Oregano (Origanum vulgare) is a highly valued aromatic herb for culinary, medicinal, and ornamental purposes. Its commercial value is largely from its essential oil (EO), which is rich in key bioactive terpenoids, such as carvacrol and thymol. Greek oregano (O. vulgare subsp. hirtum) subspecies is particularly prized for its high EO content. In this study, we generated a high-quality genome assembly of Greek oregano to investigate its evolutionary trajectory and the genetic basis of terpenoid biosynthesis. The assembly spans 709.74 Mb and is anchored to 15 chromosomes, with a scaffold N50 of 46.36 Mb. Comparative genomic analysis revealed a whole-genome duplication event, estimated at ~59.93 million years ago, which likely contributed to the diversification of terpenoid biosynthesis pathways within the Lamiaceae family. Using a rapid screening approach, we identified Greek oregano mutants with higher EO content. Integrated genomic and transcriptomic analysis of a high-EO mutant highlighted the importance of α-linolenic acid metabolism/jasmonic acid (JA) biosynthesis pathways in EO production. Exogenous JA treatment led to upregulation of key EO biosynthetic genes and higher EO content. Furthermore, a JA-inducible bHLH transcription factor, OvbHLH13, was identified as a central regulator of terpenoid biosynthesis. Through Y1H, transcriptional activation, and EMSA assays, we demonstrated that OvbHLH13 directly bound to and transactivated the promoter of OvSDR1, which encodes a critical enzyme in thymol and carvacrol production. Collectively, this genomic resource provides valuable insights into the genetic and regulatory network controlling terpenoid biosynthesis and establishes a critical genomic foundation for molecular breeding of Greek oregano.

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