Development and Characterization of Soybean with Improved Fatty Acid Profile Through Mutation Breeding and Genome Editing

Soybean (Glycine max (L.) Merrill) is considered a key crop of modern agriculture due to its seed’s high protein and oil content. However, the high percentage of polyunsaturated fatty acids in soybean oil lim¬its its stability and shelf life. Modification in the fatty acid composition can improve its stability and nutritional quality. Mutagenesis is a useful tool to induce genetic and phenotypic variation for trait improvement and discovery of novel genes. A mutant population of soybean cultivar JTN-5203 (MG V) was generated using ethyl methane sulfonate (EMS) mutagenesis and was for screening novel fatty acid alleles by using forward and reverse genetics approaches. Optimum concentration of EMS was used to treat 15,000 bulk JTN-5203 seeds producing a total of 1,820 M2 population. Fatty acid profiles such as oleic acid, linoleic, and linolenic acid were measured in more than 5900 M2 plants using near-infrared spectroscopy. DNA was extracted, normalized and pooled from these individuals, and specific primers were designed for genes involved in fatty acid biosynthesis pathway for further analysis using Sanger and next-generation sequencing technologies. Forward genetics screening revealed mutants that contained either an elevated oleic acid or a reduction in linoleic or linolenic acid content, and mutants that carried all the three important phenotypes. Single nucleotide polymorphisms were observed in the genes for fatty acid biosynthesis pathway including, FAD2-1B, FAD2-2B, and FAD2-2C. Mutants with a range of genetic variations were also identified. In addition, the high throughput mutation discovery through TILLING-by-Sequencing approach has been successfully applied. Several mutations were detected that have consequences on FAD2-1A and FAD2-1B gene functions. This includes GC to AT transition mutations that consist of 15.89% in FAD2-1A and 69% in FAD2-1B. Through mutagenesis and high-throughput sequencing technologies, novel alleles underlying the mutations observed in mutants with reduced polyunsaturated fatty acids will be identified, and these mutants can be further used in breeding soybean lines with improved fatty acid profile thereby developing an improved and heart-healthy-soybeans. Moreover, a modified version of Stupar’s whole plant transformation from half-seed explant in soybean has been used for genome editing, and putative transformants were successfully recovered