BACKGROUND
the fabaceae  is the third largest angiosperm family, containing c.  <dig>  species attributed to  <dig> genera  <cit> .
legumes provide major benefits to cropping systems and the environment, due to the ability to perform symbiotic nitrogen fixation.
in comparison to cereals, for which a broad range of genetic and genomic resources are available, genomic databases for legumes are generally still underdeveloped.
however, recent advances in sequencing and genotyping technologies offer the opportunity to rapidly ameliorate the status of given species at relatively low cost  <cit> .
major efforts are currently being directed towards the development of species-specific genomic tools and datasets.
as an example, the whole genome sequence of soybean, a warm-season grain legume, has recently been determined http://www.phytozome.net/soybean <cit> .
cool-season food legumes within the hologalegina clade of the fabaceae sub-family papilionoideae, which includes lentil, chickpea, field pea and faba bean , are important food and fodder crops, especially in developing countries such as those of the indian sub-continent  <cit> .
these species are important components of farming systems across western asia, the middle east, north africa, the indian sub-continent, north america and australia.
in australia, pulses are sown over c.  <dig> million hectares and produce c.  <dig>  million tonnes of grain with a commodity value of over au$  <dig> million  <cit> .
despite close phylogenetic relationships, pulse species vary considerably in aspects of biology such as genome size, fundamental chromosome number, ploidy level, and degree of reproductive self-compatibility.
the genome size of chickpea is relatively small , but pulses of the vicieae tribe  exhibit much larger genome sizes .
recently, generation of large-scale lentil transcriptome data by our group has substantially increased the volume of publicly available genomic data for this species  <cit> .
similar strategies have been pursued for field pea and faba bean in the current study.
field pea, which is the third most globally important grain legume crop  after soybean and common bean , is a self-pollinating diploid  species with a genome size of c.  <dig> gbp  <cit> .
various studies have been performed to determine the genetic basis of multiple phenotypic traits in field pea  <cit>  and to quantify diversity between different pea cultivars  <cit> .
recently, a comprehensive transcriptome analysis of field pea has been performed using second-generation sequencing technologies  <cit>  that will contribute significantly to the enrichment of genomics resources for field pea.
in contrast, faba bean has not been widely adopted on a global basis.
in terms of cultivation area, this species ranks fourth among the cool-season food legumes  after field pea, chickpea and lentil http://faostat.fao.org.
faba bean has been traditionally cultivated in the mediterranean basin, the nile valley, ethiopia, central and east asia, latin america, northern europe, north america and australia  <cit> .
faba bean is a diploid taxon , and exhibits facultative cross-pollination at frequencies ranging from 4-84%.
the nuclear genome size of faba bean is one of the largest yet described among crop legumes, at c.  <dig> gb.
formal genetic analysis of faba bean, such as through genetic linkage mapping and identification of quantitative trait loci , has so far been hindered by these aspects of biology  <cit> .
conventional breeding methods based on phenotypic assessment are currently in use for breeding line selection in field pea and faba bean.
such methods are logistically demanding and time-consuming, especially for traits that require specific biotic or abiotic challenges, such as resistance to individual diseases.
in addition to this, when breeding for types eaten as immature seed, quality testing adds considerable complexity to the relevant programs.
there is consequently a major requirement for species-specific molecular genetic markers and derived linkage maps for field pea and faba bean, to enable germplasm advancement through genomics-assisted selection.
current publicly available genetic and genomic tools for field pea and faba bean are limited in extent  <cit> , comprising  <dig>  and  <dig>  ests, respectively that are available in genbank.
in addition to this, a recently sequenced pisum sativum transcriptome generated a total of  <dig>  unigenes that are also available for download as a fully annotated fasta format  <cit> .
second-generation dna sequencing systems such as the roche  <dig> massively-parallel pyrosequencing platform are capable of rapidly producing species-specific genomic resources to address these short-comings.
this system can generate 4- <dig> ×  <dig> bp from each run, with individual read lengths of 400- <dig> bp  <cit> , and is suitable for de novo sequencing of small genomes  <cit> , whole genome resequencing  <cit> , snp detection  <cit> , and in particular, sequencing of transcriptomes  <cit> .
ests obtained from the latter activity provide valuable resources for gene discovery, large-scale expression analysis, improved genome annotation, elucidation of phylogenetic relationships and facilitation of breeding programs for both plants and animals through provision of ssr and single nucleotide polymorphism  genetic markers  <cit> .
ssr loci have been widely used for improvement of a range of crop species  <cit> .
only a limited number of ssrs are available in public domain for field pea and faba bean, creating an incentive for further discovery and validation.
in comparison with genomic dna-derived ssrs, those located in ests are functionally associated with genic regions, and support potential diagnostic genetic marker development  <cit> .
this study describes the development, de novo assembly and gene annotation of a transcriptome dataset derived from cdna samples obtained from several tissues at various stages of development of multiple field pea and faba bean genotypes.
clustering and annotation to generate a unigene set has permitted computational identification of ssr loci, and the design and evaluation of a set of est-ssr marker-directed primer pairs.
materials and methods
plant material
seeds of field pea were obtained from the australian temperate field crops collection  held at the department of primary industries, horsham, victoria, australia.
faba bean seeds were obtained from the australian faba bean breeding program at the university of adelaide, south australia, australia.
three to four seeds from each variety of field pea  and faba bean  were selected based on the criteria of genetic diversity and significant agronomic variation, and were sown into commercial potting mix.
these genotypes were also potential parents for the genetic mapping populations of field pea and faba bean, to be used to dissect various traits of interest.
germinated plantlets were grown to maturity under glasshouse conditions with natural light at the department of primary industries, bundoora, victoria, australia.
selected plant tissues were harvested for rna isolation from plants at various stages of development, including leaf , stem, flowers, immature pods, mature pods and immature seeds.
a total of 4- <dig> seeds were also germinated in petri dishes in order to provide material for harvest of seedling root and shoot samples.
all of the vegetative plant tissues  were pooled for rna isolation and designated ls  tissue.
all of the reproductive organs including flowers, immature pods, mature pods and immature seeds were also pooled for rna isolation and designated fs  tissue.
the seedling-derived root  and shoot  samples were used separately for rna isolation.
rna isolation and cdna preparation
total rna isolation and cdna synthesis were performed as described in an equivalent study performed for lentil  <cit> .
est sequence generation, assembly and annotation
cdnas obtained from the four distinct rna pools  were combined in equimolar ratio before proceeding to gs flx library preparation.
approximately  <dig> μg of bulked cdna was sheared by nebulisation at  <dig> kpa for 2- <dig> min.
the gs flx titanium shotgun libraries were constructed following manufacturer's instructions .
