Determination of the genetic diversity of vegetable soybean [Glycine max (L.) Merr.] using EST-SSR markers

Determination of the genetic diversity of vegetable soybean [ Glycine max (L.) Merr.] using EST-SSR markers 1 first-author 1 2 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:gongym07@126.com †E-mail:gongym07@126.com gongym07@126.com Institute of Vegetable Research, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China Institute of Vegetable Research, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China Center of Analysis and Measurement, Zhejiang University, Hangzhou 310029, China Center of Analysis and Measurement, Zhejiang University, Hangzhou 310029, China The development of expressed sequence tag-derived simple sequence repeats (EST-SSRs) provided a useful tool for investigating plant genetic diversity. In the present study, 22 polymorphic EST-SSRs from grain soybean were identified and used to assess the genetic diversity in 48 vegetable soybean accessions. Among the 22 EST-SSR loci, tri-nucleotides were the most abundant repeats, accounting for 50.00% of the total motifs. GAA was the most common motif among tri-nucleotide repeats, with a frequency of 18.18%. Polymorphic analysis identified a total of 71 alleles, with an average of 3.23 per locus. The polymorphism information content (PIC) values ranged from 0.144 to 0.630, with a mean of 0.386. Observed heterozygosity ( H o ) values varied from 0.0196 to 1.0000, with an average of 0.6092, while the expected heterozygosity ( H e ) values ranged from 0.1502 to 0.6840, with a mean value of 0.4616. Principal coordinate analysis and phylogenetic tree analysis indicated that the accessions could be assigned to different groups based to a large extent on their geographic distribution, and most accessions from China were clustered into the same groups. These results suggest that Chinese vegetable soybean accessions have a narrow genetic base. The results of this study indicate that EST-SSRs from grain soybean have high transferability to vegetable soybean, and that these new markers would be helpful in taxonomy, molecular breeding, and comparative mapping studies of vegetable soybean in the future. Expressed sequence tag (EST) Simple sequence repeat (SSR) Genetic diversity Microsatellites Vegetable soybean Introduction 1 Soybean [ Glycine max (L.) Merr.], the world’s most important cultivated legume crop, can be divided into two categories: vegetable soybean, which is harvested between reproductive stages 6 (R6) and 7 (R7) of growth when the seeds have developed to fill 80%‒90% of the pod, and grain soybean, which is harvested at reproductive stage 8 (R8) when the pod has reached full maturity (Yinbo et al., 1997 1997 ; Young et al., 2000 2000 ). Grain soybean is primarily used for manufacturing oil and protein products. Vegetable soybean, however, is consumed mainly as a vegetable or snack. Like grain soybean, vegetable soybean is rich in protein, oil, and other nutritious constituents, but there are many differences between them. For example, vegetable soybeans are usually larger (over 30 g/100 seeds) than grain soybeans (less than 25 g/100 seeds), and have many advantages in terms of sensory attributes over grain soybeans, such as green color, soft texture, sweet taste, higher protein utilization, and higher contents of vitamin A, vitamin C, sucrose, and starch (Saldivar et al., 2010 2010 ; Keatinge et al., 2011 2011 ). China is the world center for vegetable soybean production and the history of vegetable soybean cultivation in China can be traced back 1 000 years (Yinbo et al., 1997 1997 ; Cornelious and Sneller, 2002 2002 ; Lu et al., 2006 2006 ). However, because of a lack of local breeding programs, the commercial cultivars used in production have mainly been introduced from Japan and Taiwan of China. To counter this