Fine Mapping Of QTL QSW4.1 Relation To 100-Seed Weight Differentiation Between Wild And Cultivated Soybeans And Analysis The Function Of Its Candidate Genes

Soybean[Glycine max（L.）Merr.],originated in China,has a long history of cultivation.Soybean is rich in oil and plant protein,it is widely used in livestock feed,food security,medical health and other aspects,and plays an important role in our national economy.100-seed weight is a quantitative trait controlled by multiple genes.During the evolution from wild soybean to cultivated soybean,great changes have taken place in100-seed weight.By analyzing the genetic basis of 100-seed weight differentiation of wild and cultivated soybean,we can not only understand the molecular mechanism of soybean domestication,but also explore the key genes controlling the increase of 100-seed weight,so as to lay a foundation for broadening the genetic basis of cultivated soybean and improving soybean yield.Therefore,in the early stage of this study,we used cultivated soybean NN1138-2 as the recipient parent and wild soybean N24852 as the donor to construct a chromosome segment substitution line population Soja CSSLP1,which covering the whole genome of wild soybean.A 100-seed weight differentiation locus qSW4.1 was detected.On the basis of previous studies,a substitution line CL2922 carrying the target wild fragment was selected and backcrossed with recurrent parent NN1138-2 to construct a secondary population qSW4.1·BDP containing 219 lines.By increasing markers’density,the target locus was verified and fine mapped.Combined with transcriptome sequencing and genome re-sequencing of cultivated soybean NN1138-2 and wild soybean N24852,two key candidate genes were screened in the fine mapping region.Meanwhile,the key candidate genes GmSW4.1 were cloned from cultivated soybean NN1138-2 and wild soybean N24852,then the plant overexpression vector was constructed for Arabidopsis thaliana transformation.The main results are as follows:1.The secondary population qSW4.1·BDP was constructed and the existence of qSW4.1 was preliminarily verified.The substitution line CL2922 carried two wild fragments Sat＿140-Satt578-Satt607-Satt646-Sct＿191 on chromosome 4 of soybean,and Satt131-Sat＿223,located on chromosome 18 of soybean.Compared with the recurrent parent NN1138-2（18.01 g）,the substitution line CL2922 had smaller 100-seed weight（15.96 g）and smaller seed length,seed width and seed thickness.By backcrossing CL2922with NN1138-2,a secondary population qSW4.1·BDP was constructed.There were significant differences in 100-seed weight among three different environments,with the ranges of 11.49-18.48 g,13.79-20.23 g and 14.63-24.79 g,respectively.Single marker analysis showed that only sat＿140-Satt578-Satt646 was associated with 100-seed weight.These results indicated that the decrease of 100-seed weight of substitution line CL2922was caused by the 100-seed weight locus qSW4.1 in the wild segment sat＿140-Satt578-Satt646.2.By increasing the density of markers,qSW4.1 of 100-seed weight was fine mapped into a 49.1 kb region of chromosome 4.For target section sat＿140-satt578-satt646,18 pairs of SSR markers and 16 pairs of Indel markers were screened out from 92 pairs of SSR markers and 39 pairs of In Del markers,which were polymorphic among parents.Using the above polymorphism markers,the markers’density are increased,and a high density genetic linkage map was constructed,which contained 34 molecular markers,with a total length of 13.4 c M.Based on the above map,combined with the phenotypic and genotypic data,qSW4.1 was mapped to SSR04-0350～SSR04-0375,with the interval length of 516.6 kb.The allele from wild soybean had the additive effect of reducing 100-seed weight by 0.79-1.15 g.There are 13 recombinant individuals in the region of SSR04-0350～SSR04-0375,which are divided into 10 categories.The qSW4.1 is reduced to between ID6898922 and ID6948063,with a length of 49.1 kb.3.Glyma.04g082500 is a key candidate gene of qSW4.1.The predicted region of qSW4.1 was narrowed to between ID6898922 and ID6948063,including 7 predicted genes;The transcriptome sequencing results of 8 different tissues from cultivated soybean NN1138-2 and wild soybean N24852 showed that only Glyma.04g081900,Glyma.04g082200,Glyma.04g082300 and Glyma.04g082500 had high expression level（FPKM>2）,and there was differential expression between parents（2-fold or more）.According to the whole genome re-sequencing data of parents,only Glyma.04g082200 and Glyma.04g082500 had sequence differences among the four differentially expressed genes.Glyma.04g082500 plays an important role in carbohydrate distribution in corn.Therefore,Glyma.04g082500 was selected as the key candidate gene and named GmSW4.1,the gene function was verified.4.Preliminary analysis the function of GmSW4.1 gene.The CDS region of GmSW4.1 gene is 1470 bp in length and encodes 489 amino acids.Gene structure and evolution analysis showed that it encodes‘tocopherol cyclase’protein The CDS of GmSW4.1 gene in cultivated soybean NN1138-2 and wild soybean N24852 was cloned.It was found that there was non synonymous mutation in this gene between parents.The promoter of GmSW4.1 gene was cloned and 14 SNPs and 3 Indel sequences were found between the parents.The difference between-1349 bp and-1424 bp resulted in the change of parental action elements.Subcellular localization showed that the gene was expressed in the nucleus and cytoplasm.The over expression experiment of Arabidopsis thaliana showed that the T₁generation plants of GmSW4.1 were growing faster than wild plants,and the effect on 100-seed weight still needed further verification.