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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2012 Oct 3;13(10):12608–12628. doi: 10.3390/ijms131012608

Genetic Diversity and Population Structure: Implications for Conservation of Wild Soybean (Glycine soja Sieb. et Zucc) Based on Nuclear and Chloroplast Microsatellite Variation

Shuilian He 1,2, Yunsheng Wang 1,3, Sergei Volis 4, Dezhu Li 1,*, Tingshuang Yi 1,*
PMCID: PMC3497291  PMID: 23202917

Abstract

Wild soybean (Glycine soja Sieb. et Zucc) is the most important germplasm resource for soybean breeding, and is currently subject to habitat loss, fragmentation and population decline. In order to develop successful conservation strategies, a total of 604 wild soybean accessions from 43 locations sampled across its range in China, Japan and Korea were analyzed using 20 nuclear (nSSRs) and five chloroplast microsatellite markers (cpSSRs) to reveal its genetic diversity and population structure. Relatively high nSSR diversity was found in wild soybean compared with other self-pollinated species, and the region of middle and lower reaches of Yangtze River (MDRY) was revealed to have the highest genetic diversity. However, cpSSRs suggested that Korea is a center of diversity. High genetic differentiation and low gene flow among populations were detected, which is consistent with the predominant self-pollination of wild soybean. Two main clusters were revealed by MCMC structure reconstruction and phylogenetic dendrogram, one formed by a group of populations from northwestern China (NWC) and north China (NC), and the other including northeastern China (NEC), Japan, Korea, MDRY, south China (SC) and southwestern China (SWC). Contrib analyses showed that southwestern China makes the greatest contribution to the total diversity and allelic richness, and is worthy of being given conservation priority.

Keywords: Glycine soja, microsatellite, genetic diversity, genetic structure, bottleneck effect

1. Introduction

Soybean [Glycine max (L.) Merrill, Fabaceae], is the world’s most important grain legume crop for its protein and oil [1,2], and its genetic diversity has been declining during processes of domestication and artificial selection [2]. Wild soybean (Glycine soja Sieb. et Zucc), the ancestor of soybean, retains useful genetic variation for breeding improvement of yield, and resistance to pests, diseases, alkali and salt, and therefore is extremely important germplasm to enrich the soybean gene pool [3].

Wild soybean is mainly distributed in the Asiatic Floristic region including most of China (53°–24°N and 134°–97°E) [4], the Korean peninsula, the main islands of the Japanese archipelago and Far Eastern Russia [5] (Figure 1). Wild soybean resources have been severely depleted in China in the last 20 years due to habitat fragmentation [6]. Comparing with surveys in 1979 to 1983, the survey conducted by the Chinese Ministry of Agriculture in 2002 to 2004 revealed large range reductions of wild soybean [7]. For example, the most important populations of wild soybean in Jixian county of Heilongjiang province in China have disappeared following land conversion for agriculture; a large population of 0.02 km2 in the Keshan county of the same province has been almost completely destroyed, and the large population in the Zhangwu county of the Liaoning province in China has disappeared, leading to the permanent loss of the white-flowered soybean type [7]. Wild soybean has been listed as a national second-class protected plant in 1999 in China [8] and the species requires urgent conservation actions.

Figure 1.

Figure 1

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Sampling populations of wild soybean.

The genetic diversity and genetic structure of wild soybean have been studied using morphological traits [3,9], isozymes [10], RFLP [11,12], cytoplasmic DNA [11,13] and SSR markers [14–18]. However, these studies were restricted to particular region(s) and most had a limited sample size [19–22]. These studies produced conflicting results with regards to the diversification of wild soybean. For example, the Korean peninsula [14], northeastern China [3], the Yangtze River region [23], and Southern China [24,25] have all been considered as the center of the species’ diversity by different studies. In order to make appropriate conservation recommendations, a study of the level and geographical structure of the genetic variation across the whole species range is urgently needed. Widely distributed across all eukaryotic genomes, the simple sequence repeat (SSR) is a marker of choice for the analysis of genetic variation [26], with more than 1,000 SSRs markers available for wild soybean [27]. We employed 20 nSSRs and five cpSSRs to study: (i) the extent and structure of genetic variation in wild soybean sampled throughout most of its natural range; and (ii) the demographic history of wild soybean to infer historical changes in population sizes.

2. Results

2.1. Equilibrium Test and Genetic Diversity

MICROCHECKER found no evidence of scoring errors, but some samples were detected to have null alleles. We failed to amply these alleles despite two to three more genotyping attempts. Mutations in the flanking region may prevent the primer from annealing to template DNA during amplification of microsatellite loci by PCR [28], but we still kept these loci for further analyses because the frequency was relatively small (<5%). All populations deviated significantly from Hardy-Weinberg equilibrium (p < 0.05), with the observed heterozygosity being lower than expected (mean observed 0.031, range 0.000–0.205 vs. expected 0.426, range 0.018–0.797).

All nSSR loci were polymorphic in all populations. The mean allele richness (AR) and Shannon’s information index (I) were 1.9 (1–3.1) and 0.793 (0.034–1.744), respectively. The fixation index was high (mean 0.913, range 0.202–1). The outcrossing rate was low (mean 8.1%, range 0%–66.4%), but three populations (SY, J2, and K2) showed atypically high outcrossing rate (>48%). The region of MDRY had the highest genetic diversity (AR = 14.0, and I = 2.349). Observed heterozygosity (HO = 0.063) and expected heterozygosity (HE = 0.881) of this region were also higher than other regions (Table 1).

Table 1.

