Based on whole genome identification and analysis of soybean LOX gene family members, to understand the taxonomic evolutionary relationships of each member, to study the expression specificity of each gene member in different tissues and their response to abiotic stress, which provided a theoretical basis for further research on the molecular characteristics, evolutionary process, and function of the LOX gene family.

Based on the LOX protein sequences of rice and Arabidopsis species in Ensembl database, BLASTP alignment of homologous LOX protein sequences in soybean whole genome database was performed, and MEGA X software was used to construct a phylogenetic tree; Using website MEME for protein conserved motif analysis; Using online software GSDS 2.0 to analyze gene structure; Using TBtools for chromosome localization drawing; Analyze soybean LOX family replication genes using McscanX; Using the PlantCARE website to predict the promoter elements of soybean LOX gene family; Draw gene expression heatmaps of soybean under different tissues and abiotic stress using TBtools, and develop molecular markers for the excellent allele variant GmLOX15A1^-G/A significantly correlated with 100-seed weight.

A total of 43 LOX genes were identified in soybean, unevenly distributed on 13 chromosomes. Collinearity analysis indicates that the GmLOX gene has undergone extensive replication during the evolutionary process. Meanwhile, 39 different types of cis regulatory elements were detected in the LOX gene promoter, indicating that they may be involved in different pathways such as growth and development, light response, stress response, and hormone induction. Expression pattern analysis revealed that the LOX gene has different levels of expression in different tissues of soybean, indicating that members of this family have tissue and spatiotemporal expression specificity. Under drought stress conditions, the GmLOX gene was significantly differentially expressed in soybean roots and leaves (P<0.05). Among them, GmLOX3A3, GmLOX7A1, GmLOX20B1, GmLOX13A1, and GmLOX20A2 were significantly upregulated or downregulated in roots and leaves, suggesting that the GmLOX gene may play an important role in response to stress. At the same time, it was found that GmLOX15A1 is highly expressed in grain tissue and there is an excellent G/A allele variation in the seventh exon of the gene coding region. Molecular markers were developed for this variant site, and the correlation between different haplotypes of GmLOX15A1 and 100-seed weight was analyzed using 1 200 soybean germplasm resources from different ecological regions over a period of 2 years. The results showed that compared to the GmLOX15A1^-A genotype, the average 100-seed weight of soybean germplasm carrying the GmLOX15A1^-G allele gene increased by 2.33 g (P<0.001).

A total of 43 members of the LOX family were identified in soybeans, which can be divided into 3 subfamilies. The promoter region of the GmLOX gene contains a large number of cis acting elements that respond to hormones and stress, playing different roles in drought stress response. Among them, GmLOX15A1 is highly expressed in grain tissue and there is an excellent G/A allele variation in the seventh exon of the coding region of this gene. Compared with the GmLOX15A1^-A genotype, the average 100-seed weight of soybean germplasm carrying the GmLOX15A1^-G allele gene is significantly increased by 2.33 g. This locus can be used as an excellent haplotype for genetic improvement of soybean grain size.

An accurate and rapid indoor evaluation system was established by using soybeans with different resistance levels to Phomopsis seed decay as test materials. And then 170 soybean germplasm accessions were employed to screened out disease-resistant varieties, so as to provide methods and material basis for high-throughput assessment of Phomopsis seed decay in soybean and cultivation of resistant varieties.

In terms of establishing a reliable evaluation method for Phomopsis seed decay, Qihuang 34, Williams, Zhongzuo 09-560, z13-631-2, ZDD26268, Chenxiqingpidou 1 and Tongxianhuangdou were selected as experimental materials. For each soybean accession, the seeds with uniform size and undamaged seed coat were germinated in the dark after disinfection. At different germination stages, the pathogen of Phomopsis seed decay was inoculated for 24 h, 48 h, 72 h and 96 h. The mycelium coverage rate and seed decay rate of seed surface under different infection time were counted to determine the optimal identification period for evaluating Phomopsis seed decay in soybean. Then, the resistance of 170 different soybean germplasms in natural population was identified by using the coverage rate of mycelium on the surface of seeds and the decay rate of seeds as evaluation indexes. The high disease resistance varieties were screened based on 5 disease resistance levels.

