Development of a real-time PCR genotyping assay to identify high oleic acid peanuts (Arachis hypogaea L.)
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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 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 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.
KeywordsReal-time PCR Oleic acid (C18:1) Peanut (Arachis hypogaea L.) Gas chromatography Indel Microsomol-oleoyl PC desaturase
This manuscript was contributed in the memorial of Professor Mike Gale FRS, John Innes Centre, Norwich, UK. The authors gratefully thank Mr. Brandon Tonnis for assistance on collecting gas chromatography data and Mr. Dave Pinnow for assistance with DNA extraction. Thanks to Drs Daniel Gorbet and Kim Moore for their comments and suggestions to improve the quality of the manuscript. Also, the authors thank Dr. Gorbet for F435 seed and plant tissue.
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
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