BACKGROUND
pigeonpea  millsp) is one of the major grain legume crops of the tropical and subtropical regions of the world  <cit> .
it is the only cultivated food crop of the cajaninae sub-tribe and has a diploid genome with  <dig> pairs of chromosomes  and a genome size estimated to be  <dig> mbp  <cit> .
the genus cajanus comprises  <dig> species most of which are found in india, australia and one is native to west africa.
pigeonpea is a major food legume crop in south asia and east africa with india as the largest producer  followed by myanmar  and kenya   <cit> .
it plays an important role in food security, balanced diet and alleviation of poverty because of its diverse usages as a food; fodder and fuel wood  <cit> .
several abiotic  and biotic  stresses, are serious challenges for sustainable pigeonpea production to meet the demands of the resource poor people of several african and asian countries.
fusarium wilt  caused by fusarium udum is an important biotic constraint in pigeonpea production in the indian subcontinent, which results in 16-47% crop losses  <cit> .
the fungus enters the host vascular system at the root tips through wounds or invasion made by nematodes, leading to progressive chlorosis of leaves, branches, wilting and collapse of the root system  <cit> .
in india alone, the loss due to this disease is estimated to be us $ <dig> million and the percentage of disease incidence varies from  <dig>  to  <dig> %  <cit> .
sterility mosaic disease  caused by pigeonpea sterility mosaic virus  is one of the wide-spread diseases of pigeonpea, which is transmitted by an eriophyid mite .
the disease is characterized by the symptoms like bushy and pale green appearance of plants followed by reduction in size, increase in number of secondary and mosaic mottling of leaves and finally partial or complete cessation of reproductive structures.
some parts of the plant may show disease symptoms and other parts may remain unaffected  <cit> .
due to the above mentioned factors combined with limited water resources to the fields in the semi-arid tropic regions, where the crop is grown, the productivity has remained stagnant at around  <dig>  t/ha during the past two decades  <cit> .
with the advent of genomic tools such as molecular markers, genetic maps, etc., conventional plant breeding has been facilitated greatly and improved genotypes/varieties with enhanced resistance/tolerance to biotic/abiotic stresses have been developed in several crop species  <cit> .
in case of pigeonpea, however, a very limited number of genomic tools are available so far  <cit> .
for instance,  <dig> microsatellite or simple sequence repeat  markers  <cit> ,  <dig> expressed sequence tags , at the time of undertaking the study, were available in pigeonpea.
for enhancing the genomic resources in pigeonpea, transcriptome sequencing to generate ests should be a fast approach.
ests, which are generated by large-scale single pass sequencing of randomly picked cdna clones, have been cost - effective and valuable resource for efficient and rapid identification of novel genes and development of molecular markers  <cit> .
further, ests have been employed in bioinformatic analyses to identify the genes that are differentially expressed in various tissues, cell types, or developmental stages of the same or different genotypes  <cit> .
in view of above facts, this study was undertaken to obtain a comprehensive resource of fw- and smd-responsive ests in pigeonpea with the following objectives:  generation of fw- and smd- responsive ests,  functional annotation of assembled unigenes,  in silico identification of putative fw- and smd- responsive genes, and  development of novel ssr and snp markers in pigeonpea.
RESULTS
root tissue is the site for fusarium udum infection, the causal fungal agent of fusarium wilt in pigeonpea.
with an objective to evaluate the transcriptional responses after infection of roots by f. udum, six unidirectional cdna libraries were constructed.
these are from each of fw- infected root tissues of resistant  and susceptible  genotypes at different stages viz.
<dig>   <dig>   <dig>   <dig>   <dig>   <dig> days after inoculation .
infected roots were examined by light microscopy upon harvest at different stages.
the severity of wilt disease in both susceptible and resistant genotype was observed in longitudinal sections of stem and root vascular region at  <dig> and  <dig> dai .
likewise for smd, leaf tissue is the specific site of infection and therefore leaf samples of smd infected genotypes, 'icp 7035'  and 'ttb 7'  were harvested at  <dig> and  <dig> days after sowing .
rna was extracted and consequently unidirectional cdna libraries were constructed .
generation of fw- and smd- responsive ests
a total of  <dig> unidirectional cdna libraries were constructed from all the four genotypes i.e.
'icpl 20102' and 'icp 2376'; 'icp 7035' and 'ttb 7' which represent parents of mapping population segregating for fw and smd, respectively.
using sanger sequencing approach,  <dig>  ests were generated from root cdna libraries of 'icpl 20102' and  <dig>  from 'icp 2376'.
similarly,  <dig>  ests were generated from each leaf cdna libraries of smd- responsive genotypes, 'icp 7035' and 'ttb 7'.
details of est generation from different cdna libraries are given in figure  <dig>  in brief, a total of  <dig>  ests were generated and after stringent screening for shorter  and poorer quality sequences,  <dig>  high quality ests were obtained, with an average varied-read length of  <dig> bp .
all est sequences were deposited in the dbest of genbank under accession numbers gr <dig> to gr <dig> and gr <dig> to gr <dig>
pigeonpea est assembly
with an objective to minimize redundancy, clustering and assembly was done for different est datasets to define unigenes for  fw-responsive ests,  smd-responsive ests,  fw- and smd-responsive ests, and  the entire set of pigeonpea ests including those from the public domain.
these unigene  sets were referred to as ug-i, ug-ii, ug-iii and ug-iv, respectively.
the ug-i comprised of  <dig>  unigenes with  <dig> contigs and  <dig>  singletons by clustering of  <dig>  high quality ests.
similarly, for ug-ii, clustering of  <dig>  high quality sequences resulted in  <dig>  unigenes .
based on clustering of all the  <dig>  high quality sequences generated in this study, the ug-iii was defined with  <dig>  unigenes .
the cluster analysis of  <dig> ests available in the public domain along with  <dig>  pigeonpea ests resulted in ug-iv that included  <dig>  unigenes with  <dig> contigs and  <dig>  singletons.
the number of ests in a contig ranged from  <dig> to  <dig>  with an average of  <dig> ests per contig.
as expected, contigs with two est members exhibited a higher percentage  than contigs with three or more est members .
comparison of pigeonpea unigenes with other plant est databases
all the four sets of unigenes i.e.
ug-i, ug-ii, ug-iii and ug-iv were analyzed for blastn similarity search against available est datasets of legume species namely chickpea , pigeonpea , soybean , medicago , lotus , common bean  and three model plant species namely arabidopsis , rice  and poplar .
an e-value significant threshold of ≤ 1e- <dig> was used for defining a hit.
detailed results of blastn analyses for all the four unigenes sets are given in table  <dig>  for instance, analysis of ug-iii found highest identity of  <dig> % with soybean, followed by cowpea , medicago , common bean , lotus , and the least identity with chickpea .
comparative blastn analysis of pigeonpea unigenes with est databases of model plant species showed high identity with poplar , followed by arabidopsis  and the least similarity with rice .
of  <dig>  unigenes,  <dig>   showed significant identity with ests of at least one plant species analysed, while  <dig>  showed significant identity across all the plant est databases in this study.
it is also interesting to note that  <dig> unigenes did not show any homology with the legume species examined.
high quality ests generated
<dig>
<dig>
<dig>
<dig>
significant similarity with ests of at least
significant similarity with ests of at least one
significant similarity across ests of all model plant
significant similarity with ests of at least one
significant similarity across ests of all plant
to identify the putative function of all the unigenes compiled in this study, the unigenes from all the four sets  were compared against the non-redundant uniprot database, using the blastx algorithm.
at a significant threshold of ≤ 1e- <dig>   <dig>   of ug-i,  <dig>  of ug-ii,  <dig>   of ug-iii and  <dig>   of ug-iv unigenes showed significant similarity with known proteins .
details of blastx and blastn analyses against uniprot database for all four unigene sets are provided in additional files  <dig>   <dig>   <dig> and  <dig>
functional categorization of pigeonpea unigenes
the unigenes from all the four sets that showed a significant hit  against the uniprot database were further categorized into functional categories.
