Identification of genes for seed isoflavones based on bulk segregant analysis sequencing in soybean natural population.

KEY MESSAGE: We identified four hub genes for isoflavone biosynthesis based on BSA-seq and WGCNA methods and validated that GmIE3-1 positively contribute to isoflavone accumulation in soybean. Soybean isoflavones are secondary metabolites of great interest owing to their beneficial impact on human health. Herein, we profiled the seed isoflavone content by HPLC in 1551 soybean accessions grown in two locations for two years and constructed two extreme pools with high (4065.1 µg g-1) and low (1427.23 µg g-1) isoflavone contents to identify candidate genes involved in isoflavone biosynthesis pathways using bulk segregant analysis sequencing (BSA-seq) approach. The results showed that the average sequencing depths were 50.3× and 65.7× in high and low pools, respectively. A total of 23,626 polymorphic SNPs and 5299 InDels were detected between both pools and 1492 genes with different variations were identified. Based on differential genes in BSA-seq and weighted gene co-expression network analysis (WGCNA), four hub genes, Glyma.06G290400 (designated as GmIE3-1), Glyma.01G239200, Glyma.01G241500, Glyma.13G256100 were identified, encoding E3 ubiquitin-protein ligase, arm repeat protein interacting with ABF2, zinc metallopeptidase EGY3, and dynamin-related protein 3A, respectively. The allelic variation in GmIE3-1 showed a significant influence on isoflavone accumulation. The virus-induced gene silencing (VIGS) and RNAi hairy root transformation of GmIE3-1 revealed partial suppression of this gene could cause a significant decrease (P < 0.0001) of total isoflavone content, suggesting GmIE3-1 is a positive regulator for isoflavones. The present study demonstrated that the BSA-seq approach combined with WGCNA, VIGS and hairy root transformation can efficiently identify isoflavone candidate genes in soybean natural population.

The GmSNAP11 Contributes to Resistance to Soybean Cyst Nematode Race 4 in Glycine max.

Soybean cyst nematode (SCN) has devastating effects on soybean production, making it crucial to identify genes conferring SCN resistance. Here we employed next-generation sequencing-based bulked segregant analysis (BSA) to discover genomic regions, candidate genes, and diagnostic markers for resistance to SCN race 4 (SCN4) in soybean. Phenotypic analysis revealed highly significant differences among the reactions of 145 recombinant inbred lines (RILs) to SCN4. In combination with euclidean distance (ED) and Δsingle-nucleotide polymorphism (SNP)-index analyses, we identified a genomic region on Gm11 (designated as rhg1-paralog) associated with SCN4 resistance. Overexpression and RNA interference analyzes of the two candidate genes identified in this region (GmPLAC8 and GmSNAP11) revealed that only GmSNAP11 significantly contributes to SCN4 resistance. We developed a diagnostic marker for GmSNAP11. Using this marker, together with previously developed markers for SCN-resistant loci, rhg1 and Rhg4, we evaluated the relationship between genotypes and SCN4 resistance in 145 RILs and 30 soybean accessions. The results showed that all the SCN4-resistant lines harbored all the three loci, however, some lines harboring the three loci were still susceptible to SCN4. This suggests that these three loci are necessary for the resistance to SCN4, but they alone cannot confer full resistance. The GmSNAP11 and the diagnostic markers developed could be used in genomic-assisted breeding to develop soybean varieties with increased resistance to SCN4.