Abstract
Soybean seed oil typically contains 18–20% oleic acid. Increasing the content of oleic acid is beneficial for health and biodiesel production. Mutations in FAD2-1 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 FAD2 genes. Although no fad2 mutants were identified using gel-based TILLING, four fad2-1A and one fad2-1B 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, FAD2 are present as two copies, FAD2-1 and FAD2-2. Two FAD2-1 members: FAD2-1A and FAD2-1B; and three FAD2-2 members: FAD2-2A, FAD2-2B, and FAD2-2C have been reported. Syntenic, phylogenetic, and in silico analysis revealed two additional members constituting the FAD2 gene family: GmFAD2-2D and GmFAD2-2E, located on chromosomes 09 and 15, respectively. They are presumed to have diverged from other FAD2-2 members localized on chromosomes 19 (GmFAD2-2A and GmFAD2-2B) and 03 (GmFAD2-2C). 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 FAD2 gene family in soybean.
Highlights
The fatty acid desaturase-2 enzyme (FAD2-1) is responsible for the conversion of oleic acid to linoleic acid in the developing soybean seeds (Okuley et al, 1994; Schlueter et al, 2007)
FAD2-2 members have not been linked to oleic acid content, FAD2-1 has been reported to control the high oleic acid content of certain soybean lines (Hoshino et al, 2010; Pham et al, 2010, 2011)
Five fatty acid desaturase (GmFAD2) gene family members were previously reported to be located on chromosomes 10 (GmFAD2-1A; Glyma.10G278000), 20 (GmFAD2-1B; Glyma.20G111000), 19 (GmFAD2-2A; Glyma.19G147300), 19 (GmFAD2-2B; Glyma.19G147400), and 03 (GmFAD2-2C; Glyma.03G144500) (Schlueter et al, 2007; Pham et al, 2010; Zhang et al, 2014)
Summary
The fatty acid desaturase-2 enzyme (FAD2-1) is responsible for the conversion of oleic acid to linoleic acid in the developing soybean seeds (Okuley et al, 1994; Schlueter et al, 2007). It has been reported that five FAD2 members are present in four different loci and constitute the fatty acid desaturase family in the soybean genome (Schlueter et al, 2007; Bachlava et al, 2008; Pham et al, 2010; Zhang et al, 2014). Many multifunctional FAD2 enzymes have been characterized These include the bifunctional hydroxylase/desaturase activity from Lesquerella fendleribroun (Broun et al, 1998), and trifunctional acetylenase from Crepis alpina, which catalyzes the formation of both trans and cis double bonds at the 12 position of oleic acid (Carlsson et al, 2004). FAD2-2 members have not been linked to oleic acid content, FAD2-1 has been reported to control the high oleic acid content of certain soybean lines (Hoshino et al, 2010; Pham et al, 2010, 2011)
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