as a result,  <dig>  of ug-i,  <dig>  of ug-ii,  <dig>  of ug-iii and  <dig>   of ug-iv unigenes were categorised into three principal go categories i.e.
biological process, molecular function and cellular component.
like in earlier studies of this nature, it was observed that one gene could be assigned to more than one principal category, thus the total number of go mappings from each category exceeded the number of unigenes analyzed.
details on full list of gene annotation for significant hits of four unigene sets are given in additional file  <dig>   <dig>   <dig> and  <dig>  for instance, of  <dig>   unigenes of ug-iii, only  <dig>  were assigned to three principle categories.
as a result, a total of  <dig> were grouped under biological process,  <dig> under molecular function and  <dig> under cellular component .
under the biological process category, cellular process accounted to  <dig>  followed by metabolic process , biological regulation  and response to stimulus .
in the cellular component category,  <dig> unigenes coded for cell part,  <dig> to organelle, and  <dig> to organelle part.
in the last category of molecular function, majority of the unigenes were involved in binding  and catalytic activity .
the remaining  <dig> unigenes which could not be classified into any of the three go categories were grouped as "unclassified".
the distribution of unigenes  along with corresponding gene ontology  categories are provided in additional file  <dig>  based on go annotation, enzyme commission ids were also retrieved from the uniprot database to get an overview of unigenes  putatively annotated to be enzymes.
the major group of unigenes are included under oxidoreductases  followed by transferases , hydrolases , lyases , ligases  and isomerases .
similar patterns of distribution were observed in all the remaining unigene sets.
in silico expression analysis
the identification of differentially expressed genes among specific cdna libraries of fw- and smd-responsive genotypes based on est counts in each contig was done using a web statistical tool ideg.
<dig>  as a result,  <dig> genes were identified to be differentially expressed between 'icpl 20102'  and 'icp 2376'  genotypes, similarly,  <dig> genes were differentially expressed between 'icp 7035'  and 'ttb 7'  genotypes .
to assess the relatedness of each library and expressed genes in terms of expression pattern, a cluster analysis on the basis of est abundance in each contig was performed  <cit> .
of the  <dig> contigs , that were subjected to r-statistics  <cit>  only  <dig> contigs were normalized with a true positive significance  and were eventually subjected to hierarchical clustering analysis .
the correlated gene expression pattern of all normalized  <dig> contigs/genes is displayed in figure  <dig>  all the  <dig> fw- derived libraries were grouped into a single cluster, while all the four smd- challenged libraries were grouped into another cluster.
about  <dig> genes were highly expressed in smd- challenged libraries than in fw- challenged libraries and can be attributed to high accumulation of defence proteins during smd infection.
in the cluster of fw- challenged libraries, the 'icpl 20102'- <dig> dai library was distantly placed between fw- susceptible challenged libraries 'icp 2376' -  <dig> dai and 'icp 2376' -  <dig> dai.
each cluster represents a different pattern of gene expression as shown in figure  <dig>  based on the clustering pattern and library specificity, clusters i and iv were further divided into sub-clusters .
the above results indicated that the pattern and percentage of genes expression varied according to severity of the stress in specific library.
in cluster i,  <dig> %  of total genes were grouped and further sub divided into two groups with each sharing  <dig> %  and  <dig> %  genes, respectively.
similarly, cluster ii and cluster iii accounted for  <dig> %  and  <dig> %  genes and the largest cluster iv, included  <dig> %  of total genes with three sub groups iva, ivb and ivc each sharing  <dig> % , 10%  and 45%  of genes, respectively.
cluster analysis also showed high level expression of genes related to chloroplast/photosystem related proteins , developmental proteins , cellular proteins , metabolic proteins , defence/stimulus responsive proteins , protein specific binding proteins  and few uncharacterized proteins .
marker discovery
est based markers can assay the functional genetic variation compared to other class of genetic markers and hence were targeted for marker development  <cit> .
the unigene set based on generated ests in this study as well as the ones available in public domain was used for development of simple sequence repeats  and single nucleotide polymorphism  markers.
identification and development of genic microsatellite markers
the entire set of  <dig>  pigeonpea unigenes derived from ug-iv was used to identify the ssrs using misa  tool  <cit> .
as a result a total of  <dig>  ssrs were identified at the frequency of 1/ <dig> bp in coding regions .
<dig> ests contained more than one ssr and  <dig>  ssrs were found as compound ssrs.
in terms of distribution of different classes of ssrs i.e.
mono-, di-, tri-, tetra-, penta- and hexa-nucleotide repeats, mononucleotide ssrs contributed to the largest proportion .
only a limited number of ssrs of other classes were found.
for instance, di- and tri- nucleotide ssrs accounted for  <dig>  and  <dig> , respectively.
on the other hand,  <dig> tetrameric,  <dig> pentameric and  <dig> hexameric microsatellites were present .
while using the criteria for class i  and class ii ssrs  as used by temnykh and colleagues  <cit>  and kantety and colleagues  <cit> , on all ssrs  <dig> ssrs represented class i while  <dig>  ssrs represented class ii .
in general, mononucleotide ssrs are not included for primer designing and synthesis.
however, as only a very limited number of ssr markers are currently available for pigeonpea in public domain and in a separate study some mononucleotide ssrs were found polymorphic  <cit> , primer pairs were designed for  <dig> ssrs including mononucleotide ssrs.
a total of  <dig> primer pairs were considered for validation after excluding the primers for monomeric ssr motifs and compound ssrs with mononucleotide repeats.
however based on repeat number criteria, such as  <dig> minimum for di-, tri-, tetra-, penta-nucleotides, primer pairs were synthesized for  <dig> ssrs.
the details of newly developed pigeonpea est-ssr primers along with corresponding ssr motif, primer sequence, annealing temperature and product size are provided in additional file  <dig>
newly synthesized  <dig> markers were analyzed on  <dig> elite pigeonpea genotypes .
as a result,  <dig>  primer pairs provided scorable amplified products and  <dig> primer pairs produced a number of faint bands indicative of non-specific amplifications.
a total of  <dig>  markers showed polymorphism with 2- <dig> alleles with an average of  <dig> alleles per marker in genotypes examined.
these markers showed a moderate pic value ranging from  <dig>  to  <dig>  with an average of  <dig>  .
to evaluate the genetic variability within a diverse collection of pigeonpea accessions which are parents of different mapping populations segregating for important agronomic traits and also to determine genetic relationship among them, phylogenetic analysis on the basis of dissimilarities was performed using ntsys software package.
the upgma cluster diagram showed clear segregation of wild and cultivated species .
snp discovery and identification of caps markers
snps are an important class of molecular markers which are becoming more popular in recent times.
to enhance the reliability of snps identification, the snp which occurred in a contig ≥  <dig> ests from more than one genotype was considered.
in silico analysis showed a total of  <dig> snps in  <dig>  contigs with a frequency of 1/ <dig> bp .
with an objective of validating these in silico identified snps, as an example,  <dig> contigs were used to generate pcr amplicons and sequence four genotypes namely 'icpl 20102', 'icp 2376', 'icp 7035' and 'ttb 7'.
while a scorable and sequenceable amplicon was obtained in case of  <dig> contigs , the scorable amplicons were not obtained in case of four contigs .
sequencing of amplicons for all the four genotypes for all the six contigs showed occurrence of snps as predicted in silico .
for instance, for contig  <dig>  a comparison of the amplified dna sequences for four genotypes  with the  <dig> est sequences coming from two genotypes  showed the occurrence of the same snp g to c between 'icp 7035' and 'ttb 7' .
in order to perform cost-effective and robust genotyping assay for the detected  <dig> snps in  <dig> contigs, efforts were made to identify the restriction enzymes that can be used to assay snps via cleaved amplified polymorphic sequence  assay.
results indicated that snps present in  <dig> contigs can be evaluated by using caps assay .
discussion
plants are known to have developed integrated defence mechanisms against fungal and viral infections by altering spatial and temporal transcriptional changes.
the est approach was successfully utilized in identification of disease-responsive genes from various tissues and growth stages in chickpea  <cit> , lathyrus  <cit> , soybean  <cit> , rice  <cit>  and ginseng  <cit> .
