Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, its dried mature peels named “Guangchenpi”, has been used as a traditional Chinese medicine to treat cough, indigestion, and lung diseases for several hundred years. However, the biosynthesis of the crucial natural products polymethoxylated flavonoids (PMFs) in CRC remains unclear. Here, we report a chromosome-scale genome assembly of CRC with the size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, we discover a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3’,4’-heptamethoxyflavone (HPMF). Based on transient overexpression and virus-induced gene silencing experiments, we propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. In addition, a potential gene regulatory network associated with PMF biosynthesis is identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.

柑橘网状品种茶枝（CRC）是一种重要的药用植物，其干燥成熟的果皮被称为“广臣皮”，数百年来一直被用作治疗咳嗽、消化不良和肺部疾病的中药。然而，CRC 中关键天然产物多甲氧基黄酮类化合物 (PMF) 的生物合成仍不清楚。在这里，我们报告了 CRC 的染色体规模基因组组装，大小为 314.96 Mb，重叠群 N50 为 16.22 Mb。利用多组学资源，我们发现了一种假定的咖啡酸O -甲基转移酶 (CcOMT1)，它可以将甲基转移到 natsudaidain 的 3-羟基上，形成 3,5,6,7,8,3',4'-七甲氧基黄酮（HPMF）。基于瞬时过表达和病毒诱导的基因沉默实验，我们提出 CcOMT1 是 HPMF 生物合成中的候选酶。此外，还确定了与 PMF 生物合成相关的潜在基因调控网络。这项研究提供了对 PMF 生物合成的见解，并可能有助于未来植物药物生物合成基因挖掘的研究。