to salinity stress. In our study, we are trying to identify ‘candidate genes’ which can significantly contribute to salt stress tolerance. A major QTL called Saltol, present on short arm of chromosome number 1, accounts for more than 40% of the salt tolerance. On the basis of differential expression of 13 transcriptions factor genes (localized in Saltol) in contrasting cultivars of rice IR64 (sensitive) and pokkali (tolerant) under salinity stress, we have chosen GATA transcription factor. Constitutive overexpression of GATA transcription factor in transgenic rice leads to higher biomass accumulation as compared to wild type (WT) plants. Under salinity stress, transgenic rice seedling overexpressing GATA transcription factor shows higher salinity tolerance, high chlorophyll content and less ion likage as compared to WT counterparts. Furthermore, at the cellular level, lesser damage of sub cellular organelles mainly chloroplast was observed in transgenic plants as compared to wild type and RNAi knock out plants. Osmolytes and other metabolites accumulation which help the plant in osmotic stress tolerance have been found to be higher in transgenic rice as compared to wild type. Taken together, these results suggest that GATA transcription factor may play a pivotal role in plant tolerance to salt stress by regulating different stress related genes in rice.

PI-16. Enhanced abiotic stress tolerance by an E3 ubiquitin ligase activity Kim S-‐R, Kim JS, Yi J, Min HJ, An G, Kim WT

Department of Life Science, Sogang University Seoul, 121-742, Korea, Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Youngin, 446-701, Korea, Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea. [email protected]

We have screened T-DNA tagging lines of E3 ubiquitin ligase genes for their cold stress tolerance. Line E3A126 showed an enhanced cold tolerance as judged by leaf wilting and plant weight after cold stress treatment. Molecular genetic analysis of Line E3A126 revealed that the line has a T-DNA insertion at downstream of an E3 ubiquitin ligase gene. The transcript of E3A126 was expressed more than 3 fold compared to that of wild type. Phylogenetic analysis of E3A126 proteins revealed that E3A126 belongs to ATL family (Arabidopsis Tóxicos en Levadura), a particular family of RING finger E3 Ubiquitin ligases. In order to know the cellular localization of E3A126 protein, we generated a recombinant sGFP-tagged E3A126. The fluorescence associated with E3A126 was localized to the membrane fraction of endoplasmic reticulum. in vitro self-ubiquitination assay showed that E3A126 has indeed E3 ubiquitin ligase activity. Microarray analysis coupled with real-time PCR showed that Line E3A126 has enhanced expression of several important genes involved in biotic and abiotic stress response.

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Poster Session I

PI-17. Study of stress responsive regulatory mechanism of Stress Associated Proteins (SAPs) in rice

Kothari KS, Giri J, Tyagi AK

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067. [email protected]

Plants being sessile have developed very specialized mechanisms to cope up with the environmental adversities and maintain their productivity. The result of these regulatory mechanisms leads to the induction of various stress responsive genes whose gene products ultimately impart tolerance to the plant against stress conditions. The knowledge of various signalling components and proteins involved in the regulation of stress responsive gene expression can help to modulate these components so as to increase the level of tolerance and hence the yield during stress. Several classes of stress responsive genes have been identified and functionally characterized till date. In the recent past, genes encoding a class of zinc finger proteins, known as Stress Associated Proteins (SAPs), have been identified to be responsive to multiple stresses and many members from different plant species have been shown to impart abiotic stress tolerance when overexpressed. Unlike most of the zinc finger proteins, these proteins have not been shown to act as transcription factors till date. Instead, they are involved in various protein-protein interactions either among themselves or with other proteins. To elucidate the stress responsive regulatory network of these proteins in rice, we attempted to identify different interacting partners of one of the SAP protein, OsSAP1, and functional characterization of some of the interacting proteins by overexpressing these genes in model plant Arabidopsis.

PI-18. Enhanced level of aconitase gene expression leads to increased salt tolerance in rice (Oryza sativa L.)

Kumar M, Lee SC, An G, Kim SR

Department of Life Science, Sogang University, Seoul 121-742, Korea, Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Youngin, 446-701, Korea. [email protected]

Salt is one of the most important environmental constraints that limit plant growth and agricultural productivity. We conducted a gain-of-function genetic screen for salt tolerance in rice to identify genes for improving the trait and to understand the underlying mechanism of salt tolerance. From a screening of activation tagging lines (ATLs) harbouring 4x35S enhancer, a salt-tolerant lines were obtained. Molecular genetic analysis of the line showed that the salt tolerance was caused by an activation of aconitase gene. Biochemical analysis showed an increase of aconitase activity in the line than in the control plant. Under ABA treatment, the line showed better growth performance than the control plant. It also showed lower H2O2 level than control by salt stress. Taken together, it is suggested that the increased expression of aconitase gene reduce the ROS level in the stressed plant, giving the stress tolerance of the plant.

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Poster Session I PI-19. Microarray analysis of genes differentially expressed in rice overexpressing a

constitutively activated OsMKK6 Kumar K1*, Sinha AK1 1Natoinal Institute of Plant Genome Research, New Delhi, India, *Present Address: Birla Institute of Technology & Science Pilani, K.K. Birla Goa Campus. [email protected]

The highly conserved mitogen activated protein kinase (MAPK) cascade, are signaling modules in eukaryotes and consist of MAP kinase kinasekinase (MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase (MAPK). In the MAPK cascade, the signal integration from several MAPKKKs to MAPKKs and signal divergence of MAPKKs into various MAPKs predicts the cross talk between different signal transduction pathways at the MAPKK level. Out of 8 MAPKKs reported in rice the function of OsMKK6 in cold and salinity stress is well established. To define total regulon of genes controlled by OsMKK6 and to identify genes acting downstream of OsMKK6, a constitutively activated OsMKK6 rice transgenic lines were established by sited directed mutagenesis. Here, we performed a microarray analysis between overexpressed constitutively active OsMKK6 and wild type rice plants to investigate the regulatory role of OsMKK6. We have identified 316 up-regulated and 198 down-regulated genes in the overexpressed constitutively activated OsMKK6 rice as compared to the wild type. The up-regulated genes were identified based on more than two times the levels in the overexpressed lines and down-regulated genes were identified based on overexpressed to wild types expression ratio less than 0.5. In the present study, regulated genes set were subdivided into five functional categories, namely genes involved in transcription, metabolism, signaling, defense and unknown function. The regulation of some of the genes was validated by quantitative real-time PCR analysis. These results suggest that OsMKK6 has an important role in modulating a wide range of gene expression, reflecting its physiological roles in different abiotic stresses.

PI-20. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 in Arabidopsis leads to enhanced salinity and osmotic stress tolerance

Tiwari LD, Mitta Dl, Grover A

Department of Plant Molecular Bilogy, University of Delhi, South Campus. [email protected]

Protease inhibitors play important role(s) in stress and developmental responses of plants.Earlier studies have shown that rice genome contains 17 putative members in chymotrypsin protease inhibitor (CPI; ranging in size from 7.21 to 11.9 kDa) gene family with different predicted localization sites. Two genes namely OCPI1 [GenBank: AY878695; not assigned locus ID in TIGR database but assigned locus ID Os01g0615050 in RAP-DB; http://rapdb.dna.affrc.go.jp/]and OCPI2 (Os01g42860) present on chromosome 1 show head-to-head arrangement. OCPI1 and OCPI2 genes show similar transcript expression pattern. In fact, the inter-genic region between OCPI1 and OCPI2 genes is shown to serve as a bidirectional promoter. The overexpression of OCPI1 gene was noted to result in improved drought tolerance in rice in an earlier study.We aimed at over-expression of OCPI2 in Arabidopsis to analyze the role of this protein in osmotic stress tolerance. Full-length 620 bplong OCPI2 cDNA(containing 219 bp-long ORF, coding for 72 amino acid-long 7.7 kDasubtilisin–chymotrypsin protease inhibitor cytoplasmic protein) obtained from Pusa basmati 1 (indica) rice seedlings, was over-expressed in Arabidopsis using CaMV35 promoter. Transgenic Arabidopsis plants with constitutive overexpression of OCPI2 gene were raised. T3 homozygous seeds of three PCR-positive transgenic lines were analyzed for phenotype under control and stress conditions. Transgenic Arabidopsis plants showed enhanced vegetative growth as compared to untransformed wild type (WT) plants. The numbers of siliques in the transgenic plants were higher as compared to WT plants. Further, transgenic Arabidopsis plants showed enhanced tolerance to salt (NaCl treatment) and osmotic (PEG and mannitol treatments) stresses. Overall, various physiological parameters showed that transgenic plants are better in growth, water retention and cell membrane stability as compared with WT plants. Chymotrypsin inhibitor activity assaysshowed that transgenic plants had stronger protease inhibitory activity than the WT plants. We conclude that OCPI2protein through its proteinase activityhas a role in imparting tolerance against salt and osmotic stresses.

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Poster Session I

PI-21. Physiological and molecular characterization of a salt hypersensitive rice mutant

Lin K-‐C1, Jwo W-‐S2, Lai M-‐H2, Wang C-‐S3, Hong C-‐Y1 1Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan, 2Taiwan Agricultural Research Institute, Wufeng, Taichung, Taiwan, 3Department of Agronomy, National Chung-Hsing University, Taichung 40227, Taiwan. [email protected]

Salt stress negatively affected crop growth and productivity. Although salt tolerance of rice has been studied before, its molecular mechanism has not been fully understood. In this study, a sodium azide induced salt-hypersensitive rice mutant (shs1) was analyzed to reveal its physiological and molecular changes under salt stress. Three days after the application of 200 mM NaCl on two-week-old seedlings, shs1 showed a reduced growth, damage of chlorophyll content, reduced seed germination and survival rate. Compared with wild type (TNG 67) rice seedlings, salt treatment reduced catalase activity but significantly increased superoxide dismutase activity in the shoots of shs1 mutant. Meanwhile, H2O2 and MDA content were increased with salt treatment in shs1 mutant. Determination of salt concentration indicated that shs1 accumulated more than two-folds of Na+ in shoot tissues when compared to the TNG67, while no significant difference was observed in root tissues. The OsSOS1, a gene crucial for Na+ efflux protein, was markedly reduced by 10-folds under salt stress in the shs1. In contrast, the OsHKT1;4, a gene responsible for retrieval of Na+ from the transpiration stream for storage in the leaf sheath tissues, was greatly increased more than 6-folds with salt treatment in shs1. Taken together, our results suggest that the hypersensitivity of shs1 to NaCl is caused by the defect of Na+ transportation or by impaired antioxidative defense system. Further studies are underway to dissect the genes control rice salt stress tolerance.

PI-22. Deep transcriptome sequencing of wild halophyte rice, Porteresia coarctata, provides novel insights into the salinity and submergence tolerance factors

Garg R, Verma M, Agrawal S, Shankar R, Majee M, Jain M

Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi - 110067, India [email protected]

Porteresia coarctata is a wild relative of rice with capability of high salinity and submergence tolerance. The transcriptome analyses of Porteresia can lead to the identification of candidate genes involved in salinity and submergence tolerance. We sequenced the transcriptome of Porteresia under different conditions using Illumina platform and generated about 375 million high-quality reads. After optimized assembly, a total of 152,367 unique transcript sequences with average length of 794 bp were obtained. Functional annotation revealed the presence of genes involved in diverse cellular processes and 2749 transcription factor encoding genes in Porteresia. The differential gene expression analyses identified a total of 15,158 genes involved in salinity and/or submergence response(s). The stress-responsive members of different transcription factor families, including MYB, bHLH, AP2-EREBP, WRKY, bZIP and NAC, were identified. We also revealed key metabolic pathways, including amino acid biosynthesis, hormone biosynthesis, secondary metabolite biosynthesis, carbohydrate metabolism and cell wall structures, involved in stress tolerance in Porteresia. The transcriptome analyses of Porteresia is expected to highlight genes/pathways involved in salinity and submergence tolerance of this halophyte species. The data can serve as a resource for unraveling the underlying mechanism and devising strategies to engineer salinity and submergence tolerance in rice.

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Poster Session I

PI-23. Physiological, cellular and molecular changes in response to trivalent and hexavalent chromium in rice seedlings

Yang L-‐H, Hong C-‐Y

Department of Agriculture Chemistry, National Taiwan University, Taipei, Taiwan, ROC. [email protected]

Trivalent- and hexavalent- chromium(Cr) are considered to have negative effects in plants. However, their toxicity mechanisms are still unclear. In this work, physiological, cellular and molecular changes in response to Cr(III) and Cr(VI) were investigated in rice (Oryza sativa L. cv. TN1) seedlings. The growth of roots and shoots of rice seedlings was significantly inhibited by 50 µMCr(VI). In contrast, only root growth was inhibited when treated with Cr(III). Cr(VI) induced the accumulation of diverse reactive oxygen species(ROS) including O2·- ,H2O2, and OH·-, whereas only low levels of OH.- was induced by Cr(III). Cellular localization of H2O2 in tobacco BY2 cell showed that Cr(VI) induced more H2O2 accumulated in mitochondria as compared to Cr(III). Further analysis of proteins crucial for ROS removal in mitochondria by western blot revealed that alternative oxidase was induced earlier by Cr(III) than Cr(VI) in rice seedlings, whereas mitochondrial complex IV was significantly inhibited by Cr(VI). Our results indicate that Cr(VI) is able to quickly interrupt electron transport of mitochondria thereby results in the highly accumulation of toxic ROS and induces cell death; On the contrary, toxic effect of Cr(III) in rice seedlings is attenuated by the activation of ROS removal system in the mitochondria which consequently causes mild toxic effect in rice seedlings as compared to Cr(VI).

PI-24. Genome-wide analysis of tetraspanin gene family in rice Balaji M, Katiyar-‐Agarwal S

Department of Plant Molecular Biology, University of Delhi South Campus. [email protected]

Rice is a staple food crop for millions across the globe. However, there is a huge gap between the production and demand of rice throughout the world, especially in Asia. One of the major factors contributing to this gap is exposure of rice plants to various types of abiotic stresses such as salinity, drought, flood and extreme temperatures. To mitigate the effects of the abiotic stresses it is important to unravel the molecular mechanism(s) underlying plant responses to these environmental cues. Both abiotic and biotic stresses are perceived at the cell surface and either interactions with a membrane localized protein(s) or changes in membrane structure trigger a downstream signal transduction cascade, which ultimately define or regulate response of plants to any kind of stress. Tetraspanins represent a large family of cell surface proteins and known to function as ‘facilitators’ of protein interaction in diverse organisms. Earlier studies in plants have shown that they may be involved in developmental process such as leaf patterning, root formation and floral organ formation. Current study was designed to elucidate the role of tetraspanins in rice in various tissues and under abiotic stresses in rice. In silico exploration of rice genome revealed the existence of 17 tetraspanin genes. We performed comprehensive sequence analysis and genomic organization studies on these rice tetraspanin genes. Further, we prepared a comprehensive expression atlas of tetraspanin genes in several rice tissues collected at different stages of development and rice seedlings exposed to plethora of abiotic stresses. Several tetraspanin genes exhibited differential expression in various tissues as well as stress conditions included in this study. On the basis of these studies it is proposed, that some of the tetraspanin genes might play an important role in abiotic stress tolerance in rice. It would be interesting to functionally characterize these stress-responsive tetraspanin genes in rice and extrapolate these results in other plants as well.

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Poster Session I

PI-25. Effect of cold stress on the alteration of fatty acids profile in roots and shoots of rice seedlings

Chang M-‐C, Tsai J-‐N

Department of Agronomy, National Taiwan University, Roosevelt road, Taipei 10617, Taiwan. [email protected]

Cold stress is a major factor that reduces rice yield and quality. Particularly, cold stress may change fatty acids (FAs) composition, degree of unsaturation, membrane fluidity and stability and affects survival of rice seedlings. To understand the role of FAs in cold stress tolerance of rice; two rice genotypes, TCN1 (cold-sensitive) and TNG67 (cold-tolerant) at 3-leaf stage, were treated under 40C and their roots and shoots were collected for comparative analysis of FAs profile by GC-MS. The percentage of saturated fatty acids (C18:0, C20:0, C22:0, C24:0 and C26:0) was declined after short-term cold stress treatment in shoots of TCN1 rice seedlings. Moreover, the levels of C18:2 and C18:3 in TCN1 roots were also significantly decreased under short- and long- term cold stress. Whereas, in TNG67 the composition of FAs was maintained as the same except C26:0 amount was increased in shoots under short-term cold stress but reduced with long-term cold treatment. The accumulation of long chain fatty acids (LCFAs) was obviously higher in the chloroplasts of TNG67 compared to those of TCN1. The thylakoid membrane of TNG67 had higher level of C18:3 and lower content of C16:0 and C18:0. Analysis of the portion and degree of unsaturated fatty acids with Unsaturated fatty acids/ Saturated fatty acids (Un. /Sa.) and Double Bond Index (DBI) indicated that the higher content of unsaturated fatty acids can maintain the integrity of membrane in shoots and roots of TNG67 as compared to TCN1. These two parameters were positively correlated with the changing of Fv/Fm values in TNG67 under cold stress. Finally, the gene expressions of ω-3 fatty acid desaturase-related genes, OsFAD7 and OsFAD8, were highly induced under short-term cold stress in the shoots of TNG67. This may lead to the gain of C18:3 which enhances JA biosynthesis and increases cold stress tolerance. In addition, instead of declined amounts of α-tocopherol and campesterol under cold stress in TCN1, TNG67 could maintain at a steady-state level. Taken together, this study suggests that these FAs can be potential biomarkers for the selection and breeding of cold stress tolerance-related rice varieties.

PI-26. Comparative amino acid profiling and physiological characterization of salt sensitive and tolerant rice genotypes

Mishra M1, Chatterjee A1, Schulze J2, Singla-‐Pareek SL3, Pareek A1 1Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, 2Molecular Structure Facility University of California, Davis 530.752.7327, 3Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnolgy, Aruna Asaf Ali Road, New Delhi 110067,India. [email protected]

Rice is the most important staple food for a large part of world’s population. Rice is a salt sensitive crop, therefore yield reduction is main issue in salinity prone areas of world. Rice production is not keeping up with continuously increasing global population due to adverse climatic conditions. Considerable variability in genetic composition and salt tolerance has been observed among different rice cultivars. Comparative studies of these varieties help in selection of candidate genes and development of salt tolerant varieties. Study of their physiological parameters can help in commenting on the level of salt tolerance of different genotypes. In the present study, we have analyzed three rice genotypes viz. IR64, Pokkali and CSR10, for their salt tolerance at seedling stage. IR64 is a salt sensitive cultivar of Oryza sativa L., Pokkali is a salt tolerant landrace (Indica) and CSR 10 is a salt tolerant, cross bred, indica rice variety. Liquid chromatography analysis revealed amino acid composition in grains of all three genotypes. Statistical analysis (ANOVA) shows significant difference for amino acid content in IR 64 vs CSR10 as well as Pokkali vs CSR10 but no significant difference between IR 64 vs Pokkali. 7 day old seedlings of these genotypes were subjected to 200mM NaCl stress for different time durations (24 hr, 48 hr and 72 hr) and their physiological parameters like root length, shoot length, relative water content, proline accumulation, K+/Na+ ratio, chlorophyll content, lipid peroxidation, electrolyte leakage etc. were taken into account. Varietal differences were observed with increase in duration of stress for almost all considered parameters. Taken together, results suggest that CSR10 show better tolerance to salinity stress among all genotypes tested here.

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Poster Session I PI-27. Over-expression of OsIF gene in Oryza sativa L. cv IR64 improves its

performance under different abiotic stresses Soda N, Pareek A, Singla-‐Pareek SL

School of Life Sciences, Jawaharlal Nehru University, New Delhi. [email protected]

Rice, one of the world’s most important staple crops, is affected detrimentally by salinity stress. Most of the agronomic traits including salinity are quantitative in nature governed by multigenes usually clustered on chromosomes as QTLs. In rice, more than 10 QTLs related to salinity tolerance have been identified. Amongst them, a major QTL Saltol, present on chromosome I, accounts for >40% of salinity tolerance. Expression studies of signaling related genes (SRPs), mapped within Saltol QTL in contrasting rice genotypes showed differential temporal transcript accumulation. OsIF is one among these genes whose transcript levels also showed up-regulation in response to different abiotic stresses such as salt, drought, heat and heavy metal etc. These observations suggested that this gene may have some role towards abiotic stress tolerance. Over-expression of OsIF in diverse organisms like bacteria, yeast and plants provides them survival advantage under different abiotic stresses. Localization studies have suggested that intermediate filaments get disrupted after 30 min treatment with 200 mM NaCl. Yeast two hybrid assay has shown its putative interaction with several other stress related proteins such as metallothionein, plasma membrane ATPase etc. thereby suggesting it to be a part of salt stress response machinery of plants. Over-expression of OsIF in rice also helps in improving the photosynthetic efficiency of transgenic lines under salt stress. This study might serve as first evidence of involvement of cytoskeletal genes towards stress tolerance in plants.

PI-28. In planta transformation with transcription factor ap37 in rice (Oryza sativa L.) improves grain yield under drought and salt stress

Maheshwari P, Kavi-‐Kishor PB

University College of Sciences, Department of Genetics, Osmania University, Hyderabad - 500 007. [email protected]

Drought is a serious treats to the sustainability of rice yields in rainfed agriculture. In Asia, 90% of the total area is under cultivation, 45% is subjected to drought atdifferent intensities and the contribution from such drought-affected areas to the total rice production is only 25%. Conventional breeding as well as emerging genetic engineering methods may be used to improve plant drought stress tolerance. Some transcription factors regulating stress responsive genes have become important target genes for improving plant drought tolerance.Transcription factors with APETELA2 (AP2) domain have been implicated in various cellular processes involved in plant development and stress responses. In our report, we functionally characterized the AP37 gene for salt and drought tolerance during the vegetative and reproductive stages of growth. The gene AP37 was inserted under the control of ABA2 promoter into pCAMBIA 1301 vector using GUS as reporter gene and hygromycin as a selectable marker. Then the vector was mobilized into Agrobacterium tumefaciens strain LBA4404. The transformed Agrobacterium containing the gene and promoter were used for rice transformation. For in planta transformation of rice seedlings were raised from transformed seeds. The putative transgenics of To generation were confirmed by PCR amplification for AP37 gene, hptII marker (Hygromycin) and ABA2 promoter. Transgenic over-expression of the AP37 gene with the ABA2 stress inducible promoter in rice increased the tolerance to high salinity up to 200 mM of NaCl. The transgenic plants ABA2:AP37 also showed significantly enhanced salt and drought tolerance at the reproductive stage, as evidenced by the increase in grain yield by 50% over controls under severe field drought conditions. Thus, our results suggest that the AP37 gene has the potential to improve drought and salt tolerance without causing undesirable phenotypes.

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Poster Session I

PI-29. Global transcriptome analysis reveals different adaptation mechanisms in traditional and modern indica rice genotypes under phosphorus deficiency

Mehra P, Pandey BK, Giri J

National Institute of Plant Genome Research, New Delhi. [email protected]

Phosphorus (P) is one of the essential macronutrient required for plant growth and development. However, most of the soil P exists in the form of organic compounds or insoluble cation complexes, not readily available to the root for uptake. Application of phosphate fertilizers can compensate for low P availability, but the global source of P, rock phosphate is non-renewable and will be exhausted in the next 50-100 years. Furthermore, only ~20 of applied P fertiliser is used by plants as it readily forms complexes with other ions in soil. P deficiency is therefore, a big problem for cultivation of important food crop like rice in many world soils. Most of the modern high yielding rice genotypes are sensitive to P deficiency whereas traditional rice cultivars are naturally compatible to low P ecosystems. These low P tolerant genotypes have evolved a number of adaptations that allow them to withstand low levels of P in soil. These mechanisms broadly include modifications in root system architecture for enhanced P uptake and improved P- use efficiency. However, the underlying genetic mechanism for low P tolerance in traditional low P tolerant genotypes remains largely unclear. To delineate the molecular mechanisms for low P tolerance, two contrasting rice genotypes, Dular and PB1 have been studied for their behaviour under low P conditions. Our morphophysiological studies established Dular and PB1 as low P tolerant and sensitive genotypes, respectively. Comparative global transcriptome analysis of root and shoot tissues of both genotypes revealed the different adaptation mechanisms under P deficient conditions. We found the efficient P utilization as a major adaptation in traditional genotype to cope with P deficiency.

PI-30. Proteomic analysis of different rice (Oryza sativa L.) genotypes under salt stress

Mishra P, Mishra V, Singh NK, Rai V

National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110012. [email protected]

Salt stress is a major abiotic factor limiting productivity of rice crop. To evaluate the genotypic variation of NaCl stress responses at protein level in rice, proteome profiling of shoot tissues was done in three rice genotypes. Rice seedlings (15 days old) of CSR13, CSR36 (salt tolerant) and MI48 (salt sensitive) varieties grown in half strength Hoagland’s medium were subjected to NaCl stress (150 mM) for 24h. Total 400 protein spots were identified among which 23, 34 and 22 are differentially expressed in CSR-36, CSR-13 and MI-48 respectively. For MALDI TOF/TOF mass spectrometric analysis 79 proteins spots were selected. Six proteins commonly present in each variety were found to be up regulated in salt tolerant genotypes. These were heat shock protein, thioredoxin peroxidase, protein disulfide isomerise, glyceraldehyde-3-phosphate dehydrogenase, chloroplast photosystem I reaction center subunit II and chalcone synthase. Among these, heat shock protein and putative thioredoxin peroxidase were involved in stress responses whereas protein disulfide isomerise, glyceraldehyde-3-phosphate dehydrogenase, chloroplast photosystem I reaction center subunit II proteins and putative chalcone synthase are involved in metabolic pathways. This study emphasizes the role of proteins involved in salt tolerance in rice.

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Poster Session I PI-31. OsiSAP1 overexpression improves water-deficit stress tolerance in

transgenic rice Dansana PK1, Kothari KS2, Vij S1, Tyagi AK1,2

1Department of Plant Molecular Biology, University of Delhi, South Campus, Benito Juarez Road, New Delhi -110021, India, 2National Institute of Plant Genome Research, ArunaAsaf Ali Road, New Delhi-110067, India. [email protected]

OsiSAP1 (O. sativa subsp. indica stress-associated protein) is an A20/AN1 zinc-finger domain containing proteins from rice. It was found to be induced in response to multiple abiotic stress conditions. Proteins with such domain architecture have been reported from several plant species and were found primarily involved in stress response. The precise mode of action for SAPs in plants is still unclear, however, recent reports suggest their involvement in diverse processes such as ubiqutination, redox-sensing, hormone metabolism and gene regulation under stress condition. We generated transgenic rice constitutively expressing OsiSAP1 driven by maize UBIQUITIN gene promoter and evaluated for water-deficit stress tolerance during various stages of growth. Improved tolerance to water-deficit conditions was observed in transgenic rice compared to non-transgenic plants. Transcriptome analysis suggests that overexpression of OsiSAP1 in transgenic rice results in altered expression of several endogenous genes including those coding for transcription factors, membrane transporters, signalling components and genes involved in metabolism, growth and development. Of the differentially regulated genes, several genes are known to be involved in stress response.

PI-32. New insights into the molecular basis of genotype-specific adaptation Subba P, Datta A, Chakraborty S, Chakraborty N


The nucleus is the fundamental component of microenvironment of cell that has been extensively expanded during the process of evolution. It not only hosts the genome but also administers its transcription and the regulated expression of proteins, thereby playing a critical role as a modulator of cellular phenotype. To better understand the role of nuclear proteins in water-deficit condition, a nuclear proteome was developed from a dehydration-sensitive rice cultivar IR-64 followed by comparison with that of a dehydration-tolerant c.v. Rasi. The 2-DE protein profiling of c.v. IR-64 coupled with MS/MS analysis led to the identification of 93 dehydration-responsive proteins (DRPs). More than three-quarters of these DRPs were predicted to be destined to the nucleus. While the detected number of protein spots in c.v. IR-64 was higher when compared with that of Rasi, the number of DRPs was found to be lesser. Fifty seven percent of the DRPs were found to be common to both, the sensitive and tolerant cultivars, indicating significant differences between the two nuclear proteomes. To elucidate stress response signaling, we constructed a functional association network of the DRPs of c.v. IR-64, which suggests that a significant number of the components are capable of interacting with each other. The combination of nuclear proteome and interactome analyses would elucidate stress-responsive signaling and the molecular basis of dehydration tolerance in plants. Water deficit that results from decreased osmotic/water potential of soil solution due to high solute concentration in the soil, as well as ion-specific stresses due to altered Na+/K+ ratios and Na+/Cl- ratios that are inimical to the plants. Physiological and biochemical changes include retarded initial growth and photosynthesis, attenuated nutrient acquisition, inhibition of cell division and expansion, disintegration of cellular membranes, inactivation of enzymes, production of reactive oxygen species (ROS) and interrupted protein or nucleic acid metabolism. Rice (Oryza sativa) is relatively sensitive to salinity stress, particularly due to its need for irrigation and continuous growth in standing water. Rice however, exhibits enormous genetic variability in sensitivity to salinity. Tolerance to salinity in rice is a complex process involving coordinated changes in the defense pathways such as the activation of membrane transporters, ROS scavenging machinery, specific transcription factors etc. In an effort to understand the diverse responses of several rice genotypes of the world to salinity stress we screened twenty different rice accessions of the USDA-Core collection representing the diverse groups like O. glaberrima and O. sativa subspecies accessions for salt sensitivity and tolerance. We selected and treated six diverse rice genotypes to salt stress, sampled tissue at 6, 24 and 48 hrs for RNA isolation and studied the expression of specific genes belonging to different signaling pathways using qPCR. The gene expression results of the signaling pathway genes will be represented in a network using ‘Beacon’ tools (http://bioinformatics.cs.vt.edu/beacon/) to represent and share with other users pathways of the rice salinity tolerance mechanisms in standardized graphical language.

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Poster Session I PI-33. Comparative genomics and transcriptomics of drought sensitive and tolerant

indica rice cultivars Agarwal P, Mathur S, Kumar S, Raghuvanshi S

Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi – 110021, India, National Institute of Plant Genome Research, ArunaAsaf Ali Marg, New Delhi-110067, India. [email protected]

Rice is an important cereal crop and serves as staple food for around 50% of the world population. Drought is a major abiotic stress that affects its growth and development. While most commercially viable cultivars are sensitive to drought several indica rice varieties exhibit significant tolerance. Since response to drought is a global molecular phenomenon, comparative analysis of drought sensitive and tolerant cultivars at genome and transcriptome level would help decipher the complicated regulatory mechanism. Thus, a global comparative analysis of the drought tolerant variety ‘Nagina 22’ and sensitive cultivar ‘IR64’ was undertaken. Whole genome re-sequencing of paired-end and mate-pair libraries of both the varieties and subsequent assembly resulted in high quality draft assemblies with over 95% coverage and more than 30X sequence depth. Further, gene annotations were transferred from japonica reference genome to Nagina22 and IR64 draft genomes. While some genic loci were 100% conserved a majority of them showed significant variations among the rice varieties. Subsequently, cDNA libraries from different tissues of control and drought treated N22 and IR64 plants were also sequenced. Transcript tags were mapped to the reference annotations to identify the expression profile. Comparison of the expression profiles led to identification of several genic loci that differ significantly in their response to drought conditions in the two varieties. Correlation of these variations in drought-induced expression with genomic variations has shed light on several interesting facts.

PI-34. Comparative study on the affect of iron availability on phosphate deficiency-mediated morphophysiological and molecular responses in rice and Arabidopsis

Sanagala RR, Rai V, Yadav S, Mishra V, Sarkar A, Kumar PA, Jain A

National Research Center for Plant Biotechnology, PUSA Campus, New Delhi. [email protected]

P availability affects Fe-deficiency responses in rice. However, the effects of Fe availability on Pi deficiency responses in rice and Arabidopsis are not known. To decipher this interaction, rice and Arabidopsis seedlings were grown hydroponically under P+, P-, Fe-, and P-Fe- conditions for 7 days. In Arabidopsis, Pi deficiency resulted in an accumulation of anthocyanins in the shoots which was attenuated under P-Fe- condition. In rice there was reduced chlorophyll content in the shoots of P- seedlings. In Arabidopsis, Pi deficiency triggered inhibition of both primary root growth and development of lateral roots. However, there were significant increases in the lengths of primary and lateral roots of P-Fe- seedlings. Although histochemical GUS staining of P- and P-Fe- roots of Arabidopsis CycB1;1::uidA seedlings showed loss of meristematic activity in their primary roots, the lateral root tips of P-Fe- seedlings showed distinct meristematic activity. Relatively the effects of these different nutrient conditions were less dramatic on the RSA of rice seedlings. The expression of auxin transporter PIN1:GFP was discrete and comparable in the primary and lateral roots of P+ and Fe- Arabidopsis seedlings, whereas it showed slightly diffused pattern in them. Further, Pi-deficiency mediated induction of several PSI genes (Pht1;1, Pht1;4, IPSI, WRKY75) were suppressed under P-Fe- condition. On the contrary, the induction in the expression of OsPT2 was comparable under P- and P-Fe- conditions, whereas the induction in the expression of OsPT3 was 2-fold higher under P-Fe- condition compared to P- condition. In addition, proteome profiling revealed 26 differentially expressed proteins in the shoots of the seedlings grown under these nutrient conditions. These results clearly showed variable effects of Fe availability on the morphophysiological and molecular responses of rice and Arabidopsis to Pi deficiency.

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Poster Session I

PI-35. A highly abundant, stress regulated rice cyclophilin (OsCyp2) confers multistress tolerance to evolutionary diverse organisms

Roy S1, Kumari S2, Singh P3, Singla-‐Pareek SL4, Pareek A1 1 Stress Physiology and Molecular Biology Laboratory; School of Life Sciences; Jawaharlal Nehru University; New Delhi, 2Sher-e-Kashmir University of Agricultural Sciences and Technology; Jammu, 3 Department of Biotechnology; Guru Nanak Dev University; Amritsar, 4 Plant Molecular Biology; International Centre for Genetic Engineering and Biotechnology; New Delhi. [email protected]

Abiotic stress is one of the core contributors in limiting the rice productivity throughout the globe including India. Gene transcript profiling in contrasting cultivars of rice under non-stress and salt stress conditions has shown differential regulation of myriad of genes. One such member screened through substractive cDNA hybridization, belong to the cyclophilin pool having peptidylprolyl cis transisomerase activity named as Oryza sativa cyclophilin 2 (OsCyp2). Genome wide analysis of cyclophilins genes, in different plant species such as Arabidopsis, Oryza sativa and Triticum aestivum indicated it to be a multigenic family. Most of the cyclophilin family members contain a typical cyclophilin like domain (CLD) with some structural variations. Till date, 28 different cyclophilins have been identified from Oryza sativa, occupying different cellular locations, forcasting their pivotal role in important cellular processes. OsCyp2 has an open reading frame of 519 bp encoding a polypeptide of 18.6 kDa and a pI of 8.61. OsCyp2 protein has catalytic efficiency of 4.5X106-1mol-1l-1s-1 in comparison to other reported plant cyclophilins. Developmental and tissue specific transcript analysis of OsCyp2 proved its differential expression. To find out the interacting partners of OsCyp2, we screened rice cDNA expression library in yeast using yeast two hybrid technique, which revealed its interaction with some stress related partners as AP2 domain containg protein, TATA binding protein transcription factor and vacuolar ATPase G subunit containing protein. Its heterologus expression in E. coli and S. cerevisiae enabled them to survive under diverse stresses viz. salinity, heat, osmotic and oxidative stress. Similarly, ectopic expression in tobacco could improve its multiple stress tolerance as established by various physiological parameters. Hence, OsCyp2 may serve as a ‘suitable candidate’ for raising transgenic crop plants for enhanced multiple abiotic stress tolerance. To relate the efficacy of this gene to a food crop, studies pertaining to it’s over expression in Oryza sativa is underway.

PI-36. Transcriptional profiling of rice seedling under potassium deprivation condition

Shankar A1 , Singh A1, Kanwar P1, Srivastava AK2, Pandey A, Suprasanna P, Kapoor S1, Pandey GK1 1Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, DhaulaKuan, New Delhi-110021, India, 2Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai-400085, India. [email protected]

Potassium is an essential plant nutrient and is required in abundance for proper growth and development. It plays a major role in different physiological processes like cell elongation, stomatal movement, ionic homeostasis and signal transduction by acting as a major osmolyte and component of the ionic environment in the cytosol and subcellular organelles. Thus, its deficiency directly affect the plant growth and hence crop yield and production. In rice, the molecular mechanism for the regulation of potassium starvation responses has not been investigated in detail unlike Arabidopsis. Here, we present a combined physiological and whole genome transcriptomic study of rice seedlings exposed to a brief period of potassium deficiency and then followed by resupply of potassium. Our results revealed that the expressions of a diverse set of genes annotated with many distinct functions were altered under potassium deprivation. Our findings highlight altered expression patterns of potassium-responsive genes majorly involved in metabolic processes, stress responses, signaling pathways, transcriptional regulation, and transport of multiple molecules including K+. Interestingly, several genes responsive to low-potassium conditions show a reversal in expression upon resupply of potassium. The results of this study indicate that potassium deprivation leads to activation of multiple genes and gene networks, which may be acting in concert to sense the external potassium and mediate uptake, distribution and ultimately adaptation to low potassium conditions. The interplay of both upregulated and downregulated genes globally in response to potassium deprivation determines how plants cope with the stress of nutrient deficiency at different physiological as well as developmental stages of plants. Further verification of these differentially expressed genes by functional genomic

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Poster Session I approaches will provide insights into the mechanistic regulation of these genes during low potassium stress conditions, which ultimately can be translated to develop plants that can grow on soil with relatively low levels of potassium without compromising yield and productivity.

PI-37. Exploring transcriptional complexity under abiotic stresses in rice via deep sequencing

Shankar R, Bhattacharjee A, Jain M

Functional and applied Genomics laboratory, National Institute of Plant Genome Research (NIPGR), ArunaAsaf Ali Marg, New Delhi-110067, India. [email protected]

Rice is one of the most important food crops providing energy to one third of total world population. However, various abiotic and biotic stresses adversely affect the rice productivity. Abiotic stresses, mainly drought and salinity cause loss of more than 50% crop production every year throughout the world. Different stress-tolerant rice cultivars/landraces have been identified through phenotypic and physiological analyses, but many of these are poor in yield. These tolerant cultivars can be used as the source of novel genes and/or genetic variations, which may help in development of stress-tolerant cultivars with high yield. Previous studies suggest that genes involved in catalytic activity, DNA binding, transcription regulatory activity, cell wall maintenance and kinase activity are highly enriched in tolerant cultivars under stress conditions. However, more comprehensive studies are required to characterize novel genes/isoforms involved in stress tolerance and understand the underlying molecular mechanism. To answer some of these questions, we sequenced the whole transcriptome of three rice cultivars with contrasting stress responses under control, drought and salinity stress conditions and identified several novel genes/isoforms and differentially regulated genes under control and stress conditions. Further, studies of genetic variations and pathway analysis revealed the differences among these cultivars. Some of these genes/pathways might be involved in stress tolerance or adaptation. Altogether, our study will help to understand the transcriptional complexity under abiotic stresses in rice.

PI-38. Study of the two component mediated signal cascade in rice operative under osmotic stress

Sharan A1, Soni P1, Singla-‐Pareeek SL2, Pareek A1 1Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, 2Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, ArunaAsaf Ali Road, New Delhi 110067. [email protected]

The global food demand is increasing due to ever growing human population whereas area of agricultural fields is rapidly decreasing with the increase in urbanization. Besides, crop plants are subjected to various environmental stresses that influence the growth and development of plants causing decline in productivity and reduced crop yield. Soil salinity is among the most serious issues that world agriculture faces, with about 20% of all cultivated land affected currently. The two component system (TCS) plays important role in the regulation of various kinds of cellular processes in response to environmental stimuli in bacteria, yeast, slime mould and higher plants. Typically, TCS consists of a sensory histidine kinase (HK) and a response regulator (RR). Multistep phosphorelay (hybrid type TCS), consists of an adaptor protein known as histidinephosphotransfer protein (Hpt) in addition to HK and RR. Rice genome contains 11 HKs, 5 HPTs and 36 RRs. To check for the possible interactions, we used yeast two hybrid (Y2H) assay, in which TCS components were expressed as bait or prey fusion proteins followed by their β- gal assay. We have found that OsPhp1, OsPhp2 and OsPhp3 interact strongly with OsRr22, OsRr26 and weakly with OsRr23. Among response regulators, OsRr21 shows strong interaction with OsRr2 and OsRr6; OsRr22 interacts strongly with OsRr2 and OsRr3; OsRr23 shows weak interaction with OsRr9; OsRr24, OsRr26 and OsRr33 interacts weakly with OsRr12. This is the first comprehensive analysis of rice TCS interactome in rice and provides starting platform to reveal the molecular mechanisms of the signal transduction through the TCS, operative under various stress responses.

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Poster Session I PI-39. Gene expression analysis of contrasting rice cultivars in response to drought

stress Sharma E, Borah P, Kaur A, Kapoor S, Khurana JP

Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. [email protected]

Drought is a major environmental stress that adversely affects rice production due to its high water requirements. However, genetic variations available within traditional accessions of rice and its wild relatives offer wide range of alleles for drought tolerance. Here we present physiological and genome wide expression analysis for one-week-old seedlings of contrasting rice cultivars, drought tolerant Dhagaddeshi and sensitive IR20. We have identified 2,963 and 3,115 genes uniquely up and down-regulated in Dhagaddeshi as compared to 474 and 558 genes in IR20, respectively, after 3 hours of water deficit stress. However, the number of genes that express differentially in both these cultivars is fairly comparable after 6 h of drought stress. At physiological level, Dhagaddeshi seedlings showed higher cell membrane stability and differential ABA accumulation pattern under water deficit stress. Since drought tolerance is a complex and quantitative trait with large number of genes each with small to medium effects controlling the outcome, correlation of the quantitative trait loci (QTLs) with expression analysis can be used to identify candidate genes for drought tolerance. In an attempt to mine stress responsive genes underlying QTLs, the microarray expression data was overlaid with known QTLs from publicly available databases and published literature pertaining to observed physiological traits of the contrasting cultivars in this study. By localization of differentially expressed gene loci (DEG) to their known positions on physical genome of rice, we could identify 10 genomic blocks with significant differential gene expression and four of these regions enriched in DEGs co-localized with QTLs being analyzed. Further, integration of the microarray expression data with metabolic pathway data available at Gramene database highlighted number of pathways involved in phytohormones, secondary metabolites, osmolytes, amines and amino acids synthesis in response to stress. Few stress responsive genes underlying QTLs, genomic blocks and metabolic pathways were validated by qPCR and may serve as candidate genes for providing drought tolerance in rice.

PI-40. Functional characterization of Stress Associated Protein (SAP) gene family in rice

Sharma G, Giri J, Tyagi AK

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India. [email protected]

Plants have evolved intricate mechanisms involving stress perception, signal transduction and appropriate response to overcome and acclimatize prevailing stress conditions. In spite of this, there is need for the identification, establishment and implementation of molecular switches governing either enhanced stress tolerance or avoidance phenomena towards sustainable crop improvement. Recently Stress Associated Proteins (SAPs), a novel class of genes from rice was identified showing stress responsive gene expression pattern. OsSAP1, one of the first member identified imparted multiple abiotic stress tolerance to model plant tobacco. Their abiotic stress responsiveness has entailed us to undertake gene family level investigations to decipher their role and modes of action in cellular context and at organism level. To functionally characterize rice SAP gene family, Arabidopsis has been chosen as model plant. It involves abiotic stress assessment of SAP gene family members using transgenic approach and phenotyping for level of stress tolerance along with their protein-protein interaction studies. Outcome of these studies in relation to functional characters of SAP gene family members would be presented.

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Poster Session I

PI-41. Expression profiling of members of two-component system in rice reveals their tissue specificity, diurnal rhythmicity and abiotic stress responsiveness

Soni P1, Sharan A1, Nongpiur R, Singla-‐ Pareek SL2, Pareek A1 1Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, 2Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, ArunaAsaf Ali Road, New Delhi 110067. [email protected]

The two-component system (TCS) is a histidine to aspartate phosphorelay based signal transduction pathway. We carried out expression analysis of members of TCS signaling system in rice as influenced by various spatial and temporal factors as well as various abiotic stresses. It was observed that OsHk3, OsHk6, OsAhp1, OsAhp2, OsRr21 and pseudo-response regulators have prominent expression in all tissues examined in mature plant as well as in seedling. Expression of OsHk1, OsHk2, OsHk4, OsHk5, pseudo-histidinephosphotransfers and type-A response regulators was low in various tissues. Among the histidine kinases, OsHk3 showed highest level of expression and its transcript abundance was high in roots of seedlings. Authentic histidinephosphotransfers, OsRr21 and pseudo-response regulators exhibit high expression in mature panicle and green parts such shoot of seedlings, leaves, flag leaf of mature plant. Analysis of EST and MPSS database also supports our results. Our data suggests that TCS members might play important role in organ development during juvenile stage and panicle development or grain filling at mature stage. Analysis of time course transcript abundance of TCS genes at seedling stage revealed their rhythmic and diurnal expression pattern. Expression of most of the TCS members showed robust oscillation with peaks at different time points of a 12h light/12h dark cycle. It indicates that transcriptional regulation of TCS genes is influenced by diurnal rhythm. Transcript profiling of TCS genes in different abiotic stresses distinguished OsHk3, OsPhp3, OsRr21 and pseudo-response regulators as ABA inducible and multiple stress responsive TCS members.

PI-42. Salinity tolerance mechanisms in diverse rice genotypes Basu S, Venkategowda R, Kumar A, Betty A, Vallelian C, Pereira A

University of Arkansas, Fayetteville, AR, USA. [email protected]

Soil salinity imposes an ionic imbalance or disequilibrium causing hyper-ionic or hyper-osmotic stress, thus disrupting the overall homeostasis or metabolic activities of plants, resulting in reduced growth and plant death. The detrimental effects of excessive salts are the consequences of: water deficit that results from decreased osmotic/water potential of soil solution due to high solute concentration in the soil, as well as ion-specific stresses due to altered Na+/K+ ratios and Na+/Cl- ratios that are inimical to the plants. Physiological and biochemical changes include retarded initial growth and photosynthesis, attenuated nutrient acquisition, inhibition of cell division and expansion, disintegration of cellular membranes, inactivation of enzymes, production of reactive oxygen species (ROS) and interrupted protein or nucleic acid metabolism. Rice (Oryza sativa) is relatively sensitive to salinity stress, particularly due to its need for irrigation and continuous growth in standing water. Rice however, exhibits enormous genetic variability in sensitivity to salinity. Tolerance to salinity in rice is a complex process involving coordinated changes in the defense pathways such as the activation of membrane transporters, ROS scavenging machinery, specific transcription factors etc. In an effort to understand the diverse responses of several rice genotypes of the world to salinity stress we screened twenty different rice accessions of the USDA-Core collection representing the diverse groups like O. glaberrima and O. sativa subspecies accessions for salt sensitivity and tolerance. We selected and treated six diverse rice genotypes to salt stress, sampled tissue at 6, 24 and 48 hrs for RNA isolation and studied the expression of specific genes belonging to different signaling pathways using qPCR. The gene expression results of the signaling pathway genes will be represented in a network using ‘Beacon’ tools (http://bioinformatics.cs.vt.edu/beacon/) to represent and share with other users pathways of the rice salinity tolerance mechanisms in standardized graphical language.

