| The soybean is very important economic crop.As early as 3,000~5,000 years ago,people have begun to domesticate soybean in China.Soybean seed size is a very important trait in the domestication process,so mining the genes control seed size has great significance to understand the evolution of soybean genes.Small marker number and low marker density in previous genome-wide association studies(GWAS)for seed size traits in soybean reduce their efficiency.The widely used methods in soybean seed size traits is single-locus GWAS,based on population structure and polygenic background controls.This is involved in multiple testing so that Bonferroni correction should be performed,which results in the missing of many significant association loci.In addition,the high level of linkage disequilibrium(LD)in soybean genomes hinders the pinpointing of the loci associated with seed size.Therefore,the studies on genes for seed size traits in soybean are limited,which are far behind those in Arabidopsis.To address this issue,in this study 286 soybean accessions,genotyped by 106013 high-density SNPs and phenotyped seed size traits in 10 groups combinations,were used to conduct GWAS using five multi-locus GWAS methods in order to dissect the genetic foundation of seed size traits in soybean and to determine their candidate genes.In addition,the elite alleles for QTNs identified in this study for seed size traits were mined for breeding by design.The main contents were as follows.Firstly,the loci detected many times were selected as robust QTNs.The intervals within 50 kb of several significant QTNs in same year and same trait were viewed as QTN region(QTR).Secondly,all the genes were picked up from the regions within 100 kb of the robust QTNs and QTRs.The differential expression analyses across soybean varieties and between early and mid-maturation stages in the seed development were conducted in order to select candidate genes associated with seed sizes.Thirdly,domesticated loci in previous studies were compared to the seed size-related loci in this study so as to obtain the domesticated loci associated with seed size traits.Finally,we mined the elite alleles of QTNs for seed size traits identified in this study,and these elite alleles were used to conduct breeding by design.The main results are as follows:1.Five Multi-locus GWAS methods was used to associate 106013 SNPs of 286 soybean accessions with 10 phenotypic datasets of seed length,width and thickness in soybean from 2008 to 2015 and 7 phenotypic datasets for soybean 100-seed weight from 20011 to 2015.With the critical LOD score of 3.0,as a result,433,406,395 and 311 QTNs were significantly associated with seed length,width,thickness and 100-seed weight,respectively.Among the above 1137 significant QTNs,59 QTNs were robust and 62 QTRs were found.Within 100 kb intervals of robust QTNs and the middles of QTRs,there were 900 genes.The QTNs detected at least 5 times for 100-seed weight and at least 7 times for seed size traits were viewed as high frequency QTNs.As a result,10 high frequency QTNs were found.2.The downloaded transcriptome datasets at 7 soybean seed development stages were used to determine high expression genes,in which their expression levels were 2 times greater than their average expression level.As a result,353 high expression genes were found.Among these genes,the differential expression analyses between two wild soybeans and two landraces(or bred cultivars)at 15,25,35 and 55 days after flowering were conducted by software DEGseq to determine candidate genes associated with seed size traits.As a result,141 genes had the significantly differential expressions at the 0.01 level,and the differential expression mainly occurred at 35 and 55 days after flowering.The 2680 differentially expressed genes between early and mid-maturation seed development stages in previous studies were compared with the above 141 genes.The results showed that 23 common genes existed.In addition,differential expression analysis and previous studies showed that,among 10 high frequency QTNs,3 were valuable.3.593 domesticated regions and loci in previous studies were compared with 59 soybean seed size QTNs in this study.As a result,17 QTNs for seed size traits in soybean were located on soybean domestication regions,being domestication loci.For the 17 domesticated QTNs,the average number of elite alleles in wild soybean,landraces and bred cultivars were 2.93,9.28 and 9.44,respectively.Among the above 23 genes for seed size traits,5 were close to the ten domestication loci.Among the five domesticated genes for seed size,3 may be related to seed development.4.Among 59 elite alleles of 59 QTNs for seed size traits in soybean,the numbers of elite alleles for seed length,width,thickness and 100-seed weight were 18,23,16 and 28,respectively,and 17 were common across several traits.Among 286 soybean accessions,the numbers of elite alleles for the above four tarits ranged 1 ~ 17,2 ~ 22,2 ~ 15 and 1 ~ 26,respectively,and the average number of elite alleles was 9.04,14.3,10.79 and 10.99,respectively.The correlation coefficients between the number of elite alleles for each tarit with its phenotypes was 0.789,0.803,0.765 and 0.75(P < 0.0001),respectively,being significant and positive correlation between the number of elite alleles and trait phenotype.The numbers of elite alleles in wild soybeans,landraces and bred cultivars were 14.5,33.97 and 35.34,respectively.Based on the above elite allele information,it was found that Yafanzaodou,Fujiandadou and Yixingwuhuangdou might be the excellent parents to improve the seed size traits.If we want to improve 100-seed weight,seed length,width and thickness,the cross combinations of Yafanzaodou × Pingyinyangyan,Yafanzaodou × Yixingwuhuangdou,Yafanzaodou × Fujiandadou and Hunanqiudou 1 × Yafanzaodou is available,which lets the number of elite alleles be 27,18,23 and 16,respectively.If we want to improve the four traits at the same time,the cross combination of Yafanzaodou × Rongxiandahuangdou is available,which let the number of elite alleles be 58. |
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