Genetic diversity parameters estimated at 20 nSSRs and 5 cpSSRs in 43 populations of wild soybean.

nSSRs cpSSRs
Pops N A Na AR I HO HE FIS t (%) A Na AR I
G1:SC 73 199 10 9.5 1.886 0.013 0.809 0.984 0.8 11 2 2.2 0.421
G1_AF 15 64 3 1.9 0.731 0.014 0.397 0.973 1.4 6 1 1.2 0.079
G1_HY 15 88 4 2.4 1.177 0.014 0.605 0.979 1.1 9 2 1.8 0.493
G1_JO 15 45 2 1.6 0.505 0.007 0.300 0.927 3.8 6 1 1.2 0.079
G1_QZ 13 56 3 1.9 0.751 0.015 0.456 0.969 1.6 6 1 1.2 0.133
G1_RY 15 36 2 1.2 0.190 0.018 0.094 0.736 15.2 7 1 1.3 0.098
G2:MDRY 75 292 15 14.0 2.349 0.063 0.881 0.929 3.7 16 3 3.1 0.742
G2_DQ 14 57 3 2.0 0.792 0.051 0.468 0.904 5.0 10 2 2.0 0.502
G2_SC 15 114 6 2.8 1.493 0.023 0.722 0.969 1.6 10 2 1.9 0.369
G2_TB 15 136 7 3.1 1.744 0.007 0.797 0.992 0.4 14 3 2.7 0.809
G2_WC 14 124 6 3.0 1.616 0.053 0.762 0.933 3.5 11 2 2.1 0.578
G2_XU 15 114 6 2.7 1.436 0.205 0.708 0.718 16.4 10 2 1.9 0.402
G3:SWC 83 199 10 9.3 1.884 0.035 0.803 0.956 2.2 18 4 3.5 0.707
G3_CK 15 57 3 2.0 0.827 0.037 0.491 0.934 3.4 7 1 1.3 0.149
G3_CY 12 38 2 1.4 0.343 0.013 0.200 0.957 2.2 7 1 1.4 0.170
G3_GH 14 62 3 1.9 0.761 0.048 0.426 0.759 13.7 7 1 1.4 0.217
G3_N1 12 59 3 1.8 0.691 0.027 0.383 0.898 5.4 12 2 2.2 0.511
G3_N2 11 52 3 1.8 0.647 0.067 0.407 0.761 13.6 9 2 1.8 0.375
G3_YJ 14 51 3 1.8 0.663 0.014 0.414 0.914 4.5 6 1 1.2 0.130
G4:NWC 75 222 11 10.3 1.740 0.026 0.719 0.964 1.8 13 3 2.6 0.447
G4_BX 15 98 5 2.4 1.162 0.028 0.570 0.962 1.9 6 2 2.0 0.487
G4_HX 14 97 5 2.5 1.196 0.071 0.598 0.835 9.0 11 2 2.1 0.475
G4_LW 15 40 2 1.5 0.428 0.007 0.265 0.978 1.1 11 1 1.0 0.000
G4_WS 15 76 4 2.2 0.974 0.017 0.520 0.968 1.6 5 2 1.7 0.342
G4_YL 15 48 2 1.6 0.517 0.010 0.302 0.905 5.0 9 2 1.6 0.284
G5:NC 86 169 8 8.1 1.633 0.009 0.730 0.989 0.6 11 2 2.2 0.598
G5_DY 15 68 3 2.2 0.934 0.018 0.526 0.973 1.4 9 2 2.0 0.518
G5_JZ 15 44 2 1.9 0.674 0.018 0.455 0.930 3.6 10 1 1.4 0.235
G5_QH 15 49 2 1.4 0.403 0.010 0.212 0.975 1.3 7 1 1.4 0.157
G5_WQ 15 81 4 2.3 1.072 0.003 0.555 0.995 0.3 7 2 1.8 0.500
G5_XH 15 48 2 1.7 0.603 0.000 0.367 1.000 0.0 9 2 1.5 0.276
G5_YT 11 23 1 1.1 0.059 0.005 0.037 0.651 21.1 8 1 1.0 0.000
G6:NEC 89 232 12 10.7 1.932 0.035 0.802 0.956 2.2 17 3 3.4 0.878
G6_HL 15 101 5 2.6 1.303 0.037 0.658 0.946 2.8 5 2 1.8 0.411
G6_JH 15 108 5 2.7 1.405 0.082 0.694 0.882 6.3 9 2 2.0 0.434
G6_KS 15 61 3 2.3 0.979 0.003 0.582 0.995 0.3 11 2 1.8 0.474
G6_LX 15 68 3 2.2 0.930 0.028 0.520 0.958 2.2 9 2 2.1 0.571
G6_QQ 15 78 4 2.1 0.946 0.003 0.504 0.995 0.3 11 2 1.7 0.283
G6_SY 14 26 1 1.1 0.118 0.061 0.076 0.202 66.4 9 1 1.2 0.120
G7:Japan 70 157 8 7.6 1.651 0.023 0.759 0.969 1.6 10 2 2.0 0.372
G7_J1 15 63 3 2.2 0.978 0.021 0.563 0.964 1.8 23 2 1.6 0.343
G7_J2 15 23 1 1.0 0.034 0.007 0.018 0.310 52.7 8 1 1.0 0.000
G7_J3 15 24 1 1.1 0.100 0.003 0.064 0.957 2.2 5 1 1.0 0.000
G7_J4 15 64 3 2.0 0.812 0.034 0.467 0.936 3.3 5 1 1.0 0.000
G7_J5 10 57 3 2.0 0.773 0.069 0.450 0.861 7.5 5 1 1.2 0.065
G8:Korea 53 204 10 10.0 1.774 0.039 0.770 0.949 2.6 23 5 4.6 0.932
G8_K1 10 30 2 1.2 0.163 0.000 0.090 1.000 0.0 5 1 1.0 0.000
G8_K2 12 25 1 1.0 0.049 0.013 0.023 0.345 48.7 5 1 1.0 0.000
G8_K3 10 34 2 1.3 0.259 0.000 0.153 1.000 0.0 5 1 1.0 0.000
G8_K4 12 125 6 2.8 1.513 0.092 0.708 0.872 6.8 16 3 2.9 0.776
G8_K5 7 90 5 2.8 1.360 0.100 0.711 0.860 7.5 16 3 3.2 0.908
Mean 14 65 3 1.9 0.793 0.031 0.426 0.913 8.1 9 2 1.6 0.297
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N: number of samples; A: number of alleles; AR: allele richness; Na: number of different alleles; I: Shannon’s information index; HE: expected heterozygosity; HO: observed heterozygosity; FIS: fixation index; t: outcrossing rate.

