Natural Variation Analysis And Candidate Gene Identification Of Isoflavone In Soybean

Soybean[Glycine max(L.)Merrill]is an important crop grown worldwide and has been used as food and dietary supplement due to its nutritional properties and functional characteristics.Soybean is the primary source of protein,oil and bioactive components such as fatty acids,sugars and isoflavones.Soybean isoflavones are secondary metabolites of great interest because of their beneficial impact on human health.In this study,we profiled the isoflavone content in soybean mini core collection consists of 1551 soybean accessions grown in three locations across two years.A set of 1168 Chinese accessions from this mini core collection was used to investigate the influence of region of origin on isoflavone accumulation.Also,a set of 333 soybean cultivars comprised of 218 Chinese and 115 USA cultivars was taken from this diverse panel and profiled for protein,oil,fatty acids,soluble sugars and isoflavone to investigate the associations among different seed nutritional traits and also the variability of these traits in both countries' cultivars.Furthermore,BSA-seq approach based on two bulk pools of soybeans accessions was to identify the candidate genes involved in isoflavone biosynthesis pathways.The main results were as follows:Significant differences in isoflavone content were observed among the accessions,accession types,years of growth and ecoregions of origin.Total isoflavone(TIF)concentration of the soybean accessions ranged from 745?g g^-1 to 5253.98?g g^-1,which represents a 7-fold difference.The highest mean TIF concentration(2689.27?g g^-1)was observed in the Huang Huai Hai Valley Region(HR)accessions,followed by accessions from the Southern Region(SR)and Northern region(NR)with TIF concentration of 2518.91?g g^-1 and 1942.78?g g^-1,respectively.Thirty-five accessions were identified as elite soybean resources based on their higher TIF concentration(4024.74?g g^-1 to 5253.98?g g^-1).Pairwise correlation analysis showed significant positive correlations between individual isoflavones and TIF concentrations.Malonyldaidzin and malonylgenistin showed the highest correlations with TIF concentration(r=0.90 and r=0.92,respectively),whereas acetyldaidzin showed the lowest correlation.The main isoflavone components had significant negative correlations with latitude and longitude,indicating that the geographical origin of the accessions influenced their seed isoflavone composition.Based on principal component analysis,glycosides and malonylglycosides of isoflavones were the major discriminative components for the soybean accessions.The present study demonstrated the geographical distribution of soybean seed isoflavone concentrations across the main ecoregion of China.The identified soybean accessions with both high and low TIF concentrations,which are desirable materials for industrial uses and could also be used as parents to breed soybean lines with improved isoflavone quantity and composition in the seeds.Significant differences(p<0.05)were observed between the cultivars,countries and maturity groups for all the nutritional traits measured.Higher levels of oil(20.5%),linoleic acid(56.4%),sucrose(58.6 mg g^-1),total soluble sugars(109 mg g^-1)and total isoflavone(3161.8?g g^-1)were observed in USA cultivars,while higher levels of protein(42.4%)and oleic acid(23.2%)were observed in Chinese cultivars.From the principal component analysis,protein,oil,oleic acid,linoleic acid and isoflavone were identified as the major discriminative components in the soybean cultivars.Furthermore,correlation analysis presented various useful associations among the different nutritional traits.The nutritional traits varied in their associations with the geographical factors(latitude,longitude and altitude)indicating that the origin of the cultivars has a pronounced effect on the nutritional compositions.The identified unique cultivars can be utilized for various industrial uses and breeding programs aimed at improving soybean nutritional components.A total of 1551 natural population panel of diverse soybean accessions was phenotyped and used to create high and low isoflavone pools.Among the two extreme pools,the high isoflavone pool includes 50 accessions with a mean total isoflavone of 4258.75?g g^-1,and the low pool includes 51 accessions with a mean total isoflavone of 1240.71?g g^-1.Both pools with high and low total isoflavone content were used in BSA analysis to identify the candidate genes and the genomic variations between these pools.Based on BSA-seq approach 1492 genes with different variations were identified in two low and high isoflavone pools.Furthermore,the coexpression network analysis and gene annotations was performed and four candidate genes,Glyma.06G290400(IE3-1),Glyma.20G122200(IE3-2),Glyma.18G274000(IE3-3),Glyma.18G235300(IE3-4)located at different chromosomes were selected for further analysis.The identified candidate genes were validated by virus-induced gene silencing(VIGS)and hairy root transformation systems.The HPLC analysis revealed a significant decrease in total isoflavone(p<0.0001)of IE3-land IE3-3 silenced plants compared to control.While,qRTPCR results also showed a significant(p<0.0001)decrease in IE3-iand IE3-3 expression.The functional validation of these candidate genes revealed that IE3-land IE3-3 are the key genes that affects isoflavone accumulation in soybean.The present study demonstrated that combining of BSA-seq approach based on natural population,VIGS and hairy root transformation system can quickly and efficiently identify candidate genes involved in isoflavone biosynthesis in soybean.