Characterization of the Gene Family in Soybean Reveals the Limitations of Gel-Based TILLING in Genes with High Copy Number.

Soybean seed oil typically contains 18-20% oleic acid. Increasing the content of oleic acid is beneficial for health and biodiesel production. Mutations in genes have been reported to increase seed oleic acid content. A subset of 1,037 mutant families from a mutagenized soybean cultivar (cv.) Forrest population was screened using reverse genetics (TILLING) to identify mutations within genes. Although no mutants were identified using gel-based TILLING, four and one mutants were identified to have high seed oleic acid content using forward genetic screening and subsequent target sequencing. TILLING has been successfully used as a non-transgenic reverse genetic approach to identify mutations in genes controlling important agronomic traits. However, this technique presents limitations in traits such as oil composition due to gene copy number and similarities within the soybean genome. In soybean, are present as two copies, and members: FAD2-1A and FAD2-1B; and three members: , and have been reported. Syntenic, phylogenetic, and analysis revealed two additional members constituting the gene family: and , located on chromosomes 09 and 15, respectively. They are presumed to have diverged from other members localized on chromosomes 19 ( and ) and 03 (). This work discusses alternative solutions to the limitations of gel-based TILLING in functional genomics due to high copy number and multiple paralogs of the gene family in soybean.