BACKGROUND
yams  are the primary agricultural commodities and major staple crop in africa.
yam tubers are nutritionally rich and a major source of dietary fiber, carbohydrates, vitamin c and essential minerals.
worldwide, yam consumption is  <dig> million tons http://www.iita.org.
in  <dig>  yam production was  <dig> million tons worldwide, of which africa produced 96%, and nigeria is the major producer  with more than  <dig> million tons  <cit> .
the consumer demand for yam is very high in sub-saharan region of africa, but the yam production is declining in this region due to factors including anthracnose disease caused by a fungus, colletotrichum gloeosporioides  <cit> , pests, and decline in soil fertility  <cit> .
yams are polyploid crop species and are propagated vegetatively from tubers .
the water yam  is the most widely cultivated species and is highly susceptible to anthracnose disease  <cit> .
the genetic improvement of yam at iita and ctcri  concentrated on the development of disease resistant and high yielding varieties.
through classical breeding, it would be very difficult to develop a resistant cultivar due to constraints such as the long growth cycle , dioecious and poor flowering nature, polyploidy, vegetative propagation and heterozygous genetic background  <cit> .
a large collection of germplasm with huge genetic variability is available, and it would be profitable to use candidate gene approach and trap the important trait information for disease and pest resistance.
colletotrichum is a large genus of ascomycete fungi, containing many species which cause anthracnose or blight on a wide range of important crops and ornamental plants  <cit> .
two genes, clk <dig> which encodes a serine/threonine kinase in c. lindemuthianum  <cit> , and cap <dig> which encodes a wall glycoprotein of c. gloeosporioides appressoria  <cit> , have been shown to have a role in pathogenicity and virulence, respectively.
necrotrophy is clearly linked to the increased expression of plant cell wall degrading enzymes such as endo-polygalacturonases  and pectin lyases.
previous studies  <cit>  showed that endo-pg, two forms of pectin lyase, a- and b-galactopyranosidase, a-arabinofuranosidase, and a protease are secreted into culture medium containing polypectate of bean cell walls.
pectin lyase activity was first observed  <dig> days after inoculation of beans with c. lindemuthianum, rising to maximum activity at  <dig> days, after which activity declined.
thus, the expression of pectin lyase activity correlates well with the onset of necrotrophy and the subsequent development of lesions  <cit> .
therefore the yam leaf material for current investigation was collected on 3rd and 7th day after inoculation with c. gloeosporioides fungus.
initial genetic inheritance studies showed that resistance to yam anthracnose in water yam is dominant and quantitatively inherited  <cit> .
a single major dominant locus controlling resistance in the breeding line tda 95- <dig> was tentatively designated dcg- <dig>  until allelism was investigated  <cit> .
lower number of molecular markers in yam limits the genetic mapping efforts.
previously developed ssrs from dioscorea tokoro  <cit> , a wild diploid yam species of east asia and japan were found not to be useful in cultivated yams d. rotundata and d. alata  <cit> .
most of the current molecular markers for the yam genome by mignouna et al.
are based on aflp and rapd  <cit> .
however, for effective gene discovery and marker-assisted breeding, it is important to develop more user-friendly, efficient, transportable and co-dominant markers such as simple sequence repeat  markers and single nulceotide polymorphism markers .
est analysis is the most efficient and effective approach for the identification of candidate genes and also assist in new molecular markers such as est-ssrs and snps  <cit> .
as of 10th october  <dig>  there were only  <dig> est sequences stored at genbank  for the genus dioscorea.
to understand the transcriptome of yam and for new marker discovery to assist in yam crop improvement programs, generation of ests would be extremely desirable.
with the objective of characterizing transcriptome in yam, initial efforts were focused on successful isolation of mrna, cdna library construction and sequencing using sanger's method  <cit> .
two hundred est sequences were generated using this approach, but reported little functional significance during blast search analysis.
to enrich and characterize expressed gene sequence information especially during the anthracnose infection for candidate gene identification and also to utilize the sequence information for new marker discovery, a collaborative effort to generate large number of ests was initiated between vsu, usa and iita, africa.
for this purpose, two anthracnose resistant germplasm lines 87- <dig>  95- <dig> and a susceptible line 95- <dig> were selected for cdna library construction from leaf tissues inoculated with the c. gloeosporioides fungus.
the roche  <dig> pyrosequencing technology was used to generate est sequence information .
