Soybean Expressed Sequence Tags Research Articles

Identification and characterization of SSRs from soybean (Glycine max) ESTs

Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies.

Identification of MicroRNAs in Wild Soybean (Glycine soja)

MicroRNAs (miRNAs) play important roles in post-transcriptional gene silencing by directing target mRNA cleavage or translational inhibition. Currently, hundreds of miRNAs have been identified in plants, but no report has been published of wild soybean (Glycine soja Sieb). We constructed a small-RNA library consisting of 2 880 sequences with high quality, in which 1 347 were 19-24 nt in length. By utilizing the miRNA, Rfam and domesticated soybean expressed sequence tag database, we have analyzed and predicted the secondary structure of these small RNAs. As a result, 15 conserved miRNA candidates belonging to eight different families and nine novel miRNA candidates comprising eight families were identified in wild soybean seedlings. All these miRNA candidates were validated by northern blot and the novel candidates expressed in a tissue-specific manner. Furthermore, putative target genes were predicted for novel miRNA candidates and two of them were verified by 5'-rapid amplification of cDNA ends experiments. These results provided useful information for miRNA research in wild soybean and plants.

Molecular Cloning and Characterization of a cDNA for Pterocarpan 4-Dimethylallyltransferase Catalyzing the Key Prenylation Step in the Biosynthesis of Glyceollin, a Soybean Phytoalexin

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(-)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-(13)C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.

Gene Polymorphism Studies in a Teaching Laboratory

I present a laboratory procedure for illustrating transcription, post-transcriptional modification, gene conservation, and comparative genetics for use in undergraduate biology education. Students are individually assigned genes in a targeted biochemical pathway, for which they design and test polymerase chain reaction (PCR) primers. In this example, students used genes annotated for the steroid biosynthesis pathway in soybean. The authoritative Kyoto encyclopedia of genes and genomes (KEGG) interactive database and other online resources were used to design primers based first on soybean expressed sequence tags (ESTs), then on ESTs from an alternate organism if soybean sequence was unavailable. Students designed a total of 50 gene-based primer pairs (37 soybean, 13 alternative) and tested these for polymorphism state and similarity between two soybean and two pea lines. Student assessment was based on acquisition of laboratory skills and successful project completion. This simple procedure illustrates conservation of genes and is not limited to soybean or pea. Cost per student estimates are included, along with a detailed protocol and flow diagram of the procedure.

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Proteomic Analysis of Highly Purified Peroxisomes from Etiolated Soybean Cotyledons

To identify previously unknown peroxisomal proteins, we established an optimized method for isolating highly purified peroxisomes from etiolated soybean cotyledons using Percoll density gradient centrifugation followed by iodixanol density gradient centrifugation. Proteins in highly purified peroxisomes were separated by two-dimensional PAGE. We performed peptide mass fingerprinting of proteins separated in the gel with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and used the peptide mass fingerprints to search a non-redundant soybean expressed sequence tag database. We succeeded in assigning 92 proteins to 70 sequences in the database. Among them, proteins encoded by 30 sequences were judged to be located in peroxisomes. These included enzymes for fatty acid beta-oxidation, the glyoxylate cycle, photorespiratory glycolate metabolism, stress response and metabolite transport. We also show experimental evidence that plant peroxisomes contain a short-chain dehydrogenase/reductase family protein, enoyl-CoA hydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-like protein and a voltage-dependent anion-selective channel protein.

SoyXpress: A database for exploring the soybean transcriptome

BackgroundExperiments using whole transcriptome microarrays produce massive amounts of data. To gain a comprehensive understanding of this gene expression data it needs to be integrated with other available information such as gene function and metabolic pathways. Bioinformatics tools are essential to handle, organize and interpret the results. To date, no database provides whole transcriptome analysis capabilities integrated with terms describing biological functions for soybean (Glycine max (L) Merr.). To this end we have developed SoyXpress, a relational database with a suite of web interfaces to allow users to easily retrieve data and results of the microarray experiment with cross-referenced annotations of expressed sequence tags (EST) and hyperlinks to external public databases. This environment makes it possible to explore differences in gene expression, if any, between for instance transgenic and non-transgenic soybean cultivars and to interpret the results based on gene functional annotations to determine any changes that could potentially alter biological processes.ResultsSoyXpress is a database designed for exploring the soybean transcriptome. Currently SoyXpress houses 380,095 soybean Expressed Sequence Tags (EST), linked with metabolic pathways, Gene Ontology terms, SwissProt identifiers and Affymetrix gene expression data. Array data is presently available from an experiment profiling global gene expression of three conventional and two genetically engineered soybean cultivars. The microarray data is linked with the sequence data, for maximum knowledge extraction. SoyXpress is implemented in MySQL and uses a Perl CGI interface.ConclusionSoyXpress is designed for the purpose of exploring potential transcriptome differences in different plant genotypes, including genetically modified crops. Soybean EST sequences, microarray and pathway data as well as searchable and browsable gene ontology are integrated and presented. SoyXpress is publicly accessible at .