All cpSSR loci showed relatively low diversity, the mean allele richness (AR) and Shannon’s information index (I) for cpSSRs were 1.6 (1–3.2) and 0.793 (0–0.908). CpSSRs indicated that Korea has the highest allelic richness (AR = 4.6) and Shannon’s information index (I = 0.932) among all regions.

For nSSRs, CONTRIB revealed no difference in regional contribution to total diversity. However, for allelic richness, the highest contribution was made by the SWC region, followed by the regions MDRY and NEC, mainly due to their high own diversity. The lowest contributions came from regions NWC and Japan. For cpSSRs, the SWC region made the greatest contribution to total diversity and allelic richness due to both diversity and differentiation. Besides, the regions of NEC, Korea and Japan made high contributions to allelic richness due to differentiation (Figure 2).

Figure 2.

Figure 2

Figure 2

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Region contribution to the total diversity and allelic richness, (a) and (b) for nSSRs; (c) and (d) for cpSSRs. (a) Contribution to total diversity (CT); (b) Contribution to allelic richness (CTR); (c) Contribution to total diversity (CT); (d) Contribution to allelic richness (CTR).

2.2. Population Structure

MCMC structure reconstruction of nSSRs showed moderate genetic structure. When Evanno’s [29] ad hoc estimator of the actual number of clusters was used, ΔK indicated modes at K = 2 (Figure 3a). The average percentages of membership for eight geographical regions of individuals in each of the two clusters were calculated. Most samples (>66%) of the group SC, MDRY, SWC, NEC, Japan and Korea were assigned to cluster 1, and most individuals of group NEC (79.4%) and NC (86.5%) to cluster 2 (Table 2, Figure 3b). No geographic structure was detected for cpSSRs. The UPGMA dendrogram of both nSSRs and cpSSRs divided the eight regions into the same two geographical clusters (Figure 4).

Figure 3.

Figure 3

Figure 3

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Inferred population structure based on 604 samples and 20 nSSRs. (a) DeltaK from STRUCTURE; (b) Genetic structure of wild soybean inferred from the admixture model.

Table 2.

Inferred population structure based on 604 samples and 20 nSSRs.

Regions No. of sample Cluster 1 Cluster 2
G1: SC 73 0.729 0.271
G2: MDRY 75 0.771 0.229
G3: SWC 83 0.669 0.331
G4: NWC 75 0.206 0.794
G5: NC 86 0.135 0.865
G6: NEC 89 0.812 0.188
G7: Japan 70 0.694 0.307
G8: Korea 53 0.772 0.228
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Figure 4.

Figure 4

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The UPGMA tree of wild soybeans from different regions. bootstrap values are indicated at each branch. (a) The cpSSR tree basing on eight groups. a1: UPGMA tree; a2: Neighbor-joining tree; (b) The nSSR tree basing on eight groups. b1: UPGMA tree; b2: Neighbor-joining tree.

Analysis of nSSRs by AMOVA revealed that 6.0% of genetic variation was due to the genetic distance between the two clusters, 46.7% among populations within clusters and 47.3% between individuals within populations. Similar results were obtained from cpSSRs (6.8%, 57.0% and 36.25%, respectively) (Table 3). A mantel test indicated a significant isolation by distance for cpSSRs (r2 = 0.021, p = 0.002), but not for nSSRs (r2 = 0.004, p = 0.074).

Table 3.

Analysis of molecular variance (AMOVA) for wild soybean.

Loci Source of variation SS VC PV (%) Fixation indices
nSSR Among two clusters 393.04 0.565 5.99 FCT = 0.060
Among populations within clusters 5106.23 4.409 46.69 FST = 0.527
Within populations 5050.64 4.469 47.32 FSC = 0.497
cpSSR Among two clusters 68.562 0.095 6.77 FCT = 0.068
Among populations within clusters 935.881 0.802 56.98 FST = 0.637
Within populations 589.753 0.510 36.25 FSC = 0.611
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The allele size permutation test rendered non-significant differences between FST and RST estimates (p = 0.004 for nSSRs and p = 0.01 for cpSSRs; 10,000 iterations), indicating RST estimates were more appropriate than RST for our data. We found high population genetic differentiation (RST) (cpSSRs: 0.499 and nSSRs: 0.622). For cpSSRs, the overall level of inferred gene flow (Nm) was 0.502 individuals per generation among the populations; and for nSSRs, the gene flow (Nm) was 0.251.

