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5 DISCUSSION

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Rice is one of the most important food crops and a major energy source

for more than half of the world population. Ever increasing population and rice

consumers put great demand for more rice production and it is estimated that

India and other Asian countries should double their rice production by 2035.

Despite the huge yield potential of existing varieties, only half of it is being

realized owing mainly due to biotic and abiotic stresses (Siddiq, 2010).

Managing these biotic and abiotic stresses is one of the important tasks to

achieve the desired levels of rice production. Hence, development of high

yielding varieties and hybrids with inbuilt durable resistance to major diseases

is one of the important breeding objectives in rice improvement programs..

Among the biotic stresses, blast is one of the most devastating fungal

diseases caused by Magnaporthe oryza, which leads to yield loss of 70 to 80%

during an epidemic (Ou, 1985), which translate into huge economic loss

globally. In India also, blast is a major disease particularly in the irrigated,

rainfed lowland hill ecosystems and north western Himalayan region,

comprising the states of Haryana, Uttar Pradesh, Jharkhand, Himachal

Pradesh, Uttaranchal and Jammu and Kashmir, where the favorable conditions

prevail for the faster multiplication of the pathogen. Though the chemicals are

being used to control this disease throughout the world, those chemicals are

not very effective. It is reported that many isolates often develop resistance to

such chemicals (Sawada et al. 2004; Suzuki and Arai, 2007) and also these

chemicals are not eco-friendly and resulting in land, air and water pollutions.

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Moreover, use of these chemicals in large quantity may increase the production

cost and considered not economical to the farmers. On the other hand, host

plant resistance is more reliable, eco-friendly, economical and not affected by

the climatic conditions. Though more than 80 blast resistance genes have been

reported (Costanzo and Jia, 2010), only few of them, such as Pi54, Pita and Pib

were found to confer durable resistance to wide range of blast isolates.

The resistance offered by Pi54 has been well documented by many

research groups (Sharma et al. 2002; Costanzo and Jia, 2010; Ramkumar et al.

2011) and the gene has been deployed in many breeding programs for blast

resistance (Srinivasarao et al. 2009). Pita was characterized by Bryan et al.

(2000) and since then the gene is being used in breeding for blast resistance

(Costanzo and Jia, 2010) due to its significant level of resistance. Pib is another

major resistance gene characterized by Wang et al. (1999) which provide

resistance to most of the Japanese races of M. oryza. All these genes are

different from each other by their nucleotide sequence and protein structure

and hence show different defense mechanisms. As they are complementary to

each other in defending the host plant from a wide blast fungus spectrum,

pyramiding them into elite rice varieties could be an effective strategy for

combating rice blast. The aim of the present study was to identify and isolate

novel/superior alleles of three major blast resistance genes viz. Pi54, Pita and

Pib from a wide range of germplasm including wild species and landraces using

the allele mining strategy.

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Selection of appropriate plant material is important for the success of

allele mining approach. In the present study, landraces were collected from

North Eastern parts of India, which is an endemic area for blast disease.

Landraces are known for their diversity and are not much domesticated than

the modern day cultivars and hence, they may harbor novel and superior

alleles (Mikami et al. 2008; Berg, 2009). Similarly wild species are known to

posses many beneficial alleles (Xiao et al., 1996; 1998; McCouch et al., 2007).

Hence, landraces and wild Oryza species were selected for this study. Before

proceeding to allele mining, screening for blast disease resistance was

performed to the selected ecotypes to know their phenotypic reaction to the

blast fungus.

The selected ecotypes (24 diverse landraces and 110 accessions

belonging to eight different wild Oryza species) were screened with differential

isolates for the selected genes. Among the materials analyzed, landraces,

Ammana Bavo and Boha thulasi joha showed complete resistance with Pi54

specific differential isolate. Konibora and Punsimutt with Pita specific

differential isolate and Sercher and Krengosa with Pib specific differential

isolate showed resistant phenotypic pattern. While, Podumoni Ahu and Bizor II,

landraces showed extreme susceptibility for all the differential isolates. In case

of wild Oryza species, which were screened with the mixture of three

differential isolates (NLR-1, ALM-2 and PNP-4 for Pi54, Pita and Pib,

respectively), three accessions (O. rufipogon Acc. No. 106507; O. barthii Acc. No.

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100931 and O. latifolia Acc. No. 101207) showed resistance, while three

accessions i.e O. rufipogon Acc. No. 106279, 106512 and 81978 showed

susceptible phenotype. The ecotypes, which showed extreme resistance and

susceptible reaction with gene specific differential isolates, were selected for

allele mining study. Hittalmani et al. (2000) also screened the plant materials

with differential isolates, which were compatible to Pi-1, Piz-5 and Pita.

Costanzo and Jia (2010) and Wang et al. (2008b) also selected the resistant

and susceptible varieties for the Pikm and Pita allele mining study, respectively.