The soybean accessions showed the most significant differences in disease resistance levels after 96 h of germination when mycelium coverage rate and seed decay rate of soybean surface were used as evaluation indexes. Further comparison of the incidence of 24 h, 48 h, 72 h and 96 h after infection showed that the difference in disease resistance between different varieties after infection for 72 h was the most obvious. Therefore, it was the most suitable period, 72 h of infection at the bud stage after 96 h of germination, for evaluating the resistance level of different soybean varieties to Phomopsis seed decay. The resistance of 170 soybean varieties to Phomopsis seed decay was identified and classified into five disease resistance grades, namely, high resistance, medium resistance, medium susceptibility, susceptibility and high susceptibility. Among them, there were 30 varieties of grade I (high resistance to disease), 51 varieties of grade Ⅱ (medium resistance to disease), 71 varieties of grade Ⅲ (medium disease susceptibility), 4 varieties of grade Ⅳ (disease susceptibility) and 14 varieties of grade V (high disease susceptibility), idicating that there are extensive variations in the resistance to Phomopsis seed decay of soybean germplasm resources in China.

In this study, the most optimum stage of disease identification was considered as soybean seeds after 96 h germination to infect the Phomopsis longicolla for 72 h. After that, the mycelium coverage rate and seed decay rate of soybean surface were counted as evaluation parameters. The evaluation system has high accuracy and reliability, which can provide an effective method for high-throughput identification of different varieties in the laboratory. And 30 highly resistant varieties were further screened to provide a material basis for the breeding of resistant varieties.

Hardness, a structural feature of seed physical dormancy, is an important trait in soybean domestication. Although hardness is beneficial for seeds to survive in unfavorable environments, it will seriously reduce the emergence rate of soybean in the field, and detrimental to yield and processing quality. Analyzing the QTL and candidate genes using bulked segregant analysis sequencing (BSA-Seq), can provide a theoretical reference for understanding the molecular mechanism of hard seededness in soybean.

The hard seed mutant M_zp661 was obtained from the seeds of Zhongpin 661 induced by ethyl methane sulfonate (EMS), and was crossed with cultivated soybean Zhonghuang 13 (male parent) to construct recombinant inbred line (RIL) population. The progeny lines were investigated for seed hardness, water absorption capacity and anatomical structure of seed coats. Two types of extreme lines in the RIL population, with hard seeds or with imbibed seeds, were selected to construct DNA mixed pools respectively, and then BSA-Seq technology was used to detect genotype differences in extreme-mixed pools and parents. Euclidean distance (ED), delta SNP-index, and delta InDel-index methods were applied to associate hard seed genetic loci of soybean. Combining with bioinformatics analysis, transcriptome data of different soybean tissues and gene annotation information, candidate genes within significant association regions were predicted.

In the progenies of M_zp661, all areas of imbibitive seeds had the penetration ability, and the seed volume increased continuously with the soaking time. However, no changes were observed for hard seeds over 36 hours. With the prolonged of soaking time, the seed coat of hard seeds began to shrink locally and gradually spread to other parts, and finally cotyledons recovered their imbibition ability. The hard seed not only has smooth and compact seed coat, but also has regular network structure of cuticle and thicker palisade layer, while numbers of stomata and loose structures, tiny cracks and thinner palisade layer were existed in the imbibed seeds. These results suggest that the seed hardness of M_zp661 may be caused by the impermeability of the seed coat. ED, delta SNP-index and delta InDel-index association analysis methods not only identified the reported seed physical dormancy locus qHS1, but also simultaneously detected the candidate region Chr.06: 45897227-47746047, which contains a total of 189 genes. Further, transcriptome data and gene annotation predicted that Glyma.06G275300, which is specifically and highly expressed in seeds, might be the candidate gene for this associated region to regulate soybean seed hardness.

Seed hardness of soybean mutant M_zp661 was caused by the impermeability of the seed coat, and Glyma.06G275300 was predicted as a candidate gene affecting the structure of seed coat using BSA-Seq.

Exploring efficient nodulation soybean germplasm adapted to the ecological conditions of the Bashang area, identifying genetic loci and candidate genes regulating soybean-rhizobium symbiotic nodulation, and improving soybean symbiotic nitrogen fixation efficiency.

This study utilized a natural population of 260 soybean germplasms as the research object, rhizobium strain USDA110 was inoculated under outdoor potted conditions in the Bashang of Hebei Province. The single plant nodule number and single plant nodule dry weight data were used as phenotypic values. Combined with genotype data of the 260 germplasms, a genome-wide association analysis was conducted to explore genes related to soybean-rhizobium symbiotic nodulation.