situation, the Chinese government has started a project to accelerate the breeding of vegetable soybean. However, due to various restrictions, no significant breakthrough has yet been achieved (Arikit et al., 2011 2011 ). In modern plant breeding, molecular markers have become important and efficient tools. Molecular markers linked to agronomic traits can increase the accuracy and veracity of selection, thereby reducing the field workload, so the selection of suitable markers has become one of the key factors in the success of molecular breeding programs. Many types of molecular markers, such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSRs), and single nucleotide polymorphisms (SNPs), have been used to study the genetic diversity and population structure of plants (Wen et al., 2009 2009 ). Among these markers, SSRs have stood out and are considered to be the most powerful tools because of their high abundance, co-dominant inheritance, multiple alleles, reproducibility, extensive genome coverage, and ease-of-detection by polymerase chain reaction (PCR) (Moe et al., 2010 2010 ). However, due to the long time and high cost of their development, the wide use of SSRs is often limited. The rapid development of studies of expressed sequence tags (ESTs) has generated a new source for developing SSRs. Compared with genomic-SSR markers, EST-SSRs have some intrinsic advantages: (1) they are less costly to identify; (2) they are directly associated with transcribed genes; and (3) they have high transferability among related species (Varshney et al., 2005 2005 ). Recently, grain soybean studies have generated a large number of ESTs in various public databases, and EST-SSR development has been carried out in some studies. For example, Hisano et al. ( 2007 2007 ) designed 6 920 primer pairs from 63 676 grain soybean ESTs and using polymorphism analysis, obtained 680 polymorphic EST-SSRs. A further genetic linkage map study indicated that 935 loci detected by these markers were successfully mapped onto 20 linkage groups, covering 2 700.3 cM of the soybean genome. Li et al. ( 2010 2010 ) developed 34 EST-SSRs from grain soybean globular embryo ESTs, and the analysis indicated that 11 of them were polymorphic. Liu et al. ( 2010 2010 ) developed 37 EST-SSRs from the grain soybean cDNA library, and these markers could be used successfully to distinguish cultivated soybean from wild soybean. In spite of these studies in grain soybean, no EST-SSR studies have yet been reported for vegetable soybean. The objective of this study was to develop EST-SSR markers for vegetable soybean, examine their polymorphism, and evaluate the genetic diversity of vegetable soybean accessions. The results might be useful for marker-assisted selection and breeding of vegetable soybean in the future. Materials and methods 1 Materials 2 Forty-eight vegetable soybean accessions, including 45 spring-sown types and 3 autumn-sown types, were used in the study. Among the 48 accessions, 43 were from China, 3 from Japan, and 2 from the USA (Table 1 1 ). DNA was extracted from 20-d old seedlings grown in a glasshouse. A total of 0.2 g of fresh leaves were used for each repeat and DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method following the manufacturer’s instructions. The relative purity and concentration of the extracted DNA were estimated with NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA). The final concentration of each DNA sample was adjusted to 20 ng/ml. No. Accession name Origin 1 LDY081011 Liaoning, China 2 LDY081014 Liaoning, China 3 LDY081015 Liaoning, China 4 LDY081018 Liaoning, China 5 LDY081019 Liaoning, China 6 LDY081021 Liaoning, China 7 LDY081023 Liaoning, China 8 LDY081025 Liaoning, China 9 ZXN081029 Zhejiang, China 10 ZXN081031 Zhejiang, China 11 ZXN081035 Zhejiang, China 12 ZXN081036 Zhejiang, China 