The selected plant materials were screened with available molecular markers to

confirm the presence/ absence of the selected genes for the allele mining study.

Earlier allele mining studies focused mainly on coding region,

presuming that the mutations at the coding region will affect the protein

sequence and consequently its function also. The allele mining study on OsC1

alleles revealed that mutation accumulated in the allelic regions were

responsible for the non production of anthocyanin pigment and hence the

phenotypic change in apiculus coloration in rice (Saitoh et al., 2004). Allele

mining on late blight resistance gene of Solanum led to the analysis of allelic

frequency of Rpi-blb1 and Rpi-blb2 genes, which revealed high conservation of

RGA1-blb allele in the analyzed Solanum species (Wang et al. 2008a). Many

more allele mining studies have been listed in Table 2.1. However, in order to

get the complete information on allelic variations of the gene, ‘true’ allele

mining should cover non-coding region including promoter and 5’ UTR and 3’

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UTR regions of the allele (Latha et al. 2004; Ramkumar et al. 2010). Many

recent studies proved that the non-coding region of the alleles also influence

the phenotype of the trait. Ten folds upregulation was observed in waxy gene

(wx) due to the mutation at 5’ splice site (Isshiki et al., 1998). Differential

expression was observed with gene - Tb1, due to the mutation at distance 5’

UTR region (Clark et al., 2006). Hence, keeping in the view of the importance of

non-coding region on the gene expression in the present study, primers were

designed to amplify both coding and non-coding region of the gene.

In case of Pi54, which has single ORF with 991 bp coding region,

primers were designed in such a way that single amplicon covers entire genic

region. However, in case of Pib, which has 5404 bp of coding region (including

introns), it was difficult to amplify entire genic region as single amplicon.

Hence, two overlapping primer sets were designed to amplify the Pib genic

regions as two overlapping amplicons. After determining the physical position

of the targeted gene at the genome sequence, the primers were designed from

the reference genome sequence, which is available at NCBI website

(www.ncbi.nlm.nih.gov/). This method can be followed for allele mining in other

crops, for which the genome sequence is not available. However, due to the

diversity and non-conservation of the primer binding sites, the targeted alleles

may not be amplified from distant genotypes, which is a limitation of this

sequence or PCR based allele mining (Latha et al. 2004). In the present study

also, Pi54 alleles could not be amplified from following wild Oryza species: O.

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coarctata (HHKK genome), O. officinalis (CC genome), O. brachyantha (FF

genome) and O. granulata (GG genome), which might be due to the above

mentioned reasons.

The length of promoter region to be analyzed for allele mining studies is

still a big question. Many groups have analyzed promoters of different length,

ranging from -100 bp to -3000 bp (Brazma et al. 1998; Molina and Grotewold,

2005; Park et al. 2002; Veerla and Hoglund, 2006) and reported that maximum

numbers of motifs were identified between -100 and -600 bp upstream of input

sequence. To increase the probability of obtaining maximum information on

TFBMs, primers were designed in such a way that they amplify 2000 bp

immediate upstream to the TSS sequence of the chosen genes. To confirm this,

the primers were designed in such a way that they amplify part of 5’ end of the

gene also. For this purpose, 500 bp flanking sequences of that two kb

upstream sequence, which includes genic portion of the targeted promoter

allele, were used as primers binding site. All the primers were BLAST searched

in NCBI to identify their unique binding sites in the rice genome

(www.ncbi.nlm.nih.gov/).

Though methods are available for sequencing the PCR products directly

(Porter et al. 1997), many disadvantages were also reported for these methods

i.e. usually primers will not produce single amplicon in PCR; primers may

produce non-specific amplicons; week primers may not amplify in sequencing

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reactions; unused primers may hinder the sequencing process

(http://seqcore.brcf.med.umich.edu/doc/dnaseq/pcr.html) and overall

sequence quality may not be good for further analysis. Hence, cloning into a

vector and sequencing the targeted genomic region is considered as better

method to get good quality sequences. In the present study, all the promoter

and allelic regions were amplified with high fidelity Taq DNA polymerase and

cloned in cloning vector (pGXT), which was used for sequencing purpose. Only

high quality sequences (Phred score >20 per base) were used to compare with

reference and other promoter/allelic sequences.

5.1. Pi54 ALLELE AND PROMOTER MINING

Landraces were screened with differential isolate, NLR-1, and the

phenotypes were noted. Among them, two highly resistant and two complete

susceptible landraces were selected for further analysis. From the wild species

also, three each from highly resistant and complete susceptible species were

collected. Other than those, in order to analyze the diversity, six more species,

which had different kind of genome kind (AA to EE genome) were also selected.