RESULTS
generation of ests
assembly process generated  <dig>  ests in tda 95-0328;  <dig>  ests in tda 95- <dig>  and  <dig>  ests in tda 87- <dig> with average sequence lengths of  <dig>   <dig> and  <dig> bases, respectively .
all the est sequences generated were submitted to genbank at ncbi and were assigned genbank accession numbers ho809681-ho <dig> with dbest id from  <dig> to  <dig> for library tda 95-0310; ho825422-ho <dig> with dbest ids from  <dig> to  <dig> for library tda 95-0328; ho <dig> to ho <dig> with dbest ids from  <dig> to  <dig> for library tda 87- <dig>  these sequences contributed to >99% of ests currently available in yam at genbank and would serve as the major resource for the yam research community.
transcriptome analysis
est sequences obtained from each individual library were first analyzed using blast2go http://blast2go.org <cit> .
blast2go provides the gene annotation information for the ests and also helps in comparison of two est datasets to find expression differences.
blast search analysis across all three libraries revealed highest homology with the sequences from oryza sativa, vitis vinifera, populus trichocarpa and other crop species.
mapping and annotation steps in blast2go program resulted in providing go ids  to individual ests in each dataset of the three libraries .
a total of  <dig>   of  <dig>  ests were annotated with  <dig>  non-redundant  go ids in the susceptible tda 95- <dig> germplasm line, while in resistant tda 95- <dig> line,  <dig>   of  <dig>  ests were annotated with  <dig>  nr go ids and in other resistant line tda 87- <dig> library,  <dig>   of  <dig>  ests were annotated with  <dig>  nr go ids.
approximately 50% of ests could not be annotated and remained unknown using blas2go.
to complement and comprehensively annotate the yam transcriptome, all the est sequences were submitted to mercator tool of mapman database.
mercator tool generated functional predictions by searching  <dig> reference databases  and assigned mapman bin ids .
still 44% of ests lacked significant homology with genes in other crops in each of the three datasets and these may include novel metabolic genes in yam.
a comparative analysis was done using the bin id information from each library to identify unique ests in each library and also the common genes shared with other libraries.
the distribution of ests with known function is presented as a venn diagram in figure  <dig>  a large subset of  <dig> ests representing  <dig> unique bin ids were shared across three libraries and these may account for housekeeping genes involved in general cellular metabolism.
the two resistant lines tda 95- <dig> and tda 87- <dig> had  <dig> and  <dig> unique ests, respectively, which may account for the tolerance against c. gloeosporioides fungus.
in tda 95- <dig> susceptible genotype, there were  <dig> unique sequences which predominantly matched genes for protein synthesis and nucleotide synthesis .
however, there were several sequences annotated as protein kinases and were known to have a definite role in disease signaling  <cit> .
another interesting feature is the presence of genes for synthesis of cell wall precursors and secondary metabolites which might be highly induced due to severe biotic stress.
there were also genes for recycling nad to maintain glycolysis and substrate level phosphorylation in the absence of oxygen in damaged leaf of susceptible genotype  <cit> .
removal of the toxic compounds like proline is very essential for plant survival under stress  <cit> .
there were unique genes for proline degradation in susceptible genotype suggesting the presence of a possible mechanism of detoxification during pathogenesis.
common sequences between tda 95- <dig> and tda 95- <dig>  were annotated as  <dig> genes representing cell wall precursors, co-factor and vitamin metabolism, tetrapyrrole synthesis, c1-metabolism, lipid metabolism, amino acid metabolism, secondary metabolism, hormone metabolism, major and minor cho metabolism, nucleotide metabolism, genes for regulation of transcription, amino acid activation, protein synthesis, protein degradation, energy metabolism, and genes for sugar and light signaling.
on the other hand, the common genes between tda 95- <dig> and tda 87- <dig> were cell wall proteins, sulfate assimilation related genes, phosphatidylserine decarboxylase, hydroxymethylpyrimidine kinase, l-galactono- <dig> -lactone dehydrogenase, genes for dicer degradation, gebp like transcription factor, and myo inositol oxygenase.
