Unveiling the Genetic Blueprint of Verbascoside Biosynthesis in <i>Olea europaea</i> : An Integrative Metabolomics, Genetics and Transcriptomics Approach

Abstract The olive tree (Olea europaea L.) is a key source of verbascoside, a potent phenylethanoid glycoside known for its antioxidant, anti-inflammatory, and neuroprotective properties. Traditional breeding for enhanced phenolic profiles is limited by the olive’s long juvenile period and complex genetics. This study dissected the unknown genetic architecture of verbascoside biosynthesis using a multi-omics strategy integrating metabolomics, Quantitative Trait Loci (QTL) mapping, and transcriptomics across an F1 population and diverse cultivars. This approach identified a major genetic locus controlling verbascoside content on chromosome 21 (LOD = 17), which explained the wide metabolomic variation. The locus includes 193 genes, of which 29 were involved in verbascoside synthesis and 47 showed differential expression. A homology-based analysis, using data from five high-verbascoside Lamiales species, narrowed the search to a small set of high-confidence candidate genes, including BAHD acyltransferases (BEAT, AHCT, HCBT, DAT acyltransferase family) and various Cytochrome P450s (CYPs). Digital PCR confirmed the differential expression of nine candidate CYP genes. The expression of both OeCYP71D55 (a premnaspirodiene oxygenase) and the precursor-producing OeACy3G peaked at the T5 stage, 165 Days After Flowering (DAF). This peak mirrored the maximum time of verbascoside accumulation, suggesting that the transcriptional regulation of these CYPs is a rate-limiting factor in the pathway. This study defines a critical genetic locus and a core set of candidate genes governing verbascoside production in olive cultivars. These discoveries provide valuable molecular tools to fast-track the breeding of new olive varieties with enhanced health benefits.