High-quality genome assembly of autotetraploid Verbena bonariensis sheds light on flower color development

Verbena bonariensis L., known for its vibrant purple inflorescences, is widely valued in ornamental gardening and ecological landscaping. However, the absence of a high-quality reference genome has hindered the comprehensive exploration of its critical functional traits. In this study, we assembled a high-quality, chromosome-scale genome of autotetraploid V. bonariensis (2n = 4X = 28), with a genome size of 1.35 Gb, contig N50 of 47.88 Mb, and BUSCO completeness evaluation of 98.3%. The assembled genome contained 16 completely gap-free chromosomes and only 21 gaps, and 106 006 protein-coding genes, including 33 205 allele genes, were identified. Genome evolution analysis estimated that V. bonariensis diverged from its close relative Petrea volubilis 38.4 million years ago (MYA), after two rounds of whole-genome duplication (WGD) prior to speciation (WGTγ ( K s = 2.05, 157.66 MYA) and recent WGD ( K s = 0.82, 63.46 MYA). Dispersed and tandem duplicated genes significantly contributed to gene family expansion in V. bonariensis . Metabolomics analysis of two Verbena accessions with deep and pale purple petals revealed that delphinidin-3-O-glucoside, cyanidin-3-O-gentiobioside, and cyanidin-3-O-(6″-O-acetyl)glucoside-5-O-glucoside were the major pigments conferring the purple petals. Notably, the key distinction between the two phenotypes was pelargonidin-3-O-(6″-O-acetyl)glucoside-5-O-glucoside (Pe3G5G) accumulation, with the deep purple petals associated with higher accumulation. Integrated metabolomics and transcriptomics analysis revealed that 37 structural genes in the anthocyanin biosynthesis pathway, including all VbDFR (2/2) and VbPAL (6/6) genes etc., and regulatory genes such as VbbHLH79 , VbMYB123 , and VbbZIP9 etc. exhibited a significant correlation (Pearson’s | r | > 0.80) with Pe3G5G accumulation, speculating that the differential expression of these genes contributed to the phenotypic divergence in petal coloration. Preliminary functional validation demonstrated that transient overexpression of VbbHLH79 induced a purple phenotype in petal tissues of white-petal chrysanthemum. Consistently, transgenic tobacco overexpressing this gene also exhibited a deeper purple petal phenotype. These results revealed that VbbHLH79 is a critical regulatory gene involved in anthocyanidin synthesis. This study provides a valuable genomic resource for comparative and functional genomic studies of Lamiales and other Verbenaceae species, and provide candidate genes for molecular breeding of ornamental flowering plants.