many earlier studies have shown that resistant genotypes have efficient mechanisms for stress perception and enhanced expression of defence-responsive genes, which maintain cellular survival and recovery  <cit> .
hence, the present study was undertaken to identify catalog of defence related genes in response to fw and smd infection in pigeonpea by generating ests from different stress challenged tissues at various time intervals.
generation of cdna libraries and unigene assemblies
roots provide a structural and physiological support for plant interactions with the soil environment by conducting transport of water, ions and nutrients.
plants are encountered with many biotic stress factors which includes bacterial, fungal and viral infection.
roots and leaves are the primary sites of infection by these organisms.
therefore, a total of  <dig> cdna libraries were generated at different time intervals to specifically target the roots infected with fusarium udum and leaves infected with smd.
in total  <dig>  high quality ests were generated from fw- and similarly  <dig>  high quality ests from smd- challenged genotypes.
earlier, at the time of analysis in november  <dig>  the public domain consisted of only  <dig> ests for pigeonpea.
thus the present study contributes approximately 10-fold increase in the pigeonpea est resource and an addition of  <dig>  pigeonpea unigenes .
functional annotation of pigeonpea unigenes
homology searches  against other plant ests and functional characterization was done for all the defined unigene datasets .
of the  <dig>  unigenes  assembled from all the pigeonpea ests,  <dig>   had significant identity with ests of at least one plant species analyzed,  <dig>  unigenes showed significant identity with ests of all analyzed plant species in the study, while  <dig>  were found to be novel to pigeonpea.
a high significant identity was observed with soybean , and the least percentage of similarity was observed with chickpea  .
a similar blastn results were observed for the remaining three unigenes sets  against the ests of plant species surveyed.
comparative analysis of newly defined ug-iii dataset  with  <dig> public domain pigeonpea ests showed that only  <dig>  shared identity and indicated that our est sequencing study identified  <dig>   new set of pigeonpea unigenes.
relatively, very low similarity of  <dig> % with lotus and  <dig> % with medicago was observed compared to soybean and cowpea than other legume species.
these observations are in accordance with phylogenetic relationships of legumes  <cit> .
the pigeonpea ests showed higher similarity to legume ests databases  of the legume species than monocot species .
comparative analysis of pigeonpea ests with monocot species like rice  showed that the percentage of significance is much lower compared to any other legume species, inspite of larger est repository.
this is clearly attributed to phylogenetic divergence between dicots and monocots in course of evolution.
these comparisons also indicate that several unigenes that were absent in analysed non-legumes but present in all legume species may be specifically confined to legumes.
blastx analyses indicated that those ests without significant identity to any other protein sequences in the existing database may be novel and involved in plant defence responses.
hence, this novel est collection represented a significant addition to the existing pigeonpea est resources and provides valuable information for further predictions/validation of gene functions in pigeonpea.
a comprehensive comparison of functionally categorized unigenes of all the four unigenes data sets  showed a similar distribution.
a large number of unigenes were involved in cell part, organelle, binding, organelle part, metabolic and cellular process among the significantly annotated ones.
these observations are consistent with the earlier reported functional categorization studies in rice  <cit> , soybean  <cit> , barley  <cit>  and tall fescue  <cit> .
however, the sequences encoding activities related to categories such as biological regulation and response to stimulus are  <dig> and  <dig> incase of fw-responsive ests compared to  <dig> and  <dig> in case of smd-responsive ests.
this was possibly due to the fact that the ests generated from fw- challenged root libraries were most abundantly involved in stimulus to pathogenesis and ests derived from smd stress are chloroplast binding proteins.
earlier studies such as lee and colleagues  <cit> , ablett and colleagues  <cit> , also reported that photosynthesis-related proteins were the most prevalent from aerial parts of the plant, which would help to make energy related activities such as cell division, growth, elongation and development.
similarly in this study, photosynthesis related genes were identified in larger proportion  in smd-responsive cdna libraries derived from leaf tissues.
in silico differential gene expression
the invasion of pathogen not only results in expression of novel genes/transcripts, but also in altering the abundances of different ests resulting in induction or repression.
this was evident from differential expression of  <dig> genes between fw-responsive genotypes and  <dig> genes between smd-responsive genotypes.
it is however, important to mention that in silico method of gene expression is not the ideal method to identify the differentially expressed genes.
nevertheless, as large scale est data were generated from fw- responsive and smd- responsive genotypes, an effort like some earlier studies  <cit>  was made to identify some putative genes differentially expressed in fw- and smd- resistant and sensitive genotypes.
validation of these candidate genes by northern analysis or real-time quantitative pcr analysis is essential before these candidate genes are deployed in some other studies.
significant number of unigene sequences related to proteins like kinases, phosphatases, peroxidases, ribonucleases, endochitinases, glucanases and hormones like abscisic acid responsive  genes were identified to be differentially expressed and are known to play a vital role in defence mechanism.
for example, the cell wall degrading enzymes like endochitinases  implicate a major defence mechanism against pathogen  <cit> .
similarly, kinases play a major role in the plant's recognition to pathogen  <cit> .
for instance, chitinase protein , a class of pathogenesis related  proteins with bi-functional role in lysozyme/chitinase activity involved in random hydrolysation of n-aetyl-beta-d-glucosaminide-beta linkages in chitin and chitodextrins during systemic acquired resistance , was expressed at higher concentrations in fw-responsive resistant genotype  compared to susceptible genotype .
the high expression levels of chitinase in resistant genotype indicate the effectiveness within a narrow range of pathogenesis  <cit> .
similarly, the protein coding for aba-responsive protein  , which is involved in stimulus mechanism and cell localization etc.
during plant development and one of the vital roles is in defence mechanism during biotic stress signaling.
this gene was identified to be expressed relatively higher in smd-resistant pigeonpea genotype 'icp 7035' compared to the susceptible genotype 'ttb 7'.
during pathogen infection aba inhibits the transcription of a basic β- <dig>  3-glucanase  that can degrade the β- <dig>  3-glucan callose, forming a physical barrier to viral spread through plasmodesmata.
this down regulation of β- <dig>  3-glucanase by aba can be termed as a resistance factor in plant pathogen interactions  <cit> .
in our study, significant expression signals were observed in smd resistant genotype 'icp 7035' during viral infection.
this positive correlation between the aba levels and disease resistance was reported in plant species like common bean  <cit> , rice  <cit>  and tobacco  <cit> .
different enzymes like methyltransferases   and dehydrogenases   are putatively involved in synthesis of lignin in cell walls.
these enzymes also play a major role in defence against pathogen interaction  <cit> .
an in silico hierarchical clustering analysis of  <dig> differentially expressed and genes across  <dig> cdna libraries using hce v  <dig>  was done to infer potential relation between the co-expressed genes.
the profiles of some of the interesting gene families and genes that could play an important role in stress stimulus were explained.
in cluster i, of the  <dig> contigs,  <dig> were identified to be highly expressed in fw- challenged libraries of susceptible genotype.
the cluster includes genes encoding proteins involved in mitochondrial dna  stability, na+/h+ exchanger and a few uncharacterized proteins.
the sub cluster ia includes two genes, which are highly expressed in 'icpl 20102' libraries .
the genes connected in sub cluster ib are highly expressed in 'icp 2376' libraries .
one of the putative proteins tar <dig> , a mitochondrial protein is known to be involved in regulation and respiratory metabolism.
over- expression of this protein suppresses the respiration-deficient petite phenotype of a point mutation in mitochondrial rna polymerase that affects mitochondrial gene expression and mtdna stability.
this dysfunction of mitochondria might occur in response to biotic or abiotic stress  <cit> .
the over-expression of these genes was observed only in  <dig> dai libraries of both the fw- responsive genotypes.
and their immediate disappearance in the later stages of infection in resistant genotype libraries and continued expression in susceptible genotypes supports a hypothesis that continual expression of this protein may lead to mitochondrial dysfunction and subsequent cell degeneracy.
