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.

Growth period is the evolution of soybean varieties adaptded to the ecological environment. Which has important effects on the yield and agronomic traits. To lay the foundation for soybean breeding in southern China, especially in Sichuan andChongqing region.the relevant genetic loci of soybean growth period were analyzed and and candidate genes were screened out 227 soybean cultivars or resources from Sichuan and Chongqing regions were identified based on 135 SSR and 107,081 effective single nucleotide polymorphism (SNP) markers. Four growth period traits (vegetative growth, reproductive growth, whole growth and the ratio of vegetative growth / reproductive growth period) were investigated in three environments (Fengyang Anhui in 2016, Zigong Sichuan in 2016,and Fengyang Anhui in 2018).The results showed that the phenotypic variation of four traits followed normal distribution, and the genetic variation was significantly affected by variety, environment and environment interaction. A total of 51 loci and 70 loci significantly associated with growth stage traits were detected by SSR markers and SNP respectively. In particular, there were stable expression related sites in BLK_29175719-29275719 and BLK_30878620-30978620 on chromosome 13, BLK_48763386-48863386 on chromosome 14 and BLK_10093551-10293551 on chromosome 16. And 11 potential candidate genes related to plant growth and development were predicted in these four regions and four genes (namely Glyma.13g177800, Glyma.13g178300, Glyma.13g178500, and Glyma.14g223300) were directly related to soybean growth period traits.

Seed weight represents a critical yield-associated quantitative trait in soybean (Glycine max) and is governed by polygenic regulatory networks. Although several seed-weight-related genes have been functionally characterized, the interactome of the major seed-weight determining gene SW16.1 remains largely unexplored. In this study, we constructed a yeast two-hybrid (Y2H) cDNA library using pooled total RNA isolated from developing seeds of the soybean cultivar Nannong 1138-2 covering five key seed-development stages. Using GmSW16.1 (cultivated Nannong 1138-2 allelic) as bait, Y2H screening was performed. Following multi-round screening, Sanger sequencing, and filtering based on functional annotation, 11 candidate interactors assigned to nine distinct functional categories were recovered. Interactions were further verified through point-to-point Y2H assays, quantitative α-galactosidase (α-Gal) activity measurements, and bimolecular fluorescence complementation (BiFC). We confirmed that metallothionein-like protein 4A-related Glyma.18G157800 (GmMT4) and the papain-like cysteine protease Glyma.08G116300 physically interact with both GmSW16.1 and GsSW16.1 (wild soybean N24852). A two-glutamine polymorphism within the PolyQ (polyglutamine) repeat tract of these two allelic proteins did not abolish protein–protein interactions, yet significantly altered their interaction strength toward the above-mentioned partners. Glyma.08G116300 displayed preferential transcript accumulation during soybean seed development, and its transcript abundance differed dynamically between the chromosome segment substitution line CSSL3068 and its cultivated parent Nannong 1138-2 across developmental stages. Heterologous overexpression of this gene in Arabidopsis thaliana significantly increased the 1000-seed weight (by 12–15%) and increased seed length and width. This study delivers a seed-development-specific Y2H library resource for soybean research. We put forward a testable working hypothesis describing a dual-layer regulatory module integrating transcriptional control and direct protein–protein interaction for the GmSW16.1-GmMT4 regulatory node. These findings advance our mechanistic understanding of how natural PolyQ allelic variation shapes soybean seed-weight performance and provides candidate gene materials for soybean molecular design breeding.

Legumes interact with rhizobium convert N ₂ into ammonia for plant use. To investigate the plant basal nitrogen fixation mechanisms induced in response to Bradyrhizobium , differential gene expression in root of inoculated and mock-inoculated soybean was analysed by RNA-Seq. A total of 55787 transcripts were aligned to soybean genome reference sequences, 280 and 316 transcripts were found to be up-and down-regulated, respectively, in inoculated relative to mock-inoculated soybean’s root at V1 stage. Gene ontology (GO) analyses detected 104, 182 and 178 genes associated with cell component category, molecular function category and biological process category, respectively. Pathway analysis revealed that 98 differentiallly expressed genes (115 transcripts) involved in 169 biological pathways. We selected 19 differentially expressed genes and analyzed their expressions in mock-inoculated, inoculated USDA110 and CCBAU45436 using qRT-PCR. The results were consistent with those obtained from rhizobia infected RNA-Seq data. These showed that the results of RNA-Seq have reliability and universality. Additionally, this study showed some novel genes associated with nitrogen fixation process comparison with the previously identified QTLs.