Chromosome‐level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis

SUMMARY Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris . However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome‐level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi‐C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris . Genome‐wide analysis identified 35 932 protein‐coding genes (PCGs), of which 59 encode enzymes involved in 2,3‐oxidosqualene biosynthesis. In addition, 10 PvOSC , 358 PvCYP , and 177 PvUGT genes were identified, of which five PvOSCs , 25 PvCYPs , and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and β‐amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris .