Annual Wild Soybean Research Articles

Genome-wide signatures of the geographic expansion and breeding of soybean.

Soybean is a leguminous crop that provides oil and protein. Exploring the genomic signatures of soybean evolution is crucial for breeding varieties with improved adaptability to environmental extremes. We analyzed the genome sequences of 2,214 soybeans and proposed a soybean evolutionary route, i.e., the expansion of annual wild soybean (Glycine soja Sieb. & Zucc.) from southern China and its domestication in central China, followed by the expansion and local breeding selection of its landraces (G. max (L.) Merr.). We observed that the genetic introgression in soybean landraces was mostly derived from sympatric rather than allopatric wild populations during the geographic expansion. Soybean expansion and breeding were accompanied by the positive selection of flowering time genes, including GmSPA3c. Our study sheds light on the evolutionary history of soybean and provides valuable genetic resources for its future breeding.

Genetic dynamics of flowering date evolved from later to earlier in annual wild and cultivated soybean in China

AbstractAt its center of origin, the Chinese soybean germplasm pool (CSGP) consists of annual wild (WA), farmers’ landrace (LR), and released cultivar (RC) populations. A representative sample (1,024 accessions) was studied for genetic dynamics of days to flowering (DTF) shortening in two evolutionary processes (WA→LR→RC and southern→northern) via direct comparisons of quantitative trait loci (QTL)–allele matrices among populations and subpopulations using a restricted two‐stage multilocus genome‐wide association analysis (RTM‐GWAS). In CSGP, 177 DTF QTL with 797 alleles explaining 95.1% phenotypic variation were identified and organized into a DTF QTL–allele matrix and its component submatrices. (i) Allele inheritance, emergence, exclusion. and recombination were major genetic drivers of DTF‐shortening for WA→LR→RC. (ii) Genetic drivers for southern→northern differed between wild and cultivated soybean [Glycine max (L.) Merr.], with less allele exclusion, more allele emergence, and slighter allele change in WA than in LR plus RC. (iii) The LR furthest south (LRIV) was recognized as the primitive LR subpopulation, as it had the most wild alleles and newly formed QTL and alleles. (iv) Combining the WA→LR→RC and southern→northern processes, the DTF shortening was divided into four periods: WASouth→WANorth, WASouth→LRSouth, LRSouth→LRNorth, and LR→RC; the per‐year allele change rate in LR→RC exceeded the others (backward in turn), and breeding effort accelerated allele emergence and exclusion (changed). (v) Out of 177 loci and 797 alleles, 83 and 207, respectively, changed in WA→LR→RC and southern→northern processes, especially eight new loci emerged in LR and RC; in total, 81 candidate genes were annotated and grouped into six flowering‐related biological processes. (vi) Further DTF shortening (4.2–11.1 d) in CSGP was predicted. This study comprehensively explored the evolutionary mechanism of DTF shortening in WA→LR→RC and southern→northern processes through QTL–allele matrix comparisons by using RTM‐GWAS.

Identifying Wild Versus Cultivated Gene-Alleles Conferring Seed Coat Color and Days to Flowering in Soybean.

Annual wild soybean (G. soja) is the ancestor of the cultivated soybean (G. max). To reveal the genetic changes from soja to max, an improved wild soybean chromosome segment substitution line (CSSL) population, SojaCSSLP5, composed of 177 CSSLs with 182 SSR markers (SSR-map), was developed based on SojaCSSLP1 generated from NN1138-2(max)×N24852(soja). The SojaCSSLP5 was genotyped further through whole-genome resequencing, resulting in a physical map with 1366 SNPLDBs (SNP linkage-disequilibrium blocks), which are composed of more markers/segments, shorter marker length and more recombination breakpoints than the SSR-map and caused 721 new wild substituted segments. Using the SNPLDB-map, two loci co-segregating with seed-coat color (SCC) and six loci for days to flowering (DTF) with 88.02% phenotypic contribution were identified. Integrated with parental RNA-seq and DNA-resequencing, two SCC and six DTF candidate genes, including three previously cloned (G, E2 and GmPRR3B) and five newly detected ones, were predicted and verified at nucleotide mutant level, and then demonstrated with the consistency between gene-alleles and their phenotypes in SojaCSSLP5. In total, six of the eight genes were identified with the parental allele-pairs coincided to those in 303 germplasm accessions, then were further demonstrated by the consistency between gene-alleles and germplasm phenotypes. Accordingly, the CSSL population integrated with parental DNA and RNA sequencing data was demonstrated to be an efficient platform in identifying candidate wild vs. cultivated gene-alleles.