similarly, tda 87- <dig> and tda 95- <dig> shared sequences related to dihydroneopterin aldolase, glycine cleavage h protein, phosphomannose isomerase, c-lectin, alcohol dehydrogenase, and malate dehydrogenase genes.
although these genes have specific role in metabolism, regulation and signaling, the significance of their sharing between yam genotypes cannot be speculated and is beyond the scope of this paper.
functional analysis
using the go id information, the ests were classified into subsets based on molecular function and is represented as a pie diagram for each library .
a major subset of ests  across all libraries was linked to binding and catalytic activity, where the remaining groups involved transporter, signal transduction, transcription factors, secondary metabolism and antioxidant activities.
based on biological process classification, a major proportion of the genes were involved in oxidation-reduction activity across all libraries and these may involve genes in energy metabolisms and other housekeeping activities.
ests were also grouped under other biological processes such as photosynthesis, membrane transport, transcription, translation, protein folding, and others.
however, a significant proportion of the expressed genes  were biotic stress and other stress responsive genes .
differential gene expression analysis
from the est datasets, it is interesting to identify genes involved in defense mechanisms that were differentially expressed in susceptible and resistant yam genotypes.
using the bin ids of ests in each library, the expression of various ests were compared from one library to the other.
these results were summarized in figure  <dig> and additional file  <dig>  the resistant genotypes had unique ests related to carbohydrate metabolism, cell wall biogenesis, lipid and amino acid metabolism, secondary and hormone metabolism, transcription factors, protein synthesis, and signaling proteins .
even though, the est datasets is not comprehensive of the entire yam transcriptome, this analysis would shed some light on the gene expression differences during anthracnose infection.
further investigation of these differentially expressed genes using microarray platforms or rna-seq or real-time quantitative pcr experiments, would assist in elucidating the underlying molecular mechanism for anthracnose tolerance in yam.
informative defense related genes during anthracnose infection
the differential gene expression analysis provided the gene information that might be potentially over-expressed or under-expressed compared to other libraries.
these results were summated in additional file  <dig>
multiple pathogenesis-related genes and host defense related genes including signaling genes were identified from the ests in the resistant lines and they include nicotiana lesion inducing proteins, erwinia induced proteins, cysteine proteases, cysteine protease inhibitors, peroxidases, extensin, wall associated kinases, brassinosteroid receptor kinases, ethylene responsive genes, phosphatidylinositol 4-kinase, leucine rich repeat  genes, serine-threonine protein kinases and others signaling proteins.
signal transduction in plants, leading to the expression of defense genes, is initiated by leucine rich repeat -type membrane receptors containing intrinsic kinase activity as in the case of fls <dig>  <cit> .
there were unique genes involved in isoprenoid and flavonoids biosynthesis and fatty acid synthesis and elongase activity in tda 87- <dig> compared to tda 95- <dig> and tda 95- <dig>  there were hits specifically showing homologies to peroxisome biogenesis protein pex <dig>  and perxisome assembly factor  in library  <dig> and  <dig>  this confirms previous reports of plant peroxisomes role in the biosynthesis of the signaling molecules like jasmonic acid, β-oxidation of indole butyric acid , and sulphur and polyamine metabolism.
moreover, evidence is emerging from recent studies that peroxisomes have important functions in specific defense mechanisms, conferring resistance against pathogen attack  <cit> .
there were homologies to 12-oxo-phytodienoic acid reductases , enzymes of the octadecanoid pathway, which convert linolenic acid to a phytohormone jasmonic acid.
fifty nine significant matches to auxin responsive proteins , auxin signaling f-box  <dig>  and auxin response transcription factors  were observed in tda 87- <dig> and tda 95- <dig>  confirming their role in host defense against pathogens.
actually plant peroxidases  transduce the extracellular signals into the redox signals that eventually stimulate the intracellular ca2+ signaling required for induction of defense responses  <cit> .
there were significant hits to different plant peroxidases in resistant libraries confirming their requirement for defense against colletotrichum fungus.
