Genome-Wide Association Studies Of Soybean Mature Seed Hardness

Soybean is one of the most important sources of nutrition,it provides plant oil and protein for humans,poultry and livestock.Food made of soybean seed is traditional in Asia and benefits Asian’s health and is becoming increasingly popular worldwide.Soybean seed hardness plays an important role in the quality of soyfood and affects the processing preparation and the taste of soyfoods.Hard seeds delay germination and inhibit processing and vegetable soybean benefit from soft seeds.Based on researching the genetic mechanisms of soybean seed hardness,breeding efficiency will be improved.To clarify the genetic basis of soybean seed hardness,we measured the seed hardness of 216 accessions from Chinese soybean mini core collection,and analyzed the changes in seed hardness,protein content and oil content over seven eco-regions and two subgroups,correlation coefficients of seed hardness and other quality traits and the factors affected seed hardness.The genome-wide studies were conducted using 1514 SNPs based on the multi-locus random-SNP-effect MLM approach to search the SNPs which were significantly associated with seed hardness.Based on the LD decay of SNP marker,we selected candidate genes and analyzed their relative expression.The main results of this research are as follows:To evaluate the soybean seed hardness,two indexes(Fm and H)were measured using the texture analyzer in 216 soybean mini core collection over four years(2011-2014).Fm was the maximum force needed to break the seeds,Hwas expressed as the gradient of the rate of change of the force and moment from 0.3-0.5 s,and they displayed normal variation basically.There were extensive phenotypic variations in the two indexes,and the range of the mean value of Fm was 17268.2N-18451.9N,the range of the mean value of H was 28702.8N/s-29559.5N/s.The Pearson correlation coefficients of Fm and H were significant in four environments and the broad heritability was greater than 86%.An analysis of variance showed significant(P<0.0001)effects of genotype(G),year(Y)and G×Y interactions on both Fm and H.The mini core collection was divided into seven eco-regions and two subgroups.The results of our study suggested that seed hardness had a positive relationship with eco-regions from north to south and with the seeding seasons from spring to autumn.The Fm and H values of subgroup 2,which mainly comprised accessions from the south region,were higher than those of subgroup 1.To explore the relationship between seed hardness and other soybean main quality traits including seed protein,oil content and moisture,we used a grain analyzer to measure these three phenotypes of the soybean mini core collection grown in 2012,2013 and 2014.The ranges of the protein content of the soybean mini core collection in the three years were 38.0-49.7%,34.5-50.2%and 35.0-47.1%,respectively;and oil content were 14.1-21.6%,13.7-24.1%,and 16.4-24.6%,respectively.The changes in protein content over seven eco-regions and two subgroups were consistent with the changes in seed hardness,while the changes in seed oil content were opposite to the changes in seed hardness.Soybean seed hardness affected by the eco-regions and subgroups,and had a significantly positive correlation with seed protein content and had a significantly negative correlation with seed oil content.Association analyses of 1514 SNPs which related to lipid metabolism with the Fm and H values of the 216 mini core collection used the mrMLM approach.A total 10 SNPs were significantly(P≤0.0002)associated with Fm.The same procedures were followed for H.Ten of these SNPs were significantly associated with H.The mrMLM approach was also used to identify SNPs associated with the oil content of the soybean seeds in 2012-2014.Six SNPs were significantly associated with the seed oil content.Based on the LD decay,we selected twenty-five candidate genes for soybean seed hardness in the region near SNP marker including map-2655,Q-15-0087770 and map-3291.The functions of these genes were predicted using the SoyBase and Phytozome database and selected 24 candidate genes based on previous studies and gene annotations.Five candidate genes were chosen to analyze their relative expression between the high and low seed hardness accessions in soybean stages of development(R5,R6 and R7).Glyma.15G147800 where Q-15-0087770 located on,and Glyma.15G149800 appeared to be related to soybean mature seed hardness.In summary,genome-wide association studies of soybean mature seed hardness were conducted in this research.The results of this study provide a comprehensive understanding of the genetic basis of our target traits and new insights into advanced marker-assisted selections for breeding soybeans for soft seed hardness.