T2T Genome and Population Resequencing Reveal OfCCD4 Alleles Orchestrate Petal Color and Scent in Osmanthus fragrans

Osmanthus fragrans is prized for its floral color and fragrance, both key targets for genetic improvement. However, the lack of a complete genome assembly and comprehensive population structure hinders gene dissection and marker development for breeding. To investigate the genetic basis of color–scent interaction, a telomere-to-telomere (T2T) genome assembly is generated, and whole-genome resequencing of 100 cultivars is performed. Integrative population and metabolomic analyses reveal a trade-off: orange-red (‘Aurantiacus’) cultivars accumulate high α/β-carotene but low aromatic apocarotenoids (α/β-ionone), while yellow-white cultivars show the opposite pattern. Divergence mapping identifies OfCCD4 as the major underlying locus. Three alleles are characterized—functional (A), partially functional (a Del ), and a frameshift null (a Stop )—with a Stop strictly co-segregating with the ‘Aurantiacus’ phenotype. Transient and stable transformations confirm that A and a Del cleave α/β-carotene into α/β-ionone, while a Stop abolishes this activity. A co-dominant PCR marker is developed based on allele-specific polymorphisms. OfCCD4 is thus established as the key regulator of the color–scent trade-off in osmanthus. The identified alleles and molecular marker enable rapid, low-cost genotyping at the seedling stage—offering particular value for marker-assisted selection in ornamental breeding, where extended juvenility makes phenotypic selection highly time- and resource-intensive.