A total of 18 SNPs significantly associated with soybean nodule number were detected, located on chromosomes 2, 7, 8, 13, 18, and 19. Among them, the significant associated locus BARC_2.01_Chr02_43161654_A_G on chromosome 2 was identified as the main locus controlling soybean nodule number (LOD=3.89). Linkage disequilibrium analysis within the 200 kb interval upstream and downstream of this locus containing BARC_2.01_Chr02_43161654_A_G identified 10 candidate genes regulating soybean nodule number. There was a significant difference in the number of nodules among the materials corresponding to different haplotypes of Glyma.02G243200 (P<0.05), the expression pattern of this gene was queried in the SoyBase database, and it was expressed in root hairs, indicating that Glyma.02G243200 may be a key gene influencing soybean nodule number. Additionally, six SNPs significantly associated with soybean nodule dry weight were identified, located on chromosomes 6, 18, and 20. Among them, the significant associated loci BARC_2.01_Chr06_6069381_G_A and BARC_2.01_Chr06_6192925_T_C on chromosome 6 were identified as the main loci controlling soybean nodule dry weight (LOD=3.49 and LOD=3.35, respectively). Linkage disequilibrium analysis within the 100 kb interval upstream of BARC_2.01_ Chr06_6069381_G_A and downstream of BARC_2.01_Chr06_6192925_T_C identified 14 candidate genes regulating soybean nodule dry weight. Haplotype analysis revealed significant differences in nodule dry weight for the genes Glyma.06G079600 and Glyma.06G079900 between different haplotype materials (P<0.01, P<0.001), the expression pattern of this gene was queried in the SoyBase database, and they were expressed in roots, indicating that these two genes may be key genes influencing soybean nodule dry weight.

This study identified a candidate gene significantly associated with nodule number on chromosome 2 and two candidate genes significantly associated with nodule dry weight on chromosome 6, providing new genetic resources and references for genetic improvement of soybean nodulation traits.

Polymerizing soybean high oil genotypes aims at breeding varieties with higher oil content to improve economic efficiency and nutritional value. It is of great significance to increase agricultural output, reduce processing costs and meet global demand for vegetable oil growth.

Glyma.18G027100 C2 gene family was identified by bioinformatic analysis method at the whole genome level. A total of 66 soybean C2 gene family members were identified, named GmC2-01.1-GmC2-20.2 according to chromosome position. Tissue pattern analysis revealed that 7 genes were highly expressed in grains among 66 C2 family genes (GmC2-03.6, GmC2-02.7, GmC2-07.2, GmC2-18.1, GmC2-18.4, GmC2-19.1 and GmC2-20.2). In order to analyze the effect sites of these genes on soybean oil content, SNP sites in the coding regions of these genes were obtained from SFGB database. Correlation analysis of oil content in two years showed that GmC2-18.1 has SNP loci that significantly affect oil content. The genetic diversity of GmC2-18.1 coding region was analyzed by 12 extreme materials. There was a G/A mutation at 2 038 273 bp in coding region of Wm82.a2.v1 version, which regulated seed oil content. It was preliminarily speculated that this gene played a role in seed development or nutrient accumulation. Then, SNP/InDel molecular markers were developed for GmC2-18.1^-G/A gene combined with InDel natural allelic variation site 225 bp upstream of the start codon of GmSWEET39, T/C natural allelic variation site at 8 381 058 bp in coding region of GmST1, A/C natural allelic variation site at the third exon of 41 854 422 bp in coding region of GmMFT. 1 200 soybean germplasm resources from three ecological regions in China were identified by markers in 2 years.

Analysis of variance showed that GmC2-18.1^-G, GmSWEET39^-Deletion, GmST1^-T and GmMFT^-A significantly increased oil content by 1.72, 1.95, 1.58 and 2.06 percentage points (P<0.01). The results showed that the average oil content of soybean seeds carrying GmC2-18.1^-G, GmSWEET39^-Deletion, GmST1^-T and GmMFT^-A high-oil allele type (PFAT-1) was 22.89%, which increased by about 4.5% compared with that carrying GmC2-18.1^-A, GmSWEET39^-Insertion, GmST1^-C and GmMFT^-C low-oil allele type (PFAT-14). 5 percentage points, the contribution rate to oil content is about 21.69%.

Based on the markers developed above, 115 PFAT-1 high oil alleles were screened.