Identifying a wild allele conferring small seed size, high protein content and low oil content using chromosome segment substitution lines in soybean.

A wild soybean allele conferring 100-seed weight, protein content and oil content simultaneously was fine-mapped to a 329-kb region on Chromosome 15, in which Glyma.15g049200 was predicted a candidate gene. Annual wild soybean characterized with small 100-seed weight (100SW), high protein content (PRC), low oil content (OIC) may contain favourable alleles for broadening the genetic base of cultivated soybeans. To evaluate these alleles, a population composed of 195 chromosome segment substitution lines (SojaCSSLP4), with wild N24852 as donor and cultivated NN1138-2 as recurrent parent, was tested. In SojaCSSLP4, 10, 9 and 8 wild segments/QTL were detected for 100SW, PRC and OIC, respectively. Using a backcross-derived secondary population, one segment for the three traits (q100SW15, qPro15 and qOil15) and one for 100SW (q100SW18.2) were fine-mapped into a 329-kb region on chromosome 15 and a 286-kb region on chromosome 18, respectively. Integrated with the transcription data in SoyBase, 42 genes were predicted in the 329-kb region where Glyma.15g049200 showed significant expression differences at all seed development stages. Furthermore, the Glyma.15g049200 segments of the two parents were sequenced and compared, which showed two base insertions in CDS (coding sequence) in the wild N24852 comparing to the NN1138-2. Since only Glyma.15g049200 performed differential CDS between the two parents but related to the three traits, Glyma.15g049200 was predicted a pleiotropic candidate gene for 100SW, PRC and OIC. The functional annotation of Glyma.15g049200 indicated a bidirectional sucrose transporter belonging to MtN3/saliva family which might be the reason that this gene provides a same biochemical basis for 100SW, PRC and OIC, therefore, is responsible for the three traits. This result may facilitate isolation of the specific gene and provide prerequisite for understanding the other two pleiotropic QTL.

Chromosome segment detection for seed size and shape traits using an improved population of wild soybean chromosome segment substitution lines.

Size and shape of soybean seeds are closely related to seed yield and market value. Annual wild soybeans have the potential to improve cultivated soybeans, but their inferior seed characteristics should be excluded. To detect quantitative trait loci (QTLs)/segments of seed size and shape traits in annual wild soybean, its chromosome segment substitution lines (CSSLs) derived from NN1138-2 (recurrent parent, Glycine max) and N24852 (donor parent, Glycine soja) and then modified 2 iterations (coded SojaCSSLP3) were improved further to contain more lines (diagonal segments) and less heterozygous and missing portions. The new population (SojaCSSLP4) composed of 195 CSSLs was evaluated under four environments, and 11, 13, 7, 15 and 14 QTLs/segments were detected for seed length (SL), seed width (SW), seed roundness (SR), seed perimeter(SP) and seed cross section area(SA), respectively, with all 60 wild allele effects negative. Among them, 16 QTLs/segments were shared by 2-5 traits, respectively, but 0-3 segments for each of the 5 traits were independent. The non-shared Satt274 and shared Satt305, Satt540 and Satt239 were major segments, along with other segments composed of two different but related sets of genetic systems for SR and the other 4 traits, respectively. Compared with the literature, 7 SL, 5 SW and 2 SR QTLs/segments were also detected in cultivated soybeans; allele distinction took place between cultivated and wild soybeans, and also among cultivated parents. The present mapping is understood as macro-segment mapping, the segments may be further dissected into smaller segments as well as corresponding QTLs/genes.

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

AbstractCytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.

Characterizing Two Inter-specific Bin Maps for the Exploration of the QTLs/Genes that Confer Three Soybean Evolutionary Traits.

