Gene Cloning And Functional Identification Of Soybean Isoflavones Based On Genome-wide Association Analysis

Isoflavone not only involved in the regulation of plant growth and defence but aslo plays an important role in human health.Soybean is regarded as the important source of natural isoflavone.In order to meet the market demand for soybean isoflavone,breeding for soybean varieties with higher content of isoflavone is one of the essential goals for breeders.Therefore,a total of 180 soybean accessions were used for content of isoflavone,daidzein,glycitein and genistein in seeds under multitude environments Harbin,Shenyang and Changchun in 2015 and 2016,and to study genotyping of high-density SNP via SLAF-seq technology.Genomewide association study(GWAS)was used to find loci associated with soybean isoflavone content.Analysis of genetic loci of isoflavones among different conditions,the beneficial alleles of isoflavones contents and even the cloning and preliminary analysis of functions of the candidate genes associated with isoflavones were well discussed.These studies would provide a theoretical basis for breeding novel high-quality soybean varieties with higher content of isoflavone.The main results were as follows,1.Evaluation and analysis of isoflavones in grains of multiple environmental soybean germplasm resourcesThe phenotypic values of soybean isoflavonesin different locations and in different years showed a continuous unimodal distribution,which basically accorded with the normal distribution characteristics.The variation range of the three kinds of isoflavones in different environments varied very little and the coefficient of variation was less affected by the environments and the years.There was a significant positive correlation between total isoflavones and daidzein,glycitein,and genistein.The increasing and decreasing rate of the accumulation of isoflavones in the high isoflavone variety ‘Zhongdou 27' and the low isoflavone variety ‘Jiunong 20' were difference,but the trends of dynamic accumulation of isoflavones in different varieties were basically the same.2.Genome-wide association anylasis of total isoflavones and the components in soybean accessionsA total of 244 SNPs associated with soybean seed isoflavones were detected.One of the associated SNP ‘rs42031764',located on Chr.08 was found associated with daidzein and total isoflavone conteng and was detected repeatedly among multiple experiments.It was believed that ‘rs42031764' was a stable locus in different environments and might be an important one in controlling the content of isoflavone in soybean.Fourteen candidate genes with a distance less than 20 Kbp to ‘rs42031764' were selected.The results showed that the expression patterns of candidate genes,including Glyma.08G309500 and Glyma.08G310300,were quite different in ‘Zhongdou27'(high isoflavone content)and ‘Jiunong20'(low isoflavone content).The gene annotations were protein kinase and zinc finger structure,respectively.It was speculated that these two genes might participate in the synthesis of isoflavones.3.Preliminary functional identification of candidate genes Glyma.08G309500 and Glyma.08G310300The isoflavone content in Glyma.08G309500 transgenic roots was significantly higher than that of in wild type.However,there was no significant difference of the isoflavones content in the Glyma.08G310300 transgenic roots compared with the control.Thus,it was supposed that the candidate gene Glyma.08G309500 might associate with isoflavone synthesis of soybean.The isoflavone content in Glyma.08G309500 transgenic roots was significantly lower than that of control.There was no significant difference in isoflavone content between the Glyma.08G310300 transgenic roots and the control.It was further confirmed that Glyma.08G309500 might be involved in the synthesis of soybean isoflavones though the study on mutant roots.4.Functional verification of Gm MPK20-6According to the results of gene sequence comparison,Glyma.08G309500 gene has high homology with Arabidopsis At MPK20 gene,therefore,bioinformatics analysis of Glyma.08G309500,also called Gm MPK20-6,were carried out and predicted that Gm MPK20-6 was related to stress and endosperm synthesis.The Gm MPK20-6 was transformed into ‘Dong Nong 50' soybean cotyledonary node by Agrobacterium tumefaciens method.Three stable transgenic lines in T3 generation were obtained.The total isoflavone content,daidzein,and genistein in the three transgenic lines were significantly higher than those in the control group,indicating that Gm MPK20-6 gene plays a positive regulatory role in the synthesis of isoflavones in soybean grains.The resistance of the three transgenic lines was significantly higher than that of the control.It was speculated that the Gm MPK20-6 gene might increase soybean resistance to biotic stress by regulating isoflavone content.