the ssdna libraries were quantified using real-time quantitative pcr.
finally, emulsion  pcr was performed using the lib-l empcr protocol .
the enriched beads obtained as a result of em-pcr were loaded onto picotitre plates for sequencing.
all of the pooled cdna libraries obtained from different genotypes of field pea and faba bean were separately sequenced on individual quarters of picotitre plates.
all sequence reads generated from different genotypes were de novo assembled using the next gene software .
the adaptor and primer sequences were removed prior to the assembly using the 'trimming' function .
de novo assembly was performed using the greedy algorithm and error correction condensation.
the greedy algorithm searches for maximum overlap between reads and extends the overlap to form large contigs and is recommended for  <dig> reads or reads with average read length >  <dig> bp.
the error correction condensation tool functions by dividing sequence reads in which homopolymers are found and at least  <dig> bases intervene between the homopolymer runs.
these shorter reads were termed keywords, and comparison of keywords between reads allowed the correct determination of the bases at the end of each keyword.
sequence reads that contain variations of low frequency were then corrected.
assembled contig outputs were deposited in the transcriptome shotgun assembly  of genbank .
contigs and singletons were compared against the m. truncatula , a. thaliana , g. max  and p. sativum  <cit>  transcriptome databases using blastn  <cit>  with a threshold e value of 10- <dig>  both field pea and faba bean unigene sets were also blastn analysed against respective est and nucleotide sequences publicly available in genbank.
blastn analysis was also performed in the non-redundant database of genbank using the tblastx algorithm to derive putative annotations of the unigene set.
gene ontology  terms were assigned to unigenes that showed hits against the arabidopsis thaliana database using the 'gene ontology at tair' tool.
discovery of est-ssrs, primer design and marker validation
detection of est-ssr loci and primer pair design was performed using the batch primer <dig> software http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer <dig> cgi.
the parameters were designed for identification of perfect di-, tri-, tetra-, penta-, and hexanucleotide motifs with minimum of repeat numbers of  <dig>   <dig>   <dig>   <dig>  and  <dig>  respectively.
primer design parameters were set as follows: length range =  <dig> to  <dig> nucleotides with  <dig> as optimum; pcr product size range =  <dig> to  <dig> bp; optimum annealing temperature = 55°c; and gc content 40-60%, with 50% as optimum.
genomic dna was extracted from target plant genotypes for est-ssr marker validation using the dneasy®  <dig> plant kit , following the manufacturer's instructions.
frozen leaf tissue from each genotype was used for each extraction and ground using a mixer mill  <dig> .
dna was resuspended in  <dig> μl of water and dilutions were performed to obtain a final concentration of  <dig> ng/μl, followed by storage at -20°c.
a collection of randomly selected est-ssr primer pairs were validated experimentally, forward primers being synthesised with addition of a bacteriophage m13-matching sequence, to enable fluorescent tail addition through the pcr amplification process
<cit> .
pcr conditions included a hot-start at 95°c for  <dig> minutes, followed by  <dig> cycles of 94°c for  <dig> s, 60-50°c for  <dig> s and 72°c for  <dig> s, followed by  <dig> cycles of 94°c for  <dig> s, 50°c for  <dig> s and 72°c for  <dig> s and a final elongation step of 72°c for  <dig> min.
pcr products were separated using an abi3730xl  according to manufacturer's instructions with the addition of the abi genescan liz <dig> size standard and amplification product sizes were determined using the genemapper® v <dig>  software .
RESULTS
est sequencing and de novo assembly
a total of  <dig>  and  <dig>  reads were generated from a range of sampled tissues from  <dig> field pea genotypes and  <dig> faba bean genotypes, respectively, using the gs flx titanium chemistry.
in addition to adaptor/primer sequence trimming, strings of 30- <dig> nucleotides from both the 5'- and 3'-termini of each sequence read were removed in order to generate high confidence data.
table  <dig> summarises the sequence output data for each species.
after clustering and assembly, a total of  <dig>  contigs and  <dig>  singletons were obtained from field pea, representing a total of  <dig>  unigenes .
in case of faba bean, a total of  <dig>  of unigenes were obtained, comprising  <dig>  contigs and  <dig>  singletons .
the unigene sets were then further assessed for quality based on read length, and any remnant sequences less than  <dig> bp were excluded from further analysis, leaving a total of  <dig>  contigs and  <dig>  singletons  and  <dig>  contigs and  <dig>  singletons .
in field pea, the length of contigs ranged from  <dig> bp to  <dig> bp, with an average of  <dig> bp, while for faba bean, contig length ranged from  <dig> bp to  <dig> bp with an average of  <dig> bp.
average contig coverage was  <dig>  fold  for field pea and  <dig>  fold  for faba bean.
the number of reads per contig for field pea varied between  <dig> and  <dig> , with an average of  <dig>  and the corresponding values for faba bean were between  <dig> and  <dig>  with an average of  <dig> .
distributions of read length and number of reads per contig are shown in figure  <dig>  the number of contigs with read length less than  <dig> bp was minimal .
most of the contigs were longer than  <dig>  kb .
in both species, the majority of the contigs were derived from less than  <dig> reads .
a total of  <dig> % field pea contigs and  <dig> % faba bean contigs were composed of more than  <dig> reads.
the length of singletons varied from 100- <dig> bp  and 100- <dig> bp .
for field pea, the largest proportion of the singletons  varied from 301- <dig> bp, while for faba bean, the majority of singletons  varied from 201- <dig> bp .
gene annotation
since m. truncatula is the model legume species that is most closely related to field pea and faba bean, consensus sequences from all contigs and singletons were preferentially compared to medicago coding sequences.
in case of field pea, a total of  <dig>  unique matches were obtained  .
the unigene set was also compared against the nr database of genbank.
a total of  <dig>  contigs and  <dig>  singletons  obtained matches at e < 10- <dig>  any query sequences that revealed a highest-ranking match against a non-plant species were removed from the list, leaving a total of  <dig>  unique hits .
finally, all of the consensus sequences were compared against the a. thaliana database.
a total of  <dig>  unique matches were obtained, consisting of  <dig>  contigs and  <dig>  singletons .
the faba bean unigene set was also compared with the m. truncatula genome and a total of  <dig>  hits were obtained  at e < 10- <dig> .
the unigene set was subsequently compared to the nr database of genbank, resulting in  <dig>  unique hits composed of  <dig>  contigs and  <dig>  singletons.
any sequence that matched a non-plant database entry was removed from the list, resulting in  <dig>  unique hits  .
the unigene set was also compared to the a. thaliana database at a threshold value of e < 10- <dig> , and a total of  <dig>  hits were obtained, consisting of  <dig>  contigs and  <dig>  singletons.
finally, the field pea and faba bean unigene sets were also compared against the g. max est sequence database that identified  <dig>  unique matches for field pea and  <dig>  for faba bean .
'the contigs and singletons obtained from field pea in the current study were also compared against the unigene set generated from transcriptome analysis of field pea performed by franssen et al.
and as a result, a total of  <dig>  overlapping hits were identified  .
in some instances, more than one contig revealed hits to the same gene, which may be due to origin of more than one contig or singleton from a single gene due either to non-overlapping sequence reads or high levels of sequence error in a single read.
this process has also demonstrated the benefits obtained from comparison between two complementary studies.
all of the ests and nucleotide sequences currently available in genbank for field pea and faba bean were also downloaded on the local server to perform blastn searches against field pea and faba bean contigs and singletons obtained from the current study.
in case of field pea, a total of  <dig>  est and  <dig>  nucleotide sequences obtained from genbank showed significant hits against unigene set generated in the current study  .
for faba bean, a total of  <dig> ests  and  <dig>  nucleotides  were found be common between genbank and transcriptome data generated from the current study .
all unique matches obtained from field pea and faba bean contigs by comparison against the a. thaliana database were annotated and go terms were further assigned.
for field pea, a total of  <dig>  gene counts and  <dig>  annotation counts were obtained, while for faba bean, these corresponding values were  <dig>  gene counts and  <dig>  annotation counts.
proportions of each unigene set attributed to major functional categories were determined .
in case of field pea, the intracellular component category of the cellular component classification class contributed the largest proportion of all annotations , followed by the cytoplasmic component , chloroplast component , membrane component , nuclear component and plasma membrane component  categories.
other components such as plastid, cytosol, mitochondria, er, golgi apparatus, cell wall, ribosome and extracellular components were represented at proportions less than 5% of total .
among the molecular function classification class, the enzyme activity, binding activity, hydrolase activity, transferase activity, molecular function and nucleotide binding categories included the majority of detected matches .
in the biological processes classification class, cellular  and metabolic processes  constituted the major categories, followed by protein metabolism  and unknown biological processes , .
similar results were obtained for faba bean.
in the cellular component classification class, the major contributors were intracellular and cytoplasmic components  .
the enzyme activity , binding activity  and unknown molecular functions  categories contributed the most in molecular function classification class  while among the biological processes classification class, cellular and metabolic processes  constituted the major categories .
est-ssr discovery
in field pea, est-ssr discovery was performed based on analysis of assembled contig templates, of which  <dig>   contained at least one repetitive motif.
a total of  <dig>  distinct loci were identified,  <dig> template contigs containing at least two ssr loci eligible for primer pair design.
a total of  <dig>  ssr primer pairs were designed from these  <dig>  distinct loci .
in the case of faba bean, a total of  <dig>  distinct loci were identified in  <dig> of  <dig>  assembled contigs , from which  <dig> ssr primer pairs were designed .
incidences of different repeat types were determined , the most abundant being trinucleotide arrays for both field pea  and faba bean .
frequencies for each array type according to repeat unit number were also evaluated , the most common class being n =  <dig> .
validation of est-ssr assays
a subset of  <dig> est-ssr primer pairs each from field pea and faba bean data sets were selected for validation of marker assay performance.
for field pea, a total of  <dig>  successfully obtained amplification products from one or more template genotypes, of which  <dig>  revealed polymorphism between  <dig> genotypes of field pea.
inclusion of a template sample from the non-domesticated species ps <dig>  permitted polymorphism detection by  <dig> additional primer pairs  .
for faba bean,  <dig> primer pairs  exhibited successful amplification, of which  <dig> detected polymorphic  between cultivated v. faba genotypes .
when the non-domesticated v. faba genotype acc <dig> was included in the analysis, polymorphism rate increased to 48% .
discussion
est assembly and gene annotation
the increasing capacity of dna sequencing technologies has permitted substantial increases in genomic resource availability for several legume crops that had been previously underdeveloped.
recently, large-scale transcriptome characterisation using the gs flx platform has been performed for both lentil and pigeonpea  <cit> .
this technology can deliver large amounts of data at considerably lower costs as compared to traditional sequencing methods, and so provides an effective means to expedite analysis of less-studied species  <cit> .
in the present study, equivalent approaches have been applied to the two vicieae species, field pea and faba bean, in order to develop a transcribed sequence database and to identify and validate est-ssrs.
gs flx sequencing has been shown to ineffectively process homopolymer regions that are longer than  <dig> bp in length  <cit> .
therefore, poly tails at mrna termini may present major challenges, and result in under-representation of the 3'-ends of transcripts.
in the present study, the problem was resolved through use of a modified primer with an interrupted polyd tail.
this contributed to an increase in the output of the total number of sequenced fragments by c. 6% .
a number of other transcriptome studies have used the same approach to overcome the homopolymer sequencing problems  <cit> .
prior to sequencing, normalisation of the cdna samples obtained from leaf and stem tissues was performed in order to increase the sequencing efficiency of rare transcripts.
the normalisation process helps to reduce over -sampling of abundant transcripts that are presentin high quantities, hence increasing confidence of detecting a larger proportion of rare transcripts.
preliminary experiments indicated that normalisation of leaf/stem cdna could increase the possibility of detecting rare transcripts by c. 10% .
similar approaches have been applied to detect rare transcripts in lentil, m. truncatula, artemisia annua and greenhouse whitefly  <cit> .
the average contig lengths for the target species in this study are comparable to those observed in other studies .
a large proportion of the reads assembled into contigs in case of field pea , which is comparable to the values observed in other studies .
in contrast, a relatively smaller proportion  of reads from faba bean assembled into contigs, resulting in lower length and depth as compared to the data derived from field pea.
this may be due to the fact that the sequencing output for faba bean was comparatively smaller than that of field pea.
similar results have been observed in other studies  <cit> .
as a result of de novo assembly, a large number of singletons were obtained both for field pea  and faba bean , also as observed for other species  <cit> .
although some singletons may arise as contaminating sequences or artefacts, the majority probably originate from transcripts expressed at low levels, and were consequently retained in the dataset.
many singleton sequences  exhibited high read quality due to matching of protein-encoding genes in the existing genic databases, and hence provide valuable sources of information.
the remaining singletons could have resulted from various reasons such as incompleteness of known databases, sequencing errors, short read lengths leading to difficulty in assembly etc.
