Development of a real - time peR genotyping assay to identify high oleic acid peanuts ( Arachis hypogaea L . )

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 respon­ sible 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 FAD2B and can be employed on DNA extracted from either seeds or leaves. The presence of the insertion (mutant allele) in fad2B causes a frameshift downstream in the coding sequence that ultimately alters the mRNA transcript N. A. Barkley· M. L. Wang (~) . R. N. Pittman USDA-ARS, Plant Genetic Resources Conservation Unit, 1109 Experiment Street, Griffin, GA 30223, USA e-mail: [email protected] K. D. C. Chamberlin USDA-ARS, Wheat, Peanut, and Other Field Crops Research Unit, 1301N, Western Stillwater, OK 74075, USA level, and thus, decreases the activity of microsomal oleoyl-PC desaturase enzyme which converts oleic acid (CI8:1) to linoleic acid (CI8:2).'1Validation of the real-time assay was carried out by quantitatively evaluating the fatty acid composition by gas chro­ matography (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.