Major Gene Mapping And Analysis For Isoflavone Accumulation In Soybean(Glycine Max L.)

Soy isoflavone,a class of polyphenolic compounds exclusively occurred in legumes,is an important bioactive compound for both plants and human beings.The contents of seed isoflavone is a typical quantitative trait,significantly influenced by both environmental and genetic factors.The QTL mapping method is the major approach to investigate the genetic background of isoflavone.However,since the deficiencies of this method and the underdeveloped DNA sequencing technology in the previous study,most reported QTL are environment dependent loci,which is hard for further research and application.The transcriptome analysis is another useful means for understanding the molecular mechanism of isoflavone accumulation.But there are two barriers in this way: high cost and lack of integration.Although the outline of isoflavone biosynthesis pathway has been mapped for several decades,some questions are still unresolved,such as the accumulation pattern of isoflavone compounds in developing soybean seeds,and the dynamic variation of major genes influencing isoflavone biosynthesis in different development stages.For solving the above problems,we initially mapped novel QTL and meta-QTL associated with isoflavone contents.Based on the cultivars ZD27(high isoflavone contents)and HF25(low isoflavone contents)as parents with the pedigree bulk single-seed descent,the ZH RIL population(F6),a recombinant inbred lines population was constructed.The ZH RIL was planted in Harbin,Hailun and Mudanjiang in 2020.The HPLC was used to determine the individual and total isoflavone contents,and the high-density genetic map with was constructed via whole genome resequencing.Based on the genetic map and phenotype data,the composite interval mapping and RQTL package were selected to map novel QTL.Meanwhile,the current research also used meta-analysis methods and Bio Mecator program to collect and integrate the reported QTL.In this study,a RIL population with 162 lines was obtained,and a high-denstiy genetic map of 2487.17 c M,including 1093273 个SNP,was constructed.A total of 27 novel QTL associated with individual and total isoflavone contents was mapped,including two stable QTL which exhibited high LODs in all experimental locations,and eight sub stable QTL which gave high LODs in 2 experimental locations.With the combination of gene functional annotation,159 candidate genes were predicted in the study.In addition,we mapped 71 meta-QTL from 291 original QTL collected from the previous studies.The interval length of these meta-QTL was obviously smaller than the origin QTL,suggesting a significantly high accuracy of meta-QTL.For the first time,the meta-analysis method was applied in investigating the genetic background of isoflavone.Additionally,the dynamic variation of isoflavone contents and whole genome expression profile were characterized in this study.We selected the parental lines and six representative lines of ZH RIL in this research.During R5 to R8,we collected the seed of these lines every seven days(ten samples of each line were collected).The HPLC was used to determine the phenotype,and the second-generation transcriptome sequencing was utilized to perform RNA-seq.Meanwhile,based on the meta-analysis method,we collected reported RNA-seq data of soybean seed from the public database.The candidate genes were generated by performing DE analysis,WGCNA and time series analysis algorithms to analyze these data,and the major genes were obtained by combining the candidate genes and the results of QTL mapping.In this study,the dynamic variation and accumulation pattern of isoflavones in developing seed were revealed based on 80 samples.A total of 28 novel major genes were also obtained based on 444.5G RNA-seq data and 700 G meta data.The research further investigates the isoflavone biosynthesis pathway,and analyzes the dynamic variation of major genes influencing isoflavone accumulation in developing seeds.This study provides theoretical basis for revealing the genetic background of isoflavones,as well as molecular design and breeding of soybean with high isoflavones.