2.3. Demographic History

Standardized differences test and Wilcoxon sign-rank test based on both SMM and TPM model showed recent reduction in seven populations: Chengkou (CK), Wuchang (WC), Tongbai (TB), Keshan (KS), Jizhou (JZ), Japan1 (J1) and Korea5 (K5). A recent bottleneck effect was also detected in three additional populations of Wuqing (WQ), Jiaohe (JH) and Japan 5 (J5) using TPM model by the Wilcoxon sign-rank test (Table 4). The mode-shift test in allele frequency attributed L-shaped distribution to all populations, which was consistent with normal frequency class distribution ranges (p > 0.05).

Table 4.

Results from bottleneck tests of nSSRs: Significance of both tests is indicated in bold.

Populations. Standardized difference test Wilcoxm sign test
TPM SMM TPM SMM
T2 P T2 P P P
G1_AF −2.655 0.0040 −3.014 0.0013 0.9914 0.9959
G1_HY 0.713 0.2381 0.307 0.3794 0.1387 0.3108
G1_JO −0.121 0.4519 −0.303 0.3809 0.5699 0.6282
G1_QZ 0.434 0.3322 0.125 0.4504 0.0715 0.2046
G1_RY −4.223 0.0000 −4.457 0.0000 1.0000 1.0000
G2_DQ 1.498 0.0670 1.288 0.0989 0.0521 0.0668
G2_SC 0.967 0.1668 0.420 0.3374 0.1081 0.2729
G2_TB 3.360 0.0004 2.991 0.0014 0.0004 0.0004
G2_WC 2.651 0.0040 2.235 0.0127 0.0007 0.0018
G2_XU 0.854 0.1965 0.308 0.3791 0.1841 0.4492
G3_CK 2.755 0.0029 2.577 0.0050 0.0014 0.0027
G3_CY 0.146 0.4421 −0.018 0.4929 0.5898 0.5898
G3_GH −0.211 0.4165 −0.451 0.3260 0.5235 0.6603
G3_N1 −1.945 0.0259 −2.236 0.0127 0.9893 0.9964
G3_N2 0.435 0.3317 0.122 0.4514 0.7387 0.2899
G3_YJ 1.164 0.1223 0.923 0.1780 0.0770 0.0982
G4_BX −1.439 0.0751 −2.067 0.0194 0.7793 0.8533
G4_HX −0.109 0.4567 −0.624 0.2665 0.4159 0.6802
G4_LW −0.069 0.4726 −0.201 0.4203 0.4816 0.4816
G4_WS −0.194 0.4233 −0.643 0.2602 0.2450 0.2839
G4_YL −0.740 0.2295 −0.965 0.1673 0.7378 0.7378
G5_DY 0.919 0.1790 0.557 0.2886 0.0844 0.1127
G5_JZ 4.755 0.0000 4.642 0.0000 0.0000 0.0000
G5_QH −5.224 0.0000 −5.543 0.0000 1.0000 1.0000
G5_WQ 1.411 0.0791 1.071 0.1421 0.0407 0.0649
G5_XH 1.371 0.0851 1.206 0.1139 0.0523 0.0523
G5_YT −0.485 0.3140 −0.512 0.3043 0.8125 0.8750
G6_HL −0.230 0.4089 −0.844 0.1993 0.5218 0.8529
G6_JH 1.447 0.0739 0.977 0.1642 0.0181 0.0570
G6_KS 4.213 0.0000 4.054 0.0000 0.0000 0.0000
G6_LX 0.935 0.1749 0.648 0.2584 0.0978 0.2090
G6_QQ −3.357 0.0004 −4.032 0.0000 0.9976 0.9994
G6_SY −0.227 0.4102 −0.378 0.3528 0.4375 0.4375
G7_J1 3.089 0.0010 2.903 0.0019 0.0001 0.0001
G7_J2 −1.720 0.0428 −1.768 0.0385 1.0000 1.0000
G7_J3 0.979 0.1637 0.910 0.1815 0.0625 0.0625
G7_J4 −0.924 0.1777 −1.336 0.0907 0.5938 0.7392
G7_J5 1.356 0.0875 1.110 0.1335 0.0327 0.0523
G8_K1 −2.512 0.0060 −2.579 0.0050 1.0000 1.0000
G8_K2 −3.161 0.0008 −3.282 0.0005 1.0000 1.0000
G8_K3 −1.969 0.0245 −2.058 0.0198 0.9480 0.9710
G8_K4 −0.755 0.2253 −1.373 0.0848 0.6079 0.7848
G8_K5 3.511 0.0002 3.299 0.0005 0.0004 0.0004
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SS: sum of squares; VC: variance component; PV: percentage of variation;

*

p < 0.001; FCT: genetic diversity between two clusters; FSC: differentiation among populations within clusters; FST: divergence among all populations. Significant for both tests are in bold.

3. Discussion

3.1. Genetic Diversity in Wild Soybean

The genetic diversity of wild soybean was studied previously using SSRs [21,25,26,30,31]. However, this is the first time a study uses both nuclear and plastid SSRs to analyze the extent and structure of genetic variation across the whole species range. Wild soybean showed a relatively high population diversity (HE = 0.426), which is similar to the result from previous studies [31,32], Considering life form and breeding system have a highly significant influence on genetic diversity [33], we compared genetic diversity of wild soybean with other predominantly self-pollinated wild species, such as wild emmer (Triticum turgidum ssp. dicoccoides) (HE = 0.19) [34], wild barley (Hordeum spontaneum) (HE = 0.138) [35], and officinal wild rice (Oryza officinalis) (HE = 0.22) [36]. This may be caused by the special seed dispersion of the wild soybean, the pod dehiscence could discharge the mature seeds to a distance of 0–5 m (up to 6.5 m) [22]. High outcrossing rate for certain populations maybe another reason for high genetic diversity in wild soybean.

