Title: Development of a real-time PCR genotyping assay to identify high oleic acid peanuts (Arachis hypogaea L.) Authors
Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 14, 2009
Publication Date: September 25, 2009
Repository URL: http://hdl.handle.net/10113/44461
Citation: Barkley, N.L., Chamberlin, K.D., Wang, M.L., Pittman, R.N. 2009. Development of a real-time PCR genotyping assay to identify high oleic acid peanuts (Arachis hypogaea L.). Molecular Breeding. DOI 10.1007/s11032-009-9338-z 25(3):541-548. Interpretive Summary: Dietary fats are essential to humans because they supply energy, help regulate body temperature, and carry fat soluble vitamins. There are many types of fats, but they are all composed of a glycerol backbone and fatty acids. 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 multiple double bonds. Due to the differences in chemical structure of the many different fatty acids, they all have unique properties. For example, oleic acid has a single double bond in its hydrocarbon chain which makes this fatty acid very stable. On the other hand, linoleic acid which has two double bonds in the hydrocarbon chain tends to break down which ultimately causes off flavors in oil or seeds containing oil. Therefore, oils or edible seeds containing oils 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. The flavor and quality of the seed or the extracted oil is dependent on the fatty acids because a high amount of oleic acid in a peanut seed provides increased shelf life over peanuts with low levels of oleic. Thus, the goal of this work was to develop a method to rapidly assess breeding lines for oleic acid, and simultaneously discard undesirable lines that have low levels of oleic fatty acid.
Technical Abstract: Oleic acid, a monounsaturated, omega-9 fatty acid found in peanut (Arachis hypogaea L.) oil is an important seed quality trait because it provides increased shelf life, improved flavor, enhanced fatty acid composition, and has a beneficial effect on human health. Hence, a concentrated effort has been put forth on developing peanut cultivars that have high oleic acid (>74%) and a low amount (<10%) of linoleic acid, a polyunsaturated omega-6 fatty acid. A main bottleneck, however, in breeding research is fast selection of the trait(s) of interest. Therefore, in an effort to expedite breeding efforts, a real-time PCR genotyping assay was developed to rapidly identify the wild type and the mutant allele that are responsible for normal or high levels of oleic acid, respectively in peanut seeds. This test utilizes two Taqman® probes to detect the presence of an indel (insertion/deletion) in ahFAD2B and can be employed on DNA extracted from either seeds or leaves. The presence of the insertion (mutant allele) in ahFAD2B causes a frameshift downstream in the coding sequence that ultimately alters the mRNA transcript level, and thus, decreases the activity of microsomal oleoyl-PC desaturase enzyme which converts oleic acid (C18:1) to linoleic acid (C18:2). Validation of the real-time assay was carried out by quantitatively evaluating the fatty acid composition by gas chromatography (GC). Overall, this real-time PCR assay facilitates the identification of progeny carrying the high oleic acid alleles, and thus, allows early elimination of undesirable non-high oleic acid lines in segregating populations.