Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek)

Limited availability of validated, polymorphic microsatellite markers in mung bean (Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard’s similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume.     

Molecular assessment of genetic diversity in mung bean germplasm

RAPD profiles were used to identify the extent of diversity among 54 accessions of mung bean that included both improved and local land races. Out of the 40 primers screened, seven primers generated 174 amplification products with an average of 24.85 bands per primer. The RAPD profiles were analysed for Jaccard's similarity coefficients that was found to be in the range from 0 to 0.48, indicating the presence of wide range of genetic diversity at molecular level. Cluster analysis was carried out based on distances (1-similarity coefficient) using neighbour-joining method in Free Tree package. The dendrogram resolved all the accessions into two major clusters, I (with 11 accessions) and II (with 43 accessions). However, the cluster was further divided into four subclusters (II A with six, II B with nine, II C with 15 and II D with 13 accessions). The distribution of the accessions in different clusters and subclusters appears to be related to their performance in field conditions for 10 morphological traits that were scored. This study indicated that the RAPD profiles provide an easy and simple technique for preliminary genetic diversity assessment of mung bean accessions that may reflect morphological trait differences among them.     

Assessment of Genetic Diversity in Ziziphus mauritiana Using Inter-Simple Sequence Repeat Markers

Genetic diversity among 47 ber accessions belonging to cultivated species (Ziziphus mauritiana Lam) and one wild accession of Ziziphus nummularia (Burm F) Willed was investigated using Inter-Simple Sequence Repeat (ISSR) markers. A total of 167 amplification products were detected with 18 ISSR primers of which 152 (89.96%) were polymorphic. Most of the primers that produced distinct bands (14 primers out of 18) contained dinucleotide repeats. Primers based on (AC)n and (AG)_n repeats produced more polymorphic bands. Genetic similarity ranging from 43.07% to 90.30% suggested that the 48 Ziziphus genotypes used in the study were divergent. Cluster analysis based on UPGMA method and Bootstrap analysis separated all the 48 genotypes in four distinct clusters. The present study has successfully distinguished morphologically similar genotypes that emphasize the use of molecular markers to the taxonomists. Morphologically similar but genetically distinct genotypes, identified using ISSR markers could be potential sources for genotype identification and to resolve controversies over misnomination of ber genotypes. Present study is the first report on the exploitation of ISSR markers in ber for genetic diversity analysis.     

Single nucleotide polymorphism discovery in common bean

Single nucleotide polymorphisms (SNPs) were discovered in common bean (Phaseolus vulgaris L.) via resequencing of sequence-tagged sites (STSs) developed by PCR primers previously designed to soybean shotgun and bacterial artificial chromosome (BAC) end sequences, and by primers designed to common bean genes and microsatellite flanking regions. DNA fragments harboring SNPs were identified in single amplicons from six contrasting P. vulgaris genotypes of the Andean (Jalo EEP 558, G 19833, and AND 277) and Mesoamerican (BAT 93, DOR 364, and Rudá) gene pools. These genotypes are the parents of three common bean recombinant inbred line mapping populations. From an initial set of 1,880 PCR primer pairs tested, 265 robust STSs were obtained, which could be sequenced in each one of the six common bean genotypes. In the resulting 131,120?bp of aligned sequence, a total of 677 SNPs were identified, including 555 single-base changes (295 transitions and 260 transversions) and 122 small nucleotide insertions/deletions (indels). The frequency of SNPs was 5.16 SNPs/kb and the mean nucleotide diversity, expressed as Halushka??s theta, was 0.00226. This work represents one of the first efforts aimed at detecting SNPs in P. vulgaris. The SNPs identified should be an important resource for common bean geneticists and breeders for quantitative trait locus discovery, marker-assisted selection, and map-based cloning. These SNPS will be also useful for diversity analysis and microsynteny studies among legume species.     

Description and analysis of genetic diversity between commercial bean lines (Phaseolus vulgaris L.)

The effectiveness of RFLP, DAMD-PCR, ISSR and RAPD markers in assessing polymorphism and relationships between 24 commercial lines of Phaseolus vulgaris L.was evaluated. We have used a Phaseolus-specific minisatellite sequence as a probe, which enabled 23 of the bean lines tested to be fingerprinted. Based on the sequence information obtained, primers corresponding to the bean-specific minisatellite core sequence were used in subsequent PCR amplifications. Our observations indicated that while the DAMD-PCR was sensitive in detecting genetic variation between bean species and between accessions of P. vulgaris, when used alone it may be limited in its ability to detect genetic variation among cultivated bean lines due to the low number of loci amplified. Only one out of the five ISSR primers tested was efficient in generating multiple band profiles, which was insufficient to distinguish all the different bean lines. Reproducible RAPD profiles were obtained, and these allowed us to differentiate all the genotypes tested with seven primers. We ultimately used only results from RFLP and RAPD markers to explore the genetic diversity among commercial bean lines. Both analyses led to the same clustering of the bean lines according to their geographical origins (United States or Europe). With respect to the European lines, the results obtained from RAPD data also enable the lines to be clustered according to their creators. Received: 15 January 2000 / Accepted: 21 March 2000     