Annual wild soybean (Glycine soja Sieb. and Zucc.), the wild progenitor of the cultivated soybean [Glycine max (L.) Merr.], is valuable for improving the later. The construction of a linkage map is crucial for studying the genetic differentiation between these species, but marker density is the main factor limiting the accuracy of such a map. Recent advances in next-generation sequencing technologies allow for the generation of high-density linkage maps. Here, two sets of inter-specific recombinant inbred line populations, named NJIRNP and NJIR4P, composed of 284 and 161 lines, respectively, were generated from the same wild male parent, PI 342618B, and genotyped by restriction-site-associated DNA sequencing. Two linkage maps containing 5,728 and 4,354 bins were constructed based on 89,680 and 80,995 single nucleotide polymorphisms, spanning a total genetic distance of 2204.6 and 2136.7 cM, with an average distance of 0.4 and 0.5 cM between neighboring bins in NJRINP and NJRI4P, respectively. With the two maps, seven well-studied loci, B1 for seed bloom; G and I for seed coat color; E2, E3, qDTF16.1 and two linked FLOWERING LOCUS T for days to flowering, were detected. In addition, two SB and two DTF loci were newly identified in wild soybean. Using two high-density maps, the mapping resolution was enhanced, e.g., G was narrowed to a region of 0.4 Mb on chromosome 1, encompassing 54 gene models, among which only Glyma01g40590 was predicted to be involved in anthocyanin accumulation, and its interaction with I was verified in both populations. In addition, five genes, Glyma16g03030, orthologous to Arabidopsis Phytochrome A (PHYA); Glyma13g28810, Glyma13g29920, and Glyma13g30710 predicted to encode the APETALA 2 (AP2) domain; and Glyma02g00300, involved in response to red or far red light, might be candidate DTF genes. Our results demonstrate that RAD-seq is a cost-effective approach for constructing high-density and high-quality bin maps that can be used to map QTLs/genes into such small enough regions that their candidate genes can be predicted.

Functional marker development of miR1511-InDel and allelic diversity within the genus Glycine.

BackgroundSingle-stranded non-protein coding small RNAs, 18–25 nucleotides in length, are ubiquitous throughout plants genomes and are involved in post-transcriptional gene regulation. Several types of DNA markers have been reported for the detection of genetic diversity or sequence variation in soybean, one of the most important legume crops in worldwide for seed protein and oil content. Recently, with the available of public genomic databases, there has been a shift from the labor-intensive development of PCR-based markers to sequence-based genotyping and the development of functional markers within genes, often coupled with the use of RNA information. But thus far miRNA-based markers have been only developed in rice and tobacco. Here we report the first functional molecular miRNA marker, miR1511-InDel, in soybean for a specific single copy locus used to assess genetic variation in domesticated soybean (Glycine max [L.] Merr) and its wild progenitor (Glycine soja Sieb. & Zucc.).ResultsWe genotyped a total of 1,669 accessions of domesticated soybean (G. max) and its wild progenitor G. soja which are native throughout the China and parts of Korea, Japan and Russia. The results indicate that the miR1511 locus is distributed in cultivated soybean and has three alleles in annual wild soybean. Based on this result, we proposed that miR-InDel marker technology can be used to assess genetic variation. The inclusion of geo-reference data with miR1511-InDel marker data corroborated that accessions from the Yellow River basin (Huanghuai) exhibited high genetic diversity which provides more molecular evidence for gene diversity in annual wild soybean and domestication of soybean.ConclusionsThese results provide evidence for the use of RNA marker, miRNA1511-InDel, as a soybean-specific functional maker for the study of genetic diversity, genotyping of germplasm and evolution studies. This is also the first report of functional marker developed from soybean miRNA located within the functional region of pre-miRNA1511.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1665-3) contains supplementary material, which is available to authorized users.

Genetic diversity of wild soybean populations in Dongying, China, by simple sequence repeat analysis.

