Plant genomes remain highly fragmented and are often characterized by hundreds to thousands of assembly gaps. Here, we report chromosome-level reference and phased genome assembly of Ophiorrhiza pumila, a camptothecin-producing medicinal plant, through an ordered multi-scaffolding and experimental validation approach. With 21 assembly gaps and a contig N50 of 18.49 Mb, Ophiorrhiza genome is one of the most complete plant genomes assembled to date. We also report 273 nitrogen-containing metabolites, including diverse monoterpene indole alkaloids (MIAs). A comparative genomics approach identifies strictosidine biogenesis as the origin of MIA evolution. The emergence of strictosidine biosynthesis-catalyzing enzymes precede downstream enzymes’ evolution post γ whole-genome triplication, which occurred approximately 110 Mya in O. pumila, and before the whole-genome duplication in Camptotheca acuminata identified here. Combining comparative genome analysis, multi-omics analysis, and metabolic gene-cluster analysis, we propose a working model for MIA evolution, and a pangenome for MIA biosynthesis, which will help in establishing a sustainable supply of camptothecin.

植物基因组仍然高度分散，通常具有成百上千个组装缺口。在这里，我们通过有序的多支架和实验验证方法报告了喜树碱生产药用植物麦芽的染色体水平参考和阶段基因组组装。具有 21 个装配间隙和 18.49 Mb 的重叠群 N50，Ophiorrhiza基因组是迄今为止组装的最完整的植物基因组之一。我们还报告了 273 种含氮代谢物，包括多种单萜吲哚生物碱 (MIA)。比较基因组学方法将胡豆苷生物合成确定为 MIA 进化的起源。胡豆苷生物合成催化酶的出现先于下游酶在 γ 全基因组三倍化后的进化，这发生在O. pumila中大约 110 Mya，并且在此处鉴定的喜树的全基因组复制之前。结合比较基因组分析、多组学分析和代谢基因簇分析，我们提出了 MIA 进化的工作模型和 MIA 生物合成的泛基因组，这将有助于建立喜树碱的可持续供应。