Distribution of restriction site polymorphism within the chloroplast genome of the genus Glycine,subgenus Soja

Summary Restriction fragment length polymorphisms (RFLPs) have been used to detect intragenic sequence diversity in Glycine subgenus soja chloroplast DNA. The distribution of these RFLPs allow Glycine max and G. soja accessions to be grouped according to cytoplasmic genetic relatedness. DNA clones from mung bean chloroplast DNA were used to locate the RFLPs to specific regions of the chloroplast genome. In the course of the experiments, several previously unobserved RFLPs were also identified. At least six molecular changes were detected, including both restriction site loss or gain and insertion/deletion events. Three of the fragment polymorphisms detected are due to changes in the juncture region between one inverted repeat region and the large single-copy region. Probes detecting polymorphisms in three representative soybean genotypes were used to screen additional cultivars and Plant Introductions. The distribution of RFLP patterns in these accessions were consistent with the patterns of previously described cytoplasmic groupings, with the exception of one accession, which formed a new plastome group.     

SSR analysis of genetic diversity and structure of the germplasm of faba bean (Vicia faba L.)

Assessing the diversity and genetic structure of faba bean (Vicia faba L.) germplasm is essential to improve the quality and yield of this economically important crop. In this study, simple sequence repeats (SSRs) were utilized to evaluate the diversity and structure of 35 faba bean genotypes originating from three different geographical regions (Northern Africa, Eastern Africa, and Near East). All 15 SSR loci generated a total of 100 alleles. The allele number per locus varied from 4 to 11, with a mean of 6.67. The expected heterozygosity (H_e) of SSR loci ranged between 0.51 and 0.81, with a mean of 0.63. The PIC value also varied from 0.44 to 0.78, with an average of 0.58. The expected heterozygosity of 22 faba bean genotypes was higher than the observed one. Interestingly, AMOVA analysis showed that much of variability resided within accessions (79.2%). A highly significant difference among regions was also evidenced, and represented 5.3% of the total variation. Moreover, cluster analysis divided the 35 faba bean genotypes into two main clusters. The first main cluster comprised all faba bean genotypes originating from the Near East region, whereas the second main cluster comprised all the genotypes originating from the Northern and Eastern Africa regions, indicating that the Northern and Eastern African faba bean genotypes were more closely related to each other than to the Near East genotypes. Structure analysis also revealed that the 35 faba bean genotypes might be assigned to two populations, in complete accordance with cluster analysis data. In conclusion, this study showed high levels of diversity in the analysed genotypes of faba bean, and could be utilized in future breeding programmes to develop new cultivars of high yield.     

Characterization of new microsatellite markers in mung bean,Vigna radiata (L.)

The present work reports the isolation and characterization of new polymorphic microsatellites in mung bean (Vigna radiata L.). Of 93 designed primer pairs, seven were found to amplify polymorphic microsatellite loci, which were then characterized using 34 mung bean accessions. The number of alleles ranged from two to five alleles per locus with an average of three alleles. Observed and expected heterozygosity values ranged from 0 to 0.088 and from 0.275 to 0.683, respectively. All seven loci showed significant deviations from Hardy–Weinberg equilibrium, whereas only one pairwise combination (GBssr‐MB77 and GBssr‐MB91) exhibited significant departure from linkage disequilibrium. These newly developed markers are currently being utilized for diversity assessment within the mung bean germplasm collection of the Korean Gene Bank.     

Selection of international molecular standards for DNA fingerprinting of <Emphasis Type="Italic">Theobroma cacao</Emphasis>

A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Development of SCAR marker linked to anthracnose resistance from Indian French bean (Phaseolus vulgaris L.) germplasm

This study was performed to identify the French bean genotypes resistant to anthracnose disease. Thirty-five RAPD primers were used for screening four resistant and nine susceptible French bean accessions. Of these, three RAPD primers, viz. OPAH16₇₀₀, OPN6₇₀₀ and OPS₉₀₀ showed polymorphic bands differentiating between resistant and susceptible genotypes. The RAPD primer OPAH16 was then selected for conversion into a SCAR marker. The polymorphic band present in the resistant line (D line) was eluted, cloned in pTZ57R/T cloning vector and was then transferred into DH5α Escherichia coli cells. The positively transformed clones were selected based on ampicillin resistance blue-white colony selection method. The plasmid DNA was isolated from transformed white colonies, sequenced and developed into SCAR marker SPAH 16. This SCAR marker SPAH 16 was then verified via PCR using the original French bean accessions.     

