Molecular Identification and Phylogenetic Analysis of the Traditional Chinese Medicinal Plant Kochia scoparia Using ITS2 Barcoding.

Kochia scoparia has high medicinal and economic value. However, with similar morphological features, adulterants and some closely related species of K. scoparia are increasingly sold in the medicinal markets, leading to potential safety risks. In this study, 128 internal transcribed spacer 2 (ITS2) sequences were collected to distinguish K. scoparia from its closely related species and adulterants. Then, sequence alignment, sequence characteristics analysis, and genetic distance calculations were performed using MEGA 6.06 software, and the phylogenetic trees were reconstructed using both MEGA 6.06 and IQ-Tree software. Finally, the secondary structure of ITS2 was modeled using the prediction tool in the ITS2 database. The results showed that ITS2 sequences of K. scoparia ranged in length from 226 to 227 bp, with a mean GC content of 55.3%. The maximum intraspecific distance was zero, while the minimum interspecific distance from closely related species and adulterants was 0.009 and 0.242, respectively. Kochia scoparia formed an independent clade in the phylogenetic trees, and its secondary structure exhibited enough variation to be separated from that of other species. In summary, ITS2 can be used as a mini-barcode for distinguishing K. scoparia from closely related species and adulterants. Its phylogenetic trees could illustrate the evolutionary process of K. scoparia in the Camphorosmeae. The phylogenetic results using ITS2 barcode further supported the internationally recognized revised classifications of Kochia and Bassia genera as a combined Bassia genus, together with the establishment of new genera Grubovia and Sedobassia, which we suggest is accepted by the Flora of China. Graphical abstract .