Barcoding Chinese Herbal Medicines: From Gene To Genome

The authenticity of the traditional Chinese medicine is the key point of its quality control, as well as the precondition of the clinical medication safety. Especially for the rare and toxic species, authentication between the official species and their adulterants becomes very important. DNA barcoding is a rapid and accurate identification method, which has been widely applied in the authentication of both original plants and crude drugs of the traditional Chinese medicine. Although many different loci have been proposed for plant species identification, none of them could work across all species, especially among closely related ones. In recent years, chloroplast genomes have been advocated as the "super barcode" in the study of phylogenetic analysis, genetic relationships and species identification. Building standard DNA barcoding reference databases is the main form of its application. In this study, we concentrated on the DNA barcoding technic. Firstly, a DNA barcode identification system was set up for the crude drugs in the Japanese pharmacopoeia; secondly, the species of Panax and Aconitum were selected as the representative of the rare and toxic medicines respectively to study the DNA barcoding application; finally, three chloroplast genomes of the Aconitum were sequenced using the high throughput sequencing platforms in order to lay the foundation of super barcode utilization, as well as to discover highly variable loci as novel molecular markers for the Aconitum. In this study, main contents and conclusions include:1. A standard barcode database based on the ITS2 region and the psbA-trnH region for Kampo crude materials was developed. This tool provides users with the basic information on each crude drug recorded in the Japanese pharmacopoeia, herbal material DNA barcoding identification, and the standard operating procedure (SOP) from sampling to data analysis. A total of 576 samples were collected to establish the database. An additional 100 samples were identified using the database.71% of test samples were identified at species level as same as the names listed on their label. The remaining 29% were correctly identified at genus level. We report the first database that includes the standard barcode sequences from 97.3% of the crude drugs recorded in the JP (16th edition), which has a beneficial demonstration effect throughout the other pharmacopoeias.2. SNPs were detected in ITS2 region from the four Panax species and intra genomic variations were studied. Intraspecific variations were analyzed based on three typical DNA barcodes (ITS2, matK and psbA-trnH). Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Results from this study revealed that among the four medicinal plants, greater intragenomic divergence is correlated with larger intraspecific variations. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species as well as the mixed powder of P. ginseng and P. quinquefolius.3. Four popular DNA barcodes (ITS, ITS2, matK and psbA-trnH) were selected to test their ability to identify the Aconitum on aspects of successful rates of amplification and sequencing, genetic distances among inter-and intra-species, barcoding gap, and phylogenetic trees. After general analysis, ITS was superior to other loci with the highest successful identification rate of 57.9% and proposed as the best DNA barcode candidate of the four barcodes for the Aconitum.4. Chloroplast genomes of Aconitum kusnezoffii, Aconitum barbatum var. puberulum and Aconitum gymnandrum were sequenced using Illumina MiSeq and PacBio RS sequencing platforms. There were 130 unique genes in the chloroplast genomes including 84 protein-coding genes,38 tRNA and eight rRNA. Genome comparison indicated that the large single copy region and the small single copy region were much more variable than the inverted repeat regions. The sequencing of the chloroplast genomes laid the foundation of super barcode utilization in the Aconitum.5. Ten highly variable intergenic regions were discovered from the three chloroplast genomes based on the multiple sequence alignment of ClustalW2 with the aim to developing unique molecular markers of the Aconitum. The ten intergenic regions were jointed in a certain order to get a highly variable marker with the length of 6kb approximately, which was considered to be a potential molecular marker for the Aconitum identification. This study gave a novel idea on authentication of closely related species.