the hits to pectate lyase were few, one each in tda 95- <dig>  and tda 87- <dig>  compared to four in tda 95/ <dig>  the contribution of a single pectate lyase gene  to the pathogenic abilities of c. magna confirmed that pectate lyase is a pathogenicity factor required for the penetration and colonization of colletotrichum species  <cit> .
cell-wall-degrading enzymes , like pectolytic enzymes, are considered to play a role in the pathogenesis of bacteria and fungi on their hosts  <cit> .
the peptidoglycan-binding lysm domain-containing protein and beta- <dig> -glucanases may help in fungal cell wall degradation  <cit>  as there were significant hits observed in the resistant genotypes.
the disruption of a single enzyme may be complemented by the activity of other cwdes  <cit> .
there was also homology to chitin binding, chitinase activity and chitin catabolic/metabolic process and were abundant in 87- <dig>  a genotype resistant to c. gloeosporioides strain sgg.
the activation of the defense response, after pathogen inoculation, is the most important factor for the success of host resistance  <cit> .
the est data from our study also suggest strength of the expression of ubiquitous early acting genes for establishment of the resistance response of the resistant genotype 87- <dig>
marker discovery
the est sequence information generated in this study also serves as a major resource to generate new molecular markers.
simple sequence repeat motifs detected in est sequences are usually termed as est-ssrs.
these are very informative markers as they exist within the gene sequence.
a total of  <dig>  ests from three genotypes, with an average length of  <dig> bp were used to evaluate for the presence of ssr motifs.
there are a total of  <dig> est sequences containing ssrs identified from three libraries.
in this work, ssrs were considered for primer design that fitted the following criteria: a minimum pattern length of  <dig> bp, excluding polya and polyt repeat, at least  <dig> repeat units in case of di-nucleotide and at least  <dig> repeat units for tri-, tetra-, penta- and hexa-nucleotide ssrs.
there are about a total of  <dig> good quality repeats containing di , tri , tetra , penta , hexa  and hepta  repeats which were used for designing primers .
the default settings for primer <dig> program: optimum temperature of 63oc and an optimum primer size of  <dig> bases were selected for designing the primers.
a total of  <dig> primer pairs were designed from  <dig> est sequences.
a subset were tested for screening genomic dna of  <dig> genotypes using simple  <dig> % agarose gel electrophoresis for polymorphism detection and found polymorphic.
these ssr primers are summarized in additional file  <dig>
identification of homologous snps is very challenging in a polyploid species such as yam.
haplosnper generates putative snp haplotype for each contig using cap <dig> generated .ace file.
therefore, we can verify whether a snp is heterozygous within each genotype or homozygous at specific loci.
if they are heterozygous within each genotype, basically they may be copies from homeologous genomes or paralogous copies.
even though multiple sequence clusters were identified with single nucleotide variations, most of them were polymorphic within a germplasm line at many loci.
usually they will be heterozygous always and exhibit no polymorphism between the genotypes.
selection of such snps usually results in false positive assays.
however, we were able to detect  <dig> candidate snps between libraries  <dig> and  <dig> that are homologous within each genotype.
these snps can be used for further genotyping and generating genetic maps in yam.
CONCLUSIONS
despite the economic importance of yam, very little genomic or transcriptome sequence data is publicly available.
this is the first large scale est generation attempt made in yam with the objective of providing a comprehensive annotated transcriptome dataset that will be publicly available, and also used as a resource for novel gene and new marker discovery for crop improvement in yam.
we have successfully sequenced transcriptomes of two resistant and one susceptible lines of yam under the conditions of anthracnose infection.
approximately 56% of total ests were annotated and analyzed for functional characterization and differential expression of genes for tolerance to anthracnose disease.
we also used this dataset as a resource to design new ssr and snp markers.
they serve as a useful tool in identifying genetic variation in the current cultivars, wild relatives and also assist in generating genetic linkage maps.
the ssr markers generated are currently being evaluated at iita for use in their yam improvement program.
the identified snp markers will be validated to identify qtl regions through the ongoing research on yam at iita, usda-ars and vsu.
the markers generated could be used on f <dig> population generated at iita to develop first generation est-ssr based qtl map for yam.

