Population structure of the wild soybean (Glycine soja) in China: implications from microsatellite analyses

Background and Aims Wild soybean (Glycine soja), a native species of East Asia, is the closest wild relative of the cultivated soybean (G. max) and supplies valuable genetic resources for cultivar breeding. Analyses of the genetic variation and population structure of wild soybean are fundamental for effective conservation studies and utilization of this valuable genetic resource. Methods In this study, 40 wild soybean populations from China were genotyped with 20 microsatellites to investigate the natural population structure and genetic diversity. These results were integrated with previous microsatellite analyses for 231 representative individuals from East Asia to investigate the genetic relationships of wild soybeans from China. Key Results Analysis of molecular variance (AMOVA) revealed that 43·92 % of the molecular variance occurred within populations, although relatively low genetic diversity was detected for natural wild soybean populations. Most of the populations exhibited significant effects of a genetic bottleneck. Principal co-ordinate analysis, construction of a Neighbor-Joining tree and Bayesian clustering indicated two main genotypic clusters of wild soybean from China. The wild soybean populations, which are distributed in north-east and south China, separated by the Huang-Huai Valley, displayed similar genotypes, whereas those populations from the Huang-Huai Valley were different. Conclusions The previously unknown population structure of the natural populations of wild soybean distributed throughout China was determined. Two evolutionarily significant units were defined and further analysed by combining genetic diversity and structure analyses from Chinese populations with representative samples from Eastern Asia. The study suggests that during the glacial period there may have been an expansion route between south-east and north-east China, via the temperate forests in the East China Sea Land Bridge, which resulted in similar genotypes of wild soybean populations from these regions. Genetic diversity and bottleneck analysis supports that both extensive collection of germplasm resources and habitat management strategies should be undertaken for effective conservation studies of these important wild soybean resources.     

Sampling strategy for wild soybean (Glycine soja) populations based on their genetic diversity and fine-scale spatial genetic structure

A total of 892 individuals sampled from a wild soybean population in a natural reserve near the Yellow River estuary located in Kenli of Shandong Province (China) were investigated.Seventeen SSR (simple sequence repeat) primer pairs from cultivated soybeans were used to estimate the genetic diversity of the population and its variation pattern versus changes of the sample size (sub-samples),in addition to investigating the fine-scale spatial genetic structure within the population.The results showed relatively high genetic diversity of the population with the mean value of allele number (A) being 2.88,expected heterozygosity (He) 0.431,Shannon diversity index (/) 0.699,and percentage of polymorphic loci (P) 100%.Sub-samples of different sizes (ten groups) were randomly drawn from the population and their genetic diversity was calculated by computer simulation.The regression model of the four diversity indexes with the change of sample sizes was computed.As a result,27-52 individuals can reach 95% of total genetic variability of the population.Spatial autocorrelation analysis revealed that the genetic patch size of this wild soybean population is about 18 m.The study provided a scientific basis for the sampling strategy of wild soybean populations.     

中国野生大豆(Glycine soja)遗传资源主要形态、遗传变异和结构

介绍了我国野生大豆遗传资源主要的形态类型、物种内遗传关系和遗传结构。进化的瓶颈不仅发生在由野生大豆到栽培大豆,也以另一种方式"分化瓶颈"出现于同性状的不同表型类型之间。野生大豆种内种子大小类型是否存在遗传分化?野生和半野生大豆的边界在哪?半野生大豆如何产生的?半野生大豆遗传上密切于栽培种还是野生种?百粒重3～4g的小粒半野生大豆与百粒重8.5g以上的特大粒半野生大豆是否有遗传差异?百粒重8.5g以上的特大粒半野生大豆是否属于栽培大豆?野生大豆的种皮色和种子大小哪个更能反映进化程度?栽培大豆基因是否已经渗入到野生大豆?对这些在学术界常年存在的疑问本文介绍了我们的研究答案。我们认为"真"半野生大豆不存在于现在中国半野生资源收集品中;一些野生大豆中的白花、灰毛、无泥膜性状来源于栽培大豆的基因渗透。     

