The modulation of light quality on carotenoid and tocochromanol biosynthesis in mung bean (Vigna radiata) sprouts.

This study aimed to identify the regulatory mechanisms of white, blue, red lights on carotenoid and tocochromanol biosynthesis in mung bean sprouts. Results showed that three lights stimulated the increase of the predominated lutein (3.2-8.1 folds) and violaxanthin (2.1-6.1 folds) in sprouts as compared with dark control, as well as ß-carotene (20-36 folds), with the best yield observed under white light. Light signals also promoted α- and γ-tocopherol accumulation (up to 1.8 folds) as compared with dark control. The CRTISO, LUT5 and DXS (1.24-6.34 folds) exhibited high expression levels under light quality conditions, resulting in an overaccumulation of carotenoids. The MPBQ-MT, TC and TMT were decisive genes in tocochromanol biosynthesis, and were expressed up to 4.19 folds as compared with control. Overall, the results could provide novel insights into light-mediated regulation and fortification of carotenoids and tocopherols, as well as guide future agricultural cultivation of mung bean sprouts.

Effect of light quality on polyphenol biosynthesis in three varieties of mung bean sprouts with different color seed coats.

KEY MESSAGE: We investigated the mechanism of the effect of different light qualities on the synthesis and regulation of mung bean sprouts. Light quality acts as a signal molecule, strongly enhancing polyphenol biosynthesis in sprouts. Mung bean (Vigna radiata) sprouts are a popular sprouting vegetable all over the world and are an excellent source of polyphenols with high antioxidant activity. This study investigated the effects of light qualities on the kinetic changes and metabolic regulation mechanism of light signal-mediating polyphenols in three mung bean sprout cultivars. Experimental results showed that three light qualities significantly enhanced the contents of caffeic acid, rutin, vitexin, genistin and delphinidin 3-glucoside. Interestingly, ferulic acid and vitexin responded selectively to blue light and red light, severally. Most genes involved in polyphenol biosynthesis were activated under different light quality conditions, resulting in an overaccumulation of phenylpropanoids. Pearson correlation analysis showed that PAL, F3H, F3'H and F3'5'H expression correlated highly with rutin, whereas ANS expression paralleled anthocyanin biosynthesis. Moreover, MYB111, MYB3, MYB4, MYB1 and MYC2 were critical regulators of polyphenol biosynthesis in mung bean sprouts. These changes were likely due to the changes in the expression of the photoreceptor genes CRY-D, PHOT2, PHYE and light response genes (PIF3 and HY5). Our results provide insights into polyphenol biosynthesis in sprouts and microgreens.

Genomic analyses of rice bean landraces reveal adaptation and yield related loci to accelerate breeding.

Rice bean (Vigna umbellata) is an underexploited domesticated legume crop consumed for dietary protein in Asia, yet little is known about the genetic diversity of this species. Here, we present a high-quality reference genome for a rice bean landrace (FF25) built using PacBio long-read data and a Hi-C chromatin interaction map, and assess the phylogenetic position and speciation time of rice bean within the Vigna genus. We sequence 440 landraces (two core collections), and GWAS based on data for growth sites at three widely divergent latitudes reveal loci associated with flowering and yield. Loci harboring orthologs of FUL (FRUITFULL), FT (FLOWERING LOCUS T), and PRR3 (PSEUDO-RESPONSE REGULATOR 3) contribute to the adaptation of rice bean from its low latitude center of origin towards higher latitudes, and the landraces which pyramid early-flowering alleles for these loci display maximally short flowering times. We also demonstrate that copy-number-variation for VumCYP78A6 can regulate seed-yield traits. Intriguingly, 32 landraces collected from a mountainous region in South-Central China harbor a recently acquired InDel in TFL1 (TERMINAL FLOWER1) affecting stem determinacy; these materials also have exceptionally high values for multiple human-desired traits and could therefore substantially advance breeding efforts to improve rice bean.

Effect of photoperiod on polyphenol biosynthesis and cellular antioxidant capacity in mung bean (Vigna radiata) sprouts.