<cit> .
blast searches against databases of model plant species provided annotation data for field pea and faba bean ests, with totals of  <dig>  and  <dig>  unique hits, respectively.
these values are very close to the estimated number of total genes  present in a typical diploid plant genome, based on data from rice , sorghum , a. thaliana and brachpodium distachyon  <cit> .
on this basis, the sequences annotated in this study are likely to represent c. 88% and c. 72% of the gene complements of field pea and faba bean, respectively.
such estimates are also supported by comparison with the m. truncatula genome, from which a total of  <dig>  unique hits obtained from field pea represented c. 49% of the known gene space, and  <dig>  unique hits from faba bean represented c. 41% of the known gene space.
comparisons were also made to g. max, which is more distantly related to the vicieae tribe species than m. truncatula, being located outside the hologalegina clade, a total of  <dig>  unique hits from field pea and  <dig>   <dig> from faba bean represent c. 35% and 30% of the known gene space respectively, based on total of predicted  <dig>  protein-coding loci in the palaeopolyploid genome of soybean.
in comparison to the genome of a. thaliana, which is more distantly related to both model and crop legume species within the dicotyledonous plants, the corresponding values were c. 25% for field pea and c. 20% for faba bean.
marker discovery and validation
one major advantage of second-generation dna sequencing technologies is the capacity for computational interrogation of transcriptome data in order to develop large numbers of gene-based genetic markers such as ssrs and snps, of which few are currently available in the public domain for either field pea or faba bean.
the est-ssr primer pair sets generated in the current study will prove directly useful for the target species, and due to likely primer site conservation, may also be readily transferable to closely related species  <cit> .
the transcriptome data generated in the current study, being derived from distinct genotypes, may potentially be also used for the detection of snp markers in field pea and faba bean, to further enrich the available genomic resources for these two species.
the relative proportions of ssr array types in field pea and faba bean were similar to those observed in other plant species  <cit> .
in theory, the frequencies of di-, tri-, tetra-, penta-, and hexanucleotide repeats should progressively decrease, based on the relative probability of replication slippage events.
however, trinucleotide repeat units were predominant, followed by tetra-, di-, hexa-, and pentanucleotide repeat units.
this observation is quite common for est-derived ssrs, as trinucleotide expansions  within translated regions are capable of maintaining reading frame and hence generating a homopolymeric amino acid run within a partially or fully active protein.
the validation results for sub-sets of est-ssr markers demonstrated that inclusion of non-domesticated genotypes in the study increased rates of polymorphism detection, consistent with the results of similar studies  <cit> .
est-ssrs generated in the present study will consequently provide a valuable tool for the understanding of global genetic diversity among both non-domesticated and cultivated pea and faba bean germplasm, as well as for dissection of the genetic control of important agronomic traits.
CONCLUSIONS
in the current study, the generation of est-datasets for field pea and faba bean has been described.
unigene sets obtained from field pea and faba bean were annotated against different genomic databases including those of m. truncatula, a. thaliana, g. max, and the nr database from genbank.
furthermore, the est dataset was used for design of est-ssrs, subsets of which were validated across a number of cultivated and wild genotypes of pea and faba bean, indicating effectiveness of polymorphism detection and cross transferability.
authors' contributions
sk and lp contributed equally to the gs flx sequencing, est dataset analysis, est-ssr marker assay design and interpretation of ssr genotyping data.
sk and jf drafted the manuscript.
ks assisted the sequence contig annotation process.
nc contributed to data analysis, interpretation and assisted in drafting the manuscript.
nc, sk, jf, tl, jp and mm co-conceptualised and coordinated the project.
mm assisted in drafting the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
consensus sequences of assembled contigs from field pea.
the data represents the consensus sequences of  <dig>  assembled contigs generated as a result of de novo assembly of field pea ests.
click here for file
additional file 2
sequence information on singletons from field pea.
the data represents the sequence information on all the singletons generated from de novo assembly of field pea ests.
click here for file
additional file 3
consensus sequences of assembled contigs from faba bean.
the data represents the consensus sequences of  <dig>  assembled contigs generated as a result of de novo assembly of faba bean ests.
click here for file
additional file 4
sequence information on singletons from faba bean.
the data represents the sequence information on all the singletons generated from de novo assembly of faba bean ests.
click here for file
additional file 5
bioinformatic annotation  of field pea unigene set against the medicago truncatula genome.
this file contains the blast results obtained as a result of comparison of field pea unigene set against the m. truncatula genome at an e value < 10- <dig>
click here for file
additional file 6
bioinformatic annotation  of field pea unigene set against the nr database of genbank.
this file contains the blast results obtained as a result of comparison of field pea unigene set against the genbank nr database at an e value < 10- <dig>
click here for file
additional file 7
bioinformatic annotation  of field pea unigene set against the arabidopsis thaliana genome.
this file contains the blast results obtained as a result of comparison of field pea unigene set against the a. thaliana genome at an e value < 10- <dig>
click here for file
additional file 8
bioinformatic annotation  of faba bean unigene set against the medicago truncatula genome.
this file contains the blast results obtained as a result of comparison of faba bean unigene set against the m. truncatula genome at an e value < 10- <dig>
click here for file
additional file 9
bioinformatic annotation  of faba bean unigene set against nr database of genbank.
this file contains the blast results obtained as a result of comparison of faba bean unigene set against the genbank nr database at an e value < 10- <dig>
click here for file
additional file 10
bioinformatic annotation  of faba bean unigene set against arabidopsis thaliana genome.
this file contains the blast results obtained as a result of comparison of faba bean unigene set against the a. thaliana genome at an e value < 10- <dig>
click here for file
additional file 11
bioinformatic annotation  of field pea and faba bean unigene sets against the glycine max genome.
this file contains the blast results obtained as a result of comparison of field pea and faba bean unigene sets against g. max genome at an e value < 10- <dig>
click here for file
additional file 12
bioinformatic annotation  of field pea and faba bean unigene sets against the pisum sativum transcriptome dataset from franssen et al  <dig>  this file contains the blast results obtained as a result of comparison of field pea and faba bean unigene sets against p. sativum transcriptome dataset at an e value < 10- <dig>
click here for file
additional file 13
blastn of field pea and faba bean contigs and singltones against genbank est and nucleotide  data.
this file contains the blastn results obtained as a result of comparison of field pea and faba bean contigs and singletons set against the est and nucleotide  databse of genbank at an e value < 10- <dig>
click here for file
additional file 14
sequence information of all of the ssr primer pairs identified and designed using batchprimer <dig> from field pea and faba bean ests.
this file contains all of the information  on ssr primer pairs designed using batchprimer  <dig>
click here for file
additional file 15
characterisation of a sub-sets of est-ssrs on wild and cultivated genotypes of field pea and faba bean.
this file represents the data on number and size of alleles amplified from screening of subsets of est-ssrprimer pairs on different genotypes of field pea and faba bean.
click here for file
acknowledgements
this work was supported by funding from the victorian department of primary industries and the grains research and development corporation, australia.

