Construction Of The High Density Linkage Map And QTL Mapping For The Major Isoflavone Components In Soybean

Soybean isoflavone is a kind of secondary metabolites and has been paid more attention because oftheir contribution to human health and plant defense systems. With the development of molecularmarkers and sequencing technology, the QTL for isoflavone is widely researched. In this study, wepredicted and analyzed the genetic variation of isoflavone–associated properties of96representativesoybean samples by using multivariate statistical analysis method. A high density genetic linkage mapwas also constructed based on the high-throughput sequencing technology in the soybean recombinantinbreed line population （RIL, F5:7-8） developed by the cross between soybean cv. LHD2（high-isoflavoneconcentration） and cv. NHZ （low-isoflavone concentration）. The QTLs associated with isoflavonecontent were detected by the ICIM mapping method. Moreover, the major hotspot gene loci forcontrolling isoflavone content were scanned based on the linkage disequilibrium （LD） associationanalysis using the SSR and SNP markers in the soybean representative core accessions. Therefore, wecan find the stable genes for controlling the isoflavone content. It can provide the guidance forisoflavone molecular marker-assisted breeding. The main results were as follows:1. Isoflavone determination using multivariate statistical analysis method in soybean seedsThe isoflavone components had abundant genetic variation in soybean seed, with a range ofcoefficient variation from33.07%to42.42%. There were significant differences in most isoflavonecomponents among different varieties, ecotype regions and years. And there also existed significantpositive correlation between years. It indicated the isoflavone content is controlled by both genetic andenvironmental factors in soybean seeds. However, the genetic factor still plays more important role insoybean breeding. Moreover, significant positive correlation was reported between isoflavone aglyconeand the corresponding components, the correlation coefficients were larger than0.897. It demonstratedthat the isoflavone components from the same biosynthetic pathway may be regulated by the commonkey enzymes, and in result that their correlations were relatively high. Otherwise, there were twoaccessions with high isoflavone content （ZDD03026and ZDD20051） and two with low isoflavonecontent （ZDD00046and ZDD00059） selected as the parents for soybean isoflavone breeding based onthe anlaysis of two years.The correlation analysis indicated that isoflavone content had significant positive correlation withplant height, effective branches, pods per plant, seeds per plant, linoleic acid, linolenic acid, whilesignificant negative correlation with oleic acid and oil content. As a result, a predictive regressionequation on isoflavone content was obtained by using principal component regression analysis as follow:y=^-1875.22+20.27×plant height+385.56×linoleic acid, which indicated the plant height and linolenicacid had a significant positive effect on isoflavone content. The equation can become a guidence for theisoflavone breeding.2. Mapping QTLs associated with major isoflavone components in RIL populationOne soybean linkage map with a total distance of3546.54cM was constructed using161 polymorphism SSR molecular markers in the RIL population. A total of14QTLs associated with themajor isoflavone components were found by the ICIM, IM and SMA mapping methods in theICIMapping3.2software under the four environments, which explained12.3933.07%of the totalphenotypic variation in soybean seeds. The three QTLs flanked by the marker interval Sat₀03Satt306,Satt070Satt122and Satt571Satt270were detected among multiple environments in more than twomethods.A high density genetic linkage map with a total distance of2255.18cM were also constructed using5785SNP markers, with an average distance between markers of0.39cM. A total of110QTLsassociated with the major isoflavone components were also found by ICIM mapping method in theICIMapping3.2software under the four environments, which explained3.6536.48%of the totalphenotypic variation in soybean seeds. The19marker intervals （6.9726.29%of the total phenotypicvariation） were recurring and6of them （8.3620.88%of the total phenotypic variation） were detectedto associate with the same component in more than two environments.3. Association analysis of hotspot gene loci for the isoflavone content in soybean representivecore accessionsWe analyzed the genetic diversity of soybean representive core accessions and made associationanalysis to locate the gene loci related to isoflavones by the GLM and MLM methods in TASSELsoftware. The17loci were associated with the same isoflavone components by both GLM and MLMmethods in Hubei. The22loci were associated with the same isoflavone components by both GLM andMLM methods in Guangxi. According to the results, there were three loci detected by both GLM andMLM methods in both sites.4. Comparaison of major QTLs among various methods in differient populaionsAccording to the results of SSR and SNP markers, we found that the marker Satt571associated withthe malonylglycitin and glycitein characters was labeled in the Mark934616Mark970087, which wasdetected to associate with the daidzein, total content of isoflavone and total content of isoflavoneaglycone characters in the E1environment. It indicated that the QTL was maybe the important locus forisoflavone biosynthesis and regulation.Accoring to both the QTLs in RIL and hotspot gene loci in natural population, we found that therewas a gene locus BARC-014285-01304associated with the malonylgenistin （MG） character labeled inthe similar marker interval Mark545859Mark57540and Mark542666Mark577437in chromosome14.It demonstrated that there was maybe existed some major genes related to the MG in chromosome14.Otherwise, in chromosome13, a locus BARC-043173-08548associated with the glycitin （GL） characterwas also labeled in the similar marker interval Mark136063Mark174156in RIL. It indicated thisinterval was maybe also the other important locus related to the GL character in soybean.