Soybean TILLING-by-Sequencing + reveals the role of novel GmSACPD members in the unsaturated fatty acid biosynthesis while maintaining healthy nodules.

Soybean is a major source of protein and is the second largest source of oil worldwide. Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Unlike other polyunsaturated fatty acids, the saturated stearic acid is desirable for nutrition because it does not exhibit cholesterolemic effects on human health. Most developed high stearic acid soybeans carry different GmSACPD-C mutated alleles. However, due to its dual role in both seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value as a result of defective nodulation. The soybean Stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis shows the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. However, GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. In the present study, we developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing +) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 EMS mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants reveals an unprecedented role of the three GmSACPD-A, GmSACPD-B, and GmSACPD-D members in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This new discovery will positively impact the development of high stearic acid lines to enhance soybean nutritional value without potential developmental tradeoffs.