This study investigated the effects of photoperiods on the metabolic regulation of polyphenol biosynthesis, as well as antioxidant activities in mung bean sprouts. Mung bean sprouts exposed to three different photoperiods: constant dark (light/dark, 0/24 h), semilight (light/dark, 12/12 h) and constant light (light/dark, 24/0 h). The results showed that the DFR, F6H, F3'5'H, F3'H, and F3H genes in the polyphenol biosynthetic pathway, as well as PHOT1/2, HY5, and MYBs, were strongly stimulated by photoperiods in sprouts. Seven polyphenol compounds (caffeic acid, rutin, vitexin, daidzin, glycitin, ferulic acid and p-coumaric acid) were detected in sprouts; the first five gradually increased with increasing illumination time, especially rutin, which showed a 547% increase. Illumination enhanced the antioxidant activities of sprouts, which was potentially related to the increases in caffeic acid, rutin and vitexin. These findings indicated that illumination would be a promising technique to improve the nutritional value of mung bean sprouts.

Construction of High-Density Genetic Map and Identification of a Bruchid Resistance Locus in Mung Bean (Vigna radiata L.).

Mung bean (Vigna radiata L.) is an economically important grain legume cultivated in Asian countries. High-density genetic linkage is a valuable and effective tool for mapping quantitative trait loci (QTL). In the current study, a high-resolution genetic map containing 4,180 single-nucleotide polymorphisms (SNPs) was assigned to 11 linkage groups (LGs) and spanning 1,751.39 cM in length was constructed for mung bean, and the average distance between adjacent markers was 0.42 cM. Bruchids (Callosobruchus spp.) cause significant damage to and loss of legume seeds. A locus for bruchid resistance was detected. The gene Vradi05g03810, encoding a probable resistance-specific protein, was found to be the most likely key candidate gene in mung beans. A 69-bp sequence deletion was identified in the coding region by comparing the cDNA sequences of bruchid-resistant and bruchid-susceptible lines. This SNP-based high-density linkage map is one of the first to be constructed across the mung bean genome. This map will not only facilitate the genetic mapping of genes or complex loci that control important agronomic traits but also offer a tool for promoting future genetics and comparative genomic studies in Vigna.

Agronomic Traits, Fresh Food Processing Characteristics and Sensory Quality of 26 Mung Bean (Vigna radiata L.) Cultivars (Fabaceae) in China.

In recent years, with the expansion of mung bean (Vigna radiata L.) planting areas and the increase of consumer demand, it has become imperative to screen high-quality mung bean cultivars. In this study, the agronomic traits, fresh bean characteristics, and sensory evaluation of boiled beans were analyzed for 26 mung bean cultivars. The results showed that the variation coefficient and genetic diversity index of six agronomic traits of mung bean ranged from 9.04% to 44.98%, 1.68 to 1.96, respectively, with abundant genetic variation, and the highest was the grain yield. Mung bean cultivars with higher grain yield had more advantage in the number of branches, number of pods per plant, and 100-seed weight. The fresh bean traits were relatively stable, with an average coefficient variation of 8.48%. The trait with the highest genetic diversity index was the number of seeds per pod (2.03). The cultivar with the highest total sensory evaluation score of boiled beans was Zhanglv 3 (75.67), which had more advantages in taste and color. Through the comprehensive evaluation of grey relational analysis, the cultivars suitable for fresh food processing were Zhonglv 3 (0.960), Jilv 11 (0.942), Zhonglv 1 (0.915), CES-78 (0.899) and Kelv 2 (0.896). Generally, the high-quality cultivars with higher yield and fresh food processing characteristics were CES-78, Kelv 2, Zhonglv 16, and Zhonglv 2. This study provided a preference for the breeding of fresh mung bean cultivars, development of new products and improvement of mung bean resource utilization.

Genetic Diversity and Environmental Influence on Yield and Yield-Related Traits of Adzuki Bean (Vigna angularis L.).

Adzuki beans are an important food legume crop in East Asia. A large number of adzuki bean accessions are maintained in the Chinese national seed genebank. A collection of 59 elite cultivars, 389 landraces, and 27 wild adzuki beans were selected and phenotyped extensively for yield and yield-related traits at two different locations (Nanning and Nanyang, China). Ten agronomic and yield-related traits were scored, and the data were subjected to analysis of variance (ANOVA), principal component analysis (PCA), correlation, and cluster analysis. Significant variation was observed for genotypes, locations, and genotype x environment interaction for most traits. Also, there were significant differences in the phenotypes among accessions of different germplasm types. The broad-sense heritability of traits studied ranged from 4.4% to 77.8%. The number of seeds per pod (77.8%), 100-seed weight (68.0%), and number of plant branches (63.9%) had a high heritability. A total of 10 traits were transformed into 3 comprehensive factors by principal component analysis, and the first three principal component factors contributed 72.31% of the total variability. Cluster analysis categorized the 475 adzuki bean accessions into five distinct groups. The results described in this study will be useful for adzuki bean breeders for the development of varieties with high end-use quality.