another protein na+/h+ exchanger  <dig>  is an ubiquitous ion transporter that serves multiple cell physiological processes such as intracellular ph homeostasis and electro neutral exchange of protons for na+ and k+ across endomembranes.
biochemical studies suggest that na+/h+ exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis during salt stress, although the above protein is expressed in high salt stressed plants, it may also be expressed during biotic stress.
its high expression in susceptible genotype 'icpl 2376' at  <dig> and  <dig> dai libraries shows the severity of stress during fungal pathogenesis.
and in the remaining fw- and smd- challenged libraries this shows normal expression.
cluster ii genes include hevamine  and leucoanthocyanidin dioxygenase  specifically expressed in 'icpl 20102'  <dig> dai library.
the important protein hevamine represents a new class of polysaccharide-hydrolyzing  <dig> barrel enzyme belonging to families of plant chitinases and lysozymes, which are vital for plant defence against pathogenic bacteria and fungi.
recent results indicate that these enzymes may be involved not only in defence-related process or general stress response but also in growth and development processes  <cit> .
the high expression of these proteins in the late phase of fusarium infection indicates their prolonged defensive role against fungal pathogenesis.
the genes connected in cluster iii are highly expressed in fw-challenged 'icp 2376' -  <dig> and  <dig> dai libraries.
the genes related to endoprotease activity, beta-glucan synthesis, carboxypeptidases, alkaloid biosynthesis, secologanin biosynthesis, beta-galactosidase activity, microtubule-stabilizing activity, nucleic acid binding protein, ribonucleoprotein and a few uncharacterized proteins were constituted in this cluster.
for instance, antifungal class proteins such as beta-glucanases  and hansenula mrakii killer toxin-resistant proteins  located in epidermal leaf cells are believed to be involved in cell differentiation and defence against fungal pathogens  <cit> .
plants deficient in these enzymes generated by antisense transformation showed markedly reduced resistance to viral and fungal infection.
similarly, another class of proteins carboxypeptidases  with diverse functions ranging from catabolism to regulating biological processes, including function as a defence against pathogen attack  <cit> .
the majority of genes  segregated in cluster iv were highly expressed in smd-responsive cdna libraries derived from leaf tissues.
in total cluster iv genes showed high gene expression in smd derived libraries.
as expected, photosynthesis related transcripts were abundantly represented in sub clusters iva, ivb and ivc, and these include putative transcripts like ribosomal proteins, mitochondrial proteins, chloroplast precursor proteins, photosystem i and ii reaction centre proteins.
the observed expression pattern of photosynthesis related proteins in this study is also consistent with experimental observations in barley  <cit> .
overall, the differentially expressed genes are involved in diverse pathways, displaying complex expression patterns.
the different clusters based on monitoring of gene expression patterns propose that various pathways in response to biotic stress exist in pigeonpea and their interaction can lead to differential stress tolerance.
the uncharacterized class of transcripts co-expressed could be repository of novel proteins and further characterization of these may reveal their significant role in plant stress responses  <cit> .
development of functional markers
one of our primary goals of our research programme is to develop molecular markers based on expressed sequences and screen them for polymorphism.
during the last decade, microsatellites or ssrs have proven to be useful markers in plant genetic research and have been used for marker-assisted breeding purposes.
the presence of ssrs in the coding region suggests their importance as functional or gene based markers  <cit> .
unfortunately, development of microsatellite markers is expensive, labor intensive, and time consuming if they are being developed from genomic libraries  <cit> .
the data mining of microsatellites markers from est data can be a cost effective option.
the cost of mining est libraries is far lower than other traditional methods, and ssr development from ests has been successful in est data mining  <cit> .
ssr motifs with repeats more than eight for di-nucleotides, six for tri-nucleotides, and five for tetra-nucleotides were considered.
dimeric repeat motifs  were relatively abundant than trimeric repeats .
in addition to this, tetra-, penta- and hexameric repeat motifs were considerably less represented.
a total of  <dig> ssr markers have been synthesized and characterized for polymorphism survey.
however, there are some distant contrasts in frequency and distribution of ssrs in ests and in genomic survey sequences .
in general di-nucleotide ssrs of all repeat lengths are more common in gsss and tri-nucleotide ssrs are common in the ests  <cit> .
as against these reports, in our findings we observed that di-nucleotide repeats are more abundant than tri-nucleotide repeat motifs  <cit> .
however this observation is not unexpected as the frequency and distribution of ssr depends on several factors such as size of dataset, tools and criteria used for ssr discovery  <cit> .
in this study, a total of  <dig> polymorphic est-ssrs primer pairs were validated and used for diversity study on forty pigeonpea genotypes representing  <dig> cultivated  and  <dig> wild species .
all markers detected at least one allele in all genotypes tested, suggesting transferability of all markers across the cajanus genus.
in addition to high transferability, est-ssrs are good candidates for the development of conserved orthologous sequence  markers for genetic analysis and breeding of different species  <cit> .
however, est-ssrs were reported to be less polymorphic than genomic ssrs in crop plants due to greater dna sequence conservation in transcribed regions  <cit> .
for instance, the  <dig> ssr loci provided only  <dig> alleles with an average of  <dig> alleles per loci and an average  <dig>  pic value.
similar kind of diversity features were observed in earlier ssr based diversity studies in pigeonpea  <cit> .
est-ssr profiles obtained on  <dig> pigeonpea genotypes were used to compute pair-wise genetic distances among different genotypes to construct a dendrogram based on upgma clustering.
the neighbor joining tree grouped  <dig> pigeonpea genotypes into three major clusters .
the cluster i comprising  <dig> genotypes  is the largest cluster followed by cluster iii containing six wild genotypes  and cluster ii is the smallest cluster with two wild genotypes belonging to c. platycarpus species revealing clear segregation of the cultivated and the wild species.
less genetic variation was detected with in cultivated species, with only nine markers detecting polymorphism and a total of  <dig> alleles.
the low genetic variability amongst cultivars when compared with the wild species genotypes suggests that natural and artificial selection has contributed to the selection of specific alleles and to changes of allelic frequencies at specific loci as reported by odeny and colleagues  <cit> .
the distinctness of c. platycarpus with c. scarabaeoides accessions observed in this study correlate well with earlier studies  <cit> .
it is also important to note that 'icpl 20097' and 'icp 2376' genotypes were found closely related with high genetic similarity as both of these genotypes belong to the same geographic region.
in conclusion, est-ssr markers developed in this study complement the currently available or ongoing efforts on development of genomic ssrs that will be a valuable resource for linkage map development and marker assisted selection in pigeonpea  <cit> .
snps and indels are an essentially inexhaustible resource of polymorphic markers for use in the high-resolution genetic map development of traits and for association studies.
although a variety of molecular markers are available snps are comparatively advantageous because of their abundance and amenability to high throughput approaches  <cit> .
in addition, snps also offer several advantages like high-throughput and cost-effective genotyping  <cit>  and identification of functional/gene-based markers for complex trait through linkage map development or association genetics  <cit> .
although snp discovery was a cost effective task in past, advances in next generation sequencing technologies have made snp discovery cheaper and faster  <cit> .
however in case for a given species, ests are available from more than one genotype, in silico mining of ests is still a very inexpensive and fast approach for snp discovery  <cit>  and therefore we used this approach for mining snps in this study.
by using in silico mining approach in a total of  <dig> contigs coming from  <dig>  ests , a total of  <dig> potential snps were identified in  <dig> contigs that were consisted of ≥  <dig> ests.
smaller contigs were not considered for snp mining as these contigs are prone to errors due to lack of read depth as reported by wang and colleagues  <cit> .
sequence analysis of pcr products for a subset of  <dig> out of  <dig> contigs confirmed the occurrence of snps in all the cases.
as pcr products could not be generated for remaining four contigs, the presence of snps could not be confirmed in those cases.
furthermore, as snp genotyping is another important criteria in breeding programmes, identification of caps markers for  <dig> contigs will facilitate snp genotyping even in low tech laboratories  <cit> .