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Poster Session I

PI-43. Developing transgenic rice plants overexpressing plasma memebrane antiporter gene SOS1 for improved salinity tolerance

Sushma MA, PrashantkumarSH, Sreevathsa R, Sashidhar VR

Department of Crop Physiology, UAS, GKVK, Bangalore. [email protected]

Soil salinity affects large areas of cultivated land, causing significant reductions in crop yields worldwide. Sodium ions in saline soils are toxic to plants because of their adverse effects on K+ nutrition, cytosolic enzyme activities, photosynthesis, and metabolism. Three mechanisms function cooperatively to prevent the accumulation of Na+ in the cytoplasm, those are salt exclusion, salt extrusion and salt compartmentation are critical to the survival of plants under saline stress. Pumping of Na+ from the root cells is mediated by the plasma membrane Na+/H+ antiporter (SOS1/NHX7) which plays an important role in preventing the accumulation of Na+ in cytosol to toxic levels. In the present study, SOS1 (NHX7), a plasma membrane Na+/H+ antiporter gene was overexpressed in rice by Agrobacterium mediated In Planta transformation technique. To screen putative T1 plants for salt tolerance, stringent salt screening test (SSST) was followed and root and shoot growth of T1 putative transformants was used as a selection criterion. Some of the transgenics showed significantly higher root and shoot growth compared to wild type. To confirm the gene integration in putative T1 transgenic plants, PCR and RT-PCR analysis was performed. The results showed that all the selected seedlings from SSST were PCR positives and four lines were positive for RT-PCR analysis. Physiological studies viz., chlorophyll estimation, membrane stability index, cell viability test, net osmotic adjustment, K+/Na+ ratio and DAB staining were also conducted to assess their levels of salt tolerance in the T1 generation. Some of the T1 transformants showed lower percent reduction in chlorophyll content, higher membrane stability, cell viability, net osmotic adjustment and maintained higher K+/Na+ ratio after NaCl treatment compared to wild type. The results clearly demonstrated that transgenic rice plants overexpressing OsSOS1 had superior salt tolerance capacity as compared to non-transformed plants.

PI-44. Drought induced 19 protein: A probable interacting partner of Aux/IAA13 in rice

Majee SM, Buttar BS, Khurana JP

Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, India. [email protected]

In recent years, auxin receptors have been identified and they represent components of E3 ligase involved in ubiquitination and 26S proteasome mediated degradation. Interestingly, one of the target protein class is encoded by auxin inducible Aux/IAA genes. Auxin plays many diverse roles in regulating plant development. The structure of Aux/IAA gene product comprises of mainly four domains; domain I has a transcriptional repressor function, domain II forms the degron and domain III and IV are involved in dimerization. Aux/IAA gene products serve as negative regulators of auxin action, thus degradation of Aux/IAA is a prerequisite for auxin response. Recently, TIR1, one of the auxin receptors, has been identified to stimulate degradation of Aux/IAA. In our study, we tried to look for other interacting partners of Aux/IAA. In rice, Aux/IAA gene family is represented by 31 members. We have chosen OsIAA13 to look for its interacting partners. This gene expresses at high levels in the young coleoptiles tissue. Screening of coleoptiles library using Y2H approach resulted in identification of Di19 protein as one of its interacting partners. The interaction has been verified by in vitro pull-down assay and in vivo BiFC and FRET analysis. Di19 protein is drought induced protein and has zinc finger motif that is suggestive of it being involved in DNA-protein or protein-protein interactions. Although, in Arabidopsis, recent studies have given a clue of it being involved in DNA-protein interaction, but its function remains to be studied in rice. Thus, our finding that Di19 interacts with Aux/IAA may provide clue towards a probable role of OsIAA13 in auxin-mediated plant development.

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Poster Session I

PI-45. Effect of salinity stress on expression of genes in salt sensitive and salt tolerant indica type rice cultivars

Nath T, Bhattacharya S, Sengupta DN

Bose Institute, Kolkata. [email protected]

Rice cultivation is affected by salinity stress in the lowland & coastal areas and also by dehydration in the upland areas. In indica type low yielder rice cultivars like Pokkali, Nonabokra are better in salt tolerance than the high yielding rice cultivars as they are known to produce ABA more than salt sensitive rice cultivar. We have started comparing the expression of genes for ABA biosynthesis in salt sensitive IR-64 and salt tolerant Nonabokra rice plants. The expression of different genes such as SAMDC, OSBZ8, DREB2 ,ZEP, NCED1, AAO after treatment with 200mM NaCl for 3 hrs and 6 hrs, were checked from roots and lamina of 12 day old plants of IR64 and Nonabokra and showed difference (after normalization with actin gene expression) within two cultivars. The transcript levels for SAMDC & DREBP2 were really higher than the other genes like OSBZ8, ZEP & NCED1 and SAMDC, DREBP2 & OSBZ8 transcripts were detected both in control and salt treated shoots & roots of both cultivars. However the level of expression of SAMDC was found to be up regulated. The transcript level of ZEP, NCED1 were very low & detected only after salinity stress in shoot and root of both the cultivars but more in Nonabokra than IR-64. The full length cDNA for S-adenosyl methionine decarboxylase (SAMDC) was amplified and introduced into IR64 through Agrobacterium mediated transformation. About 40 transgenic lines (T0) from four independent transgenic events have been selected and confirmed by genomic PCR using hygromycin (hpt) and 35S: SAMDC primers. The T1 plants after germination on hygromycin medium have been transferred to green house for further analysis of transgene introgression and expression. These results will be presented and discussed.

PI-46. Identification and characterization of a novel chloroplasts/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice

Wu T-‐M, Lin W-‐R, Kao Y-‐T, Hsu Y-‐T, Yeh C-‐H, Hong C-‐Y, Kao CH

Department of Agricultural Chemistry, NTU. [email protected]

Glutathione reductases (GRs) are important components of the antioxidant machinery that plants use to respond against abiotic stresses. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. In this work, we describe the cloning and characterization of the full-length cDNA encoding OsGR3, a chloroplast-localized GR that up to now was considered as a non-functional enzyme due to assumed lack of N-terminal conserved domains. Expression of OsGR3 in E. coli validated that it can be translated to a protein with GR activity. OsGR3 presents 76% and 53% identity with OsGR1 (chloroplastic) and OsGR2 (cytosolic), respectively. Phylogenetic analysis revealed 2 chloroplastic GRs in Poaceae species, including rice, sorghum and branchypodium, but only one chloroplastic GR in dicots. A plastid transit peptide is located at the N-terminus of OsGR3 and genetic transformation of rice with a GR3-GFP fusion construct further confirmed its localization in chloroplasts. Furthermore, OsGR1 and OsGR3 are also targeted to the mitochondria suggesting a combined antioxidant mechanism in both organelles. On the other hand, the genes of both isoforms present distinct response to salinity. Expression of OsGR3 but not that of OsGR1 is induced by salt stress. In addition, transcript levels of OsGR3 were greatly increased upon treatment with salicylic acid but were not significantly affected by methyl jasmonate, dehydration or cold stress. Our results provide new clues about possible roles of a functional OsGR3 in salt stress and biotic stress tolerance.

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Poster Session I

PI-47. Overexpression of OsAKR4C15, a novel aldose reductase gene, enhances tolerance to sorbitol in transgenic rice

Wu C-‐Y1, 2, Hong C-‐Y2, Huang W-‐L1 1Department of Agronomy, National Chiayi University, 2Department of Agricultural Chemistry, National Taiwan University. [email protected]

In the present study, expression patterns and function of a novel rice aldo-ketoreductase gene-AKR4C15 were investigated. The OsAKR4C15 belongs to AKR4C subfamily. The OsAKR4C15 mRNA is primarily expressed in rice seeds and is induced by various abiotic stresses such as dehydration and low temperature. To explore the role of OsAKR4C15 in stress tolerance, OsAKR4C15 was driven by maize Ubiquitin1 promoter and transformed into rice. The results revealed that overexpression of OsAKR4C15 in rice significantly enhanced the tolerance to sorbitol. Under sorbitol stress, overexpression of OsAKR4C15 retained more chlorophyll contents and reduced the accumulation of H2O2 in leaf tissue as compared to WT. These results indicate that OsAKR4C15 plays a pivotal role in osmotic stress tolerance and could be used for improvement of osmotic stress tolerance in rice.

PI-48. RNA-Seq analysis of rice transcriptome under cadmium stress Oono Y, Yazawa T, Kawahara Y, Kanamori H, Yamagata H, Hosokawa S, Wu J, Handa H, Itoh T, Matsumoto T

National Institute of Agrobiological Sciences. [email protected]

Heavy metal ions are highly reactive and toxic to living cells, and their accumulation in plants is a major agricultural problem. In particular, cadmium (Cd) is absorbed by the roots from the soil and transported to the shoot negatively affecting nutrient uptake and homeostasis in plants, even at low concentrations. It is also known to adversely impact various biochemical and physiological processes including changes in the transcriptome of plants, resulting in inhibited root and shoot growth and, ultimately, reduced yield. Furthermore, ingestion of accumulation of Cd in the edible parts of plant such as seed grains with accumulation of Cd could cause highly toxic effects on human health. Thus, it is important to study the mechanisms of plant responses and defenses to Cd exposure to overcome this problem. Rice seedlings (cultivar Nipponbare) were hydroponically treated with 50 µM Cd for 24 hours. The transcriptome during Cd exposure was analyzed by mRNA-seq method using Ilumina Genome Analyzer. Totally ~60,000 expressed transcripts, including transcripts that could not be characterized by microarray-based approaches, were evaluated. Upregulation of various ROS-scavenging enzymes, chelators and metal transporters demonstrated the appropriate expression profiles to Cd exposure. qRT-PCR analyses of randomly selected Cd responsive metal transporter transcripts under various metal ion stresses suggested that the expression of Cd responsive transcripts might be easily affected by other ions. The network of Cd stress response and tolerance, which plants have developed as an adaptation to stress, could serve to acclimate the effects of exposure to non-essential metals. Exploitation of the transcriptional networks in response to Cd exposure could provide useful information on the molecular mechanisms of adaptation to abiotic stress and the improvement of stress tolerance in crop species.

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Poster Session I

PI-49. Comparative transcriptomics analysis for rice seedlings of contrasting salt-tolerant cultivars

Yang Y-‐W, Chang M-‐C, Chen H-‐C

Department of Agronomy, National Taiwan University. [email protected]

From phenotype and physiological analysis after salt treatment of TNG67 (japonica) and TCN1 (indica) seedlings, we found that TNG67 exhibited more salt-tolerant than TCN1. For further study of salt-tolerance mechanism, we use microarray to compare the gene profiling of seedlings between two contrasting salt-tolerant cultivars. The global gene expression analysis under salt stress revealed that ABA, ethylene, cytokinin and polyamine may make greater contribution than other secondary metabolites in regulation of the mechanism of salt tolerance. In addition, the major transcription factors (TFs) expressed differentially in shoots under salt stress are bZIP and ERF genes, but in roots, the major TFs are NAC genes. Otherwise, the WRKY genes expressed dominantly in roots under salt stress but much more weakly in shoots. The shoot- and root-specifically expressed TFs further indicated that both shoots and roots play important roles through different mechanism in mediating salt responsiveness in rice seedlings to overcome salt stress. Furthermore, there may exist some communications between shoot and root to confer salt tolerance of rice seedlings. Time-series design of experiments in our study can find some TFs that expressed differently only in TNG67 under salt stress but not in TCN1at early stage may be the candidate key genes for salt tolerance. Taken together, this study identifies some important hormone-related genes and TFs that may participate in the salt tolerance of TNG67.

PI-50. Functional genomics of nitrate response and its interaction with light in rice Raghuram N, Jangam AP, Pathak RR

School of Biotechnology, GGS Indraprastha University, New Delhi 110 078, India. [email protected]

Biotechnological approaches for N-use efficiency improvement in rice are severely limited by the poor molecular characterization of its response to different sources of N and its interaction with other factors. As a part of our effort to study the genomewide N response, we used rice whole transcriptome (Agilent 44K) microarrays to identify genes that are differentially expressed in response to nitrate in etiolated and green leaves of 10-day old seedlings of the rice cultivar Panvel-1. In light-grown leaves, nitrate treatment up-regulated 806 genes (geomean >0.8) and down-regulated 317 genes (geomean <-0.8), together designated as 1123 light-dependent nitrate responsive genes. In etiolated leaves, nitrate up-regulated 1040 genes and down-regulated 324 genes (with similar geomean cutoffs), together designated as 1364 etiolation-specific nitrate responsive genes. A Venn selection between the two categories revealed that 222 genes were light-independent nitrate responsive, i.e. “true” nitrate responsive genes, since these responded only to nitrate and are not affected by presence or absence of light. These data indicated that there are major differences in nitrate response in etiolated and green plants. Functional classification of these gene lists and comparison with various transcriptomes available on Ricexpro revealed that nitrate response spans a wide range of metabolic pathways, stress response, biosynthetic pathways, hormonal response, binding, signaling and other cellular processes across all tissues and developmental stages, indicating critical role in plant physiology and development. Some of the light-dependent nitrate responsive genes have been shortlisted from nitrate and amino acid metabolism, transporters, hormones, transcription factors, signaling enzymes and miscellaneous categories for further studies.

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Poster Session I

PI-51. Cloning, expression and functional characterization of a putative Cu/Zn Superoxide dismutase coded by locus Os03g11960 of Oryza sativa

Sanyal RP, Saini A, Jawali N

Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085 INDIA. [email protected]

Superoxide dismutases (SODs) are important and ubiquitous antioxidant metalloenzymes, involved in dismutation of highly toxic and reactive superoxide radical (O2˙¯) generated through various physiological activities and stress responses. In eukaryotes the SODs are classified into three types, Cu/Zn-, Mn-, and Fe-SOD based on metal cofactor required for the activity. Rice genome encodes four Cu/Zn SODs localized in different cellular compartments. Locus Os03g11960 of rice genome codes for a putative Cu/Zn SOD enzyme, and a few studies show the evidence for expression of this gene at transcript level. The full length cDNA of Os03g11960 gene was isolated, cloned and sequenced. Analysis of this sequence with other monocot and dicot Cu/Zn SOD sequences indicated that this locus codes for a putative peroxisomal Cu/Zn SOD. The Os03g11960 cDNA was overexpressed in E. coli BL21 (DE3) cells and recombinant protein was purified by affinity purification approach. The protein was analyzed for biochemical properties and it was found to be substantially thermostable and enzymatically active over a broad pH range with pH optima of 10.5. Further analysis using SOD inhibitors viz., Diethyl dithiocarbamate (DETC), hydrogen peroxide (H2O2), and sodium azide further confirm that the locus Os03g11960 codes for a Cu/Zn SOD and it is likely to be peroxisomal in nature. Detailed results on analysis of Cu/Zn SOD encodes by locus Os03g11960 of rice will be discussed in the presentation.

PI-52. RNA seq based comparative transcriptome analysis of cold stress response at seedling stage in contrasting rice genotypes

Pradhan SK, Nayak DK, Behera L, Dash SK, Das A, Mohapatra T

Central Rice Research Institute, Cuttack (Odisha) 753 006, India, [email protected]

Rice is a cold-sensitive plant and its exposure to low temperature during seedling stage results in slow seedling growth, yellowing, withering, reduced tillering and stunted growth. The present investigation aims to compare transcriptome of cold susceptible variety ‘Sahabhagidhan’ (CSV) and cold tolerant variety ‘Geetanjali’ (CTV) using RNA seq approach during cold stress and recovery conditions to gain insight into different gene expression that might provide clues to the underlying principle of cold tolerance. Both genotypes were grown at 250C in a growth chamber and cold stress was imposed by exposing the plant to 40C. RNA was isolated from leaf samples collected from control, 6, 12, 24 and 48h of cold stress and after 48h of stress recovery. The pair end sequencing libraries for RNA-seq were prepared using illumina TruSeq® RNA Library Preparation Kit. RNA-Seq was performed using illumina HiSeq2000 to generate 30 million 2x100 bp reads for each sample. Rice genome and gene information for reference cultivar Nipponbare was used for comparison. The results clearly indicated that the down-regulated genes in CSV were more than the up-regulated genes while the reverse was observed in CTV. It was observed that there were 24529 differentially expressed genes (DEGs) in the two genotypes in which 13930 and 10599 DEGs were present in CSV and CTV, respectively under stress and recovery compared with the control. The down-regulated genes declined during recovery in CSV and CTV, while the up-regulated genes were more in number during recovery of CTV from cold stress. Gene ontology classification revealed a significant role of transcription regulation, oxygen, lipid binding, catalytic and hydrolase activity. Absence of photosynthesis related genes, storage of products like starch and the synthesis of many classes of molecules which are used for energy production and as raw material for the synthesis of other molecules are observed in the CSV during cold stress condition. In biological process category, secondary metabolic process, response to abiotic stimulus, response to biotic stimulus and lipid metabolic process indicated the same biological function getting influenced in both the genotypes except signal transduction, regulation of cellular process, biological regulation, photosynthesis, generation of precursor metabolites and energy and carbohydrate metabolic process observed in the tolerant variety. Kyoto Encyclopedia of Genes and Genomes pathway annotation indicated that tolerant variety had more number of DEGs that involved in different pathways, thereby making the variety tolerant against stress condition.

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Poster Session I PI-53. Development of drought tolerant transgenic indica rice by stress inducible

transcription factor AtDREB1A Balachandran SM, Kumar GV, Manimaran P, Sundaram RM, Voleti SR, Subrahmanyam D

Directorate of Rice Research, Rajendranagar, Hyderabad 110236 [email protected]

Rice (Oryza sativa L.), a major food crop of the world, uses 30% of the fresh water worldwide whose productivity is greatly affected by drought. A wide spread research is conducted for identification of QTLs, stress regulated genes and transcription factors towards developing climate resilient crop plants in general and rice in particular. A transcription factor DREB1A identified from the model plant Arabidopsis thaliana, has been reported to enhance the stress tolerance against drought stress. We developed transgenic rice plants with AtDREB1A in the background of indica rice cultivar Samba Mahsuri (BPT 5204) through Agrobacterium mediated transformation. Ten stable, independent transgenic plants were developed and selected lines advanced to T5 generation to get homozygous lines. Expression of AtDREB1A was observed to be induced by drought stress in the transgenic rice lines, which were highly tolerant to severe water stress in both vegetative and reproductive stages without affecting their agromorphological traits. Further, expression of AtDREB1A during water stress was associated with increased accumulation of proline, maintenance of chlorophyll, increased relative water content (RWC) and decreased ion leakage under drought stress and showed significantly higher grain yield and spikelet fertility as compared to their non-transgenic control and drought tolerant check varieties Vandana and Rasi. Most of the homozygous lines were highly tolerant to drought stress and showed significantly higher grain yield and spikelet fertility than non-transgenic control plants under both stressed and unstressed conditions. The transgenic lines of BPT5204 did not show any yield penalty under normal conditions and displayed high level of tolerance to drought under stress. Hence, these lines can provide yield stability to farmers cultivating rice in drought prone areas.

PI-54. Root transcriptome: Insights into P deficiency tolerance among rice genotypes adapted to acidic soils

Wricha T, Yumnam JS, Dkhar F, Dohling AK, Rai M

School of Crop Improvement, College of PG Studies, Central Agricultural University, Umroi Road, Umiam, Meghalaya-793103 [email protected]

Majority of the soils in North East Hill Region of India are acidic and this leads to phosphorus (P) being limiting for rice growth because of high soil P-fixation capacity. For reported genes other than OsPTF1, PsTOL1, it is not clear whether their role is similar in rice varieties tolerant and susceptible to low P. So there is a need to generate and evaluate novel molecular and breeding resources. In order to elucidate the molecular basis of response to P limitation, we performed mRNA sequencing of two rice genotypes showing similar growth response to P limitation as indicated by the shoot and root biomass but differing in Pup1 locus. Approximately, 121 million sequence reads were obtained and a set of 780 and 2578 transcripts from each cultivar respectively were found to be responsive under P limitation. Total 1,090 transcripts were significantly (p value >0.05) expressed under low P levels in roots. A set of differentially expressed genes were targeted for development of candidate gene based markers for generation of polymorphic markers. An understanding of functional role of novel candidate genes in P deficiency tolerance is required. The markers generated need validation in a set of P deficiency tolerant lines. The result will help to understand the mechanism of P deficiency tolerance. The information generated will also help increase P deficiency tolerance of cultivars that may or may not already contain the Pup1 locus using gene pyramiding strategies.

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Poster Session I PI-55. Molecular engineering of “ascorbate-glutathione pathway” into rice for

adaptation to multiple stresses Mohan V, Achary M, Reddy CS, Reddy GM, Pandey P, Gupta K, Gupta G, Pandey S, Kaul T, Reddy MK

Plant Molecular Biology Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India

Environmental stresses lead to generation of ROS or oxidative stress in cells posing as a major limiting factor in plant productivity. ROS is ubiquitously produced in cells at low levels during normal growth conditions. In stress, metabolic disturbances are accompanied by high ROS generation and the inherent anti-oxidative machinery is incompetent in rapid ROS removal. Excess ROS results in peroxidation of membrane lipids, membrane disintegration and eventually cell death. In plants, crucial ROS-scavenging enzymes of ascorbate-glutathione pathway majorly contribute in combating oxidative stress by deactivation of ROS released during multiple redox reactions. Therefore, to eliminate or alleviate ROS production during adverse stress conditions, we propose to overexpress the vital enzymes of “ascorbate-glutathione pathway” such as Superoxide dismutase (SOD) Ascorbate peroxidase (APX), Monodehydroascorbate reductase (MDHAR), Dehydroascorbate reductase (DHAR), Glutathione reductase (GR) driven by a stress-inducible promoter (Rd29A) in rice plants. T7RNA-polymerase gene based system has been utilized for high expression levels of all transgenes in rice. All the gene cassettes have been pyramided in a single plant transformation vector by site-specific homologous recombination. Putative transgenic rice lines were analyzed and transgene integration in different chromosomes was confirmed. Expression of transgenes in these transgenic lines was confirmed by RT-PCR. Leaf disc assay with methyl viologen (100 µM) revealed that these transgenic lines were tolerant to oxidative stress. Moreover, no morphological anomaly was observed in these transgenic lines.

PI-56. Improving protein turn-over and protection by co-expression of HSF4, p68 and Pg47 genes (RNA Helicase) to enhance drought tolerance in aerobic rice (Oryza sativa) by maintaining cellular tolerance

Ramu SV, Veerashree, V, Udayakumar, M.

Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore. India. [email protected]

Aerobic cultivation practice is gaining prominence as a potential water saving technology. In this method, rice crop is cultivated on soils that are maintained at near 100% field capacity without puddling. Under semi-irrigated aerobic method, the crop is grown with reduced frequency of irrigation which could save irrigation water up to 60%. However, the crop may experience several constraints, which may significantly affect growth and productivity. Therefore, efforts need to be made to bridge the yield loss if the advantages of this water saving technology are to be exploited. This can be achieved by improving traits that are associated with maintenance of tissue water relations and cellular metabolism under decreasing water status. The emphasis in this study is to combine two important drought adaptive traits mainly plant water relations (improved water mining and WUE) and cellular level tolerance mechanisms. AC39020 rice genotype with superior water mining/WUE was chosen as a recipient genotype to develop transgenics co-expressing genes regulating cellular level tolerance mechanism (CT). Maintaining protein synthesis, turnover, folding and protection has phenomenal relevance in stress adaptation. From this context, the objective is to co-express a few relevant regulatory genes governing protein turnover and protection. The emphasis is to develop multigene cassettes with PgHSF4 (Heat shock factor 4), Pg47 and p68 (RNA helicases) which are known to play crucial role in protein protection, protein folding, and turnover respectively. The co-expression cassette having PsAKR1 as selectable marker against glyphosate with Pubiquitin-PgHSF4:: P2x35SCaMV-Pg47 :: PRBCS-P68 was developed using modified gateway technology, subsequently developed rice transgenics in the genotype AC39020. The T1 glyphosate resistant plants were confirmed for integration of all the genes using different primer combinations. The T2 transgenic lines showed improved tolerance to moisture stress and NaCl stress, besides showed tolerance against heavy metal stress and accelerated aging mechanisms.

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Poster Session I PI-57. Co-expression of CAP2 and CIPK improves root growth and impart stress

tolerance in rice (Oryza sativa L) Nagarjuna KN, Ramu SV, Sreevathsa R, Rama N, Chattopadhyay D, Udayakumar M, Nataraja KN,

University of Agricultural Sciences, GKVK Campus, Bangalore, India, [email protected]

Plants are exposed to various kinds of abiotic stresses. Drought stress limits growth and development leading to a decline in crop productivity. Plants have evolved various strategies and mechanisms to withstand drought. WUE, water conservation and water mining are among the important drought adaptive traits. The major emphasis of the present study is to increase the water mining ability by improving root characteristics to increase the water uptake under stress conditions. In this perspective, an attempt has been made to co-express genes related to root traits. The genes, CIPK (Calcineurin B-like [CBL] protein interaction protein kinase), responsive to various abiotic stresses, CAP2 (Cicer arietinum AP2) that encodes a protein with an AP2 DNA-binding domain that plays an important role in increasing leaf cell size, leaf surface area and number of lateral roots and stress tolerance along with EPSPS as a plant selectable marker for glyphosate tolerance were co-expressed using modified gateway cloning strategy. The rice cv. KMP175 was transformed this multigene construct by in planta transformation technique. Based on the selectable marker EPSPS, a high throughput technique was used to screen the T1 generation seedlings for selection of putative transformants on glyphosate medium. The selected transgenic lines were advanced upto T3 generation and analysed for integration and expression of the transgenes. The selected transgenic lines portrayed a normal phenotype. Further, there was a significant improvement in root growth as evidenced by root length, root volume and root biomass compared to wild type. Transgenic lines also exhibited tolerance to NaCl and PEG induced stress at the seedling stage. In addition to stress tolerance, transgenic lines exhibited improvement in seed yield than wild type at time of harvest. Taken together the data suggests that co-expression of CIPK and CAP2 can improve the root growth and impart stress tolerance in rice.

PI-58. Co-expression of EcNAC1, EcbZIP60 and EcMYC57 genes from fingermillet (Eleusine coracana) enhanced drought tolerance in rice (Oryza sativa) by improving cellular tolerance

Babitha KC, Ramu SV, Nataraja KN, Sheshshayee MS, Udayakumar M

University of Agricultural Sciences, GKVK, Bangalore. India. [email protected]

The increasing scarcity of water threatens the sustainability of the irrigated rice production system. Hence growing rice under semi-irrigated aerobic condition has phenomenal relevance. However, in this ecosystem the crop experiences decreased water availability, high VPD which affects growth and productivity. From this context, it is important to improve adaptation of rice under aerobic condition by improving water relations and cellular level tolerance mechanisms. The scientific strategy is to co-express relevant regulatory genes in the background of a genotype having superior water relation traits. Three stress responsive transcription factors from different families i.e EcNAC1, EcMYC57, EcbZIP60 were cloned from fingermillet stress cDNA library and validated their relevance for stress tolerance in model system tobacco. EcMYC57 and EcNAC1 upregulated dehydrin group of genes and EcbZIP60 upregulated unfolded protein responsive genes and thereby improved abiotic stress tolerance. A co-expression of these two groups of genes facilitated improved protein turnover and stability in tobacco transgenics. Subsequently, rice transgenics were developed co-expressing EcNAC1, EcMYC57 and EcbZIP60 in genotype AC39020 having superior water relation traits. Desirable transformants were identified based on desiccation response. Molecular characterization of transformants showed the integration and expression of all the three genes. The transgenics expressing these transcription factors showed improved tolerance to oxidative, osmotic and DTT induced ER stress conditions. Further, the transgenics showed improved growth under semi-irrigated aerobic conditions and many promising transgenic lines showed reduced spikelet sterility and higher yield compared to wild type plants. Under drought stress the transgenics showed higher expression of both dehydrin group of genes and ER stress responsive genes resulting in increased stress tolerance. The study provides proof of concept that field level tolerance can be improved by combining both cellular tolerance mechanisms and water mining traits.

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Poster Session I

PI-59. Co-expression of Pg47, OseIF4E and PgHSF4 to combat abiotic stress in rice (Oryza sativa L.)

Patil M, Rama N, Sreevathsa R, Reddy PC, Nataraja KN, Prasad TG, Udayakumar M

University of Agricultural Sciences, GKVK, Bangalore, [email protected]

In semi-arid tropics, high temperature and reduced soil moisture levels affects crop growth and productivity. The impending climate change characterized by an increase in frequency and severity of drought and elevated temperature has accentuated negative impact on the crop productivity. Plants have evolved various strategies and mechanisms to overcome abiotic stresses where there is a general decline and reprogramming of protein synthesis. Extensive modulation of gene expression occurs at various levels viz., transcription, translation and post-translational modifications. We hypothesize that by regulating these three processes, plants can maintain growth and development under stressful conditions. To achieve this, an attempt has been made to overexpress three regulatory genes i.e. Pg47 (Pennisetum glaucum 47, an RNA helicase), OseIF4E (Oryza sativa translational initiation factor 4E) and PgHSF4 (Pennisetum glaucum Heat shock factor4), which are known to play crucial role in transcription, translation and protein folding respectively. The multigene construct harbouring Pg47, OseIF4E, PgHSF4 and IgrA genes with different promoters and UTRs was developed by modified gateway cloning strategy and IgrA was used as plant selection marker for tolerance to glyphosate. T0 transformants were developed by Agrobacterium mediated in planta transformation technique using rice germplasm line AC39020. The putative transformants were screened on glyphosate medium and selected once were advanced to T2 generation. The transgenics showed integration of all genes as analysed by PCR. Further, selected lines were analysed for cellular level tolerance by exposing them to salinity (NaCl) and heavy metal [Cd (NO3)2] stress at seedling level. Transgenics exhibited improved tolerance and growth rate under aerobic condition when compared to wild type suggesting that, overexpression of Pg47, OseIF4E and PgHSF4 improves cellular level tolerance under abiotic stress.

PI-60. NGS based transcriptome profiling of drought tolerant and susceptible introgression lines derived from the cross Swarna x O. nivara

Mesapogu S1, Kadiri S1, Pushpalatha G1, Babu AP 1, Ram T1, Rao LVS1, Subrahmanyam D1, Brajendra1, Sarla N1, Rai V2 1Directorate of Rice Research, Rajendranagar, Hyderabad-500030, 2National Research Centre on Plant Biotechnology, IARI, New Delhi-110012, India. [email protected]

High yielding BC2F8 backcross inbred lines derived from Swarna x O. nivara were evaluated at different developmental stages for their water stress tolerance in hydroponics, pots and field under direct seeded and transplanted conditions using susceptible and tolerant checks. S14-3 was identified as the most drought tolerant line and S137 the most sensitive based on IRRI score and other data. Next Generation Sequencing (NGS)–based Applied Biosystems SOLiD 4 Technology was used to identify differentially expressed genes (DEGs), novel splice junctions and novel transcripts after 24h of water stress in 14 days old seedlings of S14-3 and S137. Only 61% reads mapped in control and 53% in stress in S14-3 and 48% in control and 42% in stress in S137. In all, 8168 DEGs were detected, 2839 (34%) in S14-3 and 5329 (65%) in S137. If only GO annotated genes were considered, S137 showed 32% more DEGs in control and 23% more in stress than S14-3. Down regulated genes (256 in S14-3 and 323 in S137) were more than up-regulated genes (219 in S14-3 and 304 in S137). Overall fold change was also maximum in S137 (1.8fold up and 7.7fold down). Under water stress, novel transcripts were reduced by 63% in S14-3 and 80% in S137. Likewise, splice junctions were reduced by 54% and 76% respectively compared to control. Oxidation-reduction, membrane transport, and ATP-, protein-, and nucleotide-binding were predominantly down regulated. qRT-PCR in roots and shoots of 57 genes of abiotic stress related pathways indicated A2X741/S-adenosylmethionine decarboxylase was highly upregulated in both shoot (264 fold) and root (126 fold) of S137 only and appears to be a high priority candidate gene for functional genomics of water stress response in these lines. We thank NAIP project Bioprospecting of genes and allele mining for abiotic stress tolerance (NAIP/Comp-4/C4/C-30033/2008-09) for funds.

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Poster Session I PI-61. The expression profiles of drought-tolerant NaN3-induced rice (Oryza sativa

L. spp. indica) mutant Shih M-‐D, Wu J-‐J, Wei F-‐J, Wu Y-‐P, Hsing YC,

Distinguished Research Fellow, Institute of Plant and Microbial Biology, Academia Sinica. Department of Agronomy, National Taiwan University, Taipei, Taiwan. Department of Agronomy, Taiwan Agricultural Research Institute, Chiayi, Taiwan. [email protected]

Rice (Oryza sativa L.) plant is highly drought sensitive. In most of cases, drought stress is the key factor to limit yield in the rice reproductive area of Asia. Hence, to improve varieties through breeding process, chemical mutagenesis was used to generate variations and to select drought-tolerant mutants. Chemical mutagenesis has been widely used to generate genetic variation for basic research and breeding programs. Besides, the development of high-throughput genotyping also provides the efficient detection of point mutations generated by chemical agents. In this study, IR64 mutated population generated by sodium azide (NaN3) treatment was used to screen for drought-tolerant individuals by treatment with 20% PEG solution. The total RNA were then extracted from both type to establish the high-throughput RNA-seq profiles. Our results suggest that two drought-tolerant individuals have totally different expression profiles. Functional analysis also suggests that both individuals have diverse GO profiles. We will discuss the possible drought-droved pathway of two drought-tolerant individuals and wild-type.

PI-62. Double herbicide tolerant “Swarna” rice as an integrated approach for sustainable weed management, high water-use efficiency and enhanced yield

Kaul T, Agarwal A, Vani K, Fartyal D, Gupta K, Achary M, Reddy MK

Plant Molecular Biology Lab, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi-110067, India. [email protected]

Rice is grown widely in water logging or flooding conditions essentially to suppress weed growth in lowland cultivation. Likewise, in upland rice cultivation weed control during early stages of crop growth is imperative for procuring yield potential. Effectiveness of this method is provisory to timely availability of water at crucial stages in the growing season. Due to ever-expanding populations of developing economies of Asia, the accessibility of water for rice production has emerged as a momentous issue due to the associated costs. Simultaneously, yield losses caused by weeds worldwide are reported at an average of 10-20 %. Manual weed control over large cultivated areas is time consuming, labor- and cost-intensive. Moreover, in early stages weeds are indistinguishable from crop plants posing a challenge for manual weeding. Circumstantially, incorporating chemical weed control for realizing superior and sustainable productivity is relevant. Currently, about 10,000 tons of harmful herbicides per annum are used in conventional cropping in Indian agriculture. Amongst them non-selective herbicides are majorly applied prior to sowing and/or pre-emergence of crop plants. Due to the restricted flexibility in the application schedule and their residual effect on the subsequent crop generations, there is limited execution of herbicide based weed management in Indian agriculture. We have developed double herbicide tolerant Swarna rice lines with dual mode of action using two systemic herbicides: glyphosate (for spraying) and sulphonylurea (for broadcasting). We isolated genes encoding for EPSPS-synthase (EPSPS) and acetolactate synthase (ALS) from rice and mutated for conferring herbicide tolerance via site-directed mutagenesis. These two mutated genes were in vitro pyramided into a single plant transformation vector by using site-specific homologous recombination cloning strategy to prevent their segregation in subsequent generations or during rice breeding program. Transgenic lines were confirmed by molecular analyses and showed high dose tolerance for specific herbicides when grown in fields within the net house. Double herbicide tolerant marker free rice lines shall pose as a powerful tool in the arena of integrated weed management in rice cultivation. It shall enable the farmers to control large numbers of weed species having highly variable life cycles and survival mechanisms via systematic rotation of these broad-spectrum non-selective herbicides whose mode of actions are entirely different from each other. This innovative strategy shall effectively prevent the development of herbicide tolerant weeds and ensure high water-use efficiency, thereby enhancing the rice productivity.

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Poster Session I PI-63. Genome-wide analysis of plant-type II Ca2+ATPases gene family from rice

and Arabidopsis: Potential role in abiotic stresses Yadav S, Huda KMK, Banu MSA, Trivedi DK, Tuteja N

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. [email protected]

The Plant Ca2+ ATPases are members of the P-type ATPase superfamily and play essential roles in pollen tube growth, vegetative development, inflorescence architecture, stomatal opening or closing as well as transport of Ca2+, Mn2+ and Zn2+. Their role in abiotic stress adaptation by activation of different signaling pathways is emerging. In Arabidopsis, the P-type Ca2+ ATPases can be classified in two distinct groups: type IIA (ECA) and type IIB (ACA). The availability of rice genome sequence allowed performing a genome-wide search for P-type Ca2+ ATPases proteins, and the comparison of the identified proteins with their homologs in Arabidopsis model plant. In the present study, we identified the P-type II Ca2+ ATPases from rice by analyzing their phylogenetic relationship, multiple alignment, cis-regulatory elements, protein domains, motifs and homology percentage. The phylogenetic analysis revealed that rice type IIA Ca2+ ATPases clustered with Arabidopsis type IIA Ca2+ ATPases and showed high sequence similarity within the group, whereas rice type IIB Ca2+ ATPases presented variable sequence similarities with Arabidopsis type IIB members. The protein homology modeling, identification of putative transmembrane domains and conserved motifs of rice P-type II Ca2+ ATPases provided information on their functions and structural architecture. The analysis of P-type II Ca2+ATPases promoter regions in rice showed multiple stress-induced cis-acting elements. The expression profile analysis indicated vital roles of P-type II Ca2+ ATPases in stress signaling, plant development and abiotic stress responses. The comprehensive analysis and expression profiling provided a critical platform for functional characterization of P-type II Ca2+

ATPases genes that could be applied in engineering crop plants with modified calcium signaling and homeostatic pathways.

PI-64. Cytoplasmic (OsClpB-c), mitochondrial (OsClpB-m) and plastidial (OsClpB-p) isoforms of rice ClpB/Hsp100 proteins differ in their ability to complement thermo-tolerance defect of Arabidopsis hot1-3 mutant

Mishra RC, Richa, Grover A

Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi-110021, India (email:[email protected])

OsClpB/Hsp100 proteins are rapidly and predominantly accumulated upon heat stress and perform the function of chaperones, mediating re-naturation of denatured proteins. Rice contains three ClpB/Hsp100 proteins localized to different cellular compartments cytoplasm/nucleus (OsClpB-c), mitochondria (OsClpB-m) and chloroplast (OsClpB-p). OsClpB-c (Os05g44340), OsClpB-m (Os02g08490) and OsClpB-p (Os03g31300) proteins are able to complement the thermo-sensitive phenotype of yeast hsp100 mutant (∆Schsp104). A great deal of ClpB-c biology has been unveiled employing hot1-3 mutant in the model plant Arabidopsis. Hot1-3 is a null mutant for ClpB-c gene, and is highly thermo-sensitive both at the seed and seedling levels. We stably transformed OsClpB-c, m and p proteins in this mutant background under the control of a constitutive CaMV 35S promoter (m and p without transit peptides). Homozygous lines for all the three transgenic types were analyzed for seed basal thermo-tolerance. OsClpB-c complemented the hot 1-3 mutant phenotype to more or less wild type level. The plastidial and mitochondrial OsClpB homologues also complemented the seed basal thermo-tolerance phenotype, though to varying degrees. At the level of seedlings, OsClpB-c showed high degree of complementation. However, the plastidial and mitochondrial homologues did not show complementation in the seedlings. We thus find that the chaperoning function of ClpB-c in Arabidopsis seeds can be performed by any of the OsClpB-c, OsClpB-m and OsClpB-p proteins. However, the chaperoning activity of ClpB-c in Arabidopsis seedlings performed by OsClpB-c is not fully substituted by OsClpB-m and OsClpB-p proteins. We have purified OsClpB-c and OsClpB-m proteins and wish to analyze their chaperoning activities in due course.

ABIOTIC STRESS

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Poster Session I

PI-65. Rice ClpB-c/Hsp100 promoter is regulated by HsfA2 proteins Lavania D, Chakraborty A, Rai A, Singh G, Saini P, Grover A

Department of Plant Molecular Biology, University of Delhi, South Campus, Benito Juarez Marg, New Delhi-110021, India. [email protected]

We have previously documented that OsHsfA2c (a) has rapid transcript induction under high temperature stress, (b) possesses transactivation activity, (c) forms homo-oligomeric configuration, (d) regulates expression from OsClpB-c promoter in heat shock element (HSE)-dependent manner and (e) interacts with OsClpB-c and OsHsfB4b proteins. In the present study, we noted that OsHsfA2c can bind with OsClpB-c promoter under antisense orientation also, thus revealing that the HSE of OsClpB-c promoter is not strand-specific. Binding of class A OsHsfs on the promoter of the plastidial isoform of OsClpB gene (OsClpB-p) was next analyzed. Only OsHsfA2c showed significant binding with OsClpB-p promoter; however, binding with OsClpB-p promoter was lower than that noted with OsClpB-c promoter. To study how much generalized the regulation mediated by OsHsfA2c protein is, promoters of ten selected heat shock protein genes, representing the major OsHsp gene families, were studied for their regulation by OsHsfA2c in a yeast monohybrid assay. Based on β-galactosidase reporter gene activity, we noted that OsHsfA2c strongly regulates transcription from Hsp26.7P, OsDjA5, OsDjB7 and cHsp70-1 promoters, somewhat weakly from Hsp17.4CI and OsHsp90-B promoters but shows no role in regulation of Hsp16.0Px, OsDjA4, cHsp70-4 and OsHsp90-A promoters. This observation highlights that OsHsfA2c has differential activity for various Hsp promoters. Direct interaction of OsHsfA2c with OsHsfA2d and OsHsfA2e is demonstrated in vivo by bimolecular fluorescence complementation assays. OsHsfA2c::OsHsfA2d and OsHsfA2c::OsHsfA2e complexes were nuclear-localized. It can be inferred that a supra-complex involving several OsHsfs regulates Hsp promoters under high temperature stress in rice. To the best of our knowledge, this is the first report showing interaction among class A OsHsfs of rice. Rice transgenic lines constitutively over-expressing OsHsfA2c have been generated to elucidate further the function of OsHsfA2c in planta. A yeast two-hybrid library screen is being performed to identify the cellular proteins that specifically interact with OsClpB-cyt.

PI-66. Functional analysis of redox-regulated components of arsenic tolerance: Implication of thiourea supplementation for reducing arsenic load from rice

Srivastava AK, Srivastava S, Suprasanna P

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085, India. [email protected]

Arsenic (As) is ubiquitously present environmental carcinogen that enters into human food chain through rice grains. In our previous research, thiourea (TU; a non-physiological thiol based ROS scavenger) application has been demonstrated to enhance salt and UV stress tolerance as well as the crop yield under field conditions. These effects were associated with TU ability to maintain plant redox homeostasis. Since, As stress also induces the redox imbalance, present research was initiated to evaluate TU efficiency for regulating As tolerance/accumulation in rice. The supplementation of TU (75 µM) to As (25 µM) improved root growth and also reduced the As level by 56% from aerial parts. That these effects were not due to the direct interaction between As and TU was confirmed from the complexation studies using HPLC-(ICP-MS)-(ESI-MS). The real-time RT-PCR based comparative expression profiling under As with/without TU treatment identified Sultr1;1 and Sultr1;2 as major redox-regulated sulphate transporters. Their specific induction in shoot coupled with enhanced root-to-shoot sulphate translocation (analyzed using 35S-sulphate, as a radiotracer) was observed under TU supplementation. Further, the level of thiolic metabolites (PC2 in roots and GSH and PC3 in shoots) and activities of sulphur metabolism enzymes (ATP sulphurylase and cysteine synthase in roots and 5’-adenylylsulfate reductase in shoot) were also increased in As+TU as compared to As treatment. Thus, the study not only signifies the redox regulated components of As tolerance but also proposes the use of TU application for generating safe rice crop having low As content.

ABIOTIC STRESS

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Poster Session I

PI-67. Searching for transcription factors involved in ammonium assimilation and root growth in rice plants

Han C-‐d, Priatama RA, Kumar V, Agung B

Gyeongsang National University. [email protected]

Ammonium ion is the major Nitrogen in rice paddy soils and utilized as the major source for N-assimilation in rice crops. In roots, transcriptional activities of ammonium uptake and assimilation genes are highly sensitive to the availability of exogenous ammonium. However, little is known about the transcription factor genes that regulated by ammonium supply and its role to roots and plant developments. To study the transcription factor genes that involved in Ammonium response, two weeks old rice seedlings treated using Ammonium from 0 to 3 hours. Total RNA collected from each sample and samples were prepared for Agilent 8x60K microarray system. Based on the microarray data, we select transcription factor genes that highly affected by ammonium and selected knock out mutant candidates that used for phenotype screening.

