Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

摘要

Background Cultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by the Δ12 fatty acid desaturase (FAD) encoded by AhFAD2A and AhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C → A:T) of AhFAD2A and an “A” insertion of AhFAD2B resulted in high-oleic acid phenotype. Detection of AhFAD2 mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detect AhFAD2 genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detection AhFAD2 genotype of large number of breeding materials. Results Here, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC4F2 seeds was aabb. Conclusions Due to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable for determining AhFAD2 genotype than other methods.