Discrimination of Indigenous and Local Grape Cultivars (Vitis vinifera L.) Grown in North Macedonia using PCR and High-Resolution Melting Analysis

Abstract Ensuring the authenticity of grapevine cultivars is crucial for traceability, fraud prevention, and consumer protection. In the Republic of North Macedonia, viticulture and wine production have a long-standing tradition and remain an important pillar of the country´s agricultural sector. Recently, the Macedonian government adopted a ten-year National Strategy for the Development of Viticulture and Winemaking to ensure authenticity and sustainability of wine production. Traditional identification of grape cultivars relied on ampelographic methods which are strongly affected by environmental conditions. The analysis of simple sequence repeat (SSR) markers by capillary electrophoresis has become the standard for grape cultivar identification, but is time-consuming and cost-intensive. In this study, we assessed nine SSR markers recommended by the Organisation Internationale de la Vigne et du Vin for their suitability to differentiate eleven grape cultivars commonly grown in North Macedonia using PCR combined with high-resolution melting (HRM) analysis. Compared to capillary electrophoresis, HRM reduces time and cost, as it is carried out directly after PCR in the same instrument without requiring any pretreatment steps. Among the markers tested, assays targeting VrZAG62 and VrZAG79 showed the highest discriminatory power, distinguishing 50 and 53 of 55 cultivar pairs, respectively. The complex melting curves indicate that the amplified regions differ not only in repeat number but also in base composition. Preliminary experiments demonstrate that the discriminatory power can be even enhanced by performing multiplex PCR-HRM. Our findings indicate that PCR-HRM is a reliable screening method for grape cultivar authentication, supporting food authenticity testing and protection of local viticulture.