Annual wild soybean (Glycine soja Sieb. et Zucc.), the ancestor of cultivated soybean (G. max), is believed to be a potential gene source for further improvement of soybean to cope with environmental stress. In this study, 10 simple sequence repeat (SSR) markers were used to evaluate the genetic diversity and population genetic structure in five wild soybean populations using 195 accessions collected from Dongying, China. Ten SSR markers yielded 90 bands, with an average of nine bands per marker. The percentage of polymorphic loci (P) was 97.78%, the distribution of expected heterozygosity (HE) was 0.1994-0.4460 with an average of 0.3262, and the distribution from Shannon's information index (I) was 0.3595-0.6506 with an average of 0.5386. The results showed that wild soybean had a high degree of genetic diversity at the species level. Nei's differentiation coefficient (FST) was 0.1533, and gene flow (Nm) was 1.3805, which indicated that genetic variation mainly existed within populations and that there was a certain level of gene exchange between populations. Some genetic differentiation occurred among populations, although this was not significant. Cluster analysis indicated that there was no significant correlation between the genetic structure of wild soybean populations and their geographic distribution, and the clustering results may be relatively consistent with the habitats of the accessions. In the present study, the genetic diversity of wild soybeans showed a broad genetic base and enables suggestions for the conservation of this plant to be made.

QTL mapping for the number of branches and pods using wild chromosome segment substitution lines in soybean [Glycine max (L.) Merr.

Annual wild soybean characterized with more number of branches and pods may contain favourable exotic genes/alleles for improving the yield potential of cultivated soybeans. To evaluate the wild alleles/segments, the chromosome segment substitution line population SojaCSSLP3 comprising 158 lines with N24852 (wild) as the donor and NN1138-2 (cultivated) as the recurrent parent was tested under three environments. The phenotypic data along with 198 simple sequence repeat markers were analysed for qualitative trait loci (QTL)/segments associated with the number of branches on the main stem (BN) and number of pods per plant (PN) using the inclusive composite interval mapping procedure (RSTEP-LRT-ADD model) of ICIM version 3.0. The analysis was carried out for individual environments due to a significant G × E interaction. A total of eight QTL/segments associated with BN and eight QTL/segments associated with PN were detected under the three environments, with all the wild segments having positive effects. Among these, two QTL/segments for each of the two traits could be detected under two or three environments and three QTL/segments could be detected for both traits. Four QTL/segments associated with BN and one QTL/segment associated with PN were identified only in SojaCSSLP3, not reported for cultivated crosses in the literature. The detected wild segments may provide materials for further characterization, cloning and pyramiding of the alleles conferring the two traits.

Identification of QTL/segments related to seed-quality traits in G. soja using chromosome segment substitution lines

Annual wild soybean characterized by low 100-seed weight (100SW), high protein content (PRC) and low oil content (OIC) may have favourable exotic genes/alleles for broadening the genetic base of the cultivated soybean. To evaluate the wild alleles/segments, a chromosome segment substitution line population comprising 151 lines with N24852 (wild) as the donor and NN1138-2 (cultivated) as the recurrent parent was analysed using single-marker analysis, interval mapping, inclusive composite interval mapping and mixed linear composite interval mapping. On 14 segments of ten chromosomes, 17 quantitative trait loci (QTL) were identified, with two segments each containing two QTL for 100SW and OIC and one segment containing two QTL for PRC and OIC, respectively. All the seven wild alleles/segments for 100SW were associated with negative effects and three were associated with positive effects, but one was associated with a negative effect for PRC, and five were associated with negative effects, but one was associated with a positive effect for OIC. Except Satt216 and Sat_224 for 100SW, the identified QTL/segments have been reported from cultivated soybean mapping populations. The detected wild segments may provide materials for further characterization, cloning and pyramiding of the alleles conferring the seed-quality traits.

Genetic Diversity of Wild Soybeans from Some Regions of Southern China Based on SSR and SRAP Markers

There are rich annual wild soybean (Glycine soja) resources in Southern China, which are the progenitor of cultivated soybean. To evaluate the genetic diversity and differentiation of G. soja in Southern China, we analyzed allelic profiles of 141 annual wild soybean accessions from Southern China and 8 core wild soybean accessions fromNorthern Chinaby using 41 simple sequence repeat (SSR) markers and 18 Sequence-related amplified polymorphism (SRAP) primer combinations. The 41 SSR markers produced a total of 421 alleles (10.27 per locus) with a mean of gene diversity of 0.825 (Simpson index) and 1.987 (Shannon-weaver index). The 18 SRAP primer combinations detected a total of 90 polymorphism bands (5 per primer combination) with a mean of gene diversity of 0.918 (Shannon-weaver index). SSR and SRAP markers detected 43 and 5 rare alleles in 149 wild soybeans, respectively. The wild soybeans from Fujian province showed the highest genetic diversity with Shannon-weaver index of 1.837 (by SSR) and 0.803 (by SRAP), and the highest allelic richness with an average of 8.8 alleles per locus and the most number of rare alleles of 0.68 per locus based on SSR data. An analysis of Molecular Variance (AMOVA) analysis showed that significant variance did exist amongHunan,Fujian, Guangxi andNorthern Chinasubpopulations based on SSR and SRAP data. The unweighted pair-group method of the arithmetic average (UPGMA) cluster analysis indicated that the wild soybeans fromFujianprovince occurred in different clusters based on both SSR and SRAP data. The above results indicated thatFujianprovince could be the major center of genetic diversity for annual wild soybean inSouthern China. In addition, Mantle test showed there was a weak positive linear correlation (r = 0.25) between SSR and SRAP analysis in the study.