13 YZT081045 Jilin, China 14 YZT081049 Jilin, China 15 YZT081051 Jilin, China 16 YZT081058 Jilin, China 17 YZT081065 Jilin, China 18 YZT081069 Jilin, China 19 TW091079 Kaohsiung, Taiwan, China 20 TW091080 Kaohsiung, Taiwan, China 21 TW091081 Kaohsiung, Taiwan, China 22 TW091084 Kaohsiung, Taiwan, China 23 TW091091 Kaohsiung, Taiwan, China 24 ZAL091110 Jiangsu, China 25 ZAL091116 Jiangsu, China 26 WJH101125 Heilongjiang, China 27 WJH101128 Heilongjiang, China 28 WJH101134 Heilongjiang, China 29 JAP101136 Hokkaido, Japan 30 JAP101139 Hokkaido, Japan 31 JAP101141 Hokkaido, Japan 32 USA101148 North Carolina, USA 33 USA101155 North Carolina, USA 34 SWK111158 Liaoning, China 35 SWK111175 Liaoning, China 36 SWK111176 Liaoning, China 37 SWK111177 Liaoning, China 38 WGP111178 Jiangsu, China 39 WGP111181 Jiangsu, China 40 WGP111186 Jiangsu, China 41 WGP111187 Jiangsu, China 42 YLF111192 Shanghai, China 43 YLF111197 Shanghai, China 44 YLF111203 Shanghai, China 45 ZLH111205 Fujian, China 46 ZLH111209 Fujian, China 47 ZLH111217 Fujian, China 48 ZXH111221 Guangdong, China Primer selection and prescreening 2 A total of 172 grain soybean EST-SSRs developed in previous studies (Song et al., 2004 2004 ; Hisano et al., 2007 2007 ; Shultz et al., 2007 2007 ; Xia et al., 2007 2007 ) were selected and prescreened on five vegetable soybean accessions (one Chinese landrace, one Chinese cultivar, and one cultivar each from Japan, Taiwan of China, and USA) to select suitable primers for further analysis. Based on the results of prescreening, 64 primer pairs were designed and synthesized to assess polymorphism. The forward primers of each pair were labeled with 6-FAM fluorescent dye. SSR amplification and PCR product analysis 2 PCRs were conducted using a Peltier thermo cycler (PTC)-225 thermal cycler from MJ Research Inc. (Waltham, MA, USA). All PCR amplifications were carried out in 20 μl reaction mixtures containing 20 ng genomic DNA template, 1× PCR buffer (containing Mg 2+ ), 0.2 mmol/L dNTPs, 0.2 mmol/L forward and reverse primers, and 1 U of Taq polymerase (TaKaRa, Dalian, China). The PCR program was as follows: 95 °C for 5 min, 36 cycles each at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 60 s, and a final extension at 72 °C for 10 min. The PCR reaction products were diluted and detected on a MegaBACE 1000 DNA analysis system as previously described (Gong et al., 2011 2011 ). Analysis of amplified fragment size was performed using methods described by Gong et al. ( 2010a 2010a ). Data and statistics 2 The variability at each locus was measured in terms of the number of alleles ( N a ), observed heterozygosity ( H o ), expected heterozygosity ( H e ), polymorphic information content (PIC), Nie’s gene diversity ( D A ) and a Chi-square test for Hardy-Weinberg equilibrium ( P HW ). The N a , H o , and H e were calculated using POPGENE software, Version 1.3 (Choudhary et al., 2009 2009 ). The PIC value was calculated using the formula developed by Anderson et al. ( 1993 1993 ). D A was determined using NTSYSpc Version 2.10 (NTSYS-PC 2.10, Applied Biostatistics, Setauket, NY, USA). A dendrogram was constructed using unweighted pair group method with arithmetic means (UPGMA). Results 1 Characteristics of EST-SSR markers in vegetable soybean 2 Based on the prescreening results, 64 primer pairs with clean amplification products were designed and explored over 48 vegetable soybean accessions. Among these primers, 34.38% (22) were polymorphic, which was higher than previous results of 9.83% and 32.35% in grain soybean (Hisano et al., 2007 2007 ; Li et al., 2010 2010 ). The number of repeats ranged from 8 to 22, with an average of 11.59. Out of all the repeat motifs, the length of SSRs ranged from 22 to 44 bp, with an average of 28.21 bp (Table 2 2 ). Among the 22 SSR loci, tri-nucleotides were the most abundant