Natural Variation Analysis And Candidate Gene Identification Of Folates In Soybean Seeds

Folate,also known as vitamin B9,plays a vital role in human cellular division and metabolism.However,folate deficiency is a common issue,especially in developing countries,and staple crops such as wheat,maize,and rice have low folate content,contributing to micronutrient malnutrition.Biofortification is a promising strategy that aims to increase the nutritional content of crops through selective breeding,genetic modification,and other strategies.This intervention could help address nutrient deficiencies and improve the overall nutritional quality of crops,making them more sustainable and cost-effective for improving nutrition in low-income areas.Soybean[Glycine max(L.)Merrill],a legume with reported health benefits,is a major source of vegetable protein and oil consumed worldwide,with Brazil,the United States,Argentina,China,and India as the highest producers.Due to soybean’s genetic diversity and the availability of molecular tools for crop improvement,it is an ideal crop for biofortification.However,the complex methods for extracting and quantitatively analysing soybean folate have significantly impeded the progress of the molecular breeding of folate in soybean.To address this,the current study developed a novel method for extracting and quantifying soybean folate.This allowed for the systematic analysis of folate content in natural and recombinant inbred line populations of soybean,leading to the identification of candidate genes that are significantly correlated with folate content.This was achieved through the use of quantitative trait loci(QTL)mapping and genome-wide association analysis(GWAS)methods.Furthermore,the study examined the folate content and changes in other nutrients in the vegetable soybean,which provides valuable information for soybean nutritional quality breeding.The main findings of the study are highlighted as follows:1.Development of a high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)method to accurately detect and quantify seven folate vitamers in soybean Folate extraction method and quantification parameters were optimised for seven folate monoglutamates in soybean.HPLC-MS/MS was used for their qualitative and quantitative analysis.The specific optimisation parameters were as follows:an extraction buffer of p H 5.5 was used to extract folate components and sample was boiled for 15 minutes to maximize extraction efficiency.Enzyme treatment is crucial for accurate folate quantification as the naturally occurring folates are mainly in the form of polyglutamates.Single-enzyme treatment using a composite enzyme from rat serum and chicken pancreas was had advantages such as fast extraction,simple process,accurate quantification,and low background noise,compared to the tri-enzyme treatment.The optimised HPLC-MS/MS method used in this study was also successfully applied for the extraction and quantification of folate in selected legumes such as Lablab purpures,Vigna radiata,Vigna unguiculata,Phaseolus multiflorus,Pisum sativum,Vigna angularis,and Vicia faba.The folate values observed for these legumes were in line with previously reported values.2.Natural variation of soybean folates in a diverse germplasm and their correlation with other seed quality traitsIn this study,1551 soybean germplasm accessions were used to systematically analyse the folate contents in soybean germplasm from two different planting environments in Beijing and Hainan using the developed HPLC-MS/MS method.The results showed that the total folate contents in both environments exhibited wide natural variations,with a 10-fold difference in folate content between high and low folate soybean germplasm in Hainan and an 8-fold difference in Beijing.Soybean seed coat colour,seed size,ecological region of origin,and germplasm type significantly affected folate contents.We also identified superior soybean germplasm with high folate content consistent in both environments.In addition,we mapped the geographic distribution of folate content in different soybean germplasm.These findings provide new theoretical references and breeding materials for soybean folate molecular breeding.3.The nutritional composition of the vegetable soybean(Maodou)This study aimed to profile the nutritional composition of 12 soybean cultivars at different stages of development and conduct RNA-seq analysis during seed development,focusing on key biosynthesis enzymes for quality traits.The results showed that vegetable soybean(maodou)contains high amounts of moisture,protein,fatty acids,minerals and micronutrients,including folate and carotenoids.The isoflavone content of soybean also varied widely between cultivars.The study also found that soybean at the vegetable stage has higher contents of folate,tocopherol,and carotenoids than the mature stage.Additionally,key biosynthesis enzymes of quality traits were found to be differentially expressed during seed development,which may contribute to variations in nutritional content at different stages of seed development.Overall,this study suggests that soybean is an important source of various essential nutrients for human health.4.Identification of Quantitative Trait Loci for Seed Folate Content in SoybeanIn this study,a high-density linkage map of soybean constructed using genotype by re-sequencing data and recombinant inbred lines(RILs)derived from parents Zhonghuang 13(ZH13)and Zhonghuang35(ZH35)were used to map QTL for folate content in soybean seeds in four different environments.Using composite interval mapping,61 QTL related to folates were identified on 12 chromosomes,with phenotypic variance(R~2)values ranging from 1.68%to 24.68%.A major QTL cluster(q Fo-05)was found on chromosome 5,with a length of 992 kb and containing 134 candidate genes.Based on gene annotation and SNP haplotype analysis of q Fo-05,seven candidate genes were identified.RNA-seq analysis revealed that the expression pattern of a hemerythrin RING zinc finger gene(Glyma.05G237500)was significantly different from other genes,suggesting that this gene may play an important role in folate accumulation in soybean.5.Genome-wide association study(GWAS)of folates in a natural soybean populationIn this study,1551 soybean accessions from different sources were used to conduct a GWAS of folate content using both resequencing-based genotype data and folate content data from three environments to identify genetic regions and candidate genes associated with folate content in soybean.The results showed that 6383 SNP loci were significantly associated with soybean folate content,with 1915 and 1357 SNPs associated with 5MTHF and total folate content,respectively.Three chromosomal regions located on chromosomes 9,15,and 17 were identified to be consistently associated with folate content across multiple environments.Further analysis identified 24 candidate genes,including a folate biopterin transporter gene and a glutathione peroxidase gene,which may be involved in folate transport and biosynthesis.Single haplotype analysis revealed two highly significant SNPs in Glyma.17G223900,one of which was located in the promoter region.The mutant alleles of this significant SNP had higher folate contents than the wild-type allele,indicating that this gene may be an important candidate gene for regulating folate accumulation.6.Integrating QTL mapping and GWAS to identify common regions and candidate genes forfolate content in soybeanBy integrating the QTL and GWAS results,overlapping regions were identified on chromosomes 5,6,and 9,which contained 56 genes.Single locus haplotyping determined that 57 SNPs were significant for folate content across multiple environments,of which 24 SNPs belonged to Glyma.06G206000.Further analysis showed this gene had two missense variants which were significantly associated with folate contents in the natural soybean population.Gene annotation revealed that Glyma.06G206000 is a member of the DEAD/DEAH box RNA helicase family,which is involved in RNA metabolism and may indirectly contribute to folate accumulation.This is the first study to report on the relationship between the DEAD/DEAH box RNA helicase family genes and folate content in soybean,providing a new avenue for further research into their role in soybean folate metabolism.