【Objective】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.【Method】The hard seed mutant Mzp661 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.【Result】In the progenies of Mzp661, 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 Mzp661 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.【Conclusion】Seed hardness of soybean mutant Mzp661 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.
【Objective】Soluble sugar content is one of the important quality traits of vegetable soybean. The genetic variation and genetic mechanism of soluble sugar content in fresh soybean seeds were studied to provide a basis for germplasm innovation and quality breeding of vegetable soybean.【Method】Using 133 soybean landraces from the Northeast region, the North region, the Huanghuaihai region and the South region, the soluble sugar content of fresh seeds was determined in the three environments of Lianjiang in spring, Fuqing in spring and autumn, in 2021. In combination with 82 187 high-quality SNP markers, the whole genome association analysis of soluble sugar content was conducted based on the mixed linear model MLM (Q+K), and the SNP loci with significantly related to soluble sugar content were identified. The candidate intervals were selected by the significant SNP loci and the extension of 119.07 kb linkage disequilibrium decay distance at both ends. The candidate genes were predicted according to the annotation and tissue expression information of the genes in the candidate intervals.【Result】The variation range of soluble sugar content in fresh seeds under three environments was 3.37-33.84 mg·g-1, the genetic variation coefficient was 24.59%-32.69%, and the heritability of soluble sugar content was 68.14%. 6, 8 and 22 SNPs were significantly associated with the soluble sugar content of fresh seeds were detected in Lianjiang in spring, Fuqing in spring and autumn, respectively, and phenotypic variation was 12.43%-29.27%. A total of 86 genes were obtained in the candidate regions of 9 significant SNP loci with higher interpretation rate of phenotypic variation, and 9 candidate genes were further screened by gene annotation and tissue expression information. These candidate genes are mainly involved in biological processes such as transcription factors, glycoprotein families and carbohydrate synthesis and transport. Among them, Glyma.01g016500, Glyma.13g042100, Glyma.16g131800 and Glyma.16g155300 were more highly expressed in soybean seeds and pods, which can be used as the most potential candidate genes for soluble sugar in fresh soybean seeds.【Conclusion】Through genome-wide association analysis, 36 SNPs significantly associated with soluble sugar content in fresh seeds were detected, and 9 candidate genes were further screened out, which may be involved in the regulation of soluble sugar content in fresh soybean seeds. Among them, Glyma.01g016500, Glyma.13g042100, Glyma.16g131800 and Glyma.16g155300 can be the key candidate genes for regulating soluble sugar content in fresh soybean seeds.
【Objective】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.【Method】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.【Result】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.【Conclusion】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.
【Objective】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.【Method】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.【Result】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.【Conclusion】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.