Development of a SCAR marker associated with salt tolerance in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>) from a semi-arid region

Durum wheat (Triticum turgidum ssp. durum) is one of the main species of cultivated wheat. In arid and semi-arid areas, salinity stress reduces durum wheat productivity. This study used 26 durum wheat accessions from semi-arid regions in Tunisia to analyze plant tolerance to salt stress. Salt stress was experimentally applied by regularly submerging pots in NaCl solution. The salt tolerance trait index (STTI) and salt tolerance index (STI) of various growth parameters were used as criteria to select for salt tolerance. Analysis of genetic relationships was carried out to determine the genetic distance between durum wheat accessions. Based on simple sequence repeats analysis, a molecular marker for salt stress resistance in durum wheat was developed. Salt-treated plants had reduced morphological traits compared to control plants. Most STTIs in all genotypes were below 100 %. Based on STI, 8 accessions were found to be salt-resistant, 16 were salt-moderate, two were salt-susceptible. Analysis of the genetic relationships among 28 Tunisian durum wheat accessions revealed that landraces of the same nominal type are closely related. Of the 94 SSR primers investigated, three were selected and used to design sequence characterized amplified region (SCAR) primers. One SCAR primer pair, KUCMB_Xgwm403_2, produced a 207 bp band that was present in salt-resistant durum wheat lines but absent in salt-susceptible lines. The results suggest that KUCMB_Xgwm403_2 could be a potential genetic tag for salt-tolerant durum wheats.     

Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) genotypes

Cultivated peanut possesses an extremely narrow genetic basis. Polymorphism is considerably difficult to identify with the use of conventional biochemical and molecular tools. For the purpose of obtaining considerable DNA polymorphisms and fingerprinting cultivated peanut genotypes in a convenient manner, start codon targeted polymorphism technique was used to study genetic diversity and relatedness among 20 accessions of four major botanical varieties of peanut. Of 36 primers screened, 18 primers could produce unambiguous and reproducible bands. All 18 primers generated a total of 157 fragments, with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22%) were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. Polymorphism per primer ranged from 14.29 to 66.67%, with an average of 36.76%. The results revealed that not all accessions of the same variety were grouped together and high genetic similarity was detected among the tested genotypes based on cluster analysis and genetic distance analysis, respectively. Further, accession-specific markers were observed in several accessions. All these results demonstrated the following: (1) start codon targeted polymorphism technique can be utilized to identify DNA polymorphisms and fingerprint cultivars in domesticated peanut, and (2) it possesses considerable potential for studying genetic diversity and relationships among peanut accessions.     

Genetic diversity analysis of maintainer and restorer accessions in upland cotton (Gossypium hirsutum L.)

Genetic diversity amongst 91 upland cotton accessions (50 maintainer, ‘B’ and 41 restorer ‘R’ lines) and three wild species viz., G. aridum, G. thurberi and G. anomalum was analyzed using SSR and RAPD markers. A total of 53 primers (30 SSR and 23 RAPD) were sampled for screening 94 accessions, of which 26 SSR and 17 RAPD primers were polymorphic. Average polymorphism detected by SSR, RAPD and SSR + RAPD markers was 72.5, 62 and 66.66 per cent, respectively. A unique marker CIR-200₂₆₀ that distinguishes G. thurberi from all upland accessions has been identified. Similarity coefficient values within and between B and R lines ranged from 0.65–0.95, 0.61–0.98 and 0.53–0.93 for SSR and 0.72–0.98, 0.73–0.97 and 0.69–0.98 for RAPD markers. UPGMA cluster analysis was consistent with the pedigree and genotypic background of the accessions. RAPD and SSR matrices showed significant positive product moment correlation (r?=?0.93 and 0.92) with the RAPD + SSR combined data matrix, respectively. The result indicates a moderate level of genetic diversity in B and R accessions of upland cotton. Genetically diverse combinations were identified to further evaluate heterotic performance. The maintainer, AKH-108, AKH-118 and AKH-2173; and restorer AKH-31 and AKH 4943 accession were identified as most distinct and divergent, could be used as candidate parental genotypes in hybrid and varietal development programme and also development of mapping population for trait mapping in cotton.     

