Fine Mapping Of Salt Tolerant-related QTL At The Seed Germination Stage And Functional Study Of GmCDF1 In Soybean

Soybean[Glycine max(L.)Merr.] is an economically important pulse crop for animal feed and human consumption.However,salt stress is one of the major abiotic factors adversely affectting the growth and development of soybean,which will significantly reduce the yield and quality of soybeans.Therefore,understanding the salt tolerance mechanism of soybean and breeding new salt-tolerant soybean varieties have become an effective strategy to solve this serious problem.Soybean salt tolerance is a complex quantitative trait affected by numerous genetic and non-genetic factors.Thus,it has taken considerable time and effort for improving the salt tolerance of soybean using traditional breeding by selection.With the rapid development of modern molecular biology,molecular marker-assisted selection(MAS)has emerged as a powerful and effective tool for studying complex quantitative traits.In this study,to understand the salt tolerance mechanism of soybean,linkage analysis and genome-wide association analysis(GWAS)were carried out to identify the genetic loci related to salt tolerance of soybean using a recombinant inbred line population(NJRIKY)and a natural population.Moreover,we determided the candidate gene and verified its function in the response to salt stress.The results are as follows:Firstly,four salt tolerance indices(ST-IR,ST-GI,ST-GP and ST-GR)of a recombinant inbred line(RIL)population drived from a cross between Kefeng No.1 and Nannong 1138-2 were used for QTL mapping.The correlation analysis showed that ST-IR was negatively correlated with ST-G,ST-GP and ST-GR significantly(P<0.01),whereas ST-GI was significantly positively correlated with ST-GP and ST-GR,and ST-GP was strongly positively correlated with ST-GR(P<0.01).A total of 31 QTLs associated with four salt tolerance indices during the soybean germination stage were detected on chromosomes 1,2,7,8,10,15,17 and 18,with LOD values ranging from 2.50 to 17.06.Except qSTGP-8-2,other QTLs related to salt tolerance on chromosome 8 with largely overlapped confidence intervals(CIs)were considered the same one,named qST-8-1.This QTL with marker Sat₁62 was detected mostly for the four salt tolerance indices in E1,E2,E3,and E4,contained the physical genetic region between the markers BE820148 and AW132402 and explained 6.25%-46.82%of the phenotypic variation.Besides QTLs on chromosome 8,three salt tolerance-related QTLs and four salt tolerance-related QTLs were detected on chromosomes 7 and 17,respectively.The phenotypic variations of these QTLs were from 9.08%?16.55%and 6.08%?16.98%.Secondly,the natural population was treated with salt stress and four salt tolerance indices(ST-IR,ST-GI,ST-GP and ST-GR)from three environments were used for GWAS with a high-density SNP array NJAU 355K SoySNP.The results showed that the salt stress had smaller effect on the ST-IR than the other three salt tolerance indices,ST-GI,ST-GP and ST-GR.The correlations between the four traits in the natural population were consistent with the recombinant inbred lines population.GWAS showed that 92 SNPs were significantly associated with four salt-tolerant traits,mainly on chromosomes 1,8,11,13,14,15,16,18,and 19.39,19,19 and 15 SNPs were detected by ST-IR and ST-GI,ST-GP and ST-GR respectively and formed a SNP cluster on chromosome 8,flanked by the SNP markers AX-93912074 and AX-93634504.Interestingly,this cluster was located in qST-8-1 identified by linkage mapping in four environments,indicating that this cluster is critical for salt tolerance of the germination stage of soybean and 71 candidate genes were in this region.Thirdly,to determine the candidate gene for salt tolerance,we performed whole-genome sequencing on Kefeng No.1(tolerant to salt stress)and Nannong 1138-2(sensitive to salt stress),which are the parents of the RIL population used in this study.We compared whole genome of Kefeng No.1 to Nannong 1138-2 and found that 273 SNPs were located on chromosome 8 between SNP marker AX-93912074 and AX-93634504.Among them,42 non-synonymous SNPs are located in the exons of 21 genes and 15 SNPs were in the 2.0-kb promoter regions of 11 genes including three identical genes.We carried out quantitative real-time PCR(qRT-PCR)to investigate whether the expressions of these 29 genes were affected by salt stresses in Kefeng No.1 and Nannong 1138-2.Results demonstrated that the expression of six genes was too low to be detected,16 genes did not change significantly in response to salt stress and seven genes could be induced by salt stress at soybean germinate stage.Among these seven genes,six of them were induced by salt stress both in Kefeng No.1 and Nannong 1138-2 and only Glyma.08gORF1was dramatically upregulated in Nannong 1138-2,but not in Kefeng No.1 under salt stress.The expression level of Glyma.08gORF1 in Nannong 1138-2 was nearly 30-folds higher than that in Kefeng No.1 treated with 150 mM NaCl,only 1-3 fold change for other six genes.Glyma.08gORF1 in the QTL qST-8-1 detected in our study might be a candidate gene involved in the regulation of salt tolerance in soybean.Bioinformatics analysis showed that Glyma.08gORF1 is a member of the cation diffusion facilitator family(named GmCDF1).We took qRT-PCR to explore the expression pattern of GmCDF1 in six representative soybean accessions including three salt tolerant accessions(Kefeng No.1,NJAU_C051 and NJAU_C204)and three salt sensitive accessions(Nannong 1138-2,NJAU_C071 and NJAU_C136)during germination stage under normal and salt stress conditions.Interesting,the fold changes of GmCDF1 in salt sensitive accessions were higher than these in salt tolerant accessions after treated with salt for 48 h and 96 h.qRT-PCR showed that GmCDF1 expressed constitutively in most soybean tissues.Higher levels of GmCDF1 transcript were detected in flowers,seeds and roots than these in leaves,pods and stems.To survey the function of GmCDF1,overexpression and RNA interference of GmCDF1 were performed in soybean hairy roots mediated by Agrobacterium rhizogenes strain K599,resulting in increased sensitivity and tolerance to salt stress,respectively.Also,results showed that GmCDF1 negatively regulated salt tolerance via maintaining K+-Na+homeostasis in soybean.In addition,GmCDF1 affected the expression of two ion homeostasis-associated genes,salt overly sensitivel(GmSOS1)and Na+/H+exchanger 1(GmNHX1),in transgenic hairy roots.Haplotype analysis showed that ten haplotypes of GmCDF1 were detected in 31 soybean genotypes.Candidate-gene association analysis showed that two single nucleotide polymorphisms(SNPs),S-671 and S605,in GmCDF1 were significantly associated with salt tolerance and Hap 1 was more sensitive to salt stress than Hap 2.Results demonstrated that the expression level of GmCDF1 was negatively correlated with salt tolerance in the 31 soybean accessions(r=-0.56,P<0.01).Taken together,these results indicate that GmCDF1 plays a negative role in soybean salt tolerance.