Hence, these sixteen genotypes were analyzed further for Pi54 allele mining

study (Table 4.3). Allelic diversity analysis of Pi54 revealed that this gene is

present in wide range of Oryza species which suggest that Pi54 might have

originated long before during evolution. The conservation of this allele across

Oryza species for long period may indicate that this gene should be a

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functional and important gene (Wang et al. 2008a), which provides resistance

to wide range of isolates. The nucleotide polymorphisms observed in the alleles

might have accumulated during the independent recombination events

between the species and intercross events.

Nucleotide substitutions in gene sequences have been successfully

used for many evolutionary and phylogenetic studies of lectins of legume

species and APA (Arcelin/Phytohemagglutinin/alpha-Amylase inhibitor) gene of

Phaseolus species (Lioi et al. 2006, 2007; Galasso et al. 2004). Hence, DNA

sequence variations like SNPs and InDels may reflect the diversification of

alleles of different species. It is known that wild ancestors generally contain

higher genetic variations than their domesticated descendants (Rakshit et al.

2007; Zhu et al. 2007). SNP and InDel analysis of Pi54 alleles in the present

study also revealed that wild species harbor more variation than the landraces;

i.e. among the wild species, diverse genome types like O. alta (CCDD genome),

O. latifolia (CCDD genome) and O. australiensis (EE genome) exhibited higher

polymorphisms than all other analyzed Pi54 alleles. These genomes might have

diverged long ago during speciation and belong to different genome types.

These species might have accumulated high polymorphism over the time. Non-

synonymous (Ka)/ Synonymous (Ks) ratio was also calculated to know the

significance of observed polymorphism at the nucleotide sequences. The Ka/Ks

ratio also revealed that O. latifolia has the highest significant diversity.

Polymorphism analysis in the upstream region and evolutionary distance

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(calculated by MEGA bioinformatic tool) also support the same concept that O.

latifolia has the highest diversity, which may be due to various factors like

evolutionary time and natural selection. More polymorphism and frame shift

mutations found in Pi54 alleles, suggest that Pi54 alleles themselves contribute

to the continuous variation in the function of the protein.

The low polymorphism among the resistant allele derived from

landraces could be due to the recurrent selective sweeps (process of reduction

or elimination of variations in nucleotide sequences by positive natural

selection) (Huang et al. 2008). Among the alleles derived from wild Oryza

species, O. rufipogon has shown the lowest polymorphism, which may be due to

the fact that O. rufipogon is the immediate ancestor of cultivated rice, O. sativa

(Huang et al. 2008). It could be assumed that compared to wild relatives,

landraces might maintain agronomical valuable alleles that have been

accumulated during their geographical expansion, even if nucleotide diversity

has been reduced by a population bottleneck during domestication (Mikami et

al. 2008).

The sequence variations observed in the various alleles had good

correlation with phenotypic data. In case of alleles derived from landraces,

Pi54AB and Pi54BTJ showed better disease resistance than Pi54Tetep. SNPs and

InDel polymorphisms found in the Pi54AB, Pi54BTJ and promoter allele

sequences might have led to positive selection and showed better resistance.

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Nucleotide and amino acid sequences of these alleles of landraces showed less

diversification with Pi54Tetep, so they might have wider adaptability. However,

other two susceptible alleles, Pi54PA and Pi54Bizor have accumulated several

polymorphisms in their nucleotides which altered their gene structure. In silico

analysis also revealed that these alleles did not code for any functional protein

and thus susceptible phenotype. The wild species O. latifolia, O. barthii and O.

rufipogon (Acc. No. 106507) were phenotypically resistant, which might be due

to two possible reasons that these species showed presence of more

polymorphic Pi54 alleles due to positive selection pressure or these species

might have different blast resistance genes, which could not be differentiated

by isolates used in this study.

Analysis of structural variation revealed that NBS and LRR regions are

more conserved than other parts of the gene, indicating the functional

significance in the resistance genes. LRR region is the major determinant of

recognition specificity for Avr factors (Jones and Jones, 1997; Elli et al. 2000).

In general, LRR region is reported to show more accumulated polymorphism

than NBS region (Jiang et al. 2007a). This may be due to the evolutionary

pressure by races of virulent pathogens on the host (Jones and Jones, 1997).

The present study also revealed that the number of polymorphism is high in

LRR region than NBS, which suggested that LRR region is continuously

evolving and it is the critical domain for its function. This observation is in

conformity with the observation made by with Huang et al. (2008), that they

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also reported more polymorphisms in LRR domain than NBS region in alleles of

Pita. Moreover, LRR domain is not only involved in resistance pathway but also

reported to involve in many basic functions like photomorphogenesis (Tameling

and Joosten 2007), which shows the importance of LRR domain in R genes.