ARCHITECTURE & DEVELOPMENT

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Poster Session II

PII-68. Starch metabolism related gene expression profile changes among genotypes

de Oliveira AC, Marini N, da Maia LC, Magalhaes A Jr, Bresolin APS

Federal University of Pelotas. [email protected]

Starch is an important energy source in the human diet. Starch properties determine the major functional and cooking rice traits. Recent studies showed that the major enzymes that act on starch biosynthesis influence somehow the sensing properties of rice, contributing directly or indirectly to starch grain production are: AGPase, SS, SBE and DBE. This work had as objective to evaluate the expression profiles of OsSSI, OsSSIIIa, OsSIVb, OsBEI, OsBEIIb OsGBSSI, OsAGPS2b, OsAGPL2, OsGPT1, OsISA1, OsISA2, OsISA3 and OsPUL involved in starch synthesis, in rice genotypes contrasting for amylose content. The experiment was performed in the Plant Genomics and Breeding Center (CGF), belonging to the Crop Science Department, Eliseu Maciel School of Agronomy, Federal University of Pelotas (UFPel), and Embrapa Temperate Climate, Capão do Leão – RS, Brasil. Three lowland rice genotypes (Oryza sativa L.) were used, an indica (BRS Pampa) with high amylose content, a stay-green tropical japonica (BRS Firmeza) with intermediate amylose content and a temperate japonica (Nipponbare) with low amylose content. Seeds were kept in germination chamber (BOD) at 26°C, with 16 h of light/8h dark photoperiod and 100% relative humidity for one week. Uniform plantlets were transferred to pots in the greenhouse where they remained until panicle harvesting. Samples were collected in the following stages: 5 days after flowering (DAF), 10 DAF, 15 DAF, 20 DAF, 25 DAF and 30 DAF. Total RNA was extracted from panicle grains, later cDNAs were obtained and analysed in qRT-PCR. From the results obtained, it were observed changes in transcription levels of starch related genes during grain filling The trends in gene activity were genotype dependent and suggests a late starch accumulation in the stay-green genotype.

PII-69. Combinatorial preferences of rice HAP gene family members in seed development

Das S, Agarwal P, Tyagi AK

National Institute of Plant Genome Research (NIPGR). [email protected]

HEME ACTIVATOR PROTEIN (HAP), also called NUCLEAR FACTOR-Y (NF-Y) or CCAAT-binding factor (CBF), is a common family of transcription factors in eukaryotes consisting of three different subunits, named as HAP2/NF-YA/CBF-B, HAP3/NF-YB/CBF-A and HAP5/NF-YC/CBF-C. These factors, except HAP2, resemble histone proteins, specifically H2A/H2B as they have the specific histone fold motifs. A heterodimer is formed between the HAP3 and HAP5 subunits, which is followed by binding of HAP2 to form a hetero-trimeric complex and this in turn binds to the CCAAT-box in the target promoter. In case of vertebrates, each of the three subunits is encoded by a single gene. However, during evolution, in terrestrial plants, these factors faced several rounds of duplication followed by functional diversification, such that each of the three classes has evolved with multiple subunits. These resemble in structure in their central conserved domains and are involved in varied aspects of plant growth and development. For example, recent study shows that in the monocot plant rice, there are 10, 13 and 16 genes encoding each of HAP2, HAP3 and HAP5 subunit family members respectively. In these multi-gene families, there is a great scope for formation of a number of complexes in different combinations, leading to the diverse roles of HAP factors in plants during various conditions. One such event is seed development, which is a vital event in the life cycle of terrestrial plants. A number of constitutive and seed-preferential genes play roles in different stages of seed development to regulate embryogenesis, seed maturation, reserve accumulation and desiccation. Since, some rice HAP gene family members have seed-preferential expressions; they may play significant roles during seed development. Interactions have been studied amongst some constitutive as well as seed-preferential/specific HAP3 and HAP5 subunits along with their sub-cellular localization, the results of which will be presented.


84

Poster Session II

PII-70. Characterization of a novel rice seed specific glyoxalase I -like protein and its promoter

Ghosh A1, Mustafiz A1, Pareek A2, Sopory SK1, Singla-‐Pareek SL1 1Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, 2Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. [email protected]

Glyoxalase pathway constitutes the major detoxification system of metabolically produced cytotoxin methylglyoxal (MG), into the corresponding non toxic metabolite D-Lactate. Glyoxalase I (GLY I), the first enzyme of this pathway has been characterized from diverse genera including prokaryotes, humans, plants, animals and protozoa. Genome wide analysis revealed the presence of 11 GLY I genes in rice. Detailed characterization of OsGLYI10 indicated that it lacks the typical GLY I activity and hence is not involved in MG detoxification. This was further substantiated by its inability to complement the growth defect of GLY I mutant of yeast in the presence of MG. Publicly available micro-array data of rice and Arabidopsis indicated that the expression of OsGLYI10 and its ortholog from Arabidopsis are highly seed specific. Gene ontology data suggests its involvement in the crucial steps of Arabidopsis seed germination. The qRT-PCR and immunoblot studies confirmed that OsGLYI10 is specifically expressed in the developing seeds. Its levels are maximum in the mature dry seeds which disappear within two-three days of germination. This indicates towards a novel role Glyoxalase-like protein, OsGLYI10, in the process of seed development and germination. To delineate the seed specificity of OsGLYI10 expression, in silico analysis of the OsGLYI10 promoter (pOsGLYI10) was carried out which indicated the presence of several seed specific motifs such as skn-1 motif, prolamin box, 2S seed protbanapa, amybox, abrelaterd, pollenelta etc. Promoter deletion study using agro-infiltration technique revealed that the seed specific motifs lie in the promoter region between -420 to -640 bp from the transcription start site.

PII-71. Cross talk between endogenous hormone signals and carbohydrate metabolism for inducing regenerable callus by osmotic stress treatment

Huang W-‐L, Lee S-‐T

National Chiayi University. [email protected]

This study aims to clarify the possible mechanism between endogenous phytohormones signal and carbohydrate metabolism during shoot organogenesis induced by osmotic stress in rice (Oryza sativa L. cv. Tainung 71; TNG71) callus. Non-regenerablecalli (NRC) derived from TNG71 immature embryos inoculated on MS basal medium containing 10 µM 2, 4-D. It will turn to highly regenerablecalli (HRC) (regeneration frequency more than 75%) when 0.6 M sorbitol was supplemented into the medium even though have lower calli fresh weight and water content. The regeneration ability is prominently decreased to 25% while an auxin transport inhibitor, 2, 3, 5-triiodobenzoic acid (TIBA), was included in the sorbitol-treated medium. It suggested that endogenous auxin signal may involve in the induction of HRC under osmotic stress treatment. Besides, it showed higher contents of glucose, sucrose, and starch and higher expression levels of Cell wall-bound invertase 1, Sucrose transporter 1 (OsSUT1) and OsSUT2 genes in HRC than in NRC at the same time. The expression levels of PIN-formed 1 and late embryogenesis abundant 1 were also consistent with the trend of carbohydrate metabolisms. We thus concluded a putative flowchart for inducing regenerablecalli by osmotic stress in rice. According to the hypothesis, osmotic stress may regulate endogenous levels of auxin interacted with ABA then affect carbohydrate metabolism to trigger callus initiation and further shoot regeneration in rice.


85

Poster Session II

PII-72. Knockdown of a floral meristem specific cytokinin oxidase - OsCKX2 in rice reduces yield penalty under abiotic stress conditions

Joshi R1, Sahoo KK1, Tripathi AK1, Kumar R1, Teotia PS1, Pareek A2, Singla-‐Pareek SL1 1Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India, 2Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India. [email protected]

Crop improvement targeting high yield and tolerance to abiotic stresses is an urgent necessity due to a variety of reasons. The phytohormone cytokinin positively regulates the activity and function of the shoot apical meristem which is a major factor determining the degree of panicle branching and hence the number of seeds. In the present study, we tested if engineering the components of cytokinin catabolism can lead to a reduced yield penalty under various abiotic stress conditions in rice. For this, we cloned and characterized CYTOKININ OXIDASE 2 (CKX2), a gene which encodes cytokinin oxidase involved in cytokinin catabolism and deactivates the hormone in a single enzymatic step. We found its expression to be highly specific to the floral meristem region and to be highly induced by its own substrate. Its knockdown via RNAi-mediated silencing resulted in an enhanced cytokinin levels in panicles. Owing to this, the knockdown rice plants had a higher number of panicles and more grains per panicle, leading to enhanced grain yield per plant. These plants maintained higher yield when challenged with various abiotic stress conditions. Our data establish that knockdown of CKX2 expression helps plants in bridging the yield gap under abiotic stress conditions. Future studies for designing of all-inclusive crops may aim gene pyramiding using OsCKX2 with other stress-regulated yield-associated genes to further the yield potential even under environmental stress conditions.

PII-73. Spatial dynamics of auxin in developing inflorescence meristems of rice Chongloi GL, Deshpande GM, Khanday I, Udhaya P, Vijayraghavan U

Indian Institute of Science, Bangalore. [email protected]

In Arabidopsis, live imaging of shoot meristems shows auxin flow, and its accumulation, specifies the site where lateral meristems, including incipient floret meristems, would emerge. That this auxin flow, followed by local auxin biosynthesis in emerging primordia is important for development of emerging meristems, is supported by genetic and physiological studies and their mathematical simulation. Rice inflorescence meristem generates two orders of lateral branches – primary and secondary, which ultimately produces a short branch meristem called spikelet meristem that differentiates into a single determinate floret meristem. In this study, we examine the normal dynamics of auxin flow in meristems at various developmental stages. We also probe the regulatory effects of developmental fate determining transcription factors like OsMADS1 and RFL on auxin dynamics. By live confocal microscopy, of developing branching inflorescence meristems from transgenic rice plants harboring the auxin-responsive synthetic promoter::reporter construct (IR4DR5::GFP), we recorded and analyzed the dynamic distribution and accumulation of auxin. High auxin accumulation specifies meristematic regions that correspond to emerging lateral primordia and apical regions of floret meristems with developing lateral organs. In differentiating floret organs, high levels of auxin are centralized in the pro-vascular strands. We have also generated transgenic plants (PIN1::PIN1mGFP) to image the orientation and localization of the auxin efflux carrier in developing live meristems. Preliminary analysis of inflorescence meristems shows the PIN1 polar orientation predicts the meristem regions of subsequent auxin accumulation where organ primordia will eventually emerge. These data suggest auxin provides patterning cues for new lateral primordia outgrowth. To co-relate the cross-talk between transcription factors regulating meristem development and auxin patterns, we are investigating the consequences of loss of OsMADS1 (a floret meristem regulator) or loss of RFL (an inflorescence branching regulator). These investigations will provide evidences on how transcription factors can modulate hormone dynamics to regulate the growth and development of rice inflorescence and its determinate florets.


86

Poster Session II

PII-74. Analysis of the fruit-weight2.2-like gene family in rice Meizhong L, Jun X, Wentao X, Baobao C, Tianze Y, Tao L, Tingting F

Huazhong Agricultural University, Wuhan 430070, China. [email protected]

Tomato fruit-weight 2.2 (FW2.2) was reported to control up to 30% fruit weight.Recent studies demonstrated that FW2.2-like (FWL) genes also play important roles in plant growth and development. For instance, a maize homolog of FW2.2, named cell number regulator 1 (CNR1), negatively regulates plant and organ size. However, FWL genes in rice have not been characterized yet. In this study, eight FWL genes were identified in rice genome and designated as OsFWL1-8. The chromosome location, gene structure, protein motif, and phylogenetic relationship of OsFWL genes were analyzed. RT-PCR result revealed that OsFWL genes exhibited diverse expression patterns. Using microarray data the detailed expression patterns of OsFWL5, 6, and 7 were found to negatively correlate with leaf growth activity. Rice protoplast transient transformation experiment showed that most OsFWL proteins locate at cell membrane but OsFWL8 is present in the nucleus. In addition, the functions of the OsFWL3 and 5 geneswere investigated by analyzing their T-DNA insertion lines. Compared with wild type the grain weight of osfwl3 mutant and the plant height of osfwl5 mutant were increased by 5.3% and 12.5%, respectively. We also found that the increase in grain length of osfwl3 mutant was due chiefly to incremental cell number, not cell size and the expression of OsFWL3 negatively correlated with glume growth activity. These results provide a comprehensive foundation for further study of OsFWL functions in rice.

PII-75. Physiological efficiency in relation to photosynthesis and productivity of some scented rice genotypes under low light environment

Baig MJ, Swain P, Behera D, Swain A

Central Rice Research Institute, Cuttack-753 006, Odisha. [email protected]

Light is one of the major constraints for optimum yield of rice in kharif (wet) season. The photosynthetic capacity is reduced due to overcast sky in wet season resulting in low dry matter production and grain yield. In the present investigation four scented rice genotypes viz. Ketakijoha, Kalajeera, Dhusara and Geetanjali along with the Swarnaprabha as check were grown in field condition under three irradiance level i.e. 100 (I100 as control), 75 (I75) and 50 (I50) percent of full sunlight in kharif season at Central Rice Research Institute, Cuttack. Net photosynthesis at 50% flowering stage was decreased in all the rice genotypes when the irradiance decreased from 100 to 50 through 75% of full sunlight. Under I100 the highest PN was recorded in Ketakijoha followed by Swarnaprabha and Geetanjali. While under I50 the same genotypes maintained higher values in the same trend. Stomatal conductance decreased in all the rice genotypes as per the decrease in the PN. It gives a positive correlation between net photosynthesis and stomatal conductance with a conclusion that the stomatal conductance governed the net photosynthetic rate independent of the quantity of solar radiation. Greater accumulation of chlorophyll b in Geetanjali under I50 predicted its shade adaptability. There was a significant decrease in soluble protein content in all the rice genotypes under I75, however significant difference in protein content was observed under I50 which was also reflected in SDS PAGE profile with the reduction in the content of polypeptides in I75 and following increase at I50 in Geetanjali and Ketakijoha. This was possibly due to reduction of light induced protein at I75 and then expression of stress induced protein when further reduction of irradiance. The yield and yield contributing parameters were recorded at harvest stage. Under normal light condition maximum total dry matter and grain yield were recorded in Ketakijoha followed by Geetanjali for total dry matter and Kalajeera for grain yield. Short grain aromatic variety Dhusara had maximum (45.86%) reduction in yield among the genotypes tested at 50% light intensity as compared to 30.28% yield reduction in the known tolerant genotype, Swarnaprabha. However the yield loss in rice genotypes was not proportional to the decrease in solar radiation.


87

Poster Session II

PII-76. Functional characterization of mediator subunits showing transcript enrichment in rice seeds

Malik N, Agarwal P, Mathur S, Tyagi AK


Mediator (MED) is a multi-protein complex consisting of 25 or more subunits in eukaryotes, which are divided among head, middle, tail and regulatory CDK-Cyclin C modules. Components of tail module interact with various transcription factors and convey message from these factors to RNA polymerase II transcription machinery through head and middle modules. Being a negative regulator of transcription, CDK8-Cyclin C module is not present with the core mediator complex during active transcription. In Arabidopsis, mediator has been shown to have role in various processes ranging from cell proliferation, flowering, embryo patterning and regulation of plant immune responses against pathogen infection. But, in rice, the role of mediator is largely unexplored. Expression profiling of MED subunit genes has unraveled several seed-preferential subunits. In case of rice, like other cereals, importance of seed is high as it serves as food source for human population world-wide. Understanding of the molecular mechanisms involved during rice seed development can be of great importance for enhancing rice yield. Keeping all these things in view, we have chosen mediator subunits, which show high expression during seed development stages, for functional characterization. Progress on functional characterization of these selected mediator subunits will be presented.

PII-77. Genome-wide in silico mining and characterization of seed storage protein encoding genes in rice

Elizebeth ME, Agarwal P

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi -110067. [email protected]

Cereal seeds remain as one of the major protein providers in human diet in spite of the relatively little protein content (10%) compared with legume seeds (40%). Nutritional quality of these cereal grains is in fact dependent on the storage protein content which generally constitutes about 50% of the total protein in mature grain. Rice, being the staple food for a majority of the world’s population, and the dramatic increase in human population coupled with protein starvation in the third world countries makes the enhancement of proteins in rice a necessity. The seed storage proteins (SSPs) in rice can be categorized as alkali-soluble glutelins, alcohol-soluble prolamins, water-soluble albumins, and saline-soluble globulins. Glutelins, accounting for about 60-80% by weight of the total seed proteins, form the major SSP in rice seed. The SSP encoding genes are characterized by their high level of synthesis in specific tissues and their expression is restricted to certain stages during seed development. The tightly regulated spatiotemporal expression is achieved by the orchestrated assembly of several transcriptional activators acting in a combinatorial manner on the SSP gene promoters. We identified the different SSP coding genes in rice by a genome-wide analysis and the encoded proteins were analyzed for the presence of signature as well as novel patterns. The presentation will focus on the major outcomes of these studies which will provide a better insight into these groups of proteins in rice.


88

Poster Session II

PII-78. Phytohormone signaling in the root growth promoting action of isoprothiolane in rice

Takashi M, Miyuki K, Itsuki M, Hideo N

Tokyo University of Agriculture. [email protected]

The plant growth was governed by variety factors those are internal and external. Among them, beside the indispensable factors such as nutrition and light, some external factors have considerable effects on the growth; they are symbiosis, environmental stresses, natural and synthetic chemicals and so on. The effects of these factors, to some extent, should utilize the same physiological mechanism of plant to regulate the growth. For example, the root growth promotion by symbiotic bacteria may use the mechanism that could be stimulated by some biologically active compound. Isoprothiolane (diisopropyl-1, 3-dithiolan-2-ylidenemalonate) is a pesticide effective against rice blast disease and have been used practically in the fields. In addition, isoprothiolane was reported to regulate the growth of rice root. The micromolar levels of isoprothiolane promoted the growth of seminal root, whereas the higher concentrations prevent the growth. The previous report indicated the involvement of ethylene and auxin, however, was lacking for evidences at the molecular level. Then we analyzed the effect of isoprothiolane on the root growth using phytohormone-related mutants of rice and Arabidopsis in combination with some phytohormone biosynthesis inhibitors. Those analyses indicated that jasmonate biosynthesis is also important for the root growth promotion by isoprothiolane. The levels of contribution of ethylene, jasmonate and auxin to the root growth are under investigation.

PII-79. Over-expression of indica rice cryptochrome 2 gene induces partial photomorphogenesis in dark, semi-dwarfism and early flowering

Mishra S, Sharma P, Kumar D, Khurana JP

Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021. [email protected]

Cryptochromes are blue/UV-A light photoreceptors that evolved from prokaryotic DNA photolyases and are widely distributed across kingdoms.Plantcryptochromes have been most extensively studied from Arabidopsis, which possesses three cryptochromes (CRY1, CRY2 and CRY3). Among these, cryptochrome 2 (CRY2) primarily regulates photoperiodic flowering, apart from its minor role in blue light-mediated inhibition of hypocotyl elongation. Besides Arabidopsis, CRY2 has been functionally characterized from pea, japonica rice, sorghum, tomato and apple. Since the indica and japonica rice differ markedly in their morphology, cooking quality and the environmental conditions under which they are cultivated, we decided to functionally characterize CRY2 from indica rice. The CRY2 cDNA from indica rice was isolated and sequenced previously in our laboratory; it is 1956 bp long and codes for a protein of ca73.8 kDa. OsiCRY2 cDNA sequence shows 8 SNPs vis-à-vis japonica CRY2 cDNA. Although OsiCRY2 transcript levels are not affected by light but its protein is degraded rapidly when exposed to blue light. For functional characterization, the OsiCRY2 gene was over-expressed in both rice and Arabidopsis and stable, homozygous lines raised for phenotypic analyses. The rice transgenic seedlings over-expressing OsiCRY2 exhibit partial photomorphogenesis in dark, i.e. seedlings develop short coleoptile and leaf emergence is advanced. Likewise, the dark-grown Arabidopsis seedlings over-expressing OsiCRY2 develop short hypocotyl and partially open apical hook.Also, the rice OsiCRY2 transgenics show early seed germination and emergence of an extra leaf at 3-week-old stage. Moreover, these rice transgenicsare semi-dwarf and display early flowering by nearly one week. The transcriptome analysis of seedlings of OsiCRY2 over-expression line vs. the wild type revealed that many genes involved in flowering, cell growth and DNA replication, hormone signaling and light-induced responses express at a higher level in the transgenics. The effect of over-expression of OsiCRY2 in Arabidopsis was more dramatic and most lines showed early flowering by 10-14 days under long day conditions in particular. These photomorphogenic effects observed in transgenics of both rice and Arabidopsis over-expressing OsiCRY2 are unique and impart new knowledge to our understanding of the developmental roles cryptochromes play in diverse plant species.


89

Poster Session II

PII-80. Deciphering the role of a class-I TCP transcription factor from rice in abiotic stress and developmental signaling

Mukhopadhyay P, Tyagi AK


Entangled in ominous environmental conditions, growth and developmental pattern of plants are reprogrammed owing to the production of stress-related proteins and suppression of growth-related genes. Under a defined set of conditions, the limit of stress tolerance and degree of survival of any plant is probably determined by an appropriate balance between the spatio-temporal expression levels of these two overlapping sets of genes. In a successful response to environmental challenges, a plant either manifests an escape phenomenon involving early flowering and seed setting, albeit small in number or withstands the entire period of stress, generally, with a reduced rate of growth and productiveness. Although molecular signaling related to development and stress response has been dealt separately for long, a few recent works have focused on identifying important key regulators bridging these two networks. During the last decade, a small group of plant-specific non-canonical bHLH transcription factors, the TCP-domain containing proteins, have been shown to be indispensible for growth and organ patterning. The interplay between two antagonizing groups of TCP transcription factors, the growth promoting Class-I and growth regulating Class-II, have been shown to serve a critical role in maintaining the orchestrated pattern of development via signal transduction pathways involving various hormones and Ca2+. Interestingly, microarray analysis indicated alterations in the usual expression level of a few of these factors under abiotic stresses. Even, constitutive overexpression of a miRNA targeting a subset of Class-II TCP regulators has been shown to provide stress tolerance although with developmental defects in a grass species. The present work is an attempt to dissect the role of a rice Class-I TCP transcription factor in stress signaling as its expression was found to increase under abiotic stresses.

PII-81. Functional characterization of HY5 homolog in rice Burman N, Bhatnagar A, Khurana JP

Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, India. [email protected]

Light is an important environmental signal which is perceived by plants to adapt to ambient conditions. To perceive this signal, plants have evolved four major classes of photoreceptors - phytochromes, cryptochromes, phototropins and UVR8. These photoreceptors perceive the light signal and pass on this signal to master regulators which in turn bring about changes in downstream components, leading to changes in gene expression. One of these master regulators is HY5 transcription factor, also known as LONG HYPOCOTYL 5, which plays an important role in photomorphogenesis. It has been found that HY5 plays a role in hormone signalling as well. A homolog of HY5, called HYH, has also been identified in Arabidopsis. Both HY5 and HYH are bZIP transcription factors which have a leucine zipper domain and basic residues for DNA binding. In dark, HY5 interacts with COP1/SPA1 and is constitutively degraded by COP9 signalosome complex. However, as soon as the cell perceives light, phytochrome moves into the nucleus where it inhibits COP1 activity by excluding it from the nucleus and targeting it to the cytoplasm. As a result, HY5 is able to activate the downstream light-responsive genes involved in photomorphogenesis. Most of the work on HY5 and its interacting partners to regulate photomorphogenesis has largely emerged through work on Arabidopsis. In our endeavour to characterize HY5 from monocots, we have identified its ortholog in rice based on presence of COP1-binding and bZIP domains. The work presented here deals with the functional characterisation of one of the HY5-homolog in rice. The overexpression and RNAi transgenics of this gene reveal that this gene is involved in controlling plant height and lateral root development. The overexpression transgenics show close to 30% reduction in height of last and second last internodes and also close to 50% sterility. The RNAi lines show an increase in plant height and more lateral root development as compared to wild type. The complementation analysis of AtHY5 mutants with this rice gene shows that it could be a functional equivalent of Arabidopsis HY5.


90

Poster Session II

PII-82. Rice WUSCHEL-related homeobox 3A (OsWOX3A) functions in leaf, spikelet, tiller, lateral root and root hair development

Paek N, Cho S, Yoo S

Department of Plant Science, Seoul National Univer. [email protected]

To understand the molecular genetic mechanisms of rice organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains. We found that narrow-curly leaves mainly resulted from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains. Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transport-related genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots. Instead, the number of root hairs increased. OsWOX3A is involved in organ development by modulating auxin-transport gene expression in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers, lateral roots and root hairs.

PII-83. Importance of MED15 and KIX domain in rice seed development Dwivedi N, Maurya A, Parida SK, Thakur JK


In eukaryotes, Mediator is a key component in the transcription of protein coding genes. It has diverse roles ranging from establishing coordination between proteins and complexes to form Pre-Initiation Complex (PIC), to fine tune the transcriptional process in response to regulatory signals. Mediator consists of four separate sub-domains termed Head, Middle, Tail and a separable Kinase domain. MED15 is a part of the tail module. In fungi and metazoans, MED15 is targeted by many transcription factors and is found to be important in several processes including fatty acid homeostasis and multidrug resistance. MED15 homologs are present in plants, but their functions and importances are not known. We are focusing on understanding the functional significance of MED15 in rice. There are four predicted paralogs for MED15 (OsMED15_1 to OsMED15_4) in rice. OsMED15_1 shows preferential expression in seeds. Yeast two hybrid screening revealed interactions of plant specific NAC transcription factor with OsMED15_1. Further analysis is in progress. MED15 harbors KIX domain which interacts with the of activation domains of various transcription factors. In rice, apart from p300/CBP and MED15, we found the KIX domain to be present also in F-box proteins suggesting that KIX domain might be involved in ubiquitin mediated degradation of proteins. Interestingly, our analysis revealed SNPs in KIX domain of OsMED15_1 and two F-box proteins that differentiated long grain rice genotypes from the short grained ones. Validation and confirmation of the role of KIX domain proteins in determination of grain size of rice is in progress.


91

Poster Session II

PII-84. Molecular dissection of contrasting root architecture of indica rice cultivars Kumar P, Barik S, Mayandi K, Sarkar AK


Rice (Oryza sativa) is one of the most important crops of the world and a monocot model plant. Crop yield and the adaptability of a plant to the changing environmental condition largely depend on its underground root system. In rice, primary root (that degenerate at later stage), nodal roots and lateral roots constitute root system architecture (RSA), which differs from that of dicot plants. Huge variation in characters is observed among different rice cultivars, which have come across domestication process. We have studied RSA trait of many indica rice cultivars and have identified contrasting trait. To uncover the molecular basis of the variation in RSA trait observed between cultivars, we have done comparative transcriptome analysis. Gene expression microarray analysis has identified differentially expressed genes predicted to be involved in transcription, signaling, development, metabolism etc. Functional characterization of selected genes is ongoing. We are also analyzing if contrasting RSA traits contribute to tolerance or sensitivity to abiotic stresses, such as salinity and drought.

PII-85. Study of a cytokinin-mediated delay in dark-induced senescence in detached leaves of rice cultivar Nagina 22 by transcriptome analysis

Ramanan R2,Panigrahy M1, Neelamraju S1 1Directorate of Rice Research, Hyderabad, 2Center for Cellular and Molecular Biology, Hyderabad. [email protected]

Functional stay green trait characterized by retention of leaf greenness till grain filling stage as a result of a slower decrease in chlorophyll content and photosynthetic activity is an important characteristic for improving rice yield potential and biomass. Treatment of a Nagina22 (N22, a drought and heat tolerant aus rice cultivar) leaves in dark with 2 mg / ml solution of 6-BA (6-benzyl adenine, a synthetic cytokinin which is known to delay senescence) for 72 h in dark results in prolonged greenness whereas untreated control leaves turn yellow. Microarray analysis of treated vs. untreated leaves was carried out using the AffymetrixGeneChip rice genome array and AvadisTM software to identify differentially expressed genes (DEG) during delayed senescence induced by 6-BA. In all 1051 DEGs were identified showing >1.5 fold change, of which 449 (57.3%) DEGs were up-regulated and 602 (42.7%) were down regulated in 6-BA treated leaves. Up-regulated genes included those involved in secondary metabolism particularly those acting in flavanoid and anthocyanin biosynthesis, cytokinin metabolism, regulation of redox state of cell, stress and defense responses, while among the down-regulated genes were several peroxidases, heat shock proteins and metallothionein-like proteins. Significant among the pathways was the sucrose degradation pathway with the upregulation of two invertase genes, two hexose transporters and a hexokinase-1 (HXK1) gene suggesting that delay in senescence by cytokinin is mediated via source-sink relations resulting from changes in carbon flux. Increased expression of a phosphofructokinase B-type carbohydrate kinase family gene and several plastid-localized photosynthetic genes such as Rubiscoactivase, Rubisco small chain, cytochrome b6-f complex, 23kDa photosystem II (PSII) protein, Chl a/b binding protein and 33kDa Oxygen-evolving protein of PSII could possibly explain the retention of green color in leaves in dark following 6-BA treatment. Biochemical and physiological parameters are being studied to substantiate these results.


92

Poster Session II

PII-86. Role of a rice anther specific F-box gene during male reproductive development

Ranjan R, Tyagi AK


Anther development is a complex and highly regulated process in flowering plants. It includes cell specification, tissue differentiation and meiosis leading to the production of pollen grains harboring male gametes. Studying anther development process in rice would not only be useful in breeding programs for hybrid seed production, but also it will give insight on male reproductive development process in monocots. Transcriptome analyses in rice have identified plethora of anther specific genes, functional characterization of them would be helpful in understanding of anther development process. We have identified an anther specific gene which putatively encodes an F-box protein. Analysis of promoter activity of this gene in transgenic rice shows its expression is restricted only to anther tissue. F-box proteins are the component of SCF E3 ubiquitin ligases. SCF is a major class of plant E3 ligase known to regulate various growth and developmental processes through selective degradation of signaling proteins. F-box protein is the most important component of this complex as it recognizes appropriate substrate targeted for the degradation. Rice genome encodes 687 putative F-box proteins, some of which have been characterized and shown to be involved in various stress responses and developmental processes including panicle and flower development. Here we are presenting the efforts on characterization of an anther specific F-box gene.

PII-87. Spatio-temporal activity of upstream regulatory regions of three anther-specific genes in rice

Khurana R, Kapoor S, Tyagi AK

Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi - 110021, India, National Institute of Plant Genome Research, ArunaAsaf Ali Marg, New Delhi 110067, India. [email protected]

Anther is the male founding lineage of angiosperms and is the site where pollen or male gametophyte develops. Development of anther/pollen is one of the most important factors for determining male fertility, which in turn determines the yield of a plant. Thus, it is essential to identify genes and promoters expressed in male gametophyte that regulate male fertility. Upstream regulatory regions (URRs) of three anther-specific genes namely OsiPK (coding for calcium-dependent protein kinases), OsbHLH (basic helix-loop-helix domain containing protein) and OsFbox (F-box protein encoding gene) were selected for their functional characterization in transgenic rice. Both GUS and GFP reporter genes were used in order to monitor the activity of these three promoters. GUS activity has been assayed by employing both histochemical and fluorometric methods. In rice, OsiPK URR shows maximum activity during anther dehiscence and is found to be active in pollen tubes as well. Both OsbHLH and OsFbox URRs confer maximum GUS/GFP expression in the meiotic anthers. The activity of OsiPK URR was found to be strongest among the three URRs (OsiPK, OsbHLH and OsFbox) investigated in rice. Anther-specific URRs characterized in this study can be used to generate male sterile lines for the production of hybrid seed. The spatial and temporal expression of these genes will be determined by the various anther-/pollen-specific cis-acting elements generally present in the 5’ URR. Yeast one-hybrid assay was used to identify a transcription factor binding to one such known pollen-specific motif. The interaction between transcription factor and the bait sequence was confirmed by electrophoretic mobility shift assay and will be presented.


93

Poster Session II

PII-88. A transcription factor family controls multiple aspects of plant architecture and grain shape in rice

Sun S, Wang L, Gong L, Zhang Q

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. [email protected]

Transcription factors regulate a wide variety of plant growth and developmental processes, including meristem maintenance, heading date, and organ size. We have conducted a series study for functional analysis of rice OsNTF gene family transcription factors. Plants overexpressing OsNTFs displayed varied phenotypes in plant architecture and grain shape. The OsNTF genes could be divided to two groups according to the phenotypes of transgenic plants. Overexpressors of group I genes including OsNTF11 and OsNTF17 exhibited dark green thick stem, reduced leaf angle, dense panicle, round and short grains. While overexpressing group II genes such as OsNTF9 and OsNTF1 showed mostly the opposite phenotype, i.e. thin stem, rolled leaf, and narrowed seeds. We crossed the OsNTF11-ox and OsNTF17-ox with OsNTF9-ox, and the hybrids showed the OsNTF9-ox phenotype indicating that OsNTF9 could suppress the function of OsNTF11 and OsNTF17. Knockdown of OsNTF11, OsNTF17, OsNTF1 or OsNTF10 by RNAi did not show any obvious changes in plant morphology, suggesting that OsNTFs function may be redundant with other family members. According to the overexpressors’ phenotypes of OsNTF11 and OsNTF17, we supposed that OsNTF11 and OsNTF17 may participate in the meristem function and maintenance. We used Y2H to detect the interaction between several NTFs and OSHs which determinate the meristem function, and found that OsNTF11 and OsNTF17 but not OsNTF9 could interact with the OSH1. No interactions were detected among OsNTFs and other homeobox genes. Hybrids between OsNTF11-OX and OSH1-OX showed additive phenotype, suggesting possible overlapping function. Further work on the target genes and interacting proteins are ongoing.

PII-89. Enhancement of plastidic lipid content in rice through over-expression of a glycerol-3-phosphate dehydrogenase (G3pDH) coding gene from Arabidopsis thaliana

Singh V, Singh S, Singh PK, Siddiqui A, Nandi AK

School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067. [email protected]

Lipids constitute 5-10% of dry weight and play diverse essential roles in plants. Plants require lipid for membrane biosynthesis, as a signalling molecule and as a form of stored energy. The plants possess two distinct pathways for glycerolipids biosynthesis; the prokaryotic pathway (alias plastidic pathway) and the eukaryotic pathway (alias endoplasmic reticulum pathway). In plastidic pathway, the second carbon of glycerol backbone preferentially incorporates 16-carbon fatty acid (FA) that further desaturates to generate 16:3 molecules. Whereas, in the eukaryotic pathway both the position of glycerol backbone preferentially incorporate 18-carbon FAs and get desaturated to generate 18:3 species. The plants like rice are referred as 18:3 plants, as in these plants eukaryotic pathway is predominant where 16-carbon species mostly remain unsaturated and the most abundant FA is 18:3. In Arabidopsis SUPPRESSOR OF FATTY ACID DESATURASE1 (AtSFD1) encodes glycerol-3-phosphate dehydrogenase enzyme for biosynthesis of glycerol-3-phosphate in plastidic compartment. Here, we report that the overexpression of AtSFD1 gene in rice (Oryza sativa L. ssp. Japonica cv Taipei 309) leads to approximately 20% of enhancement of total lipid content. The enhancement of total lipid is mostly attributed towards increase in the plastidic lipid like MGDG, DGDG and PG without affecting ER lipid. Our results also suggest that the enhancement of plastidic lipid content in transgenic rice plants is not due altered flow of lipid biosynthesis through prokaryotic pathway but due to enhancement of existing eukaryotic pathway for plastidic lipid biosynthesis.


94

Poster Session II

PII-90. The rice NARROW LEAF1 (NAL1) regulates adventitious root development Cho S-‐H, Yoo S-‐C, Paek N-‐C

Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. [email protected]

To improve our understanding of the molecular-genetic mechanisms governing leaf and root organ development in rice (Oryza sativa), we investigated narrow leaf1 (nal1) pleiotropic mutant, not only displaying semi-dwarfism with short and narrow leaves but also producing reduced number of adventitious (or crown) roots. By sequence analysis we found that narrow leaf5 (nal5), displaying similar defects in leaf and root development, is allelic to nal1. nal5 contains a missense mutation while nal1 harbors a 30-bp deletion mutation in the coding region of NAL1. Quantitative real-time PCR analysis revealed that expression levels of OSHB, YABBY, and PIN-FORMED genes associated with leaf development and auxin transport were markedly altered in nal1. In addition, expression levels of CROWN ROOTLESS (CRL) genes were severely down-regulated. These results indicate that NAL1 controls the genes for both leaf and adventitious root development at the transcriptional level. Notably, exogenous auxin treatment nearly rescued the reduced number of adventitious roots in nal1. Taken together, our results indicate that NAL1 plays important roles in regulating the adventitious root development in rice.

PII-91. Improving the photosynthetic capacity of rice by molecular engineering of minimal C4-pathway

Islam T, Manna M, Singh J, Donald J, Reddy MK, Kaul T

International Centre for Genetic Engineering &Biotechnology, New Delhi, India. [email protected]

Rice (Oryza sativa L.) is undoubtedly, the world’s most important staple crop amongst the three major cereals in terms of the number of individuals dependent upon it as their major source of calories and nutrition. Unlike wheat and maize that find place in poultry feed and industry, rice with a gross annual production of 600 million tons is entirely consumed by humans. For the layman, rice production is about providing food in a sustainable fashion, economically, socially and environmentally. Rice production as perceived by scientists is about transforming maximum fraction of solar energy in to maximum amount of chemical energy in grain within the minimum amount of time, land, water and fertilizer. Over the past 40 years, its production has been abreast with the enhancement in world population. However, in the past decade, it is evident that the fruits of the “first green revolution” are majorly impoverished or depleted. Annual gains in cereal yields due to conventional breeding have stagnated between 1997-2007, decreasing to less than a third of the annual gains between 1960 and 1988 (FAO, 2008). Engineering the genes of C4 photosynthetic pathway into C3 crops like rice has a huge potential to alleviate the yields of this major cereal crop especially rice and others in a drastic manner, thus, ushering an era of “second green revolution”. High throughput technologies and next generation sequencing have provided an innovative opening to comprehend the developmental and evolutionary processes of C4 photosynthesis in two-cell and single-cell systems. We developed transgenic rice expressing a minimal-C4 pathway by in vitro pyramiding of three C4 pathway genes on a single vector and their expression tightly coordinated by T7RNA polymerase. Our hypothesis involves CO2-fixation to be facilitated in cytoplasm by Carbonic Anhydrase or CA (from Pennisetum) in the form of bicarbonate ions. Phosphoenolpyruvate Carboxylase or PEPC (from Pennisetumglaucum) shall fix the bicarbonate ions as malate within cytoplasm. Subsequently, the malate would diffuse into chloroplasts, wherein this malate would be decarboxylated by PhosphoenolpyruvateCarboxy-Kinase or PEPCK (from Panicummaxicum) at the site of Rubisco enzyme, thereby enhancing its carboxylation efficiency in rice and in turn improving the photosynthetic capacity of rice.


95

Poster Session II

PII-92. Genome-wide analysis of histone chaperones in rice indicates their role in plant development and stress response

Tripathi AK1, Singh K1, Pareek A2, Singla-‐Pareek SL1 1Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, ArunaAsaf Ali Marg, New Delhi-110067, India, 2Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India. [email protected]

Histone chaperones regulate gene expression via modulating and maintaining, chromatin architecture and hence, epigenetic information. In contrast to their animal and yeast counterparts, not much is known about histone chaperones in plants. In the present study, we have carried out a comprehensive genome wide analysis of rice and identified thirty-six histone chaperones. These could be classified into seven different families, viz. NAP1, CAF-I, ASF1, HIRA, FACT, NASP, and SPT6, which are conserved from yeasts to humans. Our phylogenetic analysis suggests plant-specific and monocot/dicot-specific expansion of NASP, CAF-I, and FACT. To attribute physiological relevance to their presence as multiple members, we have analyzed their expression at various developmental stages, across different tissues, and biotic and abiotic stress conditions using microarray and qRT-PCR. We found tight transcriptional regulation of several of the histone chaperones across various development stages suggesting their role in genetic reprogramming during plant development. Further, we found significant differential regulation of eight of the histone chaperones under one or more environmental stress conditions. Considering that stress response and adaptation require altered expression of thousands of genes, our results suggest that the stress-responsive histone chaperones may serve as one of the key regulators in stress physiology by controlling the expression of some stress-associated genes, possibly via altering nucleosome dynamics at the corresponding loci. Taken together, our findings present new prospects for the physiological roles of histone chaperones in plants. Future studies on these lines may aim functional validation of the differentially expressed histone chaperones vis-à-vis development and abiotic stress adaptation in plants.

PII-93. Proteomics analysis shows association of GBSS-1 protein with grain chalkiness in rice

Mishra V, Mishra P, Rai V, Singh AK, Singh NK

National Research Centre on Plant Biotechnology, IARI, New Delhi. [email protected]

Grain chalkiness in rice is an undesirable trait that determines its market value. It is due to presence of numerous air spaces between loosely packed starch granules resulting in change in light reflection that degrades the visual appearance and cooking quality of milled rice. For identification of proteins related to chalkiness in rice milled rice grains of Pusa 1266 (high chalky), Jaya (partial chalky) with extreme chalky and translucent phenotypes from population of 310 recombinant inbred lines (RIL) were used for proteome profiling. A total of about 800 protein spots were observed in 2D gel, out of which 12 significant differentially expressed protein spots were detected between Pusa 1266 and Jaya which also appeared between chalky and non chalky bulks, were selected for MALDI TOF/TOF mass spectrometry analysis. Proteins identified were Granule-bound starch synthase-1 (GBSS-1), mRNA cap guanine-N7 methyltransferase 2, Ammonium transporter 1 member 1, Autophagy-related protein 8C, 1-Cys peroxiredoxin A, 19 kDa globulin, Zinc finger A20 and AN1 domain-containing stress-associated protein 11. Among all identified proteins 4 spots were identified as different isoforms of GBSS-1 which was significantly detected in Jaya and non chalky bulk, but absent in PUSA-1266 and chalky bulk. For validation of association of GBSS-1with chalkiness, protein of chalky and non chalky parts were analyzed from the same variety and GBSS-1 protein was prominent in non chalky and absent in chalky part of endosperm. Thus the presence of GBSS-1 proteins was correlated with the translucent part of rice seed endosperm.


96

Poster Session II

PII-94. Functional validation of PP12, M2 and RP6 inflorescence-specific promoters of rice

Kumar VS, Deveshwar P, Swapna L, Sharma R, Singh MRK, Kapoor S, Rao KV

Centre for Plant Molecular Biology, Osmania University, Hyderabad, 500007, Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi. [email protected]

Development of male gametophyte of flowering plants provides an excellent system to study many fundamentally important biological processes. To understand the complex developmental control of gene expression during male gametogenesis, it is necessary to study promoters/genes specifically expressed in different parts of the anther. The present study describes the functional validation of three inflorescence-specific promoters of rice, viz., PP12, M2 and RP6, and the importance of cis-regulatory elements to drive the anther-specific expression. The recombinant binary vectors of pCAMBIA-bar-PP12, pCAMBIA-bar-M2 and pCAMBIA-bar-RP6 were constructed by cloning 3.7kb, 4.2kb and 4.0kb fragments belonging to the expression cassettes of PP12, M2 and RP6 respectively, at HindIII & EcoRI site of pCAMBIA3300. Samba Mahsuri (SM) rice transformants were produced using the Agrobacterium strain LBA4404 containing pCAMBIA-PP12, M2 and RP6 expression units, employing embryogeniccalli derived from mature embryo. Transformants of PP12, M2 and RP6 were confirmed through Basta, PCR, Southern and histochemical GUS analyses in T0, T1 and T2 generations. Five single copy insertion containing homozygous lines, each of PP12, M2 and RP6 transformants, were selected in T2 generation and were subjected to histochemical and fluorometric GUS analyses. Functional analysis of PP12, M2 and RP6 promoters revealed unique expression patterns during panicle development. Histochemical GUS analysis of PP12, M2 and RP6 transformants showed GUS expression in anthers, glumes and glumes & anthers of developing panicle, respectively. The average specific GUS activity in different PP12, M2 and RP6 transformants, ranged from 7.54±0.65 to 49.67±0.68, 10.79±0.34 to 46.30±0.53 and 7.31±0.32 to 58.38±0.36 nmole 4MU/hour/mg of protein, respectively. These promoters can potentially be employed to manipulate gene expression in the pollen without the detrimental effects that may arise from undesirable gene expression in other sporophytic tissues.


97

Poster Session II

PII-95. Transcriptome analysis of two CRRI rice hybrids, Ajay and Rajalaxmi using RNA-Seq

Katara JL*, Rao RN, Varma RL, Behera L, Samantaray S, Singh ON and Mohapatra T

Central Rice Research Institute, Cuttack (Odisha)-753006, [email protected]

Heterosis is a biological phenomenon in which hybrids exhibit superior phenotypes as compared to their parental lines. It has been widely exploited and used in crop improvement. However the molecular mechanism of this phenomenon is still not fully understood. Therefore, an understanding of its molecular basis is imperative to exploitation of its full potential. The recent advent of RNA sequencing technologies (RNA-Seq) provides an opportunity to conduct in-depth transcript profiling in plants. Using RNA-Seq technology, the transcriptome of hybrid rice Ajay (based on WA-cytoplasm), Rajalaxmi (based on Kalinga-cytoplasm) and their respective parents were analyzed at the stages of panicle initiation (PI) and grain filling (GF). Around 293 and 302 million hi-quality paired-end reads of Ajay and Rajalaxmi, respectively were generated and aligned against the Nipponbare reference genome. Transcriptome profiling of Ajay revealed 2814 and 4819 differentially expressed genes (DEGs) at PI and GF stages, respectively as compared to its parents. In case of Rajalaxmi, 660 and 5264 DEGs were identified at PI and GF stages, respectively. KEGG pathway analysis indicated significant DEGsenriched in energy metabolism pathways such as photosynthesis, oxidative phosphorylation and carbon fixation at panicle initiation stage while at grain filling stage, carbohydrate metabolism related pathways such as glycolysis and starch and sucrose metabolism were significantly involved. Moreover, many genes involved in photosynthesis, exhibited up-regulation at PI stage whereas these genes involves in carbohydrate biosynthesis had higher expression in GF stage. DEGs might probably be the reason for higher yield and vigor in rice hybrid. By correlating DGHP with yield-related quantitative trait loci (QTL), a potential relationship between differential gene expression and phenotypic changes was revealed. The present study would be useful to understand the molecular mechanism underlying heterosis in rice hybrids studied.

PII-96. Unraveling the intricacies of MAPK cascade in rice roots Singh P, Mohanta TK, Sinha AK

National Institute of Plant Genome Research, [email protected]

The root system is an indispensable component for plant survival, involved in water and nutrient acquisition from the soil for plant growth and development as well as its anchorage. Any subtle change in the root system of the plant may lead to drastic change in its productivity. Rice root system emerges as a target for breeders to improve the ability of the plant to exploit mineral and water resources of the soil. The acclimation of rice plant to its environment requires the concerted optimization of its morphology as well as its signal transduction pathway. One of the major signaling cascade facilitating such allocation is the Mitogen Activated Protein Kinase (MAPK) cascade, a three tier kinase signaling module that is evolutionarily conserved across eukaryotes. In this study transcript profiling by qRT-PCR of entire gene family of MAPK and MAPKK in rice was undertaken to identify the potential MAPK signaling component involved during root development. Further, the activity of MAPKs was investigated using immuno kinase assay. As auxin and cytokinin play a pivotal role during root development the transcript profiling of PIN proteins (the auxin efflux carriers) were done on the roots grown in the presence of these phytohormones. Differential regulation of OsMAPKs and OsMAPKKs during different stages of root development was observed. OsMPK3 and OsMPK6 emerged as potential candidates showing a large variability in transcript level. The transcript level of PIN proteins showed strong regulation during phytohormone treatment hinting at the putative crosstalk between the MAPK cascade and phytohormone signaling components. The above study may help in unraveling the intricacies of MAPK cascade and its plausible cross-talk with auxin and cytokinin signaling components.This may further lead to the development of new varieties which are better adapted to adverse conditions more potential in yield and more sustainable.


