Identification Of Tiny Seed And Indirubin Containing Chinesem Edicinal Materials By DNA Barcoding

China has abundant medicinal plant and animal resources. More than 600 kinds of traditional Chinese medicine have been listed in pharmacopoeia of the People’s Republic of China. However, due to geographical and historical causes, the utilization of traditional Chinese medicine was very disordered. This problem seriously affected the safety use and curative effect of traditional Chinese medicine. DNA barcoding has its own unique advantages in solving this problem. In this study,tiny seed and indirubin containing Chinese medicinal materials were selected to discuss the standard operation procedure and actual application value of this approach. The main results in this study are as follows:(1) In this study, 144 samples of 28 species of Plantaginis Semen, Celosiae Semen, Hyoscyami Semen, Descurainiae and Lepidii Semen, Cuscutae Semen and Vaccariae Semen and their adulterants were selected to be the objects. The genomic DNA was extracted from 144 samples including six kinds of tiny seed Chinese medicinal materials and their adulterants by DNA extraction kit reagent kit method. Their internal transcribed spacer 2(ITS2)sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using Codon Code Aligner v 4.25. The genetic distances, variable sites and theneighbor-joining(NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-parameter(K2P) model. We found that the ITS2 sequences ranged from 194 bp to 221 bp in length and were easy to be amplified and sequenced. According to the results of BLAST1, the reliability of the identification of ITS2 was 88.9%. The maximum intraspecific genetic distances of Plantago depressa, Plantago asiatica, Celosia argentea, Hyoscyamus niger, Lepidium apetalum, Descurainia Sophia, Cuscuta chinensis, Cuscuta australis and Vaccaria hispanica were 0.005, 0, 0.009, 0.005, 0.010, 0.021, 0.005, 0.034 and 0, respectively, which were smaller than inter-specific distance among the nine species; The maximum intra-specific genetic distances of P. depressa, P. asiatica, L. apetalum, D. sophia, C. chinensis, C. australis and H. niger were smaller than inter-specific genetic distance between these species and their adulterants. Only the minimum inter-specific genetic distance of C. argentea and C. cristata was equal to the maximum intraspecific one. The NJ phylogenetic tree showed that except P. depressa and C. argentea, each species which we studied was clustered into one monophyletic branch, and clearly separated with other species. Although the results of NJ tree indicated that ITS2 can not be used to identify P. depressa, the results of BLAST1 and nearest distance were positive.Therefore, the ITS2 sequence was able to identify five kinds of tiny seed Chinese medicinal materials and their adulterants to ensure the safety of medicines, except Celosiae semen.(2)Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of Chronic Myelocytic Leukemia. Due to misidentification and similar name,In conclusion, DNA barcoding, as a novel molecular identification can be used to distinguish five kinds of thiny semen Chinese medicinal materials and their adulterants. And a new method which combines DNA barcoding with HPLC was established to identify indirubin-containing Chinese medicinal materials and their adulterants. This study provides an important evidence for other Chinese material medica in the pharmacopioea of the People’s Republic of China.