The nSSRs showed that MDRY region has the highest diversity, which is consistent with several previous studies. For example, Shimamoto et al. [13] reported the highest diversity in the Yangtze River region using RFLP markers. Southern China (including regions of MDRY, SWC and SC) was proposed as the wild soybean center of genetic diversity in a study by Wen et al. [37] using a combination of SSRs and morphological traits. The same region was also pointed as origin and center of diversity using SSR markers and nucleotide sequences in a study by Guo et al. [25]. Compared with previous results, our study applied more detailed regional division, and the center of diversity was similar, but not as obviously different than in previous studies.

Compared with nSSRs (AR = 1.9; I = 1.794), the cpSSRs showed less diversity (AR = 1.6; I = 0.932), which is congruent with Powell et al. [38], who used both nSSRs and cpSSRs of wild soybean samples from a germplasm bank. Similar results have been observed in other studies using both types of SSR markers in other species [39–41]. This is consistent with low substitution rate of plant chloroplast cpDNA sequences compared with nDNA [42]. The cpSSRs could offer unique insights into ecological and evolutionary processes in wild plant species in some situation [43], Differing from that of nSSRs, cpSSRs revealed that Korea has the highest wild soybean genetic variation.

3.2. Genetic Structure of Wild Soybean

Breeding system, life form, effective population size, genetic drift and gene flow are the major evolutionary effects on population genetic structure, with the effect of breeding system being the predominant one [44,45]. Populations of self-fertilizing species are expected to have lower allelic diversity, lower levels of heterozygosity, and high differentiation among populations than populations from outbreeding species [45]. Here, both nSSRs and cpSSRs showed high inter-population genetic differentiation and low gene flow, as expected in the predominantly selfing wild soybean, combined with low seed and pollen dispersal ability. The seed dispersal distance of wild soybean is short, and 95%, 99%, and 99.9% of the produced seeds disperse within 3.5, 5.0, and 6.5 m, respectively after natural pod dehiscence [22], and nearly 81.4% of the loci were found to be positively correlated in the first two distance classes (0–10 m) [6]. Low pollen dispersal ability can be surmised from the estimates of outcrossing rate in wild soybean, which varied from 2.3% (range 2.4%–3.0%) [46] to 13% (range 9.3%–19%) [47] using allozymes and 3.4% (range 0%–37.4%) applying nSSRs [21]. We found a higher mean outcrossing rate (8.1%), with extremely high values in some populations (G5_YT: 21.1%; G6_SY: 66.4%; G7_J2:52.7%; G8_K2: 48.7%). Despite high selfing rates, occasional outcrossing rate can be subsequent. Occasional high outcrossing was detected in other predominant self-pollinated species such as wild barley (t = 25.1%) [48]. The high outcrossing rate in some populations of a predominantly selfing species can be a consequence of rare or sporadically occurring specific environmental conditions (temperature, humidity, wind, insect pollination, etc.) [48]. In this study, the populations with high outcrossing rate were found in different habitats from all eight eco-regions, and could not be ascribed to a particular abiotic environmental factor, which can suggest an importance of some biotic factor such as high pollinator visiting activity [6], more studies should be carried out to fully resolve this issue.

The UPGMA and Neighbor-joining dendrogram based on Nei’s genetic distance and assignment test revealed two clusters of wild soybean in both nSSRs and cpSSRs. One cluster was formed by the NC and NWC regions, and the other one was formed by six geographic regions including NEC, SWC, SC, MDRY, Korea and Japan. The absence of differentiation among East China, Southern Japan and the Korean Peninsula (CJK region) is surprising. Fluctuations in sea level among the CJK region throughout the Quaternary (or even in the mid-late Tertiary) provided abundant opportunities for population fragmentation and allopatric speciation at the CJK region. Applying nDNA and cpDNA sequences, the previous phylogeographic studies on Croomia japonica [49], Kirengeshoma palmata [50], and Platycrater arguta [51] suggested deep allopatric-vicariant differentiation of disjunct lineages in the CJK region [52]. Wild soybean might have seen a continuous distribution throughout the CJK region through the exposed East China Sea (ECS) basin when the sea level fell by 85-130/140 meters during Last Glacial Maximum (LGM; 24,000–18,000 years before present) [53,54], the disjunct distribution among this region formed following the submergence of ECS land bridge, and there may be insufficient time for lineage sorting and differentiation. Wild soybean has salt resistance [55], and could grow easily in the salty conditions of a sea shore hence they have more chance to migrate along the land bridge among the CJK regions during glacial periods. We could not totally exclude the possibility of exchange of wild soybean among the CJK region via long distance dispersal due to disappear of the ECS land bridge. However, it is just a speculation and will need further studies.

3.3. Conservation Implications

In this study, a bottleneck effect was detected in seven populations: Chengkou (CK), Wuchang (WC), Tongbai (TB), Keshan (KS), Jizhou (JZ), Japan 1 (J1) and Korea 5 (K5). The CK and JZ populations are from undisturbed habitats with very small population sizes, while another five populations are situated in disturbed habitats: populations WC, J1 and K5 are along roadsides; population AF is beside an abandoned railway; populations KS and TB are along the ridge of some fields. Population KS is a relic from a larger population predating farming reclamation, and only limited individuals are left. In brief, the five populations were significantly affected by anthropological activities. Wild soybean can adapt to a wide variety of habitats with adequate water. However population size of wild soybean will rapidly decrease in the habitat, with subsequent degradation of genetic diversity and allelic richness. Conversation of wild soybean is therefore a priority, and should focus on regions already affected genetically.

