Deciphering genetic basis of phenylethanoid glycoside production through integrative multi-omics analysis in Callicarpa nudiflora

Callicarpa nudiflora is a medicinal plant rich in phenylethanoid glycosides (PhGs), particularly verbascoside, but the genetic basis of PhG biosynthesis and natural variation in its content remain largely unexplored. Here, we assembled a chromosome-level genome of C. nudiflora (511 Mb, contig N50 = 12.78 Mb) with 39 317 annotated protein-coding genes and 99.5% BUSCO completeness and quantified its bioactive compounds and gene expression across multiple tissues. Using the dataset, we elucidated the complete biosynthetic pathway of verbascoside and demonstrated multiple paralogous enzymes catalyzing one step, a functional redundancy that may contribute to the high verbascoside accumulation observed in this species. Population genomic analysis, together with quantification of verbascoside content of 95 samples grown in a common garden over three consecutive years, revealed that Southcentral Hainan (HN1) was identified as the genetic diversity center and exhibited the the highest content of verbascoside. Selective sweep analysis comparing high- and low-content individuals identified CnUGT79G7, a key glycosyltransferase in the verbascoside pathway, as being under positive selection. Two major haplotypes of CnUGT79G7 were identified: Hap1, a high-expression haplotype prevalent in the top-geoherb region (77% frequency), and Hap2, a low-expression haplotype fixed in other populations. Although the two haplotypes encode proteins with similar catalytic activity, Hap1 is associated with significantly higher CnUGT79G7 expression and elevated verbascoside content, suggesting that cis-regulatory variation underlies chemotypic differentiation. Collectively, this work identifies key molecular components for verbascoside biosynthesis, establishes a genomic and metabolic framework for understanding top-geoherbalism, and provides valuable resources for the conservation and molecular breeding of medicinal plants.