DNA Barcoding Identification Of Dendrobii Caulis, Menispermi Rhizoma, Cinnamomi Cortex And The Study On Chloroplast Genome Of Dendrobium Officinale

After more than ten years’development, DNA barcoding is widely applied in the field of identification of traditional Chinese medicine (TCM).This study discussed the operation process and actual application of DNA barcoding identification of Dendrobium species, Menispermi Rhizoma, Cinnamomi Cortex and their adulterants, and screening DNA barcodes to identify rare and endangered Dendrobium species based on the chloroplast genome sequence of D. officinale. The study came to the following results:(1) 173 samples of 40 Dendrobium species were collected to evaluate the identification ability of ITS2 sequence. The results showed that the ITS2 of Dendrobii officinalis Caulis could be amplified by the specific primer DO-F/R. The intra-specific variation of all Dendrobium ITS2 sequences was much lower than the inter-specific one and the identification efficiencies were 15.12% and 61.27% calculated by BLAST and Nearest Distance methods, respectively. Most of the species of the genus could be distinguished by ITS2 sequence.The chloroplast genome of D. officinale was sequenced by Illumina Miseq. Genome structure, gene order, GC content and codon usage were analyzed. Compared with the other chloroplast genomes of Orchidaceae, DNA markers for species identification were evaluated and screened by the calculation of K2P genetic distance and the construction of the neighbor-joining tree. The results showed that petA-psbJ and psbK-psbl could be the candidate DNA barcodes to identify Orchidaceae.According to the results of chloroplast genome analysis,34 samples belonging to 17 species of Dendrobium were used to verify the identification ability of psbK-psbl. The results showed that the efficiencies of amplification and sequencing were 100%, and the psbK-psbI sequences were conservative within species and highly variable among species. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.(2) 49 samples belonging to five species, including 34 samples of M. dauricum, from different locations and herb markets in China were collected and subjected to DNA barcoding analysis using the ITS2 and psbA-trnH. The sequences of all 34 samples of Menispermi Rhizoma were highly consistent. The intra-specific genetic distance was much smaller than the inter-specific one. Phylogenetic analysis showed that both sequences allow the successful identification of all species. Nearest distance and BLAST methods for both ITS2 and psbA-trnH regions indicated 100% identification efficiency. All the commercial materials were effectively identified and the results were consistent with the medicinal drug labels. Our research showed that DNA barcoding could effectively distinguish Menispermi Rhizoma from its adulterants.(3)The psbA-trnH sequences of 59 samples from eight species of Cinnamomum were amplified and sequenced successfully. The Kimura 2-parameter (K2P) distances were calculated and phylogenetic tree was constructed using software MEGA 5.0. The psbA-trnH intra-specific genetic distance of C. cassia was highly conservative, which was smaller than the inter-specific one. The NJ tree indicated that Cinnamomi Cortex could be distinguished from its adulterants unambiguously. The chloroplast psbA-trnH intergenic region can be used to authenticate medicinal and edible materials of C. cassia, which provide technical guarantee to ensure medication and food safety standards.