Title: A real-time PCR genotyping assay to detect FAD2A SNPs in peanuts (Arachis hypogaea L.) Authors
Submitted to: Electronic Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 13, 2010
Publication Date: January 15, 2011
Repository URL: http://hdl.handle.net/10113/49701
Citation: Barkley, N.L., Wang, M.L., Pittman, R.N. 2011. A real-time PCR genotyping assay to detect FAD2A SNPs in peanuts (Arachis hypogaea L.). Electronic Journal of Biotechnology. 14(1). Available: http://www.ejbiotechnology.info/index.php/ejbiotechnology/issue/view/65. Interpretive Summary: Fatty acids are characterized by the number of carbon and hydrogen atoms along with the absence or presence of double bonds in the fatty acid hydrocarbon chain. Saturated fatty acids contain no double bonds; whereas, unsaturated fatty acids have at least one or several double bonds in the hydrocarbon chain. Due to the differences in chemical structure of the many different fatty acids, they all have unique properties. For example, oleic acid which is one of the most predominant fatty acids found in nature has a single double bond in its hydrocarbon chain which makes this fatty acid very stable. Therefore, oils or edible seeds containing fatty acids are more stable if they are composed of a high amount of oleic acid and low amounts of linoleic acid. Peanut seeds and peanut oil are comprised of both oleic and linoleic fatty acids as well as up to 10 other fatty acids. The flavor and quality of the seed or the extracted oil is dependent on the fatty acid composition. Thus, the goal of this work was to develop a method to rapidly assess breeding lines and peanut germplasm for variation in a gene that is linked to high oleic acid accumulation.
Technical Abstract: The high oleic (C18:1) phenotype in peanuts has been previously demonstrated to result from a homozygous recessive genotype (ol1ol1ol2ol2) in two homeologous fatty acid desaturase genes (FAD2A and FAD2B) with two key SNPs. These mutant SNPs, specifically G448A in FAD2A and 442insA in FAD2B, significantly limit the normal function of the desaturase enzyme activity which converts oleic acid into linoleic acid by the addition of a second double bond in the hydrocarbon chain. Previously, a genotyping assay was developed to detect wild type and mutant alleles in FAD2B. A real-time PCR assay has now been developed to detect wild type and mutant alleles (G448A) in FAD2A using either seed or leaf tissue. This assay was demonstrated to be applicable for the detection of homozygous and heterozygous samples. The FAD2A genotyping assay was validated by employing gas chromatography to determine total fatty acid composition and by genotyping peanut lines that have been well characterized. Overall, development of rapid assays such as real-time PCR which can identify key genotypes linked to important agronomic traits such as oleic acid, will improve breeding efficiency by targeting desirable genotypes/phenotypes at early stages of development.