Genetic diversity and population structure of Korean wild soybean (Glycine soja Sieb. and Zucc.) inferred from microsatellite markers

Korea is considered one of the centers of genetic diversity for cultivated as well as wild soybeans. Natural habitats of wild soybeans are distributed across the Korean mainland and the islands surrounding the Korean peninsula. In this study, the genetic diversity of 100 mainland Korean wild soybean accessions was evaluated by using 42 simple sequence repeat markers covering 17 soybean chromosomes. All analyzed loci were polymorphic and a total of 114 alleles were found. The observed average genetic diversity was low (0.4). The results showed that the 100 selected accessions did not exactly follow the geographical distribution. These results were further confirmed by the phylogeny inferred from five morphological characteristics (i.e., leaf shape, leaf area, plant shape, seed area, and 100-seed weight). Together, the genetic and morphological evaluations suggested conclusively that the selected population did not follow the geographical distribution pattern. The present study could provide useful information for the ex situ conservation and exploitation of wild soybean accessions in soybean improvement stratagems, and will aid in further understanding about the phylogeography of the species in the Korean center of diversity.     

Importance of genetic characterization and conservation of plant genetic resources: The breeding system and genetic diversity of wild soybean (Glycine soja)

Abstract Plant genetic resources play an important role in the improvement of cultivated plants. To characterize and evaluate the ecological and reproductive features of wild soybean ( Glycine soja Sieb. et Zucc.), which is the most probable ancestor of cultivated soybean ( G. max (L) Merr.), the breeding system and genetic diversity of G. soja were investigated. The extent of natural cross-pollination of G. soja was estimated in four populations along the Omono River in Akita Prefecture, Japan by examining allozyme variation. Although it has been previously believed that G. soja is autogamous, as is cultivated soybean, the mean multilocus outcrossing rate ( t m) estimate was 13%. These values are much higher than the outcrossing rate previously reported for both G. soja and G. max . Frequent visits by honeybees and carpenter bees to flowers were also observed, which supported this conjecture. Furthermore, to evaluate the genetic variation of G. soja as a genetic resource, the genetic structure of 447 populations over Japan were analyzed. Wild soybean populations had a higher degree of variation of isozyme loci. The G ST coefficient of gene differentian values among the sites within the district were particularly high, revealing that the isozyme genotype was greatly different among site populations and homogeneous within the sites. The genetic differentiation among nine districts was observed in the allele frequencies of a few loci, indicating that geographic isolation in the wild soybean population was effectively created through the distance between the districts. The difference in the allele frequency among the districts may be produced under genetic drift. Finally, the importance of the preservation of natural plant populations and the habitats of wild progenitors (i.e. the in situ conservation of plant genetic resources) was emphasized.     

Environmental versus geographical effects on genomic variation in wild soybean (Glycine soja) across its native range in northeast Asia

A fundamental goal in evolutionary biology is to understand how various evolutionary factors interact to affect the population structure of diverse species, especially those of ecological and/or agricultural importance such as wild soybean (Glycine soja). G. soja, from which domesticated soybeans (Glycine max) were derived, is widely distributed throughout diverse habitats in East Asia (Russia, Japan, Korea, and China). Here, we utilize over 39,000 single nucleotide polymorphisms genotyped in 99 ecotypes of wild soybean sampled across their native geographic range in northeast Asia, to understand population structure and the relative contribution of environment versus geography to population differentiation in this species. A STRUCTURE analysis identified four genetic groups that largely corresponded to the geographic regions of central China, northern China, Korea, and Japan, with high levels of admixture between genetic groups. A canonical correlation and redundancy analysis showed that environmental factors contributed 23.6% to population differentiation, much more than that for geographic factors (6.6%). Precipitation variables largely explained divergence of the groups along longitudinal axes, whereas temperature variables contributed more to latitudinal divergence. This study provides a foundation for further understanding of the genetic basis of climatic adaptation in this ecologically and agriculturally important species.     

