Artemisia annua is the major natural source of artemisinin, an anti-malarial medicine commonly used worldwide. Here, we present chromosome-level haploid maps for two A. annua strains with different artemisinin contents to explore the relationships between genomic organization and artemisinin production. High-fidelity sequencing, optical mapping, and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A. annua. Approximately 50,000 genes were annotated for each haplotype genome, and a triplication event that occurred approximately 58.12 million years ago was examined for the first time in this species. A total of 3,903,467–5,193,414 variants (SNPs, indels, and structural variants) were identified in the 1.5-Gb genome during pairwise comparison between haplotypes, consistent with the high heterozygosity of this species. Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-diene synthase genes. This correlation was further confirmed by resequencing of 36 A. annua samples with varied artemisinin contents. Circular consensus sequencing of transcripts facilitated the detection of paralog expression. Collectively, our study provides chromosome-level allele-aware genome assemblies for two A. annua strains and new insights into the biosynthesis of artemisinin and its regulation, which will contribute to conquering malaria worldwide.

青蒿是青蒿素的主要天然来源，青蒿素是一种全球常用的抗疟疾药物。在这里，我们展示了两个A 的染色体水平单倍体图谱。 不同青蒿素含量的青蒿菌株探讨基因组组织与青蒿素生产之间的关系。高保真测序、光学作图和染色质构象捕获测序用于组装异质和重复基因组并解析A的单倍型。 青蒿. 每个单倍型基因组注释了大约 50,000 个基因，并且在该物种中首次检查了大约 5812 万年前发生的三倍体事件。在单倍型之间的成对比较期间，在 1.5-Gb 基因组中鉴定出总共 3,903,467–5,193,414 个变体（SNP、插入缺失和结构变体），与该物种的高杂合性一致。基因组分析揭示了青蒿素浓度与amorpha-4,11-二烯合酶基因拷贝数之间的相关性。对 36 A. annua重新测序进一步证实了这种相关性青蒿素含量不同的样品。转录本的循环共识测序有助于检测旁系同源物表达。总的来说，我们的研究为两种青蒿菌株提供了染色体水平的等位基因感知基因组组装，并为青蒿素的生物合成及其调控提供了新的见解，这将有助于在全球范围内战胜疟疾。