98

Poster Session II

PII-97. Roles of OsMlo6 in pollen germination and fertilization Yi J, An G

Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. Department of Plant Systems Biotech, Kyung Hee University, Yongin 446-701, [email protected]

Fast pollen tube elongation is essential for fertilization of ovule. Hydration of pollen grain and tube germination is starting point for it. Here, we show a male gametophyte mutant with the segregation distortion. The mutant pollen of heterozygous plants is morphologically normal as follows; the grains successfully achieved the tri-nuclear stage and were in viable state via fluorescein diacetate staining. However, they did not germinate in vitro and in vivo and it seemed not to hydrate. The silenced gene by T-DNA is MLO6, a member of the plant specific Mildew resistance locus o (MLO) family discovered in barley. We identified a domain in OsMLO6 that mediated a calcium-dependent interaction with calmodulin in vitro. The calcium signaling is known to play a crucial role in pollen germination and pollen tube growth. Our result suggests that OsMLO6 contribute for it through interaction with calmodulin.

PII-98. Functional analysis of FBK, a rice F-box protein, a component of the Ubiquitin-26S proteasome system reveals its role in regulating plant development

Borah P, Rydquist SW, Khurana JP

Department of Plant Molecular Biology & Interdisciplinary Centre for Plant Genomics, University of Delhi South Campus, New Delhi 110021, India [email protected]

Proteolysis of proteins takes place as a regulated response to distinct environmental and developmental cues so that abundance of crucial cellular regulators could be controlled. This process is particularly important for plants as they require a high degree of plasticity in their developmental processes owing to their sessile nature. Proteins targeted for proteolysis are ubiquitinated for turn-over by the 26S proteasome with the concomitant release of ubiquitin for reuse. The SCF complexes (Skp1-Cullin-RBX1-F-box) form a core group of the Ubiquitin-26S proteasome system and are involved in substrate identification and ubiquitination. The substrate adaptor, F-box protein, is one of the largest groups of proteins found in plants. The present work deals with characterization of a rice F-box protein, FBK. In silico analyses and experimentations by Y2H and co-immunoprecipitation (Co-IP) showed that this protein is a part of an SCF complex and could be a part of a novel SCFFBK E3 ligase. Expression analysis revealed its higher transcript abundance in the panicle. Rice transgenic over-expressing FBK and RNAi plants clearly reveal the role of FBK in root development. Keeping in view this phenotype and its abundant expression in anthers, the putative interacting partners were identified from root and anther Y2H libraries and their interactions validated by Bimolecular Fluorescence Complementation and Co-IP assays. Presently, experiments are underway to determine the role of FBK in root and anther development as well as the ubiquitination status of the interacting partners. The transgenic studies complemented with the protein studies would be instrumental in understanding the role of FBK in rice root and panicle development which would further enable us to amalgamate such studies with crop productivity, a major concern in crop research.


99

Poster Session II

PII-99. Dissecting the rice lignocellulosic pathway regulatory network Mihani R, Basu S, Ambavaram MM, Krishnan A, Pereira A

University of Arkansas, Fayetteville, AR, USA. [email protected]

Cellulose from plant biomass is the largest renewable energy resource of carbon fixed from the atmosphere, which can be converted into fermentable sugars for production into bioethanol. Rice straw is the major crop waste that is often burnt or needs to be removed from fields, and can alternatively be developed as a sustainable feedstock for bioethanol production. However, the cellulose present as lignocellulosic biomass is embedded in a hemicellulose and lignin matrix from which it needs to be extracted for efficient processing. To understand the basis of lignocellulose regulation in rice, as a model for grasses, we analyzed rice genotypes overexpressing the Arabidopsis transcription factor (TF) SHINE (AtSHN) that showed a 34% increase in cellulose and 45% reduction in lignin content with no negative effects on plant performance. Using a detailed systems-level analysis of global gene expression in rice, we developed a model of the SHN regulatory network, with SHN as a master-regulator coordinating down-regulation of lignin biosynthesis and up-regulation of cellulose and other cell wall biosynthesis pathway genes. To validate the network, affinity tagged AtSHN protein was expressed in rice and used to identify rice TF promoters bound by the AtSHN protein using ChIP-qPCR. To demonstrate transcriptional regulation of downstream TF genes, AtSHN expression constructs were co-transformed into rice protoplasts with candidate rice TF promoters driving the firefly Luciferase (LUC) gene, and could identify up- and down- regulated TF promoters. Subsequently, regulation of lignin biosynthesis genes was shown in experiments of rice lignocellulose TF expression constructs, co-transformed with lignin biosynthetic pathway gene promoters driving the LUC gene. These experiments demonstrate the role of AtSHN acting as master regulator of rice lignocellulose TFs that directly regulate the expression of lignin biosynthetic pathway, and support the development of crop lignocellulosic wastes for utilization as biofuels.

PII-100. OsMYC2-1 is a rice orthologue of AtMYC2 involved in blue light signaling and disease defense.

Giri MK1, Prasad VBR2, Chattopadhyay S2, Nandi AS1

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067. Department of Biotechnology, National Institute of Technology, Durgapur 713209, India. [email protected]

AtMYC2, a basic helix loop helix transcription factor is involved in regulation of several aspects of growth, development and stress responses in Arabidopsis. AtMYC2 homologues have been identified from several plants like tomato, tobacco, banana etc. In spite of its direct role in stress responses and photomorphogenesis, orthologues of AtMYC2 are not identified yet from rice. In this study through basic local alignment search tool (BLAST), we identified a putative orthologue of AtMYC2 from rice OsMYC2-1 which show 47% sequence similarity with AtMYC2 at amino acid level. AtMYC2 functions as a negative regulator of blue light-mediated inhibition of hypocotyl elongation in Arabidopsis. In addition, AtMYC2 also negatively regulates the expression of CHLOROPHYLL A/B BINDING PROTEIN1 (CAB1) and RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN 1A (RBCS1A) in blue light dependent manner. We show here that like AtMYC2, OsMYC2-1 acts as a negative regulator of blue light-mediated inhibition of coleoptile elongation in rice. Furthermore, OsMYC2-1 efficiently complements blue light signaling in Arabidopsis both in terms of seedling phenotype and expression of CAB1 and RBCS1A. Like atmyc2 mutant plants, OsMYC2-1 siRNA lines are resistant to bacterial pathogen Xanthomonas oryzae pv oryzae. The expression of OsMYC2-1 increases during Xanthomonas oryzae and fungal pathogen Rhizoctonia solani infection. Furthermore OsMYC2-1 also seems to complement the disease defense phenotype of atmyc2 mutant. Taken together our results suggest that OsMYC2-1 is the functional orthologue of AtMYC2.


100

Poster Session II

PII-101. Unraveling the cell wall and metabolic functions of a Grass-Diverged Acyltransferase, AT5, and a Rice-Diverged Acyltransferase, AT15

Bartley L, Peck M, Lin F, Ebert B, Gjersing E, Skyes R, Thibivilliers S, Zhao K, Foster S, Scheller H

University of Oklahoma, Norman, OK, USA. Joint BioEnergy Instititue, Emeryville, CA, USA. National Renewable Energy Lab, Golden, CO, USA. [email protected]

Phenylpropanoids called hydroxycinnamic acids (HCAs) in grass cell walls influence nutrient accessibility of feed, antioxidant properties of whole grains, and biochemical conversion of grass biomass to fuels. Hence, this project seeks to understand the functions of a group of BAHD acyltransferases (ATs) that have greatly expanded numbers in grasses and other monocots compared with dicots, and at least some of which appear to function in cell wall HCA-incorporation. Our recent paper reported that rice AT10 likely functions to incorporate the HCA, p-coumarate, into the hemicellulose arabinoxylan, as AT10 over expression leads to an increase of this molecule in rice leaf cell walls (Bartley et al. Plant Phys 161:1615). Here we present data that AT10 over expression also increases grain HCA content. In addition when another acyltransferase, AT5, is over expressed, the amount of another HCA, ferulic acid, esterified to the cell wall also increases, but the change does not appear to be in arabinoxylan. This and sequence similarity to the biochemically characterized AT4, which acts as a p-coumarate monolignol transferase, has prompted us to test the hypothesis that AT5 is a feruloyl monolignol transferase with NMR and mass spec cell wall analyses. Another acyltransferase, AT15, is part of a rice-expanded subclade. AT15 over expression decreases cell wall ferulate content, suggesting that the primary AT15 target is another molecule and that the cell wall phenotype is secondary. Gene network analysis connects AT15 to wax biosynthesis genes, a hypothesis we are working to test with metabolic analyses. These results provide target genes that can be used to improve the usefulness of rice and other grasses for feed, food, and biofuels and provide a framework for revealing the functions of genes in metabolic processes.

PII-102. OsMADS29 homodimerizes and also forms heterodimers with 19 other seed expressing MIKC MADS-box proteins in rice

Nayar S1, Kapoor M2, Kapoor S1 1Department of Plant Molecular Biology & Interdisciplinary Centre for Plant Genomics, University of Delhi South Campus, New Delhi 110021, India. 2 University School of Biotechnology, Guru Gobind Singh Indraprastha University, Dwarka, New Delhi, 110078, India. [email protected]

Protein-protein interactions are important for complex formation and many proteins require forming these higher order complexes for their functioning. MADS box transcription factors are known to form these complexes in the form of dimer, ternary and quaternary structures. MADS29 is a recently characterized MIKCC protein in rice seed development whose functional partners are still unknown. We found that the intracellular localization of MADS29 monomer was not specific to the nucleus, however its homodimer was exclusively nuclear-localized. Deletion analysis revealed that K domain is essential for dimerization, mutations in the NLS and leucine zipper motifs affect nuclear localization but are not involved in the dimerization function and the MADS box also is not involved in protein-protein interactions. MADS29 was also found to form heterodimers with different classes of MADS box proteins such as A class, C class and E class along with SVP group, B-sister and AGL12 like proteins. It was significant to note that MADS29 interacted with seven E class proteins. These interactions were confirmed using bimolecular fluorescence and yeast two hybrid assays. These data suggest that for its functioning MADS29 requires to form hetero-dimers with various classes of MIKC proteins thus suggesting it to be an important player during seed development.


101

Poster Session II

PII-103. Characterization of gamma-ray induced rice mutant reveals the alteration in plant morphology, chloroplast structure, thylakoid organization and expression of transcripts involved in chlorophyll biosynthesis

Das P1, Singla-‐Pareek SL2 and Pareek A1∗

1Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India. 2Plant Molecular Biology Laboratory, International Center of Genetic Engineering and Biology, New Delhi-110067, India. ∗Email: [email protected]

Mutant analysis is a vital and powerful approach used to analyze and understand the function of nuclear gene that plays role in complex biological processes in plants. Rice (Oryza sativa L.) is a model plant for studying functional genomics in cereals. We irradiated Oryza sativa L. with gamma rays to screen heat tolerant mutants and identified a unique mutant line that exhibits phenotype of chlorophyll deficiency and dwarfism. Here, we report that in the chlorophyll deficient (ChlD) mutant, albino phenotype was observed for up to five days old stage and then entered into a transit phase where newly expanded leaves gradually turned green. Chlorophyll abundance in five days old ChlD mutant seedlings was quite low which increased gradually with the age of the plant. When the plant reached to twenty days, the level of chlorophyll content attained the level identical to that of wild type (WT). Transmission electron microscopy analysis revealed that at early stage of development in ChlD seedlings, the number and structure of chloroplasts are distinguishable from WT. It is also observed that at early stage, ChlD seedlings contained defectively developed chloroplasts which lack well-stacked granum and contained less stroma lamellae in comparison to WT. But interestingly, the structure and the organization of chloroplasts of mutant seedlings turned to normal as in WT with the increase in age of the plant. These results suggested that in this novel ChlD mutant line, mutation may delay the light induced thylakoid assembly by suppressing the activation of a/some important gene/genes at early stage of development. Electron transport activity and transcriptional expression analysis of key genes involved in chlorophyll biosynthetic pathway has been carried out further as an evidence for switching off and/or on of gene/genes at particular stages of development in ChlD mutant.

PII-104. Apomeiosis in rice: a first step towards apomixes Emmanuel G, Delphine M, Sylvie J, Laurence C, Lucie P, Maud R, Pauline M, Gaétan D, Brigitte C, Raphaël M


Apomixis, or asexual clonal reproduction through seeds, would facilitate the spread out of vigor hybrid in farmer’s field. Apomictic seed development is a two-step process: apomeiosis, i.e. production of unreduced spores bearing the maternal chromosome complement; followed by the triggering of a parthenogenetic embryo development. As a first step towards apomixis, our strategy was to generate in rice the MiMe apomeiotic mutant (spo11/rec8/osd1) originally developed in Arabidopsis. To do so, we first identified three genes : rec8 (kinetochore orientation), pair1 (absence of recombination) and osd1 (absence of second meiosis division); as well as their corresponding single mutants exhibiting similar phenotypes to those observed in Arabidopsis. We then crossed heterozygous rec8 and pair1 mutant lines and validated the homozygous double mutant phenotype. Finally, the heterozygous double mutant was crossed with heterozygous osd1 mutant. We successfully generated in rice the homozygous triple mutant (pair1/rec8/osd1), which exhibit replacement of meiosis by a mitotic-like division. Indeed, pair1 and rec8 mutations led to a mitotic-like first division instead of a normal meiotic one. Moreover, the presence of osd1 prevented the occurrence of a second division. Importantly, the MiMe rice plants were morphologically identical to wild-type plants and fertile. Self-progenies were tetraploid. Thus, we developed a model of induced apomeisosis in rice thereby opening the possibility to implement apomixis in this species.


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PII-105. Analysis of the targets of ubiquitin-mediated protein degradation machinery during sporophytic-to-gametophytic transition during male gametophyte development in rice

Chawla M, Deveshwar P, Kapoor M1, Kapoor S

Department of Plant Molecular Biology & Interdisciplinary Centre for Plant Genomics, University of Delhi South Campus, New Delhi 110021, India. 1 University School of Biotechnology, Guru Gobind Singh Indraprastha University, Dwarka, New Delhi, 110078, India. [email protected]

The onset of meiosis is a landmark event in the life cycle of higher plants that marks the transition from the sporophytic to the gametophytic phase of development. This transition occurs both in male and female reproductive organs leading to the development of male and female gametophytes, respectively. A previously carried out transcriptome analysis of four stages of anther development in rice revealed specific up-regulation of genes involved in the regulation of transcription, signal transduction and protein metabolism during this transition. Of the genes involved in the function of protein metabolism, a large number of genes belonged to the family of F-box genes, which are mainlyknown for their involvement in the E3 ligase (SCF) complex that ubiquitinates specific proteins destined for degradation by the 26S proteasome complex. Twenty three pre-meiosis and meiosis stage-specific F-box genes have been selected for analyses of interaction with the co-expressing SKP genes by one-to-one Y2H assays. Simultaneously, these genes are being used as baits to screen the meiotic anther cDNA library to identify other interacting proteins. The analyses involving eight such F-box genes have revealed that only two of them interact with meiosis-specific SKPs, suggesting their involvement in the SCF complex. One of them has also been found to interact with a WD-40 domain containing protein, which could very well be its target for degradation. These data are suggestive of the involvement of meiosis-specific F-box genes in not only E3-ligase but also other, as yet unknown, functions.

PII-106. FZP promotes expression of MADS box genes that determine floral identity and suppresses RFL and LAX1 to regulate panicle architecture in rice

Bai X, Mao D, Wen M*, Zhang L, Yan W, Shen G and Xing Y

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. * Present address: Institute of Subtropical Agriculture, Chinese Academy of Sciences. Changsha 410125, China. [email protected]

FRIZZLE PANICLE (FZP) is a major regulator to panicle architecture. To elucidate its role in panicle branching and spikelet formation, expression analyses of the branching related genes and genes involved in the ABCDE model for floral development were made in the new fzp mutants (fzp-11) and FZP-overexpressing plants. The fzp-11 had a single base mutation (A-T) in the AP2/ERF domain caused an amino acid substitution. In the fzp-11 panicle, the panicle branching related genes of RFL and LAX PANICLE1 (LAX1) were up-regulated, whereas they were down-regulated in FZP-overexpressing plants. These results suggested that FZP suppresses the expression of RFL (Rice FLO/LFY) and LAX1 to regulate panicle branching. In the fzp-11 panicle, all the three class B genes, two AGL6 gene (OsMADS6 and OsMADS17) and one of the class E (OsMADS7) in “ABCDE model” were down-regulated. And whereas these genes were up-regulated in FZP-overexpressing plants. These results suggested that FZP participates in the“ABCDE” pathway to regulate the spikelet formation. Fourteen wild rice accessions carrying several haplotypes produce normal panicles. FZP of wild rice could not recover the phenotypes of the fzp mutant. These indicated that FZP has difference functions for panicle development between cultivar and wild rice.


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PII-107. Capturing the rice metabolic responses under varying conditions Kundu SM1, Poolman G2, Shaw R1, Chatterjee A1, Huma B1 and Fell DA2

1Department of Biophysics, Molecular Biology, and Bioinformatics, Calcutta University, Kolkata 700009, India. 2Department of Biology and Medical Science, Oxford Brookes University, Headington, Oxford OX3OBP, United Kingdom. [email protected]

Rice (Oryza sativa) is a major cereal crop which makes up nearly 20% of the total caloric intake of the world human population. To assist the efforts of rice biotechnologists to design more efficient rice cultivars, our aim is to understand the rice genome scale metabolism and its system level responses under varying conditions. Here, we shall present a partially compartmentalized genome scale metabolic model of rice. It is primarily built based on the RiceCyc database after sevral levels of curation. The flux balance analysis shows that our model can produce biomass precursors (in experimentally reported proportions) from its inorganic nutrients with the help of light energy. We have estimated the values for quantum demand and Assimilation Quotient. While we simulate the model at varying light intensities, we observe the redox shuttles between the chloroplast, cytosol and mitochondrion at low light levels. The results also indicate that the photorespiration can act to dissipate excess energy at high light levels. It suggests that the inhibition of this pathway might not be wholly beneficial to the plant. A variation in the mitochondrial metabolism is also observed. Interestingly the model can capture a range of realistic behaviour of plant metabolism. Moreover, further progress on chlorophyll biosynthesis pathway would also be presented. The in-silico reaction deletion studies of our metabolic model indicate that while deleting non-essential reactions there are alternative possible metabolic pathways to produce the biomass in the fixed proportion; however, sometimes the photon demands vary significantly. Thus, we can mimick the several potential posibilities of rce metabolism, which can help the rice biotechnologists.

PII-108. An insight into differential Fe accumulation in developing rice grain through high throughput RNA-seq

Das A, Sharma S, Mohapatra T

Central Rice Research Institute, Cuttack – 753006, Odisha, INDIA. [email protected]

Rice (Oryza sativa L.) is the main staple food for nearly half of the world’s population. Cereals, especially rice is deficient in micronutrients such as Fe. However, rice genotypes differ in grain Fe concentration. The present study is focused on identification of gene(s) involved in Fe accumulation in developing rice grain through high throughput RNA-seq technology and to understand the basis of differential Fe accumulation in developing rice grain. Two cultivars of rice viz. Sharbati (high Fe) and Lalat (low Fe) differing in grain iron concentration were used in the study. Root and grain transcriptome sequences of these two cultivars (at mid-grain filling stage) were generated using RNA-Seq (Illumina Hiseq 2000 platform). For each genotype, on an average 9.7 and 7 Gb data was generated for grain and root samples, respectively. The short reads were aligned against the Nipponbare reference genome (IRGSP build 5.0), thereby successfully mapping 95% and 67% of the reads from grain and root samples, respectively. The upregulated differentially expressed genes (DEGs) in cv. Sharbati with respect to cv. Lalat were 26% and 20% in root and grain, respectively. In the grain, 3685 and 3281 genes and in the root, 1573 and 11799 genes exclusively expressed in cv. Sharbati and Lalat, respectively. The GO classification of grain transcriptome of both the genotypes showed nearly similar representation of genes in cellular component, molecular function, and biological process categories. Genes known to be involved in Fe metabolism were analyzed for expression. The S-adenosyl-L-methionine synthetases (OsSAMS1, 2) were found to be highly expressed in root of both the cultivars, corroborating a major role of these genes in producing the precursor (SAM) which is utilized to produce phytosiderophore by the action of OsNAS, OsNAAT and OsDMAS genes. As compared to Lalat, OsNAS1 and OsNAAT2 genes showed higher expression in the grains of Sharbati while OsDMAS1 had higher expression in its root. Of the 18 yellow-stripe like (YSL) genes in rice, only 11 were found to be expressed in the two cultivars. Out of these, 2 (OsYSL2 and OsYSL 8) were up-regulated (FPKM values of 3.41-3.81) in the grain of Sharbati highlighting their importance in the uptake of Fe from soil and its accumulation in the developing grain. Rice metal-NA transporter, OsYSL2 is required for long-distance transport of Fe and assumed to be responsible for delivery of Fe into developing grain. There was no expression of OsYSL8 in the root. OsYSL15, the primary transporter responsible for uptake of Fe-PS from the rhizosphere and known to be


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up-regulated in response to Fe deficiency, was not represented in the cultivars. Ferric chelate reductase (OsFRO1) was found to be expressed in the grain of both the cultivars and in the root of Sharbati alone and this might have provided additional advantage to this cultivar for Fe uptake. Further, the high Fe containing cv. Sharbati also had higher expression of OsIRT1 (Fe2+ transporter gene) in its root. Our findings suggest that higher grain Fe concentration of cv. Sharbati might be due to higher expression of key Fe transporters (viz. OsYSL2, OsYSL8 and OsITR1) and root membrane bound OsFRO1 which gives an edge in absorption, transport and assimilation of Fe in the developing grain. Interestingly, expression of ferritin gene was much higher in the root than grain and while root of Sharbati had higher expression of ferritin, the developing grain of Lalat showed higher expression of the gene. Thus, Fe concentration and ferritin expression in grains are not positively correlated.

PII-109. Isolation and characterization of EMS induced mutants for chlorophyll biosynthesis process in upland rice variety Nagina22

Lima JM1, Nath M1, Mandol N1,Kulkarni KP1, Dokku P1, Viswakarma C1, Sahu S1, Kumar P1, Chinnusamy V2, Robin S3, Sarla N4, Seshashayee M5, Singh K6, Singh AK2, Mohapatra T1,7, Sharma RP1, Amitha Mithra SV1 and Singh NK1*

1NATIONAL RESEARCH CENTER ON PLANT BIOTECHNOLOGY, NEW DELHI-110012 INDIA, New Delhi; 2Indian Agriculture Research Institute, New Delhi -110012, India. 3 Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, 641003, India, 4 Directorate of Rice Research-ICAR, Rajendranagar, Hyderabad, Andhra Pradesh, 500030, India. 5

University of Agricultural Sciences, GKVK, Bangalore. India. 6 Punjab Agriculture University, Ludhiana; 7Central Rice Research Institute, Cuttack (Orissa) 753 006, Cuttack. India. [email protected]

Chlorophyll biosynthesis is one of the major biological processes that support life on the planet. Induced mutations serve as a valuable tool for isolation of desired mutants and functional characterization of genes in plants. The knowledge on chlorophyll biogenesis has been accumulating since two decades. In this study, an effort was made to identify rice mutants showing differential morphology for chlorophyll biosynthesis in the EMS induced mutant populations of cv. Nagina22 available at NRCPB, IARI, New Delhi. Among the mutants grown in the research field of IARI, six exhibited variation in leaf colour across early seedling stage to late vegetative stage. They are albino seedling mutants (M-81 and M-282), white-yellow revert green mutant (M-745), white stripe leaf mutants (M-507 and M-423) and yellow leaf mutant (M-57). Mutants M-81 and M-282 showed albino phenotype at seedling stage which later became green. M-745 showed white leaf with yellow stripe at early seedling stage which turned green at late seedling stage. Interestingly, it reverted to the same phenotype again at early tillering stage and became green at late tillering stage. M-507 and M-423 showed green leaves at early seedling stage and white stripes appeared at late seedling stage which remained throughout their life. M-57 was green at early seedling stage and became yellow during the tillering period. Eighteen genes known to be responsible for chloroplast biogenesis or chlorophyll content in leaves were selected and primers designed using CODDLE software for finding alleles in gene sequences. TILLING was employed to detect SNP changes in these mutants for all 18 genes and is being sequenced for confirmation. Some of the genes related to chlorophyll biosynthesis showed SNP variations in mutants as compared to their wild type Nagina22. This array of mutants identified for chlorophyll biosynthesis and preliminary genetic characterization attempted in this study will be useful resources for chloroplast biogenesis studies in future.

COMPARATIVE & EVOLUTIONARY GENOMICS

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PIII-111. Prokaryotic proteins preserve their distinct feature in rice and Arabidopsis Singh A, Singla-‐Pareek SL, Pareek A, Kushwaha HR

School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi -110067, India. [email protected]

Evolution of genes in various organisms take place due to number of processes such as duplication, gene fusion etc. which led to the evolution of new genes and gene families. Large number of genes has transferred from the earlier prokaryotic species to various eukaryotes. In the current analysis, we have attempted to identify the old “prokaryotic” genes present in rice and arabidopsis genome, and highlight the role(s) of these proteins in the two genomes. The number of old “prokaryotic” genes in rice was found to be relatively higher, than in arabidopsis. Analysis shows that large numbers of these old “prokaryotic” genes are present in rice but have acquired new functions. Contrastingly in Arabidopsis, the functions of old “prokaryotic” genes largely remained the same as in the ancestor prokaryotes. The identification of large fraction of these old “prokaryotic” genes on various endo-symbiotic organelles further strengthened the endo-symbiotic hypothesis. The data shows that arabidopsis evolved through subfunctionalization while rice has evolved through the neofunctionalization process. The analysis of domain architecture of both old and new proteins in rice and arabidopsis revealed their similar distribution. The analysis of process involving old “prokaryotic” proteins showed the functional polarity of the proteins in rice in response to selective selection pressure while in arabidopsis; these proteins maintain their ancestral function. Our data shows that the old “prokaryotic” proteins maintained their distinctness in both rice and arabidopsis. The domain architecture diversity of old proteins and new proteins were found to be similar, leading towards an assumption that these domain architecture have evolved in a conserved manner.

PIII-112. Genome-wide identification and analysis of Japonica and Indica cultivar-preferred transcripts in rice using public microarray

Bhatnagar N, Jung K-‐H

Department of Plant Molecular Systems Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. [email protected]

Accumulation of genome-wide transcriptome data provides new insight on a genomic scale which cannot be gained by analyses of individual data. The majority of rice (O. sativa) species are japonica and indica cultivars. Genome-wide identification of genes differentially expressed between japonica and indica cultivars will be very useful in understanding the domestication and evolution of rice species. In this study, we analyzed 983 of the 1866 entries in the Affymetrix array data in the public database: 595 generated from indica and 388 from japonica rice cultivars. To discover differentially expressed genes in each cultivar, we performed significance analysis of microarrays for normalized data, and identified 490 genes preferentially expressed in japonica and 104 genes in indica. Gene Ontology analyses revealed that defense response-related genes are significantly enriched in both cultivars, indicating that japonica and indica might be under strong selection pressure for these traits during domestication. In addition, 36 (34.6%) of 104 genes preferentially expressed in indica and 256 (52.2%) of 490 genes preferentially expressed in japonica were annotated as genes of unknown function. Biotic stress overview in the MapMan toolkit revealed key elements of the signaling pathway for defense response in japonica or indica eQTLs. The percentage of screened genes preferentially expressed in indica was 4-fold higher (34.6%) and that in japonica was 5-fold (52.2%) higher than expected (11.1%), suggesting that genes of unknown function are responsible for the novel traits that distinguish japonica and indica cultivars. The identification of 10 functionally characterized genes expressed preferentially in either japonica or indica highlights the significance of our candidate genes during the domestication of rice species. Functional analysis of the roles of individual components of stress-mediated signaling pathways will shed light on potential molecular mechanisms to improve disease resistance in rice.


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PIII-113. Genetics distance bias on domesticated gene region among rice accessions collected in Taiwan

Wei F-‐j, Hour A-‐l, Lin H-‐y, Tsai Y-‐c, Lai M-‐h, Chang S-‐j, Lin Y-‐r, Hsing Y-‐iC

Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Department of Agronomy, National Taiwan University, Taipei, Taiwan. [email protected]

Domestication is the selection process depending on human being’s demand. The domesticated genes are the heredity unit to represent these selected traits. We would like to study how diversity the domestication genes and their nearby regions among the rice landraces with widely different characters but all been adopted in Taiwan. Totally 20 rice accessions , ranged from thousands of years to modern varieties which may be classified as landraces, upland races, old varieties and modern elite varieties, of Indica or Japonica rice. After quality control, mapping quality and variant calling quality, the mapped NGS data and BAM files were then transformed to variant calling format, the VCF files. Then we further processed decreasing false negative possibility on SNP-relative non-variant variety. The generic distance among whole genome and domesticated gene region were calculated and analyzed. A total of 25 domesticated genes, including heading day, grain and plant morphology, abiotic tolerance, etc., were examed. The genetic distances of genome-wide, chromosome-wide or regions around domestication genes showed ib difference between them. The variation of phylogenetic clades revealed the evolutionary changes during domestication along genomics. Integration and comparison of resulted phylogeny gave more information about the genetic resources of domestication.

PIII-114. Evolution of salinity tolerance in African rice Meyer R, Barretto A, Amas J, Gregorio G, Purugganan M

New York University Center for Genomics and Systems Biology, New York, NY USA 10003. [email protected]

High salinity affects 50% of global irrigated land area. Farmers in the Atlantic Coast region stretching from Senegal to Liberia plant indigenous crops that are tolerant to saline conditions, and this project focuses on one such crop, African rice (Oryza glaberrima Steud.). African rice is irrigated by rivers with a conspicuous salinity gradient. Thus, the agricultural system developed for African rice implies that varieties have been arranged with matched fitness to local saline levels. However, farmers also practice agriculture in unique ways including dyking of mangrove swamps, soil overturning, and sometimes desalination, which spurs questions about the nature of selection on African rice. How has the O. glaberrima genome been modified to tolerate salinity? Do tolerant varieties more frequently occur in coastal saline areas? We selected 100 African rice landraces from upland and coastal West African environments and screened landraces for salinity tolerant phenotypes. Phenotypes were correlated with each other and with location. We then performed whole genome sequencing on these 100 landraces using Illumina HiSeq aiming for 10x coverage. Twenty percent of landraces were highly tolerant to saline conditions, although leaf relative Na+/K+ concentrations suggest there are multiple strategies for tolerance. We built a SNP map for O. glaberrima and identified DNA regions that distinguish landraces from different environment types and with different levels of salinity tolerance. Herein we discuss different approaches taken to perform mapping and to impute SNPs. There was no conspicuous correlation between location and genotype or phenotype, suggesting trade and soft selection pressures have influenced geographic patterns. Nonetheless, preliminary candidate regions that confer tolerance have been found.


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PIII-115. The dynamic evolution of Em-like locus in cereal plants Shih M-‐D, Hsing Y-‐IC

Distinguished Research Fellow, Institute of Plant and Microbial Biology, Academia Sinica. [email protected]

Late embryogenesis abundant (LEA) proteins have been proposed to associate with desiccation tolerance. Basing on the consensus motifs and sequence similarity, LEA proteins are classified into at least 5 different groups. Up to date, over hundred of group I LEA, or termed as Em, proteins have been identified from plants, bacteria, and animals. Moreover, each haploid genome of angiosperms only contains 1 to 3 copies in two conserved loci. Hence, Em proteins may be a useful tool to study the evolution of desiccation stress-related proteins. Em-like genes are the third type of group I LEA genes. Their coding products lack 20-mer motif that is the significant signature to Em1 and Em6 type proteins. Although the evolution procedure is still unknown, the results of the similarity alignments show that Em-like genes only exist in Poaceae plants. Em-like locus in rice genome is a 193 kb segment locating on chromosome 1. Such segment also contains a shorten duplication without Em-like gene in chromosome 5. To investigate the evolution of Em-like locus, the sequences of 2 O. sativa varieties (IR64 and Nipponbare) and 9 OMAP-selected Oryza spp. (from 7 diploid and 2 tetraploid species), and 6 cereal species, were used to compared their dynamic alteration. The analysis focus on the synteny disruption, transposable element insertion, gene disruption, and gene/gene fragment movement. Our results reveal the structural variation and dynamic evolution of this region. We will also discuss the possible mechanism of Em-like locus evolution.

PIII-116. Phylogenetic and expression analysis of FCS-like zinc finger family in Poaceae

Muhammed JK, Laxmi A

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi. [email protected]

FCS type zinc finger (zf-FCS) containing proteins are a ubiquitous family of zinc finger proteins which are present in virus, bacteria and eukaryotes. In animals, they are found to be associated with proteins involved in development and mutations in them cause serious disorders such as myeloproliferative diseases. zf-FCS is a C2-C2 type zinc finger characterized by a conserved phenylalanine and serine residue associated with third cysteine. In animals they are found as single zinc finger or in tandem repeats up to ten zinc fingers. The animal zf-FCSs are found to be very diverse in structure and function; the different subclasses are involved in DNA-protein, RNA-protein and protein-protein interaction. Protein topology analysis suggests that plants have only a single subclass of zf-FCS. We named this plant specific domain as FCS-Like Zinc-finger (FLZ). The FLZ is relatively less diverged with highly conserved alpha-beta-alpha topology. The FLZ-domain containing proteins are poorly studied so far. Recently, we identified that FLZ domain is involved in protein-protein interaction. In this study, we conducted a genome wide survey of FLZ-domain containing proteins from the sequenced genomes of Poaceae. Phylogenetic analysis identified that the FLZ-domain containing proteins are a less diverged class of proteins and both whole genome and local gene duplication resulted in a rapid expansion of members from lower plants to higher plants. The gene expression analysis of rice FLZ genes from publically available microarray data showed a varied expression pattern suggesting divergence in the expression domain. The expression of rice FLZ family is compared with Arabidopsis FLZ proteins. The co-expression analysis is showed that rice FLZ genes are co-expressed primarily with genes involved in primary and secondary metabolism. This study will be helpful in the functional characterization of FLZ domain containing proteins in future.


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PIII-117. Genetic diversity and population structure of rice landraces from Eastern and North Eastern states of India

Prasad M , Basabdatta D, Samik S, Roy B, Ghosh M, Kumar PS, Kumar GT

Division of Plant Biology, Bose Institute, Main Campus, Kolkata, Department of Horticulture, Institute of Agricultural Science, University of Calcutta, Kolkata, National Institute of Plant Genome Research, New Delhi. [email protected]

Rice (Oryza sativa L.) comprises of more than 120,000 diverse accessions including landraces, which serves as crucial genetic resources for agronomic and quality traits. In India the rice landraces of the states West Bengal, Assam, Mizoram, Manipur and Nagaland are worthy candidates for genetic assessment, and considering this, 91 rice accessions were screened using microsatellite markers to identify 182 alleles which included 51 rare and 27 null alleles. The average PIC value was 0.7467/marker. The non-aromatic landraces from West Bengal was most diverse with 154 alleles and an average PIC value of 0.8005/marker, followed by the aromatic landraces from West Bengal with 118 alleles and an average PIC value of 0.6524/marker, while the landraces from North East ranked third with 113 alleles and an average PIC value of 0.5745/marker. Dendrogram analysis showed distinct clusters consisting of predominantly aromatic landraces and predominantly North East Indian landraces. The non-aromatic landraces from West Bengal were interspersed within these two clusters. The accessions were moderately structured, showing four sub-populations (A-D) with an Fst value of 0.398, 0.364, 0.206 and 0.281, respectively. The assigned clustering of accessions conforms in both distance-based and model-based approaches. Hence, these observations on grouping of accessions based on their agronomic traits would expedite molecular breeding for improvement of rice cultivars. Further, in-situ preservation of the landraces will also assist in protecting biodiversity and cultural heritage.

PIII-118. Comparative genomic analysis of the parents of an elite rice hybrid Yao W, Xie W, Zhang Q

National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. [email protected]

Rice is a staple food crop and an ideal model for functional genome research of monocot. Utilization of heterosis has contributed tremendously to the increased productivity of rice. We report the next generation sequencing of Zhenshan 97 and Minghui 63, which are the parents of an elite rice hybrid Shanyou 63. Both Zhenshan 97 and Minghui 63 were sequenced with three different sequencing libraries (insert size of 280 bp, 5 kb and 10 kb) for more than 200× sequencing coverage. Reads were aligned to the Nipponbare genome to detect SNPs, indels and structural variations (SVs). Totally, 971884 SNPs and 198153 indels were detected between Zhenshan 97 and Minghui 63. About 27% of the SNPs and 22% of the indels were in the genic region. In addition, 719 genes were absent in the genome of Zhenshan 97 but present in Minghui 63 and 808 genes were absent in the genome of Minghui 63 but present in Zhenshan 97 based on the predicted SVs. The expression pattern of 111 of these 1527 genes were extracted from a previous published expression profile of Zhenshan 97 and Minghui 63 and were found to be in accordance with the absence/presence of these genes in the genome of Zhenshan 97 and Minghui 63. The presence variation of these 1527 genes in an “immortalized F2” population derived from Shanyou 63 were deduced based on the gene presence variation between the genome of Zhenshan 97 and Minghui 63. As a result, varieties of the “immortalized F2” population were founded to contain 1137 (ranging from 719 to 1335) of these 1527 genes on average.


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PIII-119. Ds transposon can provide 4 splice donor sites for exonization in transgenic rice

Yang H-‐C, Hsu L-‐H, Liu L-‐Y, Charng Y-‐C

[email protected]

Insertion of transposable elements into introns can cause the alteration of splicing sites, called exonization, which can enrich the complexity of transcriptome and proteome. To mimic transposable elements inserting into a plant gene, in this study, mini Ds was inserted into 7 introns of a modified rice marker gene EPSPS (5’-enolpyruvylshikimate-3’-phosphate synthase), forwardly and reversely. These 14 mimic constructs were transformed into rice. Novel transcripts were identified by RT-PCR with specific primers. The mini Ds inserted in the forward and reverse directions into the EPSPS intron 4, offering 1 and 3 transcripts patterns, respectively. Also, mini Ds inserted in the reverse direction into the EPSPS intron 6, offering 2 transcripts patterns. Exonization of mini Ds in EPSPS was biased towards providing splice donor sites of the inserted mini Ds sequence. These results agreed with previous observations in transgenic tobacco. Furthermore, the frequency of each conserved spliced donor site is different from previous study. Information on these exonization events may enhance transcript divergence and functional genomic studies.

PIII-120. Sequence similarity of genes predicted from the rice genome with 48 different model organisms and construction of a tree of life based on 103 conserved genes

Jayaswal PK, Dogra V, Sharma AS, Sharma TR, Singh NK

National Research Centre on Plant Biotechnology (NRCPB), IARI, Pusa, New Delhi-110012, India, Banasthali Vidyapith, Rajasthan-304022, India. [email protected]

Comparative genomics approach is widely used for the study of genes conserved between different species and it facilitates evolutionary studies. With the development of next generation sequencing chemistries for the large scale genome sequencing huge amount of data are deposited in the public domain repositories which are available for the comparative genome analysis. The latest versions of the rice chromosome pseudomolecule sequence are predicted to have 66,338 CDS genes. In this study our aim was to identify the genes and which are uniquely expressed in rice and show homology with the expressed genes in 48 different organisms. These organisms included 17 plant species, 10 vertebrates, 13 invertebrates and 8 fungi (source: http://www.ncbi.nlm.nih.gov/unigene, http://asia.ensembl.org/info/data/ftp/index.html). We found that out of the 53,627 rice gene sequence including splicing site variants 11,500 of which were unique to rice. Of these expressed rice genes, Zea mays showed the highest similarity of 32,457 (60.67 % ), followed by Hordeum vulgare 31,504 (58.89%) and wheat 30,994 (57.94%) . On the basis of comparative genome analysis we found 103 gene sequences which were conserved among all the 48 organisms. Out of the 53,492 expressed rice gene sequence 1,600 (2.99 %) and 1,502 (2.80%) were commonly expressed in fish as well as in chicken. These commonly expressed genes in fish as well as in chicken in respect of rice show the presence of large number of common proteins between vegetarian and non-vegetarian food diet. A neighbor-joining (NJ) based method for the phylogenetic tree construction demonstrated close association between the six different mammals and shared average 1,622 (3.03%) of rice genes showing similarity with mammalian genes. In the same way, eight fungi shared homology with average 1260 (2.10%) of the rice genes . Particularly, Aspergillus oryzae which is important for the traditional fermented foods production in japan shared 1067 genes with rice and highly destructive Puccinia graminis showed the lowest number of matches ( i.e. 887 genes) in the fungus group with rice. This comparative genomic analysis suggest that sorgham and maize show closer relation to rice as compared to wheat and barley. The 103 conserved gene based phylogenetic tree helps to explore the inter as well as intra genomic sequence evolution.


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PIII-121. The origin of an Oryza-specific system s5, and its subsequent evolution in rice lineages inducing reproductive isolation

Ouyang Y, Li G, Jiaming M, Zhang Q

National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. [email protected]

The killer-protector system at S5 locus encoded by tightly linked ORF3, ORF4 and ORF5 controls indica-japonica hybrid sterility in rice, which provides opportunity to understand the mechanisms driving speciation and maintaining reproductive barriers. Here, we present direct evidence for the evolution process of such Oryza-specific system, which originated after the divergence of Oryza genus. Both sequence comparison and phylogenic analysis indicate that the ancestral combination at S5 locus is ORF3+ORF4+ORF5+. The subsequent evolutionary process of S5 is associated with a selection sweep, possibly driven by positive selection in ORF3+ORF4-ORF5+. The typical japonica genotype of ORF3-ORF4+ORF5- emerged in the lineage carrying ORF3+ORF4+ORF5-, which reached to high frequency in japonica rice. Therefore, reproductive isolation was established between the populations carrying ORF3+ORF4-ORF5+ and others with ORF3-ORF4+ORF5-. These data demonstrate, for the first time, how a novel system quickly evolved complex counter-interaction networks promoting genetic differentiation and maintaining reproductive barriers. Besides, the wide-compatibility genes (WCGs) provide a suppression force for reproductive isolation and speciation, and four types of WCGs that identified at S5 locus might have significant implications for crop improvement.

PIII-122. DNA barcoding for establishing genetic identity of species and phylogenomics: a case study in rice

Yadav MC1*, Rajkumar S1, Tiwari S1, Pani DR2, Latha M3, Dikshit N4 1Division of Genomic Resources, National Bureau of Plant Genetic Resources, New Delhi-110012, 2NBPGR, Regional Station, Cuttack, Odisha, 3NBPGR, Regional Station, Thrissur, Kerala, 4NBPGR, Regional Station, Akola, Maharashtra, India. [email protected]

Phylogenomics deals with the inference of evolutionary relationships among species and higher taxonomic entities using DNA sequence data from conserved genomic regions. DNA bar-coding is the use of a short DNA sequence or sequences from a standardized locus (or loci) as a species identification tool. The chloroplast gene rbcL has been widely used for inferring phylogeny, codes for larger sub-unit of the enzyme ribulose-1,5-bisphosphate carboxylase/ oxygenase (RUBISCO) which is an important enzyme in photosynthesis. This single copy gene is approximately 1431 bp in length, is free from length mutations except at the 3’ end and has a fairly conservative rate of evolution. We amplified and sequenced partial rbcL gene fragment of 599 bp lengths using universal primers from 100 rice accessions belonging to 16 different species of genus Oryza. Similarly, trnH-psbA spacer region of chloroplast genome was also amplified and sequenced from these rice accessions using psbA3F and trnHR primers. In these partial rbcL gene regions, SNPs were observed at 10 nucleotide positions in multiple sequence alignments, which could distinguish different genome species namely O. brachyantha, O. eichingeri, O. australiensis, O. grandiglumis, O. granulata, O. latifolia, O. minuta, O. officinalis and O. sativa f. spontanea. The AA-genome species namely O. nivara, O. rufipogon, O. barthii, O. longistaminata, O. meridionalis and O. glumaepatula grouped in the same clade in the phylogenetic tree and could not be differentiated among themselves. Nevertheless, these species were separated from other genome species of rice. An interesting polymorphism in mono-nucleotide repeats region in trnH-psbA spacer was observed along with InDels at three sites. A 6-bp InDel clearly distinguished O. brachyantha from other Oryza species. Thus, DNA bar-coding markers could be used to broaden our understanding of both phylogenetic signal and detection of different genomes in rice for efficient management and use of crop biodiversity.


111

Poster Session III

PIII-123. Diversity of cysteine-rich antimicrobial-like peptides in some Oryza AA genome species

Shenton M, Kurata N , Ohyanagi H, Toyoda A, Fujiyama A ,Nagata T

Plant Genetics Laboratory, National Institute of Genetics, Japan [email protected]

Defensive and reproductive protein genes undergo rapid evolution. Small, cysteine rich secreted peptides (CRPs) act as antimicrobial agents and function in plant intercellular signalling and are over-represented among proteins expressed in reproductive tissues. As defensive and/or reproductive proteins, what patterns of evolution can we observe among Oryza CRP families? How diverse are CRPs in closely related Oryza species and what is the source of the variation? How are they expressed? We surveyed the CRP gene sequences of six Oryza genomes comprising Oryza sativa ssp. japonica and ssp. indica, Oryza glaberrima and three accessions of Oryza rufipogon. The main cause of variation among Oryza CRPs appears to be gene loss or duplication, with considerable turnover within CRP gene families. In a total of 1009 CRP ortholog groups, 382 had a gene in each of the six genomes; 411 were present in more than one, but not all, and 216 were specific to only one of the genomes. We examined Nipponbare CRP expression patterns in flower tissues and in public RNAseq data in various tissues as well as under biotic and abiotic stress. Defensin-like and Thionin-like genes were predominantly expressed in flower tissues, and were not stress-responsive, presumably indicating that their proteins have evolved specialized roles in reproductive signalling, although they are related to antimicrobial proteins. Other CRPs displayed varied expression patterns, with highly-conserved genes more likely to be highly and broadly expressed. Oryza genomes, like other plant genomes, seem to accumulate large reservoirs of CRP sequences, in contrast to antimicrobial CRPs in animal genomes. This could be driven by the need for more and varied antimicrobials, in the absence of an adaptive immune system. Reproductive signaling also requires specific signaling molecules. The necessary phenotypic variation could be achieved by gene gain/loss caused by genome plasticity within CRP sequence containing regions.

