Fallopia multiflora (Thunb.) Harald, a vine belonging to the Polygonaceae family, is used in traditional medicine. The stilbenes contained in it have significant pharmacological activities in anti-oxidation and anti-aging. This study describes the assembly of the F. multiflora genome and presents its chromosome-level genome sequence containing 1.46 gigabases of data (with a contig N50 of 1.97 megabases), 1.44 gigabases of which was assigned to 11 pseudochromosomes. Comparative genomics confirmed that F. multiflora shared a whole-genome duplication event with Tartary buckwheat and then underwent different transposon evolution after separation. Combining genomics, transcriptomics, and metabolomics data to map a network of associated genes and metabolites, we identified two FmRS genes responsible for the catalysis of one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA to resveratrol in F. multiflora. These findings not only serve as the basis for revealing the stilbene biosynthetic pathway but will also contribute to the development of tools for increasing the production of bioactive stilbenes through molecular breeding in plants or metabolic engineering in microbes. Moreover, the reference genome of F. multiflora is a useful addition to the genomes of the Polygonaceae family.

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