 the fabaceae is the third largest angiosperm family, containing c. species attributed to genera. legumes provide major benefits to cropping systems and the environment, due to the ability to perform symbiotic nitrogen fixation. in comparison to cereals, for which a broad range of genetic and genomic resources are available, genomic databases for legumes are generally still underdeveloped. however, recent advances in sequencing and genotyping technologies offer the opportunity to rapidly ameliorate the status of given species at relatively low cost. major efforts are currently being directed towards the development of species-specific genomic tools and datasets. as an example, the whole genome sequence of soybean, a warm-season grain legume, has recently been determined http://www.phytozome.net/soybean. cool-season food legumes within the hologalegina clade of the fabaceae sub-family papilionoideae, which includes lentil, chickpea, field pea and faba bean, are important food and fodder crops, especially in developing countries such as those of the indian sub-continent. these species are important components of farming systems across western asia, the middle east, north africa, the indian sub-continent, north america and australia. in australia, pulses are sown over c. million hectares and produce c. million tonnes of grain with a commodity value of over au$ million. despite close phylogenetic relationships, pulse species vary considerably in aspects of biology such as genome size, fundamental chromosome number, ploidy level, and degree of reproductive self-compatibility. the genome size of chickpea is relatively small, but pulses of the vicieae tribe exhibit much larger genome sizes. recently, generation of large-scale lentil transcriptome data by our group has substantially increased the volume of publicly available genomic data for this species. similar strategies have been pursued for field pea and faba bean in the current study. field pea, which is the third most globally important grain legume crop after soybean and common bean, is a self-pollinating diploid species with a genome size of c. gbp. various studies have been performed to determine the genetic basis of multiple phenotypic traits in field pea and to quantify diversity between different pea cultivars. recently, a comprehensive transcriptome analysis of field pea has been performed using second-generation sequencing technologies that will contribute significantly to the enrichment of genomics resources for field pea. in contrast, faba bean has not been widely adopted on a global basis. in terms of cultivation area, this species ranks fourth among the cool-season food legumes after field pea, chickpea and lentil http://faostat.fao.org. faba bean has been traditionally cultivated in the mediterranean basin, the nile valley, ethiopia, central and east asia, latin america, northern europe, north america and australia. faba bean is a diploid taxon, and exhibits facultative cross-pollination at frequencies ranging from 4-84%. the nuclear genome size of faba bean is one of the largest yet described among crop legumes, at c. gb. formal genetic analysis of faba bean, such as through genetic linkage mapping and identification of quantitative trait loci, has so far been hindered by these aspects of biology. conventional breeding methods based on phenotypic assessment are currently in use for breeding line selection in field pea and faba bean. such methods are logistically demanding and time-consuming, especially for traits that require specific biotic or abiotic challenges, such as resistance to individual diseases. in addition to this, when breeding for types eaten as immature seed, quality testing adds considerable complexity to the relevant programs. there is consequently a major requirement for species-specific molecular genetic markers and derived linkage maps for field pea and faba bean, to enable germplasm advancement through genomics-assisted selection. current publicly available genetic and genomic tools for field pea and faba bean are limited in extent, comprising and ests, respectively that are available in genbank. in addition to this, a recently sequenced pisum sativum transcriptome generated a total of unigenes that are also available for download as a fully annotated fasta format. second-generation dna sequencing systems such as the roche massively-parallel pyrosequencing platform are capable of rapidly producing species-specific genomic resources to address these short-comings. this system can generate 4- × bp from each run, with individual read lengths of 400- bp, and is suitable for de novo sequencing of small genomes, whole genome resequencing, snp detection, and in particular, sequencing of transcriptomes. ests obtained from the latter activity provide valuable resources for gene discovery, large-scale expression analysis, improved genome annotation, elucidation of phylogenetic relationships and facilitation of breeding programs for both plants and animals through provision of ssr and single nucleotide polymorphism genetic markers. ssr loci have been widely used for improvement of a range of crop species. only a limited number of ssrs are available in public domain for field pea and faba bean, creating an incentive for further discovery and validation. in comparison with genomic dna-derived ssrs, those located in ests are functionally associated with genic regions, and support potential diagnostic genetic marker development. this study describes the development, de novo assembly and gene annotation of a transcriptome dataset derived from cdna samples obtained from several tissues at various stages of development of multiple field pea and faba bean genotypes. clustering and annotation to generate a unigene set has permitted computational identification of ssr loci, and the design and evaluation of a set of est-ssr marker-directed primer pairs. materials and methods plant material seeds of field pea were obtained from the australian temperate field crops collection held at the department of primary industries, horsham, victoria, australia. faba bean seeds were obtained from the australian faba bean breeding program at the university of adelaide, south australia, australia. three to four seeds from each variety of field pea and faba bean were selected based on the criteria of genetic diversity and significant agronomic variation, and were sown into commercial potting mix. these genotypes were also potential parents for the genetic mapping populations of field pea and faba bean, to be used to dissect various traits of interest. germinated plantlets were grown to maturity under glasshouse conditions with natural light at the department of primary industries, bundoora, victoria, australia. selected plant tissues were harvested for rna isolation from plants at various stages of development, including leaf, stem, flowers, immature pods, mature pods and immature seeds. a total of 4- seeds were also germinated in petri dishes in order to provide material for harvest of seedling root and shoot samples. all of the vegetative plant tissues were pooled for rna isolation and designated ls tissue. all of the reproductive organs including flowers, immature pods, mature pods and immature seeds were also pooled for rna isolation and designated fs tissue. the seedling-derived root and shoot samples were used separately for rna isolation. rna isolation and cdna preparation total rna isolation and cdna synthesis were performed as described in an equivalent study performed for lentil. est sequence generation, assembly and annotation cdnas obtained from the four distinct rna pools were combined in equimolar ratio before proceeding to gs flx library preparation. approximately μg of bulked cdna was sheared by nebulisation at kpa for 2- min. the gs flx titanium shotgun libraries were constructed following manufacturer's instructions. the ssdna libraries were quantified using real-time quantitative pcr. finally, emulsion pcr was performed using the lib-l empcr protocol. the enriched beads obtained as a result of em-pcr were loaded onto picotitre plates for sequencing. all of the pooled cdna libraries obtained from different genotypes of field pea and faba bean were separately sequenced on individual quarters of picotitre plates. all sequence reads generated from different genotypes were de novo assembled using the next gene software. the adaptor and primer sequences were removed prior to the assembly using the'trimming' function. de novo assembly was performed using the greedy algorithm and error correction condensation. the greedy algorithm searches for maximum overlap between reads and extends the overlap to form large contigs and is recommended for reads or reads with average read length > bp. the error correction condensation tool functions by dividing sequence reads in which homopolymers are found and at least