A Simple Allele-Specific PCR Assay for Detecting FAD2 Alleles in Both A and B Genomes of the Cultivated Peanut for High-Oleate Trait Selection

In cultivated tetraploid peanut (2n = 4x = 40, AABB), the conversion of oleic acid to linoleic acid is mainly catalyzed by the Delta(12) fatty acid desaturase (FAD). Two homoeologous genes (FAD2A and FAD2B) encoding for the desaturase are located on the A and B genomes, respectively. Abolishing or reducing the desaturase activity by gene mutation can significantly increase the oleic acid/linoleic acid ratio. F435-derived high-oleate peanut cultivars contain two key mutations within the Delta(12) fatty acid desaturase gene which include a 1-bp substitution of G:C -> A:T in the A genome and a 1-bp insertion of A:T in the B genome. Both of these mutations contribute to abolishing or reducing the desaturase activity, leading to accumulation of oleate versus linoleate. Currently, detection of FAD2 alleles can be achieved by a cleaved amplified polymorphic sequence marker for the A genome and a real-time polymerase chain reaction (PCR) marker for the B genome; however, detection of these key mutations has to use different assay platforms. Therefore, a simple PCR assay for detection of FAD2 alleles on both genomes was developed by designing allele-specific primers and altering PCR annealing temperatures. This assay was successfully used for detecting FAD2 alleles in peanut. Gas chromatography (GC) was used to determine fatty acid composition of PCR-assayed genotypes. The results from the PCR assay and GC analysis were consistent. This PCR assay is quick, reliable, economical, and easy to use. Implementation of this PCR assay will greatly enhance the efficiency of germplasm characterization and marker-assisted selection of high oleate in peanut.