Genome-wide identification and expression profiles of AP2/ERF transcription factor family in mung bean (Vigna radiata L.).

Mung bean (Vigna radiata L. Wilczek) is an economically important grain legume crop in Asia, with high nutritional quality and potential in other parts of the world particularly arid and semiarid regions. Considering the potential adverse effects of drought, high salt, and other abiotic stresses on crop yield, significant efforts have been made to understand the underlying molecular mechanisms of tolerance to these abiotic stresses in legumes. In this study, a total of 186 putative AP2/ERF genes were identified, which were named VrERF1-186. These VrERF genes were classified into four main subfamilies according to the number of AP2 domains and sequence similarity, including 24 AP2 gene members, 81 ERF gene members, 79 DREB gene members, and 2 RAV members. VrERF genes are scattered across all 11 chromosomes and form small gene clusters on chromosomes due to segmental or tandem duplication. Promoter analysis revealed various cis-acting elements related to light, hormones, and stress responsiveness processes. The expression profiles of the VrERF genes in tissues during development and in response to abiotic stresses were assessed by transcriptome sequencing, and the selected reference genes were validated by qRT-PCR. A total of 174 VrERF genes were expressed in at least one of five tissues, while others showed distinct expression patterns in different tissues or under specific abiotic stress treatments, which indicates that VrERF genes are involved in developmental and environmental stress responses in V. radiata. In conclusion, the genome localization, genome-wide characterization, gene duplication, phylogenetic relationships, and expression pattern of VrERF genes in V. radiata were analyzed, and these results will lay the foundation for further functional analysis of these genes and improve stress tolerance to adverse conditions in plants.

Effect of photoperiod on vitamin E and carotenoid biosynthesis in mung bean (Vigna radiata) sprouts.

Light affects the accumulation of vitamin E and carotenoids in many crops. This study investigated the impact of photoperiods on the metabolic regulation of vitamin E and carotenoids in mung bean sprouts considering their dietary health benefits. Mung beans were germinated under three different photoperiods: constant light, semilight and constant dark. Results revealed that the semilight photoperiod was optimum for vitamin E and carotenoid accumulation in mung bean sprouts. DXS was activated in the constant dark and was inhibited by constant light. GGPPS and HPT were sensitive to semilight photoperiod in the vitamin E biosynthetic pathway, playing dominant roles in vitamin E accumulation. The PSY, LCYE, LUT5, LUT1 and ZE genes, which are associated with carotenoid biosynthesis, were activated under semilight treatment and significantly regulated the accumulation of carotenoids. This knowledge improves knowledge on light-mediated regulation of vitamin E and carotenoids in mung bean sprouts.

The social stigma of infertile women in Zhejiang Province, China: a questionnaire-based study.

BACKGROUND: Infertile women often face stigmatization worldwide. This study aimed to investigate the stigma against infertile women in China and to analyze its influencing factors. METHODS: Of 270 women who were randomly selected from patients receiving adjuvant fertility treatment in Zhejiang Province, China, 254 successfully completed the general information questionnaire, disease information questionnaire, and Chinese version of the infertility stigma scale (ISS). The ISS contained 27 positively worded items, each of which was graded on a 5-point Likert-type scale. RESULTS: The total stigma score of female infertility patients was 66.39 ± 21.96. By dividing the number of items, the average score for each ISS item was 2.13 ± 0.81, indicating the presence of stigma. Among the four ISS factors, the social withdrawal score was the highest (2.64 ± 1.05), whereas the family stigma score was the lowest (1.88 ± 0.88). Multiple stepwise regression analysis further revealed that the duration of infertility and monthly income were important predictors of the stigma of infertile women. CONCLUSIONS: Infertile women experience moderate to high levels of stigma in Zhejiang, China. Thus, supportive psychological interventions and public education are required to change patients' cognition and assist patients in coping with negative experiences.

Construction of a High-Density Genetic Map and Its Application for QTL Mapping of Leaflet Shapes in Mung Bean (Vigna radiata L.).

