Comparison of Population Genetic Structures between Asian and American Mungbean Accessions Using SSR Markers

The purpose of this study was to evaluate the genetic diversity and population structure of 65 mungbean accessions collected from East and Southeast Asia, the United States and Guatemala using 15 simple sequence repeat (SSR) markers. In total, 47 alleles were detected, the number of the alleles per locus range from two to six, with an average of 3.13. The mean major allele frequency (MAF), expected heterozygosity (HE), and polymorphic information content (PIC) of the 15 SSR loci were 0.76, 0.05, and 0.28, respectively. Of the 47 alleles, 17 (36.2%) were common, with a frequency of 0.05– 0.5; 16 (34.0%) were rare (frequency < 0.05) and 14 (29.8%) were abundant (frequency > 0.5). On the basis of the UPGMA dendrogram, most of the accessions were clustered into two main groups. The first group (Group I) included seven accessions and the second comprised 58 accessions, which were further divided into four subgroups. Four subpopulations were detected by model-based structure analysis. Fifty-five accessions (84.6%) showed a clear relation to each cluster based on their inferred ancestry value (>75%), while the remaining 10 accessions (15.4%) were categorized as admixtures. Mungbean accessions from US distributed to almost all clusters and 2 accessions shared genetic constituents showing it derived from mixed ancestry with Asean accessions. These results could be useful in identifying mungbean germplasms and facilitating their improvement programs.


Introduction
Mungbean (Vigna radiata L. Wilczek), which originated from India, belongs to the family Leguminosae, subgenus Ceratotropis.Due to its protein-rich edible seeds, its ability to fix nitrogen, drought tolerance, and early maturity, it is widely planted in various cropping systems (Tangphatsornruang et al., 2009).Mungbean, which provides two of the most important and inexpensive sources of dietary protein to the people of Asia and Africa, is a tropical legume species (Somta et al., 2008).Mungbean sprouts are a common food in some Asian countries and are an excellent source of protein, calcium, and vitamin C. It is commonly used as a vegetable accompaniment to a meal (Rehman, Ali, Saleem, & Tadesse, 2010).Mungbean is characterized by a short growth period and early maturity, which allows adaption to multiple cropping systems of the lowland tropics and subtropics (Gwag, 2008).

Plant Material and DNA Extraction
A total of 65 accessions of mungbean collected from Korea (Nos.1-23), the Philippines (Nos.24-39), Vietnam , the United States (Nos.56-64), and Guatemala (No. 65) were obtained from the National Agrobiodiversity Center (NAC) of Korea (Table 1).Plant young leaves were sampled and DNA was extracted using a Qiagen DNA extraction kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop ND-1000 UV-Vis spectrophotometer (Dupont Agricultural Genomics Laboratory, Newark, DE, USA).Finally, the DNA of each sample was prepared at concentration of 20 ng/ µL.

SSR Analysis
Fifteen SSR primers developed by Gwag (2008) were used for genotyping.A three-primer system (Schuelke, 2000) was used to determine the size of PCR products.In the system, unlike the normal reverse primer, the forward primer was composed of normal primer concatenate with a universal M13 oligonucleotide (TGTAAAACGACGGCCAGT) labeled with one of three fluorescent dyes (6-FAM, NED, or HEX) that allowed PCR products to be triplexed during electrophoresis.PCR amplification was performed in a total volume of 20 µL containing 50 ng of template DNA, 0.2 mM of each dNTP, 1× PCR buffer, 1U Taq DNA polymerase, 8 pmol of each reverse and fluorescent labeled M13 (-21) primer, and 2 pmol of forward primer with M13 (-21) tail at its 5'-end.The conditions for the PCR amplification were as follows: 94°C for 3 min, 30 cycles of [94°C for 30 sec, 60°C for 45s, 72°C for 1 min], 10 cycles of [94°C for 30 sec, 53°C for 45s, 72°C for 1 min], and a final extension step of 72°C for 10 min.Microsatellite alleles were resolved using a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with GENESCAN 3.7 software (Applied Biosystems) and sized precisely against 6-carboxy-X-rhodamine (ROX) molecular size standards using GENOTYPE 3.7 software (Applied Biosystems).