repeats, accounting for 50.00%, followed by di-nucleotides (45.45%) and tetra-nucleotides (4.55%). GAA was the most common motif among the tri-nucleotide repeats, with a frequency of 18.18%, followed by AGC, CAA, GTC, GGC, ATG, GAT, AGA, and AAG, each with a frequency of 9.09%. The dominant repeat motif of the di-nucleotides was AT with a frequency of 40.00%, followed by TA (20.00%), and TC, AC, AG, and GT, each accounting for 10.00% (Table 3 3 , Fig. 1 1 ). More details about the different repeat motifs are listed in Table 2 2 . Locus name Corresponding ID from references Primer sequence (5′–3′) Motif Annealing Temp. (°C) Expected size (bp) Practical size (bp) Number of alleles Reference or information source 2 Gmp-009 2 AW620774 Forward: TCACCCACCCAATACACAAA 2 (GAA) 9 2 56 2 174 2 166–181 2 2 2 Song et al., 2004 2004 Reverse: TTCGCAACTTGTTACAACGG 2 Gmp-017 2 CG819919.1 Forward: ACCTCTTCCCCCATTCAGTT 2 (AT) 12 2 55 2 236 2 198–236 2 3 2 Shultz et al., 2007 2007 Reverse: ACCTCTTCCCCCATTCAGTT 2 Gmp-045 2 CSSR381 Forward: AACACCGTTGGTTTTCATGC 2 (AGC) 8 2 55 2 167 2 156–169 2 2 2 Xia et al., 2007 2007 Reverse: TTGCAGTTTGGGTTTGAACA 2 Gmp-046 2 CSSR385 Forward: AACCCTTCTTCCACTTCCGT 2 (CAA) 9 2 55 2 201 2 197–211 2 3 2 Xia et al., 2007 2007 Reverse: AACCCTTCTTCCACTTCCGT 2 Gmp-048 2 CSSR391 Forward: CCGCCGAAGTACGAAGTAGA 2 (GTC) 9 2 54 2 260 2 247–263 2 4 2 Xia et al., 2007 2007 Reverse: CCGCCGAAGTACGAAGTAGA 2 Gmp-049 2 CSSR400 Forward: CTTCTCTCAGCACCCTCCAC 2 (TC) 18 2 54 2 269 2 250–283 2 3 2 Xia et al., 2007 2007 Reverse: AACCCTTCTTCCACTTCCGT 2 Gmp-050 2 CSSR405 Forward: AACAACAACAGCCACCACAA 2 (CAA) 8 2 54 2 219 2 197–238 2 6 2 Xia et al., 2007 2007 Reverse: CTGGCATTGACACTGTTGCT 2 Gmp-058 2 CSSR443 Forward: GTATGGATTTCATGGGTGGG 2 (GGC) 8 2 54 2 124 2 110–160 2 6 2 Xia et al., 2007 2007 Reverse: GTTCCTCGGAGACAGCAAAG 2 Gmp-059 2 CSSR449 Forward: GAAATGACAATAATGCCGGG 2 (AT) 21 2 54 2 183 2 177–182 2 3 2 Xia et al., 2007 2007 Reverse: TTCCATTCAAAGCAGAAGCA 2 Gmp-066 2 CSSR472 Forward: GGTTACGGCACTTCCTACCA 2 (AAC) 9 2 55 2 225 2 202–235 2 2 2 Xia et al., 2007 2007 Reverse: AATTTTTGCGTTGTTGAGGG 2 Gmp-072 2 CSSR498 Forward: GAGATTCATGAGAAGGGCCA 2 (ATG) 9 2 56 2 160 2 144–165 2 4 2 Xia et al., 2007 2007 Reverse: CTCCCCGTGTTAGGTGTTGT 2 Gmp-088 2 CSSR540 Forward: GAGGTTGGTGCCTGGAGATA 2 (GAT) 9 2 56 2 211 2 197–235 2 3 2 Xia et al., 2007 2007 Reverse: TGGCGAGTTACGAGGCTATT 2 Gmp-112 2 GMES0255 Forward: TCACCCACCCAATACACAAA 2 (AT) 11 2 56 2 154 2 140–156 2 4 2 Hisano et al., 2007 2007 Reverse: TTCGCAACTTGTTACAACGG 2 Gmp-116 2 GMES0633 Forward: GCCTGTGGTTGGTCTTCATT 2 (AT) 22 2 54 2 189 2 172–192 2 3 2 Hisano et al., 2007 2007 Reverse: AAAACCATATGCTTGCGGAC 2 Gmp-122 2 GMES0644 Forward: AGATTGGAAGAGCCATCCCT 2 (AGA) 12 2 54 2 294 2 294–308 2 4 2 Hisano et al., 2007 2007 Reverse: ACTTCTCGCCCTCGTTCTTT 2 Gmp-128 2 GMES0687 Forward: CATCCTAACCGGTCAAAAACA 2 (ATTA) 7 2 55 2 152 2 138–154 2 4 2 Hisano et al., 2007 2007 Reverse: GGAACTTCTCCCTTGGGTTC 2 Gmp-133 2 GMES0709 Forward: ACAGGTTGTGGGACGGTAAA 2 (ACA) 9 2 55 2 217 2 197–221 2 3 2 Hisano et al., 2007 2007 Reverse: ACCAAATAGCTGGAATCCCC 2 Gmp-141 2 GMES0799 Forward: CCCTTCCCTCTTCTCCTGTT 2 (TA) 11 2 54 2 133 2 107–128 2 2 2 Hisano et al., 2007 2007 Reverse: ATGCAAACAAGAATCCTGGC 2 Gmp-149 2 GMES1455a Forward: ACCAGCACCGCGTAATAAAT 2 (AG) 12 2 54 2 180 2 197–210 2 2 2 Hisano et al., 2007 2007 Reverse: AACCAGGACAACCGAGTCAC 2 Gmp-166 2 GMES3937 Forward: GGAGAAGGCCTCATCAACAG 2 (AAG) 9 2 56 2 186 2 183–186 2 2 2 Hisano et al., 2007 2007 Reverse: AGGTACTGGACACTCGGTGG 2 Gmp-197 2 GMES4774 Forward: AGGATCACATACCAGGCACC 2 (TA) 18 2 56 2 277 2 253–285 2 4 2 Hisano et al., 2007 2007 Reverse: AGGATCACATACCAGGCACC 2 Gmp-199 2 GMES6145 Forward: CTCCGATGCTTATCCCAAAA 2 (GT) 15 2 56 2 184 2 168–182 2 2 2 Hisano et al., 2007 2007 Reverse: AAAACCCTCCAAAATCAGGG 3 SSR motif 3 Motif type 15 