CONCLUSIONS
this study has contributed a new and significant set of  <dig>  ests that together with  <dig> public domain ests provides a unigene set of  <dig>  sequences for pigeonpea.
detailed analysis of these datasets have provided several important features of pigeonpea transcriptome such as conserved genes  as well as possible pigeonpea specific genes, assignment of pigeonpea genes to different go categories, identification of differentially expressed genes in response to fw- and smd- stresses, etc.
in terms of applied aspect of developed resource in breeding, this study has demonstrated development and application of gene-based molecular markers i.e ssrs, snps and caps.
in summary, it is anticipated that this study is a significant contribution to enhance genomic resources in a so called orphan legume crop that will eventually impact pigeonpea breeding  <cit> .

 pigeonpea millsp) is one of the major grain legume crops of the tropical and subtropical regions of the world. it is the only cultivated food crop of the cajaninae sub-tribe and has a diploid genome with pairs of chromosomes and a genome size estimated to be mbp. the genus cajanus comprises species most of which are found in india, australia and one is native to west africa. pigeonpea is a major food legume crop in south asia and east africa with india as the largest producer followed by myanmar and kenya. it plays an important role in food security, balanced diet and alleviation of poverty because of its diverse usages as a food; fodder and fuel wood. several abiotic and biotic stresses, are serious challenges for sustainable pigeonpea production to meet the demands of the resource poor people of several african and asian countries. fusarium wilt caused by fusarium udum is an important biotic constraint in pigeonpea production in the indian subcontinent, which results in 16-47% crop losses. the fungus enters the host vascular system at the root tips through wounds or invasion made by nematodes, leading to progressive chlorosis of leaves, branches, wilting and collapse of the root system. in india alone, the loss due to this disease is estimated to be us $ million and the percentage of disease incidence varies from to %. sterility mosaic disease caused by pigeonpea sterility mosaic virus is one of the wide-spread diseases of pigeonpea, which is transmitted by an eriophyid mite. the disease is characterized by the symptoms like bushy and pale green appearance of plants followed by reduction in size, increase in number of secondary and mosaic mottling of leaves and finally partial or complete cessation of reproductive structures. some parts of the plant may show disease symptoms and other parts may remain unaffected. due to the above mentioned factors combined with limited water resources to the fields in the semi-arid tropic regions, where the crop is grown, the productivity has remained stagnant at around t/ha during the past two decades. with the advent of genomic tools such as molecular markers, genetic maps, etc., conventional plant breeding has been facilitated greatly and improved genotypes/varieties with enhanced resistance/tolerance to biotic/abiotic stresses have been developed in several crop species. in case of pigeonpea, however, a very limited number of genomic tools are available so far. for instance, microsatellite or simple sequence repeat markers, expressed sequence tags, at the time of undertaking the study, were available in pigeonpea. for enhancing the genomic resources in pigeonpea, transcriptome sequencing to generate ests should be a fast approach. ests, which are generated by large-scale single pass sequencing of randomly picked cdna clones, have been cost - effective and valuable resource for efficient and rapid identification of novel genes and development of molecular markers. further, ests have been employed in bioinformatic analyses to identify the genes that are differentially expressed in various tissues, cell types, or developmental stages of the same or different genotypes. in view of above facts, this study was undertaken to obtain a comprehensive resource of fw- and smd-responsive ests in pigeonpea with the following objectives: generation of fw- and smd- responsive ests, functional annotation of assembled unigenes, in silico identification of putative fw- and smd- responsive genes, and development of novel ssr and snp markers in pigeonpea.  root tissue is the site for fusarium udum infection, the causal fungal agent of fusarium wilt in pigeonpea. with an objective to evaluate the transcriptional responses after infection of roots by f. udum, six unidirectional cdna libraries were constructed. these are from each of fw- infected root tissues of resistant and susceptible genotypes at different stages viz. days after inoculation. infected roots were examined by light microscopy upon harvest at different stages. the severity of wilt disease in both susceptible and resistant genotype was observed in longitudinal sections of stem and root vascular region at and dai. likewise for smd, leaf tissue is the specific site of infection and therefore leaf samples of smd infected genotypes,'icp 7035' and'ttb 7' were harvested at and days after sowing. rna was extracted and consequently unidirectional cdna libraries were constructed. generation of fw- and smd- responsive ests a total of unidirectional cdna libraries were constructed from all the four genotypes i.e.'icpl 20102' and'icp 2376';'icp 7035' and'ttb 7' which represent parents of mapping population segregating for fw and smd, respectively. using sanger sequencing approach, ests were generated from root cdna libraries of'icpl 20102' and from'icp 2376'. similarly, ests were generated from each leaf cdna libraries of smd- responsive genotypes,'icp 7035' and'ttb 7'. details of est generation from different cdna libraries are given in figure in brief, a total of ests were generated and after stringent screening for shorter and poorer quality sequences, high quality ests were obtained, with an average varied-read length of bp. all est sequences were deposited in the dbest of genbank under accession numbers gr to gr and gr to gr pigeonpea est assembly with an objective to minimize redundancy, clustering and assembly was done for different est datasets to define unigenes for fw-responsive ests, smd-responsive ests, fw- and smd-responsive ests, and the entire set of pigeonpea ests including those from the public domain. these unigene sets were referred to as ug-i, ug-ii, ug-iii and ug-iv, respectively. the ug-i comprised of unigenes with contigs and singletons by clustering of high quality ests. similarly, for ug-ii, clustering of high quality sequences resulted in unigenes. based on clustering of all the high quality sequences generated in this study, the ug-iii was defined with unigenes. the cluster analysis of ests available in the public domain along with pigeonpea ests resulted in ug-iv that included unigenes with contigs and singletons. the number of ests in a contig ranged from to with an average of ests per contig. as expected, contigs with two est members exhibited a higher percentage than contigs with three or more est members. comparison of pigeonpea unigenes with other plant est databases all the four sets of unigenes i.e. ug-i, ug-ii, ug-iii and ug-iv were analyzed for blastn similarity search against available est datasets of legume species namely chickpea, pigeonpea, soybean, medicago, lotus, common bean and three model plant species namely arabidopsis, rice and poplar. an e-value significant threshold of ≤ 1e- was used for defining a hit. detailed results of blastn analyses for all the four unigenes sets are given in table for instance, analysis of ug-iii found highest identity of % with soybean, followed by cowpea, medicago, common bean, lotus, and the least identity with chickpea. comparative blastn analysis of pigeonpea unigenes with est databases of model plant species showed high identity with poplar, followed by arabidopsis and the least similarity with rice. of unigenes, showed significant identity with ests of at least one plant species analysed, while showed significant identity across all the plant est databases in this study. it is also interesting to note that unigenes did not show any homology with the legume species examined. high quality ests generated significant similarity with ests of at least significant similarity with ests of at least one significant similarity across ests of all model plant significant similarity with ests of at least one significant similarity across ests of all plant to identify the putative function of all the unigenes compiled in this study, the unigenes from all the four sets were compared against the non-redundant uniprot database, using the blastx algorithm. at a significant threshold of ≤ 1e- of ug-i, of ug-ii, of ug-iii and of ug-iv unigenes showed significant similarity with known proteins. details of blastx and blastn analyses against uniprot database for all four unigene sets are provided in additional files and functional categorization of pigeonpea unigenes the unigenes from all the four sets that showed a significant hit against the uniprot database were further categorized into functional categories. as a result, of ug-i, of ug-ii, of ug-iii and of ug-iv unigenes were categorised into three principal go categories i.e. biological process, molecular function and cellular component. like in earlier studies of this nature, it was observed that one gene could be assigned to more than one principal category, thus the total number of go mappings from each category exceeded the number of unigenes analyzed. details on full list of gene annotation for significant hits of four unigene sets are given in additional file and for instance, of unigenes of ug-iii, only were assigned to three principle categories. as a result, a total of were grouped under biological process, under molecular function and under cellular component. under the biological process category, cellular process accounted to followed by metabolic process, biological regulation and response to stimulus. in the cellular component category, unigenes coded for cell part, to organelle, and to organelle part. in the last category of molecular function, majority of the unigenes were involved in binding and catalytic activity. the remaining unigenes which could not be classified into any of the three go categories were grouped as "unclassified". the distribution of unigenes along with corresponding