When selecting conservation sites one must also consider a population’s contribution to total diversity and allelic richness. The SWC region was inferred to have greatest contribution to total diversity and allelic richness with both nSSRs and cpSSRs. Wild soybean in this region shows an unusually small population size, combined with a fragmented distribution: several populations from Ninglang county of Yunnan province and Chayu county of Xizang province are separated from the main populations by as much as 400 km. Furthermore, both previous ex situ and in situ conservation initiatives have paid little attention to this region, and only dozens (from a total of 6172) of wild soybean seed accessions from this region have been collected and stored in the Chinese Crop Germplasm Resources databank (http://icgr.caas.net.cn/cgrisintroduction.html). This region deserves high conservation priority.

4. Experimental Section

4.1. Samples Collection, DNA Extraction and Microsatellite Genotyping

A total of 604 wild soybean individual leaf samples were obtained from 43 populations across most of the species distribution (Figure 1). Five populations represented two countries, Korea and Japan, and 5 to 6 populations represented each of six regions of China (Table 5). Total genomic DNA was extracted from silica gel-dried leaves using the CTAB method of Doyle and Doyle [56]. The extracted DNA was resuspended in 0.1× TE buffer (10 mmol/L Tris-HCl, PH 8.0, 1 mmol/L EDTA) to a final concentration of 50–100 ng/μL.

Table 5.

Locations and habitats of sampled wild soybean populations.

Geographical region Population name Location of sampling Longitude Latitude Altitude (m) Habitat
G1: SC Population AF Anfu county, Jiangxi province 27.388 114.602 85 Beside road
Population JO Jianou county, Fujian province 26.962 112.153 126 Beside river
Population HY Hengyang county, Hunan province 27.024 118.293 123 Beside river
Population RY Ruyuan county, Guangdong province 25.872 110.862 510 Beside road
Population QZ Quanzhou county, Guangxi province 24.919 113.136 722 Beside road
G2: MDYR Population WC Wuchang district, Hubei province 30.549 119.972 15 Beside road
Population XU Xuanwu district, Jiangsu province 31.314 117.128 Waste land
Population DQ Duqing county, Zhejiang province 32.370 113.400 15 Beside canal
Population SC Shucha county, Anhui province 30.521 114.395 45 Beside road
Population TB Tongbai county, Henan province 32.045 118.861 33 Beside road
G3: SWC Population CK Chengkou county, Chongqing 31.983 108.667 805 Valley
Population YJ Yinjiang county, Guizhou province 30.996 104.349 458 Valley,
Population GH Guanghan city, Sichuan province 28.000 108.406 458 Beside river
Population CY Chayu county, Xizang province 28.600 97.400 1685 Unknown
Population NL1 Ninglang county, Yunnan province 27.455 100.758 2600 Beside filed
Population NL2 Ninglang county, Yunnan province 27.340 100.954 2550 Beside filed
G4: NWC Population BX Bingxian county, Shaanxi province 35.040 108.077 835 Valley,
Population HX Huixian county, Gansu province 33.893 105.826 1126 Canal
Population LW Lingwu county, Ningxia province 38.146 106.326 1103 Canal
Population WS Wenshui county, Shanxi province 37.417 112.017 759 Beside canal
Population YL Yulin city, Shanxi province 38.281 109.738 1051 Along river
G5: NC Population JZ Jizhou county, Hebei province 37.574 118.524 23 Beside road
Population DY Dongying city, Shandong province 37.742 115.686 6 Beside ditches
Population WQ Wuqing district, Tianjing 39.808 119.432 −6 Beside ditches
Population XH Xuanhua county, Hebei province 39.449 117.249 601 Beside river
Population QH Qinghuangdao city, Hebei province 40.593 115.021 18 Beside river
Population YT Yantai city, Shandong province 37.485 121.453 10 Wasteland
G6: NEC Population LX Lanxi county, Heilongjiang province 41.893 123.411 139 Beside pond
Population JH Jiaohe county, Jinlin province 45.849 132.762 126 Beside river
Population KS Keshan county, Heilongjaing province 43.808 127.237 325 Aside field
Population QQ Qiqihaer city, Heilongjiang province 48.283 125.498 304 Beside river
Population HL Hulin city, Heilongjiang province 46.218 126.338 73 Beside filed
Population SY Shenyang, Liaoning province 47.341 123.940 wasteland
G7: Japan Population J1 Kanagawa, Japan 34.960 137.160 12 Wet Land
Population J2 Tokyo, Japan 34.828 135.770 35 Wet Land
Population J3 Hirakata, Osaka, Japan 34.810 135.480 11 Wet Land
Population J4 Okazaki, Japan 34.959 137.139 37 Wet Land
Population J5 Kyushu University, Fukuoka, Japan 33.597 130.215 Unknown
Population K1 Gangwon-do, South Korea 37.625 128.492 520 Wet Land
Population K2 Gangwon-do, South, Korea 38.031 128.639 340 Wet Land
Population K3 Incheon, South Korea 37.533 126.497 11 Wet Land
Population K4 Yeongcheon-si city, Korea 36.113 128.982 102 Along road
Population K5 Moonkyeong-si city, Korea 36.721 128.358 77 Along road
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G1: SC, south China; G2: MDYR, Middle and lower reaches of Yangtze River; G3: SWC, southwestern China; G4: NWC, northwestern China; G5: NC, north China; G6: NEC, northeastern China.