Occurrence 14 Number of repeats 2 Total 6 7 8 9 10 11 12 13 14 15 16 17 18 >18 7 Di- AT 1 1 2 4 TC 1 1 AC 1 1 TA 1 1 2 AG 1 1 GT 1 1 Total 2 3 1 2 2 10 11 Tri- GAA 1 1 2 AGC 1 1 CAA 1 1 GTC 1 1 GGC 1 1 ACA 1 1 ATG 1 1 GAT 1 1 AGA 1 1 AAG 1 1 Total 3 7 1 11 Tetra- ATTA 1 1 17 Total 0 1 3 7 0 3 3 0 0 1 0 0 2 2 22 Polymorphism of EST-SSRs 2 The 22 EST-SSRs detected a total of 71 alleles in 48 vegetable soybean accessions (Table 1 1 ). For each locus, the number of alleles ranged from 2 (Gmp-009, Gmp-045, Gmp-066, Gmp-141, Gmp-149, Gmp-166, and Gmp-199) to 6 (Gmp-050 and Gmp-058), with an average of 3.23 (Table 4 4 ). The most frequent number of alleles per locus was 2. This result was slightly lower than the value for wild soybean (3.80) (Liu et al., 2010 2010 ). PIC values varied from a high value of 0.630 (Gmp-050) to a low value of 0.144 (Gmp-046), with a mean value of 0.386, which was similar to the average value of 0.400 in grain soybean (Hisano et al., 2007 2007 ). As a strictly self-fertilizating crop, vegetable soybean was expected to have lower heterozygosity than hybrid crops. However, H o values varied widely among these markers, ranging from 0.0196 to 1.0000, with an average of 0.6092. Also, H e values varied significantly, ranging from 0.1502 to 0.6840, with a mean of 0.4616, which was significantly higher than in grain soybean (0.0140) and wild soybean (0.0690) (Li et al., 2008 2008 ; Liu et al., 2010 2010 ). In addition, 16 EST-SSRs (72.73%) had significantly higher levels of H o than did H e . All loci except Gmp-116, Gmp-149, and Gmp-166, showed significant deviations from the Hardly-Weinberg equilibrium ( P < 0.01). Considering that the accessions used in this study included cultivars and landraces from China, Japan, and USA, these results were not surprising. denotes significant deviation from Hardy-Weinberg equilibrium ( P < 0.01) Locus name H o H e PIC P HW Gmp-009 0.1923 0.3107 0.262 ** ** Gmp-017 0.7000 0.5850 0.495 ** ** Gmp-045 1.0000 0.5000 0.375 ** ** Gmp-046 0.0400 0.1502 0.144 ** ** Gmp-048 0.9038 0.6433 0.570 ** ** Gmp-049 0.3409 0.5338 0.473 ** ** Gmp-050 0.3529 0.6840 0.630 ** ** Gmp-058 0.8654 0.5775 0.486 ** ** Gmp-059 0.1429 0.4638 0.399 ** ** Gmp-066 0.9804 0.5438 0.441 ** ** Gmp-072 0.8431 0.4931 0.372 ** ** Gmp-088 0.7885 0.4776 0.364 ** ** Gmp-112 0.6905 0.5558 0.456 ** ** Gmp-116 0.3462 0.2862 0.245 Gmp-122 0.0196 0.1624 0.152 ** ** Gmp-128 1.0000 0.6248 0.554 ** ** Gmp-133 1.0000 0.5185 0.403 ** ** Gmp-141 0.9615 0.4993 0.375 ** ** Gmp-149 0.2500 0.2188 0.195 Gmp-166 0.2400 0.2112 0.189 Gmp-197 1.0000 0.5000 0.375 ** ** Gmp-199 0.7451 0.6146 0.533 ** ** Assessment of genetic diversity in 48 vegetable soybean accessions 2 To evaluate the usefulness of these EST-SSRs, a genetic variation study was carried out in the 48 accessions. A cluster graph produced by two-dimensional principle coordinate analysis indicated that cultivars 21, 22, and 32 (within the circle in Fig. 2 2 ), which were autumn-sown types, were clustered in the lower-left side and separated from other accessions. Consistent with this result, phylogenetic tree analysis showed that at a coefficient of 0.76, all accessions were clustered into two main groups: spring-sown type (I) and autumn-sown type (II) (Fig. 3 3 ). At a coefficient of 0.80, accessions of the spring-sown type were clustered into five groups. Most Chinese cultivars and landraces fell into Group 1. Group 2 was comprised of two cultivars, one from Kaohsiung, Taiwan, China (No. 19) and the other from Fujian, China (No. 45). Group 3 contained three cultivars, two from Hokkaido, Japan (Nos. 29 and 30) and the other from Kaohsiung, Taiwan, China (No. 23). Groups 4 and 5 each had only one accession, from Jiangsu, China (No. 24) and North Carolina, USA (No. 33), respectively. Group 1 was further divided into two subgroups at a coefficient