Sampling strategy for wild soybean ( Glycine soja ) populations based on their genetic diversity and fine-scale spatial genetic structure

A total of 892 individuals sampled from a wild soybean population in a natural reserve near the Yellow River estuary located in Kenli of Shandong Province (China) were investigated. Seventeen SSR (simple sequence repeat) primer pairs from cultivated soybeans were used to estimate the genetic diversity of the population and its variation pattern versus changes of the sample size (sub-samples), in addition to investigating the fine-scale spatial genetic structure within the population. The results showed relatively high genetic diversity of the population with the mean value of allele number (A) being 2.88, expected heterozygosity (He) 0.431, Shannon diversity index (I) 0.699, and percentage of polymorphic loci (P) 100%. Sub-samples of different sizes (ten groups) were randomly drawn from the population and their genetic diversity was calculated by computer simulation. The regression model of the four diversity indexes with the change of sample sizes was computed. As a result, 27–52 individuals can reach 95% of total genetic variability of the population. Spatial autocorrelation analysis revealed that the genetic patch size of this wild soybean population is about 18 m. The study provided a scientific basis for the sampling strategy of wild soybean populations. __________ Translated from Journal of Fudan University (Natural Science), 2006, 45(3): 322–327 [译自: 复旦学报 (自然科学版)]     

Genetic characterization of a leaf margin necrosis mutant in wild annual soybean (Glycine soja)

A leaf margin necrosis mutant was observed in a wild annual soybean (Glycine soja Sieb. & Zucc.) population from South Korea. Genetic studies showed that it was controlled by a single recessive nuclear gene, designatedlmn. TheLmn locus segregated independently of theAp, Dial, Dia3, Idh2, Pgi1 andTi isozyme loci.     

Fine scale genetic structure in the wild ancestor of maize (Zea mays ssp. parviglumis)

Analysis of fine scale genetic structure in continuous populations of outcrossing plant species has traditionally been limited by the availability of sufficient markers. We used a set of 468 SNPs to characterize fine‐scale genetic structure within and between two dense stands of the wild ancestor of maize, teosinte (Zea mays ssp. parviglumis). Our analyses confirmed that teosinte is highly outcrossing and showed little population structure over short distances. We found that the two populations were clearly genetically differentiated, although the actual level of differentiation was low. Spatial autocorrelation of relatedness was observed within both sites but was somewhat stronger in one of the populations. Using principal component analysis, we found evidence for significant local differentiation in the population with stronger spatial autocorrelation. This differentiation was associated with pronounced shifts in the first two principal components along the field. These shifts corresponded to changes in allele frequencies, potentially due to local topographical features. There was little evidence for selection at individual loci as a contributing factor to differentiation. Our results demonstrate that significant local differentiation may, but need not, co‐occur with spatial autocorrelation of relatedness. The present study represents one of the most detailed analyses of local genetic structure to date and provides a benchmark for future studies dealing with fine scale patterns of genetic diversity in natural plant populations.     

Allelic differentiation of Kunitz trypsin inhibitor in wild soybean (Glycine soja)

Soybean Kunitz trypsin inhibitor (SKTI) has several polymorphic types, which are controlled by co-dominant multiple alleles at a single locus. Of these types, Tia and Tib are predominant types, and there are nine differences in amino acids between Tia and Tib. Recently, an intermediate transitional type (Tib ( i5 )) between them was detected. However, other transitional types have not been detected despite surveys of many cultivated and wild soybeans. One of the reasons why other transitional variants have not been found is inferred to be due to the difficulty of the detection of SKTI protein variants by polyacrylamide gel electrophoresis (PAGE). To detect novel variants of SKTI, nucleotide sequence analysis in addition to PAGE was carried out. Four new variants were found from many Japanese wild soybeans. Of these variants, three (designated as Tia ( a1 ), Tia ( a2 ), Tia ( b1 )) were detected through gene sequence analysis on wild soybeans having the same electrophoretic mobility as Tia, and one (Tig) was detected through PAGE. The Tig variant showed a slightly lower electrophoretic mobility than Tic. The nucleotide sequences of Tig were identical to those of Tib except for one T --> C transitional mutation at position +340. The sequences of Tia ( a1 )and Tia ( a2 ) genes were identical to those of Tia with the exception of a G --> A mutation at position +376 and a T --> C mutation at +404, respectively. The sequence of Tia ( b1 ) differed from Tia by three nucleotides: C --> A at position +331, T --> C at +459 and A --> G at +484. Of the three nucleotide changes, two were common to Tia ( b1 ), Tib ( i5 ) and Tib, suggesting that Tia ( b1 ) is an intermediate transitional type between Tia and Tib. Our results suggest that Tib type has been differentiated through a series of mutations from Tia before the domestication of cultivated soybean.     

