Chromosome‐level assembly of basil genome unveils the genetic variation driving Genovese and Thai aroma types

SUMMARY Basil, Ocimum basilicum L., is a widely cultivated aromatic herb, prized for its culinary and medicinal uses, predominantly owing to its unique aroma, primarily determined by eugenol for Genovese cultivars or methyl chavicol for Thai cultivars. To date, a comprehensive basil reference genome has been lacking, with only a fragmented draft available. To fill this gap, we employed PacBio HiFi and Hi‐C sequencing to construct a homeolog‐phased chromosome‐level genome for basil. The tetraploid basil genome was assembled into 26 pseudomolecules and further categorized into subgenomes. High levels of synteny were observed between the two basil subgenomes but comparisons to Salvia rosmarinus show collinearity quickly breaks down in near relatives. We utilized a bi‐parental population derived from a Genovese × Thai cross to map quantitative trait loci (QTL) for the aroma chemotype. We discovered a single QTL governing the eugenol/methyl chavicol ratio, which encompassed a genomic region with 95 genes, including 15 genes encoding a shikimate O ‐ hydroxycinnamoyltransferase (HCT/CST) enzyme. Of them, only ObHCT1 exhibited significantly higher expression in the Genovese cultivar and showed a trichome‐specific expression. ObHCT1 was functionally confirmed as a genuine HCT enzyme using an in vitro assay. The high‐quality, contiguous basil reference genome is now publicly accessible at BasilBase, a valuable resource for the scientific community. Combined with insights into cell‐type‐specific gene expression, it promises to elucidate specialized metabolite biosynthesis pathways at the cellular level.