Association Analysis between Agronomic-Processing Traits and SSR Markers and Genetic Dissection of Specific Accessions in Chinese Wild Soybean Population

Association analysis is potential in genetic dissection of germplasm accessions and breeding materials for designing crosses and improving selection efficiencies. The present study was aimed at finding elite QTLs/alleles as well as their carriers through genetic dissection of agronomic-processing traits in Chinese annual wild soybean (Glycine soja Sieb. et Zucc.) population for improvement and broadening the genetic background of modern soybean cultivars. The genotypic data of 204 simple-sequence repeat (SSR) markers on 174 wild accessions sampled from and evenly distributed in all the wild soybean eco-regions in China were used and analyzed for association with six agronomic and processing traits under TASSEL GLM (general linear model) program based on the population structure analysis. The QTLs significantly associated with the traits were analyzed further for their allele effects. The results showed: (1) Fifty-one SSR loci (times) associated with the six agronomic-processing traits were identified in the wild population. There were a few markers/loci associated with two or more traits simultaneously, which might be the genetic bases of correlation among the traits. Sixteen of fifty-one associated loci (times) were in agreement with mapped QTLs from linkage mapping procedure. (2)There existed only a few association loci in wild population coincided with those in landrace and released cultivar populations, indicating the difference of genetic structure among the three kinds of populations. (3) A set of elite alleles of detected loci and their carrier materials were screened out. Alleles for loci associated with several traits had different phenotypic effects in different traits, e.g. GMES5532a-A332 had positive phenotypic effect for both 100-seed weight and seedling death rate under submergence, while GMES5532a-A344 had negative effect on 100-seed weight but positive effect on seedling death rate under submergence. (4)There showed great difference of the genetic structure among the tested materials with extreme phenotypic value. The extreme accessions possessed the alleles with bigger effects, such as N23349 containing four alleles with bigger positive effects having its 100-seed weight as high as 9.08 g, while N23387 containing four alleles with bigger negative effects having its 100-seed weight only 0.75 g. The above results implied that association mapping could offer further genetic information complementary to linkage mapping, especially the information of multiple alleles of QTL on whole genome could be used in cross design for pyramiding elite alleles and marker-assisted selection in breeding for soybean.

Genome-wide genetic dissection of germplasm resources and implications for breeding by design in soybean

“Breeding by Design” as a concept described by Peleman and van der Voort aims to bring together superior alleles for all genes of agronomic importance from potential genetic resources. This might be achievable through high-resolution allele detection based on precise QTL (quantitative trait locus/loci) mapping of potential parental resources. The present paper reviews the works at the Chinese National Center for Soybean Improvement (NCSI) on exploration of QTL and their superior alleles of agronomic traits for genetic dissection of germplasm resources in soybeans towards practicing “Breeding by Design”. Among the major germplasm resources, i.e. released commercial cultivar (RC), farmers’ landrace (LR) and annual wild soybean accession (WS), the RC was recognized as the primary potential adapted parental sources, with a great number of new alleles (45.9%) having emerged and accumulated during the 90 years’ scientific breeding processes. A mapping strategy, i.e. a full model procedure (including additive (A), epistasis (AA), A × environment (E) and AA × E effects), scanning with QTLNetwork2.0 and followed by verification with other procedures, was suggested and used for the experimental data when the underlying genetic model was usually unknown. In total, 110 data sets of 81 agronomically important traits were analyzed for their QTL, with 14.5% of the data sets showing major QTL (contribution rate more than 10.0% for each QTL), 55.5% showing a few major QTL but more small QTL, and 30.0% having only small QTL. In addition to the detected QTL, the collective unmapped minor QTL sometimes accounted for more than 50% of the genetic variation in a number of traits. Integrated with linkage mapping, association mappings were conducted on germplasm populations and validated to be able to provide complete information on multiple QTL and their multiple alleles. Accordingly, the QTL and their alleles of agronomic traits for large samples of RC, LR and WS were identified and then the QTL-allele matrices were established. Based on which the parental materials can be chosen for complementary recombination among loci and alleles to make the crossing plans genetically optimized. This approach has provided a way towards breeding by design, but the accuracy will depend on the precision of the loci and allele matrices.