PIII-124. Development and validation of a 50k SNP genotyping chip based on conserved single copy and domestication related rice genes

Singh N1, Jaiswal P1, Panda K1, Mandal P1, Mishra S1, Singh B1, Singh R1, Sharma TR1, Gupta A2, Singh NK 1 1 National Research Centre on Plant Biotechnology, IARI, Pusa, New Delhi, 110012, India.2Rayat & Bahra Institute of Engineering & Bio-Technology, Mohali, Punjab 140104, [email protected]

Single nucleotide polymorphisms (SNPs) are the most abundant DNA markers in the plant genomes. These are usually biallelic, co-dominantly inherited, sequence tagged, and occur more frequently in the non-coding than the coding region of the genome. SNPs have tremendous applications and prospects in rice genetics and breeding as they are very useful for creating high-density linkage map, tagging of economically important genes, fingerprinting, background selection and evolutionary studies. The present study reports development and validation of a genome-wide high density SNP genotyping chip based on conserved single copy rice genes which are conserved between wheat and rice, unique to rice genome and cloned agronomically important rice genes. Due to their conserved nature the single copy genes give clues to the origin of wheat and rice from a common ancestor. To design the 50K Affymetrix genotyping array, high quality SNPs, were identified from the oryzaSNP and other rice SNP databases, in 4,659 single copy rice genes conserved between wheat and rice, 14,966 single copy rice genes that were unique to rice and 162 cloned agronomically important and domestication related rice genes were selected. Assay was designed by taking 35 bases of sequence from either side of the SNP, and then validated in silico by Affymetrix assay design tool having higher success rate. We are using this array for diversity analysis of wild rice germplasm, evolution of wild rice population and their relationship with cultivated rice. This assay is also being used for background selection in marker-assisted backcross breeding program for transfer of major QTLs for abiotic stress tolerance into mega rice varieties.


112

Poster Session III

PIII-125. Morphological and genetic characterization of wild rice germplasm collected from different states of India

Singh B1, Mishra S 1, Singh N1, Panda K1, Singh BP1, Rai V1, Bhawmick PK2, Singh AK 2, Singh NK1 1 National Research Centre on Plant Biotechnology, IARI, Pusa, New Delhi, 110012, India.2 Division of Genetics, Indian Agriculture Research Institute, New Delhi -110012, India [email protected]

The impending climate change is anticipated to have drastic impact on plant biodiversity and crop production with severe consequences for global food security. Rice is one of the most important food crops, which is grown and consumed worldwide, hence it will play a major role in ensuring global food security. To enhance the rice productivity under adverse climate we need to discover agronomically superior alleles from rice germplasm, more importantly from their wild relatives. The versatile environmental adaptability of wild rice, O. rufipogon and O. nivara, is expected to have useful genes/alleles to cope up with the adverse effects of climate changes. Gene from wild rice have already had great impact on hybrid rice revolution, biotic stress tolerance viz. bacterial blight resistance gene Xa21, Grassy stunt virus resistance, rice blast resistance Pi-9t. Proper characterization of wild rice germplasm is important for their successful utilization. In view of this present study was conducted to collect and characterize about 300 wild rice accessions, from five different geographically distinct states of India. Along with the seed samples, complete geographical location information, passport data were collected and photographs of the samples and habitat were also taken. To characterize these accessions, 46 morphological markers, 24 SSR markers, 21 p-SINE markers and 48 SNPs markers were used. Clustering based on morphological markers differentiated all the accessions into two major groups, SSR clustered them into three, while p-SINE differentiated all the accessions into three groups annual, intermediate and perennial type. Based on 48-plex SNP genotyping all the accessions studied revealed two major groups of population structure and a third group which is intermediate between both the populations was also present. Genetic characterization revealed huge variability among the Indian wild rice accessions signifying a rich source of agronomically important genes. All the accessions are being charactering for tolerance to abiotic stresses including drought, flooding and salinity for future utilization.

PIII-126. TRIM transposons and their evolutional and biological functions in Oryza genomes

Gao D1, Chen M2, Meyers B3, Wing R4, Jackson S1

1 Center for Applied Genetic Technologies, University of Georgia, 111 Riverbend Road, Athens, GA 30602, USA. 2 State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,China. 3 Department of Plant & Soil Sciences, and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA. 4 Arizona Genome Institute, University of Arizona, 1657 E. Helen Street, Tucson, AZ 85721, USA. [email protected]

Terminal-repeat retrotransposons in miniature (TRIMs) are unique elements distinguished from other LTR retrotransposons by their small size, high sequence conservation and frequent presence in genic regions. Owing to their tiny length, TRIMs are difficult to identify, only a few TRIM families have been reported in flowering plants, thus the evolutionary impact and transposition mechanism of TRIMs are still poorly understood. We developed a bioinformatics pipeline for discovering TRIMs by combining de novo annotation and homology searches. We identified 15 TRIM families in the genomes of cultivated rice and its relatives. The TRIM elements are shared within the Oryza genus and/or across the grass family. They are distributed throughout the genomes and are often located within or near genes. We also found that new TRIM insertions alter both gene structure and expression. The putative autonomous element was identified for a TRIM in rice and provides clues as to their evolutionary origins and transposition mechanisms.

SMALL RNA & EPIGENOMICS

113

Poster Session IV

PIV-127. Methylome profiling during the anther development in rice Singh RKM, Kapoor M, Kapoor S

Department of Plant Molecular Biology, UDSC. [email protected]

A number of previous studies have shown that DNA methylation patterns vary between different genotypes and also among different stages of development. However, the extent to which these changes translate into differential gene expression has not been well understood. Furthermore, DNA methylation changes during male gametophyte development have also not been examined. With the aim to understand methylome dynamics and its influence on differential gene expression, in the current study, we have examined different stages of anther development; pre meiotic anther (PMA), meiotic anther (MA), anther with single celled pollen (SCP) and anther with tri-nucleate pollen (TPA) by using MBD-seq. The data obtained from this approach revealed that 22.8—29.1% of genes were methylated within their transcribed regions, while 8.9—14.8% of the promoter regions showed methylation within 200 bp upstream of the transcription start site. We observed strong inverse correlation of gene expression and DNA methylation at genic ends (±300 bp of the TSS and TTS), suggesting that active genes either did not require or got rid of methylation at genic ends. However, we did not find any appreciable change in the genic-end methylation levels of genes with different expression levels, suggesting that DNA methylation was more likely to be involved in switching the genes ‘on’ or ‘off’, rather than modulating their expression levels.

PIV-128. Comparative analysis of miRNA mediated spatiotemporal and drought responsive signaling networks in contrasting indica rice cultivars

Balyan S, Kumar M, Raghuvanshi U, Raghuvanshi S

Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi – 110021, India. [email protected]

miRNA mediated post-transcriptional regulatory networks are involved in the reprogramming of gene expression controlling developmental transitions, hormone signaling, nutrient homeostasis, biotic and abiotic stress responses. Also within a particular plant species, evolution has led to conservation as well as divergence of these regulatory schemas, resulting into distinct response to similar stimulus. In the present study, we investigated miRNA mediated regulatory networks, which are involved in the spatiotemporal and drought responsive signaling in drought tolerant N22 (Nagina 22) and sensitive PB1 (Pusa Basmati 1) indica rice cultivars. Genome wide small RNA datasets were generated from flag leaf and spikelets of both control and drought stressed field grown plants of N22 and PB1 at ‘heading’ stage of development. miRNA population displayed a spectrum of response ranging from stable to very variable in nature between flag leaf and spikelet. Moreover, these spatiotemporal expression profiles showed distinct cultivar bias as some miRNAs displayed flag leaf enrichment in N22 while spikelet enrichment in PB1. Drought stress adversely affect rice yield and is one of major obstacle in the present and future scenario towards achieving sustainable food. We compared the small RNA datasets in response to drought of N22 and PB1 with a view to identify miRNAs that play nodal role in the stress tolerance/ avoidance response of rice. This identified miRNA groups with similar profiles or inverse profiles between N22 and PB1. Interestingly a group of miRNA named ‘Drought associated miRNAs’ (DTA-miRNAs) showed characteristic up-regulation in drought tolerant cultivars (Nagina 22 and Vandana) and down-regulation in drought sensitive cultivar (Pusa Basmati 1 and IR64).


114

Poster Session IV

PIV-129. Differential expression of microRNAs in rice genotypes under heat stress Sailaja B, Subrahmanyam D, Voleti S, Sarla N, Viraktamath BC, Mangrauthia SK

Directorate of Rice Research. [email protected]

Abiotic stress is one of the important limiting factors that affects yield and results in the loss of rice production. To combat the stress conditions, plants adapt at cellular and molecular level through altered expression of certain transcripts/genes. During heat stress, enhanced expression of heat-shock proteins under the control of heat stress transcription factors, and increased expression of osmoprotectants and antioxidants is a well-known adaptive strategy of plants. In addition, recent reports showed that plant miRNAs have significant regulatory impact by controlling the expression of transcription factors and metabolic genes during abiotic stress conditions. In this study, expression analysis of stress associated miRNAs was performed in shoot and root tissues of heat tolerant and susceptible rice cultivars under various heat stress treatments. Based on the previous reports, 9 miRNAs were chosen for expression study. Heat tolerant rice cultivar showed down regulation of majority of miRNAs in root and shoot tissue suggesting the upregulation of their target genes during heat stress. Further, miR169 showed tissue specific expression during short and long duration stress. Interestingly, all miRNAs showed down regulation during stress treatments in root tissue. The expression pattern of these miRNAs was analyzed in heat susceptible rice cultivar also. Taken together, the differential expression pattern of miRNAs in root and shoot tissue of tolerant and susceptible rice cultivars suggest the regulatory role of miRNAs during heat stress. Further studies of these miRNAs may provide better understanding on the mechanisms of genotypic variation in heat stress tolerance and may facilitate to find alternate avenues for rice improvement.

PIV-130. Functional characterization of cytosine DNA methyltransferases in rice (Oryza sativa)

Malik G, Kapoor M

University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector 16C Dwarka, New Delhi. India. [email protected]

C5-cytosine DNA methylation is one of the most widespread epigenetic modifications that is established and maintained by a family of highly conserved proteins known as cytosine DNA methyltransferases (MTases). Cytosine DNA methylation plays an important role in regulating gene expression and is crucial for proper growth and development in plants. To elucidate the role of DNA methylation in regulating rice development, we analyzed the consequence of genome-wide hypomethylation on rice seed germination and seedling development. A decrease of ~50% global DNA methylation levels was observed to severely affect seed germination and the seedlings formed remained stunted and did not develop beyond the coleoptile stage. To identify and functionally characterize the core components of the DNA methylation machinery, we searched the TIGR-RGAP database and identified a total of 10 cytosine DNA methyltransferases encoded in the rice genome. These genes were observed to cluster with the four known subfamilies of DNA methyltransferases in land plants namely, MET, CMT, DRM and DNMT2. Microarray based gene expression studies at different stages of rice development and under abiotic stress showed that out of the ten MTases, six genes are differentially expressed at different stages of development, while four genes did not show any expression during panicle and seed formation. Subcellular localization studies revealed that most of the MTases are localized specifically in the nucleus. In order to investigate the biological function of OsCMT2 and OsMET1-2, we employed artificial microRNA (amiRNA) technique to generate knock-down mutant lines. The amiRNA-based silencing of OsCMT2 resulted in growth retardation during initial stages of vegetative development in T1 plants. On the other hand, osmet1-2 plants displayed severe phenotypic aberrations in both vegetative and reproductive stages which included growth retardation, short stature, defective root development, delayed flowering, weak and shrivelled panicles with low seed set. These findings suggest that DNA methylation plays an important role in maintaing growth, vigour and reproductive competence in rice during vegetative and reproductive stages.


115

Poster Session IV

PIV-131. miRNA expression patterns regulating rice development in heat and salt stress

Mishra NS

Plant Molecular Biology division, International Center for Genetic Engineering and Technology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India, [email protected]

Global climate change accompanied by rising temperature and increasing soil salinity are limiting growth and yield of rice production. microRNAs (miRs) have been established, as ubiquitous regulators of gene expression and a key defense against environmental stresses. Thus, understanding of the regulatory miR networks under multiple stresses is necessary to modify crop plants to sustain and/or increase their productivity under unfavourable conditions. Analysis of NGS data (Illumina, GA) of several cDNA libraries of different indica rice varieties representing the young tissues from normal and stressed rice plants, accompanied by computational predictions identified several known and novel miRs from leaf, root and flower tissues of local basmati variety. Molecular approaches were employed to validate these predictions. By in depth profiling of expression patterns we could associate the expression of miRs to specific organs under normal and stressed growth conditions. Specific miRs that are highly deregulated by high-temperature stress or salt stress have also been identified using the NGS data. The spatial and temporal accumulation of these miRs and their predicted targets was analyzed. Selected molecules are now being functionally analysed by raising over-expression transgenics in rice using the artificial miR (amiR) approach. The sequenced libraries were also searched for presence of mirtrons, which represent a special class of miRs that are processed directly from the spliced out introns. The study was aided by predictions from an in house webserver, Mirtronpred. The predictions from server followed by additional stringent selection identified 15 mirtronic sequences in rice. The prediction accuracy was subsequently validated by molecular analysis of a predicted Os-mirtron-109.

PIV-132. Global transcriptome analysis reveals interplay of TCP family transcription factor and miRNA164c in control of direct regeneration in rice

Raghavendrarao S1,2, Phanindra MLV1, Nain V1, KumariRBD2, Singh VK1, Aggarwal D1, Raman VK1, Dhandapani G1, Solanke AU1, Kumar AP1

1National Research Centre on Plant Biotechnology, New Delhi, India, 2Department of Plant Science, Bharathidasan University, Tiruchirappalli,India. [email protected]

Developmental stage shift of multicellular organisms depends on complex network of gene regulatory pathways. Direct rice regeneration from callus tissue involves a transition from undifferentiated to differentiation stage which requires a tight regulation of hormonal signalling and gene expression. To study this transition phase, we have employed global transcriptome analysis in rice to find out which genes/regulatory networks play a vital role in regulation of rice regeneration. Microarray analysis carried out with the affymetrix 57k Rice Genome Genechip revealed 2396 up regulated and 902 down regulated genes. Analysis of transcription factors revealed that TCP family (7/32) and YABBY family (3/5) proteins were most differentially regulated. These protein families were implicated in shoot apical meristem differentiation and their role in direct regeneration from callus was investigated. Further, to explore the role of microRNAs in rice regeneration miRNA profiling using miRCURY array was performed which identified a subset of miRNAs that are differentially expressed in the regenerating callus. Among the differentially regulated miRNAs, mir164c and miR319a were highly up and down regulated, respectively in the regenerating callus. The expression patterns of representative genes were further confirmed by qRT-PCR and Northern blotting. The differential regeneration rates observed across the cultivars can also be linked to differential expression of TCP protein and hence differential regulation of miR164c and miR319a. These results provide interesting insights that will significantly improve our understanding of direct regeneration in rice from callus tissue.


116

Poster Session IV

PIV-133. Investigating the crosstalk between the microRNA and MAP kinase pathways

Raghuram B, Sinha AK


The versatility of the MAP kinase pathways in plants extends from various biotic and abiotic stresses to the very important developmental processes of the plant. Our knowledge till date about the plant MAP kinases suggests that these pathways are functionally regulated by their respective MAP kinase upstream kinases and MAP kinase phosphatases at the protein level. However, at the transcriptional level, there are no reports which explain the transient differential expression of the MAP kinase transcripts in different biotic and abiotic stress conditions. Due to their very sensitive effects, signaling components are being considered as the ideal targets for the miRNA mediated regulation. Studies from animal systems, especially in C. elegans have clear evidences showing MAP kinase transcripts being targets of miRNA mediated regulation. It is also known in human systems that TRBP, a dsRNA binding protein is phosphorylated and activated by Erk2 – a MAP kinase, thus stabilizing its interaction with Dicer and accumulating mature miRNAs. The microRNA and MAP kinase pathways being very basic to the cellular processes of eukaryotes, the conservation of the interactions and crosstalk between these two pathways among all eukaryotes seems very reasonable. Therefore, it is very interesting to study the similar kind of regulation in plants, which would reveal exciting details about the conservation of the two major regulatory pathways and the underlying nexus in plant and animal kingdoms. To address these questions, it was started by computational prediction of the microRNAs targeting MAP kinase transcripts in rice and further validated by qRT-PCR analysis. MicroRNA biogenesis factors in rice were identified, cloned and their functions were studied. Also to unfold the crosstalk between MAP kinase and microRNA pathways, miRNA expression profiles were studied in MAP kinase mutants.

PIV-134. RNA polymerase III-transcribed novel non-coding RNAs in rice Yukawa Y

Graduate School of Natural Sciences, Nagoya City University, Japan. [email protected]

After the completion of rice genome project, vast numbers of non-coding RNAs (ncRNAs) have been found in rice genome by means of recent post-genome studies. However, little is known about their functions except for miRNA, siRNA and their relatives. Previously, we have successfully found long ncRNAs transcribed by RNA polymerase III (Pol III) from Arabidopsis. These RNA genes have highly conserved cis-regulatory elements, known as upstream element (USE) and TATA-like box, at proximal upstream of transcribed region, therefore we could find such unknown RNA genes from genome data. The same strategy was applied to rice genome in this study. As a result we found 17 novel rice ncRNA gene candidates. All these gene candidates included USE, TATA-like box and Pol III terminator composed by T stretch, and 76 to 347 nucleotides of transcribed regions were predicted. Then we tried to confirm by Pol III-dependent in vitro transcription system with tobacco nuclear extracts and Northern blot analysis. Some of them were substantially expressed.


117

Poster Session IV

PIV-135. Identification of arsenic-stress responsive miRNAs and their targets from rice (Oryza sativa L.)

Sharma D, Tiwari M, Lakhwani D, Asif MH, Tripathi RD, Trivedi PK

CSIR-National Botanical Research Institute. [email protected]

MicroRNAs (miRNAs) are a novel class of endogenous, non-coding small RNAs that hybridize with their target messenger RNAs (mRNAs) and repress their expression post-transcriptionally in both plants and animals. They negatively regulate genes involved in organ polarity, morphogenesis, flowering, hormone signalling and adaptation to environment. Arsenic (As) is a non-essential heavy metal that is highly toxic to organisms including plants. High As level in crops such as rice imposes serious threat to human health worldwide and understanding the molecular mechanisms of As transport and accumulation in rice may provide promising solutions to the problem. Previous studies utilizing microarray led to identification of numerous differentially expressed genes in rice in response to As stress however, involvement of miRNAs during As stress is not known. To understand the likely association of miRNAs in As-stress, we have been carried out profiling of miRNAs in contrasting As accumulating rice cultivars using Affymetrix GeneChip miRNA Array that covers set of miRNAs of 131 organisms listed in Sanger database release 15. Out of total 496 miRNAs of rice present on array, a set of miRNAs were differentially expressed during As(III) and As(V) stress in contrasting varieties. Amongst these miRNAs, members of miR396, miR408, miR1846, miR2102 and miR2907 families were up-regulated. In addition, members of miR164, miR171, miR395, miR529, miR820, miR1861 families were significantly down-regulated. Analysis also suggests differential expression of set of miRNAs in contrasting rice cultivars for As stress. The predicted targets of these miRNAs include mostly transcription factors, protein kinases, DNA and ATP-binding proteins that are linked with various vital cellular, metabolic processes and stress responses. These observations revealed the role of miRNAs during As-stress. Future characterization of these miRNAs would be helpful to understand molecular insight of regulatory networks of As uptake and assimilation in rice.

PIV-136. Identification and characterization of novel miRNAs from drought tolerant indica rice variety Nagina 22

Mutum RD, Chhillar-‐Balyan S, Kansal S, Kumar S, Kumar M, Raghuvanshi S

Department of Plant Molecular Biology, UDSC, Benito Juarez Road, New Delhi -110021, India. [email protected]

MicroRNAs are endogenous small non-coding RNAs that play crucial role in post-transcriptional gene regulation via target degradation or translational inhibition. To identify novel miRNAs in a drought-tolerant indica rice variety Nagina 22 (N22), deep sequencing data of 5 small RNA populations from different tissue and growth conditions were analyzed. Putative novel miRNAs have been identified in N22 based on their precursor hairpin structure generation, mapping pattern of small RNA reads on precursor sequence, presence of star sequence and conservation in other published small RNA libraries of rice. The putative novel miRNAs were confirmed by performing Taqman RT-PCR analysis. Further analysis identified several novel miRNAs that show differential expression under either developmental or environmental cues (drought stress). Comparison of small RNA data from N22 and PB1 (Pusa Basmati 1) also identified novel miRNAs that have a variety specific expression profile. In order to understand the functional implications of miRNAs, identification of miRNA-target pairs is crucial. Thus ‘degradome’ libraries were prepared from flag leaf and spikelets at heading and anthesis stage of development. Sequencing and analysis of the library led to the identification of targets of known miRNAs as well as targets of novel miRNAs.


118

Poster Session IV

PIV-137. MicroRNA expression profile during flowering in indica rice variety Nagina22

Kansal S, Raghuvanshi S


MicroRNAs are small regulatory molecules that have a major impact on plant development. They respond to developmental and abiotic signals by targeting specific genes thereby bringing about their regulatory effect on the plant. In the current study, we have studied the small RNA population at heading and anthesis stages of panicle development under control and field drought conditions. These stages represent the panicle exertion, flowering and fertilization stages of the rice plant. Moreover we have compared the data from drought and heat tolerant indica rice variety Nagina22 with that of relatively sensitive PB1. Expression of about one-fifth of the miRNA population shows significant dynamic changes during transition from heading to anthesis. Similarly, drought at anthesis stage modulates the expression of about 25% of the miRNA population. This significant regulation of miRNAs seems to encompass nearly all the classes of cellular proteins including transcription factors, structural protein and metabolic enzymes. Quantitative RT-PCR results reveal that some of these differential changes in miRNAs are specific to variety as they show different expression trends in N22 and PB1. We also show here that miRNAs expression also responds to changes in light conditions. Interestingly, some miRNAs are up-regulated by dark-treatment by are significantly down-regulated in etiolated rice seedlings indicating that miRNAs are themselves under a complex regulatory schema comprising of both developmental and environmental cues.

PIV-138. Identification and characterization of salt responsive miRNA-SSRs in rice (Oryza sativa)

Mondal TK, Ganie SA

National Bureau of Plant Genetic Resource. [email protected]

Salinity is an important abiotic stress that affects agricultural production and productivity. It is a complex trait that is regulated by different molecular mechanisms. Micro-RNAs are non-coding RNAs which are highly conserved and regulate gene expression. Simple sequence repeats (SSRs) are robust molecular markers for studying genetic diversity. Although several SSR markers are available now, challenge remains to identify the trait-specific SSRs which can be used for marker assisted breeding. In order to understand the genetic diversity of salt responsive-miRNA genes of rice, SSR markers were mined from 130 members of salt-responsive miRNA genes of rice and validated among the contrasting panels of tolerant as well as susceptible rice genotypes, each with 12 genotypes. Sixteen miRNA-SSRs were found to be polymorphic that classified the rice genotypes according to their known phenotypes. It had also been found that miRNA genes were more diverse in susceptible panel than the tolerant one (as indicated by polymorphic index content) which might interferes to form the stem-loop structure of premature miRNA and reduce miRNA genesis in susceptible genotypes. Thus, we concluded that length variations of the repeats in salt responsive miRNA genes may be responsible for a possible sensitivity to salinity adaptation. This is the first report of characterization of trait specific miRNA derived SSRs in plants.


119

Poster Session IV

PIV-139. JMJ705-mediated demethylation of histone H3 lysine 27 is required for defense-related gene activation and disease resistance in rice

Li T, Chen X, Zhong X, Zhao Y, Liu X, Zhou S, Zhou D-‐X

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China, Institut de Biologie des Plantes, Université Paris-sud 11, 91405 Orsay, France. [email protected]

Histone methylation is an important epigenetic modification in gene regulation. Di- or tri-methylation of histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remained unclear. Here, we demonstrate that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase specifically demethylate H3K27me2/3. The expression of JMJ705 is induced by jasmonic acid (JA) and infection of the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae (Xoo). Over-expression of the gene reduces the resting level of H3K27me2/3 resulting preferential activation of biotic stress-responsive genes and enhances the rice plant resistance to Xoo. Mutation of the gene reduces the resistance. Data from analysis of over-expression, mutant and RNAi plants treated with JA indicate that JMJ705 is required for JA-induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a stress-responsive H3K27me2/3 demethylase that may be implicated in plant immunity by erasing H3K27me3 from the chromatin of defense-related genes and potentiating their higher expression upon pathogen infection.

INFORMATICS & SYSTEMS

120

Poster Session V

PV-140. HapRice: SNP haplotype database and web tool for rice Yonemaru J-‐i, Ebana K, Yano M

National Institute of Agrobiological Sciences. [email protected]

Single nucleotide polymorphisms (SNPs) are the most frequent polymorphisms in the rice genome. Next-generation sequencing technology has accelerated the resequencing of diverse rice cultivars in Asia and the discovery of genome-wide SNPs. These SNPs are promising tools for elucidation of the genetic diversity of rice and for genetic analysis of traits with economic value. To enhance the potential utility of genome-wide SNPs, analytical tools useful to geneticists and breeders need to be developed. “Haplotype”, which is common patterns of SNP alleles in the genome, have important implications for identifying traits. To define the haplotypes in the rice genome, we determined the allele frequencies of 3334 SNPs within 76 world accessions and 3252 SNPs within 177 Japanese accessions. The SNP haplotypes were defined by the allele frequencies in world rice and Japanese rice cultivar groups. Based on these data, we constructed “HapRice”, which consists of an SNP haplotype database and a Web tool for both SNP selection and marker design. The SNP haplotype database has two interfaces: “SNP Information Table” and “SNP Genome Viewer”. The SNP Information Table was implemented to provide the genome position and source of SNP discovery. The SNP Genome Viewer, which can be used to visualize the allele frequency of each SNP in the rice genome, was constructed on the same platform as the rice QTL database Q-TARO and the rice functional annotation gene database OGRO (both available at http://qtaro.abr.affrc.go.jp/). Therefore, users can easily examine the differences in SNP haplotypes among subgroups close to QTLs or functionally annotated genes. The Web tool has functions both for selecting SNPs polymorphic between any two rice accessions and for primer design to construct cleaved amplified polymorphic sequence markers at any selected SNPs. This database will enable precise haplotype analysis using large SNP data sets obtained for the genomes of many Asian rice accessions.

PV-141. A web enabled database of wild rice germplasm collected from different eco-geographical niches of India

Panda K, Singh B, Singh N, Mishra S, Rai V, Sharma TR, Singh N K

National Research Centre on Plant Biotechnology, IARI, New Delhi 110012, India. [email protected]

Getting authentic and comprehensive information on the vast resources of wild rice germplasm is essential for efficient sharing of this valuable source of genes for utilization in crop improvement. Due to their unprotected survival in nature for millions of years, the wild rice genotypes are expected to harbour genes for resistant to various diseases and extreme environmental conditions. For the detailed study of wild rice genotypes, collection has been initiated from different parts of India with photographs and videos along with basic passport data. Further this germplasm is being evaluated at a single location to generate additional data on these lines. There is a need to develop comprehensive and authentic information modules of electronic database for the benefit of target user communities like basic science researchers and rice breeders. Hence, a web-enabled “Indian WildriceGermplasm Database” (nksingh.nationalprof.in) has been created with the objective that it will be helpful to all the end users. The database contains the basic information on geographical location like village, block, district, state, longitude, latitude and photographs of each of the wild rice accessions. Till now total 300 wild rice accessions are included in this database. This database contains 46 morphological descriptors like plant height, leaf colouretc, of total 218 genotype. In addition, general information on 24 recognized wild rice species available literature are also included in the database, including their genome constitution, global distribution, habitat, morphological characters and specific trait information. Further, we are planning to add genotyping data on each accession. It is hoped that this database will help end –users in mining agronomically important genes from the wild rice resource.


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Poster Session V

PV-142. Manually curated database of rice proteins Gour P, Garg P, Jain R, Joseph SV, Tyagi AK, Raghuvanshi S


Significant amount of scientific experimental data is available as published literature. While suitable for manual reading the articles are otherwise written in a machine non-readable format. Thus, text-mining of literature is essential to catalogue the knowledge into machine-readable format that can be rapidly searched and analyzed. Here we demonstrate the feasibility of directly digitizing the experimental data, instead of mining the text. MCDRP (Manually Curated Database of Rice Proteins) is a comprehensive resource for browsing and retrieving knowledge on rice proteins, which has been manually curated from rice literature and stored in a protein-centric manner. This has been done with the help of logical data models that split the experimental details into small logical units, each encoded with the help of various newly defined and existing ontology terms like plant ontology, environmental ontology, trait ontology and gene ontology terms. So far, we have curated data for around 1800 rice proteins from over 400 research articles. The digitization of the experimental data offers a seamless integration of experimental data and renders it amenable to computerized search such that exact data from multiple articles can be searched/retrieved rapidly. This biocurationendeavour will create knowledge nests, which are precise and comprehensive, and present an up-to-date picture of the current biological information and thus provide the users an easy access to detailed and highly cross-linked information which can be seamlessly traced back to its source.

PV-143. Data curation by semantic digitization of experimental data: strengths and possibilities

Garg P, Gour P, Jain R, Joseph SV, Tyagi AK, Raghuvanshi S


Semantic digitization of experimental data renders it amenable to computer-based analysis. This is in contrast to usual text-mining based literature curation, which draws information from the text of the research article. The digitized data can be seamlessly integrated from different perspectives to draw interesting conclusions. The database titled ‘Manually Curated Database of Rice Proteins’ offers digitized experimental data from published research articles on rice proteins. The curation process utilizes in-house developed curation models to convert pictorial or graphical experimental data into digital format. Since the data has been curated with the help of universal ontologies and notations, information from different experiments is naturally correlated. Here we attempt to demonstrate the flexibility and depth of information gathered as a result of digitization of experimental data. Some of the most interesting correlations can be drawn by analyzing proteins that share a common ‘Trait’ or ‘Molecular function’. Integration of data from over 400 research articles has identified several traits that are regulated by more than one rice protein and visa-versa, resulting into a complex network of associations. The current release of data has over 600 different traits associated to ~280 rice proteins out of which around 100 traits are associated with more than one rice protein. Analysis of these networks has identified several putative and yet unknown functional associations between rice proteins. Moreover, these networks can also be overlaid with information such as the associated tissue or molecular functions.


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Poster Session V

PV-144. Dissecting the gene regulatory networks underlying morphogenesis of architecture and organ size in rice using a systematic approach

Wang L, Sun S, Xie W, Xu C, Xiao J, Zhang Q

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. [email protected]

Plants show a wide range of architectures that are determined by lateral branches, stem length, meristem determinacy, organ angle and size. Some genes controlling the branch of panicle were recently elucidated in rice, and most of them encoded transcription factors (TFs). To dissect the gene regulatory network underlying rice panicle development, we collected 39 tissues/organs including four consecutive stages of young panicles, from two indica varieties covering the entire life cycle to analyze the global gene expression profiles using Affemetrix GeneChip Rice Genome Array. The results showed that many genes showed dynamic expression patterns during panicle development, and two groups of genes exhibited opposite expression trends from early to late stages. Among them, genes encoding TFs were enriched. TFs of two families which were targeted by two types of miRNAs showing panicle-predominant expression patterns were selected for further functional analysis. Panicle branches were greatly changed in the mutants or RNAi lines of these TFs and in the corresponding miRNA overexpressors (miRNAox), while opposite phenotypes were observed in the target-mimic transformation lines of the miRNAs and TFs overexpressors. Panicles at very early stages were sampled from wild type and miRNAox to compare their global transcriptomes, again, expression levels of many TFs including some known panicle regulators such as RFL1 and LAX1 were dramatically changed in miRNAox. Also we demonstrated that these two known genes were the direct targets of our interrogated TFs. Double mutants further showed that those known genes genetically interact with the miRNAs and TFs. Furthermore, we found these genes regulated the organs especially seed size, which provided novel clues to understand the linkage and balance between the number of branches or flowers and the final size of seeds.

PV-145. Network-guided predictions and characterization of genes governing PRR-mediated immunity in cereals

Caddell D1, Daudi A1, Hwang S3, Yang B2, Krasileva K2, Oh T1, Wise R4, Dubcovsky J2, Marcotte E3, Ronald P1 1Dept Plant Pathology and the Genome Center, University of California, Davis, CA 95616, 2Dept Chemistry and Biochemistry, University of Texas, Austin, TX 78712, 3Dept Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011. [email protected]

With the sequencing of many plant genomes finished and dozens more underway, diverse proteomics and genomics datasets are increasingly becoming available. We previously reported the construction of RiceNet, an experimentally validated genome-scale functional gene network of rice genes, reconstructed from quantitative integration of heterogeneous genomics and proteomics datasets. Datasets from five different species were integrated into a statistical framework that allowed for the prediction of functional linkages between pairs of genes using guilt-by-association. We aim to use this resource to tackle a fundamental question in plant biology on a genome-wide scale: What are the genetic networks that govern cereal pattern recognition receptor (PRR)-mediated immune responses? This project will lead to in-depth characterization of subnetworks governing PRR-mediated immunity in cereals. The PRRs studied in this project fall into the non-RD class of kinases and include rice Xa3, Xa21, Pi-D2, wheat WKS1, and barley Rpg1. Integration of results from this study will contribute to prioritizing a list of high-confidence genes governing PRR-mediated immune responses. To validate the quality of these predictions, we will confirm the in vivo function of the selected genes in rice, wheat, and barley. Results will be made broadly available, allowing for generation of novel hypotheses and biotechnological applications, and serve as a basis for comparative genomics studies.

BIOTIC STRESS

123

Poster Session VI

PVI-146. Sub-cellular localization and molecular characterization of rice blast resistance gene Pi54-of cloned form Oryza officinalis

Devanna BN, Rai AK, Vijayan J, Sharma TR

National Research Centre on Plant Biotechnology, IARI, PUSA Campus, New Delhi 110012. [email protected]

The management of rice blast disease through host plant resistance has lead to the cloning of 19 rice blast resistance (R) genes. Rice blast resistant gene Pi54 cloned from indica rice cv. Tetep has alleles and orthologues in different rice cv. and wild rice species, respectively. A Pi54 orthologue Pi54-of isolated from rice blast resistant wild rice species Oryza officinalis was found to be Magnaporthe oryzae inducible. For molecular characterization of Pi54-of gene product, the Pi54-of protein was expressed in vitro in pET29a vector system. For this, ORF (1.14kb) of Pi54of was PCR amplified and cloned in pET29a vector at KpnI and BamHI site. The construct was transferred into E. coli BL21 expression cells for in vitro expression. The total protein isolated from IPTG treated and un-treated positive BL21 clones were analyzed by SDS-PAGE and result shows the induction of Pi54-of protein product (~43 kDa) in IPTG treated clones. Further confirmation of Pi54-of protein was performed by western blot hybridization using polyclonal antibodies developed for Pi54 protein and MALDI-TOF-TOF. In order to study the sub-cellular localization of Pi54-of protein, it was fused with the C- terminal amino acid of GFP protein (pGFP100) and transferred into onion epidermis cells for transient expression analysis. Through confocal microscopic observation the localization of Pi54-of protein was found to be cytoplasmic. The Pi54-of gene is pathogen inducible and can be used for the development of rice blast resistance plants.

PVI-147. Identification of sheath specific Rhizoctonia solani responsive promoter from rice

Kumari A, Kamboj R, Sharma TR

NRC on Plant Biotechnology, IARI, New Delhi-110012. [email protected]

Sheath blight of rice caused by Rhizoctonia solani is one of the important diseases of rice affecting its yield. Due to the semi-saprophytic nature of R. solani, it infects a large number of plant species. Absolute resistance to R. solani is not available in any of the rice germplasm grown worldwide, however, quantitative resistance in some rice lines have been reported. For isolation of sheath specific R. solani responsive promoter, we inoculated sheath blight resistant and susceptible line with standard inoculums of the pathogen. Sheath samples were collected at different hours of inoculation for RNA isolation. High quality RNA samples were used for preparing cDNA libraries for transcriptome profiling using Illumina platform. Corresponding cDNA samples were used for expression studies of three genes reported to be associated with sheath blight resistance in rice plant. The domain analysis of these three genes showed that they contain VHS and GAT domain containing protein (LOC_Os05g39760), kri protein (LOC_Os05g41100), and PX domain containing protein (LOC_Os05g50660). Quantitative real-time expression analysis revealed upregulation of two genes. An increase of 2.4 fold has been obtained in LOC_Os05g41100 while LOC_Os05g50660 showed 4 fold increase at 24 hours after pathogen infection. We have also found important pathogen responsive cis regulatory elements viz. GCC box, W1 box, EIRE and MeJA responsive element in the upstream regions of the PX domain containing protein (LOC_Os05g50660). Sheath blight specific promoter proposed to be cloned in this study can be used for specific expression of defense response chitinase genes cloned from rice and would be helpful in developing sheath blight resistance plants.

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124

Poster Session VI

PVI-148. Effects of endophytic colonization with Azospirillum strain on disease resistance in rice plants

Nakashita H1,2, Yasuda M2, Kusajima M1,2, Shima S2, Minamisawa K1,2 1Tokyo University of Agriculture, 2RIKEN Innovation Center, 3Tohoku University. [email protected]

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, the expression of pathogenesis-related (PR) genes was not induced by interaction with this bacterium. The levels of SA were not influenced by the colonization with strain B510, however, the colonization suppressed the induction Os-PR1a but slightly induced PBZ1 due to unknown reason. These results indicated the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.

PVI-149. Analysis of positional effect of blast resistance gene Pi54 on its phenotypic expression in transgenic rice lines

Arora K1, Rai AK1, Narula A2, Sharma TR1 1National Research Centre on Plant Biotechnology, PUSA CAMPUS, New Delhi, 2JAMIA HAMDARD. [email protected]

Rice blast caused by Magnaporthe oryzae, an obligate, intracellular, hemibiotropic fungal pathogen is a serious constraint in rice production at global level. Pi54 gene is associated with resistance to rice blast and is effective against different M. oryzae populations. Three different transgenic lines containing Pi54 and its orthologue Pi54rh alleles had earlier been shown to be resistant to different races of M. oryzae. In order to determine the precise location of Pi54 gene in the genome of these transgenic lines, inverse PCR was performed using a set of specifically designed PCR primers. Our analysis showed that in two transgenic lines, Pi54 gene has been integrated on chromosomes 6 and 10 at 12.94 Mb and 22.3 Mb, respectively. Similarly, Pi54rh allele was located on chromosome 1 at 16.25 Mb. The Pi54 gene present on chromosome 6 was found to be in a non-coding region with retrotransposon gene and ZIM binding domain genes in the adjoining region. On the contrary, in another TP-Pi54 line, the gene was introgressed in between the coding region of SAP domain gene. The Pi54rh allele was also found to be present in the non coding region flanked by the retrotransposon genes. Positional changes of Pi54 gene did not result in significant effect on the phenotype except the height and days to flowering were considerably affected in the line containing Pi54rh allele. However, no significant differences were obtained on their reaction to M. oryzae. Disruption of genes and effect of adjoining genes could be attributed as molecular basis of variable phenotypes of transgenic lines harbouring Pi54 gene at different locations of rice genome.

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125

Poster Session VI

PVI-150. Development of transgenic rice with herbicidal and insecticidal resistance: By in vitro gene pyramiding of EPSP synthase, Glyphosate acetyl transferase (GAT) and Allium sativum leaf agglutinin (ASAL) encoding genes

Chandrasekhar K1,2, Vani K1, Thirulogachandar V1, Muvvavijayalakshmi3, Reddy MK1 1Plant Molecular Biology,ICGEB,New Delhi -110067, 2Department of Biotechnology, Acharya Nagarjuna University,Ngarjuna Nagar,Guntur, A.P, India – 522510, 3Department of Botany & Microbiology, Acharya Nagarjuna University, Ngarjuna Nagar,Guntur, A.P, India – 522510. [email protected]

Biotic stress is one of the major constraints that limit plant growth and crop productivity worldwide. Various insect pests and weeds account for the two-third of crop losses and thereby increase crop production cost by 30-40%. Hence, there is an urgent need for simultaneous development of herbicide tolerant and insect resistant crop plants for sustainable crop productivity. Weeds are the most serious biological constraint in crop production and manual weed control over large areas is not feasible from the point of labour supply and monetary costs. If the herbicide resistant cultivars are developed, then non-selective herbicides can be used to kill all types of weeds without causing any injury to the crop. With the availability of sound knowledge about the target site and mode of action of non-selective herbicide glyphosate, we modified the target site of rice EPSP synthase gene to confer resistance to the herbicide glyphosate and also introduced glyphosate-detoxifying gene, glyphosate acetyl transferase (GAT) for glyphosate tolerance. Similarly to develop resistance to sap sucking pests we introduced Allium sativum leaf agglutinin (ASAL) encoding gene. By in vitro gene pyramiding we linked glyphosate resistant genes, EPSP synthase, GAT and sap sucking pest resistant genes ASAL onto a single plant transformation vector and simultaneously transformed into rice cultivar IR-64. The molecular analysis of transgene(s) integration, expression in rice transgenic lines and their tolerance and/or resistance to glyphosate and sap sucking pests are discussed.

PVI-151. Functional characterization of rice multidrug and toxic compound extrusion proteins, OsMATE1 and OsMATE2, in Arabidopsis suggest role in plant growth, development and biotic stress

Tiwari M, Sharma D, Trivedi PK

CSIR-National Botanical Research Institute. [email protected]

Multidrug and Toxic compound Extrusion proteins (MATE) are a group of secondary active transporters with ubiquitous occurrences in all domains of life. This is a newly characterized gene family in plants with limited information about functional significance. In plants, studies suggest that MATE proteins participate in secondary metabolite sequestration, aluminium tolerance and biotic stresses. However, limited information about this gene family is available from rice. In present study, we carried out genome-wide identification of members of this family from rice and functionally characterized two members, OsMATE1 and OsMATE2. Analysis revealed that rice genome contains 53 members of this gene family. Heterologous expression of OsMATE1 and OsMATE2 in Arabidopsis altered growth and morphology of transgenic plants. Phenotypic alternations include size and pattern of rosette leaves, early flowering, reduced flower size, increased silique and seed numbers in transgenic Arabidopsis lines compared to WT plants. Genome-wide expression and qRT-PCR analyses revealed modulation of genes involved in plant growth, development and biotic stress in transgenic lines. A set of genes responsible for pathogen perception, amplification and those involved in signaling cascade were down-regulated. Consequently, enhanced pathogen susceptibility in transgenic lines expressing both MATEs was observed. Sub-cellular localization revealed that both MATEs are plasma-membrane localized. Promoter analyses of both the MATEs in Arabidopsis suggest tissue-specific expression mainly in root, shoot apices and reproductive organs. Our results infer that OsMATE1 and OsMATE2 are functionally redundant to each other and regulate plant growth and development. We also report that these proteins are negative regulator of defense process and highlight their role in disease resistance in plants.

BIOTIC STRESS

126

Poster Session VI

PVI-152. Role of ethylene signaling in induced disease resistance in rice Kusajima M1,2, Shima S2, Nakashita H1,2 1Tokyo University of Agriculture, 2RIKEN Innovation Center. [email protected]

Induced disease resistance, activated by some microbes and chemicals, protects the whole plant from the attacks by various types of pathogens such as bacteria, fungi and viruses. Systemic acquired resistance (SAR) induced by pathogen infection through salicylic acid (SA) accumulation has been practically utilized in rice fields by exploiting the plant activators capable of inducing of SAR. Another induced resistance activated by symbiotic bacteria has been speculated to involve unknown signaling pathway. In this study, we analyzed the requirement of ethylene signaling for induction of these resistances. To analyze the influences of lack of ethylene signaling, EIN2 (ethylene-insensitive 2) RNAi transgenic plants were established. The transgenic plants lacked the ethylene response when they are treated with 1-aminocyclopropane-1-carboxylic acid (ACC). SAR induction in the transgenic plants was analyzed by phytopathology and molecular biology techniques with a plant activator benzisothiazole, which revealed that ethylene signaling was not necessary for SAR. On the other hand, the symbiotic bacteria-induced resistance was not developed in the transgenic plants, suggesting an unknown signaling pathway for induced resistance other than SA-mediated one. To clarify the role of ethylene signaling in disease resistance in rice, the interaction between ethylene signaling and other phytohormons during the induction of induced resistance is under investigation.

PVI-153. Molecular diversity analysis of Avr-Pita gene in Indian field isolates of Magnaporthe oryzae

Singh PK1, Thakur S1, Mahato AK1, Rathour R2, Mukund V3, Prashanthi SK4, Singh AK5, Singh UD5, Singh NK1, Sharma TR1

1National Research Centre on Plant Biotechnology, IARI, New Delhi-110012, 2CSK Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh-176062, 3Central Rainfed Upland Rice Research Station, CRRI, Hazaribagh, Jharkhand-825301, 4University of Agricultural Sciences, Dharwad, Karnataka-58001, 5Indian Agricultural Research Institute, New Delhi-110012. [email protected]

The extensive use of blast-resistant rice cultivars has led to the emergence of new virulent race of Magnaporthe oryzae leading to resistance breakdown. Race-cultivar specificity in the rice blast fungus follows the gene-for-gene system. The race-specific resistance gene, Pita is being extensively used in blast resistance breeding programs worldwide, including India. For better understanding of Pita mediated resistance triggered by Avr-Pita gene, we studied nucleotide sequence variations within the Avr-Pita alleles cloned from 80 Indian field isolates of M. oryzae collected from major rice blast hotspots of the country using allele mining approach. A Pot3 transposon insertion was found in the promoter region of Avr-Pita gene identified in the virulent isolates of M. oryzae. A total of 22 haplotypes were identified from 38 functional Avr-Pita variants. The coding region of all the Avr-Pita alleles showed maximum nucleotide diversity as compared to other parts of the gene like introns, 5′ and 3′ UTRs. Evolutionary study of these alleles showed a significant deviation from the neutral evolution. These results indicate that Avr-Pita is following purifying selection to favor a major allele in the Indian population of M. oryzae. Our findings indicate that transposons can influence the stability of Avr-Pita and might be one of the molecular mechanisms for defeating resistance genes in the Indian rice cultivars.