Enhanced Oleic Acid Content in the Soybean Mutant M23 Is Associated with the Deletion in the Fad2‐1a Gene Encoding a Fatty Acid Desaturase

AbstractOleic acid is one of the three major unsaturated fatty acids in soybean oil. Expression of the embryo‐specific Fad2‐1 gene correlates with fatty acid biosynthesis and oil deposition in developing seeds. Elevated levels of oleate in the X‐ray‐induced mutant line M23 have been shown to be associated with the deletion of a Fad2‐1 gene. In soybean, two homologs of the Fad2‐1 gene, termed Fad2‐1a (L43920) and Fad2‐1b (AY611472), presumably encode functional embryo‐specific fatty acid desaturase. The objectives of this investigation were to determine which copy of the Fad2‐1 gene is associated with the increased oleate content in the mutant line and what is the relative transcript abundance of these two embryo‐specific genes. PCR and DNA blot analyses showed that increased oleate content in M23 mutant was associated with the deletion of Fad2‐1a. These results were further validated using five independent soybean populations developed by crossing mid‐oleate and normal‐oleate parents. Investigation of the soybean expressed sequence tag database and reverse transcription PCR analyses revealed that Fad2‐1b is the predominantly transcribed copy of the Fad2‐1 gene. We hypothesize that null mutation in Fad2‐1b in the fad2‐1a mutant background should further elevate the oleic contents of soybean oil.

A sequence based synteny map between soybean and Arabidopsis thaliana

BackgroundSoybean (Glycine max, L. Merr.) is one of the world's most important crops, however, its complete genomic sequence has yet to be determined. Nonetheless, a large body of sequence information exists, particularly in the form of expressed sequence tags (ESTs). Herein, we report the use of the model organism Arabidopsis thaliana (thale cress) for which the entire genomic sequence is available as a framework to align thousands of short soybean sequences.ResultsA series of JAVA-based programs were created that processed and compared 341,619 soybean DNA sequences against A. thaliana chromosomal DNA. A. thaliana DNA was probed for short, exact matches (15 bp) to each soybean sequence, and then checked for the number of additional 7 bp matches in the adjacent 400 bp region. The position of these matches was used to order soybean sequences in relation to the A. thaliana genome.ConclusionReported associations between soybean sequences and A. thaliana were within a 95% confidence interval of e-30 – e-100. In addition, the clustering of soybean expressed sequence tags (ESTs) based on A. thaliana sequence was accurate enough to identify potential single nucleotide polymorphisms (SNPs) within the soybean sequence clusters. An EST, bacterial artificial chromosome (BAC) end sequence and marker amplicon sequence synteny map of soybean and A. thaliana is presented. In addition, all JAVA programs used to create this map are available upon request and on the WEB.

The FAD2 Gene Family of Soybean: Insights into the Structural and Functional Divergence of a Paleopolyploid Genome

The ω‐6 fatty acid desaturase (FAD2) gene family in soybean [Glycine max (L.) Merr.] consists of at least five members in four regions of the genome and are responsible for the conversion of oleic acid to linoleic acid. Here we report the identification of two new ω‐6 fatty acid desaturase (FAD2) gene copies from soybean expressed sequence tags (ESTs). Four bacterial artificial chromosomes (BACs) containing five FAD2 genes were sequenced to investigate structural and functional conservation between duplicate loci. Sequence comparisons show that the soybean genome is a mosaic, with some duplicate regions retaining high sequence conservation in both genic and intergenic regions, while others have only the FAD2 genes in common. Genetic mapping using SSRs from within the BAC sequences showed that two BACs with high sequence homeology mapped to linkage groups I and O; these groups share syntenic markers. Another BAC mapped to linkage group L. The fourth BAC could not be mapped. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis of the five FAD2 genes showed that the FAD2‐2B and FAD2‐2C copies were the best candidates for temperature‐dependent expression changes in developing pod tissue. Semiquantitative RT‐PCR confirmed these results, with FAD2‐2C showing upward of an eightfold increase in expression in developing pods grown in cooler conditions relative to those grown in warm conditions. The implications of these results suggest a candidate gene for controlling the levels of linoleic acid in developing pods grown in cooler climates.