Genotyping was performed using 20 nSSRs representing all 20 wild soybean linkage groups corresponding to the 20 chromosomes, and five cpSSRs from intergenic regions. All the 25 loci are polymorphic and have been used in previous studies [15,21,57] (Table 6). PCR reactions were performed in 15 μL reactions containing 30–50 ng genomic DNA, 0.6 μM of each primer, 7.5 μL 2× Taq PCR MasterMix (Tiangen Biotech, Beijing, China). PCR amplifications were conducted under the following conditions: 94 °C for 2 min; 35 cycles at 94 °C for 30 s, 50 °C for 40 s, and 72 °C for 1 min; followed by a final extension step at 72 °C for 7 min. Primers are shown in Table 6. All the SSR markers were polymorphic based on electrophoresis performed on an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Fragment length sizes were scored automatically using the program GeneMapper (Applied Biosystems).

Table 6.

Characteristics of the 25 microsatellite loci for wild soybean. Forward (F) and reverse (R) primer sequences, repeat motifs, size range and linkage group are given.

Primer name Primer sequence (5′ to 3′) Repeat motif Size range linkage group
gmcp1 F:TCGATTCTATGCCCCTACTT
R:AGACTCCCAAGTTTTCAGTCG		 (T)12 124–126 TrnT/trnL
gmcp3 F:GCTTCAGAATTGTCCTATTTA
R:ATCAAATAACGCCTCATCTA		 (A)12CG(T)11 103–113 TrnT/trnL
gmcp4 F:TATCACTGTCAAGATTAAGAG
R:CTTTTATATGTATGGCGCAAC		 (A)11 127–136 atpB/rbcL
RD19 F:CTAAATATTACAAAATGGAATTCT
R:ACCAATTCAAAAAATCGAATA		 (A)14 149–151 rps19
SOYCP F:CATAGATAGGTACCATCCTTTTT
R:CGCCGTATGAAAGCAATAC		 (T)13(G)10 90–98 trnM
Satt126 F:ATAAAACAAATTCGCTGATAT
R:GCTTGGTAGCTGTAGGAA		 (ATT)18 109–172 B2
Satt135 F:TTCCAATACCTCCCAACTAAC
R:CACGGATTTTAAATCATTATTACAT		 (ATT)19 141–204 D2
Satt215 F:GCGCCTTCTTCTGCTAAATCA
R:CCCATTCAATTGAGATCCAAAATTAC		 (ATT)11 114–221 J
Satt216 F:TACCCTTAATCACCGGACAA
R:AGGGAACTAACACATTTAATCATCA		 (ATT)20 137–251 D1b
Satt221 F:GCGGCAAACCATTATCTTCATT
R:GCGATTGTACCACTAAAAACCATAG		 (ATT)23 109–224 D1a
Satt231 F:GGCACGAATCAACATCAAAACTTC
R:GCGTGTGCAAAATGTTCATCATCT		 (ATT)32 160–328 E
Satt233 F:AAGCATACTCGTCGTAAC
R:GCGGTGCAAAGATATTAGAAA		 (ATT)16 169–238 A2
Satt270 F:TGTGATGCCCCTTTTCT
R:GCGCAGTGCATGGTTTTCTCA		 (ATT)16 183–249 I
satt277 F:GGTGGTGGCGGGTTACTATTACT
R:CCACGCTTCAGTTGATTCTTACA		 (ATT)40 128–255 C2
satt288 F:GCGGGGTGATTTAGTGTTTGACACCT
R:GCGCTTATAATTAAGAGCAAAAGAAG		 (ATT)17 195–273 G
Satt294 F:GCGCTCAGTGTGAAAGTTGTTTCTAT
R:GCGGGTCAAATGCAAATTATTTTT		 (ATT)23 237–303 C1
Satt373 F:TCCGCGAGATAAATTCGTAAAAT
R:GGCCAGATACCCAAGTTGTACTTGT		 (TAT)21 210–279 L
Satt423 F:TTCGCTTGGGTTCAGTTACTT
R:GTTGGGGAATTAAAAAAATG		 (ATT)19 225–351 F
Satt463 F:CTGCAAATTTGATGCACATGTGTCTA
R:TTGGATCTCATATTCAAACTTTCAAG		 (ATT)19 100–214 M
satt509 F:GCGCAAGTGGCCAGCTCATCTATT
R:GCGCTACCGTGTGGTGGTGTGCTACCT		 (ATT)30 119–242 B1
Satt530 F:CCAAGCGGGTGAAGAGGTTTTT
R:CATGCATATTGACTTCATTATT		 (ATT)12 201–279 N
satt555 F:GCGGTTGGCTTTGATGATGT
R:TTACCGCATGTTCTTGGACTA		 (ATT)13 234–312 K
Satt568 F:CGGACACCGGTCTACTAGGAAAGTAA
R:GCGGAATAATCCAATTCAATTTA		 (ATT)17 212–275 H
satt572 F:GCGGAGCATGTAAATCCAGCCTATTGA
R:GCGGGCTAACTTATGTTACTAAACAAT		 (ATT)14 130–241 A1
satt581 F:CCAAAGCTGAGCAGCTGATAACT
R:CCCTCACTCCTAGATTATTTGTTGT		 (ATT)11 130–196 O
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4.2. Microsatellite Validation and Diversity