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127

Poster Session VI

PVI-154. Identification and mapping of new blast resistance gene from japonica rice Liziangxintuanheigu (LTH)

Rathour R, Singh H, Kapila RK, Sharma KD, Sharma TR

Department of Agricultural Biotechnology, HP Agricultural University, Palampur-176062. [email protected]

A new blast resistance gene was identified and genetically mapped from japonica genotype Liziangxintuanheigu (LTH), which has been used by IRRI as susceptible background for developing international blast differentials. Based on genetic analysis of an F2 progeny of cross Dular/Lijiangxintuanheigu, the blast resistance in LTH was found to be controlled by a single dominant gene. Based on bulked segregant analysis and subsequent linkage mapping of 200 susceptible F2 individuals, the gene was localized on long arm of chromosome 11. To further fine map the resistance gene, 80 sequence tagged site markers spanning 27.67 to 28.92 Mb of chromosome 11 were developed using the sequence information of the reference genotype Nipponbare. Among these, seven polymorphic markers were used for genetic mapping of the resistance gene. Based on linkage analysis with these markers, the gene was localized to a 360 Kb region flanked by markers K-37 and SSR-21.Identification of a new blast resistance gene from LTH suggests the need to revisit the monogenic status of the blast differential lines developed by IRRI-JIRCAS network.

PVI-155. Rice tungro bacilliform virus (RTBV) based transient gene silencing in rice: A versatile tool for rice gene silencing

Kant R, Dasgupta I

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021. [email protected]

The availability of gene sequences of rice has made it necessary to design easy-to-use technologies to decipher their functions. Virus-induced gene silencing (VIGS) technology is a promising reverse genetic tool for high-throughput, rapid and robust functional analysis of plant genes. It exploits host RNAi machinery to silence the expression of target genes through a modified viral vector. Functional analysis of multiple genes across many dicots and few monocot species revealed through VIGS indicate the significance and applicability of this tool. Rice tungro bacilliform virus RTBV, a plant pararetrovirus contains a dsDNA genome, which has been earlier modified in our laboratory in a partial dimer form and cloned in a binary vector to develop a VIGS vector for rice, has been used to silence a marker gene, phytoenedesaturase (pds) in rice. To optimize the silencing by RTBV-VIGS, the effect of orientation of target gene in viral vector on silencing efficiency was investigated, using chlH subunit of Magnesium chelatase gene of rice. The gene was PCR-amplified from rice cDNA using gene-specific primers and cloned in sense, antisense and hairpin orientation in RTBV-VIGS vector. They were agro-inoculated in 15 day-old rice plants and were analyzed for phenotypic expression and relative transcript level at 20 days post-inoculation. Mg chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX, the last intermediate for chlorophyll biosynthesis. Preliminary results for gene silencing using sense and antisense chlH indicate that the phenotype as yellowing of the emerging leaves. Real-time PCR analysis indicated a 30-90% reduction in relative transcript levels of chlH, as compared to mock inoculated plants. In addition, we are investigating the comparative silencing efficiency using sense, antisense and hairpin constructs of chlH simultaneously using RTBV-VIGS system. Thus RTBV-VIGS promises to be a powerful tool for investigating the functions of unknown genes contributing to higher rice yield and stress resistance and provides high-throughput platform for functional genomics in rice.

BIOTIC STRESS

128

Poster Session VI

PVI-156. A group C MAP Kinase, OsMPK7 imparts resistance against Xanthomonas oryzae infection in rice

Jalmi SK, Ara H, Sinha AK

National Institute of Plant Genome Research, ArunaAsaf Ali Marg, New Delhi-110067. [email protected]

MAPK cascade comprises of MPKKKs, MPKKs and MPKs, which transduces signal from upstream receptor to downstream target. In this study the role of OsMPK7, a group C Mitogen-Activated Protein Kinase was assessed. Yeast two hybrid analysis of OsMKK3 showed its interaction with OsMPK7 in rice. Through Real time PCR analysis OsMPK7 was seen to be induced in heat stress and Xanthomonas oryzae infection. To study involvement of OsMPK7 in plant-X. oryzae interaction, transiently transformed rice leaves with OsMPK7 were infected with X. oryzae. MPK7 transiently expressed leaves showed less lesions compared to wild type leaves, indicating its role in X. oryzae infection. Similarly involvement of OsMPK7 in reducing cell death after X. oryzae infection was studied by carrying out Agrobacterium mediated transient transformation in rice roots followed by infection with X. oryzae. Roots transiently transformed with OsMPK7 showed less cell death as compared to the roots transformed with empty pCAMBIA vector, whereas roots transformed with OsMPK7 RNAi construct showed more cell death. To further investigate the possible role of OsMPK7 in X. oryzae infection, callose deposition assay, HR response assay and study of induction of defence response genes like PR genes in MPK7 transiently transformed samples were carried out. Also the role of its upstream kinase MKK3 during in X. oryzae infection will be discussed.

PVI-157. Rice senescence associated OsSAG12 genes play negative roles in senescence and pathogen induced cell death

Singh S, Giri MK, Singh PK, Siddiqui A, Nandi AK

School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067. [email protected]

Leaf senescence is a highly complex but ordered process involving expression of large numbers of senescence associated genes (SAGs). Arabidopsis SAG12 (AtSAG12) gene codes for a papain-like cysteine protease. Through BLAST search using AtSAG12 amino acid sequences as query, a few putative homologues from rice genome (OsSAGs; Oryza sativa SAGs) were identified. OsSAG12-1 and OsSAG12-2 homologues were found to be closest to AtSAG12 with 64% and 65% similar amino acid composition respectively. Expression of OsSAG12-1 is enhanced during senescence and pathogen-induced cell death. To evaluate the possible role of OsSAG12-1, RNAi transgenic lines were generated in Japonica rice cultivar TP309. The transgenic lines developed early senescence at varying levels and showed enhanced cell death when inoculated with bacterial pathogen Xanthomonas oryzaepv. oryzae. Sub-cellular localization of OsSAG12-1 is not restricted in vacuolar compartment as shown for other vacuolar processing protease including AtSAG12. To find out the physiological role of another homologue, OsSAG12-2, under-expression lines were generated using artificial microRNA (amiRNA) technology. The OsSAG12-2 amiRNA lines also supported a similar negative regulation in senescence for this cysteine protease in rice. Under-expressed OsSAG12-2 plants showed higher susceptibility against Xanthomonas oryzaepv. oryzae with reduced expression of Oryza sativa Pathogenesis Related (OsPRs) proteins PR1b and PR10. Bacterial expression of OsSAG12-2 suggested that, this peptide undergoes autolysis to generate functional protease. The matured OsSAG12-2 protease showed 40% trypsin equivalent activity on azo-casein substrate. The present study revealed that OsSAG12 genes are negative regulator of cell death in rice. The results altogether suggest that it may be possible to generate long living disease resistant transgenic lines by over expressing these OsSAG12 genes in rice.

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Poster Session VI

PVI-158. Evaluation of bio-control agents and chemical elicitor salicylic acid for sheath blight management in rice (Oryza sativa L.)

Shukla A, Bahuguna RN, Kumar J

G.B.Pant University of Agriculture and Technology, Pantnagar-263145, India bCrop and Environmental Sciences Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines ,G.B.Pant University of Agriculture and Technology,Pantnagar-263145,India. [email protected]

Disease management with the use of conventional pesticides is emerging as major threat to our environment and health safety. Moreover, growing cost of pesticides and their use in major crops like rice is economically unsustainable. Induction of systemic resistance through the application of bio control agents or chemical elicitor such as salicylic acid (SA) as a resilient strategy of disease management in rice holds promise. Powder talcum formulation of two local strains of bio control agents, Trichoderma harzianum (Th38) and Pseudomonas fluorescens (Pf28) and chemical elicitor SA were used for inducing systemic resistance against sheath blight disease in basmati rice, variety Vasumati. Higher lipid peroxidation, chlorophyll a/b ratio, proline and gas exchange parameters were indicative of disease severity even before the detection of visible lesions. Both bioagents and SA efficiently activated the different components of systemic resistance with significantly higher levels of phenylalanine ammonia lyase, lipoxygenase, superoxide dismutase, peroxidase, hydrogen peroxide and total phenolics over control. Comparative analysis with systemic fungicide carbendazim revealed comparable potential of bio agents and SA against sheath blight disease. However, bioagents treatments overrule SA treatment with higher growth and yield both under normal and pathogen infection. Besides advocating use of bioagents as an alternative to conventional pesticides, various physiological traits that were signatory to disease severity and systemic resistance assume special significance to screen or develop rice varieties through breeding for disease tolerance.

PVI-159. Gene mining in rice traditional landraces of Karnataka, India for blast resistance

Prashanthi, Ingole KD, Krishnaraj PU, Bhat S

Department of Biotechnology, University of Agricultural Sciences, Dharwad, [email protected]

Rice germplasm are the reservoirs of valuable traits. Rice is grown in diverse ecosystems characterized by varied climatic, method of cultivation, edaphic and topographical factors. India with several such rice growing ecosystems, is a rich source of rice diversity. Landraces are niche-specific and they also have several specialty uses apart from being tolerant to biotic and abiotic stresses. Since the landraces have evolved and adapted to diverse ecosystems, they could be the potential sources of valuable genes/alleles to fight against various stresses. Therefore, mining the resistant genes in landraces might be important foundational work in the breeding program. In the present study three well characterized genes, viz.,Pi1, Pi2 (Piz-5) and Pi5 that are known to confer broad spectrum resistance to blast (Magnaporthe oryzae) were mined from landrace collections of Karnataka state, India, by using PCR (polymerase chain reaction) based molecular markers. The results indicated the presence of Pi-1 gene in 39 landraces, accounting for 45.24% of 84 landraces analysed. Pi-z5 and Pi-5 gene were present in 60 and 59 landraces accounting the distribution frequency of 71.43% and 70.24% respectively. Few landraces had of all the three genes mined in this study and showed blast resistance (0-3 grade) consistently over five years of testing in Uniform Blast Nursery at Rice Research Station,U.A.S.Dharwad. They also showed resistance in different hotspots tested in India and hence deserve the attention as the natural gene pyramids. They serve as important gene pool for future breeding programme for blast resistance.

BIOTIC STRESS

130

Poster Session VI

PVI-160. Pyramiding of major Pi genes in rice for durable resistance against diverse blast races of the North Eastern Hill Region of India

Rai M, War GF, Najiar MRJ, Kumari B, Tyagi W

School of Crop Improvement, College of Post Graduate Studies, Central Agricultural University, Umiam, Meghalaya- 793103. [email protected]

Marker Assisted Backcrossing (MAB) is an effective approach to introgress and pyramid blast resistance genes in rice to achieve durable resistance against rice blast that causes upto 46 percent yield loss in the North Eastern Hill Region (NEHR) of India. Six advanced backcross generations (BC3F1/BC2F1) obtained from crossing recipients Shahsarang, CAUR1 (lowland genotypes) and Bhalum 3 (upland rice genotype) with donors IRBL9W (carrying Pi9 gene) and DHMAS (carrying Pi-1, Pita and Pi54 genes) were screened for donor foreground by using linked markers for Pi54, Pi1, Pita and Pi9 polymorphic between parents. Out of the 428 BC3F1/BC2F1 progenies screened, 103 plants were found to carry at least one donor foreground allele. Seventeen backcross plants of Shahsarang and DHMAS were found to have two genes and three progeny plants carried three gene combination. On the basis of marker assisted foreground selection, disease score, and background reconstitution as revealed by SSR markers and DUS characters BC3F1/BC2F1 lines for all the cross combinations were selected for further pyramiding. Also, twenty blast isolates collected from various locations of the NEHR were pathotyped against 17 monogenic differential lines to find out their virulence status with respect to individual resistance genes. The backcross progenies identified in the current study along with the pathotyping data obtained using lowland blast isolates will be helpful in designing future breeding strategies against blast disease relevant to rice production in lowland ecosystem of NEHR.

PVI-161. Phytohormones and atmospheric variation upregulates the expression of rice blast resistance gene Pi54

Rai AK1, Gupta SK1, Chand D2, Sharma TR1* 1 National Research Centre on Plant Biotechnology, IARI, Pusa, New Delhi, 110012, India. 2 Department of Biotechnology, Himachal Pradesh University, Shimla, 171005, India, [email protected])

Outcome of the encounter between plant and a pathogen depends on the balance between the ability of a pathogen to suppress the plant’s immune system and the ability of the plant to detect the pathogen and subsequently activate its defense system. Phytohormones like salicylic acid (SA) and Jasmonic acid (JA) are the primary regulator of the defense network that converts the pathogen induced early signaling events into specific activation of effective defense responses. Understanding the expression pattern of R genes under different types of stress conditions and hormone treatments is a basic requirement to explain the behavior of resistance genes in natural field conditions. The expression pattern of Pi54 gene had been reported to be different from most of the other plant disease resistance genes. In this study, the expression of Pi54 gene was found to be induced by exogenous application of Salicylic acid (4.98 fold) and Jasmonic acid (2.67 fold). Abiotic stress conditions like increased temperature and humidity were also found to upregulate the expression of Pi54 gene by 10 fold and 5 fold, respectively. The Pi54 gene has also been shown to activate a cascade of plant defense response genes in transgenic rice lines in response to Magnaporthe oryzae inoculation. Results obtained in this study suggest that Pi54 gene might play an important role in detection of the pathogen as well as activation of defense response pathways mediated by Salicylic acid and Jasmonic acid. Responsiveness to changes in environmental conditions seems to equip rice plants for successful management of blast infection in variable atmospheric conditions. Hence, while the Pi54 gene is itself induced by the blast pathogen, phytohormones, as well as by rise in temperature and humidity, its enhanced expression leads to activation of genes involved in callose deposition and disease resistance. These features make it quite unique among NBS-LRR class of resistance genes of rice.

BIOTIC STRESS

131

Poster Session VI

PVI-162. Skp1 of E3 Ubiquitin Ligase is necessary for development of appressoria in the rice blast fungus Magnaporthe oryzae

Prakash C and Chattoo BB

Centre for Genome Research, Department of Microbiology and Biotechnology Centre, Maharaja Sayajirao University, Vadodara 390 002, Gujarat, India. Contact: [email protected]

The rice blast fungus Magnaporthe oryzae is a serious threat to rice plant worldwide. Infection occurs when the spore lands on rice leaf surface and germinates to give rise to a specialized structure, the appressorium. The morphogenesis of the appressorium is controlled by cell cycle progression. Skp1(S-phase associated kinase protein 1) is a core component within the SCF E3 ubiquitin ligase complex necessary for protein degradation by 26S proteaosomal pathway. Timely degradation of proteins is critical to cell cycle progression. Magnaporthe oryzae has single MoSkp1 (MGG_04978). Various RNAi and Antisense transformants of MoSkp1 showed reduced sporulation, defective spore morphology, less septation and diffused nucleus, and inability to form appressoria on the inductive surface. Further they showed the formation of elongated germ tubes which do not penetrate host tissue. Analysis of knock down mutants showed defective cell cycle during initial spore germination. MoSkp1 is mobilized from spore to germ tube and abundantly expressed in appressoria, indicating its involvement in the early stages of pathogenic development. MoSkp1 complemented Skp1 function in the fission yeast mutant Skp1 A7 by restoring normal length of yeast cells at a restrictive temperature. Yeast two hybrid assay showed MoSkp1 interacts with putative F-box protein (MGG_06351), revealing its protein complex forming ability. Current investigation suggests that reduction in MoSkp1 activity is associated with reduced total protein ubiquitination and defective cell cycle and appressorial development.

MOLECULAR BREEDING

132

Poster Session VII

PVII-163. Allele mining for drought responsive transcription factors SNAC1 and DREB1F genes in Indian wild rice

Singh BP1, Singh B1, Singh AK2, Rai V1, Singh NK1 1National Research Center on plant Biotechnology New Delhi-110012, 2Division of Genetics IARI, New Delhi -110012. [email protected]

Rice is one of the most important staple food crops in the world. With an increasing global population, the demand for rice will continue to rise, which raises challenges for the breeding of high-yielding rice cultivars. Wild relatives of crop plants hold a wealth of alleles that can help break yield barriers and enhance tolerance to environmental stresses. The cultivated varieties of rice are likely to have only a small proportion of this variability due to their narrow genetic base as compared to the wild rice counterparts due to breeding and domestication bottlenecks. Drought is a worldwide problem, seriously constraining global rice production. There is a need for new stress tolerant rice varieties that can prepare India’s agriculture for a drier future. In rice a large number of abiotic stress responsive genes have been reported. These genes induced during stress conditions function not only in the protection of cells from stress by production of important metabolic proteins, but also in the regulation of genes, e.g. transcription factors (TFs), involved in signal transduction during stress. SNAC1 and DREB1F are two potential transcription factor genes for the development of drought tolerant rice varieties. An attempt has therefore was made for allele mining of these two important genes in 89 wild rice and two cultivated rice varieties, N22 (drought tolerant) and IR64 (Succeptible). We are doing drought phenotyping of wild rice accessions in a rain out shelter facility. We have identified haplotypes of these transcription factor genes by re-sequencing approach. Amplicons of both the genes were sequenced by targeted sequencing in ion torrent sequencer. Sequence analysis revealed SNAC1 gene has five high quality SNPs, four in exonic regions and one in intronic region; while intronless DREB1F gene has three SNPs. Positive association of this variation with drought tolerance will provide potential alleles for drought tolerance in rice.

PVII-164. A genetical genomics analysis of the metabolome exemplified using a rice population

Gong L1,4, Chen W1,4, Gao Y1,4, Liu X2, Zhang H3, Xu C1, Yu S1, Zhang Q1,2,5, Luo J1,2,5 1National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China, 2College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China, 3Key Laboratory of Horticultural Plant Biology (Ministry of Education), Huazhong Agricultural University, Wuhan 430070, China, 4Contributed equally. [email protected]

Plant metabolites are crucial for both plant life and human nutrition. Despite recent advance in metabolomics, genetic control of plant metabolome remains largely unknown. Here we performed a genetical genomics analysis of the rice metabolome which provided high resolution for over 2800 quantitative trait loci (QTLs) for 900 metabolites. Distinct and overlapped accumulation was observed and complex genetic regulation of metabolism was revealed in two different tissues. Data mining has associated 24 candidate genes with various mQTLs, including ones controlling and/or regulating important morphological traits and biological processes, and the corresponding pathways were reconstructed by updating in vivo functions of previously identified and newly assigned genes. This study demonstrated a powerful tool and provided vast amount of high-quality data for understanding plant metabolome which may help bridge the gap between the genome and phenome.

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Poster Session VII

PVII-165. Elite Swarna x O. nivara introgression lines for functional genomics of yield

Haritha G, Naga D N, Monika S, Swamy BPM, Kaladhar K, Sarla N


Oryza nivara the closest wild progenitor of O. sativa is a good source for introgressing yield related traits into popular rice varieties. A major yield QTL yldp2.3 was identified earlier in BC2F2 of Swarna x O. nivara IRGC81848. At least 10 BC2F8 Introgression Lines (ILs) were identified as elite lines for yield after field evaluation for atleast 3 years in DRR field. They differed from Swarna in several traits eg height, tiller number, days to flowering, number of secondary branches, and grain size and colour but showed higher yield than Swarna. One IL, IET 21542 (RPBio4918-248S) IC594146 showed 25% higher yield over irrigated medium duration check varieties Jaya in 2009, 2010 and NDR359 in 2011 in 3 years of multilocation testing giving a mean yield of 5.7, 5.0 and 5.9 t/ha respectively and was released as DRRDhan40 in Maharashtra, Tamilnadu and West Bengal. It has segments of 3 yield QTLs from O. nivara on chromosomes 2, 8 and 9. A total of 514 SSR markers were used to screen ILs 65S and 7K, 470 SSR markers in ILs 14S, 14-3S, 148S, 24K and 350 SSR markers in ILs 70S, 75S, 166-30S and 248S for polymorphism between ILs and recurrent parent Swarna. The introgressions from O. nivara ranged from 1.7% in 248S to 15.3% in 65S. These ILs RPBIO4918-7K, 24K, 14S, 14-3S, 65S, 70S, 75S, 148S, 166-30S and 248S were also screened for markers within yldp2.3 to identify QTL-NILs. Only 14-3S and 65S showed one yield sub QTL from O. nivara IRGC81848. ILs 7K and 24K showed O. nivara IRGC81832 alleles for yield QTL yldp2.1 and tiller number QTL nt2.3 respectively. Two F2 mapping populations 7K/Swarna and Swarna/65S have been grown to map/fine map QTLs for yield and related traits from O. nivara and identifying trait-linked genes and markers.

PVII-166. Genetic dissection of markers within QTL yld 2.1 from O. rufipogon and their linkage with yield traits in rice

Haritha G, Naga D N, Sudhakar T, Gowthami C, Krishnam RA, Subhasisa B, Tripura V VGN, Nisha R, Jyothi B, Sarla N


Wild species are an important source of yield enhancing QTLs. One major yield QTL yld 2.1 from O. rufipogon was identified earlier in BC2 test cross progeny at DRR. 90 F2 plants derived from introgression line (IL) 50-7 x KMR3 (set1) were screened for six SSR and 3 gene specific markers within yld 2.1. Single marker analysis based on F2, F3 and F4 phenotype and genotype data showed that 4 SSR markers RM 3874, RM3688, RM 1920 and RM 6318, were significantly linked to number of secondary branches, yield per plant, panicle weight, and two gene specific markers NSH3, NSH8 were linked to spikelet fertility and yield per plant respectively. Further, as part of a fine mapping project 1200 F2 plants derived from IL50-7 and KMR3 (set2) were grown and plant height, number of tillers/productive tillers and grain yield were recorded. Segregation of 5 polymorphic markers within sub QTL 3 (RM3688-RM3762) of yld2.1 were analyzed in 1076 F2 and 750 F3 families. QS15 was significantly linked to plant height, tiller number and yield/plant and KFM10 was significantly linked to height, tiller number and yield/plot. Thus, the five genes between QS15 and KFM10 within sub QTL3 are candidate genes for yield. Based on grain yield, 33 high yielding and 33 low yielding families in F3 which showed similar yield in F2 also were advanced to F4 generation for selective genotyping. Interestingly there was a preponderance of heterozygotes in high yielding extremes and homozygotes in low yielding extremes at KFM3 locus. ILs with tiller number, 1000grain weight and yield higher than parents were also identified in F4 for evaluating in multilocation trials. Thus selective genotyping of extreme phenotypes for yield traits will help to discover linked genes/markers, to develop near isogenic lines and to identify the functions of genes.

MOLECULAR BREEDING

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Poster Session VII

PVII-167. Genetic architecture of the natural variation in heading date among Asian rice cultivars

Kiyosumi H1, Yasunori N1,2, Nozomi O2, Kaworu E1, Kazuki M1, Taeko S1, Tsuyu A1,2, Shuichi F1, Toshio Y1, Masahiro Y1 1National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan, 2Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 205-0854, Japan. [email protected]

Heading date is one of crucial factors determining regional and seasonal adaptation in rice (Oryza sativa L.). Heading date has therefore been a major target of selection in breeding programs. We previously reported a QTL mapping study by using twelve F2 populations derived from crosses of the japonica rice cultivar ‘Koshihikari’, a common parental line, with diverse cultivars originating from various regions in Asia. These analyses successfully detected one to four QTLs with large effect in each F2 population. However, this study demonstrated that a limited number of loci cannot explain whole of the varietal differences in heading date. It is very likely that additional QTLs are also involved in the phenotypic variation in these populations. To detect these hidden QTLs, we developed advanced backcrossed populations derived from the same crosses with the F2 populations. QTL analyses consisting of over 12,000 backcrossed individuals revealed 178 QTLs, which dispersed widely across the rice genome. 52 of the detected QTLs corresponded well with the genomic positions of the rice flowering time genes, which were detected in other studies, such as Hd1, Hd3a, RFT1, Hd17, Hd6, Hd16, Ghd7, DTH8, Hd2 and Ehd1. The other 126 QTLs found on different genomic positions with the above flowering time genes, although genetic effects of these QTLs were relatively small. These results indicate that both of the major effect QTLs (flowering time genes) and the minor effect QTLs are contributing to the natural variation in heading date in rice. The large number of QTLs allows us to catalog allelic variations of rice flowering time genes and QTLs, and to understand genetic diversity of flowering time in Asian rice cultivars.

PVII-168. Quantitative trait loci (QTL) associated with resistance against Cuttack population of brown plant hopper in rice

Behera L, Mohanty SK, Panda RS, Mohapatra SL, Jena M, Sahu RK, Sahu SC, Mohapatra T

Central Rice Research Institute, Cuttack. [email protected]

Brown plant hopper (Nilaparvatalugens Stal.) (BPH) has emerged as the most destructive pests of rice throughout Asia. Chemical measures to control BPH, though available, are associated with serious environmental and health issues. Host plant resistance has been the only durable and environmentally feasible option for BPH control. Till date, 27 monogenically controlled resistance genes imparting resistance against BPH have been identified in cultivated and wild rice. Several QTLs have also been reported to be associated with resistance. The insect being highly adaptive and migratory along with short generation period, overcomes the resistance in a short time as evident. Therefore, there is a strong need to identify new resistant donors with novel resistance genes/QTLs for BPH management. Molecular marker-based genetic analysis was conducted with ‘Salkathi’, a highly BPH-resistant land race from Odisha. Three hundred F8 generation recombinant inbred lines developed from a cross between ‘TN1’ (susceptible) and ‘Salkathi’ (resistant) were phenotyped for their reaction against BPH. Percent mortality of RILs due to BPH infestation showed high heritability (83.63). RILs with SES score of 1, 3 and 5 when grouped under resistance category and those with scores 7 and 9 under susceptible type, 1:1 phenotype segregation ration was inferred that suggested involvement of a single major locus with a few modifiers in resistance. Of the 514 primers used, eighty three were found polymorphic between the parents and thus were used to genotype RILs. QTL analysis identified two loci for mortality percentage. One was a major locus flanked by markers RM 6329 and RM565 on chromosome 3 that explained 72.7% phenotypic variance. The other QTL located between the markers RM 551 and RM335 on chromosome 4 was a minor locus explaining 5.1% phenotype variance. The major QTL coincided with the region where Bph14 and bph11(t) have already been mapped from the introgressed lines derived from wild rice O. officinalis. As in the present study, the donor is a land race belonging to O. sativa ecotype indica, the major QTL identified might be a novel gene that can be deployed for BPH resistance in marker assisted breeding programmes.

MOLECULAR BREEDING

135

Poster Session VII

PVII-169. Evaluation of inter sub- specific rils of rice for high yield and better quality using principal component analysis

Sohgaura N, Mishra DK, Koutu GK, Singh SK

Department of Plant Breeding & Genetics, J.N.K.V.V., Jabalpur (M.P) 482004. [email protected]

Improved grain yield and grain quality are two important objectives of rice breeding programmes in developing countries. Thus the present aim of this study is dissecting yield and quality related inter-componental traits to obtain precise information and to rank genotypes based on combination of phenotypic traits through principal component analysis using 61 RILs population derived from a cross between two contrasting cultivars, JNPT 100 (Tropical japonica) and HMT (indica) planted in RCBD with three replications at Seed Breeding Farm of Plant Breeding & Genetics, JNKVV, Jabalpur (M.P) in kharif 2011. On the basis of PCA analysis, only 5 principal components exhibited more than 2 eigen value and showed about 74.17 % variability are selected. From the first five PCs it was cleared that the PC1 was highly related to quality attributing traits whereas PC3 and PC4 were highly resembled to yield attributing traits. The second PC shared few traits regarding yield and quality. Thus, a good breeding programme can be initiated by selecting the RILs from these PCs to improve yield and quality traits. Principal component scores suggested that out of 61 RILs under study, RIL 7-36, 7-38 and 7-16 are the best for both yield and quality and can be utilised directly for cultivation programme. However, RIL 7-47 followed by 7-16, 7-38, 7-35, 7-39, 7-22, 7-41, 7-31, 7-10 and 7-26 performed well for yield traits whereas RIL 7-18 followed by 7-36, 7-38, 7-19, 7-17, 7-37, 7-55, 7-13, 7-2 and 7-16 exhibited better performance for quality traits. All these RILs might be utilised for crossing programme to develop superior hybrids having high yield and quality traits.

PVII-170. Development of PCR-based markers that can distinguish WA-CMS lines and their cognate isogenic maintainer lines of hybrid rice and their use in seed purity assessments

Karnati P, Viraktamath BC, Hariprasad AS, Balachandran SM, Hajira SK, Jayaram, Madhav MS, Revathi P, BalachiranjeeviCh, Sundaram RM

Directorate of Rice Research (DRR), Hyderabad. [email protected]

Development of PCR-based markers that can distinguish WA-CMS lines and their Cognate Isogenic Maintainer Lines of hybrid rice and their use in seed purity assessments Maintenance of genetic purity of parental lines of three-line system in hybrid rice seed production is of utmost importance to realize the complete yield potential of hybrids. The admixture of CMS line with its maintainer line or other pollen shedders can result in higher level of contaminants during hybrid seed production. In the present study, comparative sequence analysis of WA-CMS mitochondria and its cognate Maintainer mitochondria was undertaken to identify polymorphic regions across the genome. Targeting the polymorphic regions, particularly in the genic regions, we have developed a set of PCR-based markers, which can distinguish all WA-CMS lines from their isogenic maintainer lines and other contaminants in dominant and co-dominant manner. In tests on mixed samples of plants containing both CMS and Maintainer lines, a PCR-based assay could correctly predict the genotypes of CMS and their maintainer lines and accurately identify contaminants, indicating that it can be used to detect the mixture of maintainer lines and other contaminants in the commercial seed stocks of the WA-CMS line. Through single seed or seedling based assay, we demonstrate the utility of the co-dominant markers discovered in this study for rapid and reliable detection of contaminants in commercial seed-lots of WA-CMS seed lots.

MOLECULAR BREEDING

136

Poster Session VII

PVII-171. Physiological characterization of wild germplasm of rice for drought tolerance

Kaur SG, Chaudhary R, Singh G, Bhatia D, Mangat GS, Kumari N, Singh K

Punjab Agricultural University, Ludhiana. [email protected]

Drought is one of the major environmental threat to rice production in rainfed rice growing areas. Nearly, 30 to 40% yield loss in rice productivity had been observed worldwide under severe drought conditions (www.irri.org). Keeping in view, the increasing demand of at least 35% more rice production by 2030, we urgently need to explore and utilize the genetic variability present in wild species of rice for enhancing/improving drought tolerance of cultivated rice. PAU, Ludhiana is maintaining an active collection of 16,000 accessions of wild species of rice, procured from International Rice Research Institute (IRRI), Philippines. Out of these, 350 accessions belonging to AA genome viz. O. nivara, O. glaberrima and O. rufipogon were evaluated for drought tolerance. These genotypes were planted in randomized complete block design with two replications constitutively for two years (2010 and 2011) under aerobic conditions in the field area of School of Agricultural Biotechnology, PAU, Ludhiana. Initial screening was done by measuring iron deficiency induced chlorosis (IDIC), SPAD value, and leaf area index at early vegetative, maximum tillering and panicle emergence stage. Based on the results of preliminary screening, 30 non-chlorotic and 10 chlorotic genotypes were selected for further physiological characterization under water deficit conditions at the booting stage. The physiological traits viz. leaf rolling, drought score, chlorophyll content, RLWC (relative leaf water content), proline content, rubisco activity, photosynthetic rate, Fv/Fm, stomatal conductance and transpiration rate were recorded for the selected genotypes . From the results obtained from physiological studies, six accessions of O. rufipogon, seventeen accessions of O. nivara and one of O. glaberrima were designated as drought tolerant genotypes. The process of transferring this useful genetic variability for drought tolerance from wild germplasm to elite rice cultivars have already been initiated. Detailed results pertaining to physiological characterization will be presented in the conference. Key words: wild rice germplasm, drought, abiotic stress.

PVII-172. Genotype specific alleles and DNA finger printing Singh SK, Koutu GK, Mishra DK

Department of Plant Breeding & Genetics, JNKVV, Jabalpur (M.P.) 482004. [email protected]

Molecular characterization of elite rice varieties is very important for varietal identification, germplasm management and DNA fingerprinting. Unique alleles provides new opportunity to search specific traits present in the material and these unique alleles may also serve as diagnostic tools for particular region of the genome of respective genotypes. The study was done with an aim to develop molecular tags for rice lines. Twenty-four microsatellite markers were studied across 25 types of rice to characterize and discriminate among different varieties. The number of alleles per locus ranged from 2 to 4 with an average of 3.33 alleles across 24 loci. A total of 56 rare alleles were detected at 20 loci, whereas 16 specific alleles were detected at 13 loci. Polymorphic information content (PIC) values ranged from 0.113 to 0.530 with an average of 0.234. For cluster analysis dendogram was generated by Neighbor-joining method implemented in PowerMarker version 3.25. The PIC values revealed that RM468 and RM 234 might be the best marker for identification and diversity estimation of rice varieties, followed by RM 42, RM438, RM276, RM223, and RM16 markers. In this study, eleven SSR markers (RM 201, RM 234, RM 468, RM 42, RM 223, RM 502, RM 236, RM 256, RM 529, RM 259 and RM 276) were identified that produced specific alleles both in the aromatic and non-aromatic rice varieties. Specific allele/alleles size may be used as a powerful tool in genotype identification and variety protection, seed-purity evaluation, germplasm characterization/diversity studies, gene and quantitative trait locus (QTL) analysis, pedigree analysis and marker assisted breeding. These markers can also be used in conjunction with pedigrees and agronomic data to help document ownership and protect intellectual property rights.

MOLECULAR BREEDING

137

Poster Session VII

PVII-173. Molecular mapping of alternate dwarfing gene(s) to the widely used green revolution gene ‘sd1’ in Rice

Jena SS, Patil S, Vemireddy LR, DurgaraniChV, Siddiq EA

Institute of Biotechnology, Acharya NG Ranga Agricultural University, Rajendranagar, Hyderabad-500030, India. [email protected]

Dwarf accessions non-allelic to sd-1 gene are identified from a set of 33 mutant dwarf accessions of rice employing a gene specific marker for sd1 gene and biochemical assay (Gibberellic acid (GA) response). Keeping the foregoing in view, the present study was initiated to find and map alternative dwarfing gene (asd1) from a F2 population derived from the cross of CN384, a mutant dwarf accessions and a tall landrace, INRC10192. The phenotypic data of 19 traits related to plant height and yield component are recorded in parents i.e., CN384 and INRC10192 and 189 F2 individuals. The parents are significantly different in all the traits measured, and are reflected in many transgressivesegregants ranging from 7.94% (plant height) to 100% (appearance of sixth internode) in F2 offspring. Most of the traits measured showed clear normal distribution except few traits. Though plant height is believed to be a quantitative trait, in the present investigation, its distribution is skewed towards tall parent type. Parental polymorphism study between the parents was done using 605 hypervariable SSR markers. Genotyping of F2 individuals was performed using 30 polymorphic SSR markers. Linkage analysis using 26 marker loci and 19 phenotypic traits data by MAPMAKER/QTL Cartographer revealed 13 QTLs. Out of the 13 QTLs identified, 7 QTLs were associated with the plant height related traits having phenotypic variance (R2 value) in the range of 8-58%. Two QTLs for plant height, three QTLs for culm height and one QTL for internode number were identified in the chromosome number 6. One QTL for internode length was also identified in the chromosome number 5.

PVII-174. Natural variation in Early flowering1 contributes to early flowering in japonica rice under natural long-day conditions

Yoo S-‐C, Kwon C-‐T, Cho S-‐H, Paek N-‐C

Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. [email protected]

Plant breeders have focused on improving plant architecture as one key means to increase crop yield. Here, we provide main-effect quantitative trait loci (QTL) and strong candidate genes for plant shape-related traits by whole genome re-sequencing (WGS) of two parental cultivars using next-generation sequencing (NGS) technology. To identify QTL influencing plant shape in rice, we analyzed six agronomic traits: plant height, tiller number, panicle diameter, panicle length, flag leaf length and flag leaf width. QTL analysis was performed with 178 F7 recombinant inbred lines derived from a cross of the japonica rice ‘SNU-SG1’ and the high-yield tongil-type rice ‘Milyang23’. Using 131 molecular markers, including 28 NGS-based insertion/deletion (NID) markers, we identified 28 QTLs for the six traits with a threshold LOD > 2.8. Fifty six candidate genes were identified for the 12 main-effect QTLs by WGS data analysis for coding sequences. By further amino acid sequence and meta-expression profile between japonica and indica analyses, we identified 15 strong candidate genes for 10 main-effect QTLs. Notably, application of WGS approach substantially enhanced the efficiency of polymorphic marker development for QTL mapping and candidate gene isolation. This yields useful genetic resources for breeding high-yielding rice cultivars with improved plant architecture.

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Poster Session VII

PVII-175. Identification of novel QTLs for salt tolerance in CSR11 / MI48 recombinant inbred lines by BSA approach

Tiwari S1, Krishnamurthy SL2, Sharma SK2, Batra V2, Kumar V2, Singh B2, Bhowmik PK3, Rai V1, Singh AK3, Singh NK1 1National Research Centre for Plant Biotechnology, Pusa Campus, New Delhi-India, 2Central Soil Salinity Research Institute, Karnal-India, 3Division of Genetics, IARI, New Delhi-India. [email protected]

Salt tolerance in rice is a quantitative trait controlled by multiple genes. Genetic mapping of the causal genes for the trait is a common method for learning about the molecular processes underlying the biological function. One method to rapidly identify markers or genomic regions linked to a trait phenotype is by bulk segregant analysis (BSA). BSA partitions a population from a single cross into two pools, or bulks, based on phenotypic performance, so that each bulk contains only a small proportion of the population with extreme phenotypes. The method is based on marker analysis of pooled genomic DNA samples from each bulk to identify association between marker and trait phenotype and thereby designate a probable location for the QTL. In the present study parental polymorphism was surveyed between a salt tolerant rice variety CSR11 and a salt sensitive variety MI48, the two parents of a RIL population of 206 lines. Out of 750 SSR markers surveyed 80 were polymorphic. Phenotyping for salt tolerance was done in controlled microplots under moderate (pH 9.5) and high sodicity (pH 9.9) conditions at CSSRI Karnal. Ten extreme tolerant and ten extreme sensitive RILs were identified on the basis of their stress susceptibility index for grain yield from three years phenotyping data. DNA was extracted from each of the extreme RILs and combined in equal quantities to prepare extreme tolerant and extreme sensitive bulks and analyzed along with the two parents using all the polymorphic markers. By applying BSA we have identified eight markers located at 5 regions on chromosomes 1, 2, 8, 9 and 10 showing tight linkage with the trait. Two of these locations match with QTLs previously mapped on chromosomes 1 and 8. Genetic map of the three novel QTLs on chromosomes 2, 9 and 10 has been prepared to identify their precise location and effect.

PVII-176. Assessment of SNP on their functions based on whole genome sequencing in rice

Kim T-‐H, Jeong I-‐S, Yoon U-‐H, Lee G-‐S, Ji H-‐S, Lee H-‐J

National Academy of Agricultural Science, Genomic Division, RDA. [email protected]

With the development of rapid sequencing technologies, whole genome sequencing is widely utilized for identification genes associated with agronomical important traits, molecular-assisted breeding and quantitative trait locus (QTL) in crop species. Whole genome resequencing of ten Korea rice accessions were generated 2,447 million raw reads which provided over 58 fold-coverage of the Nipponbare genome. We mapped a large number of short reads from ten accessions onto Nipponbare sequence as reference genome. We detected 3,144,016 SNPs, which estimated to be one per 2.2Kb on average. Among 553,527 SNPs in cording region (17.6% of total SNPs), 287,386 SNPs were non-synonymous SNP (1.69 SNPs/gene). By detected potential SNPs, we observed genetic variants between ten accessions in genes that have an influence on rice flowering time and bacterial blight resistance. We also offered analysis of rice gene families on the basis of ratios of non-synonymous to synonymous SNPs in each gene family. Gene families which would be related to essential functions tended to have lower ratio than whole genome average, whereas gene families that had functions in regulatory processes and signal recognition had higher ratios. Unmapped region against Nipponbare genome occupied about 1 ~ 2% in each accession, over 50% were repeat regions. The length of the gap was from 1bp in all accessions to 45,967bp in Ilpum, on average 182bp. We also identified genes absent in some accessions. By unmapped regions against the reference genome, we observed 3,497 possible genes loss events. The frequency of loss genes in each chromosome ranged from 33 genes on chromosome 5 to 913 genes on chromosome 11. The genetic difference obtained from our results should be valuable to identify genes associated with diverse traits of agronomical importance for molecular breeding.

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Poster Session VII

PVII-177. Identification of high water use efficient rice genotypes using physiological, anatomical and molecular analysis

Roja V, Patil S, Deborah DAK, Kiranmayi SL, Siddiq EA, srividhya A, Vemireddy LR


Experimental findings suggest that without much yield penalty, rice can be successfully grown with still reduced water requirement. Water use efficiency (WUE) reported to be varied substantially among rice genotypes. To this end, we made an attempt to identify high WUE rice genotypes employing both physiological and molecular marker analysis. In all, 48 rice genotypes comprising drought tolerant lines, NERICA varieties, landraces, and modern cultivars were screened for high water use efficiency based on various physiological traits like relative water content (RWC), specific leaf area (SLA), anatomical features like no of stomata, size of each stomata and distance between stomata. In addition, molecular markers associated with the above traits were identified using association mapping by TASSEL 2.1. The rice genotypes considered high WUE based on physiological (high RWC and low SLA), anatomical (less number of stomata, large stomata and more distance between stomata) and molecular marker analysis (low genotypic diversity) Using this criteria we have identified the rice genotypes viz., INRC10192, Solumpiket, Azucena, Vandana, Bala and N22 as donors for higher water use efficiency. Also, based on the same criteria we found that the modern cultivars like IR64, BPT5204, Swarna, Tellahamsa showing low water use efficiency. Based on molecular marker analysis also we found that these high and low WUE genotypes clustered together indicating the suitability of these donors and recipients as parents in marker-assisted improvement of the WUE trait. Further, employing association mapping approach with reported molecular markers linked to WUE traits revealed association of RM555 with stomata on upper surface, RM9 with RWC and the loci RM219, RM185, RM21 with stomata on lower surface with phenotypic variance range of 11.4 and 16.4%. Employing physiological, anatomical and molecular marker analysis, in the present investigation, we have identified rice genotypes INRC10192, Solumpiket, Azucena, Vandana, Bala and N22 as higher WUE varieties and have potential to be used as donors for development of high WUE varieties through MAB.

PVII-178. Development of rice core germplasm set for precision breeding Mikkilineni V, Roy N, Udhan K, Alwaris A, Jha S, Reddy M, Patil P, Varakuti M, Govindaraj K, Char B

Maharashtra Hybrid Seeds Company Limited, Dawalwadi, Jalna, MS 431203, India. [email protected]

Rice is a key cereal crop around the world and hence understanding and utilizing the genetic variation in Rice germplasm is essential for improving the crop. Studying the extent of variability among genotypes would enable us to choose diverse parental lines for hybridization in order to develop new lines and for improving the chances of heterosis in a hybrid breeding program. In other words, a good understanding of genetic variability among the germplasm lines will enable precision breeding. Additionally, understanding genetic variability would help in identification of desirable alleles and aid in identifying the marker-trait associations. Here, we present the results of genetic variability study of 1248 in-house Rice germplasm lines where both phenotypic traits and molecular markers were used to analyze the germplasm. Thirteen phenotypic traits were selected for the study. Microsatellite and SNP markers distributed across the genome were used in molecular analysis. We have found the overall diversity of the germplasm to be in the range of 15% and 96%. Phylogenetic tree has been constructed that has shown that the germplasm lines were grouped into major and minor classes. From the genetic diversity data, we can conclude that sufficient genetic variation exists between Rice genotypes. Based on genetic diversity estimates and also based on agronomic and phenotypic traits, a core germplasm set will be identified and the efficiency of the core set will be further validated in experimental crosses.

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Poster Session VII

PVII-179. An eQTL guided co-expression analysis for constructing regulatory network using a rice RIL population

Wang J, Yu H, Weng X, Xie W, Xu C, Li X, Xiao J, Zhang Q

National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research. [email protected]

The ability to reveal the regulatory architecture of the genes at the whole genome level by constructing the regulatory network is critical for understanding the biological processes and developmental programs of the organism. Here we conducted an eQTL guided function-related co-expression analysis for identifying the putative regulators and constructing gene regulatory network. We performed an eQTL analysis of 210 RILs derived from a cross between two indica rice lines, Zhenshan 97 and Minghui 63, the parents of an elite hybrid, using data obtained by hybridizing RNA samples of flag leaves at heading stage with Affymetrix whole genome arrays. Making use of an ultra-high density SNP bin map constructed by population sequencing, 13,647 eQTLs for 10,725 e-traits were detected, including 5079 cis-eQTLs (37.2%) and 8568 trans-eQTLs (62.8%). The analysis revealed 138 trans-eQTLs hotspots, each of which apparently regulates the expression variations of many genes. Co-expression analysis of functionally related genes within the framework of regulator-target relationships outlined by the eQTLs led to the identification of putative regulators in the system. The usefulness of the strategy was demonstrated with the genes known to be involved in flowering. We also applied this strategy to the analysis of QTLs for yield traits, which also suggested the likely candidate genes. The eQTL-guided co-expression analysis may provide a promising solution for outlining a framework for the complex regulatory network of an organism.

PVII-180. Markers assisted recurrent selection for hybrid rice parental line improvement

Revathi P, Singh AK, Suryendra J, Senguttuvel P, Kemparaju K B, Sundaram R M, Hari Prasad A S, Viraktamath BC

Directorate of Rice Research-ICAR, Rajendranagar, Hyderabad, Andhra Pradesh, 500030, [email protected]

Population improvement programme involving male sterility have made little use of molecular markers, even though they could be very helpful in targeted selection of the desirable recombinants. In the present study a DRCP-102 restorer population, sterile plants were crossed with donors of biotic (BLB, Blast and BPH) stress resistance and abiotic stress tolerance (salinity and drought). The F1s plants were validated and confirmed for the presence of resistant genes with the help of molecular markers. The harvested F2 seeds from each crosses of equal seed quantity were bulked and raised in isolation for random mating. The seeds only from the sterile plants (out crossed) were harvested and bulked to constitute a recurrent selection cycle. In the same way recurrent selections were continued up to 4th cycles. In the fourth cycle around five hundred plants were screened for the presence of BLB, blast, BPH, salinity, drought and fertility restorer Rf genes by MAS. The seeds harvested from the sterile plants (out crossed) with multiple biotic and abiotic stress resistance genes were bulked and advanced for further recurrent selection for various traits improvement. Harvested male fertile plants identified to carry biotic and abiotic stress resistance/tolerance genes are being handled by pedigree method of breeding for restorer line development with multiple biotic stress resistance.

