Using the ITS2 sequence-structure as a DNA mini-barcode: A case study in authenticating the traditional medicine“Fang Feng”

Abstract An ideal DNA barcode should be short enough to be easily amplified. However, barcodes that are too short may lack discriminatory efficacy, and how additional phylogenetic information can be obtained and used without the addition of nucleotides is a major concern. In this study, we selected 135 individuals that represented 97 species from 21 genera to explore the possibility of employing the short ITS2 fragment as a barcode to identify the traditional medicine “Fang Feng” using sequence and structure information. The results revealed that all of the ITS2 sequences had a common four-helix folding structure and some shared motifs. This conserved structure facilitated the alignment of ambiguous sequences from highly divergent taxa. The sequence-structure data yielded a maximum parsimony (MP) tree in which the species referred to as “Fang Feng” was successfully identified. Additionally, we compared the species-level discriminatory powers of the sequence-structure ITS2 and the full-length ITS (ITS1, 5.8S, ITS2), and found that their efficacies were very similar. These findings support the possibility of incorporating the ITS2 secondary structure into DNA barcode analysis. Therefore, we propose that ITS2 sequence-structure information could be used as an alternative mini-barcode when a full-length ITS is not present.