 yams are the primary agricultural commodities and major staple crop in africa. yam tubers are nutritionally rich and a major source of dietary fiber, carbohydrates, vitamin c and essential minerals. worldwide, yam consumption is million tons http://www.iita.org. in yam production was million tons worldwide, of which africa produced 96%, and nigeria is the major producer with more than million tons. the consumer demand for yam is very high in sub-saharan region of africa, but the yam production is declining in this region due to factors including anthracnose disease caused by a fungus, colletotrichum gloeosporioides, pests, and decline in soil fertility. yams are polyploid crop species and are propagated vegetatively from tubers. the water yam is the most widely cultivated species and is highly susceptible to anthracnose disease. the genetic improvement of yam at iita and ctcri concentrated on the development of disease resistant and high yielding varieties. through classical breeding, it would be very difficult to develop a resistant cultivar due to constraints such as the long growth cycle, dioecious and poor flowering nature, polyploidy, vegetative propagation and heterozygous genetic background. a large collection of germplasm with huge genetic variability is available, and it would be profitable to use candidate gene approach and trap the important trait information for disease and pest resistance. colletotrichum is a large genus of ascomycete fungi, containing many species which cause anthracnose or blight on a wide range of important crops and ornamental plants. two genes, clk which encodes a serine/threonine kinase in c. lindemuthianum, and cap which encodes a wall glycoprotein of c. gloeosporioides appressoria, have been shown to have a role in pathogenicity and virulence, respectively. necrotrophy is clearly linked to the increased expression of plant cell wall degrading enzymes such as endo-polygalacturonases and pectin lyases. previous studies showed that endo-pg, two forms of pectin lyase, a- and b-galactopyranosidase, a-arabinofuranosidase, and a protease are secreted into culture medium containing polypectate of bean cell walls. pectin lyase activity was first observed days after inoculation of beans with c. lindemuthianum, rising to maximum activity at days, after which activity declined. thus, the expression of pectin lyase activity correlates well with the onset of necrotrophy and the subsequent development of lesions. therefore the yam leaf material for current investigation was collected on 3rd and 7th day after inoculation with c. gloeosporioides fungus. initial genetic inheritance studies showed that resistance to yam anthracnose in water yam is dominant and quantitatively inherited. a single major dominant locus controlling resistance in the breeding line tda 95- was tentatively designated dcg- until allelism was investigated. lower number of molecular markers in yam limits the genetic mapping efforts. previously developed ssrs from dioscorea tokoro, a wild diploid yam species of east asia and japan were found not to be useful in cultivated yams d. rotundata and d. alata. most of the current molecular markers for the yam genome by mignouna et al. are based on aflp and rapd. however, for effective gene discovery and marker-assisted breeding, it is important to develop more user-friendly, efficient, transportable and co-dominant markers such as simple sequence repeat markers and single nulceotide polymorphism markers. est analysis is the most efficient and effective approach for the identification of candidate genes and also assist in new molecular markers such as est-ssrs and snps. as of 10th october there were only est sequences stored at genbank for the genus dioscorea. to understand the transcriptome of yam and for new marker discovery to assist in yam crop improvement programs, generation of ests would be extremely desirable. with the objective of characterizing transcriptome in yam, initial efforts were focused on successful isolation of mrna, cdna library construction and sequencing using sanger's method. two hundred est sequences were generated using this approach, but reported little functional significance during blast search analysis. to enrich and characterize expressed gene sequence information especially during the anthracnose infection for candidate gene identification and also to utilize the sequence information for new marker discovery, a collaborative effort to generate large number of ests was initiated between vsu, usa and iita, africa. for this purpose, two anthracnose resistant germplasm lines 87- 95- and a susceptible line 95- were selected for cdna library construction from leaf tissues inoculated with the c. gloeosporioides fungus. the roche pyrosequencing technology was used to generate est sequence information.  