Identification and Characterization of New Soybean Sources of Significantly Elevated Seed Stearic Acid with Resistance to Soybean Cyst Nematode Using Forward and Reverse Genetic Screening

Stearoyl-acyl carrier protein desaturase (SACPD), which has four isoforms in soybean: SACPD-A, -B, -C and –D, converts stearic acid into oleic acid, influencing and controlling the accumulation of stearic acid. The available soybean lines with high level of seed stearic acid are mostly susceptible to soybean cyst nematode (SCN), one of the most devastating pests in soybean production. We developed a new ethyl methanesulfonate (EMS) mutagenized SCN-resistant soybean cv. Forrest M2 population, which displayed many kinds of significant morphological phenotypes. A subset of the mutagenized population containing  about 1000 families (M3 and M4 lines), was screened using TILLING to identify  mutants within the SACPD genes, we identified three missense SACPD-D mutants: F2360 (G263E), F2374 (E37K), and F2487 (T297M), and all the three lines were measured to contain the same level of seed stearic acid as the wild type Forrest. Using a forward screening approach, we were able to identify four mutants: F605, F620, F714, and F813, all of which contain high level of seed stearic acid with an increased level of up to 2.4 times compared to the wild type parent Forrest used to develop the mutagenized population. Each of these four mutants was identified to carry one missense or nonsense mutation of SACPD-C: Q83* (F605), L79F (F620), D77N (F714), and P102L (F813), among them, Q83*, L79F, and P102L are the new SACPD-C alleles identified in soybean. However, none of the four mutants exhibited mutations in SACPD-A, SACPD-B, and SACPD-D except that two of them (F605 and F813) showed one identical silent mutation (P80=) in SACPD-D. The four mutants were all identified to be resistant to SCN.

In this study, we report for the first time three new missense and one nonsense mutations localized in the first side chain of the SACPD-C enzyme between the conserved E_105/76 and H_146/117 residues, which are essential for iron ion interaction. Furthermore, the alignment of SACPD-C protein sequences of these four mutants with those of other soybean lines and other plant species indicated that all the four SACPD-C mutations we identified in this work are localized in the highly new conserved amino acids positions. Put together, only the mutations of SACPD-C perfectly correlate with the alteration of soybean seed stearic acid phenotype, suggesting SACPD-C is most likely responsible for the accumulation of stearic acid in soybean seed. The identified mutants can be used as new SCN resistant soybean sources with high seed stearic acid contents for further breeding purposes.