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Molecular dissection of Breadmaking quality, is the thesis on towards identification of QTLs governing breadmkaing quality. it involves genetic linkage group contruction, genetic mapping, QTL identification and Mixograph analysis

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MOLECULAR DISSECTION OF BREADMAKING QUALITY IN WHEAT (Triticum aestivum L ) L.)A thesis submitted to the University of Pune For the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY ELANGOVAN MANI Plant Molecular Biology Unit Division of Biochemical Sciences National Chemical Laboratory Pune 411008 (INDIA)

October 2007

INTER-INSTITUTIONAL COLLABORATIVE RESEARCH EFFORTResearch work embodied in this thesis was carried out at

NATIONAL CHEMICAL LABORATORY, PUNEIn collaboration with AGHARKAR RESEARCH INSTITUTE (ARI) (ARI), PUNE DIRECTORATE OF WHEAT RESEARCH (DWR), KARNALand

INSTITUTE OF PLANT GENETICS AND CROP PLANT RESEARCH (IPK), GERMANY Scientist involved from each instituteNCL: DR V S NCL DR. V.S. GUPTA ARI: DR. V.S. RAO DWR: DR. R. TIWARI & DR. R.K. GUPTA IPK: DR. MARION S RDER

CERTIFICATECertified that the work in the Ph.D. thesis entitled Molecular dissection of breadmaking quality in wheat (Triticum aestivum L.) submitted by

Mr. Elangovan Mani was carried out by the candidate under my supervision. The material obtained from other sources has been duly acknowledged in the thesis.

Date:

Dr. Vidya S. Gupta (Research Advisor)

DECLARATIONI hereby declare that the thesis entitled MOLECULAR DISSECTION OF BREADMAKING QUALITY IN WHEAT (Triticum aestivum L.) submitted for Ph.D. degree to the University of Pune has not been submitted by me for a degree at any other university.

Date:

Elangovan Mani National Chemical Laboratory Pune: 411008

CONTENTS TitleAcknowledgements List of Abbreviations List of Figures List of Tables

Page NumberIV V-VII VIII-IX X 1-35

Chapter 1: Introduction - Wheat for Bread making1.1 1.2 1.3 1.4 1.5 Wheat: A global perspective Wheat production in India: some facts and concerns Wheat: A model system for polyploid genetics Wheat: Suitable for various end products Wheat and bread making quality 1.5.1 Bread making: A classical way 1.5.2 Methods to evaluate bread making process Gluten and breadmaking quality Quality mapping using molecular markers 1.7.1 Methodology of QTL mapping in plants using DNA markers 1.7.2 Marker assisted selection for crop improvement 1.7.3 Mapping and tagging of quality traits in wheat 1.7.4 MAS in Wheat 1.7.5 Fine mapping / Map based cloning in Wheat Advances in wheat genomics: RFLPs to RNAi Wonder wheat: A future path in wheat molecular research Genesis of my thesis

1.6 1.7

1.8 1.9 1.10

Chapter 2: Materials and methods2.1 Chemicals, enzymes and oligonucleotides 2.2 Plant material 2.3 DNA extraction 2.3.1 CTAB DNA extraction method 2.3.2 Rapid DNA extraction 2.4 DNA quantification 2.5 PCR amplification using various DNA primers 2.5.1 ISSR analysis 2.5.2 SSR analysis 2.6 Resolution of PCR products using various methods

36-52

2.7 2.8

2.9

2.10

2.6.1 Agarose gel electrophoresis 2.6.2 Poly-acrylamide gel electrophoresis 2.6.3 Automated laser flourescence gel separation Extraction of HMW and LMW glutenins 2.7.1 Polyacrylamide gel electrophoresis (PAGE) of glutenin proteins Evaluation of phenotypic traits 2.8.1 Test weight (Tw) 2.8.2 Thousand grain weight (Tgw) 2.8.3 Grain protein content (Gpc) 2.8.4 Sedimentation volume (Sv) 2.8.5 Mixograph 2.8.6 Loaf volume Frame work map construction 2.9.1 Scoring of marker data 2.9.2 Linkage group construction Statistical analysis, QTL mapping and QTL clusters 2.10.1 The Analysis of variance 2.10.2 QTL mapping 2.10.3 QTL clusters 53-60

Chapter 3: Results3.1

3.2 3.3

3.4 3.5

3.6

Framework map construction 3.1.1 Parental survey analysis 3.1.2 Population Survey 3.1.3 Linkage group construction Phenotypic characteristics of the parents and the progeny population Correlation among yield and BMQ traits 3.3.1 Correlation among Tw, Tgw, Gpc and Sv 3.3.2 Correlation among Lv data recorded at different locations 3.3.3 Correlation among mixograph traits AMMI analysis Identification of QTLs associated with yield and BMQ related traits 3.5.1 QTLs for Tw 3.5.2 QTLs for Tgw 3.5.3 QTLs for Sv 3.5.4 QTLs for Gpc 3.5.5 QTLs for Mixograph traits 3.5.6 QTLs for Lv QTL clusters formed by QTLs affecting yield, dough rheology and BMQ traits

II

Chapter 4: Discussion4.1 4.2 4.3 4.4 4.5 4.6

61-78

Features of framework map Precise phenotyping at multiple locations during multiple years Normal distribution of BMQ and yield traits Corrrelation between the traits GxE interaction contribute higher than environment alone for Lv QTL mapping for various traits under study 4.6.1 Tgw QTLs 4.6.2 Tw QTLs 4.6.3 Sv QTLs 4.6.4 Gpc QTLs 4.6.5 Mixograph QTLs 4.6.6 Lv QTLs 4.7 Co-locating QTLs and QTL clusters 4.8 Future strategy 79-81 82-110 111