Interestingly, the resistant allele Pi54AB, also had one AA substitution at NBS

region, which might be positive evolution of the allele. Whereas the susceptible

alleles derived from landraces did not code for any functional protein. The

susceptible alleles derived from wild Oryza species, Pi54rufi3 had one AA

substitution at NBS region, while Pi54rufi4 had two and one AA substitution at

LRR and NBS region, respectively. Probably, these mutations at this crucial

region might have affected the protein specificity towards the recognition of the

AVR factors which might have led to susceptibility.

Phylogenetic analysis based on the allelic sequence polymorphisms also

revealed interesting results. It grouped most of the protein coding alleles in one

group and two alleles of sucesptible landraces (Bizor II and PA), which could

not code for any functional protein, were accommodated in another group (Fig.

4.3). In the protein coding allele cluster, Pi54Tetep was grouped with landraces

Pi54BTJ and Pi54AB, which indicated the analogous of the sequence similarity of

the alleles with the reference allele. The alleles, Pi54long, Pi54bar, Pi54rufi3 and

Pi54rufi4 were grouped together, which belong to the AA genome and observed

accumulated similar kind of mutations. The alleles derived from O. alta, O.

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australiensis and O. minuta had higher polymorphism than other ecotypes at

nucleotide level and these ecotypes formed a group (cluster IIb).

The outcome of the allele mining study will lead to various applications;

among them, development of allele specific markers is the major one. In this

study also, an attempt was made to develop functional marker to differentiate

resistance and susceptible Pi54 alleles. The marker developed in this study was

more accurate in genotyping than other linked markers, as it is based on 144

bp InDel, which is located in the exonic region of Pi54 gene. This marker can be

separated in low percentage of agarose gel in less time, since the size difference

between the resistance and susceptible allele amplicons is more than 100 bp.

(Ramkumar et al. 2011).

Promoter region is crucial region, which controls the gene regulation by

influencing the gene expression. Transcription factor binding motifs (TFBMs)

are known to play major role in gene expression. Alteration and

presence/absence of these TFBMs influence the gene regulation, which lead to

differential expression of the gene. Many studies were reported illustrating the

significance of the upstream region. Other than the report on xa13 promoter

mutation (Chu et al. 2006), blast resistance gene Pi54 also showed significant

difference in the phenotypic pattern due to SNP in the promoter region

(Madhav et al. 2005). Grain filling (G1F1) gene also showed a reduced

expression due to the accumulated mutation in the promoter region, in

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comparsion to wild rice species (Wang et al. 2008d). Hence, nucleotide

sequences of promoter regions were analyzed for TFBM or simply motif by

using the TFBM data base, PLACE. Many defense responsive motifs were

distributed all over the upstream regions of most of the promoter alleles

analyzed, which may illustrate the role of TFBMs in the resistance genes. For

instance, BOXLCOREDCPAL, which is involved in elicitor response;

DPBFCOREDCDC3, which is involved in abscisic acid response in Arabidopsis

thaliana were such elements found in the upstream region of all the alleles.

Significant difference was observed between the resistance and susceptible

promoter alleles of Pi54 by showing presence/ absence of ten motifs in either

resistant or susceptible alleles. This also indicates that positive selection for

the resistance alleles was taken place not only at coding region, but also at the

promoter region. However, no common polymorphism was found among the

promoter alleles derived from wild Oryza species, which might be due to high

divergence of the alleles and also that the selected ecotypes belong to different

kind of genomes (AA to EE genome).

5.2. Pita ALLELE AND PROMOTER MINING

Pita gene was identified by Kiyosawa et al. (1971) in indica variety,

which showed consistent resistance reaction to wide spectrum of M. gresea

isolates, which indicates the significance of resistance of this gene. In this

study, Pita alleles were amplified, cloned and sequenced from a four landraces

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and one wild Oryza species (O. latifolia). However, Pita alleles could not be

amplified in other selected wild Oryza species, and this could be due to non

conservation of primer binding sites or limited distribution of Pita allele in

selected rice species.

In contrary to the expectation Pitalati had relatively lower SNPs and

InDels than few landraces. Pita allele of O. latifolia and reference allele from

Tadukan might have originated from same source and positive selection by

nature (selective sweep) might have resulted in less polymorphism. Among the

resistant alleles, PitaKonibora showed the least polymorphism and Ka/Ks ratio

value whereas, Punsimutt (resistant allele) showed higher SNPs at the

nucleotide level; however, this allele shwoed very less polymorphism at amino

acid level (only three amino acid substitutions). Hence both alleles had

undergone positive selection of the nature. Phylogenetic analysis based on the

polymorphisms of Pita alleles also revealed that Pitalati was grouped with the

reference sequence. Since, the PitaPunsimutt and PitaBizor showed higher

polymorphism and also that these two alleles shared many common

polymorphism in comparison with PitaTadukan, they grouped together in the

phylogenetic analysis.

