The Feasibility Of ITS2 Secondary Structure In DNA Barcoding Of Medical Plants

Accurate species identification is the prerequisite for ensuring the efficacy and safety of traditional Chinese medicine(TCM).Traditional TCM identifications,relying on base source,morphological characters,microscopic structure and physicochemical property,are of strong subjectivity and poor stability.Alternatively,DNA barcoding technology has been widely used in TCM identification and the internal transcribed spacer region 2(ITS2)of rDNA has even been added into the Chinese Pharmacopoeia as the standard barcode.Although ITS2 has showed the highest resolving power among the most commonly used markers,it still lacks enough information in some certain herbs.Therefore,it is still necessary to explore more phylogenetic information to help TCM identification.In cells,the activity of ITS2 depends on its secondary structure.Recently,ITS2 secondary structure has been increasingly used in phylogenetic study because it includes information not found in nucleotide sequence.However,until recently it is remain unclear what extent could ITS2 improve species resolution with its secondary structure information.With the prediction,alignment and coding technology of RNA secondary structure gradually maturing,it is possible to explore the phylogenetic value of ITS2 secondary structure.In this study,ITS/ITS2 was obtained through DNA sequencing or GenBank downloading and identified and delimited based on the GenBank annotation or Hidden Markov models.The ITS2 matrix of a genus was imported in LocARNA software to generate secondary structures and their alignment.Finally,the improved RNAstat software was used to transform secondary structure into phylogenetic information and maximum parsimony analysis was performed into PAUP to reconstruct phylogenetic trees.We firstly tested this approach in the specific herbs of Solanum plants and TCM "Mu tong".After verifying its success,we expanded our work to 100 genera.Our results showed that:(1)Adding ITS2 secondary structure into phylogenetic,species resolution rate increased 33%and thus "Mu tong" and the common used Solanum herbs were successfully separated from their adulterants and substitutes.(2)The common used sequence alignment methods(e.g.Clustal X)may have some limitation to align the structured ITS2,because its length of aligment,gap sizes and positions all have some differences with the secondary structure guide alignments(e.g.LocARNA)in most of our taxa.(3)In seed plants,the alignment length increased 31%,the number of variable character increased 46%and the number of parsimony information character increased 45%when ITS2 secondary structure information was incorporated in phylogenetic.In sequence-structure tree,all bootstrap values were increased.In total 23 genera improved their species resolution and averagely the species identification rate increased 8%.Therefore,the shorter ITS2 and its secondary structure was equal to its widely used larger alternatives,such as ITS,matK+ITS2 and trnH-psbA+ITS2 in term of the species resolution rate.Based on these results,we suggest that ITS2 secondary structure information should be incorporated into medical plants DNA barcode.The combination of nucleotides and structure information make up for the insufficient phylogenetic information of ITS2 and improve species resolution ability.Therefore,the sequence-structure method in DNA barcoding will improve the system of TCM molecular identification,and provide a promising tool for TCM identification.