Genome-wide Association Study Of Isoflavone Content In Soybean Seeds Based On High-density SNP Chip

Isoflavones,with great value in foods,medicine,cosmetics,animal feeds and agriculture,are the product of secondary metabolites in higher plants.Selecting and breeding specific accessions with high isoflavone content is one of the main goals for soybean quality breeding.However,soybean isoflavone content is a complicated quantitive trait controlled and regulated by multiple genes and was easily affected by environmental factors.Thus,it is difficult to achieve breakthrough by applying traditional phenotypic-based breeding methods.The emergence and application of high-throughput genotyping technologies provide us with significant and powerful tools to investigate the molecular and genetic mechanism underlie the regulation and synthesis of soybean isoflavone.Meanwhile,these technologies are also important premise for the improvement of soybean isoflavone content on molecular level.In this study,we designed and developed a high-density SNP genotyping chip,NJAU 355K SoySNP,covering the whole genome.This high-density SNP array was evaluated and validated in 367 soybean accessions for genotyping.We identified SNPs related to isoflavone content in soybean seeds by performing genome-wide association study.GWAS was conducted by utilizing the natural population composed by 196 cultivated soybeans derived from those 367 accessions in two environments during one year.Meanwhile,we also predicted candidate genes and performed preliminary functional investigation.The aim of these researches was to explore the genetic basis of isoflavone biosynthesis of soybean seeds.Main results are as follows:Firstly,the high-density SNP array NJAU 355K SoySNP contains 355,595 SNPs covering the whole soybean genome.The 355,595 SNPs were validated in the 367 individuals(including 105 wild accessions and 262 cultivated accessions and the result of genotypic analysis shows that after filtering of quality control process,the distribution of the remaining 292,053 SNP markers was uneven along chromosomes.Exactly 39.47%SNPs were targeted to pericentromeric regions and the rest 60.53%were targeted to the arms of the 20 soybean chromosomes.Of the 292,053 SNPs,291,962 SNPs were mapped to chromosomes and the rest 91 SNPs were targeted to unanchored sequence scaffolds.Of the 291,962 chromosomal SNPs,the average SNP spacing was approximately 3.3 kb along the 20 chromosomes of soybean(975 Mb)and the inter-SNP spacings of 95.2%SNPs were shorter than 9 kb.Only 66 SNPs had a marker spacing of>100 kb.A total of 90,088(30.86%)of the 291,962 chromosomal SNPs resided in genes.We identified 13,444 non-synonymous SNPs in 11,407 genes,including 994 nonsense SNPs in 977 genes that resulted in start codon changes,premature stop or elongation of the transcripts.High quality SNP markers,accounting for 68.98%of the 54175 predicted genes,covered a total of 37,370 genes.215,657 of the 292,053 SNPs(73.84%)had MAF values greater than 10%among the soybean accessions.With the genotyping results,we conducted a population genetic analysis and we observed that cultivated soybeans originated from the northern and middle regions of China,from where it spread to other regions.The genome-wide scan revealed that altogether 1,128 genes exist in the regions with signals of artificial selection and all these genes are related to important agronomic traits concerning domestication.Secondly,we performed genome-wide association study of isoflavone content in soybean seeds by applying the high-density chip developed in present study and the SNP genotyping data derived from 196 cultivated accessions.The results revealed that extensive phenotypic variations existed in this natural population.For glycitin content,the maximum is 173 times the minimum.Although the effect of genotype,environment and the interaction between genotype and environment influence the content of isoflavone and other components significantly,the heritability of total isoflavone content,daidzin content,glycitin content and genistin content was high,from 67.8%to 83.8%,meaning that most of the phenotypic variations were related to genetic factors.The correlation analysis of total isoflavone content and three main components revealed that the content of those components showed significantly positive correlation other than the correlation between genistin content and glycitin content.By utilizing compressed mixed linear model(cMLM),we detected 43 significant SNP-trait associations,related to a total of 33 SNPs.We detected 40 significant SNP-trait associations in Nanjing,which is significantly greater than 3 associations in Nantong.Meanwhile,SNP-trait associations in Nanjing were different from those in Nantong.The associated SNPs were located on chromosome 5,6,11 and 20.Moreover,these SNPs formed clusters on chromosome 11 and 20.We detected that 15 SNPs were significantly associated with total isoflavone content,ten SNPs were significantly associated daidzin content and 18 SNPs were significantly associated with glycitin content.What's more,ten SNPs significantly associated with daidzin content were co-associated with total isoflavone content.Unfortunately,significant associations about genistin content were not detected.The contribution rate of these SNPs range from 10.54%to 14.53%.We performed annotation for the genes in the LD regions of these SNPs and found that identified structure genes of isoflavone sythesis pathway were not detected.However,we observed different types of transcription factors.What's more,only one SNP was detected to locate in the gene region and this SNP was co-associated with total isoflavone content and dadzin content,locating in the 5'-UTR of GmMYB29 transcription factor.This gene was orthologous to a trascription factor regulating isoflavone synthesis detected in Lotus,indicating that it is possible that this gene involves in the regulation of soybean isoflavone biosynthesis pathway.Thirdly,the preliminary functional investigation of the gene of GmMYB29 trascription factor showed that the gene located in the nucleus.In the leaves treated by GSH and common cutworm,the induced expression pattern of GmMYB29 and IFS2 was the same.IFS2 is one of the key enzymes involved in isoflavone synthesis.In different tissues of soybean,the accumulation of isoflavonoids closely associated with the expression of GmMYB29 and IFS2.Dual luciferase reporter assay system and transient expression revealed that GmMYB29 can incease the expression activity of IFS2 and CHS8.Promoter deletion analysis revealed that an element related to the full activation of the promoter located in the 208bp region(from-885 to-1093)of IFS2 promoter.Cis-acting elements prediction revealed that only one MYB binding element MYBCORE,indicating that GmMYB29 probably activate the expression of IFS2 by identifying and binding this cis-acting element.To further verify the function of GmMYB29 during the synthesis of isoflavone,we constructed over expression vector and RNAi interference vetor for GmMYB29 and then transformed to soybean hairy roots respectively.We then detected the increase of total isoflavone content in hairy roots that overexpress GmMYB29 while we observed the decrease of total isoflavone content in hairy roots with the RNAi interference vetor.The change was significant although the greatest change fold was 3.3.This provides us with the direct evidence that GmMYB29 regulates isoflavone synthesis.