Mung bean (Vigna radiata L.) is an important but understudied food legume in Asia and now worldwide. Genetic studies may help to accelerate the exploitation of new genes for breeding in this crop. Here, we used a recombination inbred line population to construct an SNP genetic linkage map by genome sequencing technology. We obtained 21,508 high-quality SNP markers integrated into 1,946 bin markers that were mapped onto 11 linkage groups (LGs) with 99-258 bin markers per LG. The total genetic length of the map was 1060.2 cM (38.76-168.03 cM per LG), with an average distance between markers of 0.54 cM. However, there were 18 gaps >5 cM, distribution on LG1, 3, 5, 7, and 9. Gene mapping for lobed and indented leaflets was conducted using the map. A major quantitative trait locus (QTL) associated with indented leaflets was detected on chromosome 10, with phenotypic variation explained (PVE) values of 39.7% and 45.4% under two different environments. Several QTLs for lobed leaflets were detected and most of them were tightly linked together on Chromosome 3. However, only one major QTL, which explained the largest phenotypic variation (27.7-69.5%), was stably detected under two different environments using both R and Q methods. In the two main stable QTLs regions on chromosomes 3 and 10, candidate genes for regulating the molecular mechanism of different leaflet shapes were detected by functional annotation. The overlap of major QTLs under different environments indicated that the present map would be good enough for precisely mapping genes, and both the QTL analysis and gene prediction were useful for investigating the mechanism of leaf development in mung bean or legumes.

Construction of a high density linkage map and genome dissection of bruchid resistance in zombi pea (Vigna vexillata (L.) A. Rich).

Zombi pea (Vigna vexillata) is a legume crop that is resistant to several biotic and abiotic stresses. Callosobruchus maculatus and Callosobruchus chinensis are serious stored-insect pests of legume crops. We constructed a high-density linkage map and performed quantitative trait loci (QTLs) mapping for resistance to these insect species in zombi pea. An F2 population of 198 individuals from a cross between 'TVNu 240' (resistant) and 'TVNu 1623' (susceptible) varieties was used to construct a linkage map of 6,529 single nucleotide polymorphism markers generated from sequencing amplified fragments of specific loci. The map comprised 11 linkage groups, spanning 1,740.9 cM, with an average of 593.5 markers per linkage group and an average distance of 0.27 cM between markers. High levels of micro-synteny between V. vexillata and cowpea (Vigna unguiculata), mungbean (Vigna radiata), azuki bean (Vigna angularis) and common bean (Phaseolus vulgaris) were found. One major and three minor QTLs for C. chinensis resistance and one major and one minor QTLs for C. maculatus resistance were identified. The major QTLs for resistance to C. chinensis and C. maculatus appeared to be the same locus. The linkage map developed in this study will facilitate the identification of useful genes/QTLs in zombi pea.

De novo transcriptomic analysis of cowpea (Vigna unguiculata L. Walp.) for genic SSR marker development.

BACKGROUND: Cowpea [Vigna unguiculata (L.) Walp.] is one of the most important legumes in tropical and semi-arid regions. However, there is relatively little genomic information available for genetic research on and breeding of cowpea. The objectives of this study were to analyse the cowpea transcriptome and develop genic molecular markers for future genetic studies of this genus. RESULTS: Approximately 54 million high-quality cDNA sequence reads were obtained from cowpea based on Illumina paired-end sequencing technology and were de novo assembled to generate 47,899 unigenes with an N50 length of 1534 bp. Sequence similarity analysis revealed 36,289 unigenes (75.8%) with significant similarity to known proteins in the non-redundant (Nr) protein database, 23,471 unigenes (49.0%) with BLAST hits in the Swiss-Prot database, and 20,654 unigenes (43.1%) with high similarity in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Further analysis identified 5560 simple sequence repeats (SSRs) as potential genic molecular markers. Validating a random set of 500 SSR markers yielded 54 polymorphic markers among 32 cowpea accessions. CONCLUSIONS: This transcriptomic analysis of cowpea provided a valuable set of genomic data for characterizing genes with important agronomic traits in Vigna unguiculata and a new set of genic SSR markers for further genetic studies and breeding in cowpea and related Vigna species.

The Fd-GOGAT1 mutant gene lc7 confers resistance to Xanthomonas oryzae pv. Oryzae in rice.

