Variation Andgenetic Dissection Of Seedpurinecontent Inchinese Germplasm Population Of Cultivated Soybean

Soybean[Glycine max(L.)Merr.]is an important food in people's life because of its high protein.But,In recent years there are some reports assert that eating soybean can cause high blood uric acid and gout,they would bring perplex to consumer,so the research about the purine content of soybean seed has the vital significance.The purine in soybean seed mainly exists in the form of nucleotide,and its composition in soybean seed includes Adenine,Guanine,Hypoxanthine and Xanthine,among them is given priority to with adenine and guanine,the content of Hypoxanthine and Xanthine is very little.So far,only a few genetic andbreeding researches related to the soybean seed purine content have been reported.In this study,a representative sample composed of 320 assessions from Chinese cultivated soybean were researched by GWAS with high-density SNP markers,to discover the loci and the corresponding favorable alleles that are significantly associated with purine content in soybean seed,which would provide useful parents and genetic resources to breeding soybean varieties with low purine content.The main results are as follow:1.By comparing different HClO4 content and temperature for hydrolysis of mashed soybean specimen and buffer solution mobile phase,a stable and accurate HPLC(High performance liquid chromatography)procedure for measuring multiple components of purines in soybean seeds was established.The optimized hydrolysis condition was hydrolyzing mashed soybean seed with 35%(v/v)of HClO4 for 60 mins at 100?.The optimized chromatography condition was using Welch Ultimate Polar RP18(4.6 mm×250 mm,5.0?m)as chromatographic column with 0.02 mol·L-1 KH2PO4 buffer solution(pH2.9)as mobile phase at flow rate of 1.0 mL·min-1.2.The purine content of 320 soybean accessions(182 landraces and 132 released cultivars)were measured by HPLC(the experiment was conducted in 2014 and 2015 with two replications within each year).The total purine content was 2348.86mg/kg on average,with the range of 1822.13 to 2886.92mg/kg,Genetic coefficient of variation(GCV)was 8.55%and the heritability was 65.19%.The average contents of Adenine,Guanine and Hypoxanthine were 1097.52mg/kg,1138.25mg/kg and 37.69mg/kg,respectively;the ranges of above composition were 859.97-1385.71mg/kg,1438.95-2886.95mg/kg and 13.36-129.88mg/kg,respectively;their GCV were 6.80%,7.08%,and 16.76%,respectively;the heritabilities were 62.26%,69.68%,and 12.76%,respectively.The average content of Xanthine was 64.55mg/kg,the range of Xanthine was 21.47-197.26mg/kg.There were wide positive correlation between different purine content.Furthermore some soybean varieties with low purine content(Jindou25,He84-5,Gui119 and so on)were selected for future soybean breeding program.3.A total of 145558 genome-wide SNPs were identified by RAD-seq,among these,106125 SNPs had a minimum allele frequency(MAF)?0.01,which were used for organized 20205 SNP linkage disequilibrium block(SNPLDB)based on the soft of Haploview,those SNPLDBs include 13482 free SNPs and 6723 haplotypes;The length of halpotype ranged from 5bp to 200000bp,included 2-11 alleles.The linkage disequilibrium(LD)decay of 320 cultivated soybean varieties was 550kb based on 106125 SNPs;Phylogenetic tree and Principal component showed that cultivated soybean varieties had widespread penetration.4.According to the joint ANOVA result,the interaction of line and environment were significant for the all traits,for the content of total purine,Adenine and Guenine,their the variance of line were also significant,however,for the content ofHypoxanthine and Xanthine,their variance of line were not significant,this result indicated that content of Hypoxanthine and Xanthine had no real difference among different varieties.Therefore the GWAS was performed only in content of total purine,Adenine and Guenine.There were 68major QTLs showed association with total purine content,and they accounted 27.23%of phenotypic variance,unmapped QTL accounted 38.23%of phenotypic variance,the interaction of genotype and environment can account 29.34%of phenotypic variance.Then under the same method,we detected75 and 79 major QTLs association with Adenine,Guanine,respectively;they accounted 26.12%and 49.47%of phenotypic variance,respectively;unmapped QTL can account 36.14%and 20.21%of phenotypic variance,respectively;at the same time,the interaction of genotype and environment can account 31.32%and 25.13%of phenotypic variance respectively.By establishing QTL-allele matrices of different kind of purine content,we can discovery both negative and positive alleles existed in a germplasm,no matter it has the lowest or largest purine content,so purine content in soybean seed is a potential trait which can aggregated positive allelic variation.The whole population matrixs were separated into six eco-region matrices from the genetic differentiation among eco-region was found.Based on the matrice of total purine content,potential optimized crosses of low purine content were estimated,the result showed that we can breeding low purine content offspring in 51040 crosses,even over 1125.5mg/kg,it is lower than the lowest parent whose purine content is 1822.13mg/kg.