Development and characterization of low α-linolenic acid Brassica oleracea lines bearing a novel mutation in a ‘class a’ FATTY ACID DESATURASE 3gene

Stacy D Singer, Randall J Weselake, Habibur Rahman

BackgroundTraditional canola (Brassica napus L.; AACC, 2n¿=¿38) cultivars yield seed oil with a relatively high proportion of ¿-linolenic acid (ALA; C18:3 cis¿9,12,15), which is desirable from a health perspective. Unfortunately, due to the instability of this fatty acid, elevated levels also result in oils that exhibit a short shelf life and problems associated with use at high temperatures. As a result, the development of cultivars bearing reduced amounts of ALA in their seeds is becoming a priority. To date, several low ALA B. napus cultivars (~2-3% ALA of total fatty acids) have been developed and molecular analyses have revealed that the low ALA phenotype of lines tested thus far is a result of mutations within two `class b¿ FATTY ACID DESATURASE 3 (FAD3) genes. Since B. napus possesses six FAD3 genes (two `class a¿, two `class b¿ and two `class c¿) and ALA levels of approximately 2-3% remain in these low ALA lines, it is likely that the mutation of additional FAD3 genes could further decrease the content of this fatty acid.ResultsIn this study, we generated low ALA (¿2%) lines of B. oleracea, which is the C genome progenitor species of B. napus, via ethyl methanesulphonate (EMS) mutagenesis. We identified a novel nonsense mutation within the `class a¿ FAD3 gene (BoFAD3-2) in these lines, which would result in the production of an encoded protein lacking 110 amino acids at its C terminus. When expressed in Saccharomyces cerevisiae, this mutant protein exhibited a drastic decline in its ¿-15 desaturase activity compared to the wild-type (wt) protein. Furthermore, we demonstrated that the expression of the mutant BoFAD3-2 gene was significantly reduced in developing seeds of low ALA lines when compared to expression in wt plants.ConclusionsGiven the additive nature of FAD3 mutations on ALA content and the ease with which B. napus can be re-synthesized from its progenitor species, the mutant isolated here has the potential to be used for the future development of B. napus cultivars exhibiting further reductions in ALA content.