bases intervene between the homopolymer runs. these shorter reads were termed keywords, and comparison of keywords between reads allowed the correct determination of the bases at the end of each keyword. sequence reads that contain variations of low frequency were then corrected. assembled contig outputs were deposited in the transcriptome shotgun assembly of genbank. contigs and singletons were compared against the m. truncatula, a. thaliana, g. max and p. sativum transcriptome databases using blastn with a threshold e value of 10- both field pea and faba bean unigene sets were also blastn analysed against respective est and nucleotide sequences publicly available in genbank. blastn analysis was also performed in the non-redundant database of genbank using the tblastx algorithm to derive putative annotations of the unigene set. gene ontology terms were assigned to unigenes that showed hits against the arabidopsis thaliana database using the'gene ontology at tair' tool. discovery of est-ssrs, primer design and marker validation detection of est-ssr loci and primer pair design was performed using the batch primer software http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer cgi. the parameters were designed for identification of perfect di-, tri-, tetra-, penta-, and hexanucleotide motifs with minimum of repeat numbers of and respectively. primer design parameters were set as follows: length range = to nucleotides with as optimum; pcr product size range = to bp; optimum annealing temperature = 55°c; and gc content 40-60%, with 50% as optimum. genomic dna was extracted from target plant genotypes for est-ssr marker validation using the dneasy® plant kit, following the manufacturer's instructions. frozen leaf tissue from each genotype was used for each extraction and ground using a mixer mill. dna was resuspended in μl of water and dilutions were performed to obtain a final concentration of ng/μl, followed by storage at -20°c. a collection of randomly selected est-ssr primer pairs were validated experimentally, forward primers being synthesised with addition of a bacteriophage m13-matching sequence, to enable fluorescent tail addition through the pcr amplification process. pcr conditions included a hot-start at 95°c for minutes, followed by cycles of 94°c for s, 60-50°c for s and 72°c for s, followed by cycles of 94°c for s, 50°c for s and 72°c for s and a final elongation step of 72°c for min. pcr products were separated using an abi3730xl according to manufacturer's instructions with the addition of the abi genescan liz size standard and amplification product sizes were determined using the genemapper® v software.  est sequencing and de novo assembly a total of and reads were generated from a range of sampled tissues from field pea genotypes and faba bean genotypes, respectively, using the gs flx titanium chemistry. in addition to adaptor/primer sequence trimming, strings of 30- nucleotides from both the 5'- and 3'-termini of each sequence read were removed in order to generate high confidence data. table summarises the sequence output data for each species. after clustering and assembly, a total of contigs and singletons were obtained from field pea, representing a total of unigenes. in case of faba bean, a total of of unigenes were obtained, comprising contigs and singletons. the unigene sets were then further assessed for quality based on read length, and any remnant sequences less than bp were excluded from further analysis, leaving a total of contigs and singletons and contigs and singletons. in field pea, the length of contigs ranged from bp to bp, with an average of bp, while for faba bean, contig length ranged from bp to bp with an average of bp. average contig coverage was fold for field pea and fold for faba bean. the number of reads per contig for field pea varied between and, with an average of and the corresponding values for faba bean were between and with an average of. distributions of read length and number of reads per contig are shown in figure the number of contigs with read length less than bp was minimal. most of the contigs were longer than kb. in both species, the majority of the contigs were derived from less than reads. a total of % field pea contigs and % faba bean contigs were composed of more than reads. the length of singletons varied from 100- bp and 100- bp. for field pea, the largest proportion of the singletons varied from 301- bp, while for faba bean, the majority of singletons varied from 201- bp. gene annotation since m. truncatula is the model legume species that is most closely related to field pea and faba bean, consensus sequences from all contigs and singletons were preferentially compared to medicago coding sequences. in case of field pea, a total of unique matches were obtained. the unigene set was also compared against the nr database of genbank. a total of contigs and singletons obtained matches at e < 10- any query sequences that revealed a highest-ranking match against a non-plant species were removed from the list, leaving a total of unique hits. finally, all of the consensus sequences were compared against the a. thaliana database. a total of unique matches were obtained, consisting of contigs and singletons. the faba bean unigene set was also compared with the m. truncatula genome and a total of hits were obtained at e < 10-. the unigene set was subsequently compared to the nr database of genbank, resulting in unique hits composed of contigs and singletons. any sequence that matched a non-plant database entry was removed from the list, resulting in unique hits. the unigene set was also compared to the a. thaliana database at a threshold value of e < 10-, and a total of hits were obtained, consisting of contigs and singletons. finally, the field pea and faba bean unigene sets were also compared against the g. max est sequence database that identified unique matches for field pea and for faba bean.'the contigs and singletons obtained from field pea in the current study were also compared against the unigene set generated from transcriptome analysis of field pea performed by franssen et al. and as a result, a total of overlapping hits were identified. in some instances, more than one contig revealed hits to the same gene, which may be due to origin of more than one contig or singleton from a single gene due either to non-overlapping sequence reads or high levels of sequence error in a single read. this process has also demonstrated the benefits obtained from comparison between two complementary studies. all of the ests and nucleotide sequences currently available in genbank for field pea and faba bean were also downloaded on the local server to perform blastn searches against field pea and faba bean contigs and singletons obtained from the current study. in case of field pea, a total of est and nucleotide sequences obtained from genbank showed significant hits against unigene set generated in the current study. for faba bean, a total of ests and nucleotides were found be common between genbank and transcriptome data generated from the current study. all unique matches obtained from field pea and faba bean contigs by comparison against the a. thaliana database were annotated and go terms were further assigned. for field pea, a total of gene counts and annotation counts were obtained, while for faba bean, these corresponding values were gene counts and annotation counts. proportions of each unigene set attributed to major functional categories were determined. in case of field pea, the intracellular component category of the cellular component classification class contributed the largest proportion of all annotations, followed by the cytoplasmic component, chloroplast component, membrane component, nuclear component and plasma membrane component categories. other components such as plastid, cytosol, mitochondria, er, golgi apparatus, cell wall, ribosome and extracellular components were represented at proportions less than 5% of total. among the molecular function classification class, the enzyme activity, binding activity, hydrolase activity, transferase activity, molecular function and nucleotide binding categories included the majority of detected matches. in the biological processes classification class, cellular and metabolic processes constituted the major categories, followed by protein metabolism and unknown biological processes,. similar results were obtained for faba bean. in the cellular component classification class, the major contributors were intracellular and cytoplasmic components. the enzyme activity, binding activity and unknown molecular functions categories contributed the most in molecular function classification class while among the biological processes classification class, cellular and metabolic processes constituted the major categories. est-ssr discovery in field pea, est-ssr discovery