generation of ests assembly process generated ests in tda 95-0328; ests in tda 95- and ests in tda 87- with average sequence lengths of and bases, respectively. all the est sequences generated were submitted to genbank at ncbi and were assigned genbank accession numbers ho809681-ho with dbest id from to for library tda 95-0310; ho825422-ho with dbest ids from to for library tda 95-0328; ho to ho with dbest ids from to for library tda 87- these sequences contributed to >99% of ests currently available in yam at genbank and would serve as the major resource for the yam research community. transcriptome analysis est sequences obtained from each individual library were first analyzed using blast2go http://blast2go.org. blast2go provides the gene annotation information for the ests and also helps in comparison of two est datasets to find expression differences. blast search analysis across all three libraries revealed highest homology with the sequences from oryza sativa, vitis vinifera, populus trichocarpa and other crop species. mapping and annotation steps in blast2go program resulted in providing go ids to individual ests in each dataset of the three libraries. a total of of ests were annotated with non-redundant go ids in the susceptible tda 95- germplasm line, while in resistant tda 95- line, of ests were annotated with nr go ids and in other resistant line tda 87- library, of ests were annotated with nr go ids. approximately 50% of ests could not be annotated and remained unknown using blas2go. to complement and comprehensively annotate the yam transcriptome, all the est sequences were submitted to mercator tool of mapman database. mercator tool generated functional predictions by searching reference databases and assigned mapman bin ids. still 44% of ests lacked significant homology with genes in other crops in each of the three datasets and these may include novel metabolic genes in yam. a comparative analysis was done using the bin id information from each library to identify unique ests in each library and also the common genes shared with other libraries. the distribution of ests with known function is presented as a venn diagram in figure a large subset of ests representing unique bin ids were shared across three libraries and these may account for housekeeping genes involved in general cellular metabolism. the two resistant lines tda 95- and tda 87- had and unique ests, respectively, which may account for the tolerance against c. gloeosporioides fungus. in tda 95- susceptible genotype, there were unique sequences which predominantly matched genes for protein synthesis and nucleotide synthesis. however, there were several sequences annotated as protein kinases and were known to have a definite role in disease signaling. another interesting feature is the presence of genes for synthesis of cell wall precursors and secondary metabolites which might be highly induced due to severe biotic stress. there were also genes for recycling nad to maintain glycolysis and substrate level phosphorylation in the absence of oxygen in damaged leaf of susceptible genotype. removal of the toxic compounds like proline is very essential for plant survival under stress. there were unique genes for proline degradation in susceptible genotype suggesting the presence of a possible mechanism of detoxification during pathogenesis. common sequences between tda 95- and tda 95- were annotated as genes representing cell wall precursors, co-factor and vitamin metabolism, tetrapyrrole synthesis, c1-metabolism, lipid metabolism, amino acid metabolism, secondary metabolism, hormone metabolism, major and minor cho metabolism, nucleotide metabolism, genes for regulation of transcription, amino acid activation, protein synthesis, protein degradation, energy metabolism, and genes for sugar and light signaling. on the other hand, the common genes between tda 95- and tda 87- were cell wall proteins, sulfate assimilation related genes, phosphatidylserine decarboxylase, hydroxymethylpyrimidine kinase, l-galactono- -lactone dehydrogenase, genes for dicer degradation, gebp like transcription factor, and myo inositol oxygenase. similarly, tda 87- and tda 95- shared sequences related to dihydroneopterin aldolase, glycine cleavage h protein, phosphomannose isomerase, c-lectin, alcohol dehydrogenase, and malate dehydrogenase genes. although these genes have specific role in metabolism, regulation and signaling, the significance of their sharing between yam genotypes cannot be speculated and is beyond the scope of this paper. functional analysis using the go id information, the ests were classified into subsets based on molecular function and is represented as a pie diagram for each library. a major subset of ests across all libraries was linked to binding and catalytic activity, where the remaining groups involved transporter, signal transduction, transcription factors, secondary metabolism and antioxidant activities. based on biological process classification, a major proportion of the genes were involved in oxidation-reduction activity across all libraries and these may involve genes in energy metabolisms and other housekeeping activities. ests were also grouped under other biological processes such as photosynthesis, membrane transport, transcription, translation, protein folding, and others. however, a significant proportion of the expressed genes were biotic stress and other stress responsive genes. differential gene expression analysis from the est datasets, it is interesting to identify genes involved in defense mechanisms that were differentially expressed in susceptible and resistant yam genotypes. using the bin ids of ests in each library, the expression of various ests were compared from one library to the other. these