Phenolics in the Seed Coat of Wild Soybean (Glycine soja) and Their Significance for Seed Hardness and Seed Germination

Hardseededness in annual wild soybean (Glycine soja Sieb. Et Zucc.) is a valuable trait that affects the germination, viability, and quality of stored seeds. Two G. soja ecotypes native to Shandong Province of China have been used to identify the phenolics in the seed coat that correlate with the seed hardness and seed germination. Three major phenolics from the seed coat were isolated and identified as epicatechin, cyanidin 3-O-glucoside, and delphinidin 3-O-glucoside. Of the three phenolics, only the change of epicatechin exhibited a significant positive correlation with the change of hard seed percentages both under different water conditions during seed development and under different gas conditions during seed storage. Epicatechin also reveals a hormesis-like effect on the seed germination of G. soja. Epicatechin is suggested to be functionally related to coat-imposed hardseededness in G. soja.

Genetic variation of SNP loci based on candidate gene for resistance to soybean cyst nematode

For clarifying the difference of genetic diversity and linkage disequilibrium (LD) level between cultivated (Glycine max (L.) Merr.) and annual wild soybean (Glycine soja Sieb. & Zucc.), genetic variation pattern of 8 SNP loci developed from soybean cyst nematode resistance candidate genes rhg1 and Rhg4 in soybean germplasm were analyzed. The results indicated that G. max population, consisted of cultivated soybean mini-core collection and modern cultivars, had a higher LD levels (R2 value is 0.216) than G. soja population. Since 100% of pairwise loci within a gene and 16.6% of pairwise loci between genes were significant in G. max population, two specific LD regions were formed for each gene. A total of 46 haplotypes were detected in 363 soybean germplasm. The population of G. soja had less number of haplotypes and higher haplotype diversity than the population of G. max. Among the 31 population-specific haplotypes, 15 haplotypes were specific for G. soja population. In addition, the frequency of two major predominant haplotypes (Hap_10 and Hap_11) in G. soja population was obviously decreased in G. max population, which might indicate that some new haplotypes were formed and some old haplotypes were lost during the G. max domesticated from G. soja.

DNA methylation polymorphism in annual wild soybean (Glycine soja Sieb. et Zucc.) and cultivated soybean (G. max L. Merr.)

To study DNA methylation polymorphism in soybean, 20 and 27 lines of annual wild and cultivated soybeans, respectively, were selected from previously established “Soybean Core Collections in China”, and subjected to methylation-sensitive amplified polymorphism (MSAP) analysis. Twenty-seven primer pairs generated 984 CG/CNG methylation sites across the 47 lines, and the data were dissected into methylation-sensitive (MS) and methylation-insensitive(MIS) polymorphisms. The calculated MSP vs. MISP for wild soybeans were 34.65 vs. 34.76%, while corresponding results for the cultivated soybeans were 47.05 vs. 47.15%, indicating higher levels of MSP and MISP in cultivated than in wild soybeans. These results were incongruent with the amplified fragment length polymorphism (AFLP) analysis of the same soybean lines, and suggested enhanced DNA methylation polymorphism due to human selection. All three markers, MSP, MISP and AFLP, enabled clustering of the soybean lines into two distinct groups each predominantly containing wild or cultivated ones. Homology search indicated that 12 out of 24 sequenced MSPs had significant similarities to known-function or predicated genes, suggesting possible functional relevance of the methylation polymorphism. No significant association between MSP and MISP or MSP and AFLP was detected. Our results suggest that DNA methylation polymorphism in soybean has been under both natural and human selections, implicating possible roles of this epigenetic modification in genome evolution and domestication.Key words: Annual wild soybean (Glycine soja), cultivated soybean (G. max), DNA methylation polymorphism, epigenetic variation, evolution, domestication

