Application Of High Resolution Melting Analysis In SNP Genotyping Of Key Enzymes Genes In The Biosynthesis Of Soybean Isoflavones

Isoflavones is a crucial secondary metabolite in metabolism of soybean， which hasimportantly impressed on human health.It plays a key role in the prevention and treatment ofosteoporosis sickness, cardiovascular disease and cancer.Different Isoflavones content were found in Different varieties of soybean,so it is veryimportant to study key enzymes genes in the biosynthesis of soybean isoflavones.SNP is animportant genome–wide genetic markers with high genetic stability.SNP is identification ofallelic variants in natural populations by Ecotilling which has become a rapid SNP discoverymethod, but it is not convenient for detection. High Resolution Melting (HRM) analysis is a rapid,sensitive, and high-throughput method that can be used to examine the types of SNP. The resultswere as follows.（1）In this study,we established the genomic library of3000different soybeans varieties andpresented an optimum method of soybean genomic DNA extraction.Comparison of threemethods of genomic DNA extraction from soybean,we finally decided to use CTAB DNAextraction method.Determination of content of Isoflavones in256different soybeansvarieties by HPLC to provide a basis for SNP Genotyping and phenotype data.（2）In this paper, HRM techniques was used in Ecotilling of256soybean cultivars to detect theSNP of IFS1exons. The results of HRM were verified by direct sequencing. The effect ofhomogenization and non-homogenization of the template on the testing was discussed andresults showed that the former had more remarkable effect. The testing effect of differentproportions of mixed samples was compared and we found that1:1and1:4mixed sampleswere optimal. The comparison between the testing results of exon regions of IFS1by HRMand results of PCR products sequencing showed the accuracy of fifth and seventh sectionsof exons were91.2%and100%, respectively.（3）Five soybean varieties with the highest content of isoflavones and five soybean varieties withthe lowest content of isoflavones were chosen from determination of content of isoflavonesin256different soybean varieties. Gene sequencing indicated thatSNPs of key enzymesgenes in the biosynthesis of soybean isoflavones.we totally found7SNPs and introduced HRM method for SNP genotyping in soybean.It demonstrates that the soybean-cultivatingEcotilling established by the use of HRM is a new method of testing SNP with highsensitivity, high throughput and low cost.