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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.

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Posidonia oceanica is an endemic seagrass species in the Mediterranean Sea. Due to its ecological importance, it has been the subject of several genetic studies aimed at increasing general knowledge of the system and helping to define appropriate management strategies. We compared the efficiency of 2 different classes of molecular markers, 'inter simple sequence repeats' (ISSRs) and 'simple sequence repeats' (SSRs) or 'microsatellites', in population genetic analysis of P. oceanica. To do this, we analyzed meadows along the coasts of the Calabria peninsula (Italy, South Tyrrhenian Sea and Ionian Sea), a putative biogeographic barrier for P. oceanica. SSR and ISSR markers detected different levels of genetic variability within P. oceanica populations. Dinucleotide microsatellites were more polymorphic than tri-and heptanucleotide microsatellites and ISSRs. Nevertheless, discriminating power analysis performed for each single locus showed that some trinucleotide microsatellite and ISSR primers were more sensitive than dinucleotides. The use of selected ISSR and SSR markers together allowed the detection of higher polymorphism than either set of marker alone. Tyrrhenian meadows were always more polymorphic than Ionian ones, according to both SSRs and ISSRs. Both markers strongly suggested the Calabria peninsula as an important biogeographic barrier between the Western and the Eastern side of the Mediterranean basin. KEY WORDS: Posidonia oceanica · ISSR · SSR · Genetic diversity · Biogeographic barrierResale or republication not permitted without written consent of the publisher