Genetic analysis using next-generation sequencing method to disclose genomic variants in grapevine cultivars

The grapevine (Vitis vinifera L.) is one of the most economically important crops globally, distinguished by its significant genetic diversity, which is reflected in a variety of cultivars with unique morphological, agronomic, and qualitative traits. Understanding the genetic basis of this variability is essential for effective grapevine management, the conservation of genetic resources, and the development of advanced breeding strategies. Recent advances in next-generation sequencing (NGS) technologies, such as genotyping-by-sequencing (GBS) and double-digest RAD sequencing (ddRADseq), have provided powerful tools for analyzing genetic variation, identifying functional genes, and characterizing loci associated with important agronomic traits. This research aims to explore the population structure, genetic diversity, and key genetic loci in a collection of clones derived from Campanian and Apulian grapevine varieties, known for their desirable traits in viticulture and winemaking. The first study analyzed a total of 138 clones from six autochthonous grapevine varieties from southern Italy, sampled in Campania and Apulia. These clones are traditionally cultivated in environments with different climatic conditions. The Campanian clones include two red-berried varieties, CAMAIOLA And AGLIANICO LASCO, and the white-berried GRECO B. The Apulian clones include two red-berried varieties, NERO DI TROIA and MALVASIA NERA DI BRINDISI, as well as the white-berried Minutolo b. Two reduced representation sequencing methods were used to extract single-nucleotide polymorphism (SNP) markers. The study revealed high genetic diversity, particularly among clones within the same variety. Population structure analysis and fixation index (FST) calculations indicated significant genetic similarity among varieties originating from the same geographical region. Approximately 200 loci were identified with divergent markers (FST ≥ 0.80) within annotated exons, many of which were linked to important traits like phenotypic adaptability and environmental responsiveness. These findings offer valuable opportunities for grapevine breeding initiatives. Additionally, the genetic variability of 35 PRIMITIVO clones from Apulia, including selected and certified clones propagated over several years, was examined using genotyping-by-sequencing. A total of 38,387 filtered SNPs were analyzed, and pairwise identity-by-state (IBS) analysis showed high variability (IBS < 0.75) among the clones. Genetic diversity analysis revealed three main groups, differentiated by geographic origin. Clones from Gioia del Colle were further divided into two distinct clusters, corresponding to variation in grape-related traits. The FST analysis identified numerous loci associated with stress responses and developmental traits, including genes involved in stress regulation, as well as adaptation to environmental conditions such as glutamate receptors and auxin and ethylene signaling. A broader analysis of five emblematic Apulian grapevine varieties (NEGRAMARO, MALVASIA NERA, PRIMITIVO, MINUTOLO, and UVA DI TROIA) using the GBS approach uncovered significant intra- and inter-varietal genetic variation. Key genes related to secondary metabolism, stress resilience, and berry quality were identified, including WRKY transcription factors and enzymes that influence wine flavor, aroma, and pigmentation. Several SNP loci were validated for varietal traceability, essential for maintaining wine authenticity and marketability. These findings not only contribute to the conservation of viticultural biodiversity but also support sustainable wine production strategies, ensuring the competitiveness of Apulian wines in the global market. The research highlights the importance of preserving and utilizing intra-varietal variability, which is essential for the sustainability and adaptability of viticulture, especially in the context of changing environmental conditions.