Population structure, genetic diversity, and clone formation in Quercus chrysolepis (Fagaceae)

Stands of canyon live oak (Quercus chrysolepis, Fagaceae) are maintained for fuelwood, fire management, recreation, and as habitat for wildlife. Information about the link between the oak's reproductive ecology and its extent of genetic diversity is important in developing land management policies that will maintain the long-term viability of populations. Basal sprouting is the primary means of reproduction following fire or cutting, and stands frequently include groups of visibly connected trees in a clustered distribution that suggests cloning. We determined the extent to which clusters of trees were clonal and defined the spatial pattern and diversity of genotypes for six populations across nearly the entire east-west extent of the San Bernardino Mountains in southern California. We mapped over 100 trees at each of five sites and genotyped each tree for allozymes at seven polymorphic loci. We identified clones using these multilocus genotypes and detected an average of 34.4 ± 7.3 (SD) clones per site, most of which had unique genotypes. In general, clustered trees belong to single clones and most clones consist of few trees (mean = 3.4 ± 0.6 trees per clone). However, clone size increased significantly with increased individual heterozygosity, suggesting that selection may favor highly heterozygous clones. Clonal diversity and evenness were high relative to reports for most other clonal species; an average of 97% of clones had distinct genotypes, and Simpson's index of diversity averaged 0.95 ± 0.02. Population genetic analyses of 319 clones from six sites revealed high genetic diversity within sites (mean HS = 0.443). Only a small proportion of the total genetic diversity was explained by variation among sites (mean GST = 0.018), which is consistent with high gene flow among sites (Nm = 9.5). We found no significant substructure among plots within sites, and fixation indices within sites were generally small, suggesting that either little inbreeding occurs, and/or few inbred progeny survive. However, spatial autocorrelation analysis of clones indicated fine-scale genetic structure at distances under 4 m, possibly due to limited seed dispersal. Our data suggest that guidelines for seed collection of canyon live oak for use in restoration can be specified in a manner similar to that recommended for conifer species within the region studied.     

中国野生大豆的遗传多样性和生态特异性分析

野生大豆（Glycine soja）是栽培大豆的祖先,为东亚特有种,大部分分布在中国。我们采用52对SSR引物和10个植物学性状,以遗传丰富度和Simpson多样性指数为指标,对来自中国3个地理生态区域涉及24个省区的196份野生大豆所构成的代表性样本的遗传变异进行了研究,以期从分子水平和表型水平两个层面上揭示中国野生大豆遗传多样性和地理生态特异性。结果表明：中国野生大豆群体SSR位点的等位基因平均丰富度（NA）和平均Simpson多样性指数（H）分别为16.1和0.852,高于栽培大豆（NA=11.4,H=0.773）,野生群体的遗传多样性明显高于栽培群体。3个地理生态群体中南方群体多样性最高（NA=12.9,H=0.842）,黄淮海群体最低（NA=11.4,H=0.805）,东北群体居中（NA=12.5,H=0.834）。群体间存在遗传分化,不同群体具有不同的特异等位基因,位点AW132402（A2连锁群）、Satt522（F）、satt150（M）、Sat_332（D1a）、Satt046（K）、sct_190（K）等的一些等位基因只在特定群体出现,表现出群体分化后的生态特异性。中国野生大豆植物学性状的群体变异丰富,平均Simpson多样性指数为0.710。地理群体间存在分化,最明显的是生育期性状的分化,反映了地理、光照和温度等生态因子的选择作用,其中南方地理群体多样性最高（H=0.671）。SSR分子标记和植物学性状所获结果相对一致,表明中国野生大豆地理群体间性状分化有其遗传分化的基础。     