Promoter alleles of Pita were amplified and cloned from one resistant

landrace (Konibora), one susceptible landrace (Bizor II), two resistant wild

Oryza species (O. latifolia (Acc. No. 101207) and O. rufipogon (Acc. No. 106507))

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and two susceptible accessions of O. rufipogon (Acc. No. 106279 and 106512).

Nucleotide level polymorphism in promoter region was very less in comparison

with allelic region of the gene. However, motif level differences were observed at

Konibora promoter allele. Similarly, the promoter allele derived from

susceptible landrace, PitaBizor, showed motif level differentiation. For instance,

MYB1AT (involved in dehydration-responsive gene rd22 of Arabidopsis), but

absent in this PitaBizor promoter allele, otherwise, it was found in all other

resistant genotypes’ promoter region. Surprisingly, none of the promoter allele

derived from wild Oryza species showed polymorphism at motif level, when

compared to the reference promoter sequence, which might be the result of

selective sweep by nature. The phylogenetic analysis also revealed that

susceptible promoter allele PitaBizor was out grouped in the phylogenetic tree, as

this the only susceptible allele, which was different from other resistant

promoter alleles derived from landraces and wild species.

5.3. Pib ALLELE AND PROMOTER MINING

Miyamoto et al. (1996) reported a high resolution molecular map for the

Pib gene, which was mapped on chromosome 2. Later, Wang et al. (1999) have

cloned and characterized the same gene, and reported that this gene also

belonged to the NBS-LRR gene family. Pib is also known for its broad spectrum

resistance to the Japanese pathogen races (Wang et al. 1999). In this study

also, the landraces and wild Oryza species, which were collected from India

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and various parts of the world (through IRRI), had higher polymorphism as

compared to the reference sequence. The divergence deviation score shows that

all the selected and sequenced Pib alleles were nearly one, which indicated high

divergence. Among the ecotypes, blast resistant ecotype, Krengosa had lowest

Ka/Ks ratio, indicating less mutation, as compared to the reference sequence.

The phylogenetic analysis also revealed that the analyzed Pib alleles were

different from the reference sequence. PibKrengosa allele was grouped with the Pib

reference allele (PibEngkatek) sequence and showed more similarity to the

PibEngkatek than other alleles.

Promoter sequence of analyzed alleles also showed considerable

sequence difference. Comparison of promoter allele sequence with reference

sequence revealed that selected alleles’ promoter regions had 1500 bp deletion

or insertion in the reference sequence. Hence, 500 bp of promoter region only

could amplify from the selected landraces. No amplicon could be generated in

the wild Oryza species. Comparison of promoter alleles revealed that PibSearcher

(resistant allele) had unique motif, related to ABA response insensitive, while

the susceptible promoter allele PibPA showed five unique motifs, which were

absent in all other analyzed Pib promoter sequences. These motif include

MYBST1 (core motif of MybSt1 (a potato MYB homolog) binding site found in

Solanum tuberosum); SREATMSD (sugar-repressive element (SRE) found in

Arabidopsis); TATCCACHVAL21 ("TATCCAC box" is a part of the conserved cis-

acting response complex (GARC), was necessary for a full GA response found in

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Hordeum vulgare); TATCCAOSAMY ("TATCCA" element, which involved in sugar

and alpha-amylase regulation in rice) and TATCCAYMOTIFOSRAMY3D

(responsible for the sugar and amylase repression). The phylogenetic analysis

also revealed that the PibEngkatek was much different from all other alleles

derived from landraces, as it was out grouped from the cluster. Moreover, there

was no significant deviation among the alleles derived from landraces, which

could be due to similar kind of accumulated mutations. The reference allele

was characterized from the japonica variety and all analyzed alleles were

derived from indica group. Hence these rice landraces might have accumulated

the same kind of mutations, which may differ from the reference sequence.

Core promoter is the minimum promoter length required for expression

of a particular gene. In contrast to the serial deletion of DNA fragment with

restriction enzymes (Better et al. 1985), the deletion analysis was performed

through structural PCR (Guo et al. 2010), because of its ease and precise

selection of promoter fragments for the expression analysis. Pita promoter of

resistant landrace, Konibora was used for this identification of core promoter.

Pita showed a good resistance for wide range of isolates and it belongs to NBS-

LRR type resistance gene. Pita promoter is a constitutive promoter and hence it

can represent a good model for wide range of resistance genes. Due to these

reasons, Pita allele was considered for the core promoter identification study. If

the core promoter can be demarked with optimal length, it can be used for

transgenic approach for the same gene as well as other resistance/ target gene.

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This core promoter can be demarked with deletion analysis (Better et al. 1985;

Guo et al. 2010), in which validation of TFBMs also can be performed.

For the core promoter identification, pCXGFP binary vector was used

since, the utility and high efficiency of this vector was proved by Chen et al.