Disease resistance is an important goal of crop improvement. The molecular mechanism of resistance requires further study. Here, we report the identification of a rice leaf color mutant, lc7, which is defective in chlorophyll synthesis and photosynthesis but confers resistance to Xanthomonas oryzae pv. Oryzae (Xoo). Map-based cloning revealed that lc7 encodes a mutant ferredoxin-dependent glutamate synthase1 (Fd-GOGAT1). Fd-GOGAT1 has been proposed to have great potential for improving nitrogen-use efficiency, but its function in bacterial resistance has not been reported. The lc7 mutant accumulates excessive levels of ROS (reactive oxygen species) in the leaves, causing the leaf color to become yellow after the four-leaf stage. Compared to the wild type, lc7 mutants have a broad-spectrum high resistance to seven Xoo strains. Differentially expressed genes (DEGs) and qRT-PCR analysis indicate that many defense pathways that are involved in this broad-spectrum resistance are activated in the lc7 mutant. These results suggest that Fd-GOGAT1 plays an important role in broad-spectrum bacterial blight resistance, in addition to modulating nitrogen assimilation and chloroplast development.

Development of Gene-Based SSR Markers in Rice Bean (Vigna umbellata L.) Based on Transcriptome Data.

Rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi) is a warm season annual legume mainly grown in East Asia. Only scarce genomic resources are currently available for this legume crop species and no simple sequence repeat (SSR) markers have been specifically developed for rice bean yet. In this study, approximately 26 million high quality cDNA sequence reads were obtained from rice bean using Illumina paired-end sequencing technology and assembled into 71,929 unigenes with an average length of 986 bp. Of these unigenes, 38,840 (33.2%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases. Furthermore, 30,170 (76.3%) could be classified into gene ontology categories, 25,451 (64.4%) into Swiss-Prot categories and 21,982 (55.6%) into KOG database categories (E-value < 1.0E-5). A total of 9,301 (23.5%) were mapped onto 118 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. A total of 3,011 genic SSRs were identified as potential molecular markers. AG/CT (30.3%), AAG/CTT (8.1%) and AGAA/TTCT (20.0%) are the three main repeat motifs. A total of 300 SSR loci were randomly selected for validation by using PCR amplification. Of these loci, 23 primer pairs were polymorphic among 32 rice bean accessions. A UPGMA dendrogram revealed three major clusters among 32 rice bean accessions. The large number of SSR-containing sequences and genic SSRs in this study will be valuable for the construction of high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.

VrDREB2A, a DREB-binding transcription factor from Vigna radiata, increased drought and high-salt tolerance in transgenic Arabidopsis thaliana.

Mung bean (Vigna radiata L.) is commonly grown in Asia as an important nutritional dry grain legume, as it can survive better in arid conditions than other crops. Abiotic stresses, such as drought and high-salt contents, negatively impact its growth and production. The dehydration-responsive element-binding protein 2 (DREB2) transcription factors play a significant role in the response to these stress stimuli via transcriptional regulation of downstream genes containing the cis-element dehydration-responsive element (DRE). However, the molecular mechanisms involved in the drought tolerance of this species remain elusive, with very few reported candidate genes. No DREB2 ortholog has been reported for mung bean, and the function of mung bean DREB2 is not clear. In this study, a novel VrDREB2A gene with conserved AP2 domains and transactivation ability was isolated from mung bean. A modified VrDREB2A protein lacking the putative negative regulatory domain encoded by nucleotides 394-543 was shown to be localized in the nucleus. Expression of the VrDREB2A gene was induced by drought, high salt concentrations and abscisic acid treatment. Furthermore, comparing with the wild type Arabidopsis, the overexpression of VrDREB2A activated the expression of downstream genes in transgenic Arabidopsis, resulting in enhanced tolerance to drought and high-salt stresses and no growth retardation. The results from this study indicate that VrDREB2A functions as an important transcriptional activator and may help increase the abiotic stress tolerance of the mung bean plant.

Development and Validation of EST-SSR Markers from the Transcriptome of Adzuki Bean (Vigna angularis).

