Mutation scanning and genotyping by High-Resolution DNA Melting Analysis in olive germplasm

The application of high-resolution melting (HRM) analysis of DNA is reported for scanning and genotyping Olea europaea germplasm. To test the sensitivity of the method, a functional gene marker, phytochrome A (phyA), was used, since this gene is correlated with important traits for the ecology of the species. We have designed a set of oligos able to produce amplicons of 307 bp to scan for the presence of single polymorphic mutations in a specific phyA fragment encompassing the chromophore attachment site (Cys323). The presence of mutations for substitution, either homozygous or heterozygous, was easily detected by melting curve analysis in a high-resolution melter. It has been established that the sensitivity of the HRM analysis can be significantly improved designing specific primers very close to the mutation sites. All SNPs found were confirmed by sequence analyses and ARMS-PCR. The method has also been confirmed to be very powerful for the visualization of microsatellite (SSR) length polymorphisms. HRM analysis has a very high reproducibility and sensitivity for detecting SNPs and SSRs, allowing olive cultivar genotyping and resulting in an informative, easy, and low-cost method able to greatly reduce the operating time.