(2009) in rice. This binary vector has GFP as reporter marker without

promoter. The reporter gene - GFP has more benefits i.e. the presence of

expressed GFP can be screen with the fluorescent microscope or simple UV

light and the same tissue can be re-used unlike the GUS assay in which the

stained tissue cannot be re-used. Expression of GFP provided a greater

precision in selection of transgenic calli, plantlets and whole plants. This

binary vector also has Hygromycin phosphotransferase (hpt) as plant selection

marker, which can be used in selection of transgenic calli.

Different length of the selected Pita promoter was derived by amplifying

the defined portion of the promoter region by PCR. Accordingly, the primers

were designed to amplify -1 to -449 bp (Del I), -1 to -935 bp (Del II) and -1 to -

1592 bp (Del III) regions and these portions were amplified individually and

inserted in pCXGFP. In order to confirm the orientation of the inserted

promoter, PCR screening method was used, for which the primers were

designed in such a way that the amplicons will be generated only if the

promoter region was in right direction. Costa et al. (1994) demonstrated the

efficiency of PCR based determination of orientation of the insert in a vector for

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the first time. With this method, two things were concluded i.e. first, the

presence or absence of recombinant vector and secondly, the orientation of the

promoter region. This particular method is advantageous over restriction

enzyme digestion i.e easy in use; rapid confirmation of the presence as well as

orientation; also saves time and cost. The confirmed recombinant plasmids -

constructs were isolated and were mobilized to Agrobacterium strain EHA 105.

Agrobacterium EHA 105, which was reported to be more efficient than the LBA

4404 (Cao et al. 1998; Zhang et al. 2008) and many research group have used

this strain for Agrobacterium mediated gene transformation to plants.

Motif analysis at these upstream sequences with motif database -

PLACE revealed the presence of diverse TFBMs at different positions. Three

different lengths of promoters had different motifs, some are common and some

are unique to the particular promoter length. The Del I had 10 unique motifs,

which were not present in -450 to -1592bp region of Pita promoter of Konibora

(Table 5.1), which includes, MYB1LEPR (involved in defense related gene

activation in Lycopersicon esculentum); IBOXCORENT (“I box core motif” which

is known to be involved in light response expression of the gene in Nocotiana

plumbaginifolia); SV40COREENHAN (SV40 core enhancer); PALBOXAPC

(elicitor or light responsive response in Petroselinum crispum) and

PYRIMIDINEBOXHVEPB1 ("Pyrimidine box" found in the barley and required

for GA induction).

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Table 5.1 Unique Motifs present in the different promoter length and their

signal sequence with position (The green color indicates the motifs present in

-1 to -449 bp; blue color indicates the motifs present in -450 to – 936; red color

indicates the motifs present in -937 to – 1592).

S. No Name Location Signal sequence

1 IBOXCORENT 19 GATAAGR

2 MYB1LEPR 73 GTTAGTT

3 TRANSINITDICOTS 152 AMNAUGGC

4 SV40COREENHAN 210 GTGGWWHG

5 PALBOXAPC 216 CCGTCC

6 PYRIMIDINEBOXHVEPB1 305 TTTTTTCC

7 SPHCOREZMC1 340 TCCATGCAT

8 LTRE1HVBLT49 407 CCGAAA

9 MYBPZM 433 CCWACC

10 POLASIG1 520 AATAAA

11 TBOXATGAPB 581 ACTTTG

12 ACGTTBOX 619 AACGTT

13 S1FBOXSORPS1L21 630 ATGGTA

14 MYB26PS 651 GTTAGGTT

15 SEF3MOTIFGM 742 AACCCA

16 PRECONSCRHSP70A 899 SCGAYNRNNNNNNNNNNNNNNNHD

17 SEBFCONSSTPR10A 908 YTGTCWC

18 NTBBF1ARROLB 913 ACTTTA

19 ANAERO3CONSENSUS 920 TCATCAC

20 MYBPLANT 941 MACCWAMC

21 REALPHALGLHCB21 992 AACCAA

22 GMHDLGMVSPB 1035 CATTAATTAG

23 WBBOXPCWRKY1 1049 TTTGACY

24 SEF1MOTIF 1072 ATATTTAWW

25 AMYBOX1 1131 TAACARA

26 GAREAT 1131 TAACAAR

27 MYBGAHV 1131 TAACAAA

28 AACACOREOSGLUB1 1132 AACAAAC

29 MYB2CONSENSUSAT 1145 YAACKG

30 MYBCOREATCYCB1 1146 AACGG

31 LTRECOREATCOR15 1169 CCGAC

32 SORLIP2AT 1398 GGGCC

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33 INTRONLOWER 1455 TGCAGG

34 INRNTPSADB 1508 YTCANTYY

35 UPRMOTIFIIAT 1565 CCNNNNNNNNNNNNCCACG

36 TATCCACHVAL21 1576 TATCCAC

37 TATCCAYMOTIFOSRAMY3D 1576 TATCCAY

38 ABRERATCAL 1580 MACGYGB

Along with above described motifs, Del II had nine unique motifs, which were

not present in either -1 to -552 or -936 to -1592. This includes TBOXATGAPB

(light-activated transcription elements in the Arabidopsis thaliana);