gene ontology categories are provided in additional file based on go annotation, enzyme commission ids were also retrieved from the uniprot database to get an overview of unigenes putatively annotated to be enzymes. the major group of unigenes are included under oxidoreductases followed by transferases, hydrolases, lyases, ligases and isomerases. similar patterns of distribution were observed in all the remaining unigene sets. in silico expression analysis the identification of differentially expressed genes among specific cdna libraries of fw- and smd-responsive genotypes based on est counts in each contig was done using a web statistical tool ideg. as a result, genes were identified to be differentially expressed between'icpl 20102' and'icp 2376' genotypes, similarly, genes were differentially expressed between'icp 7035' and'ttb 7' genotypes. to assess the relatedness of each library and expressed genes in terms of expression pattern, a cluster analysis on the basis of est abundance in each contig was performed. of the contigs, that were subjected to r-statistics only contigs were normalized with a true positive significance and were eventually subjected to hierarchical clustering analysis. the correlated gene expression pattern of all normalized contigs/genes is displayed in figure all the fw- derived libraries were grouped into a single cluster, while all the four smd- challenged libraries were grouped into another cluster. about genes were highly expressed in smd- challenged libraries than in fw- challenged libraries and can be attributed to high accumulation of defence proteins during smd infection. in the cluster of fw- challenged libraries, the'icpl 20102'- dai library was distantly placed between fw- susceptible challenged libraries'icp 2376' - dai and'icp 2376' - dai. each cluster represents a different pattern of gene expression as shown in figure based on the clustering pattern and library specificity, clusters i and iv were further divided into sub-clusters. the above results indicated that the pattern and percentage of genes expression varied according to severity of the stress in specific library. in cluster i, % of total genes were grouped and further sub divided into two groups with each sharing % and % genes, respectively. similarly, cluster ii and cluster iii accounted for % and % genes and the largest cluster iv, included % of total genes with three sub groups iva, ivb and ivc each sharing %, 10% and 45% of genes, respectively. cluster analysis also showed high level expression of genes related to chloroplast/photosystem related proteins, developmental proteins, cellular proteins, metabolic proteins, defence/stimulus responsive proteins, protein specific binding proteins and few uncharacterized proteins. marker discovery est based markers can assay the functional genetic variation compared to other class of genetic markers and hence were targeted for marker development. the unigene set based on generated ests in this study as well as the ones available in public domain was used for development of simple sequence repeats and single nucleotide polymorphism markers. identification and development of genic microsatellite markers the entire set of pigeonpea unigenes derived from ug-iv was used to identify the ssrs using misa tool. as a result a total of ssrs were identified at the frequency of 1/ bp in coding regions. ests contained more than one ssr and ssrs were found as compound ssrs. in terms of distribution of different classes of ssrs i.e. mono-, di-, tri-, tetra-, penta- and hexa-nucleotide repeats, mononucleotide ssrs contributed to the largest proportion. only a limited number of ssrs of other classes were found. for instance, di- and tri- nucleotide ssrs accounted for and, respectively. on the other hand, tetrameric, pentameric and hexameric microsatellites were present. while using the criteria for class i and class ii ssrs as used by temnykh and colleagues and kantety and colleagues, on all ssrs ssrs represented class i while ssrs represented class ii. in general, mononucleotide ssrs are not included for primer designing and synthesis. however, as only a very limited number of ssr markers are currently available for pigeonpea in public domain and in a separate study some mononucleotide ssrs were found polymorphic, primer pairs were designed for ssrs including mononucleotide ssrs. a total of primer pairs were considered for validation after excluding the primers for monomeric ssr motifs and compound ssrs with mononucleotide repeats. however based on repeat number criteria, such as minimum for di-, tri-, tetra-, penta-nucleotides, primer pairs were synthesized for ssrs. the details of newly developed pigeonpea est-ssr primers along with corresponding ssr motif, primer sequence, annealing temperature and product size are provided in additional file newly synthesized markers were analyzed on elite pigeonpea genotypes. as a result, primer pairs provided scorable amplified products and primer pairs produced a number of faint bands indicative of non-specific amplifications. a total of markers showed polymorphism with 2- alleles with an average of alleles per marker in genotypes examined. these markers showed a moderate pic value ranging from to with an average of. to evaluate the genetic variability within a diverse collection of pigeonpea accessions which are parents of different mapping populations segregating for important agronomic traits and also to determine genetic relationship among them, phylogenetic analysis on the basis of dissimilarities was performed using ntsys software package. the upgma cluster diagram showed clear segregation of wild and cultivated species. snp discovery and identification of caps markers snps are an important class of molecular markers which are becoming more popular in recent times. to enhance the reliability of snps identification, the snp which occurred in a contig ≥ ests from more than one genotype was considered. in silico analysis showed a total of snps in contigs with a frequency of 1/ bp. with an objective of validating these in silico identified snps, as an example, contigs were used to generate pcr amplicons and sequence four genotypes namely'icpl 20102','icp 2376','icp 7035' and'ttb 7'. while a scorable and sequenceable amplicon was obtained in case of contigs, the scorable amplicons were not obtained in case of four contigs. sequencing of amplicons for all the four genotypes for all the six contigs showed occurrence of snps as predicted in silico. for instance, for contig a comparison of the amplified dna sequences for four genotypes with the est sequences coming from two genotypes showed the occurrence of the same snp g to c between'icp 7035' and'ttb 7'. in order to perform cost-effective and robust genotyping assay for the detected snps in contigs, efforts were made to identify the restriction enzymes that can be used to assay snps via cleaved amplified polymorphic sequence assay. results indicated that snps present in contigs can be evaluated by using caps assay. discussion plants are known to have developed integrated defence mechanisms against fungal and viral infections by altering spatial and temporal transcriptional changes. the est approach was successfully utilized in identification of disease-responsive genes from various tissues and growth stages in chickpea, lathyrus, soybean, rice and ginseng. many earlier studies have shown that resistant genotypes have efficient mechanisms for stress perception and enhanced expression of defence-responsive genes, which maintain cellular survival and recovery. hence, the present study was undertaken to identify catalog of defence related genes in response to fw and smd infection in pigeonpea by generating ests from different stress challenged tissues at various time intervals. generation of cdna libraries and unigene assemblies roots provide a structural and physiological support for plant interactions with the soil environment by conducting transport of water, ions and nutrients. plants are encountered with many biotic stress factors which includes bacterial, fungal and viral infection. roots and leaves are the primary sites of infection by these organisms. therefore, a total of cdna libraries were generated at different time intervals to specifically target the roots infected with fusarium udum and leaves infected with smd. in total high quality ests were generated from fw- and similarly high quality ests from smd- challenged genotypes. earlier, at the time of analysis in november the public domain consisted of only ests for pigeonpea. thus the present study contributes approximately 10-fold increase in the pigeonpea est resource and an addition of pigeonpea unigenes. functional annotation of pigeonpea unigenes homology searches against other plant ests and functional characterization was done for all the defined unigene datasets. of the unigenes assembled from all the pigeonpea ests, had significant identity with ests of at least one plant species analyzed, unigenes showed significant identity with ests of all analyzed plant species in the study, while were found to be novel to pigeonpea. a high significant identity was observed with soybean, and the least percentage of similarity was observed with chickpea. a similar blastn results were observed for the remaining three unigenes sets against the ests of plant species surveyed. comparative analysis of newly defined ug-iii dataset with public domain pigeonpea ests showed that only shared identity and indicated that our est sequencing study identified new set of pigeonpea unigenes. relatively, very low similarity of % with lotus and % with medicago was observed compared to soybean and cowpea than other legume species. these observations are in accordance with phylogenetic relationships of legumes. the pigeonpea ests showed higher similarity to legume ests databases of the legume species than monocot species. comparative analysis of pigeonpea ests with monocot species like rice showed that the percentage of significance is much lower compared to any other legume species, inspite of larger est repository. this is clearly attributed to phylogenetic divergence between dicots and monocots in course of evolution. these comparisons also indicate that several unigenes that were absent in analysed non-legumes but present in all legume species may be specifically confined to legumes. blastx analyses indicated that those ests without significant identity to any other protein sequences in the