Resource Screening And Genome-wide Association Analysis Of Traits Related To Low-sulfur Stress In The Seedling Of Soybean

Cultivated soybean[Glycine max（L.）Merr.]is originated from China.Wild soybean[Glycine soja Sieb.et Zucc.],the wild relative species of cultivated soybean,distributes in east Asia and preserves more resistance and genetic diversity.Soybean is rich in protein and oil,which is an important grain and oil crop in China.However,the content of sulfurcontaining amino acids such as cysteine and methionine in soybean protein is relatively low,which reduces the nutritional value of soybean and its processed food.High efficiency utilization of sulfur can obviously increase soybean yield and improve soybean quality.At present,there are few researches about the utilization of sulfur in soybean,and many important biological problems from metabolism to function have not been solved.Due to the complex metabolism process,strict regulation and multiple functions of sulfur element,and in view of the present situation of agricultural production in China,there is insufficient sulfur content in soil.In addition,there is a high-sulfur demand for soybeans,in order to enhance the absorption and utilization efficiency of plants under sulfur deficiency stress,it is necessary to study its genetic mechanism.Moreover,there is little research on low-sulfur tolerance resources of soybean at home and abroad.In order to excavate abundant and excellent low-sulfur tolerance materials and candidate genes in soybean,this study evaluated the tolerance of low-sulfur stress for 121 wild soybeans and 219 cultivated soybeans by indoor hydroponic experiments with normal sulfur treatment and low-sulfur treatment,and the extreme materials were screened.Genome-wide association analysis of sulfur stressrelated traits is conducted by using 292053 SNP markers covering the whole genome of soybean for 121 wild soybeans and 219 cultivated soybeans,and the candidate genes within significant association sites were predicted.The main study results as follows:Firstly,we measured 12 phenotypic data of plant height,root length,fresh weight and dry weight of shoot,fresh weight and dry weight of root,root/shoot ratio of length,root/shoot ratio of fresh weight and root/shoot ratio of dry weight,sulfur content,sulfur assimilation efficiency,sulfur utilization efficiency from 121 wild soybean accessions.Among them,the first nine indexes are indirect indexes of soybean sulfur utilization,and the latter three traits are directly related to soybean sulfur utilization.Descriptive statistical analysis showed that the phenotypic values in the low-sulfur environment were lower than in the normal sulfur environment except for sulfur utilization efficiency and plant height.There were extensive genetic variation among different traits,and the coefficient of variation of the other traits was more than 30%except for root length.In addition,these indicators showed a nearly normal and continuous distribution under different sulfur treatments,which accorded with the characteristics of quantitative traits controlled by multiple genes.The fresh weight of root had the highest broad-sense heritability 90%and 97%in the normal sulfur environment and the low-sulfur environment,respectively.And there was a significantly positive correlation among biomass of root,biomass of shoot and sulfur assimilation efficiency,while sulfur utilization efficiency was significantly negative correlated with sulfur content and sulfur assimilation efficiency.Analysis of variance showed that there were significant differences among genotypes,genotype and treatment interactions in three environments.Except for the three traits directly related to soybean sulfur utilization had significant differences between treatments,the remaining traits were greatly affected by the environment,which led to have different differences between treatments in different years.The ratio of the traits in the lowsulfur environment to the normal sulfur environment was used as the low-sulfur tolerance index.The S value of each material calculated by the weighting method was used to screen the resources of low-sulfur sensitivity and low-sulfur tolerance.In all three environments,the accession with low-sulfur sensitivity was only No.109（zyd5181）,and the accession with low-sulfur tolerance was only No.91（zyd4299）.Secondly,a total of 107 significant SNPs were detected by genome-wide association analysis for the traits indirectly related to sulfur utilization,and four SNPs were detected simultaneously by the same trait in different environments or different traits in the same environment.There were significant SNP markers on all other chromosomes except for chromosome 12,mostly on chromosomes 3,8 and 17.The traits directly related to sulfur include sulfur content,sulfur assimilation efficiency and sulfur utilization efficiency,when the ratio of the traits in the low-sulfur environment to the normal environment as the lowsulfur tolerance index,a total of 75 SNPs were detected to be significantly associated with these three traits.Taking the traits under the normal sulfur and the low-sulfur environments as indicators,23 and 5 repeat sites were detected respectively,and 10 repeat sites were screened under different treatment conditions.Under normal sulfur treatment conditions,AX-94068642,AX-93772107 and AX-94068643 sites on chromosome 9 were significantly associated with sulfur assimilation efficiency in two environments,and AX-93812076 and AX-93911940 sites on chromosome 13 were also significantly associated with this trait in three environments.Under low-sulfur treatment conditions,no repeat sites were detected in multiple environments.Analysis of the phenotypes of the haplotypes of each loci found that there were 7 SNPs in the phenotype showing significant differences among the haplotypes,and a total of 43 genes were predicted by predicting candidate genes in the chromosome regions of 80kb before and after these sites.It is predicted that these genes are mainly related to repeat proteins,hypothetical proteins,domain proteins,auxin binding proteins,disease resistance proteins,serine/threonine protein kinases and so on.In addition,AX-93944415 marker was labeled in the coding region of glysoja₀35985 mainly used as MOB kinase activator.And AX-93812076 was located in the 3’UTR region of glysoja₀32469 mainly used as the synthesis of PHD finger proteins.The function of these candidate genes needs to be further studied.Thirdly,we measured 13 phenotypic data of plant height,root length,fresh weight and dry weight of shoot,fresh weight and dry weight of root,SPAD value of the second and third upper leaves of plants on day 6,SPAD value of the second and third upper leaves of plants on day 10,root/shoot ratio of length,root/shoot ratio of fresh weight and root/shoot ratio of dry weight from 219 cultivated soybeans accessions.Descriptive statistical analysis showed that the phenotypic values in the low-sulfur environment were lower than in the normal sulfur environment.There were extensive genetic variation among different traits,and the coefficient of variation of the other traits was more than 30%except for root length,SPAD value of the second and third upper leaves of plants on day 6 and SPAD value of the second and third upper leaves of plants on day 10.In addition,these indicators showed a nearly normal and continuous distribution under different sulfur treatments,which accorded with the characteristics of quantitative traits controlled by multiple genes.The root/shoot ratio of length had the highest broad-sense heritability 84%and 78%in the normal sulfur environment and the low-sulfur environment,respectively.And there was a significantly positive correlation between biomass of root and biomass of shoot.Analysis of variance showed that there were significant differences among genotypes,genotype and treatment interactions in three environments.Except for plant height,root length,SPAD value of the second and third upper leaves of plants on day 6 and SPAD value of the second and third upper leaves of plants on day 10 had significant differences between treatments,the remaining traits were greatly affected by the environment,which led to have different differences between treatments in different years.The ratio of the traits in the low-sulfur environment to the normal sulfur environment was used as the low-sulfur tolerance index.The S value of each material calculated by the weighting method was used to screen the resources of low-sulfur sensitivity and low-sulfur tolerance.Varieties of three low sulfur sensitivity were screened which were 3,168 and 189 accessions,and varieties of seven low sulfur tolerance were screened which were 7,89,91,102,127,159 and 175 accessions in all three environments.The genome-wide association analysis was performed by using the lowsulfur tolerance index of traits and markers.A total of 325 significantly associated SNPs were detected,of which 10 SNPs were significantly associated with the same traits in different environments or different traits in the same environment.Comparing the results of association analysis between wild soybeans and cultivated soybeans,we found that the AX94131947 locus associated with wild soybeans is located in a SNP cluster associated with cultivated soybeans,and there were 17 candidate genes in the SNP cluster region.