Characterization of the Soybean Genome Using EST-derived Microsatellite Markers

We generated a high-density genetic linkage map of soybean using expressed sequence tag (EST)-derived microsatellite markers. A total of 6920 primer pairs (10.9%) were designed to amplify simple sequence repeats (SSRs) from 63 676 publicly available non-redundant soybean ESTs. The polymorphism of two parent plants, the Japanese cultivar ‘Misuzudaizu’ and the Chinese line ‘Moshidou Gong 503’, were examined using 10% polyacrylamide gel electrophoresis. Primer pairs showing polymorphism were then used for genotyping 94 recombinant inbred lines (RILs) derived from a cross between the parents. In addition to previously reported markers, 680 EST-derived microsatellite markers were selected and subjected to linkage analysis. As a result, 935 marker loci were mapped successfully onto 20 linkage groups, which totaled 2700.3 cM in length; 693 loci were detected using the 668 EST-derived microsatellite markers developed in this study, the other 242 loci were detected with 105 RFLP markers, 136 genome-derived microsatellite markers, and one phenotypic marker. We examined allelic variation among 23 soybean cultivars/lines and a wild soybean line using 668 mapped EST-derived microsatellite markers (corresponding to 686 marker loci), in order to determine the transferability of the markers among soybean germplasms. A limited degree of macrosynteny was observed at the segmental level between the genomes of soybean and the model legume Lotus japonicus, which suggests that considerable genome shuffling occurred after separation of the species and during establishment of the paleopolyploid soybean genome.

Isolation of a novel nodulin: a molecular marker of osmotic stress in Glycine max/Bradyrhizobium japonicum nodule

Symbiotic N(2) fixation of legume crops is highly sensitive to drought, which results in a dramatic drop of N accumulation and yield. The symbiosis between soybean (Glycine max) and Bradyrhizobium japonicum, because of its extreme sensitivity to drought, was chosen as a model to analyse the response to drought stress at a molecular level. The mRNA differential display technique was performed to isolate cDNA markers differentially expressed in well-watered [100% of N(2) fixation capacity (NFC)] and drought-stressed nodules (40% NFC). One gene noted, G93, appeared strongly down-regulated by drought and fully recovered after rehydration. In situ hybridization showed that G93 transcripts were localized in N(2)-fixing cells of mature nodules, indicating that G93 could be considered as a late nodulin. However, G93 expression was not directly correlated to N(2) fixation but mainly responded to osmotic stress. Other stresses that lead to decrease of N(2) fixation did not affect G93 expression. Sequence analyses showed that G93 presented a strong homology with two soybean expressed sequence tags (ESTs) and with the ZR1 protein of Medicago sativa. Putative roles of this nodulin in adaptation of soybean nodule to osmotic stress are proposed.

Identification and phylogenetic analysis of sequences of Bean pod mottle virus, Soybean mosaic virus, and Cowpea chlorotic mottle virus in expressed sequence tag data from soybean

Field- or greenhouse-grown plants are naturally exposed to microbes. Sequence data derived from these plants can therefore inadvertently include microbe sequences. We have examined the extent of viral sequences in public soybean (Glycine max and Glycine soja) sequence data. A collection of 303 149 soybean expressed sequence tags (ESTs) was computationally analyzed for sequences from 12 viruses infecting soybean: Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea chlorotic mottle virus (CCMV), Cowpea mosaic virus (CPMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Soybean mosaic virus (SMV), Tobacco ringspot virus (TRSV), and Tobacco streak virus (TSV). A total of 1652 sequences matching BPMV (1097 sequences), SMV (132 sequences), and CCMV (423 sequences) were discovered in the soybean EST collection. The viral sequences were assembled into contiguous sequences, and sequence tracts in common were used in a parsimony analysis of the phylogenetic relationship of putative viral genotypes. Our results include representative sequences from two BPMV subgroups and, surprisingly, many variants of BPMV nucleotide sequences. Furthermore, the results suggest that BPMV viral strains may be mixes of different BPMV RNA-1 and RNA-2 genotypes. This study presents the largest collection of sequence data available to date for the BPMV, SMV, and CCMV viruses and represents the first report of this magnitude of varieties of BPMV nucleotide sequences.

