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Glycine soja is the wild relative species of cultivated soybean. In this study, we investigated the population divergence and genetic basis of the local adaptation of wild soybean in China using genome-wide single-nucleotide polymorphisms (SNPs) of a population of 72 G. soja accessions. Using phylogenetic analysis, we observed that G. soja accessions clustered into three distinct groups, each corresponding to a specific geographic region, the northeastern region (NER), central region (CR), and southern region (SR), consistent with previous studies. Notably, we found a significant positive correlation between genetic and geographic distances. Further population structure analysis revealed each group was associated with an ancestral population and a specific geographic area. By utilizing the genome sequencing data of accessions from 16 different locations, we inferred the population history of these wild soybean groups. Our results indicate that the three groups diverged ~25,000 years ago, coinciding with the time of the last glacial maximum. The effective population size of the SR group expanded first, and subsequently, the NER and CR groups expanded approximately 5000 and 2500 years ago, respectively. Moreover, 83, 104, and 101 significant associated loci (SALs) were identified using genome-wide association analysis for annual mean temperature, annual precipitation, and latitude, respectively. Functional analysis of genes located in SALs highlighted candidate genes related to local adaptation. This study highlights the significant role of geographic isolation and environmental factors in shaping the genetic structure and adaptability of wild soybean populations. Furthermore, it emphasizes the value of wild soybean as a crucial genetic resource for enhancing the adaptability of cultivated soybeans, which have experienced a loss of genetic diversity due to domestication and intensive breeding practices. The insights gained from our research provide valuable information for the protection, conservation, and utilization of this important genetic resource.

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Powdery mildew (PMD), caused by the pathogen Microsphaera diffusa, leads to substantial yield decreases in susceptible soybean under favorable environmental conditions. Effective prevention of soybean PMD damage can be achieved by identifying resistance genes and developing resistant cultivars. In this study, we genotyped 331 soybean germplasm accessions, primarily from Northeast China, using the SoySNP50K BeadChip, and evaluated their resistance to PMD in a greenhouse setting. To identify marker-trait associations while effectively controlling for population structure, we conducted genome-wide association studies utilizing factored spectrally transformed linear mixed models, mixed linear models, efficient mixed-model association eXpedited, and compressed mixed linear models. The results revealed seven single nucleotide polymorphism (SNP) loci strongly associated with PMD resistance in soybean. Among these, one SNP was localized on chromosome (Chr) 14, and six SNPs with low linkage disequilibrium were localized near or in the region of previously mapped genes on Chr 16. In the reference genome of Williams82, we discovered 96 genes within the candidate region, including 17 resistance (R)-like genes, which were identified as potential candidate genes for PMD resistance. In addition, we performed quantitative real-time reverse transcription polymerase chain reaction analysis to evaluate the gene expression levels in highly resistant and susceptible genotypes, focusing on leaf tissues collected at different times after M. diffusa inoculation. Among the examined genes, three R-like genes, including Glyma.16G210800, Glyma.16G212300, and Glyma.16G213900, were identified as strong candidates associated with PMD resistance. This discovery can significantly enhance our understanding of soybean resistance to PMD. Furthermore, the significant SNPs strongly associated with resistance can serve as valuable markers for genetic improvement in breeding M. diffusa-resistant soybean cultivars.

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KEY MESSAGE: Here, a novel pleiotropic QTL qSS14 simultaneously regulating four seed size traits and two consistently detected QTLs qSW17 and qSLW02 were identified across multiple years. Seed-related traits were the key agronomic traits that have been artificially selected during the domestication of wild soybean. Identifying the genetic loci and genes that regulate seed size could clarify the genetic variations in seed-related traits and provide novel insights into high-yield soybean breeding. In this study, we used a high-density genetic map constructed by F10 RIL populations from a cross between Glycine max and Glycine soja to detect additive QTLs for seven seed-related traits over the last three years. As a result, we identified one novel pleiotropic QTL, qSS14, that simultaneously controlled four seed size traits (100-seed weight, seed length, seed width, and seed thickness) and two consistently detected QTLs, qSW17, and qSLW02, in multiple years of phenotypic data. Furthermore, we predicted two, two and three candidate genes within these three critical loci based on the parental resequencing data and gene function annotations. And the relative expression of four candidate genes GLYMA_14G155100, GLYMA_17G061000, GLYMA_02G273100, and GLYMA_02G273300 showed significant differences among parents and the extreme materials through qRT-PCR analysis. These findings could facilitate the determination of beneficial genes in wild soybean and contribute to our understanding of the soybean domestication process.

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Seed coat color is a typical evolutionary trait. Identification of the genetic loci that control seed coat color during the domestication of wild soybean could clarify the genetic variations between cultivated and wild soybean. We used 276 F10 recombinant inbred lines (RILs) from the cross between a cultivated soybean (JY47) and a wild soybean (ZYD00321) as the materials to identify the quantitative trait loci (QTLs) for seed coat color. We constructed a high-density genetic map using re-sequencing technology. The average distance between adjacent markers was 0.31 cM on this map, comprising 9,083 bin markers. We identified two stable QTLs (qSC08 and qSC11) for seed coat color using this map, which, respectively, explained 21.933 and 26.934% of the phenotypic variation. Two candidate genes (CHS3C and CHS4A) in qSC08 were identified according to the parental re-sequencing data and gene function annotations. Five genes (LOC100786658, LOC100801691, LOC100806824, LOC100795475, and LOC100787559) were predicted in the novel QTL qSC11, which, according to gene function annotations, might control seed coat color. This result could facilitate the identification of beneficial genes from wild soybean and provide useful information to clarify the genetic variations for seed coat color in cultivated and wild soybean.

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In this study, the genetic diversity and population structure of 205 wild soybean core collections in Northeast China from nine latitude populations and nine longitude populations were evaluated using SSR markers. A total of 973 alleles were detected by 43 SSR loci, and the average number of alleles per locus was 22.628. The mean Shannon information index (I) and the mean expected heterozygosity were 2.528 and 0.879, respectively. At the population level, the regions of 42°N and 124°E had the highest genetic diversity among all latitudes and longitudes. The greater the difference in latitude was, the greater the genetic distance was, whereas a similar trend was not found in longitude populations. Three main clusters (1N, <41°N-42°N; 2N, 43°N-44°N; and 3N, 45°N->49°N) were assigned to populations. AMOVA analysis showed that the genetic differentiation among latitude and longitude populations was 0.088 and 0.058, respectively, and the majority of genetic variation occurred within populations. The Mantel test revealed that genetic distance was significantly correlated with geographical distance (r = 0.207, p < 0.05). Furthermore, spatial autocorrelation analysis showed that there was a spatial structure (ω = 119.58, p < 0.01) and the correlation coefficient (r) decreased as distance increased within a radius of 250 km.

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The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

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Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.

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