Genetic Analysis Of Sulfur Containing Amino Acids Related Traits And Functional Study Of Genes Encoded Cystathionine β-lyase In Soybean

Soybean[Glycine Max（L.）Merr.],one of the most important food and oil crops,provides essential proteins and amino acids for humans and animals.The requirement is increasing as the life quality is improving.The proportion of protein in soybean is approximately 35-42%,however,soybean is not a perfect protein source as the content of sulfur containing amino acids is very low.It is the key method for soybean nutritional quality improvement to enhance the content of sulfur eontaining amino acids in soybean.Soybean is originated from China and there are abundant germplasm resources.Soybean protein content is effected by environment and genetic factors.Thus,it is not worked to improve protein content using traditional breeding methods and breeders focus on marker-assisted breeding（MAS）which identify QTLs linked to the interesting traits using molecular markers.In this study,to identify genetic loci associated sulfur containing amino acids content,linkage analysis and genome-wide association study（GWAS）were performed for mutiple traits about sulfur containing amino acids using a recombinant inbred lines population（NJRIKY）,which contained 184 lines,and a natural population,which contained 262 accessions.Meanwhile,we identified the candidate gene and clarity its function in the process of synthesis for sulfur containing amino acids.The main results in this study as follows:Firstly,QTL mapping was performed for 1115 7S,115+7S（SGC）and 115/7S（RGC）in three years using NJRIKY population which derived from a cross between Kefeng No.1 and Nannong 1138-2 and the mean value of three years was analyzed as a single environment.Additive QTLs and epistatic QTLs were identified using WinQTLCart 2.5 and QTLNetwork 2.0.A total of 36 additive QTLs,3 pairs of epistatic QTLs and 5 major chromosome regions were identified.2 of 6 epistatic QTLs（q11S-6-5,q11S-20-3）were also detected as additive QTLs indicating that they not only were involved in epistatic interactions but also had additive effects.Compared with genes encoded glycinin and β-conglycinin,we found that genes encoding the subunits of β-conglycinin were localized to marker intervals sat₄18-satt650 and sat₁96-sat₃03,which xvere linked to RGC and SGC;marker sat₃18,associated with 11S,7S,and SGC,was located near Glyma10g04280（Gy4）,which encodes a subunit of glycinin.These results,which take epistatic interactions into account,will improve our understanding of the genetic basis of 11S and 7S contents and will lay a foundation for marker-assisted selection breeding of soybean and improving the quality of soybean products.Secondly,QTL mapping was performed for Met/PC,Cys/PC,Met+Cys/PC（SAA/PC）and protein content（PC）in two years using NJRIKY population and the mean value of two years was analyzed as a single environment.The distribution of the four traits exhibited a wide variation and an approximately normal distribution.The broad-sense heritability was 14.76-73.90%and protein content of that was the highest.The correlation analysis showed that Met/PC and Cys/PC significant positive correlated with SAA/PC and negative correlated with PC.Met/PC was significant positive correlated with Cys/PC.A total of 35 QTLs were detected for the four traits with four major chromosome regions containing and distributed on Chr 3,8,11 and 20,respectively.Thirdly,we measured Met/PC,Cys/PC,SAA/PC and PC for 262 accessions and performed GWAS for them using a high-density SNP array.The correlations between the four traits were accordant with linkage analysis using NJRIKY.The broad-sense heritability of PC was the highest with the value of 68.40%.111,14,65 and 123 SNPs were detected by Met/PC,Cys/PC,SAA/PC and PC in the natural population and mainly distributed on Chr 4,7,8,12,16 and 17 with SNP clusters.Comparing the results of linkage analysis with GWAS,two regions located on Chr 3 and 10,respectively,were identified by both of studies.Then,we detected one gene,Glyma03g28530,which encoded the key enzyme of sulfur containing amino acids synthesis,cystathionine β-lyase,as a candidate gene.Fourthly,to verify the function of candidate gene,we performed functional investigation test for Glyma03g28530（GmCBL1）and its orthologous,Glymal9g31280（GmCBL2）.The result of expression level analysis showed that GmCBL1 and GmCBL2 were expressed in all tissues and were higher in pods and roots.The gene expression levels were induced by drought,cold,salt and ABA treatment.To further verify the function of GmCBL1 and GmCBI.2 during the synthesis of sulfur containing amino acids,we constructed overexpression vectors and transformed to soybean hairy roots,respectively.We detected that increased of Cys and Met in the soybean hairy roots overexpressed GmCBL1 and increased of Met in that overexpressed GmCBL2.The results suggested that GmCBLl and GmCBL2 are important for synthesis of sulfur containing amino acids.Glyma20g28660,encoded the subunit of β-conglycinin,and Glyma06g09670,belonged to bHLH subfamily,were identified to interact with GmCBL1 through yeast two-hybrid assay.This indicated that glycinin and β-conglycinin were related with sulfur containing amino acids in soybean.The sequence analysis between the promoters of GmCBL1 and GmCBL1 showed that the sequence closed to ATG was relatively conservative and both of them could drive the expression of GUS.Fifthly,the expression levels of GmCBL1 and GmCBL2 were measured for NJRIKY and the natural population in two years and eQTLs were identified.The results showed that the expression level of GmCBL1 in both of population was significantly positive correlated with Met/PC and Cys/PC,the expression level of GmCBL2 in the natural population was positive correlated with Met/PC.These results were accordant with the variation trend of the sulftur containing amino acids in soybean hairy roots overexpressed GmCBL1 and GmCBL2.This is a powerful proof to suggest that both of genes were involved in the synthesis of sulfur containing amino acids.The wide variation was detected in the gene expression levels in both of populations and showed an approximately normal distribution.A total of 14 eQTL were identified in linkage analysis and 198 SNPs were detected in GWAS for GmCBL1 and GmCBL2.3 SNPs（AX-93887959,AX-93887966,AX-93974283）were detected by both expression of genes.3 SNPs（AX-93994591,AX-93698337,AX-93994607）located on Chr 3 were identified by GmCBL1 expression in two years and 1 SNP（AX-94274378）was located within the promoter of GmCBL1.2 SNPs（AX-93974283 and AX-93644909）were detected by GmCBL2 expression in two years.Comparing the results of linkage analysis with GWAS,we detected that GmCBL1 was controlled by both cis-and trans-eQTLs while GmCBL2 was controlled only by trans-eQTL in this study.