Deciphering centuries of selection: genetic structure and reproductive screening in Basilicata and Calabria's ancient...

The olive tree (Olea europaea L.) is a cornerstone of Mediterranean biocultural heritage, yet the shift toward intensive monoculture threatens its vast genetic diversity. This study presents a comprehensive molecular characterization of 347 olive accessions, including 172 monumental trees, from the Calabria and Basilicata regions of Southern Italy. Utilizing 10 nuclear SSR markers and chloroplast (cp-SSR) genotyping, we explored genetic diversity and maternal inheritance. Furthermore, we employed a novel diagnostic marker targeting the GA2ox-S gene, the functional determinant of the diallelic self-incompatibility (DSI) system, to assign genotypes to reproductive groups G1 and G2. Our results identified 114 unique genotypes, representing a significant reservoir of uncatalogued traditional germplasm. Notably, we discovered three instances of putative polyploidy in indigenous Calabrian olives, a rarity for the species typically considered diploid. Comparative analysis with an international dataset revealed a highly admixed genetic structure, with local genotypes showing strong affinity to Algerian and Greek pools while remaining distinct from Western Mediterranean and Middle Eastern clusters. Systematic genotyping of both canopies and rootstocks provided genetic evidence consistent with historical grafting practices in 34 instances. We observed a clear structural and maternal partition: cultivated canopies predominantly belonged to the E1 (Eastern) lineage, while uncultivated rootstocks preserved the indigenous E2 (Central-Western) maternal heritage. Reproductive screening revealed a regional dichotomy: the G1 group dominates in Basilicata, while the G2 group is more prevalent among Calabrian landraces. These findings underscore how historical agricultural practices, grafting elite clones onto resilient wild oleasters, clonal propagation, and DSI-driven obligate outcrossing have synergistically shaped the high heterozygosity of Southern Italian groves. This work provides valuable genetic resources for conservation and future breeding programs aimed at exploring resilience against climate change and emerging pathogens.