Genetic diversity of the azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi) gene pool as assessed by SSR markers

To facilitate the wider use of genetic resources including newly collected cultivated and wild azuki bean germplasm, the genetic diversity of the azuki bean complex, based on 13 simple sequence repeat (SSR) primers, was evaluated and a core collection was developed using 616 accessions originating from 8 Asian countries. Wild germplasm from Japan was highly diverse and represented much of the allelic variation found in cultivated germplasm. The SSR results together with recent archaeobotanical evidence support the view that Japan is one center of domestication of azuki bean, at least for the northeast Asian azuki bean. Cultivated azuki beans from China, Korea, and Japan were the most diverse and were genetically distinct from each other, suggesting a long and relatively isolated history of cultivation in each country. Cultivated azuki beans from eastern Nepal and Bhutan were similar to each other and quite distinct from others. For two primers, most eastern Nepalese and Bhutanese cultivated accessions had null alleles. In addition, wild accessions from the Yangtze River region of China and the Himalayan region had a null allele for one or the other of these primers. Whether the distinct diversity of azuki bean in the Himalayan region is due to introgression or separate domestication events requires further study. In contrast, western Nepalese azuki beans showed an SSR profile similar to that of Chinese azuki beans. The genetic distinctness of cultivated azuki beans from Vietnam has been revealed for the first time. The specific alleles indicate that Vietnamese azuki beans have been cultivated in isolation from Chinese azuki beans for a long time. Wild germplasm from the Himalayan region showed the highest level of variation. Based on the results, Himalayan germplasm could be considered a novel gene source for azuki bean breeding. A comparison with mungbean SSR analysis revealed that the mean gene diversity of cultivated azuki bean (0.74) was much higher than that of cultivated mungbean (0.41). The reduction in gene diversity due to domestication, the domestication bottleneck, in azuki bean is not strong compared with that in mungbean.     

Cyclodextrins increase phytosterol and tocopherol levels in suspension cultured cells obtained from mung beans and safflower

In this work, suspension‐cultured cells of mung beans and safflower were used in order to analyze the effect of methyl jasmonate and/or cyclodextrins, on bioactive compound production such as phytosterols and tocopherols. The results indicated that mung bean suspension‐cultured cells produced higher amount of total phytosterols and tocopherols. In particular, mung bean suspension‐cultured cells produced almost 220‐fold higher levels of tocopherols than safflower suspension‐cultured cells in the best conditions. However, while cyclodextrins were able to enhance extracellular production of phytosterols, in the case of tocopherols, they only increased their intracellular accumulation. Our results showed that mung bean cells could be used as a highly efficient system for the production of phytosterols and tocopherols which have a wide range of biological activities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1662–1665, 2017     

Identification and Association Analysis of Castor Bean Orthologous Candidate Gene-Based Markers for High Oil Content in Jatropha curcas

Jatropha (Jatropha curcas) and castor bean (Ricinus communis) possess several taxonomic similarities, and their seeds contain a high proportion of oil (up to 40%) which has been used in various industrial products, including diesel oil. Thirty-two candidate genes responsible for fatty acid biosynthesis were identified in the castor bean genome sequence. Testing of 48 primer pairs from candidate gene regions, including 12 SSRs from castor bean on 54 genotypes of J. curcas, 65% amplified successfully on Jatropha out of which 20% showed polymorphisms. Jatropha genotypes, categorized for oil content, were used in association analysis of candidate gene regions with high oil content. One marker–trait association for the oil trait was identified. Stearoyl desaturase amplicon (700 bp) consisting of intron and exon (P?=?0.00013) showed association with high oil content in Jatropha genotypes. Sequencing of the 1.3-kb amplicon, including the 700-bp fragment of stearoyl desaturase, which had shown association with the high oil content, revealed SNPs in the exonic region. The SNPs resulted in substitution of leucine with glutamine in the open reading frame of stearoyl desaturase of low oil content genotypes. The molecular marker is expected to be useful in marker-assisted breeding of high oil content genotypes in Jatropha.     

Thermal phase transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relation to chilling injury

The phase behaviour of leaf polar lipids from three plants, varying in their sensitivity to chilling, was investigated by differential scanning calorimetry. For the lipids from mung bean (Vigna radiata L. var. Berken), a chilling-sensitive plant, a transition exotherm was detected beginning at $\text{10 ± 2°}\text{C}$. No exotherm was evident above 0°C with polar lipids from wheat (Triticum aestivum cv. Falcon) or pea (Pisum sativum cv. Massey Gem), plants which are insensitive to chilling. The enthalpy for the transition in the mung bean polar lipids indicated that only about 7% w/w of the lipid was in the gel phase at ?8°C. The thermal transition of the mung bean lipids was mimicked by wheat and pea polar lipids after the addition of 1 to 2% w/w of a relatively high melting-point lipid such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol or dimyristoylphosphatidylcholine. Analysis of the polar lipids from the three plants showed that a dipalmitoylphosphatidylglycerol was present in mung bean (1.7% w/w) and pea (0.3% w/w) but undetected in wheat, indicating that the transition exotherm temperature of 10°C in mung bean, 0°C in pea and about ?3°C in wheat correlates with the proportion of the high melting-point disaturated component in the polar lipids. The results indicate that the transition exotherm, observed at temperatures above 0°C in the membranes of chilling-sensitive plants, could be induced by small amounts of high melting-point lipids and involves only a small proportion of the membrane polar lipids.     

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.