Platycodon grandiflorus has been widely used in Asia as a medicinal herb and food because of its anti-inflammatory and hepatoprotective properties. P. grandiflorus has important clinical value because of the active triterpenoid saponins in its roots. However, the biosynthetic pathway of triterpenoid saponins in P. grandiflorus remains unclear, and the related genes remain unknown. Therefore, in this study, we assembled a high-quality and integrated telomere-to-telomere P. grandiflorus reference genome and combined time-specific transcriptome and metabolome profiling to identify the cytochrome P450s (CYPs) responsible for the hydroxylation processes involved in triterpenoid saponin biosynthesis. Nine chromosomes were assembled without gaps or mismatches, and nine centromeres and 18 telomere regions were identified. This genome eliminated redundant sequences from previous genome versions and incorporated structural variation information. Comparative analysis of the P. grandiflorus genome revealed that P. grandiflorus underwent a core eudicot γ-WGT event. We screened 211 CYPs and found that tandem and proximal duplications may be crucial for the expansion of CYP families. We outlined the proposed hydroxylation steps, likely catalyzed by the CYP716A/72A/749A families, in platycodin biosynthesis and identified three PgCYP716A, seven PgCYP72A, and seven PgCYP749A genes that showed a positive correlation with platycodin biosynthesis. By establishing a T2T assembly genome, transcriptome, and metabolome resource for P. grandiflorus, we provide a foundation for the complete elucidation of the platycodins biosynthetic pathway, which consequently leads to heterologous bioproduction, and serves as a fundamental genetic resource for molecular-assisted breeding and genetic improvement of P. grandiflorus.

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