Genetic diversity and peculiarity of annual wild soybean (G. soja Sieb. et Zucc.) from various eco-regions in China

Annual wild soybean (Glycine soja Sieb. et Zucc.) is believed to be a potential gene source for future soybean improvement in coping with the world climate change for food security. To evaluate the wild soybean genetic diversity and differentiation, we analyzed allelic profiles at 60 simple-sequence repeat (SSR) loci and variation of eight morph-biological traits of a representative sample with 196 accessions from the natural growing area in China. For comparison, a representative sample with 200 landraces of Chinese cultivated soybean was included in this study. The SSR loci produced 1,067 alleles (17.8 per locus) with a mean gene diversity of 0.857 in the wild sample, which indicated the genetic diversity of G. soja was much higher than that of its cultivated counterpart (total 826 alleles, 13.7 per locus, mean gene diversity 0.727). After domestication, the genetic diversity of the cultigens decreased, with its 65.5% alleles inherited from the wild soybean, while 34.5% alleles newly emerged. AMOVA analysis showed that significant variance did exist among Northeast China, Huang-Huai-Hai Valleys and Southern China subpopulations. UPGMA cluster analysis indicated very significant association between the geographic grouping and genetic clustering, which demonstrated the geographic differentiation of the wild population had its relevant genetic bases. In comparison with the other two subpopulations, the Southern China subpopulation showed the highest allelic richness, diversity index and largest number of specific-present alleles, which suggests Southern China should be the major center of diversity for annual wild soybean.

Genetic diversity and ecological differentiation of Chinese annual wild soybean (Glycine soja)

Genetic diversity and ecological differentiation of Chinese annual wild soybean (<i>Glycine soja</i>)

Discovery of new male-sterile cytoplasm sources and development of a new cytoplasmic-nuclear male-sterile line NJCMS3A in soybean

Most of the cytoplasmic-nuclear male-sterile (CMS) lines of soybean were developed only from a limited cytoplasm sources and performed not as good as required in hybrid seed production, therefore, to explore new male-sterile cytoplasm sources should be one of the effective ways to improve the pollination and hybridization for a better pod-set in utilization of heterosis of soybeans. In the present study, total 80 crosses between 70 cultivated and annual wild soybean accessions and three maintainers (N2899, N21249, and N23998) of NJCMS1A were made for detecting potential new sources with male-sterile cytoplasm. The results showed that in addition to the crosses with N8855.1 (the cytoplasm donor parent of NJCMS1A) and its derived line NG99-893 as cytoplasm parent, there appeared three crosses, including N21566 × N21249 and N23168 × N21249, with male-sterile plants in their progenies. According to the male fertility performance of backcrosses and reciprocal crosses with the tester N21249, the landrace N21566 and annual wild soybean accession N23168 were further confirmed to have male-sterile cytoplasm. Accordingly, it was understood that the source with male-sterile cytoplasm in soybean gene pool might be not occasional. The results also showed that the genetic system of male sterility of the newly found cytoplasm source N21566 was different from the old cytoplasm source N8855.1, while N23168 was to be further studied. Based on the above results, the derived male-sterile plants from [(N21566 × N21249) F1 × N21249] BC1F1 were back-crossed with the recurrent parent N21249 for five successive times, and a new CMS line and its maintainer line, designated as NJCMS3A and NJCMS3B, respectively, were obtained. NJCMS3A had normal female fertility and stable male sterility. Its microspore abortion was mainly at middle uninucleate stage, earlier than that of NJCMS1A and NJCMS2A. The male fertility of F1s between NJCMS3A and 20 pollen parents showed that 7 accessions could restore its male fertility and other 13 could maintain its male sterility. The male sterility of NJCMS3A and its restoration were controlled by one pair of gametophyte male-sterile gene according to male fertility segregation of crosses between NJCMS3A and three restorers. The nuclear gene(s) of male sterility in NJCMS3A appeared different from the previously reported CMS lines, NJCMS1A and NJCMS2A. The development of NJCMS3A demonstrated the feasibility to discover new CMS system through choosing maintainers with suitable nuclear background.