existing database may be novel and involved in plant defence responses. hence, this novel est collection represented a significant addition to the existing pigeonpea est resources and provides valuable information for further predictions/validation of gene functions in pigeonpea. a comprehensive comparison of functionally categorized unigenes of all the four unigenes data sets showed a similar distribution. a large number of unigenes were involved in cell part, organelle, binding, organelle part, metabolic and cellular process among the significantly annotated ones. these observations are consistent with the earlier reported functional categorization studies in rice, soybean, barley and tall fescue. however, the sequences encoding activities related to categories such as biological regulation and response to stimulus are and incase of fw-responsive ests compared to and in case of smd-responsive ests. this was possibly due to the fact that the ests generated from fw- challenged root libraries were most abundantly involved in stimulus to pathogenesis and ests derived from smd stress are chloroplast binding proteins. earlier studies such as lee and colleagues, ablett and colleagues, also reported that photosynthesis-related proteins were the most prevalent from aerial parts of the plant, which would help to make energy related activities such as cell division, growth, elongation and development. similarly in this study, photosynthesis related genes were identified in larger proportion in smd-responsive cdna libraries derived from leaf tissues. in silico differential gene expression the invasion of pathogen not only results in expression of novel genes/transcripts, but also in altering the abundances of different ests resulting in induction or repression. this was evident from differential expression of genes between fw-responsive genotypes and genes between smd-responsive genotypes. it is however, important to mention that in silico method of gene expression is not the ideal method to identify the differentially expressed genes. nevertheless, as large scale est data were generated from fw- responsive and smd- responsive genotypes, an effort like some earlier studies was made to identify some putative genes differentially expressed in fw- and smd- resistant and sensitive genotypes. validation of these candidate genes by northern analysis or real-time quantitative pcr analysis is essential before these candidate genes are deployed in some other studies. significant number of unigene sequences related to proteins like kinases, phosphatases, peroxidases, ribonucleases, endochitinases, glucanases and hormones like abscisic acid responsive genes were identified to be differentially expressed and are known to play a vital role in defence mechanism. for example, the cell wall degrading enzymes like endochitinases implicate a major defence mechanism against pathogen. similarly, kinases play a major role in the plant's recognition to pathogen. for instance, chitinase protein, a class of pathogenesis related proteins with bi-functional role in lysozyme/chitinase activity involved in random hydrolysation of n-aetyl-beta-d-glucosaminide-beta linkages in chitin and chitodextrins during systemic acquired resistance, was expressed at higher concentrations in fw-responsive resistant genotype compared to susceptible genotype. the high expression levels of chitinase in resistant genotype indicate the effectiveness within a narrow range of pathogenesis. similarly, the protein coding for aba-responsive protein, which is involved in stimulus mechanism and cell localization etc. during plant development and one of the vital roles is in defence mechanism during biotic stress signaling. this gene was identified to be expressed relatively higher in smd-resistant pigeonpea genotype'icp 7035' compared to the susceptible genotype'ttb 7'. during pathogen infection aba inhibits the transcription of a basic β- 3-glucanase that can degrade the β- 3-glucan callose, forming a physical barrier to viral spread through plasmodesmata. this down regulation of β- 3-glucanase by aba can be termed as a resistance factor in plant pathogen interactions. in our study, significant expression signals were observed in smd resistant genotype'icp 7035' during viral infection. this positive correlation between the aba levels and disease resistance was reported in plant species like common bean, rice and tobacco. different enzymes like methyltransferases and dehydrogenases are putatively involved in synthesis of lignin in cell walls. these enzymes also play a major role in defence against pathogen interaction. an in silico hierarchical clustering analysis of differentially expressed and genes across cdna libraries using hce v was done to infer potential relation between the co-expressed genes. the profiles of some of the interesting gene families and genes that could play an important role in stress stimulus were explained. in cluster i, of the contigs, were identified to be highly expressed in fw- challenged libraries of susceptible genotype. the cluster includes genes encoding proteins involved in mitochondrial dna stability, na+/h+ exchanger and a few uncharacterized proteins. the sub cluster ia includes two genes, which are highly expressed in'icpl 20102' libraries. the genes connected in sub cluster ib are highly expressed in'icp 2376' libraries. one of the putative proteins tar, a mitochondrial protein is known to be involved in regulation and respiratory metabolism. over- expression of this protein suppresses the respiration-deficient petite phenotype of a point mutation in mitochondrial rna polymerase that affects mitochondrial gene expression and mtdna stability. this dysfunction of mitochondria might occur in response to biotic or abiotic stress. the over-expression of these genes was observed only in dai libraries of both the fw- responsive genotypes. and their immediate disappearance in the later stages of infection in resistant genotype libraries and continued expression in susceptible genotypes supports a hypothesis that continual expression of this protein may lead to mitochondrial dysfunction and subsequent cell degeneracy. another protein na+/h+ exchanger is an ubiquitous ion transporter that serves multiple cell physiological processes such as intracellular ph homeostasis and electro neutral exchange of protons for na+ and k+ across endomembranes. biochemical studies suggest that na+/h+ exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis during salt stress, although the above protein is expressed in high salt stressed plants, it may also be expressed during biotic stress. its high expression in susceptible genotype'icpl 2376' at and dai libraries shows the severity of stress during fungal pathogenesis. and in the remaining fw- and smd- challenged libraries this shows normal expression. cluster ii genes include hevamine and leucoanthocyanidin dioxygenase specifically expressed in'icpl 20102' dai library. the important protein hevamine represents a new class of polysaccharide-hydrolyzing barrel enzyme belonging to families of plant chitinases and lysozymes, which are vital for plant defence against pathogenic bacteria and fungi. recent results indicate that these enzymes may be involved not only in defence-related process or general stress response but also in growth and development processes. the high expression of these proteins in the late phase of fusarium infection indicates their prolonged defensive role against fungal pathogenesis. the genes connected in cluster iii are highly expressed in fw-challenged'icp 2376' - and dai libraries. the genes related to endoprotease activity, beta-glucan synthesis, carboxypeptidases, alkaloid biosynthesis, secologanin biosynthesis, beta-galactosidase activity, microtubule-stabilizing activity, nucleic acid binding protein, ribonucleoprotein and a few uncharacterized proteins were constituted in this cluster. for instance, antifungal class proteins such as beta-glucanases and hansenula mrakii killer toxin-resistant proteins located in epidermal leaf cells are believed to be involved in cell differentiation and defence against fungal pathogens. plants deficient in these enzymes generated by antisense transformation showed markedly reduced resistance to viral and fungal infection. similarly, another class of proteins carboxypeptidases with diverse functions ranging from catabolism to regulating biological processes, including function as a defence against pathogen attack. the majority of genes segregated in cluster iv were highly expressed in smd-responsive cdna libraries derived from leaf tissues. in total cluster iv genes showed high gene expression in smd derived libraries. as expected, photosynthesis related transcripts were abundantly represented in sub clusters iva, ivb and ivc, and these include putative transcripts like ribosomal proteins, mitochondrial proteins, chloroplast precursor proteins, photosystem i and ii reaction centre proteins. the observed expression pattern of photosynthesis related proteins in this study is also consistent with experimental observations in barley. overall, the differentially expressed genes are involved in diverse pathways, displaying complex expression patterns. the different clusters based on monitoring of gene expression patterns propose that various pathways in response to biotic stress exist in pigeonpea and their interaction can lead to differential stress tolerance. the uncharacterized class of transcripts co-expressed could be repository of novel proteins and further characterization of these may reveal their significant role in plant stress responses. development of functional markers one of our primary goals of our research programme is to develop molecular markers based on expressed sequences and screen them for polymorphism. during the last decade, microsatellites or ssrs have proven to be useful markers in plant genetic research and have been used for marker-assisted breeding purposes. the presence of ssrs in the coding region suggests their importance as functional or gene based markers. unfortunately, development of microsatellite markers is expensive, labor intensive, and time consuming if they are being developed from genomic libraries. the data mining of microsatellites markers from est data can be a cost effective option. the cost of mining est libraries is far lower than other traditional methods, and ssr development from ests has been successful in est data mining. ssr motifs with