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Poster Session VII

PVII-181. Development and use of foxtail millet genetic and genomic resources for comparative mapping in cereals and bioenergy grasses

Prasad M, Muthamilarasan M, Kumari K, Pandey G, Gupta S, Venkata SB, Parida SK, Chattopadhyay D


Foxtail millet (Setaria italica L.) is a tractable experimental model crop for studying functional genomics of millets, cereals and bioenergy grasses. But the limited availability of genetic and genomic resources is significantly impeding its improvement and hence, the whole genome NGS data of foxtail millet was analyzed to develop 21,294 genomic simple sequence repeat (SSR) markers and physically mapped onto foxtail millet genome. The higher percentage (~89%) of cross-genera transferability across millet and non-millet species signified the importance of these genomic SSR markers in studying the related grass species. Comparative mapping of physically mapped SSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~17%), maize (~15%), rice (~7%) and Brachypodium (6%). Further, we developed 447 EST-derived simple sequence repeats (eSSRs) markers which showed a high-level of cross-genera amplification at an average of ~88% in millets and non-millet species. In silico comparative mapping showed the sytenic relationships between sorghum (~68%), maize (~61%), rice (~42%) and it suggested the nested chromosome fusion frequently observed in grass genomes. In addition, 5123 intron length polymorphic (ILP) markers were developed and it showed high-level of cross-genera amplification at an average of ~85% in millets and non-millet species. In silico comparative mapping of physically mapped ILP markers demonstrated sequence-based orthology and syntenic relationship between foxtail millet and sorghum (~50%), maize (~46%), rice (~21%) and Brachypodium (~21%). Further, we constructed the Foxtail millet Marker Database (FmMDb; http://www.nipgr.res.in/foxtail.html), a comprehensive online database for information retrieval, visualization and management of large-scale marker datasets with unrestricted public access. FmMDb is the first comprehensive database which provides complete marker information to the plant science community attempting to produce elite cultivars and would promisingly expedite the research on genetic diversity, phylogenetic relationships, construction of high-density genome maps, mapping of useful genes, comparative genome mapping and marker-assisted selection for crop improvement of millets, cereals and bioenergy grasses, thus addressing global food insecurity.

PVII-182. Marker assisted Introgression of Pi-z5 into elite indica rice variety, Sambha Mahsuri (BPT-5204) for blast resistance

Prashanthi SK, Yashavantha KJ, Patil SS, Shridevi AJ, Hanamaratti G, Singh UD, Rathour R, Variar M, Singh AK, Sharma TR

University of Agricultural Sciences, Dharwad Department of Biotechnology, [email protected]

Rice blast disease, caused by the fungus Magnaporthe oryzae is the most destructive biotic stress of rice in all rice growing regions of India. Use of resistant cultivars is the effective, economical and environmentally friendly way for control of blast disease. Samba Mahsuri (BPT-5204) is a medium slender grain indica rice variety that is very popular with farmers and consumers across India because of its high yield and excellent cooking quality. The susceptibility of Samba Mahsuri to blast is the major limiting factor for its popularity. Considering the seriousness of the disease and importance of the variety, major gene Piz-5 (Pi-2) was introgressed to improve BPT-5204 for blast resistance. The gene is known for its broad spectrum resistance to majority of blast isolates in India. Marker assisted backcross breeding (MABB) approach was employed to introgress Piz-5 from the donor line C101A51 into the genetic background of BPT-5204. Foreground selection for the target gene Piz-5 was effected using molecular marker, AP5930 in each generation. Backcrossing was accomplished up to BC3 generations and homozygous plants with target gene were identified in BC3F2 generation. Selected lines were advanced to BC3F4 generation. Polymorphic Markers for background selection were identified and homozygous lines were subjected limited background selection along with precise phenotypic selection to identify superior lines. Advanced Introgressed lines were evaluated in two different locations and top five lines coupled with blast resistant were selected considering grain type and their high yield over check (BPT-5204).

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Poster Session VII

PVII-183. EMS induced mutation in the SRS3 gene associated with the grain size variation in rice (Oryza sativa L.)

Umakanta N, Robin S, Sarla N, Seshashayee M, Singh AK, Singh K, Srinivasan R, Singh NK1, Sharma RP1, Mohapatra T

Central Rice Research Institute, Cuttack (Orissa) 753 006, India, 1National Research Centre on Plant Biotechnology, IARI, Pusa, New Delhi, 110012, India

Rice is one of the most important staple food crops of the world’s population. With rapid expansion of population and increase in demand for food grain, increasing rice productivity is utmost important. To achieve this, cloning and characterization of new gene/QTL for yield is foremost important steps in rice breeding. In this study, a short and bold grain mutant (M40) obtained by EMS treatment of the upland indica cultivar Nagina22 (N22) was used to locate the candidate gene responsible for the mutant phenotype. Exogenous application of auxin, cytokinin, gibberellin and brassinosteroid in this mutant revealed a lack of effect on grain size. The cells were shorter and narrower, and lesser in number in the mutant as compared to the wild type. Reduced rate of grain filling and less amount of dry matter accumulation in the endosperm during the peak grain filling stage of panicle development were observed in the mutant. The candidate genomic region was identified on short arm of chromosome no. 5 to an interval of 250 kbs using two F2 mapping populations (M40 X IR64 and M40 X Pusa1121) through Graphical Genotyping and linkage mapping. Composite interval mapping revealed this to be a major gene explaining 16.2% of grain length (GL) variation between M40 and IR64 with a very high likelihood (LOD 50.75). Among the 60 protein coding genes present in 250 kb genomic region, one (srs3) was already reported to control grain size in rice. It encodes kinesin 13 family proteins with a major role in mediation of microtubule organisation during mitosis. Sequencing of 11 kb srs3 gene region revealed a substitution of C to T in the coding region of the mutant (M40) leading to conversion of glutamine to a stop codon in the motor domain, translating only 459 codons and giving a truncated protein product as against the 1100 long amino acid in the wild type (N22). The post translational effect of the truncated protein in the mutant was further strengthened by similar expression pattern of gene in both the mutant and the wild type. The relationship between the mutation and the phenotype was validated by sequencing the segregants from M40 X IR64 population having contrasting parent type phenotypes. Sequencing of 96 germplasm lines including short and long grains in the kinesin motor domain region of the gene did not reveal the presence of this mutation, indicating creation of a new allele of srs3 gene by EMS mutagenesis.

PVII-184. Association mapping for grain size traits in rice Deborah DAK, Patil S, Roja V, Anuradha G, Siddiq EA, Vemireddy LR

Institute Of Biotechnology, Acharya N. G. Ranga Agricutural University, Hyderabad 500030, India. [email protected]

Introduction Grain size, an important quality trait of rice is affected by length, breadth and length-breadth ratio influenced by multiple genes. Association mapping is a powerful tool for genetically dissecting complex traits controlled by multiple quantitative trait loci (QTLs). In the present study, an attempt was made to identify markers associated with kernel size, kernel weight, grain size and grain weight by candidate gene based association mapping. Methodology In all, 96 rice genotypes were used for recording grain size traits viz., length(L), breadth(B), L/B ratio and grain weight for both kernel and polished rice and for screening with molecular markers associated with the grain size QTLs/genes from previous reports. Association mapping was done using GLM and MLM models of TASSEL 2.1. Results Of the markers used, the locus RM18616 explained maximum phenotypic variance (14.6%) followed by RM430 (10.42%). The locus RM18582 linked to the major QTL for grain size on chromosome 5 was found to be associated with all the grain size traits and grain weight both in kernel and polished form of rice with a phenotypic variance range of 2-10%. The markers linked to the known genes viz., GW5, qSS7, qGW8 and SRS5 also showed association with the majority of the grain size traits but with low phenotypic variance ranging from 2.0-8.44%. Discussion and Conclusion From the present study, RM18582 and RM18616 loci found to be strongly associated with grain size and have the potential to be used in marker assisted breeding of grain size traits.

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Poster Session VII

PVII-185. iMAGIC – A multiparent population derived from EMS induced isogenic mutants for dissecting high order interaction of genes governing complex traits

Gopala-‐Krishnan S, Singh N, Singh AK

Division of Genetics, Indian Agricultural Research Institute, Pusa campus, New Delhi - 110 012, India

Functional genomics aimed at understanding the function of specific genes, key steps in their regulation and their role in metabolic pathways, exploits several approaches including mutagenesis, genetic transformation, and transcriptome analysis. Among these, an effective strategy to identify gene function is the comparative analyses of the mutant and wild type. In rice, there exists a large number of mutant resources among which, EMS induced point mutations has been widely used to generate mutant library in rice. EMS induced mutagenesis in combination with next generation sequencing popularly known as ‘Mutmap’ approach has helped in characterizing mutation for specific trait. Complex traits such as grain yield is determined by different component traits such as biomass, productive tillers/ plants, filled grains/panicle, grain size, grain weight, etc. Over 30 genes governing these component traits have been cloned and characterized. However, there are large gaps in understanding the basic processes governing yield traits and there is a need for a systematic approach to understand the regulatory networks controlling a complex trait such as yield. Isogenic lines have been used to study such interactions between component traits, but it still suffers from background effects due to minor genomic variations retained in them. EMS induced mutants by virtue of its common genetic background except for the point mutation completely rules out any such background noise. A multiparent advanced generation intercross population can be produced by crossing individual mutants functionally characterized for each of the component traits governing complex traits such as yield in a definite pattern, which will produce large number of different combinations of point mutations in a single genetic background. Functional genomics of such a population will help in dissecting the expression of specific genes and higher order interaction between them in complex biological processes in a systematic manner.

PVII-186. Studies on cold tolerance in rice (Oryza sativa L.) using gene specific markers

Chetukuri A, Srinivas D, Sivasankar A, Sujatha M, Jamaloddin Md, Swathi G,

Institute of Biotechnology, Acharya N. G. Ranga Agricutural University, Hyderabad 500030, India. [email protected]

Rice (Oryza sativa L.) is the most important staple food crop for more than 60% of the global population and forms the cheapest source of food and energy. Low temperature is a major abiotic stress for growing rice. Low temperature stress is an important factor affecting growth and development of rabi grown rice particularly in Telangana region of A.P where night temperature in winter falls below 15 °C. This coincides with important stage of tillering and panicle emergence, which has a direct effect on yield. Damages of cold include injury to seedling, delayed heading etc. leading to yield reduction. In the present study, 76 rice genotypes were evaluated for cold tolerance by employing morphological traits and SSR markers. A molecular basis of selection in segregating breeding material using molecular markers for cold tolerance would be accurate and dependable. This tool would be of great value to breeders engaged in breeding for cold tolerance. Analysis for cold tolerance through molecular markers was carried out on 76 rice genotypes employing SSR markers. The number of alleles ranged from 2 to 8 with a mean of 4.08 indicating greater magnitude of diversity among the genotypes. The PIC value ranged from 0.688 to 0.943 with an average of 0.855, conformed the markers used were highly informative. Association studies were done with cold tolerant QTL linked markers. Cluster analysis using SSR markers revealed a dendrogram with range of 0.51 to 0.96 similarity indicating considerable amount of diversity among the genotypes. Based on the present study, cold tolerant (Tellahamsa, IR-83222-174, Emergency and Pusa superfast) and susceptible genotypes were identified. The findings of this study can be utilised in Breeding programmes and marker assisted Breeding for development of cold tolerant varieties for the benefit of the farmers.

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Poster Session VII

PVII-187. Mapping QTLs for iron and zinc concentration in unpolished rice of Jalmagna × Swarna RILs

Tripura Venkata VGN, Kiranmayi SL, Manorama K, Radhika K, Cheralu C, Rao KV, Viraktamath BC, Sarla N

Acharya NG Ranga Agricultural University, Hyderabad; Directorate of Rice Research, Hyderabad; ANGRAU, Regional Agricultural Research Station (RARS), Warangal. [email protected]

Identifying QTLs for iron and zinc in rice grains can help in gene discovery and biofortification programs. 126 F7 Recombinant inbred lines (RILs) derived from Jalmagna (J) x Swarna (S) were used to map QTLs for iron and zinc concentration in unpolished rice. These lines were grown at DRR and RARS, iron and zinc concentrations in unpolished rice were estimated using Atomic Absorption Spectroscopy. At DRR and RARS, the iron concentration was 40 and 39ppm in Jalmagna, and 32 and 28ppm in Swarna and zinc concentration was 24 and 22ppm in Jalmagna and 15 and 14ppm in Swarna respectively. In RILs, iron concentration ranged from 4 to 187ppm at DRR and 6 to 270ppm at RARS and zinc concentration from 14 to 28ppm at DRR and 14 to 31ppm at RARS. Iron concentration showed significant positive correlation with zinc concentration at RARS. Zinc concentration was also positively correlated with seed/grain width at both locations. Segregation analysis of 82 SSR and 2 gene specific markers which were polymorphic between J and S showed that 40% loci were homozygous for J, 32% for S, 12% heterozygous and 16% null. MapDisto and QTL Cartographer were used for linkage and QTL analysis. In all, 11 QTLs for iron and 8 QTLs for zinc were identified with LOD 2.4 to 70 on 9 linkage groups using CIM and MIM. R2 varied from 2 to 13% using CIM. In DRR, 5 iron and 4 zinc QTLs and in RARS 6 iron and 4 zinc QTLs were mapped. QTLs for iron showed conspicuously high additive and dominant effects. qFe7.3 at RARS was colocated with qZn7.2 at DRR. Ten RILs were identified with iron ranging from 58 to 270ppm and zinc from 27 to 31ppm for use in gene discovery and biofortification programs.

PVII-188. Transfer of a major QTL for high grain number qGN4 -1, into twelve different Indian rice mega varieties through marker–assisted backcrossing

Singh VK1, Sharma A1, Singh AK2, Nagarajan M3, Singh BD4, Singh NK1 1Rice Genome Laboratory, National Research Center on Plant Biotechnology, New Delhi-110012 India. 2Division of Genetics, Indian Agriculture Research Institute, New Delhi -110012, India. 3 Indian Agriculture Research Institute, Regional Centre, Aduthurai-612101 India.4School of Biotechnology, BHU, Varanasi-221005, [email protected]

Rice is the world’s most important staple food crop feeding more than half of the human population. The demand for rice is expected to increase further with the increase in the global human population, but the annual gain in yield potential through breeding has slowed down. Large number of well-filled grains per panicle is an important yield component trait in rice. Identification of a major QTL for grain number on chromosome 4 (qGN4-1) has provided the opportunity to apply marker assisted backcrossing (MAB) to develop rice cultivars with high grain number. Successful examples of QTL transfer to rice are introgression of saltol and sub1 loci in rice for enhanced salt and flooding tolerance, respectively. We are transferring the qGN4-1 in twelve rice mega varieties namely CSR 30, CR-1009, HUR-105, IR-64, MTU-1010, Pusa-44, Pusa Basmati-1, Pusa Basmati-1121, Ranjit, Sambha Mahsuri, Sarju-52 and Swarna . Donors for the transfer of qGN4-1 QTL are: HG-28, HG-67 (RILs from pusa 1266/Pusa Basmati 1 cross). The flanking SSR markers to qGN4-1 namely, nkssr 04-19, RM3276, Hvssr 04-49 and RM2441were evaluated for polymorphism among the selected parents. Each of the crosses were confirmed by the presence of donor alleles. A total of 300 BC1F1 seeds were generated for each cross and presence of QTL qGN4-1 was confirmed by analysis of nkssr 04-11, nkssr 04-19 and RM-3276 markers. The generation was advanced to BC2F1 for more background recovery of the recurrent parent and 300 BC2F1 seeds were produced for each cross and planted for further validation and generation advancement. The study will lead to development of a series of near isogenic lines with and without the grain number QTL qGN4-1 in twelve different genetic backgrounds mega rice varieties for studying the stability of this QTL.

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Poster Session VII

PVII-189. Improvement of popular rice variety ADT 43 for blast resistance through marker assisted backcross breeding

Divya B, Robin S, Rabindran R and John JA.

Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, 641003. * Directorate of Rice Research-ICAR, Rajendranagar, Hyderabad, Andhra Pradesh, 500030, India. [email protected]

Marker assisted backcrossing breeding has become one of the essential tool in transferring novel genes for improvement of adapted varieties. This study was conducted to improve blast resistance in the popular rice variety ADT 43 due to its high yield, acceptable grain quality and wide adaptation in irrigated areas. But this variety is highly susceptible to blast disease with potential disease incidence of 86.74 %. Three gene pyramided (Pi1, Pi2 and Pi33) CO 39 NIL viz., CT13432-3R was selected as the donor and ADT 43 as recurrent parent in this breeding programme. Marker assisted backcrossing was carried out to introgress the three blast resistance genes at each generation of the cross ADT43/CT13432-3R. Each advanced back cross generation were monitored for blast resistance both phenotypically under blast nursery and genotypically using reported linked SSR markers. The selected three gene pyramided lines in each generation were further back crossed to recurrent parent ADT 43 upto BC4F1/BC3F2 generation. Back ground analysis was done using 125 genome wide SSR markers to assess the recovery of the recurrent parent genome in the back cross progenies. In the BC3F1 generation three gene pyramided lines viz., AC-B3-11-7, AC-B3-11-36 AC-B3-11-57, AC-B3-11-83 were identified to be very promising. They showed a genome recovery upto 94.90 percent and were advanced to further generations. The morphological, yield and grain quality traits were assessed and they were significantly similar to ADT43. The introgressed lines with three gene combinations were highly resistant to the blast pathogen than genotypes with single genes or the susceptible check varieties under natural blast nursery screening at two epiphytotic locations of Coimbatore and Gudalur. The selected gene pyramided back cross lines in the desirable back ground were advanced to obtain an improved ADT 43 with blast resistance. The Senior Research Fellowship provided by the ICAR to *D.B, Funds from NPP & DRR is acknowledged.

PVII-190. Allele mining of high affinity potassium transporter genes in Indian wild rice germplasm (O. nivara and O. rufipogon) accessions

Mishra S1, Singh B1, Panda K1, Singh N1, Rai V1, Singh AK2, Singh NK 1 1NRC on Plant Biotechnology IARI New Delhi 110012, India.2Division of genetics, IARI, New Delhi 110012, [email protected]

Soil salinity is a major constraint to rice production in inland and coastal saline agri-ecosystems. Wild rice growing naturally in the hotspots bear continuous environmental pressures both biotic and abiotic stresses. Therefore, they are likely to carry genes responsible to combat these stresses. Mining of superior alleles of known genes can be helpful in the crop improvement programme for breeding superior rice varieties. Here rice accessions collected from wild habitat were screened under controlled conditions in hydroponics for their salinity tolerance using 150 mM sodium chloride. Tolerance of the accessions were measured against six cultivated varieties used as controls viz. FL478, CSR11, CSR27 (salt tolerant), MI48 (moderately salt tolerant), Pusa 44 (salt susceptible) and VSR 156 (highly salt susceptible) and categorized according to standard evaluation system after 15 days of salt treatment. Out of 128 accessions of wild rice screened four accessions viz. Oryza rufipogon 336719, O. nivara 336724, O. nivara 330644 obtained from National Bureau Of Plant Genetic Resources, New Delhi and NKSWR048 collected from Nakahara village of Ballia district in U.P., India were found to be salt tolerant, 18 were moderately salt tolerant, while 19 accessions were salt susceptible and rest 87 accessions were highly salt susceptible. None of the accessions could be classified as highly salt tolerant. To find association between sequence variations in the salt stress responsive genes, high affinity potassium transporters (HKT 2;1 and HKT 2;4) were amplified, sequenced and a minimum-evolution tree of sequenced region was constructed. Significant variation was observed in the sequence of HKT 2;1 and HKT 2;4 among the wild rice accessions some of which were associated with their salt tolerance level. Once validated, these alleles can be introgressed into cultivated rice varieties for improving their salt tolerance.

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Poster Session VII

PVII-191. Allele minning of SKC1 and SOS1 genes for salinity stress tolerance in landraces of rice

Singh D1, Singh AK2, Singh NK1 1 National Research Centre on Plant Biotechnology, IARI, Pusa, New Delhi, 110012, India.2Division of Genetic, Indian Agriculture Research Institute, New Delhi -110012, [email protected]

Rice (Oryza sativa L.) is the principal staple food and over fifty percent of the world’s population depends on rice for their food requirements. It is grown under diverse eco-geographical conditions mainly in tropical and subtropical countries. Global climatic changes impose a grave threat to rice production by creating abiotic stress environment of which drought, flood and salinity are considered to be the most serious constraints. Modern high yielding rice varieties are susceptible to such stress environments while the traditional varieties and landraces are very resistant to different types of abiotic stresses as they harbors several stress tolerance genes. So, their proper utilization, conservation and disclosing their hidden genetic potential are of utmost importance. In this study, a total of 75 rice genotypes, including modern high yielding varieties and landraces were screened for salinity tolerance under controlled conditions in hydroponics taking VSR 156 as sensitive; IR74, moderately tolerant, Pokkali and FL 478 as tolerant control. In which Narendra User 1, Narendra User 2 and A-69-1 showed highest tolerance to salt stress, others were are either moderately tolerant or susceptible to salt stress. For molecular analysis we plan sequence two well characterized genes responsible for salt tolerance namely SKC1 and SOS1. SKC1 maintained K+ homeostasis in the salt-tolerant varieties under salt stress where as SOS1 is involved directly in the transport of sodium ions across the plasma membrane. It contributes to plasma membrane Na+/H+ exchange, where SOS2 and SOS3 regulate SOS1 transport activity. Primers to amplify SOS1 and SKC1 genes have been designed and amplification is in progress. The amplified product will be used for ion torrent based targeted resequencing and data will be used to get novel alleles of these genes and their possible association with the salt tolerance.

PVII-192. Pyramiding of bacterial blight and blast resistance genes in the Basmati rice variety Pusa Basmati 6 through marker assisted backcross breeding

Ellur RK, Pathania S, Krishnan GS, Bhowmick PK, Vinod KK, Singh UD, Mondal KK, Rathour R, Prabhu KV, Singh AK

Division of Genetics, Indian Agricultural Research Institute, New Delhi-110012. [email protected]

Pusa Basmati 6 is a semi-dwarf, superior quality Basmati rice variety with tolerance to lodging, excellent grain, cooking quality and very strong aroma. However,it is highly susceptible to bacterial blight caused by Xanthomonasoryzaepv. oryzae and blast disease caused by Magnaporthegrisea, which affects both yield as well as grain quality. Marker assisted simultaneous but step-wise backcross breeding (MASS-BB) was used to incorporate the bacterial blight resistance genes xa13 and Xa21 from a common donor Improved Pusa Basmati 1 and two blast resistance genes, Piz5 and Pi54 from Pusa 1602 and Pusa 1603 respectively, into PB6. The isogenic lines carrying individual blast resistance genes were inter-crossed and followed by pedigree selection to develop Pusa 1884 homozygous for both the genes. Gene linked markers were used for foreground selection followed by rigorous phenotypic and background selection to accelerate the recovery of both the recurrent parent phenome and genome. Marker aided selection in combination with phenotypic selection helped in the development of Pusa 1728 (xa13 + Xa21), Pusa 1726 (PB6+ Piz5) and Pusa 1727(PB6 + Pi54) with RPG recovery of 97.7%, 94.2 % and 93.5% estimated using 19, 54 and 59 STMS markers representing genome wide coverage, respectively. Pusa 1728 (PB6+xa13+Xa21) was found resistant against the virulent BB isolates collected from Basmati growing areas of the country. Similarly, Pusa 1884(PB6+Piz5+Pi54) exhibited resistance to blast disease under artificial inoculation with respective diagnostic isolates. Further, these lines were also found resistant when evaluated under natural epiphytotic conditions of UBN-Malan. The performance of improved lines was on par with PB6 for agronomic and cooking quality traits. These improved lines will drastically reduce the use of pesticides rendering the produce organic. Further, these lines are invaluable sources for disease resistance genes in Basmati rice improvement program as well as in functional genomics studies.

MOLECULAR BREEDING

147

Poster Session VII

PVII-193. Development of aerobic rice lines with intact Basmati grain characteristics by marker assisted selection

Kharb A1*, Promila2, Jindal K3, Kumar J4, Sandhu N5, Kumar J6, Mehla BS6, Jain S7 and Jain RK8 1,2,3,4,5,6,8Department of Biotechnology and Molecular Biology, CCS Haryana Agricultural University, Hisar 125004 6Rice Research Station, Kaul, CCS Haryana Agricultural University, Hisar 125004. 7Bioinformatics Section, CCS Haryana Agricultural University, Hisar 125004. India [email protected]

Water scarcity and drought have seriously threatened the sustainability of irrigated lowland rice ecosystem in several parts of the world including India. Development of aerobic rice varieties which combine the drought-resistant characteristics of upland varieties with the high-yielding traits of lowland varieties, could be an effective strategy for saving water. In the present investigation, crosses were made between Basmati (PUSA 1121 and Pusa Basmati 1460) and aerobic (MAS25 and MASARB25) rice varieties with the objective to select high yielding aerobic rice lines with intact Basmati rice traits. Hybrid status of F1 plants was confirmed by microsatellite marker analysis. Polymorphism among the parental rice varieties has been worked out for >100 microsatellite markers including the markers specific for BAD2A aroma locus and QTLs for aerobic root morphology. Promising F2/F3 plants of two crosses were selected based on field evaluation data on yield and yield components, pothouse data on root morphology and molecular allelic profile. In 2012, experiments were conducted to evaluate selected Pusa 1121 x MAS25 F4 and Pusa Basmati 1460 x MASARB25 F3 populations. In these filial populations, enormous variation was observed for plant height, panicle length, effective number of tillers per plant, root morphology (length, thickness and biomass), grain length/breadth ratio and yield per plant. Out of 62 plants selected from two crosses, 29 plants had Basmati specific allele (homozygous condition), 14 plants had aerobic specific allele (homozygous condition) and 19 plants had both Basmati and aerobic allele (heterozygous condition) at BAD2 aroma locus. As many as 39 promising F3 and F4 plants from the two crosses have been selected, which had higher grain yield, root length and biomass greater than the respective aerobic rice parent and Basmati specific allele at BAD2 locus (either in homozygous or heterozygous condition) for further progeny analysis. Most of these selected plants also possessed QTLs identified earlier for grain yield and root length under aerobic conditions on chromosome 1 and 8 respectively.

PVII-194. Identification of heat tolerance associated alleles in wild introgression lines and mutants of rice

Vishnu Prasanth V, Tripura Venkata VGN, Suchandranath Babu M, Rama Devi SJS, Venkateswara Rao Y, Mangrauthia SK, Voleti SR, Sarla N

Directorate of Rice Research, Hyderabad, India. [email protected]

Temperatures higher than the optimum affect pollen and spikelet fertility adversely leading to decreased yield. High yielding introgression lines derived from wild species and also mutants are a good resource for favorable alleles for heat tolerance. 48 lines including 4 parents, 4 checks 17 KMR3-O.rufipogon ILs (set1), 15 Swarna- O. nivara ILs (set2) and 4 EMS induced mutants of N22 (set3) were evaluated for several heat tolerance related traits in pots and field in irrigated conditions at DRR. These lines were also analyzed for presence of 52 SSR alleles reported to be linked to spikelet fertility under heat stress. 13 out of 52 loci showed clear polymorphism between heat tolerant checks Bala, IR64 and N22 (positive alleles) and heat susceptible check Azucena (negative alleles). Most of the lines in set 1 and set 2 showed positive alleles for heat tolerance at 8 and 4 loci respectively. 10 lines out of 17 in set 1 showed positive alleles for flanking markers of heat resistance QTL qhr3.1. In set 3, NH219 and NH686 showed positive alleles at 5 and 4 loci respectively. Nine lines in set 1, 3 in set2 and 2 in set3 identified as heat tolerant based on spikelet fertility under heat stress also had positive alleles for atleast 3 loci. Positive allele of RM527 was present in all the 3 sets and of RM229 in set 2 and 3 only. These lines are being used to identify new QTLs for heat tolerance and gene expression studies. The work was supported by ICAR-NICRA (National Initiative on Climatic Resilient Agriculture) project at DRR. The introgression lines and N22 mutants were developed as part of DBT Network projects on functional genomics at DRR.

MOLECULAR BREEDING

148

Poster Session VII

PVII-195. Heterosis prediction in japonica and indica derived hybrids in rice (Oryza sativa L.)

Soni SK, Sundaram MK, Yadav VK, Bhadana VP, Pratap N, Yadav RK, Hajira S, Balachiranjeevi CH, Prasanna LB, Mahadeva HKS 1Biotechnology Laboratory, Crop Improvement Section, DRR, Rajendranagar, Hyderabad- 500 030 (A.P.) India 2Deptt.of Genetics & Plant Breeding, C. S. Azad. Univ. of Agri. & Tech., Kanpur-208 002 (U.P.) India 3Deptt.of Genetics & Plant Breeding, N.D. Uni. of Agri. & Tech., Faizabad- 224 229(U.P.) India.110392 [email protected]

To meet the rice production requirement for 2050, adoption of hybrid rice breeding technology is one of the most feasible options. Hybrids derived from Indica × Japonica crosses are known to possess higher levels of heterosis. In the present study, a set of 30 hybrids derived from crosses between 10 tropical japonica lines and 3 indica were analyzed for their heterosis and molecular marker amplification pattern. The standard heterosis for grain yield of the hybrids varied from -80.73% (TJ-39050 × PB-1) to 14.95% (TJ-11010 × PB-1) over the standard check NDR-359. The maximum heterosis for grain yield per plant was recorded for the hybrid TJ-11010 × PB-1 (12.5%) and TJ–11010 × CSR-36 (9.52%) showed least heterosis over the standard check NDR-359. The parental lines of all the hybrids were then characterized at molecular level using a set of 40 hypervariable microsatellite markers, these markers amplified a total of 131 allelic fragments with an average of 3.28 alleles per locus indicating a high degree of polymorphism among the rice lines analyzed. PIC value ranged from 0.231 (HRM 17201) to 0.775 (HRM 25796) with an average PIC value of 0.542. The similarity coefficients among the genotypes ranged from 0.58 to 0.83. The Maximum and minimum similarity value of 83% was observed between the genotypes TJ-5769 and TJ-25966 and 53% in TJ-16081 and TJ-64897 respectively. An index called coefficient of marker polymorphism (CMP) was calculated based on extent of polymorphism among the parental lines for the hybrids and it was correlated with the standard heterosis for grain yield. The correlation between CMP and heterosis for 23 hybrids was positive for all the hybrid combinations with an average value of r = 0.234 indicating the possible utility of the SSR markers considered for the present study for prediction and validation of heterosis.

PVII-196. Precise identification of introgression and mapping of broad spectrum blast resistance gene(s) from wild species of Oryza

UmaKanth B1, Rama Devi SJS1, Prasad MS2, Singh K3, Viraktamath BC1, Madhav MS1 1Biotechnology lab, Crop Improvement section, Directorate of Rice Research,2Plant pathology Section, Directorate of Rice Research, 3School of Biotechnology, Punjab Agricultural University. [email protected]

Rice blast caused by Magnaportheoryzae is one of the most important disease that affect yield upto 25% in India. Except two genes, wild Oryza species were not explored efficiently in mining of novel genes for blast resistance. To exploit Oryza species, about 368 (BC3F5) introgression lines (ILs) of different wild species made in PR114 background were subjected to blast phenotyping at DRR and found 50 extreme resistant ILs. To know the existence of important blast genes gene profiling was carried out for 10 known major blast resistance genes using tightly linked markers. Among 50 ILs, 9 ILs have shown the absence of known genes which may indicate the presence of novel gene(s). IL-295 (derived from O. nivara) was selected for current study to map the novel genes since it showed broad spectrum resistance at multiple hotspot locations of India. To map the loci involved in resistance, polymorphic study of IL-295 have been carried out with its recurrent parent and donor line using 676 SSR markers covering whole genome, which led to the identification of possible introgression region(s). The introgressed region is present at two loci i.e., from 1.5 to 2.1Mb (0.6Mb) and 6.0 to 12.3Mb (6.3Mb) on chromosome 8 and the percentage of introgressed genome is around 2%. The region of introgressed genome might be unique as this region does not have any of the reported major genes. For mapping and simultaneous transfer of these loci, three different F2 mapping populations were developed by crossing IL-295 with BPT-5204, Swarna and CO-39 and identified the SSR markers linked to these loci. Additional mapping efforts certainly lead to development of high resolution molecular map of these loci and identification of broad spectrum blast resistance gene(s).

MOLECULAR BREEDING

149

Poster Session VII

PVII-197. Development of near isogenic lines for functional genomics of blast resistance in Basmati rice

Khanna A, Shikari AB, Gopalakrishnan S, Singh UD, Sharma TR, Rathour R, Variar M, Prashanthi SK, Nagarajan M, Singh AK

Division of Genetics, Indian Agricultural Research Institute, New Delhi. [email protected]

Isogenic lines and near-isogenic lines (NILs) are invaluable tools in functional genomics, since it rules out the effects of genomic background noise.Therefore, construction of panel of NILs with well characterized genes governing a specific trait can provide valuable molecular insights in understanding gene function. Basmati rice is premium quality export rice from India, whichis prone to various biotic stresses among whichthe blast disease caused by Magnaporthe grisea is one of the most deleterious pathogen affectingboth yield and grain quality. Pusa Basmati 1, a popular Basmati rice variety was used as recurrent parent for development of NILs for seven broad spectrum blast resistance genes namely Pi54, Pita, Pi1, Pi9, Pi5, Pib and Piz5 using non-Basmati rice donors DHMAS70Q 164-2a, IRBL9-W, IRBL5-M, IRBLb-B and IRBLz5-CA, respectively. Gene linked/ gene based markers were used for foreground selection and rigorousphenotype selection for agronomic and cooking quality parameterswas done to accelerate the recovery of recurrent parent phenome. Recombinant selection in the carrier chromosomes and background selection using markers across the rice genome was carried out for recovery of recurrent parent genome, which ranged from 95.0% to 97.3% in the NILs. Artificial screening with virulent isolates and screening in the uniform blast nursery at two hotspot locations confirmed the resistance in the NILs. Intercrosses of the NILs was effected to develop gene pyramided NILs with 2, 3 and 4 genes in different combinations in the background of Pusa Basmati 1. These monogenic and pyramided NILs will not only serve as an excellent resource for functional genomic analyses for blast resistance but also serve as valuable donors for blast resistance genesin Basmati rice improvement. Further, the deployment of pyramided lines as improved cultivars will help to achieve durable resistance to rice blast in Basmati growing regions of India.

PVII-198. Linkage mapping of QTLs and genetic variability for micronutrient content in PAU201 x Palman 579 F2 population in rice (Oryza sativa L.)

Kumar J1*, Brar B2, Jain S3 and Jain RK4

1, 2, 4 Department of Molecular Biology and Biotechnology, CCS Haryana Agricultural University, Hisar 125004, India. 3 Bioinformatics Section, College of Basic Sciences and Humanities, CCS Haryana Agricultural University, Hisar 125004, India* [email protected]

Biofortification for iron and zinc in rice can be a potent solution for health related problems caused by micronutrient malnutrition. We assessed the variability for iron and zinc content in dehusked rice grains of a collection of 220 rice genotypes and identified iron (HKR 95-130, HKR 95-157 and Palman 579) and zinc (Taraori Basmati, TNG67 and Pusa 1460) rich genotypes. Crosses were made between Palman 579 and PAU201 (high yielding) rice genotypes, F1 hybrids confirmed by microsatellite marker analysis and F2 population raised. PAU201 x Palman 579 F2 population displayed large variation for various physio-morphological traits including grain yield per plant (1.93 - 41.53 g, PAU201 – 44.97 g, Palman 579 – 24.54 g), iron content (18.6 - 475.4 µg/g, PAU201 – 56.43 g, Palman 579 – 382.36 µg/g) and zinc content (4.4 - 157.4 µg/g, PAU201 – 16.5 µg/g, Palman 579 - 21.6 µg/g). Transgressive segregation for grain iron and/or zinc contents was noticed in some F2 individuals with one of the F2 plants having exceptionally higher iron (475.4 µg/g) as well as zinc (157.4 µg/g) contents. Grain iron content showed significant positive correlation (r=0.523) with grain zinc content indicating the feasibility of improving iron and zinc levels simultaneously in rice grain. Seventy six polymorphic microsatellite markers were used to map the QTLs associated with mineral content in grains using PAU201 x Palman 579 F2 plant harvests displaying the entire range of variation in Fe and Zn contents. Composite interval mapping (CIM) analysis by Win QTL cartographer 2.5 revealed a total of eleven QTLs for mineral content in rice grains on chromosome 2, 3, 7, 10 and 12. Eight QTLs for Fe content were mapped on chromosome 2 (qFE1, qFE2 and qFE3 at map positions 54.4, 138.6, 159.2 cM respectively), chromosome 3 (qFE4 at map position 54.6cM), chromosome 7 (qFE5 at map position 31.3 cM), chromosome 10 (qFE6 and qFE7 at map positions 25.9, 108.0 cM respectively) and chromosome 12 (qFE8 at map position 60.3 cM). In addition, three QTLs (qZN1, qZN2 and qZN3) for Zn content were identified on chromosome 2 (qZN1) and 10 (qZN2 and qZN3) with the map positions of 65.5, 23.8 and 115.9 cM respectively.

MOLECULAR BREEDING

150

Poster Session VII

PVII-199. Improvement of locally adapted rice cultivars to abiotic stress tolerance through marker assisted backcross breeding

Singh ON, Jena PPP, Kumar SB, Katara JL, Marndi BC, Swain P, Sarkar RK, Singh DP, Reddy JN, Mohapatra T

Central Rice Research Institute, Cuttack (Orissa) 753 006, India

Rice is sensitive to a variety of abiotic stresses including drought, submergence and salinity. Recently, to manage drought stress, two large effective QTLs, DTY2.1 and DTY3.1 have been identified. Using these two QTLs, Swarna-Sub1, a popular rice variety in flood prone areas, is being improved through marker assisted backcrossing (MABC) in the present study. To transfer these QTLs into Swarna-Sub1, four SSR markers (RM521, RM324, RM416 and RM520) were used. Among these markers, RM 521 and RM 520 gave clear polymorphism for DTY 2.1 and DTY 3.1, respectively between susceptible (Swana-Sub1) and tolerant (IR 81896-B-B-195) parents. A major QTL called Submergence 1 (Sub1) has provided the opportunity to develop submergence tolerant version of Swarna rice cultivar. Using Swarna-Sub1 as donor, two popular rice varieties of Odisha namely, Pooja and Pratikshya are being improved for submergence tolerance through MABC. Among the four primers (IYT1, IYT3, Sub1A203 and Sub1BC2) tested, Sub1BC2 has given clear polymorphism between susceptible and tolerant genotypes for submergence tolerance. In case of salinity tolerance, an excellent source has been identified in a Pokkali landrace and the locus in this line has been mapped and designated as Saltol. Using FL478 carrying Saltol as donor, a popular rice variety, Gayatri is being improved for salinity tolerance through MABC in the present study. Among the four primers (RM493, RM8094, AP3206 and RM3412) used, RM8094 and RM3412 have given clear polymorphism between susceptible and tolerant genotypes. Hence in this programme, two rice varieties (Pooja and Pratikshya) are being improved for submergence tolerance, one (Gayatri) for salinity tolerance and one (Swarna-sub1) already having submergence tolerance is being improved for drought tolerance by MAS. The selected markers on the basis of polymorphism survey have been used to advance the breeding material through marker-assisted backcross breeding (MABC) approach. At present the breeding material for submergence and salinity tolerance has been advanced to BC2F1 generation. Similarly, Swaran-Sub1 has been advanced to BC3F3 by introgression of two yield QTLs under drought, DTY2.1/DTY3.1. These improved genotypes, tolerant to different abiotic stresses will be useful in the unfavorable lowland areas to increase the production and productivity in the target areas.

TRANSLATIONAL GENOMICS

151

Poster Session VIII

PVIII-200. Rapid and efficient Agrobacterium mediated transformation of indica rice variety Pusa Sugandh 2. A platform for gene functional validation

Raman KV1, Aggarwal D1, Rao SR1, Dhandapani G1, Solanke AU1, Sreevathsa R1, Kumar PA1, Abdin MZ2, Mohapatra T3, Pattanayak D1 1National Research Centre on Plant Biotechnology, New Delhi 110012, 2Jamia Hamdard, New Delhi 110012, 3Central Rice Research Institute, Cuttack, Odisha 753006. [email protected]

Rice, one of the major food crops, is grown worldwide to feed about half of the global population. The completion of rice genome sequencing project has generated incredible amount of information about genes and the genome architecture. It is imperative to study the function of unknown genes for the better understanding of the growth and development mechanism for crop improvement. Transgenics play a crucial role in such gene functional studies. Progress in the rice Agrobacterium mediated transformation system has enabled introduction of important agronomical traits and functional validation of rice genes. The existing protocols using mature embryo derived calli are lengthy with low transformation frequency due to the continues subculturing of primary calli for the secondary callus production to be used for the transformation. In the present study, we have developed a rapid and highly efficient transformation protocol using early scutellum derived calli of the rice variety Pusa Sugandh 2. We compared the competency of the 3- day, 4- day and 5- day old primary calli with 21 days old secondary calli by the Agrobacterium (strain EHA 105) mediated transformation of a modified pCAMBIA 1304 harbouring GFP- GUS fusion gene driven by maize ubiquitin promoter. It was observed that 5 day old primary calli showed high competency with the stable transformation frequency (40%) when compared to 21- day old secondary calli. Transformation process was monitored through the GUS and GFP expression at transient and stable level. Molecular analysis confirmed the stable integration of transgene. With the use of 5- day old primary calli, the transgenic plants were developed in a two month period unlike the transformation of secondary calli taking four months to develop the transgenics. This protocol will be useful for the development of large scale transgenics for locus- phenotype correlation studies and crop improvement.

PVIII-201. Genome-wide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance

Jain M*, Moharana KC, Shankar R, Kumari R, GargR

Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi - 110067, India. [email protected]

Next generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole-genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought-tolerant Nagina 22 and salinity-tolerant Pokkali). More than 356 million 90 bp paired-end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1,784,583 non-redundant SNPs and 154,275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401,683 and 662,509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One-fourth of the SNPs and InDels were detected in genic regions and about 3.5% of the total SNPs resulted in non-synonymous changes. Large effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high-throughput genotyping and molecular breeding for abiotic stress traits in rice.


152

Poster Session VIII

PVIII-202. Gene targeting of OsMADS1 in rice by using the positive/negative selection strategy

Kannan P1, Majhi BB1, Basu D1, Kartha RV2, Vijayraghavan U2, Veluthambi K1 1Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai-625021, India, 2Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India. [email protected]

Gene targeting (GT) via homologous recombination is an effective method to precisely modify a gene in its native site. In higher plants, the frequency of targeted integration of a transgene into the endogenous homologous sequence is very low in the order of 10-5 to 10-3 in comparison to random integrations. In our study, targeted disruption of the OsMADS1 gene was attempted by Agrobacterium-mediated transformation involving the ‘positive/negative selection’ strategy. Rice OsMADS1 is a flower-specific gene, which controls identity of floret meristem and its determinate development. Together with other floret organ identity genes OsMADS1 contributes to organ development. In the GT vector, the positive selectable marker hygromycinphosphotrasferase gene (hph) is flanked by a 4.28-kb 5’sequence of OsMADS1 and a 4-kb 3’sequence of OsMADS1. The diphtheria toxin-A subunit gene (DT-A) is placed on both ends of the T-DNA to eliminate the plants with ectopic T-DNA integrations. Rice transformation of 2,643 scutellum-derived calli with the GT vector yielded three independent hph resistant, transgenic plants. The T0 plants were analysed by Southern blotting. The plant DNA was digested with HindIII or SacI and the blot was probed with the hph gene. The expected 11.5-kb (upon HindIII digestion) and 6.5-kb (upon SacI digestion) fragments hybridized. This result confirmed that the hph gene was integrated as a single copy in the OsMADS1 locus and no ectopic T-DNA integration had occurred. Further analysis was done by using the 5’ part of the OsMADS1 and the 3’ part of the OsMADS1 as probes. The results confirmed that the T0 transgenic plants were hemizygous in the OsMADS1 locus. The T0 plants, upon selfing and forwarding to the T1 generation, displayed 3:1 segregation of hygromycin-resistant (HygR) and -sensitive (HygS) plants. The homozygous, knock-out T1 plants were identified by semi-quantitative Southern blotting using the 3’ part of the OsMADS1 gene as the probe. The GT frequency in our experiment was 0.1 %. The phenotypes of flowers in the gene targeted, homozygous plants will be presented.

PVIII-203. Identification of candidate genes underlying a QTL cluster governing drought tolerant traits employing RNA-Seq approach in rice

Patil S, Roja V, Deborah DAK, Jena SS, Srividhya A, Siddiq EA, Vemireddy LR


Identification of candidate genes influencing drought tolerance would help to develop drought tolerant rice varieties through marker-assisted breeding. In the present study, an attempt has been made to identify the candidate genes underlying a QTL cluster governing drought tolerant traits employing next-generation-based transcriptomic approach i.e., RNA-Seq. A QTL cluster on chromosome 8 between RM38 and RM331 governing drought related traits viz., Root Dry Weight and Root Shoot Ratio has been identified from a RIL population of INRC10192/IR64 cross. In order to identify the candidate genes underlying the QTL cluster, RNA-Seq using Ion torrent platform (ABI) has been sought from 20day old root tissues of the parental lines. RNA-Seq analysis of the roots shows 4 million and 3.1 million high quality reads, in IR64 and INRC10192 respectively and aligned against the Nipponbare reference genome. A total 9795 and 6901 genes are expressed exclusively in IR-64 and INRC-10192 respectively in addition 52 differentially expressed genes were found. Out of which 18 genes were found to be located within the QTL cluster region on chromosome 8 in the marker interval of RM38 and RM331. RNA-Seq, a next-generation based root transcriptome of parental lines INRC10192 and IR64 revealed 18 differentially expressed genes within a QTL cluster governing drought tolerant traits on chromosome 8. Functional validation of these genes will help to identify of the candidate gene(s). Introgression of the candidate gene(s) of the QTL region into drought susceptible varieties will aid to develop drought tolerant rice varieties.