Application of comparative genomics in developing molecular markers tightly linked to the virus resistance geneRsv4 in soybean

The Rsv4 gene confers resistance to all the known strain groups of soybean mosaic virus in soybean (Glycine max (L.) Merr.). To construct a fine genetic map near Rsv4 in soybean, we employed a comparative genomics approach that used genome sequence information of the model legume Lotus japonicus. Sequences of the soybean expressed sequence tags (ESTs) AI856415 and BF070293 mapping to one side of the Rsv4 gene showed high similarity with gene sequences of the transformation-competent artificial chromosome (TAC) clone LjT32P24 of Lotus. LjT32P24 is tightly linked to another sequenced TAC clone, LjT26I01, in Lotus. A new marker, AW307114A, developed from soybean EST AW307114, which is homologous to a Lotus gene within LjT26I01, was mapped to the other side of the Rsv4 gene. The identification of the microsyntenic relationship facilitated the development of additional 2 EST markers between BF070293-S and AW307114A bracketing the Rsv4 gene. Several other markers developed in this study were mapped to putative homoeologous or duplicated chromosomal regions in soybean. Alignment between the soybean maps indicated that Rsv4 is located near a local chromosomal rearrangement. This targeted comparative mapping serves to provide a foundation for marker-assisted selection and cloning of the Rsv4 gene.

Identification and characterization of a novel heat shock transcription factor gene, GmHsfA1, in soybeans (Glycine max)

Plants have a large family of HSFs with different roles in the heat shock response that mediate the expression of HSP regulated genes. The HSF encoding genes are easily identified by their highly conserved modular structure and motifs. In the present study, a putative GmHsfA1 was identified and characterized from the soybean expressed sequence tag (EST) database by sequence comparison with the functionally well-characterized LpHsfA1 and rapid amplification of cDNA ends (RACE). Multiple alignment showed that the amino acid sequence of GmHSFA1, matching best with LpHSFA1 (52.2% similarity), was obviously different from that of each of several HSFA1s from other plant species. The GmHsfA1 has a constitutive expression profile in the different tissues examined. The overexpression of GmHsfA1 in transgenic soybean plants led to the activation of GmHsp70 under normal temperature and the overexpression of GmHsp70 under high temperature. Furthermore, transgenic soybean plants with GmHsfA1 overexpression showed obvious enhancement of thermotolerance under heat stress in comparison with non-transgenic plants. The experimental results suggested that GmHSFA1 is a novel and functional heat-shock transcription factor.

Identification and characterization of an isopentenyltransferase ( IPT) gene in soybean ( Glycine max L.)

A putative isopentenyltransferase ( IPT) encoding gene was identified from the soybean expressed sequence tag (EST) database, and the full-length gene was cloned with RACE. The gene was most similar to AtIPT5, an isopentenyltransferase gene in Arabidopsis, with respect to expression profile and sequence alignment. Southern hybridization showed that there were two copies (isoforms) of GmIPT in the soybean genome. Transgenic study with tobacco in the present study indicated that the protein encoded by the GmIPT1 gene could produce active cytokinins (CKs) in plants. RT-PCR revealed that the GmIPT1 gene may be expressed constitutively in several tissues. Promoter– GUS fusion revealed highly specific expression sites of GmIPT1 in roots, shoots and leaves, although these were not detected by Northern blot analysis, due to very low expression status. The isolated upstream regulatory sequence of GmIPT1 shared common transcription factor binding motifs with the corresponding regulatory regions of AtIPTs and OsIPTs. GmIPT1 expression was up-regulated by other phytohormones and chemicals, as indicated by the results of quantitative PCR and GUS activity examination in transgenic Arabidopsis. This is consistent with finding putative phytohormone and stress-responsive motifs in the promoter region. Involvement of putative signals and/or other factors in regulating GmIPT1 expression, and related control of CK production in higher plants are discussed.

Characterization of soybean genomic features by analysis of its expressed sequence tags.