repeats more than eight for di-nucleotides, six for tri-nucleotides, and five for tetra-nucleotides were considered. dimeric repeat motifs were relatively abundant than trimeric repeats. in addition to this, tetra-, penta- and hexameric repeat motifs were considerably less represented. a total of ssr markers have been synthesized and characterized for polymorphism survey. however, there are some distant contrasts in frequency and distribution of ssrs in ests and in genomic survey sequences. in general di-nucleotide ssrs of all repeat lengths are more common in gsss and tri-nucleotide ssrs are common in the ests. as against these reports, in our findings we observed that di-nucleotide repeats are more abundant than tri-nucleotide repeat motifs. however this observation is not unexpected as the frequency and distribution of ssr depends on several factors such as size of dataset, tools and criteria used for ssr discovery. in this study, a total of polymorphic est-ssrs primer pairs were validated and used for diversity study on forty pigeonpea genotypes representing cultivated and wild species. all markers detected at least one allele in all genotypes tested, suggesting transferability of all markers across the cajanus genus. in addition to high transferability, est-ssrs are good candidates for the development of conserved orthologous sequence markers for genetic analysis and breeding of different species. however, est-ssrs were reported to be less polymorphic than genomic ssrs in crop plants due to greater dna sequence conservation in transcribed regions. for instance, the ssr loci provided only alleles with an average of alleles per loci and an average pic value. similar kind of diversity features were observed in earlier ssr based diversity studies in pigeonpea. est-ssr profiles obtained on pigeonpea genotypes were used to compute pair-wise genetic distances among different genotypes to construct a dendrogram based on upgma clustering. the neighbor joining tree grouped pigeonpea genotypes into three major clusters. the cluster i comprising genotypes is the largest cluster followed by cluster iii containing six wild genotypes and cluster ii is the smallest cluster with two wild genotypes belonging to c. platycarpus species revealing clear segregation of the cultivated and the wild species. less genetic variation was detected with in cultivated species, with only nine markers detecting polymorphism and a total of alleles. the low genetic variability amongst cultivars when compared with the wild species genotypes suggests that natural and artificial selection has contributed to the selection of specific alleles and to changes of allelic frequencies at specific loci as reported by odeny and colleagues. the distinctness of c. platycarpus with c. scarabaeoides accessions observed in this study correlate well with earlier studies. it is also important to note that'icpl 20097' and'icp 2376' genotypes were found closely related with high genetic similarity as both of these genotypes belong to the same geographic region. in conclusion, est-ssr markers developed in this study complement the currently available or ongoing efforts on development of genomic ssrs that will be a valuable resource for linkage map development and marker assisted selection in pigeonpea. snps and indels are an essentially inexhaustible resource of polymorphic markers for use in the high-resolution genetic map development of traits and for association studies. although a variety of molecular markers are available snps are comparatively advantageous because of their abundance and amenability to high throughput approaches. in addition, snps also offer several advantages like high-throughput and cost-effective genotyping and identification of functional/gene-based markers for complex trait through linkage map development or association genetics. although snp discovery was a cost effective task in past, advances in next generation sequencing technologies have made snp discovery cheaper and faster. however in case for a given species, ests are available from more than one genotype, in silico mining of ests is still a very inexpensive and fast approach for snp discovery and therefore we used this approach for mining snps in this study. by using in silico mining approach in a total of contigs coming from ests, a total of potential snps were identified in contigs that were consisted of ≥ ests. smaller contigs were not considered for snp mining as these contigs are prone to errors due to lack of read depth as reported by wang and colleagues. sequence analysis of pcr products for a subset of out of contigs confirmed the occurrence of snps in all the cases. as pcr products could not be generated for remaining four contigs, the presence of snps could not be confirmed in those cases. furthermore, as snp genotyping is another important criteria in breeding programmes, identification of caps markers for contigs will facilitate snp genotyping even in low tech laboratories.  this study has contributed a new and significant set of ests that together with public domain ests provides a unigene set of sequences for pigeonpea. detailed analysis of these datasets have provided several important features of pigeonpea transcriptome such as conserved genes as well as possible pigeonpea specific genes, assignment of pigeonpea genes to different go categories, identification of differentially expressed genes in response to fw- and smd- stresses, etc. in terms of applied aspect of developed resource in breeding, this study has demonstrated development and application of gene-based molecular markers i.e ssrs, snps and caps. in summary, it is anticipated that this study is a significant contribution to enhance genomic resources in a so called orphan legume crop that will eventually impact pigeonpea breeding. 
functional categorization of the annotated unigenes sequences showed that genes were assigned to cellular component category, to biological process, and in molecular function. similarly, blastx similarity results showed that only out of total unigenes correspond to known proteins in the uniprot database. modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. validation of identified biotic stress responsive genes should provide candidate genes for allele mining as well as candidate markers for molecular breeding. the pigeonpea est dataset generated here provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. blastn analysis of unigenes showed a significant identity with ests of different legumes, rice, arabidopsis and poplar. availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. with an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of fw- and smd- responsive expressed sequence tags. pigeonpea millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses are serious challenges for sustainable crop production. sequence analyses of this dataset have showed conservation of a considerable number of pigeonpea transcripts across legume and model plant species analysed as well as some putative pigeonpea specific genes. recognition sites for restriction enzymes were identified for snps in contigs that indicates possibility of assaying snps in genes using cleaved amplified polymorphic sequences assay. further, genes were identified differentially expressed between fw- responsive genotypes and between smd- responsive genotypes. generated ests were compiled together with ests available in public domain, at the time of analysis, and a set of unigenes were defined that were used for identification of molecular markers in pigeonpea. as expected, pigeonpea ests are more closely related to soybean and cowpea ests than other plant ests.
 pigeonpea millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses are serious challenges for sustainable crop production. modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. with an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of fw- and smd- responsive expressed sequence tags.  a total of cdna libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to fw and smd and a total of ests were generated and deposited in dbest of genbank under accession numbers gr to gr and gr to gr clustering and assembly analyses of these ests resulted into unique sequences including contigs and singletons. blastn analysis of unigenes showed a significant identity with ests of different legumes, rice, arabidopsis and poplar. as expected, pigeonpea ests are more closely related to soybean and cowpea ests than other plant ests. similarly, blastx similarity results showed that only out of total unigenes correspond to known proteins in the uniprot database. functional categorization of the annotated unigenes sequences showed that genes were assigned to cellular component category, to biological process, and in molecular function. further, genes were identified differentially expressed between fw- responsive genotypes and between smd- responsive genotypes. generated ests were compiled together with ests available in public domain, at the time of analysis, and a set of unigenes were defined that were used for identification of molecular markers in pigeonpea. for instance, simple sequence repeat motifs were identified in unigenes and primer pairs were designed. assessment of a set of primer pairs on elite pigeonpea lines showed polymorphism with markers with an average of four alleles per marker and an average polymorphic information content value of. similarly, in silico mining of contigs with ≥ sequences detected single nucleotide polymorphisms in contigs. as an example, a set of contigs were used for confirming in silico predicted snps in a set of four genotypes using wet lab experiments. occurrence of snps were confirmed for all the contigs for which scorable and sequenceable amplicons were generated. pcr amplicons were not obtained in case of contigs. recognition sites for restriction enzymes were identified for snps in contigs that indicates possibility of assaying snps in genes using cleaved amplified polymorphic sequences assay.  the pigeonpea est dataset generated here provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. sequence analyses of this dataset have showed conservation of a considerable number of pigeonpea transcripts across legume and model plant species analysed as well as some putative pigeonpea specific genes. validation of identified biotic stress responsive genes should provide candidate genes for allele mining as well as candidate markers for molecular breeding. 