The possible origin of thick stem in Chinese wild soybean (Glycine soja)

Thicker, erect stem and enlarged seeds are characteristic of the domestication of cultivated soybeans (Glycine max) from its progenitor, wild soybean (G. soja). Wild soybeans have different stem thicknesses but the thick stem as defined here appears in a small number of small-seeded wild soybeans (≤2.0 g/100-seeds) in China. However, little attention has been paid to this phenomenon in considering the origin of thick stem in wild soybean. Here, we addressed this question through the study of a mixed field of wild, semi-wild and cultivated soybeans. Thick-stemmed samples had lower sensitivity to light period, higher mean genetic diversity (H _e = 0.090, H = 0.535) and higher mean multilocus outcrossing rate (t _m = 9.77 %), while thin-stemmed plants were the opposite (H _e = 0.029, H = 0.416) and lower mean outcrossing rate (t _m = 5.88 %). F statistics calculations indicated that there was genetic differentiation between the thin and thick stems. UPGM cluster analysis showed that not only were thick-stemmed wild soybeans genetically different from thin-stemmed ones, but they were also genetically closer to semi-wild soybean, to varying degrees completely dependent on seed size. These data strongly implied that the plants with thick stems had more complicated genetic backgrounds than the thin-stemmed ones, and that they were related to cultivated soybeans. This study suggests that if plants have distinctly thick stems (an average 2.5-fold thicker than other thin-stemmed plants) or stems similar to semi-wild plants and/or near to local soybeans in a natural wild population adjacent to farmlands, such plants could be cultivar-introgressive offspring.     

Population genetic structure of the Korean threadsail filefish (Stephanolepis cirrhifer) based on microsatellite marker analysis

The threadsail filefish Stephanolepis cirrhifer is one of the most important commercial fish resources in Korea. However, its natural populations have declined drastically over the last several decades. To investigate the genetic characteristics of the threadsail filefish for conservation and management purposes, its genetic variation was analyzed in Korean fishing grounds using 11 microsatellite loci. All populations showed moderate genetic diversity (mean number of alleles (N_A) = 8.6, expected heterozygosity (He) = 0.73), representing a slightly lower level of genetic variability than other congeneric species. The presence of a weak genetic population substructure was detected (F_ST = 0.023, R_ST = 0.030, P < 0.001), but this substructure did not feature significant isolation based on the distance between populations. This differentiation may be primarily attributable to genetic differences between populations from Geomundo and other localities, based on relative low level of gene flow, a high level of pairwise F_ST and R_ST or the position of this population in a phylogenetic tree. An analysis of molecular variance (AMOVA) also revealed a greater part of the variation within the population and genetic differentiation among the samples analyzed and identified two genetic clusters. The possibility of a recent genetic bottleneck was observed in some populations of S. cirrhifer. Given the prevailing ocean currents and the intensity of threadsail filefish harvesting activities in Korea, the possibility that human harvest and stock enhancement activities may have partially contributed to our detected genetic pattern cannot be excluded. Management strategies that take these findings into account might lower the risk of harmful genetic change in the species.     

Fine-scale genetic structure enhances biparental inbreeding by promoting mating events between more related individuals in wild soybean (Glycine soja; Fabaceae) populations