of 0.81: one group comprised 24 accessions mainly from the northern regions of China, and the other group included 14 accessions mainly from the southern regions of China. These results indicated that the average genetic similarity among Chinese accessions was high, meaning that, although some accessions are far from each other in terms of geographic distance, they have a similar genetic background. However, the genetic relationships among Chinese, Japanese, and USA accessions are distant. The numbers represent different accessions as listed in Table 1 1 The numbers represent different accessions as listed in Table 1 1 Discussion 1 In this study, a total of 172 grain soybean EST-SSR markers were selected and screened for useful markers for vegetable soybean. Finally, we obtained 64 primer pairs with clean amplification products. Among these primers, 22 loci were polymorphic with a polymorphism percentage of 34.38%, which was higher than previous results of 9.83% and 32.35% in grain soybean (Hisano et al., 2007 2007 ; Li et al., 2010 2010 ). These results indicated that grain soybean EST-SSRs have high transferability to vegetable soybean and could be a potential resource for the development of vegetable soybean EST-SSR markers. Further analysis showed that tri-nucleotides were the most abundant motif in vegetable soybean (Table 3 3 ). It has been suggested that the abundance of tri-nucleotide SSRs in plants might be attributed to the absence of frameshift mutations, due to the variety of tri-nucleotide repeats (Metzgar et al., 2000 2000 ). Also, the predominance of tri-nucleotide repeats in EST-SSRs is due to the suppression of non-trinucleotide SSRs in coding regions, which could reduce the occurrence of frameshift mutations within transcribed genes. Morgante et al. ( 2002 2002 ) reported that the high frequency of tri-nucleotide repeats in plant ESTs is due to mutation pressure and putative selection for specific amino acid stretches. Previous studies indicated that tri-nucleotides were the most dominant repeat motif in legume crops, such as grain soybean, wild soybean, pea and faba bean (Kuroda et al., 2009 2009 ; Gong et al., 2010a 2010a ; 2010b 2010b ; Liu et al., 2010 2010 ). Our results support those findings. GAA was found to be the most abundant of the tri-nucleotide repeats, consistent with previous results for grain soybean (Roy et al., 2004 2004 ). SSRs with long motifs are usually ignored during generation of markers because of their scarcity in plant genomes (Wang et al., 2010 2010 ). However, one EST-SSR with a tetra-nucleotide repeat motif was found to be polymorphic in our study, which suggested that long motif SSRs may also be useful for generating informative markers. Generally, N a , PIC, H o , and H e are important indexes of the polymorphism of SSRs. In this study, a total of 71 alleles were detected in 22 loci, with an average of 3.23 per locus, equivalent to about 18% of genomic SSRs in grain soybean (Li et al., 2008 2008 ). The differences in the number of alleles per locus between EST-SSRs and genomic SSRs are due mainly to the origin of the sequences, which are more conserved in coding regions than in non-coding regions. Thus, the N a values of EST-SSRs are usually lower than those of genomic SSRs (Temnykh et al., 2000 2000 ). Liu et al. ( 2010 2010 ) reported that 37 EST-SSR markers from grain soybean detected a total of 142 alleles in wild soybean, with a mean value of 3.8, which is slightly higher than the value found in our results. The PIC value is the most important index of molecular marker polymorphism. In our study, PIC values among the 22 loci ranged from 0.144 to 0.630, with an average of 0.386, similar to the mean value (0.400) of grain soybean (Hisano et al., 2007 2007 ). As a strictly self-fertilizating crop, vegetable soybean is expected to have low