The adzuki bean (Vigna angularis (Ohwi) Ohwi and Ohashi) is an important grain legume of Asia. It is cultivated mainly in China, Japan and Korea. Despite its importance, few genomic resources are available for molecular genetic research of adzuki bean. In this study, we developed EST-SSR markers for the adzuki bean through next-generation sequencing. More than 112 million high-quality cDNA sequence reads were obtained from adzuki bean using Illumina paired-end sequencing technology, and the sequences were de novo assembled into 65,950 unigenes. The average length of the unigenes was 1,213 bp. Among the unigenes, 14,547 sequences contained a unique simple sequence repeat (SSR) and 3,350 sequences contained more than one SSR. A total of 7,947 EST-SSRs were identified as potential molecular markers, with mono-nucleotide A/T repeats (99.0%) as the most abundant motif class, followed by AG/CT (68.4%), AAG/CTT (30.0%), AAAG/CTTT (26.2%), AAAAG/CTTTT (16.1%), and AACGGG/CCCGTT (6.0%). A total of 500 SSR markers were randomly selected for validation, of which 296 markers produced reproducible amplicons with 38 polymorphic markers among the 32 adzuki bean genotypes selected from diverse geographical locations across China. The large number of SSR-containing sequences and EST-SSR markers will be valuable for genetic analysis of the adzuki bean and related Vigna species.

Assessment of genetic diversity and population structure of mung bean (Vigna radiata) germplasm using EST-based and genomic SSR markers.

Mung bean is an important legume crop in tropical and subtropical countries of Asia and has high nutritional and economic value. However the genetic diversity of mung bean is poorly characterized. In this study, our goal was to develop and use microsatellite simple sequence repeat (SSR) markers for germplasm evaluation. In total, 500 novel expression sequence tag EST-based SSRs (eSSRs) and genomic SSRs (gSSRs) were developed from mung bean transcriptome and genome sequences. Of these, only 58 were useful for diversity evaluation in a panel of 157 cultivated and wild mung bean accessions from different collection sites in East Asia. A total of 2.66 alleles were detected on average per locus which shows that polymorphism is generally low for the species. The average polymorphic information content (PIC) of gSSRs was higher than eSSRs and most of the polymorphic gSSRs were composed of di- and tri-nucleotide repeats (52.4% and 38.1% of all loci, respectively). The genotypes were differentiated into nine subgroups by cluster analysis, and the wild mung bean accessions separated well from the cultivated accessions. Analysis of molecular variance indicated that 22% of variance was observed among populations and 78% was due to differences within populations. Clustering, population structure analyses showed that non-Chinese cultivated and wild mung bean accessions were separated from Chinese accessions, but no geographical distinctions existed between genotypes collected in China. Interestingly, the average PIC value of cultivated mung bean (0.36) was higher than that of wild mung bean (0.25) showing that further collecting and wide crosses are necessary for mung bean improvement.

Transcriptome sequencing of mung bean (Vigna radiate L.) genes and the identification of EST-SSR markers.

Mung bean (Vigna radiate (L.) Wilczek) is an important traditional food legume crop, with high economic and nutritional value. It is widely grown in China and other Asian countries. Despite its importance, genomic information is currently unavailable for this crop plant species or some of its close relatives in the Vigna genus. In this study, more than 103 million high quality cDNA sequence reads were obtained from mung bean using Illumina paired-end sequencing technology. The processed reads were assembled into 48,693 unigenes with an average length of 874 bp. Of these unigenes, 25,820 (53.0%) and 23,235 (47.7%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases, respectively. Furthermore, 19,242 (39.5%) could be classified into gene ontology categories, 18,316 (37.6%) into Swiss-Prot categories and 10,918 (22.4%) into KOG database categories (E-value < 1.0E-5). A total of 6,585 (8.3%) were mapped onto 244 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. Among the unigenes, 10,053 sequences contained a unique simple sequence repeat (SSR), and 2,303 sequences contained more than one SSR together in the same expressed sequence tag (EST). A total of 13,134 EST-SSRs were identified as potential molecular markers, with mono-nucleotide A/T repeats being the most abundant motif class and G/C repeats being rare. In this SSR analysis, we found five main repeat motifs: AG/CT (30.8%), GAA/TTC (12.6%), AAAT/ATTT (6.8%), AAAAT/ATTTT (6.2%) and AAAAAT/ATTTTT (1.9%). A total of 200 SSR loci were randomly selected for validation by PCR amplification as EST-SSR markers. Of these, 66 marker primer pairs produced reproducible amplicons that were polymorphic among 31 mung bean accessions selected from diverse geographical locations. The large number of SSR-containing sequences found in this study will be valuable for the construction of a high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.