Soybean is the second largest source of oil worldwide. Developing soybean varieties with high levels of oleic acid is a primary goal of the soybean breeders and industry. Edible oils containing high level of oleic acid and low level of linoleic acid are considered with higher oxidative stability and can be used as a natural antioxidant in food stability. All developed high oleic acid soybeans carry two alleles; GmFAD2-1A and GmFAD2-1B. However, when planted in cold soil, a possible reduction in seed germination was reported when high seed oleic acid derived from GmFAD2-1 alleles were used. Besides the soybean fatty acid desaturase (GmFAD2-1) subfamily, the GmFAD2-2 subfamily is composed of five members, including GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E. Segmental duplication of GmFAD2-1A/GmFAD2-1B, GmFAD2-2A/GmFAD2-2C, GmFAD2-2A/GmFAD2-2D, and GmFAD2-2D/GmFAD2-2C have occurred about 10.65, 27.04, 100.81, and 106.55 Mya, respectively. Using TILLING-by-Sequencing+ technology, we successfully identified 12, 8, 10, 9, and 19 EMS mutants at the GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E genes, respectively. Functional analyses of newly identified mutants revealed unprecedented role of the five GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E members in controlling the seed oleic acid content. Most importantly, unlike GmFAD2-1 members, subcellular localization revealed that members of the GmFAD2-2 subfamily showed a cytoplasmic localization, which may suggest the presence of an alternative fatty acid desaturase pathway in soybean for converting oleic acid content without substantially altering the traditional plastidial/ER fatty acid production.

Soybean [Glycine max (L.) Merr.] seed is a valuable source of protein and oil worldwide. Traditionally, the natural variations were heavily used in conventional soybean breeding programs to select desired traits. However, traditional plant breeding is encumbered with low frequencies of spontaneous mutations. In mutation breeding, genetic variations from induced mutations provide abundant sources of alterations in important soybean traits; this facilitated the development of soybean germplasm with modified seed composition traits to meet the different needs of end users. In this study, a total of 2366 ‘Forrest’-derived M2 families were developed for both forward and reverse genetic studies. A subset of 881 M3 families was forward genetically screened to measure the contents of protein, oil, carbohydrates, and fatty acids. A total of 14 mutants were identified to have stable seed composition phenotypes observed in both M3 and M4 generations. Correlation analyses have been conducted among ten seed composition traits and compared to a collection of 103 soybean germplasms. Mainly, ethyl methanesulfonate (EMS) mutagenesis had a strong impact on the seed-composition correlation that was observed among the 103 soybean germplasms, which offers multiple benefits for the soybean farmers and industry to breed for desired multiple seed phenotypes.

Resistance to soybean cyst nematodes (SCN) in “Peking-type” resistance is bigenic, requiring Rhg4-a and rhg1-a. Rhg4-a encodes a serine hydroxymethyltransferase (GmSHMT08) and rhg1-a encodes a soluble NSF attachment protein (GmSNAP18). Recently, it has been shown that a pathogenesis-related protein, GmPR08-Bet VI, potentiates the interaction between GmSHMT08 and GmSNAP18. Mutational analysis using spontaneously occurring and ethyl methanesulfonate (EMS)-induced mutations was carried out to increase our knowledge of the interacting GmSHMT08/GmSNAP18/GmPR08-Bet VI multi-protein complex. Mutations affecting the GmSHMT08 protein structure (dimerization and tetramerization) and interaction sites with GmSNAP18 and GmPR08-Bet VI proteins were found to impact the multi-protein complex. Interestingly, mutations affecting the PLP/THF substrate binding and catalysis did not affect the multi-protein complex, although they resulted in increased susceptibility to SCN. Most importantly, GmSHMT08 and GmSNAP18 from PI88788 were shown to interact within the cell, being potentiated in the presence of GmPR08-Bet VI. In addition, we have shown the presence of incompatibility between the GmSNAP18 (rhg1-b) of PI88788 and GmSHMT08 (Rhg4-a) from Peking. Components of the reactive oxygen species (ROS) pathway were shown to be induced in the SCN incompatible reaction and were mapped to QTLs for resistance to SCN using different mapping populations.

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing⁺ (TbyS⁺), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS⁺ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS⁺, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.