was performed based on analysis of assembled contig templates, of which contained at least one repetitive motif. a total of distinct loci were identified, template contigs containing at least two ssr loci eligible for primer pair design. a total of ssr primer pairs were designed from these distinct loci. in the case of faba bean, a total of distinct loci were identified in of assembled contigs, from which ssr primer pairs were designed. incidences of different repeat types were determined, the most abundant being trinucleotide arrays for both field pea and faba bean. frequencies for each array type according to repeat unit number were also evaluated, the most common class being n =. validation of est-ssr assays a subset of est-ssr primer pairs each from field pea and faba bean data sets were selected for validation of marker assay performance. for field pea, a total of successfully obtained amplification products from one or more template genotypes, of which revealed polymorphism between genotypes of field pea. inclusion of a template sample from the non-domesticated species ps permitted polymorphism detection by additional primer pairs. for faba bean, primer pairs exhibited successful amplification, of which detected polymorphic between cultivated v. faba genotypes. when the non-domesticated v. faba genotype acc was included in the analysis, polymorphism rate increased to 48%. discussion est assembly and gene annotation the increasing capacity of dna sequencing technologies has permitted substantial increases in genomic resource availability for several legume crops that had been previously underdeveloped. recently, large-scale transcriptome characterisation using the gs flx platform has been performed for both lentil and pigeonpea. this technology can deliver large amounts of data at considerably lower costs as compared to traditional sequencing methods, and so provides an effective means to expedite analysis of less-studied species. in the present study, equivalent approaches have been applied to the two vicieae species, field pea and faba bean, in order to develop a transcribed sequence database and to identify and validate est-ssrs. gs flx sequencing has been shown to ineffectively process homopolymer regions that are longer than bp in length. therefore, poly tails at mrna termini may present major challenges, and result in under-representation of the 3'-ends of transcripts. in the present study, the problem was resolved through use of a modified primer with an interrupted polyd tail. this contributed to an increase in the output of the total number of sequenced fragments by c. 6%. a number of other transcriptome studies have used the same approach to overcome the homopolymer sequencing problems. prior to sequencing, normalisation of the cdna samples obtained from leaf and stem tissues was performed in order to increase the sequencing efficiency of rare transcripts. the normalisation process helps to reduce over -sampling of abundant transcripts that are presentin high quantities, hence increasing confidence of detecting a larger proportion of rare transcripts. preliminary experiments indicated that normalisation of leaf/stem cdna could increase the possibility of detecting rare transcripts by c. 10%. similar approaches have been applied to detect rare transcripts in lentil, m. truncatula, artemisia annua and greenhouse whitefly. the average contig lengths for the target species in this study are comparable to those observed in other studies. a large proportion of the reads assembled into contigs in case of field pea, which is comparable to the values observed in other studies. in contrast, a relatively smaller proportion of reads from faba bean assembled into contigs, resulting in lower length and depth as compared to the data derived from field pea. this may be due to the fact that the sequencing output for faba bean was comparatively smaller than that of field pea. similar results have been observed in other studies. as a result of de novo assembly, a large number of singletons were obtained both for field pea and faba bean, also as observed for other species. although some singletons may arise as contaminating sequences or artefacts, the majority probably originate from transcripts expressed at low levels, and were consequently retained in the dataset. many singleton sequences exhibited high read quality due to matching of protein-encoding genes in the existing genic databases, and hence provide valuable sources of information. the remaining singletons could have resulted from various reasons such as incompleteness of known databases, sequencing errors, short read lengths leading to difficulty in assembly etc.. blast searches against databases of model plant species provided annotation data for field pea and faba bean ests, with totals of and unique hits, respectively. these values are very close to the estimated number of total genes present in a typical diploid plant genome, based on data from rice, sorghum, a. thaliana and brachpodium distachyon. on this basis, the sequences annotated in this study are likely to represent c. 88% and c. 72% of the gene complements of field pea and faba bean, respectively. such estimates are also supported by comparison with the m. truncatula genome, from which a total of unique hits obtained from field pea represented c. 49% of the known gene space, and unique hits from faba bean represented c. 41% of the known gene space. comparisons were also made to g. max, which is more distantly related to the vicieae tribe species than m. truncatula, being located outside the hologalegina clade, a total of unique hits from field pea and from faba bean represent c. 35% and 30% of the known gene space respectively, based on total of predicted protein-coding loci in the palaeopolyploid genome of soybean. in comparison to the genome of a. thaliana, which is more distantly related to both model and crop legume species within the dicotyledonous plants, the corresponding values were c. 25% for field pea and c. 20% for faba bean. marker discovery and validation one major advantage of second-generation dna sequencing technologies is the capacity for computational interrogation of transcriptome data in order to develop large numbers of gene-based genetic markers such as ssrs and snps, of which few are currently available in the public domain for either field pea or faba bean. the est-ssr primer pair sets generated in the current study will prove directly useful for the target species, and due to likely primer site conservation, may also be readily transferable to closely related species. the transcriptome data generated in the current study, being derived from distinct genotypes, may potentially be also used for the detection of snp markers in field pea and faba bean, to further enrich the available genomic resources for these two species. the relative proportions of ssr array types in field pea and faba bean were similar to those observed in other plant species. in theory, the frequencies of di-, tri-, tetra-, penta-, and hexanucleotide repeats should progressively decrease, based on the relative probability of replication slippage events. however, trinucleotide repeat units were predominant, followed by tetra-, di-, hexa-, and pentanucleotide repeat units. this observation is quite common for est-derived ssrs, as trinucleotide expansions within translated regions are capable of maintaining reading frame and hence generating a homopolymeric amino acid run within a partially or fully active protein. the validation results for sub-sets of est-ssr markers demonstrated that inclusion of non-domesticated genotypes in the study increased rates of polymorphism detection, consistent with the results of similar studies. est-ssrs generated in the present study will consequently provide a valuable tool for the understanding of global genetic diversity among both non-domesticated and cultivated pea and faba bean germplasm, as well as for dissection of the genetic control of important agronomic traits.  