results were summarized in figure and additional file the resistant genotypes had unique ests related to carbohydrate metabolism, cell wall biogenesis, lipid and amino acid metabolism, secondary and hormone metabolism, transcription factors, protein synthesis, and signaling proteins. even though, the est datasets is not comprehensive of the entire yam transcriptome, this analysis would shed some light on the gene expression differences during anthracnose infection. further investigation of these differentially expressed genes using microarray platforms or rna-seq or real-time quantitative pcr experiments, would assist in elucidating the underlying molecular mechanism for anthracnose tolerance in yam. informative defense related genes during anthracnose infection the differential gene expression analysis provided the gene information that might be potentially over-expressed or under-expressed compared to other libraries. these results were summated in additional file multiple pathogenesis-related genes and host defense related genes including signaling genes were identified from the ests in the resistant lines and they include nicotiana lesion inducing proteins, erwinia induced proteins, cysteine proteases, cysteine protease inhibitors, peroxidases, extensin, wall associated kinases, brassinosteroid receptor kinases, ethylene responsive genes, phosphatidylinositol 4-kinase, leucine rich repeat genes, serine-threonine protein kinases and others signaling proteins. signal transduction in plants, leading to the expression of defense genes, is initiated by leucine rich repeat -type membrane receptors containing intrinsic kinase activity as in the case of fls. there were unique genes involved in isoprenoid and flavonoids biosynthesis and fatty acid synthesis and elongase activity in tda 87- compared to tda 95- and tda 95- there were hits specifically showing homologies to peroxisome biogenesis protein pex and perxisome assembly factor in library and this confirms previous reports of plant peroxisomes role in the biosynthesis of the signaling molecules like jasmonic acid, β-oxidation of indole butyric acid, and sulphur and polyamine metabolism. moreover, evidence is emerging from recent studies that peroxisomes have important functions in specific defense mechanisms, conferring resistance against pathogen attack. there were homologies to 12-oxo-phytodienoic acid reductases, enzymes of the octadecanoid pathway, which convert linolenic acid to a phytohormone jasmonic acid. fifty nine significant matches to auxin responsive proteins, auxin signaling f-box and auxin response transcription factors were observed in tda 87- and tda 95- confirming their role in host defense against pathogens. actually plant peroxidases transduce the extracellular signals into the redox signals that eventually stimulate the intracellular ca2+ signaling required for induction of defense responses. there were significant hits to different plant peroxidases in resistant libraries confirming their requirement for defense against colletotrichum fungus. the hits to pectate lyase were few, one each in tda 95- and tda 87- compared to four in tda 95/ the contribution of a single pectate lyase gene to the pathogenic abilities of c. magna confirmed that pectate lyase is a pathogenicity factor required for the penetration and colonization of colletotrichum species. cell-wall-degrading enzymes, like pectolytic enzymes, are considered to play a role in the pathogenesis of bacteria and fungi on their hosts. the peptidoglycan-binding lysm domain-containing protein and beta- -glucanases may help in fungal cell wall degradation as there were significant hits observed in the resistant genotypes. the disruption of a single enzyme may be complemented by the activity of other cwdes. there was also homology to chitin binding, chitinase activity and chitin catabolic/metabolic process and were abundant in 87- a genotype resistant to c. gloeosporioides strain sgg. the activation of the defense response, after pathogen inoculation, is the most important factor for the success of host resistance. the est data from our study also suggest strength of the expression of ubiquitous early acting genes for establishment of the resistance response of the resistant genotype 87- marker discovery the est sequence information generated in this study also serves as a major resource to generate new molecular markers. simple sequence repeat motifs detected in est sequences are usually termed as est-ssrs. these are very informative markers as they exist within the gene sequence. a total of ests from three genotypes, with an average length of bp were used to evaluate for the presence of ssr motifs. there are a total of est sequences containing ssrs identified from three libraries. in this work, ssrs were considered for primer design that fitted the following criteria: a minimum pattern length of bp, excluding polya and polyt repeat, at least repeat units in case of di-nucleotide and at least repeat units for tri-, tetra-, penta- and hexa-nucleotide ssrs. there are about a total of good quality repeats containing di, tri, tetra, penta, hexa and hepta repeats which were used for designing primers. the default settings for primer program: optimum temperature of 63oc and an optimum primer size of bases