Microsatellite High-Resolution Melting (SSR-HRM) to Track Olive Genotypes: From Field to Olive Oil.

The need to support food labelling has driven to the development of PCR-based techniques suitable for food analysis. DNA-based markers have been successfully employed for varietal tracing in Protected Designation of Origin (PDO) olive oils. In this study, we report a fast, sensitive, and easy-to-use strategy for PDO olive varietal identification. To achieve this aim four different DNA extraction methods were tested and compared, based on initial volume, extraction time, the gDNA concentration, and quality ratios. The optimized DNA extraction protocol from extra virgin olive oils, based on CTAB-hexane-chloroform extraction, proved to be the most effective. High-resolution melting (HRM) DNA assay was developed based on nuclear microsatellites (gSSR) and plastid DNA (cpDNA) aiming an accurate identification of the olive varieties present in the olive oil samples. After PCR reproducibility evaluation, six molecular markers: three SSRs and three cpDNA loci were chosen based on their discrimination power. The SSR-HRM strategy assays were designed to target UDO99-011, UDO99-039, UDO99-024, and ssrOeUA-DCA16 loci. All SSR-PCR products generated from these primers were analyzed by capillary electrophoresis (CE) for HRM data validation. The SSR coupled with HRM melting curve analysis generated 14 HRM profiles sufficient to genotype all varieties, highlighting their potential use for varietal discrimination. The locus ssrOeUA-DCA16 generated a specific melting curve that allow a high-throughput discrimination of the Picual and Cobrançosa varieties in olive oil samples. Further, the UDO99-024 was also tested by SSR-HRM assay in commercial olive oil samples with promising results. Considering time, cost, and performance SSR-HRM proved to be a reliable method suitable for varietal tracing of olive oils. PRACTICAL APPLICATION: Olive oil authenticity is a form of protecting producers and consumers against fraudulent practices. Herein, we present a DNA barcode suitable for the identification of olive varieties, allowing an accurate identification of the olive varieties in olive oil samples using SSR-HRM assay. Its applicability in commercial olive oil samples is viable. This methodology can be used as a tool for Extra Virgin Olive Oil (EVOO) adulterations detection.