Chapter 5: Summary and Future directionsBibliography Bio-data

III

AcknowledgementsI would like to express my sincere gratitude to Dr. Vidya S. Gupta, NCL, Pune my Ph.D. advisor for her academic guidance and support throughout the course of my Ph.D. programme. I would also like to thank Dr. Meena D. Lagu for her advice in planning the research work and constant encouragement during my Ph.D. programme. I thank Dr. Mohini Sainani for her help in terms of arranging the consumables required for research work. I express my special thanks to Dr. Marion S Rder, for her academic guidance and help during my research work in IPK, Germany. I thank Dr. VS Rao, Dr. Subhada and Dr. Manoj, ARI, Pune, for their generous help while performing the Mixograph experiments and extending their field facilities. Dr. RK Gupta & Dr. Ratan Tiwari, DWR, Karnal, are gratefully acknowledged for developing the plant material required for this study and phenotyping the BMQ traits. I sincerely express my thanks to Mr. Chavan, Dr.Bhagwat, Dr. Hon Rao (Hol Farm, ARI, Pune) and Ms. Prashisti (DWR, Karnal) for their extreme sincerity during harvest and phenotype evaluation. I feel happy to express my thanks to Dr. Sivaram, Director, NCL for providing me the research facilities and my colleagues Ajay, Reddy, Suhas, Rashmi, Shashi and Dr. Abhay for teaching me molecular biology techniques. I would like to thank Dr. Bhushan Dholakia and Dr. Neeta Madan, the postdocs in our wheat group for their help in data analysis and proof reading my manuscripts & thesis. I express my sincere thanks to Dr. Narendra Kadoo, for helping me in running QTL softwares, Dr. Ashok Giri for his help during my Germany trip, Dr. MI Khan and Dr. Sushma for helping in RP-HPLC analysis of gliadin proteins. I am grateful to Ms. Richa Rai, for her sincerity, support and help in my research work and Dr. Savita, Ms. Sofia Banu, Mrs. Ramya Dixit for proof reading this thesis. Friends are big part of my life, I convey my special thanks to Thiru, Geetha, Lalitha, Aditi, Marivel, Balaji, Manje, Krishna, Poonam, Rasika, Varsha, Ram, Ashwini, Charu, Ajit, Atul, Pawan and Laxmi for supporting me throughout my Ph.D. I would also like to thank Gauri, Manasi T, Vaiju, Pawan, Radhika, Sagar, Hemangi, Manasi M, Jidnyasa, Arun, Priya, Neha, Prashant, Suchitra, Rakesh, Nagraj, Ashwini, Sameer for their help and motivation. I am thankful to my father Mr. Mani and my mother Mrs. Prema, for giving me great support and encouragement during my Ph.D. I also thank my sisters Mrs. Vidya, Mrs. Devi and Ms. Uma for their kindness and affection. I thank Prof. D.V.R Seshadri, IIM, Bangalore for awakening the entrepreneurship attitude in myself. The month long stay in Chennai during Technology Led Entrepreneurship programme, was the most memorable period during my Ph.D. and I thank Dr. Avinash Dwivedi and Dr. MG Kulkarni, CSIR, India for organizing this leadership programme. I express my gratitude to IAESTE foundation for awarding me 6 months fellowship to work in IPK, Germany. I thank CSIR, India for supporting my research work through Junior and senior research fellowships.

Elangovan Mani IV

LIST OF ABBREVIATIONSC AACC AFLP APS AS-PCR BAC BMQ bn, mn bp cM cm CTAB cv DH DNA dNTPs DT DWR EDTA EST g/gm, mg, mg, ng Gpc h HCl ha HMW Tw IAA ISSR ITMI K2O Kb, Mb Degree centigrade American association of cereal chemists Amplified fragment length polymorphism Ammonium per sulphate Allele specific PCR Bacterial artificial chromosome Bread making quality Billion, Million base pair centimorgan centimeter cyltrimethyl ammonium bromide cultivar doubled haploid deoxyribonucleic acid Deoxy ribonucleotide tri phosphate ditelosomic Directorate of wheat research ethylenediamine tetra acetate expressed sequence tags gram, milligram, microgram, nanogram Grain protein content hour hydrochloric acid hectare High molecular weight Test weight or Hectolitre weight Iso-amyl alcohol Inter simple sequence repeat International triticeae mapping initiative Potassium oxide Kilobase pair, Megabase pairV

KCl kDa/kD kg Kg/ha KH L, mL, L LMW LOD M, mM, M MAS MgCl2 Mha min M mm mM mmole, mmole mRNA MT Mt MW N2 NaCl NaOH NIL NIR nM NT PAGE PCR pmoles PVP QTL RAPD

Potassium chloride kilo Dalton kilogram Kilogram per hectare Kernel hardness liter, milliliter, microliter Low- molecular weight Log of the odd (Base 10 logarithm of the likelihood ratio) molar, millimolar, micromolar Marker Assisted Selection Magnesium chloride Million hectare minute micromolar millimeter millimolar millimole, micromole messenger RNA Metric tonnes million tonnes Molecular Weight Nitrogen sodium chloride sodium hydroxide Near isogenic line Near infrared reflectance nanomoles nulli-tetrasomic Polyacrylamide gel electrophoresis Polymerase chain reaction picomoles Polyvinyl pyrrolidone Quantitative trait loci Random amplified polymorphic DNAVI

RFLP RIL RNA rpm RT-PCR s SCAR SDS SSR STMS STS TAE TEMED Tgw Tm Tris var

Restriction fragment length polymorphism Recombinant inbred line Ribonucleic acid revolutions per minute Reverse transcriptase-polymerase chain reaction second Sequen