heterozygosity. However, in this study, the mean value of 48 vegetable soybean accessions was 0.6092, which was significantly higher that the average values of grain soybean (0.0140) and wild soybean (0.0690) (Li et al., 2008 2008 ; Liu et al., 2010 2010 ). In addition, 19 (86.36%) loci exhibited significant deviations from Hardy-Weinberg equilibrium corrected for multiple comparisons ( P < 0.01), which is similar to the value for wild soybean (Liu et al., 2010 2010 ). These results indicate that these markers are highly informative and could be used to differentiate genotypes and cluster them for genetic diversity analysis. Assessment of genetic diversity within a population is essential to characterize germplasm and provides insights into evolutionary aspects, conservation, utilization, and establishment of breeding programs (Li et al., 2011 2011 ). In this study, genetic variation in 48 vegetable soybean accessions was analyzed using principal coordinate analysis and phylogenetic tree. Both results (Figs. 2 2 and 3 3 ) showed that cultivars with the same origin tended to be clustered. These results indicated that the genetic relationship among vegetable soybean accessions was related to a great extent to their geographic distribution, which was similar to the results for wheat (Li et al., 2008 2008 ). In China, southeastern coastal provinces, including Zhejiang, Jiangsu, Shanghai, and Fujian, are the main cultivation regions for vegetable soybean, while the northeastern provinces, such as Liaoning, Jilin, and Heilongjiang, are the main cultivation regions for grain soybean. The ecological and geographical conditions differ greatly between these areas, and accessions originating from those areas should show high genetic differentiation. However, the results of the cluster analysis in our study (Fig. 3 3 ) suggested that most accessions from China were clustered into the same groups. These results suggest that genetic variation among Chinese populations is low and that vegetable soybean in China has a narrow genetic base, forming the major constraint to vegetable soybean breeding programs. However, genetic diversity among Chinese, Japanese, and American accessions was high, indicating that they have a distant relationship. Zhou et al. ( 2002 2002 ) suggested that grain soybean collections from China, Japan, and USA have different genetic bases, and that they could be used to increase the diversity in Chinese breeding programs. Mimura et al. ( 2007 2007 ) reported that the genetic background of vegetable soybean accessions from Japan and China was different, and the same result was found in grain soybean. Our results were consistent with these previous studies. So, more foreign germplasm needs to be introduced into Chinese breeding programs to broaden the genetic base of vegetable soybean. This will benefit vegetable soybean breeding and variety improvement in the future. In summary, this paper is the first report concerning the development of EST-SSRs in vegetable soybean. Twenty-two EST-SSR markers from grain soybean showed high polymorphism in vegetable soybean and were successfully used to investigate the genetic diversity among 48 vegetable soybean accessions from China, Japan, and the USA. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1200243 Project supported by the National Natural Science Foundation of China (Nos. 31101538, 31000942, and 31000676), the Grand Science and Technology Special Project of Zhejiang Province (Nos. 2010 C02006, 2012C12903-4-1, and 2012C12903-6-3), the Zhejiang Provincial Natural Science Foundation of China (No. LY12C15004), the Public Welfare Project of Zhejiang Province (No. 2011C22011), and the Shaoxing Important Science and Technology Projects (No. 2012A22008), China 2012-09-09 2013-03-16 2013-03-03 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 14