Outcrossing between genetically distant individuals in a plant population enhances allelic heterozygosity-an important source for genetic diversity and adaptive evolution. Fine-scale spatial genetic structure (FSGS) can interfere with outcrossing by promoting mating between more related individuals. To test the influence of FSGS on outcrossing, FSGS and outcrossing rates were analyzed with four wild soybean (Glycine soja) populations from different habitats, using simple sequence repeat (SSR) fingerprints. Spatial autocorrelation analysis indicated variable FSGS (15.44-25.87 m) in all four populations. Multilocus mixed-mating analysis of 1605 progeny indicated substantial variation in single-locus outcrossing (T(s) = 6.3-12.6%) although the total outcrossing rates as estimated by multilocus outcrossing (T(m) = 12.8-17%) did not vary significantly among populations. The comparison between FSGS and outcrossing rates demonstrated that strong FSGS with large genetic patch size can enhance biparental inbreeding by promoting mating between more related individuals in a population. The results suggest that patch size management can aid in situ conservation by avoiding formation of strong FSGS and encouraging true outcrossing among individuals.     

Lack of specific binding of labeledRhizobium japonicum lipopolysaccharides to wild soybean (Glycine soja) roots

Summary Lipopolysaccharides (LPS) were extracted from two strains ofRhizobium japonicum (61A76NS and 3I1b110-I). The extracted LPS was purified by gel filtration column chromatography and the amount of 2-keto-3-deoxyoctonate (KDO) was determined. Column purified LPS from both strains were conjugated to rhodamine isothiocyanate on celite to examine binding of this purified, labeled surface component to aseptically grownGlycine soja (wild soybean) seedlings as a basis for symbiotic specificity using fluorescent microscopy. Rhodamine conjugated LPS from both strains ofRhizobium japonicum did not exhibit specific binding to wild soybean seedling roots.Paper no. 8130 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Extensive spatial genetic structure revealed by AFLP but not SSR molecular markers in the wind-pollinated tree, Fagus sylvatica

Studies of fine-scale spatial genetic structure (SGS) in wind-pollinated trees have shown that SGS is generally weak and extends over relatively short distances (less than 30-40 m) from individual trees. However, recent simulations have shown that detection of SGS is heavily dependent on both the choice of molecular markers and the strategy used to sample the studied population. Published studies may not always have used sufficient markers and/or individuals for the accurate estimation of SGS. To assess the extent of SGS within a population of the wind-pollinated tree Fagus sylvatica, we genotyped 200 trees at six microsatellite or simple sequence repeat (SSR) loci and 250 amplified fragment length polymorphisms (AFLP) and conducted spatial analyses of pairwise kinship coefficients. We re-sampled our data set over individuals and over loci to determine the effect of reducing the sample size and number of loci used for SGS estimation. We found that SGS estimated from AFLP markers extended nearly four times further than has been estimated before using other molecular markers in this species, indicating a persistent effect of restricted gene flow at small spatial scales. However, our SSR-based estimate was in agreement with other published studies. Spatial genetic structure in F. sylvatica and similar wind-pollinated trees may therefore be substantially larger than has been estimated previously. Although 100-150 AFLP loci and 150-200 individuals appear sufficient for adequately estimating SGS in our analysis, 150-200 individuals and six SSR loci may still be too few to provide a good estimation of SGS in this species.     

Comparison of saponin composition and content in wild soybean (Glycine soja Sieb. and Zucc.) before and after germination

Eight wild soybean accessions with different saponin phenotypes were used to examine saponin composition and relative saponin quantity in various tissues of mature seeds and two-week-old seedlings by LC–PDA/MS/MS. Saponin composition and content were varied according to tissues and accessions. The average total saponin concentration in 1?g mature dry seeds of wild soybean was 16.08?±?3.13?μmol. In two-week-old seedlings, produced from 1?g mature seeds, it was 27.94?±?6.52?μmol. Group A saponins were highly concentrated in seed hypocotyl (4.04?±?0.71?μmol). High concentration of DDMP saponins (7.37?±?5.22?μmol) and Sg-6 saponins (2.19?±?0.59?μmol) was found in cotyledonary leaf. In seedlings, the amounts of group A and Sg-6 saponins reduced 2.3- and 1.3-folds, respectively, while DDMP?+?B?+?E saponins increased 2.5-fold than those of mature seeds. Our findings show that the group A and Sg-6 saponins in mature seeds were degraded and/or translocated by germination whereas DDMP saponins were newly synthesized.