Abstract:The identification of Perilla frutescens and its varieties, Perilla fructus and its adulterants was studied based on ITS2 primary sequence combined with its secondary structure, providing a more scientific classification method for the usage of Perilla frutescens as a medicinal plant. After the collection of Perillae fructus and its adulterants, Perilla frutescens and its varieties, their DNA was isolated for PCR amplification. The ITS2 sequences of all species were obtained through the combination with the sequences in GenBank. MEGA 7.0 software was used for analysis of sequence, calculation of intra-species and inter-species genetic distances, and construction of NJ cluster tree. The prediction of ITS2 secondary structure of each species was based on ITS2 database. 4Sale software was used to compare the primary structure with the second structure matrix, and ProfDistS software was used to construct the evolutionary tree of PNJ. ITS2 sequence had high success rate of amplification. Except P. frutescens var. frutescens, ITS2 sequences of Perilla frutescens and other varieties had significant barcoding gap. NJ tree showed that ITS2 could distinguish Perilla frutescens and P. frutescens var. frutescens from other varieties. Perilla frutescens and P. frutescens var. frutescens differed in the secondary structures of ITS2. The ITS2 sequences of Perilla fructus and its adulterants were quite different, and there was significant genetic distance between various species. NJ tree and PNJ tree showed that Perilla fructus and its adulterants were clustered into separate branches with higher bootstrap values. Based on ITS2 barcode, Perillae fructus and its adulterants could be accurately identified, providing a reliable method for standardizing the pharmacopeial herbs market and ensuring the clinical medication safety. However, the identification of Perilla frutescens and its variety P. frutescens var. frutescens should be combined with the secondary structure of ITS2. It is also suggested that Perilla frutescensa and P. frutescens var. frutescens could be combined into one species to provide reference for the classification of Perilla genus.