in the current study, the generation of est-datasets for field pea and faba bean has been described. unigene sets obtained from field pea and faba bean were annotated against different genomic databases including those of m. truncatula, a. thaliana, g. max, and the nr database from genbank. furthermore, the est dataset was used for design of est-ssrs, subsets of which were validated across a number of cultivated and wild genotypes of pea and faba bean, indicating effectiveness of polymorphism detection and cross transferability. authors' contributions sk and lp contributed equally to the gs flx sequencing, est dataset analysis, est-ssr marker assay design and interpretation of ssr genotyping data. sk and jf drafted the manuscript. ks assisted the sequence contig annotation process. nc contributed to data analysis, interpretation and assisted in drafting the manuscript. nc, sk, jf, tl, jp and mm co-conceptualised and coordinated the project. mm assisted in drafting the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 consensus sequences of assembled contigs from field pea. the data represents the consensus sequences of assembled contigs generated as a result of de novo assembly of field pea ests. click here for file additional file 2 sequence information on singletons from field pea. the data represents the sequence information on all the singletons generated from de novo assembly of field pea ests. click here for file additional file 3 consensus sequences of assembled contigs from faba bean. the data represents the consensus sequences of assembled contigs generated as a result of de novo assembly of faba bean ests. click here for file additional file 4 sequence information on singletons from faba bean. the data represents the sequence information on all the singletons generated from de novo assembly of faba bean ests. click here for file additional file 5 bioinformatic annotation of field pea unigene set against the medicago truncatula genome. this file contains the blast results obtained as a result of comparison of field pea unigene set against the m. truncatula genome at an e value < 10- click here for file additional file 6 bioinformatic annotation of field pea unigene set against the nr database of genbank. this file contains the blast results obtained as a result of comparison of field pea unigene set against the genbank nr database at an e value < 10- click here for file additional file 7 bioinformatic annotation of field pea unigene set against the arabidopsis thaliana genome. this file contains the blast results obtained as a result of comparison of field pea unigene set against the a. thaliana genome at an e value < 10- click here for file additional file 8 bioinformatic annotation of faba bean unigene set against the medicago truncatula genome. this file contains the blast results obtained as a result of comparison of faba bean unigene set against the m. truncatula genome at an e value < 10- click here for file additional file 9 bioinformatic annotation of faba bean unigene set against nr database of genbank. this file contains the blast results obtained as a result of comparison of faba bean unigene set against the genbank nr database at an e value < 10- click here for file additional file 10 bioinformatic annotation of faba bean unigene set against arabidopsis thaliana genome. this file contains the blast results obtained as a result of comparison of faba bean unigene set against the a. thaliana genome at an e value < 10- click here for file additional file 11 bioinformatic annotation of field pea and faba bean unigene sets against the glycine max genome. this file contains the blast results obtained as a result of comparison of field pea and faba bean unigene sets against g. max genome at an e value < 10- click here for file additional file 12 bioinformatic annotation of field pea and faba bean unigene sets against the pisum sativum transcriptome dataset from franssen et al this file contains the blast results obtained as a result of comparison of field pea and faba bean unigene sets against p. sativum transcriptome dataset at an e value < 10- click here for file additional file 13 blastn of field pea and faba bean contigs and singltones against genbank est and nucleotide data. this file contains the blastn results obtained as a result of comparison of field pea and faba bean contigs and singletons set against the est and nucleotide databse of genbank at an e value < 10- click here for file additional file 14 sequence information of all of the ssr primer pairs identified and designed using batchprimer from field pea and faba bean ests. this file contains all of the information on ssr primer pairs designed using batchprimer click here for file additional file 15 characterisation of a sub-sets of est-ssrs on wild and cultivated genotypes of field pea and faba bean. this file represents the data on number and size of alleles amplified from screening of subsets of est-ssrprimer pairs on different genotypes of field pea and faba bean. click here for file acknowledgements this work was supported by funding from the victorian department of primary industries and the grains research and development corporation, australia.
a significant enrichment of genomic resources is consequently required in order to understand the genetic architecture of important agronomic traits, and to support germplasm enhancement, genetic diversity, population structure and demographic studies. for field pea, primer pairs successfully obtained amplification products from one or more template genotypes, of which 59% revealed polymorphism between genotypes. comparison to the genome of soybean resulted in unique hits for field pea and unique hits for faba bean, corresponding to c. 35% and 30% of the known gene space, respectively. consensus sequences were compared against the genome of the model legume species medicago truncatula gaertn., as well as that of the more distantly related, but better-characterised genome of arabidopsis thaliana l.. in comparison to m. truncatula coding sequences, and unique hits were obtained from field pea and faba bean. simple sequence repeat -containing expressed sequence tags were identified from consensus sequences, and totals of and primer pairs were designed for field pea and faba bean. in the case of faba bean, primer pairs displayed successful amplification, of which 48% detected polymorphism. the subsets from these primer pairs proved highly useful for polymorphism detection within pisum and vicia germplasm. est-ssr loci were detected at incidences of 14-17%, permitting design of comprehensive sets of primer pairs. field pea and faba bean are cool-season grain legume species that provide rich sources of food for humans and fodder for livestock. cdna samples obtained from various tissue types of specific field pea and faba bean genotypes were sequenced using roche gs flx titanium technology. a total of and reads for field pea and faba bean, respectively, were de novo assembled to generate sets of and unigenes. totals of field pea and faba bean unigenes were subsequently annotated from genbank. subsets of est-ssr markers were screened for validation across modest panels of field pea and faba bean cultivars, as well as related non-domesticated species. the generation of est datasets for field pea and faba bean has permitted effective unigene identification and functional sequence annotation.
 field pea and faba bean are cool-season grain legume species that provide rich sources of food for humans and fodder for livestock. to date, both species have been relative'genomic orphans' due to limited availability of genetic and genomic information. a significant enrichment of genomic resources is consequently required in order to understand the genetic architecture of important agronomic traits, and to support germplasm enhancement, genetic diversity, population structure and demographic studies.  cdna samples obtained from various tissue types of specific field pea and faba bean genotypes were sequenced using roche gs flx titanium technology. a total of and reads for field pea and faba bean, respectively, were de novo assembled to generate sets of and unigenes. consensus sequences were compared against the genome of the model legume species medicago truncatula gaertn., as well as that of the more distantly related, but better-characterised genome of arabidopsis thaliana l.. in comparison to m. truncatula coding sequences, and unique hits were obtained from field pea and faba bean. totals of field pea and faba bean unigenes were subsequently annotated from genbank. comparison to the genome of soybean resulted in unique hits for field pea and unique hits for faba bean, corresponding to c. 35% and 30% of the known gene space, respectively. simple sequence repeat -containing expressed sequence tags were identified from consensus sequences, and totals of and primer pairs were designed for field pea and faba bean. subsets of est-ssr markers were screened for validation across modest panels of field pea and faba bean cultivars, as well as related non-domesticated species. for field pea, primer pairs successfully obtained amplification products from one or more template genotypes, of which 59% revealed polymorphism between genotypes. in the case of faba bean, primer pairs displayed successful amplification, of which 48% detected polymorphism.  the generation of est datasets for field pea and faba bean has permitted effective unigene identification and functional sequence annotation. est-ssr loci were detected at incidences of 14-17%, permitting design of comprehensive sets of primer pairs. the subsets from these primer pairs proved highly useful for polymorphism detection within pisum and vicia germplasm. 