We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products of these unigenes were annotated according to their homology with the categorized proteins of Arabidopsis. Genes corresponding to cell growth and/or maintenance, enzymes and cell communication belonged to the slow-evolving class, whereas genes related to transcription regulation, cell, binding and death appeared to be fast-evolving. Soybean unigenes with no match to genes within the Arabidopsis genome were identified as soybean-specific genes. These genes were mainly involved in nodule development and the synthesis of seed storage proteins. In addition, we also identified 61 genes regulated by salicylic acid, 1,322 transcription factor genes and 326 disease resistance-like genes from soybean unigenes. SSR analysis showed that the soybean genome was more complex than the Arabidopsis and the Medicago truncatula genomes. GC content in soybean unigene sequences is similar to that in Arabidopsis and M. truncatula. Furthermore, the combined analysis of the EST database and the BAC-contig sequences revealed that the total gene number in the soybean genome is about 63,501.

Genomic characterization of the S-adenosylmethionine decarboxylase genes from soybean.

A full-length gene GmSAMDC1, encoding the S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in polyamine biosynthesis, was identified from soybean expressed sequence tags and was characterized. GmSAMDC1 encoded a peptide of 355 amino acids. When compared with other plant SAMDCs, the GmSAMDC1 protein had several highly conserved regions including a putative pro-enzyme cleavage site and a PEST sequence. The 5' leader sequence of the the GmSAMDC1 mRNA contained two additional open reading frames (ORFs), which may regulate the translational process. The genomic sequence of the GmSAMDC1 gene contained three introns in the 5' leader sequence, but no intron in the 3'-UTR or the main pro-enzyme ORF. A simple sequence repeat (SSR) was found in intron 2, and the GmSAMDC1 gene was mapped to linkage group D1 using this SSR. The genomic organization of the GmSAMDC1 gene in the subgenus Glycine and the subgenus Soja was found to be different by Southern-blot and PCR analysis. A pseudogene, GmSAMDC2, was also identified. This gene contained no intron and lost its two uORFs. Northern-blot analysis showed that the GmSAMDC1 gene expression was induced by salt, drought and cold, but not induced by wounding; suggesting that the gene was implicated in response to multiple-stress conditions.

Characterization of a plant homolog of hop, a cochaperone of hsp90.

The 90-kD molecular chaperone hsp90 is the key component of a multiprotein chaperone complex that facilitates folding, stabilization, and functional modulation of a number of signaling proteins. The components of the animal chaperone complex include hsp90, hsp70, hsp40, Hop, and p23. The animal Hop functions to link hsp90 and hsp70, and it can also inhibit the ATPase activity of hsp90. We have demonstrated the presence of an hsp90 chaperone complex in plant cells, but not all components of the complex have been identified. Here, we report the isolation and characterization of soybean (Glycine max) GmHop-1, a soybean homolog of mammalian Hop. An analysis of soybean expressed sequence tags, combined with preexisting data in literature, suggested the presence of at least three related genes encoding Hop-like proteins in soybean. Transcripts corresponding to Hop-like proteins in soybean were detected under normal growth conditions, and their levels increased further in response to stress. A recombinant GmHop-1 bound hsp90 and its binding to hsp90 could be blocked by the tetratricopeptide repeat (TPR) domain of rat (Rattus norvegicus) protein phosphatase 5. Deletion of amino acids 325 to 395, adjacent to the TPR2A domain in GmHop-1, resulted in loss of hsp90 binding. In a minimal assembly system, GmHop-1 was able to stimulate mammalian steroid receptor folding. These data show that plant and animal Hop homologs are conserved in their general characteristics, and suggest that a Hop-like protein in plants is an important cochaperone of plant hsp90.

A compilation of soybean ESTs: generation and analysis.

Whole-genome sequencing is fundamental to understanding the genetic composition of an organism. Given the size and complexity of the soybean genome, an alternative approach is targeted random-gene sequencing, which provides an immediate and productive method of gene discovery. In this study, more than 120000 soybean expressed sequence tags (ESTs) generated from more than 50 cDNA libraries were evaluated. These ESTs coalesced into 16928 contigs and 17336 singletons. On average, each contig was composed of 6 ESTs and spanned 788 bases. The average sequence length submitted to dbEST was 414 bases. Using only those libraries generating more than 800 ESTs each and only those contigs with 10 or more ESTs each, correlated patterns of gene expression among libraries and genes were discerned. Two-dimensional qualitative representations of contig and library similarities were generated based on expression profiles. Genes with similar expression patterns and, potentially, similar functions were identified. These studies provide a rich source of publicly available gene sequences as well as valuable insight into the structure, function, and evolution of a model crop legume genome.