PRECONSCRHSP70A (consensus sequence of plastid response element in

Chlamydomonas reinhardtii); SEBFCONSSTPR10A (binding site of the potato

silencing element binding factor); NTBBF1ARROLB (required for tissue-specific

expression and auxin induction in Agrobacterium rhizogenes);

ANAERO3CONSENSUS (found and involved in the fermentative pathway of Zea

mays and Arabidopsis thaliana and Pisum sativum).

The Del III also had 19 unique motifs along with the motifs present in

Del I and Del II. They are: GMHDLGMVSPB (required for high level, light

regulated, and tissue specific expression in Oryza sativa); REALPHALGLHCB21

("REalpha" and Lhcb21 gene promoter found in Lemna gibba);

WBBOXPCWRKY1 (WRKY proteins bind specifically to the DNA sequence in

Arabidopsis thaliana); SEF1MOTIF (observed in soybean beta-conglicinin);

GAREAT (GA-responsive element at Arabidopsis thaliana); MYBGAHV (central

element of gibberellin (GA) response complex in rice); AACACOREOSGLUB1

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(core of AACA motifs found in rice glutelin genes, involved in controlling the

endosperm-specific expression); MYB2CONSENSUSAT (MYB recognition site

found in the promoters of the dehydration-responsive gene of Arabidopsis

thaliana). MYBCOREATCYCB1 ("Myb core" activation of reporter gene in

Arabidopsis thaliana); LTRECOREATCOR15 (core of low temperature

responsive element (LTRE) and ABA responsive element in A. thaliana);

TATCCACHVAL21 (GA-responsive element (GARE) at Hordeum vulgare);

TATCCAYMOTIFOSRAMY3D ("TATCCAY motif" found in rice and responsible

for sugar repression) and ABRERATCAL ("ABRE-related sequence" and found at

Ca(2+)-responsive upregulated genes in Arabidopsis).

The constructs for Del I, Del II and Del III with above mentioned unique

motifs and motifs, which were present in more than one place or constructs,

were transferred to rice plant (Taipei 309) by Agrobacterium mediated

transformation method. The positive plants harboring Del constructs were

screened using PCR targeting the GFP gene. The positive plants were further

confirmed by the Pita promoter length specific primers. In order to check the

expression of GFP gene, the positive plants with different constructs were

observed under fluorescent microscope. Surprisingly the plants with all the

three constructs were observed with green fluorescent, which suggested that

these three constructs with their promoter length were enough to express the

GFP gene. This may be due to the presence of basic elements of TATA box,

CAAT box, -300 element, GATA box, etc. in the Del I constructs itself. As these

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elements were also present in Del II and Del III constructs, they also expressed

the GFP gene. Total RNA was isolated from leaves of transgenic plants

harboring these three different promoter length promoter constructs without

any aritificial pathogen inoculation since it is a constitutive promoter, which

will not affect by the pathogen interaction and expressed throughout the plant.

From this total RNA, cDNA was prepared. The expression of GFP was confirmed

from the cDNA, by amplifying the GFP gene using GFP specific primers, which

was located in exonic region of the gene and hence amplified in the cDNA also.

As all the constructs could express GFP gene, in order to check whether

there was any difference in the level of GFP expression with different

constructs, real time PCR analysis was performed with the actin as internal

control. Actin was selected as internal control, as it is a housekeeping gene;

expressing constantly throughout the cell life. The real time PCR analysis

revealed that there was no significant difference between Del I and Del II as the

expression level was equal while Del III with the maximum motif showed higher

expression than the Del I and II. The CT value of these constructs revealed that

the Del III construct could express the gfp two fold higher than other two

constructs. This may be because of the presence of important motifs like

GMHDLGMVSPB, which is required for high level expression of genes in rice

(Lescot et al., 2002) and LTRECOREATCOR15 which was low temperature

responsive gene expression in A. thaliana; which were absent in other two

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constructs. Hence, the presence of these motifs would lead to the better

expression of this Del III construct in comparison to Del I and Del II constructs.

Guo et al. (2010) also attempted to identify the core promoter of CBL1

gene (calcium sensor gene that regulates drought, cold and salt signals in

Arabidopsis) by selecting five different promoter lengths from -1659, -1414, -

1048, -296 to -167 and concluded that all these promoter lengths could

express the gene and also reported that full length promoter (-1659 bp) was

expressing better than all other promoters. Twell et al. (1991) also

characterized the two promoters of tomato genes (LAT52 and LAT59) and

reported that less than 200 bp promoter region is enough to express the genes.

