Genetic Analysis Of Soybean Isoflavone Contents And Its Resistance To Soybean Mosaic Virus

Soybean[Glycine max（L.）Merr.]is originated in China and is widely planted all over the world.It is one of the important food and cash crops.It not only provides human with high-quality protein and high oil,but also contains isoflavones.Isoflavones are secondary metabolites from the metabolic pathway of phenylpropane,which mainly found in legumes,especially in soybean.For human,isoflavones have the functions of estrogen,preventing and treating various diseases and beauty.For plants,isoflavones are phytoantitoxins and are resistant to multiple pathogens.Isoflavones are also involved in the interaction between plants and microorganisms,and induce the formation of soybean nodule.Therefore,it is of great significance to increase the content of soybean isoflavones.Soybean mosaic virus（SMV）is a disease that seriously affects soybean yield and quality.Therefore,it is very important to improve the resistance of soybean to SMV.Many studies had shown that isoflavones can enhance plant resistance to pathogens,but the relationship between isoflavones and the resistance of soybean to SMV is not clear,so further research is needed.In this study,in order to mine target trait-related genes and analyze the relationship between soybean isoflavones and soybean resistance to SMV,phenotypic analysis,correlation analysis,and genetic analysis were performed on the isoflavone content in seeds,the isoflavone content in leaves of inoculated and non-inoculated SMV,and the disease rate of leaves in 211 cultivated soybeans.To create a high isoflavone material and further clarify the relationship between soybean isoflavones and soybean resistance to SMV,isoflavone competition pathway genes GmF3H1,GmF3H2 and GmFNSII-1 in soybean were simultaneously mutated using CRISPR/Cas9 multiple editing system.The main results of this study are as follows:1.Phenotypic data of total isoflavone content（TIC）and individual isoflavone（daidzein DAC,genistein GEC and glycitin GLC）of 211 cultivated soybeans seeds in five environments showed that the content of isoflavones in soybean seeds varied widely.The traits were normal or approximately normal distribution,and were affected by genotype,environment and the interaction between genotype and environment.Except that genistein and daidzein were not significantly correlated,there were significant positive correlations among other components.The results of genome-wide association analysis（GWAS）showed that total isoflavone content and individual isoflavone content involved 77 significant SNPs,mainly distributed on chromosomes 1,4,5,6,11,15,17,19 and 20.There were 26 significant SNPs on chromosome 11 that were repeatedly associated in different environments,of which5 SNPs were repeatedly associated in three environments.Excellent haplotype and SNP identification of these five SNPs revealed that AX-93936943-G and AX-93788689-T were excellent SNPs,and Hap1（GT）was an excellent haplotype.Gmgly1 may be a candidate gene related to isoflavone content by functional annotation and gene expression pattern analysis of the candidate genes.2.Phenotypic data of total isoflavone content（TIC）and individual isoflavone（DAC,GEC,GLC）which were non-inoculated and inoculated with SMV and disease rate（DR）of leaves in three environments showed that all traits had a wide range of phenotypic variations.These traits were affected by genotype,environment,genetype and environment interactions.In addition,isoflavone content was also affected by SMV treatment.The results of correlation analysis showed that there was no significant correlation between daidzein and genistein after SMV inoculation,while the others showed significant positive correlations.There were significant positive correlation between soybean seed total isoflavone content,daidzein,genistin and genistin in leaves.Compared with non-inoculated SMV,there was a significant correlation（p<0.01）between total isoflavone content and disease rate at the third week.The correlation between genistein content and disease rate at the third and fourth weeks was extremely significant（p<0.01）,showing that SMV induced synthesis of isoflavones.The GWAS results of the isoflavone content in the leaves of inoculated and non-inoculated SMV showed that total isoflavone content and individual isoflavone involved 100significant SNPs,which were mainly distributed on chromosomes 2,3,4,6,7,8,9,10,13,15 and 18.The 58 SNPs located on chromosome 10 were distributed in clusters and were significantly associated with all the traits of multiple environments.Functional annotation was performed on the candidate regions of repeated associations,and potential genes related to isoflavone synthesis were found:genes of the P450 family,UDP-glycosyltransferase family,glutathione S-transferase family,transcription factors,and R genes related to disease-resistant function,heat shock protein,etc.The GWAS results of the disease rate showed that 115 significant SNPs were involved in three environments and BLUP values.57 significant SNPs has been repeatedly detected in different environments.There are 56 SNPs located in chromosome 15 and showed a clustered distribution,and four SNPs were simultaneously detected in E2 and E3 and BLUP.The genes function annotation of candidate region included genes of the heat shock protein family and the UDP-glycotransferase family,etc.There was an SNP on chromosome 13,which was significantly associated with the disease rate of E3 environment and BLUP of three environments in the fourth week.3.Isoflavone competition pathway genes GmF3H1,GmF3H2 and GmFNSII-1 were simultaneously edited in soybean using the CRISPR/Cas9 multiple editing system.The single gene editing efficiency in the T₀ transgenic material was as high as 81.48%,and the mutative frequency of three genes was as high as 44.4%,and the mutations could be stably transmitted to the descendants.The metabolome data showed that the isoflavone content of the triple mutants was significantly higher than that of the wild type.The results of phenotypic identification of progeny showed that the isoflavone content of the T₃ homozygous triple mutant lines of SMV inoculated or non-inoculated significantly increased compared with that of the wild type.Transcriptome data showed that compared with non-inoculated SMV,genes related to isoflavone biosynthesis were significantly up-regulated in both wild-type and two homozygous triple mutant lines,suggesting that SMV could induce the expression of genes related to isoflavone biosynthesis.After inoculation of SMV,compared with wild type,Glutathione（GSH）metabolism related genes of two triple mutant lines were significantly upregulated.Further analysis showed that the SMV CP protein content of the two triple mutant lines were significantly decreased,indicating that the knockout of the isoflavone competition pathway genes increased the biosynthesis of soybean isoflavone,which induced the expression of genes related to GSH synthesis,and thus improved soybean resistance to SMV.