Data Analysis
Basic statistics for diversity measurements at each microsatellite locus, including the total number of alleles, allele frequency, major allele frequency (MAF), gene diversity (GD), and polymorphic information content (PIC value; Yu et al., 2003), were calculated using the genetic analysis package PowerMarker ver.3.25 (Liu & Muse 2005).The variability at each locus was measured in terms of number of alleles, expected heterozygosity (H E ), and genetic distance between each pair of accessions using the genetic analysis package POPGENE 1.31 (Yeh, Yang, & Boyle, 1999).
The unweighted pair group method with an arithmetic mean (UPGMA) tree from shared allele frequencies was constructed using the MEGA 4.0 program (Tamura et al., 2007), which is embedded in PowerMarker.The model-based program STRUCTURE 2.2 (Pritchard, Stephens, & Donnelly, 2000) was used to identify population structure of the accessions implementing a Bayesian clustering approach.In this model, the population genetic structure was characterized by admixture model and correlated allele frequencies.Five independent replications were performed for each run with K ranging from two to ten using a burn-in of 10000.The most probable value of ΔK was detected by using K as a criterion and the status of each location was established based on Pritchard et al. (2000).A location would be considered to attached with one cluster by having more than 75% membership probabilities.

SSR Polymorphisms
All 15 SSR markers showed polymorphisms and a total of 47 alleles were identified (Table 2).The size of the PCR products ranged from 110 to 321 bp (Table 2).Polymorphism of SSR markers was measured in terms of the numbers of observed alleles (NA), the number of rare alleles, major allele frequency (MAF), expected heterozygosity (H E ), and the polymorphism information content (PIC).To assess the value of PIC the following was utilized: Here, pi is the allele frequency of the ith alleles of the locus (Yu et al., 2003).Alleles with a frequency less than 5% were defined as rare alleles, whereas common alleles and abundant alleles were defined as those alleles with a frequency between 5% and 50% and more than 50%, respectively.The numbers of observed alleles ranged from two to six with means of 3.13 per locus while the numbers of rare alleles varied from zero to four with means of 1.07 per locus.A total of 16 rare alleles comprised 34.0% of all alleles were identified at all 15 loci.Common and abundant alleles comprised 36.2% and 29.8%, respectively, of the total.The major allele frequency per locus and the expected heterozygosity in all the accessions varied from 0. 49 to 0. 98 and 0 to 0. 22, with averages of 0. 76 and 0. 05, respectively.The genetic diversity and PIC value ranged from 0. 10 to 0. 54 and 0. 10 to 0. 51, with averages of 0. 33 and 0. 28, respectively.Positive relationships were found between genetic diversity and PIC values.For some loci, however, the degree of polymorphism showed no correlation with the number of alleles (Table 2).

Distance-based Phylogeny
The genetic relationships of populations in the present study was analysed by calculating the shared allele frequencies.A dendrogram (Figure 1) was generated using MEGA 4 (Tamura et al., 2007) embedded in the PowerMarker program (Liu & Muse, 2005).Similarity coefficients generated by PowerMarker varied from 0 to 0.8, with an average of 0.33, and were used to construct an UPGMA dendrogram using MEGA 4 software.Most of the accessions studied were clustered into two main groups by the UPGMA dendrogram (Figure 1).The first group (Group I) included seven accessions from Korea.The second group consisted of 58 accessions and was further divided into four subgroups.Subgroup GII-I included four accessions from Korea.GII -II consisted of five accessions (two from the United States and three from Southeast Asia).GII-III (26 accessions) and GII-IV (23 accession) both consisted of accessions from East Asia, Southeast Asia, and the United States.The resulting phylogram (Figure 2) revealed a complex accession distribution pattern.Asia, Southeast Asia and America.The colour of the blocks correspond to those of the three regions

Population Structure Analysis
A model-based approach was used to infer the population structures and detect the ancestral and hybrid forms within accessions in the present study (Pritchard et al., 2000).Results of five independent replications for each run with K ranging from two to ten were consistent, but the distribution of LnP(D) revealed a continuingly increasing curve without a clear maximum for the true K.The ad hoc quantity (ΔK) was used to estimate the real

Figure 1 .
Figure 1.UPGMA dendrogram showing phylogenetic relationships among 65 mungbean accessions from East Asia, Southeast Asia and America.The colour of the blocks correspond to those of the three regions Figure 3 used to b

Table 1 .
The details information of 65 mungbean accessions used in this study

Table 2 .
Overall diversity statistics at 15 SSR loci in 65 mungbean accessions a: Number of alleles; b: Number of rare alleles; c: Major allele frequency; d: Gene diversity; e: Expected heterozygosity; f: Polymorphism information content.

Table 3 .
Number of mungbean accessions, number of alleles, major allele frequency, genetic diversity, polymorphic information content according to originated region/country.
In conclusion, present result pointed out that Mungbean accessions from US, with high heterogeneity, genetically related with Asean accessions.