Tebbutt and Lonsdale (1995) did a deletion analysis to characterize the

promoter region of tobacco gene - pollen specific polygalacturonase (Npg1) and

segmented the upstream sequence into four different regions i.e. modulation

domain, basic promoter, core promoter and minimal promoter. Swapna et al.

(2011) also attempted to characterize and analyze the putative regulatory

motifs of pollen allergen gene (OSIPA) by deletion analysis using different

lengths of promoters (-1567, -1272, -966, -617 and -199 bp) and revealed that

all the constructs could express the gene; however, -1567 bp length promoter

could express the gene with maximum expression. In the present study also,

all the three constructs could express the gene while Del III (-1592 bp promoter

region) construct could express the gene with maximum expression.

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In conclusion, allele mining of blast resistance genes has led to many

interesting findings. Superior alleles of Pi54 were identified in the landraces

from Ammano Bavo and Boha Tulasi Joha, which showed better resistance

than Tetep. The observed polymorphisms at ORF and promoter region of these

alleles might have led to the positive selection of these alleles. Functional

marker for Pi54 was developed to differentiate the Pi54 resistant and

susceptible genotypes. Different sources of resistance were identified for Pita

(Konibora and Punsimutt) and Pib (Sercher and Krengosa). About 16, 5 and 3

novel alleles of Pi54, Pita and Pib respectively, were isolated from different

landraces and wild Oryza species. Allele mining in various landraces and wild

species led to the understanding of diversity of alleles and possible utility of

these alleles from landraces. Promoter mining of Pi54 alleles led to the

identification of Pi54 specific cis elements which differentiates the resistance

and susceptible promoter alleles i.e. motifs AGMOTIFNTMYB2 (AG-motif, which

was found at the promoter region of defense responsive gene - phenylalanine

ammonia lyase which can be induced by various stresses),

ANAERO2CONSENSUS (motif found in the anaerobic genes), ERELEE4

(ethylene responsive element, found in Lycopersicon chilense),

HEXMOTIFTAH3H4 (type 1 element, which plays important role in replication

in Triticum aestivum), MYBPLANT (plant MYB binding site at promoters of

phenylpropanoid biosynthetic genes of tobacco), TGACGTVMAMY ("TGACGT

motif" found in the Vigna mungo). The above mentioned cis elements were

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present only in the reference and resistance promoter alleles while they were

absent in susceptible promoter allele. Promoter mining of other blast resistance

genes led to the identification of putative and novel TFBMs and information on

motifs diversity, which will help in understanding their regulation. The novel

TFBMs identified by reliable and widely used motif prediction tools, are a good

source for motif validation experiments like CHiP, which are time consuming

and costly. Identification of core promoter led to the demarcation of length of

Pita core promoter and identification of motifs, which were probable response

for the higher expression of Del III Pita promoter.

5.4. FUTURE PROSPECTS

The identified superior alleles derived from landraces (Pi54AB and

Pi54BTJ) can be validated for their efficacy by transferring these alleles to the

susceptible genotype. After confirmation of their strong resistance, these alleles

can be used in molecular breeding with/ without the Pi54 allele derived from

Tetep for developing broad and durable resistant varieties. The identified novel

resistance alleles of Pib and Pita also can be good resource for blast resistance

breeding programs.

Upon the comparison of susceptible and resistance Pi54 allelic

sequences revealed the presence of 45 SNPs and five InDels in the coding

region and 30 SNPs and 7 InDels in the non-coding region, which differentiate

the resistant and susceptible Pi54 alleles. Among them, the largest InDel (144

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bp) was validated in this study for its presence/ absence in the diverse

genotypes, which revealed that this InDel differentiated most of the resistant

and susceptible cultivars. Similarly, the other observed nucleotide changes

(SNPs and InDels) present in the coding and non-coding regions can be used

for knowing the different haplotypes distribution and their haplotype structure

among population.

The candidate novel motifs like ACCCCAGG (unknown function), which

differentiates the resistance and susceptible Pi54 promoter alleles by its

presence only in the resistance alleles, leads to difference in gene expression

due to presence/ absence of this motifs, can be validated through motif

deletion analysis. Similarly, CGAGGG, CAGCGCAG (function not known)

motifs, which found only in the resistant promoter alleles of Pita can be also

validated for its role in the expression of defense reaction. The other motifs

found in the Pi54 and Pita promoter alleles, which are reported for non-defense

response role, can also be validated for its role in resistance reaction. For

finding the rice based constitutive promoters, the expression of constructs with

different Pita promoter length can be compared with expression of CMV 35s

promoter, so as to replace the 35s promoter with rice specific plant promoter in

trans-gene expression studies.