Microsatellite data from each population was tested for amplification errors and null alleles, large allele dropout or stuttering using 1000 randomizations in MICROCHECKER v.2.2.3 [58]. Genepop v. 3.4 online [59] was used to check for deviation from Hardy-Weinberg expectations and between loci in each population using exact tests with 10,000 dememorizations, 100 batches and 1000 iterations. Significance level was adjusted using the sequential Bonferroni correction for multiple comparisons [60]. For nSSRs, the number of alleles per locus (A), the numbers of different alleles (Na), the observed heterozygosities (HO), expected heterozygosities (HE), fixation index (FIS) and Shannon’s information index (I) were calculated using GenALEx v. 6.4 [61]; allelic richness (AR) was calculated by FSTAT 2.9.3.2 [62]; outcrossing rate (t) was calculated from the fixation index using the equation t = (1 − FIS)/(1 + FIS) [63]. For cpSSR, the number of alleles per locus (A), the numbers of different alleles (Na) and Shannon’s information index (I) were calculated using GenALEx v. 6.4 [61]. In each individual, genetic variants at all cpSSR and nSSR sites were combined into haplotypes. Then, each region was characterized for its plastid DNA diversity using the number of haplotypes detected and gene diversity estimated using the program CONTRIB [64]. Contribution of each region to total diversity (CT) and to total allelic richness (CTR) were calculated according to Petit et al. [65].

4.3. Population Spatial Structure

Genetic differentiation was investigated using the model based clustering method STRUCTURE 2.1 [66,67] for nSSRs. Burn-in time and replication number were set to 100,000 and 100,000 (further generation following the burn in) for each run, respectively. The number of populations (K) in the model was systematically varied from 1 to 10. In order to decrease the margin of error, the average value of 20 simulations performed for each K was used. We used the ΔK method [29] representing the highest median likelihood values to assign wild soybean accessions using the online tool Structure Harvester [68]. For the chosen K value, the run that had the highest likelihood estimate was adopted to assign individuals to clusters. The 10 runs with the lowest DI values for the selected K-value were retained, and their admixture estimates were averaged using CLUMPP v. 1.1.1 [69], applying the greedy algorithm with random input order and 1,000 permutations to align the runs and calculate G’ statistics. Results were visualized using DISTRUCT 1.1 [70].

Nei’s genetic distance (D) and Goldstein’s distance [(δμ)2] are commonly used for microsatellite. Considered Goldstein’s distance (δμ)2 showed bias at small sample sizes and the bias was directly related to the number of alleles and range in allele size [71], a dendrogram based on Nei’s (1978) [72] genetic distance (D) between groups was constructed using the UPGMA method implemented in the PHYLIP v. 3.68 [73]. In order to make sure the results of UPGMA method, a neighbor joining tree also was constructed using PHYLIP v. 3.68. A hierarchical analysis of molecular variance (AMOVA) [74] implemented in Arlequin v. 3.11 [75] was used to partition the observed genetic variation into among clusters, among populations within a cluster and among individuals within a population.

Two commonly estimators of population differentiation are FST, based on allele identity, and RST, which incorporates microsatellite-specific mutation models. We used the allele size permutation test in SPAGeDI [76] to test whether allele sizes were informative in wild soybean microsatellite data set, which would indicate that mutatioin has contributed to differentiation. Because RST was shown to be most appropriate for our data, (see results), the global RST across all samples was calculated in Arlequin v. 3.11 [75]. Gene flow were quantified using the approach of transform estimates of RST into indirect estimates of the average number of migrants exchanged per generation among populations (Nm) [77]. Gene differentiation (RST) was calculated using AMOVA analyses based on population levels, gene flow was estimated from RST (nSSRs: Nm = 0.25(1 − RST)/RST; cpSSRs: Nm = 0.5(1 − RST)/RST).

4.4. Demographic History

We assessed demographic history based on microsatellite data using different and complementary methods. Heterozygosity excess test [78] and mode-shift test [79] from BOTTLENECK 1.2.02 [80] were used to detect the recent population bottleneck. This program conducts tests for recent (within the past 2Ne to 4Ne generations) population bottlenecks that severely reduce effective population size (Ne) and produce an excess in heterozygosity. Heterozygosity excess test was performed under two mutation models: stepwise mutation model (SMM) and two-phase mutation model (TPM). The model of TPM include both 95% single-step mutations and 5% multiple-step mutations, as recommended by Piry [80]. Heterozygosity excess was detected using the one-tailed Wilcoxon sigh-rank test and standardized differences test on 20 nSSR loci [80]. Significance was determined also by the standardized differences and Wilcoxon tests. Mode-shift test detects allele frequency to investigate whether allele frequency distort from the expected L-shaped distribution. During a bottleneck, the loss of rare alleles occurs more rapidly than the associated decrease in expected heterozygosity, as rare alleles do not contribute to HE as much as common alleles, and thus distort the allele frequency distribution from its expected L-shaped distribution [78].

5. Conclusions

In summary, our results show a relatively high level of genetic diversity and genetic differentiation in wild soybean. Two major genetic clusters were revealed by both structure and phylogenetic reconstruction. The MDRY and Korea regions contain the highest genetic diversity, and SWC contributes the most to total diversity and allelic richness. Significant genetic bottlenecks have affected five populations with obvious human disturbance. Based on these results, conversation of wild soybean should reduce habitat loss by human interference, and the SWC region should be conserved with priority.

Acknowledgements

This study was supported by grants from National Natural Science Foundation of China (project No. is Y01C541211), and the grant of the Talent Project of Yunnan Province (grant No. 2011CI042). This study was conducted in the Key Laboratory of the Southwest China Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences.

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