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

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 theΔ12 fatty acid desaturase (FAD) encoded byAhFAD2AandAhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C→A:T) ofAhFAD2Aand an "A" insertion ofAhFAD2Bresulted in high-oleic acid phenotype. Detection ofAhFAD2mutation 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 detectAhFAD2genotype 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 detectionAhFAD2genotype 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 BC4F2seeds was aabb. Conclusions: Due to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable fordeterminingAhFAD2genotype than other methods.

Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.

The cultivated peanut is a valuable source of dietary oil and ranks fi fth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthase (I, II, III), β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.