153

Poster Session VIII

PVIII-204. Development of transgenic male sterile and restorer lines for production of hybrids in rice (Oryza sativa L.)

Rao SKG1, Kumar VS1, Khurana R2, Deveshwar P2, Tyagi AK3, Kapoor S2, Rao KV1 1Centre for Plant Molecular Biology, Osmania University, Hyderabad, 500007, 2Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, 110021, 3National Institute of Plant Genome Research, ArunaAsaf Ali Marg, New Delhi, 110 067. [email protected]

Male sterility, the inability of a plant to produce fertile pollen, is a useful tool for hybrid breeding and hybrid seed production. In order to introduce inducible male sterility in rice, the coding region of N-acetyl L-Ornithinase (argE) was fused to OSIPA promoter sequence known to function exclusively in the anther. Similarly, for engineering nuclear male sterility the coding region of Brassica napus cysteine protease (BnCysP1) was fused to PP12 promoter sequence which drives the gene expression predominantly in tapetal cells of the anther. For the fertility restoration, a 590 bp sequence of the BnCysP1 was used in both sense and antisense orientation separated by Gus intron. The recombinant binary vectors of pCAMBIA-hyg-OSIPA-argE, pCAMBIA-bar-PP12-BnCysP1 and pANDA-hyg-BnCysP1Si were constructed and mobilized into Agrobacterium strain EHA105. Samba Mahsuri (SM) rice transformants of argE, BnCysP1 and BnCysP1Si were produced using embryogeniccalli derived from mature embryo. Putatively transformed calli were selected on the MS medium containing hygromycin (50-70 mg/l)/ phosphinothricin (5-7 mg/l), and were regenerated on the MS medium with BAP and NAA. The putative transgenic plants were confirmed by PCR and Southern analyses. In T1 generation, the selfed-seed collected from argE transformants showed 3 hygromycin tolerant (T): 1 susceptible (S) ratio. In order to confirm the lethal effects of N-acetyl L-Ornithinase (N-ac-Pt), pollen from the argE transformants were tested for their sensitivity to N-ac-Pt. The pollen from different argE transformants exhibited 3:1 ratio for N-ac-Pt sensitivity and tolerance, respectively. Transgenic rice plants expressing BnCysP1 failed to produce functional pollen. To obtain seed, these plants were cross-pollinated with pollen from control plants. Seeds collected from these plants showed 1 (T):1 (S) ratio when germinated on the MS medium with 5 mg/l phosphinothricin confirming that the male sterility has been successfully engineered in rice.

PVIII-205. Nagina22 mutants for functional genomics of Phosphorus use efficiency in rice

Yugandhar P, Panigrahy M, Rao DN, Kumari BR, Krishnamurthy P, Voleti SR, Sarla N

Directorate of Rice Research-ICAR, Rajendranagar, Hyderabad, Andhra Pradesh, 500030, India. yugandhar12345@ gmail.com

Mutants are an excellent resource to study function and regulation of genes for complex and important traits such as Phosphorus use efficiency (PUE). 2500 EMS induced mutants in M3 to M6 generations were screened in normal (15 Olsen Value) and low P (1.8 Olsen Value) field conditions simultaneously at DRR. In N22 and mutants flowering was delayed by 10-15 days in low P compared to normal P. On the basis of survival and seed set in low P field, 54 Nagina 22 (N22) mutants were identified as low P tolerant and 7 as susceptible. In addition, several other screening methods such as hydroponics with and without sand, pot experiments with low P soil and addition of normal P and double P were used for further characterization of selected mutants for morphological, physiological and biochemical traits related to P uptake and transport. Twenty mutants were evaluated in low P field for height, P content in shoot, root, grain, fresh/dry weight of shoot and root, days to flowering, tiller number and grain yield. The mutants were categorized as 1. low P tolerant (13) if their yield in low P field was higher than N22, 2. moderately tolerant (2) if yield was lower than N22, 3. moderately susceptible (43) if they survive up to 100 days but do not flower and 4. susceptible (7) if they die within 70 days of transplanting and also do not flower. The 3 mutants NH686, 363, 787 and N22 gave yield of 8,6,6,1g per plant in low P and 29,28,28,20 g in normal P. NH686 and 787 had higher photosynthetic rate, SOD, CAT activity than N22 in young leaves and POD activity in flag leaf in low P. Root acid phosphatase activity was higher than N22 in long term P stress. NH776 and 710 show longer roots, more biomass, higher shoot/root P ratio and higher grain P than N22 indicating these are gain of function mutants for PUE and 814 had lowest P content in shoot. NH349 was confirmed as gain of function mutant for both uptake and transport to shoot. F2 from crosses between N22 and selected mutants are being used to identify causal genes for P related traits using Mutmap approach. qRT PCR using 13 transporter genes is in progress using these mutants.


154

Poster Session VIII

PVIII-206. Identification and charecterization of ems induced mutants of cultivar nagina22 of rice for productivity traits and bacterial blight resistance

Kaur S1, Kaur A1, Bhatia D1, Vikal Y1, Komal R1, Sahi G1, Lore JS 2, Hunjan M 2, Sharma RP3, Singh AK3, Mahopatra T4, Sarla N5, Sheshshayee MS 6, Singh K1 1School of Agricultural Biotechnology, 2Deparment of Plant Breeding & Genetics, Punjab Agricultural University, 3Indian Agricultural Research Institute, New Delhi, 4Central Rice Research Institute, Cuttack, 5Directorate of Rice Research, Hyderabad, 6University of Agricultural Sciences, Banglore, India

Mutants are one of the best and effective resources for interpreting gene function. Chemical mutagen Ethyl Methyl Sulphonate (EMS) has been extensively used to induce large number of functional variations in rice and other crops. A set of 800 EMS based M1 mutants of cultivar Nagina22 were received from IARI, New Delhi under a network project sponsored by DBT. Visual mutants for productivity traits were tagged and single plant from each M2 progenies were carried forward to M8. Selected mutants were genotyped with a set of 24 polymorphic markers containing two markers from each chromosome for confirmation that mutants belong to N22 and not an off type or mixture. Evaluation of all 800 M8 mutants for all productivity related traits revealed a significant major and micro variation in mutants as compared to N22. After inoculation of all the progenies at M4 with Xoo pathotype, a single M4 progeny was found to be resistant to Bacterial leaf blight (BB) and carried forward upto M8, each time selecting resistant by inoculation. Two F2 populations were generated, one by crossing BB mutant with PAU201, a high yielding cultivar of Punjab, and other for short grain (SG) mutant with IR64, to analyze the regions responsible for these phenotypes. To detect BB resistant region, bulked segregant analysis (BSA) was performed, by pooling the DNA from 8 extreme individuals. Three markers, RM23736, RM23742, RM23914, on chromosome 9 confirmed the BSA and maker interval is chosen for population analysis. F2 population of SG and its F3 progeny was evaluated for productivity traits and SSR markers were used for genotyping. Association of phenotype and genotype was revealed using QTL cartographer v2.5. Two QTLs for grain width (qGW3.1, qGW3.2) and three QTLs for grain length (qGL3.1, qGL3.2 and qGL7.1) were found on chromosome 3 and 7.

PVIII-207. Marker aided pyramiding of nine genes/ QTLs for management of biotic and abiotic stresses in rice Rao GJNl , Das Gl, Prasad Dl,2 , Das KM1, Reddy JNl, Marndi BC1, Kar MKl, Varier M3, Prakash A1 and Mohapatra T 1Central Rice Research Institute, Cuttack-753006, India. 2Kaveri seed Company Ltd., Secunderabad-500003, India 3CRURRS, Hazaribagh- 835301, India

Rice (Oryza sativa L.), grown in a wide range of climatic conditions, suffers severe yield losses due to biotic stresses like bacterial blight, blast and gall midge and abiotic stresses like submergence and salinity. One of the most efficient methods to minimize the yield losses against these stresses is enhancement of host resistance, an economical and environment friendly approach. Pyramiding of multiple resistance/tolerance genes/QTLs into elite genotypes using marker technology is an effective strategy to manage several stresses at the same time. This report presents the pyramiding of nine genes into improved Swarna and IR 64, two mega varieties of rice each having three BB resistant genes (Xa21+xa13+xa5) already. The donors for blast are C1O1A51 (Pi2) and WHD-1S-75-1-127 (Pi9), Kavya (Gm1) and Abhaya (Gm4) for Gall midge, FR13A/Swarna Sub1 (Sub1) for submergence and FL478 (Saltol) for salinity. Marker assisted backcross breeding strategy was employed to pyramid these genes/QTLs into the Swarna and IR 64. These parents were inter crossed in different combinations for several cycles and selections from these crosses resulted in plants with gene/QTL combinations likeGm1+Gm4+Pi2+ Pi9+Sub1+Saltol+ (Xa21+xa13+ xa5),Gm1+Gm4+(Xa21+xa13+xa5), Pi2+Pi9+(Xa21+ xa13 +xa5), Sub1+ Saltol+(Xa21+ xa13+xa5) and also all other possible combinations. The gene pyramids exhibited desirable levels of resistance/tolerance against the target stresses thus demonstrating the utility of MAS approach in development of resistant/tolerant cultivars in minimum time with high precision. The methodology, morphological and quality characterization of gene pyramids and the results of the screening against different stresses will be presented in detail.


155

Poster Session VIII

PVIII-208. Understanding the gene machineries underlying adaptive response of rice to heat stress

Sarkar N and Grover A

Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, DhaulaKuan, New Delhi-110021, India (email:[email protected])

Protein folding and disaggregation are crucial processes for survival of cells under unfavorable conditions. A network of molecular chaperones supports these processes. Gene machineries underlying response of rice to sub-lethal levels of heat stress were analysed in terms of specific Hsps and the overall transcriptomeprofilings in this study. Collaborative action of Hsp70 and Hsp100 proteins is an important component of the chaperone network in living cells. Our search of the published rice genome sequence has revealed that there are 32 Hsp70 genes in rice genome. We suggest that rice Hsp70 superfamily genes are represented by 24 Hsp70 family and 8 Hsp110 family members. J-proteins/DnaJ members as co-chaperones assist Hsp70. RiceJ-protein family is extraordinary large with 104 genes. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-independent manner in preventing aggregation of cellular proteins and in their subsequent refolding. We identified 40 α-crystallindomain containing genes in rice andphylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. Transcript expression analysis showed that 19 sHsp genes were upregulated by high temperature stress. To obtain the complete picture of other genes that are associated with response of rice to heat stress, global transcriptome of rice leaf tissues was performed by microarray to investigate specifically the early events in the heat stress and recovery response. The transcriptome profile was significantly modulated within 10 min of heat stress: strikingly, the number of up-regulated genes was higher than the number of down-regulated genes in 10 min of heat stress. The enrichment of transcription factor binding sites for HSFs, bZIPs and DREBs coupled with up-regulation of transcription factors of different families suggests that the heat stress response in rice involves integration of various regulatory networks. Co-expression network analysis is useful tool for identification of functional association of coexpressed genes. We developed a co-expression network of rice from heat stress transcriptome data. The interpretation of microarray data in the context of co-expression network analysis identified several functionally correlated genes consisting of previously documented heat up-regulated genes as well as new genes that can be implicated in heat stress. Based on the findings on parallel analysis of growth of seedlings, associated changes in transcripts of selected Hsps, genome-wide microarray profiling and the co-expression networkanalysis, our study is a step forward in understanding heat response of rice.

PVIII-209. SpotTSS: A transcription start site predictor for plant promoters using structural features and core motifs

Haque T1, Shahid S1, Raj S2, Zaman S1, Elias SM1, Razzaque S1, Biswas S1, Zaman F2, Seraj ZI1,* 1Department of Biochemistry and Molecular Biology, University of Dhaka; 2Bio-Bio-1 Research Foundation. [email protected]

Accurate delineation of Transcription start site (TSS) in genome is an elusive task but this site is decisive to understand differential gene expression profiles. Many computational methods have been proposed to address this problem using both the physical properties of DNA and the regulatory elements; but are mostly specific for mammalian promoters. As plant promoter features vary significantly from mammalian ones therefore, a good predictor for plant genomes may need different strategies for TSS identification compared to existing ones. Furthermore, better TSS prediction can advance the structural annotation of plant genomes.

Analysis of average structural profiles and core motif of promoter, coding and intergenic sequences of plant shows that the nucleotides around TSS have some distinct features that can be exploited for TSS prediction. We implemented these distinct features to develop a plant TSS prediction program, SpotTSS, using a binary classifier, Support Vector Machine. In a careful evaluation study, we have compared SpotTSS to state-of-the-art algorithms that have been used for plants: PromPredict, TSSP-TCM and EP3. SpotTSS performs better in all evaluation analyses for various plant species, hence shows a better all-round performance. Furthermore, we demonstrated that structural profiles of constitutive and stress-inducible promoters vary significantly around their TSS and can be used as potential discriminatory elements to differentiate between these promoters.

156

Author Index

A

Abdin MZ ............................ 151 Abdullah AD ......................... 53 Abreu I ............................ 16, 28 Achary M ........................ 76, 79 Adachi S ............................... 43 Afif H .................................... 53 Agarwal A ............................. 79 Agarwal P ..... 13, 25, 65, 83, 87 Aggarwal D ........... 52, 115, 151 Agrawal S ............................. 59 Agung B ............................... 82 Ahmadi N ............................... 5 ain M .............................. 51, 67 Ali N ...................................... 46 Almeida D ............................. 28 Alwaris A ............................ 139 Amarasinghe V ..................... 34 Amas J ......................... 26, 106 Ambavaram MM ............. 14, 99 Ambavaram MRM ................ 16 An G ............... 7, 21, 56, 57, 98 Anand D ............................... 13 Anderson S ...................... 8, 33 Anil Grover .................... iv, i, 15 Anuradha G .................. 44, 142 Ara H .................................. 128 Ariyadasa TU ....................... 51 Arora K ............................... 124 Arul L .................................... 53 Asif MH ............................... 117

B

Babitha KC ........................... 77 Babu AP ............................... 78 Bahuguna RN ..................... 129 Bai X ................................... 102 Baig MJ ................................ 86 Baisakh N ............................. 14 Bajaj D .................................. 13 Bakshi S ............................... 15 Balachandran SM ... 40, 75, 135 Balachiranjeevi CH ............. 148 BalachiranjeeviCh .............. 135 Balaji M ................................ 60 Balaji S ................................. 53 Balasubramanian P .............. 53 Baldrich Patricia ................... 11 Balyan S ............................. 113 Bandopadhyay SA ............... 28 Bansal KC ............................ 36 Banu MSA ............................ 80

Baobao C ............................. 86 Baranwal V ........................... 18 Barik S .................................. 91 Barretto A ..................... 26, 106 Barros P ............................... 28 Bartley L ............................. 100 Basabdatta D ..................... 108 Basak P ................................ 28 Basu D ......................... 45, 152 Basu S ................ 14, 16, 69, 99 Batlang U ............................. 14 Batra V ............................... 138 Behera D .............................. 86 Behera L ................. 74, 97, 134 Betty A .................................. 69 Bhadana VP ....................... 148 Bhat S ................................ 129 Bhatia D ..................... 136, 154 Bhatnagar A ......................... 89 Bhatnagar N ................. 50, 105 Bhattacharjee A .............. 51, 67 Bhattacharya S ..................... 71 Bhawmick PK ..................... 112 Bhowmick PK ..................... 146 Bhowmick R ......................... 24 Bhowmik PK ................. 24, 138 Bian C .................................. 26 Biswas S ............................ 155 Bonneau R ........................... 35 Borah P .......................... 68, 98 Brajendra .............................. 44 Brajendra P .......................... 44 Brar B ................................. 149 Brar DS ................................ 42 Bresolin APS ........................ 83 Brigitte C ............................ 101 Burman N ............................. 89 Buttar BS .............................. 70

C

Caddell D ........................... 122 Cai J ..................................... 26 Campo Sonia ....................... 11 Casal C ................................ 42 Chakraborty A ...................... 81 Chakraborty N ................ 17, 64 Chakraborty S ...................... 64 Chand D ............................. 130 Chander S ............................ 28 Chandrasekhar K ............... 125 Chang M-C ............... 52, 61, 73 Char B ................................ 139 Chatterjee A ................. 61, 103 Chattoo BB ................... 47, 131 Chattopadhyay D ......... 77, 141

Chattopadhyay S .................. 99 Chaudhary R ...................... 136 Chauhan MS ........................ 36 Chawla M ........................... 102 Chen H-C ....................... 52, 73 Chen L .................................. 23 Chen M ........................... 3, 112 Chen W .......................... 7, 132 Chen X ............................... 119 Chen Y ..................... 11, 23, 52 Chen Y-A .............................. 11 Chen Y-J .............................. 52 Cheralu C ........................... 144 Chetukuri A ........................ 143 Chhillar-Balyan S ............... 117 Chinnusamy V .................... 104 Cho S ..................... 90, 94, 137 Cho S-H ....................... 94, 137 Chongloi GL ..................... 4, 85 Christie SFM ........................ 24 Chuba T ............................... 20 Conrad L .......................... 8, 33 Cordeiro A ............................ 28 Costa de Oliveira A .............. 34 Costa M ................................ 28 Cotton C ............................... 35 Courtois B .............................. 5 Cruz D .................................. 27

D

da Maia LC ........................... 83 Dangwal M ........................... 32 Dansana PK ......................... 64 Das A ........................... 74, 103 Das G ................................. 154 Das KM .............................. 154 Das P ................................. 101 Das S ................. 13, 36, 38, 83 Dasgupta I .................... 39, 127 Dash SK ............................... 74 Datta A ................................. 64 Datta K ................................. 46 Datta SK ............................... 46 Daudi A .............................. 122 de Oliveira A ............... 3, 34, 83 de Oliveira AC ...................... 83 Deborah DAK 44, 139, 142, 152 Delphine M ................... 53, 101 Deshpande G ............. 4, 22, 85 Deshpande GM .................... 85 Devanna BN ....................... 123 Deveshwar P ........ 96, 102, 153 Dey A ................................... 49 Dhandapani G ...... 52, 115, 151 Dharmawardhana P ............. 34

157

Author Index

Diévart A ................................. 5 Dikshit N ............................. 110 Divol F ..................................... 5 Divya B ............................... 145 Dkhar F ................................. 75 Dogra V ............................... 109 Dohling AK ............................ 75 Dokku P .............................. 104 Donald J ................................ 94 Donaldo M ............................ 53 Dong Y .................................. 26 Dubcovsky J ....................... 122 Duitama J .............................. 27 DurgaraniChV ..................... 137 Dwivedi N ........................ 25, 90

E

Ebana K .............................. 120 Ebert B ................................ 100 Elias SM .............................. 155 Elizebeth ME ......................... 87 Ellur RK ......................... 45, 146 Emmanuel G ............ iii, 53, 101 Endo M ................................. 46

F

Farias DR .............................. 34 Farmer A ............................... 27 Fartyal D ............................... 79 Fell DA ................................ 103 Ferreira L .............................. 28 Figueiredo D ......................... 28 Foster S .............................. 100 Fu B ...................................... 26 Fujiyama A .......................... 111

G

Gaétan D ............................ 101 Ganie SA ............................ 118 Gao D1 ............................... 112 Gao Y .............................. 7, 132 Garg P ................................ 121 Garg R .................................. 59 Gautam RK ........................... 24 Ghosh A ................................ 84 Ghosh M ............................. 108 Gilroy S ................................. 16 Giri J ......................... 57, 63, 68 Giri MK .......................... 99, 128 Gjersing E ........................... 100 Gong L ...................... 7, 93, 132

Gopala-Krishnan S ....... 45, 143 Gopalakrishnan S ............... 149 Gorska A ............................... 28 Goto S ............................. 12, 39 Gou X ................................ 8, 33 Gour P ................................ 121 Govindaraj K ....................... 139 Gowthami C ........................ 133 Gregorio G .................... 26, 106 Grover A ........... 58, 80, 81, 155 Guiderdoni E ........................... 5 Gupta A ............................... 111 Gupta B ........................... 18, 54 Gupta BK .............................. 54 Gupta G ................................ 76 Gupta K ........................... 76, 79 Gupta P ................................. 54 Gupta S ....................... 130, 141 Gupta SK ............................ 130

H

Hafemeister C ....................... 35 Hajira S ................. 40, 135, 148 Hajira Sk ....................... 40, 135 Han B ................................ 3, 10 Han C-d ................................ 82 Hanamaratti G .................... 141 Handa H ................................ 72 Hanumappa M ...................... 24 Haque T .............................. 155 Hari Prasad A S .................. 140 Hariprasad AS .................... 135 Haritha G ............................ 133 Hayashi N ............................. 39 Henry R ................................... 3 Henry S ................................... 5 Hideo N ................................. 88 Hirasawa T ............................ 43 Hirochika H ........................... 39 Holloway-Phillips M-M .......... 35 Hong C-Y ............ 59, 60, 71, 72 Hosokawa S .......................... 72 Hsing YC ..................... 3, 30, 79 Hsing Y-I ....................... 11, 107 Hsing Yue-Ie ......................... 11 Huang L-T ............................. 11 Huang W-L ...................... 72, 84 Huda KMK ............................ 80 Hunjan M ............................ 154 Hwang S ............................. 122

I

Ignacio JC ............................. 42

Ingole KD ............................ 129 Islam T .................................. 94 Ismail AM .............................. 42 Itoh T ............................... 27, 72 Itsuki M ................................. 88

J

Jackson S ........................... 112 Jain A .................................... 65 Jain M .............. 51, 59, 67, 151 Jain R ................. 121, 147, 149 Jain RK ...................... 147, 149 Jain S ......................... 147, 149 Jain Sunita .......................... 147 Jaiswal P ................ 24, 34, 111 Jalmi SK .............................. 128 Jamaloddin Md ................... 143 Jangam AP ........................... 73 Jawali N .......................... 47, 74 Jayaram .............................. 135 Jayaswal PK ....................... 109 Jean-Luc V ............................ 53 Jena KK ................................ 43 Jena M ................................ 134 Jena PP ....................... 50, 150 Jena PPP ............................ 150 Jena SS ..................... 137, 152 Jeong I-S ............................ 138 Jha S ................................... 139 Ji H-S .................................. 138 Jiaming M ........................... 110 Jiang C-J ............................... 39 Jie L ........................................ ii Jindal K ............................... 147 John JA ............................... 145 Johnson C ......................... 8, 33 Jones D ............................. 8, 33 Joseph SV .......................... 121 Joshi R .................................. 85 Jun X ..................................... 86 Jung K-H ...................... 50, 105 Jwo W-S ............................... 59 Jyothi B ............................... 133

K

Kadiri S ................................. 78 Kaladhar K .......................... 133 Kamble NU ........................... 49 Kamboj R ............................ 123 Kanamori H ........................... 72 Kannan P ............................ 152 Kansal S .................... 117, 118 Kant R .......................... 39, 127

158

Author Index

Kanwar P .............................. 66 Kao CH ................................. 71 Kapila RK ........................... 127 Kapoor M ....... 21, 32, 100, 102,

113, 114 Kapoor S 13, 18, 21, 32, 66, 68,

92, 96, 100, 102, 113, 153 Kar MK ............................... 154 Karnati P ............................ 135 Kartha RV ........................... 152 Katara JL ...................... 97, 150 Katiyar-Agarwal S ................ 60 Kaul T ....................... 76, 79, 94 Kaur A .......................... 68, 154 Kaur C .................................. 51 Kaur S ........................ 136, 154 Kaur SG ............................. 136 Kavi-Kishor PB ..................... 62 Kawahara Y .................... 27, 72 Kaworu E ............................ 134 Kazuki M ............................ 134 Kemparaju K B ................... 140 Khanday I ......................... 4, 85 Khanna A ........................... 149 Kharb A .............................. 147 Kharb Anju ......................... 147 Khurana JP .. 68, 70, 88, 89, 98 Khurana R .................... 92, 153 Kim B-G ................................ 50 Kim JS .................................. 56 Kim S-R ................................ 56 Kim SR ................................. 57 Kim T-H .............................. 138 Kim WT ................................ 56 Kimbrel J .............................. 24 Kiranmayi SL .............. 139, 144 Kiyosumi H ......................... 134 Kobayashi T ........................... 6 Kohli A .................................. 20 Kokiladevi E ......................... 53 Komal R ............................. 154 Kothari KS ...................... 57, 64 Koutu GK .................... 135, 136 Krasileva K ......................... 122 Kretzschmar T ...................... 42 Krishnam RA ...................... 133 Krishnamurthy P ................. 153 Krishnamurthy SL ......... 24, 138 Krishnan A ................ 14, 16, 99 Krishnan GS ....................... 146 Krishnaraj PU ..................... 129 Kruger MM ........................... 34 Kulkarni K ....................... 9, 104 Kulkarni KP ........................ 104 Kumagai M ........................... 27 Kumar A ........... 25, 41, 69, 115 Kumar AP ........................... 115 Kumar BS ............................. 50

Kumar D ............................... 88 Kumar GT ........................... 108 Kumar GV ............................ 75 Kumar J ........ 50, 129, 147, 149 Kumar K ......................... 53, 58 Kumar KK ............................. 53 Kumar M ............... 57, 113, 117 Kumar P .. 52, 65, 91, 104, 108,

151 Kumar PA ............... 52, 65, 151 Kumar PS ........................... 108 Kumar R ............................... 85 Kumar S ............... 65, 117, 150 Kumar SB ........................... 150 Kumar V .... 25, 36, 82, 96, 138,

153 Kumar VS ..................... 96, 153 Kumari A ............................ 123 Kumari B .................... 130, 153 Kumari BR .......................... 153 Kumari K ............................ 141 Kumari N ............................ 136 Kumari R ............................ 151 Kumari S .............................. 66 Kundu SM .......................... 103 Kurata N ......................... 3, 111 Kusajima M ................ 124, 126 Kushwaha HR .............. 55, 105 Kwon C-T ........................... 137 Kwon Y-I ............................... 46

L

Laha GS ............................... 40 Lai M-H ........................... 11, 59 Lakhwani D ........................ 117 Lakshmanan M ..................... 35 Latha GM ............................. 52 Latha M .............................. 110 Laurence C ......................... 101 Lavania D ............................. 81 Laxmi A .............................. 107 Lee D-Y .......................... 21, 35 Lee G-S .............................. 138 Lee H-J ............................... 138 Lee SC ................................. 57 Lee S-T ................................ 84 Lee Y-S ................................ 21 Li G ..................................... 110 Li L ....................................... 26 Li T ..................................... 119 Li X ......................... 22, 26, 140 Lian X ..................................... 7 Lima JM .............................. 104 Lin F ................................... 100 Lin K-C ................................. 59 Lin W-R ................................ 71

Liu H ..................................... 20 Liu X ....................... 7, 119, 132 Liu Y-G ................................. 41 Liu YG .................................. 23 Lore JS ............................... 154 Lourenço T ........................... 16 Lucie P ............................... 101 Luo J .............................. 7, 132 Lyu K ...................................... 7

M

Mackill DJ ............................. 42 Madabula FP ........................ 34 Madhav MS .......... 40, 135, 148 Maeda S ............................... 39 Magalhaes A Jr ................... 83 Mageswari L ......................... 53 Mahadeva HKS .................. 148 Mahato AK ......................... 126 Maheshwari P ...................... 62 Mahopatra T ....................... 154 Maia LC .......................... 34, 83 Majee M ......................... 49, 59 Majee SM ............................. 70 Majhi BB ....................... 45, 152 Majumder AL ........................ 28 Malick N ............................... 13 Malik G ......................... 32, 114 Malik N ................................. 87 Mandal P ............................ 111 Mandol N ............................ 104 Mangat GS ......................... 136 Mangrauthia SK ......... 114, 147 Manica-Berto R .................... 34 Manimaran P ........................ 75 Manna M .............................. 94 Manorama K ....................... 144 Mao D ................................. 102 Marcotte E .......................... 122 Marini N ................................ 83 Marndi BC .................. 150, 154 Masahiro Y .............. iii, i, ii, 134 Mathur S ......................... 65, 87 Matsumoto T ........................ 72 Maud R ............................... 101 Maurya A .............................. 90 Mayandi K ............................ 91 Mehla B.S. .......................... 147 Mehla BS ............................ 147 Mehra P ................................ 63 Meizhong L ........................... 86 Mertz LM .............................. 34 Mesapogu S ......................... 78 Meyer R ........................ 26, 106 Meyer RS ............................. 26 Meyers B ........................ 7, 112

159

Author Index

Mihani R ................................ 99 Mikkilineni V ........................ 139 Min HJ ................................... 56 Minamisawa K .................... 124 Mishra DK ................... 135, 136 Mishra M ......................... 18, 61 Mishra NS ........................... 115 Mishra P .......................... 63, 95 Mishra RC ............................. 80 Mishra S 88, 111, 112, 120, 145 Mishra V .............. 24, 63, 65, 95 Mitta Dl .................................. 58 Miyuki K ................................ 88 Mohan V ............................... 76 Mohanta TK .......................... 97 Mohanty B ............................. 35 Mohanty SK ........................ 134 Mohapatra S ................. 36, 134 Mohapatra SL ..................... 134 Mohapatra T .. 9, 50, 52, 74, 97,

103, 104, 134, 142, 150, 151, 154

Moharana KC ...................... 151 Monaco MK ........................... 34 Mondal KK .......................... 146 Mondal TK .......................... 118 Monika S ............................. 133 Mori A ................................... 46 Mori M ................................... 39 Muhammed JK .................... 107 Mukherjee A .......................... 28 Mukherjee R ......................... 28 Mukherjee S .......................... 28 Mukhopadhyay P .................. 89 Mukund V ............................ 126 Mustafiz A ....................... 51, 84 Muthamilarasan M .............. 141 Mutum RD ........................... 117

N

Naga D N ............................ 133 Nagarajan M ............... 144, 149 Nagarjuna KN ....................... 77 Nagata T ............................. 111 Nain V ................................. 115 Naithani S ............................. 34 Najiar MRJ .......................... 130 Nakanishi H ............................ 6 Nakanishi-Itai R ...................... 6 Nakashita H ................ 124, 126 Nandi AK ....................... 93, 128 Nandi AS ............................... 99 Naoki Y ................................. 39 Narula A .............................. 124 Nataraja KN .................... 77, 78 Nath M ................................ 104

Nath T ................................... 71 Nayak DK .............................. 74 Nayar S ......................... 21, 100 Neelamraju S ........................ 91 Ngangkham U ......................... 9 Nicotra AN ............................ 35 Nisha R ............................... 133 Nishizawa NK ......................... 6 Nongpiur R ...................... 30, 69 Nozomi O ............................ 134 Nutan KK ........................ 54, 56

O

Oard J ................................... 27 Oda K .................................... 39 Oh T .................................... 122 Ohtsuki N .............................. 46 Ohyanagi H ......................... 111 Ojha GC ................................ 36 Oliveira M ........................ 16, 28 Oliveira MM ........................... 28 Oono Y .................................. 72 Osakabe K ............................ 46 Ouyang Y ............................ 110

P

Pachauri A ............................ 36 Paek N .................... 90, 94, 137 Paek N-C ...................... 94, 137 Panaud O .......................... 3, 10 Panda K ...... 111, 112, 120, 145 Panda RS ........................... 134 Pandey A .............................. 66 Pandey BK ............................ 63 Pandey G ................ 18, 66, 141 Pandey GK ..................... 18, 66 Pandey P .............................. 76 Pandey S .............................. 76 Pandey V .............................. 18 Pandit A ................................ 24 Pani DR .............................. 110 Panigrahy M .................. 91, 153 Pareek A 18, 30, 54, 55, 56, 61,

62, 66, 67, 69, 84, 85, 95, 101, 105

Parida A ................................ 17 Parida SK .......... 13, 25, 90, 141 Pathak RR ............................ 73 Pathania S .......................... 146 Patil M ................................... 78 Patil P ................................. 139 Patil S .. 44, 137, 139, 141, 142,

152

Patil SS ............................... 141 Patra BC ............................... 36 Pattanayak D ............... 52, 151 Paul S ................................... 46 Pauline M ............................ 101 Peck M ................................ 100 Pegoraro C ........................... 34 Pelayo MA ............................ 42 Pereira A ...... 14, 16, 55, 69, 99 Périn C .................................... 5 Peris-Peris Cristina ............... 11 Petla BP ................................ 49 Pham G ................................. 35 Phanindra MLV ................... 115 Plessis A ............................... 35 Prabhu KV .......................... 146 Pradhan SK .......................... 74 Prakash A ........................... 154 Prakash C ........................... 131 Prasad D ............................. 154 Prasad M ........... 108, 141, 148 Prasad MS .......................... 148 Prasad TG ............................ 78 Prasad VBR .......................... 99 Prasanna LB ....................... 148 Prashanthi .. 126, 129, 141, 149 Prashanthi SK .... 126, 141, 149 Pratap N .............................. 148 Preece J ................................ 34 Priatama RA ......................... 82 Priya MRV ............................. 40 Puig J ...................................... 5 Purkayastha A ...................... 39 Purugganan M ....... 26, 35, 106 Pushpalatha G ................ 44, 78

Q

Qifa Z .............................. iii, i, iv

R

Rabindran R ........................ 145 Radhika K ........................... 144 Raghavendrarao S .............. 115 Raghuram B ........................ 116 Raghuram N ......................... 73 Raghuvanshi S ..... 32, 65, 113,

117, 118, 121 Raghuvanshi U ................... 113 Rai A ...... 36, 81, 123, 124, 130 Rai AK ................ 123, 124, 130 Rai M ........................... 75, 130 Rai V ..... 24, 44, 63, 65, 78, 95,

112, 120, 132, 138, 145

160

Author Index

Raj S .................................. 155 Rajkumar S ........................ 110 Ram T ................................. 78 Ram T .................................. 44 Rama Devi SJS .......... 147, 148 Rama N .......................... 77, 78 Ramakrishna K ................. 4, 22 Raman KV .................... 52, 151 Raman VK .......................... 115 Ramanan R .......................... 91 Ramu SV ........................ 76, 77 Rania BS .............................. 53 Ranjan R .............................. 92 Rao DN .............................. 153 Rao GJN ............................ 154 Rao KV ................. 96, 144, 153 Rao LVS ............................... 78 Rao NN .................................. 4 Rao RN ................................ 97 Rao SKG ............................ 153 Rao SR ......................... 52, 151 Raorane M ........................... 20 Raphaël M .......................... 101 Rastogi K .............................. 53 Rathour R .. 126, 127, 141, 146,

149 Razzaque S ........................ 155 Reddy AN ............................. 55 Reddy CS ............................. 76 Reddy GM ............................ 76 Reddy JN ............. 50, 150, 154 Reddy M .... 29, 76, 79, 94, 125,

139 Reddy MK .. 29, 76, 79, 94, 125 Reddy PC ............................. 78 Revathi P .................... 135, 140 Richa .................................... 80 Robin S .......... 9, 104, 142, 145 Roja V ................ 139, 142, 152 Rombaldi CV ........................ 34 Ronald P ............................ 122 Roy B ................................. 108 Roy N ................................. 139 Roy S ................................... 66 Russell S .......................... 8, 33 Rydquist SW ........................ 98

S

Sage A ................................. 24 Sahi G ................................ 154 Sahoo K ................... 18, 54, 85 Sahoo KK ....................... 18, 85 Sahu RK ............................. 134 Sahu S ....................... 104, 134 Sahu SC ............................. 134 Saibo N .......................... 16, 28

Saika H ................................. 46 Sailaja B ............................. 114 Saini A ............................ 47, 74 Saini P .................................. 81 Samantaray S ...................... 97 Samik S .............................. 108 San Segundo Blanca ........ i, 11 Sanagala RR ........................ 65 Sandhu N ........................... 147 Santos AP ............................ 28 Santos RS ............................ 34 Santos T ............................... 28 Sanyal RP ............................ 74 Sapeta H ........................ 16, 28 Saran A ................................ 30 Sarawgi AK .......................... 36 Sarkar A ................... 51, 65, 91 Sarkar AK ....................... 51, 91 Sarkar N ............................. 155 Sarkar RK ........................... 150 Sarla N .... 9, 78, 104, 114, 133,

142, 144, 147, 153, 154 Sarwade R ........................... 45 Sashidhar VR ....................... 70 Scheffler B ............................ 27 Scheller H ........................... 100 Schulze J .............................. 61 Sen SK ................................. 49 Sengupta DN .................. 15, 71 Sengupta S .......................... 28 Senguttuvel P ..................... 140 Senoura T .............................. 6 Septiningsih E ................ 35, 42 Septiningsih EM ................... 42 Seraj ZI ............................... 155 Serra T ........................... 16, 28 Seshashayee M ..... 9, 104, 142 Shah JM ............................... 45 Shaheen R ........................... 36 Shahid S ............................. 155 Shankar R .............. 59, 67, 151 Sharan A ........................ 67, 69 Sharma A ................... 109, 144 Sharma AS ......................... 109 Sharma D ................... 117, 125 Sharma E ............................. 68 Sharma G ............................. 68 Sharma KD ......................... 127 Sharma P ............................. 88 Sharma R .. 9, 21, 96, 104, 142,

154 Sharma RP ..... 9, 104, 142, 154 Sharma S ....... 24, 36, 103, 138 Sharma SK ............. 24, 36, 138 Sharma TR .. 37, 109, 111, 120,

123, 124, 126, 127, 130, 141, 149

Shaw R ............................... 103

Shen G ............................... 102 Shenton M .......................... 111 Sheshshayee MS ......... 77, 154 Shih M-D ................ 30, 79, 107 Shikari AB .......................... 149 Shima S ...................... 124, 126 Shimoda FS ......................... 12 Shobha Rani N ..................... 40 Shridevi AJ ......................... 141 Shuichi F ............................ 134 Shukla A ............................. 129 Siddiq E A ............................ 44 Siddiq EA ... 137, 139, 142, 152 Siddiqui A ..................... 93, 128 Sihi S .................................... 15 Singh A .... 9, 13, 18, 24, 36, 45,

66, 95, 104, 105, 112, 126, 132, 138, 140, 141, 142, 143, 144, 145, 146, 149, 154

Singh AK 9, 13, 24, 45, 95, 104, 112, 126, 132, 138, 140, 141, 142, 143, 144, 145, 146, 149, 154

Singh B 24, 111, 112, 120, 132, 138, 144, 145

Singh BD ............................ 144 Singh BP .................... 112, 132 Singh D ...................... 146, 150 Singh DP ............................ 150 Singh G ........................ 81, 136 Singh H .............................. 127 Singh J ................................. 94 Singh K ...... 9, 42, 95, 104, 136,

142, 148, 154 Singh MRK ........................... 96 Singh N 9, 24, 63, 95, 104, 109,

111, 112, 120, 126, 132, 138, 142, 143, 144, 145, 146

Singh N K ........................... 120 Singh NK ..... 9, 24, 63, 95, 104,

109, 111, 112, 126, 132, 138, 142, 144, 145, 146

Singh ON ................ 50, 97, 150 Singh P ..... 66, 93, 97, 126, 128 Singh PK .............. 93, 126, 128 Singh R ................ 36, 111, 113 Singh RKM ......................... 113 Singh S ... 36, 93, 128, 135, 136 Singh SK .................... 135, 136 Singh SP .............................. 36 Singh UD .... 126, 141, 146, 149 Singh V ........... 36, 93, 115, 144 Singh VK .................... 115, 144 Singla- Pareek SL ................ 69 Singla-Pareeek SL ............... 67

161

Author Index

Singla-Pareek SL ..... 18, 30, 51, 54, 55, 56, 61, 62, 66, 84, 85, 95, 101, 105

Sinha AK ... 29, 58, 97, 116, 128 Sinha S ................................. 24 Sivasankar A ....................... 143 Skyes R .............................. 100 Soda N ............................ 30, 62 Sohgaura N ......................... 135 Solanke AU ........... 52, 115, 151 Soni P ....................... 30, 67, 69 Soni SK ............................... 148 Sonti RV .......................... 12, 40 Sopory SK ........... 18, 51, 54, 84 Srala N .................................. 44 Sreenu K ............................... 44 Sreevathsa R ..... 52, 70, 77, 78,

151 Srinivas D ........................... 143 Srinivasan K .......................... 36 Srinivasan R ....................... 142 Srivastava AK ................. 66, 81 Srivastava S .................... 55, 81 Srividhya A .......................... 152 Stein J ................................... 34 Subba P ................................ 64 Subbarao LV ................... 36, 44 Subhasisa B ........................ 133 Subrahmanyam D .... 44, 75, 78,

114 Suchandranath Babu M ...... 147 Sudhakar D ........................... 53 Sudhakar T ......................... 133 Suetsugu M ........................... 12 Sugano S .............................. 39 Sujatha M ............................ 143 Sukumar M ........................... 44 Sullivan C .............................. 24 Sun J ..................................... 43 Sun S ............................ 93, 122 Sundaram MK ..................... 148 Sundaram R M .................... 140 Sundaram RM ......... 40, 75, 135 Sundaresan V ................... 8, 33 Sunkar R ............................... 25 Suprasanna P ................. 66, 81 Suryendra J ........................ 140 Sushma MA .......................... 70 Swain A ................................. 86 Swain P ......................... 86, 150 Swamy BPM ....................... 133 Swapna L .............................. 96 Swathi G ............................. 143 Sylvie J ............................... 101

T

T hein SZM ........................... 42 Taeko S .............................. 134 Takashi M ............................. 88 Takatsuji H ...................... 12, 39 Tanabata T ........................... 43 Tao L ..................................... 86 Teotia PS .............................. 85 Thakur JK ....................... 25, 90 Thakur S ............................. 126 Thibivilliers S ....................... 100 Thirulogachandar V ............ 125 Thomas J .............................. 55 Tianze Y ................................ 86 Tingting F .............................. 86 Tiwari LD ............................... 58 Tiwari M ...................... 117, 125 Tiwari S ................. 24, 110, 138 Tohme J ................................ 27 Toki S .................................... 46 Torres E ................................ 27 Toshio Y .............................. 134 Toyoda A ............................ 111 Tripathi AK ................ 18, 85, 95 Tripathi RD .......................... 117 Tripura V VGN .................... 133 Tripura Venkata VGN . 144, 147 Trivedi DK ............................. 80 Trivedi PK ................... 117, 125 Tsai C-N ................................ 52 Tsai J-N ................................ 61 Tsakispaloglou N .................. 20 Tsuyu A ............................... 134 Tuteja N ................................ 80 Tyagi AK 13, 18, 21, 57, 64, 68,

83, 87, 89, 92, 121, 153 Tyagi W ............................... 130

U

Udayakumar M ............... 77, 78 Udhan K .............................. 139 Udhaya P .............................. 85 Ueno Y .................................. 12 Uga Y ...................................... 4 Umakanta N ........................ 142 UmaKanth B ....................... 148

V

Vallelian C ............................. 69 Vani K ........................... 79, 125 Varakuti M ........................... 139 Variar M ...................... 141, 149

Varier M .............................. 154 Varma RL .............................. 97 Veeraghattapu Roja .............. 44 Veluthambi K ............... 45, 152 Vemireddy LR ..... 44, 137, 139,

142, 152 Venkata SB ......................... 141 Venkategowda R 14, 16, 55, 69 Venkateswara Rao Y .......... 147 Verma M ............................... 59 Vij S ...................................... 64 Vijayan J ............................. 123 Vijayraghavan U 4, 22, 85, 152 Vikal Y ................................. 154 Vinod KK ............................. 146 Viraktamath BC ..... 36, 40, 114,

135, 140, 144, 148 Vishnu Prasanth V .............. 147 Viswakarma C ..................... 104 Voleti S ........ 75, 114, 147, 153 Voleti SR .............. 75, 147, 153

W

Walid BR ............................... 53 Wang B ................................. 23 Wang C-S ............................. 59 Wang J ................................ 140 Wang L ........................ 93, 122 Wang W .................................. 3 War GF ............................... 130 Ware D .................................. 34 Wei F-J .................... 11, 30, 79 Wei S .................................... 34 Wen M ................................ 102 Weng X ............................... 140 Wentao X .............................. 86 Westhoff P .............................. 6 Wilkins O ............................... 35 Wing R ............................ 3, 112 Wing RA .................................. 3 Wise R ................................ 122 Wricha T ............................... 75 Wu HB .................................. 23 Wu J ................... 27, 30, 72, 79 Wu J-J ............................. 30, 79 Wu Ming-Tsung .................... 11 Wu T-M ................................. 71 Wu Y-P ........................... 30, 79

X

Xiao J ................... 37, 122, 140 Xie W ............. 7, 108, 122, 140 Xing Y .......................... 20, 102

162

Author Index

Xiong L ................................... 5 Xu C ................... 122, 132, 140

Y

Yadav AK ............................. 18 Yadav MC1 ........................ 110 Yadav RK ........................... 148 Yadav S ...................... 4, 65, 80 Yadav SR ............................... 4 Yadav VK ........................... 148 Yamagata H ......................... 72 Yamamoto T ......................... 43 Yamanouchi U ...................... 43 Yan W .......................... 20, 102 Yang B ............................... 122 Yang L-H .............................. 60 Yang Y-W ....................... 52, 73

Yano M ........................... 4, 120 Yao W ................................ 108 Yashavantha KJ ................. 141 Yasuda M ........................... 124 Yasunori N ......................... 134 Yazawa T ............................. 72 Yi J ........................... 21, 56, 98 Yokoi-Nishizawa A ............... 46 Yonemaru J .................. 43, 120 Yonemaru J-i ...................... 120 Yoo S ..................... 90, 94, 137 Yoo S-C ........................ 94, 137 Yoon U-H ........................... 138 Yoshida R ............................. 12 Yu H ................................... 140 Yu S ............................... 7, 132 Yugander A .......................... 40 Yugandhar P ...................... 153 Yukawa Y ........................... 116 Yumnam JS .......................... 75

Z

Zaman F ............................. 155 Zaman S ............................. 155 Zhang D ................................. 8 Zhang H ............................. 132 Zhang J ................................ 20 Zhang L .............................. 102 Zhang Q ...... 3, 7, 93, 108, 110,

122, 132, 140 Zhang Y ................................ 26 Zhao K ................................ 100 Zhao Y .......................... 31, 119 Zhong X .............................. 119 Zhou D-X ............................ 119 Zhou S ................................ 119 Zhou X .................................. 20 Zhu X ...................................... 5 Zilberman D .......................... 13

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