were selected for designing the primers. a total of primer pairs were designed from est sequences. a subset were tested for screening genomic dna of genotypes using simple % agarose gel electrophoresis for polymorphism detection and found polymorphic. these ssr primers are summarized in additional file identification of homologous snps is very challenging in a polyploid species such as yam. haplosnper generates putative snp haplotype for each contig using cap generated.ace file. therefore, we can verify whether a snp is heterozygous within each genotype or homozygous at specific loci. if they are heterozygous within each genotype, basically they may be copies from homeologous genomes or paralogous copies. even though multiple sequence clusters were identified with single nucleotide variations, most of them were polymorphic within a germplasm line at many loci. usually they will be heterozygous always and exhibit no polymorphism between the genotypes. selection of such snps usually results in false positive assays. however, we were able to detect candidate snps between libraries and that are homologous within each genotype. these snps can be used for further genotyping and generating genetic maps in yam.  despite the economic importance of yam, very little genomic or transcriptome sequence data is publicly available. this is the first large scale est generation attempt made in yam with the objective of providing a comprehensive annotated transcriptome dataset that will be publicly available, and also used as a resource for novel gene and new marker discovery for crop improvement in yam. we have successfully sequenced transcriptomes of two resistant and one susceptible lines of yam under the conditions of anthracnose infection. approximately 56% of total ests were annotated and analyzed for functional characterization and differential expression of genes for tolerance to anthracnose disease. we also used this dataset as a resource to design new ssr and snp markers. they serve as a useful tool in identifying genetic variation in the current cultivars, wild relatives and also assist in generating genetic linkage maps. the ssr markers generated are currently being evaluated at iita for use in their yam improvement program. the identified snp markers will be validated to identify qtl regions through the ongoing research on yam at iita, usda-ars and vsu. the markers generated could be used on f population generated at iita to develop first generation est-ssr based qtl map for yam. 
cdna libraries were constructed from anthracnose fungus challenged leaf tissues for transcriptome characterization, and differential gene expression analysis. gene annotations were used to characterize the transcriptome in yam and also perform a differential gene expression analysis between the resistant and susceptible est datasets. therefore, this collaborative project was developed for genetic improvement and germplasm characterization of yams using molecular markers. availability of high quality sequence information is necessary for designing molecular markers associated with resistance. however, very limited sequence information pertaining to yam is available at public genome databases. the current investigation is focused on studying gene expression, by large scale generation of ests, from one susceptible and two resistant yam genotypes challenged with the fungus. mining for ssrs in the ests revealed unique sequences containing ssrs and ssr markers were designed using those sequences.
 anthracnose is a major limiting factor in the production of yam worldwide. availability of high quality sequence information is necessary for designing molecular markers associated with resistance. however, very limited sequence information pertaining to yam is available at public genome databases. therefore, this collaborative project was developed for genetic improvement and germplasm characterization of yams using molecular markers. the current investigation is focused on studying gene expression, by large scale generation of ests, from one susceptible and two resistant yam genotypes challenged with the fungus. total rna was isolated from young leaves of resistant and susceptible genotypes and cdna libraries were sequenced using roche technology.  a total of est sequences were generated from the cdna libraries of the resistant and susceptible genotypes. greater than 56% of ests were annotated using mapman mercator tool and blast2go search tools. gene annotations were used to characterize the transcriptome in yam and also perform a differential gene expression analysis between the resistant and susceptible est datasets. mining for ssrs in the ests revealed unique sequences containing ssrs and ssr markers were designed using those sequences.  we have developed a comprehensive annotated transcriptome data set in yam to enrich the est information in public databases. cdna libraries were constructed from anthracnose fungus challenged leaf tissues for transcriptome characterization, and differential gene expression analysis. thus, it helped in identifying unique transcripts in each library for disease resistance. these est resources provide the basis for future microarray development, marker validation, genetic linkage mapping and qtl analysis in dioscorea species. 