[Study on high throughput sequencing identification of Fructus Arctii and five counterfeit species mix power].

Fructus Arctii is a traditional Chinese medicine. The main counterfeit species are the seeds of Arctium tomentosum, Onopordum acanthium, Silybum marianum, Saussurea costus, Amorpha fruticosa. Traditional identification methods or molecular barcoding techniques can identify Fructus Arctii and its counterfeit species. However, the identification of the mixture of it and its spurious species is rarely reported. In this paper, we sequenced the ITS2 sequences of Fructus Arctii and 5 kinds of spurious species mix powder by high-throughput sequencing to identify the mixed powder species and providing new ideas for the identification of Fructus Arctii mix powder. The total DNA in mixed powder was extracted, and the ITS2 sequences in total DNA was amplified. Paired-end sequencing was performed on the DNA fragment of the community using the Illumina MiSeq platform. The sequence was analyzed by the software FLASH, QIIME and GraPhlAn etc. The results showed that the high quality ITS2 sequences of 39910 mix samples were obtained from the mixed samples, of which the total ITS2 sequence of the samples genus was 34 935. Phylogenetic analysis showed that the samples contained Fructus Arctii, A. tomentosum, O. acanthium, S. marianum, S. costus and A. fruticosa. Using ITS2 sequences as DNA barcodes, high-throughput sequencing technology can be used to detect the Fructus Arctii and its spurious specie in mixed powder, which can provide reference for the quality control, safe use of medicinal materials of Fructus Arctii and the identification of mixed powder of traditional Chinese medicine.

[DNA Barcoding of Mongolian Oxytropis Medicinal Materials].

Objective: To identify Oxytropis medicinal materials using ITS2 sequence. Methods: The second internal transcribed spacer( ITS2) of Oxytropis fetissovii, Oxytropis myriophylla and Oxytropis grandiflora medicinal material samples was amplified by PCR and sequenced. To expand scope of the research topic,ITS2 sequences of related species were downloaded from Gen Bank. Sequences assembly and consensus sequence generation were performed by ITS2 Database. The related data analysis and processing was performed using software MEGA 5. 10 and the NJ tree was constructed. The ITS2 secondary structure was predicted using ITS2 web server, and the differences of the ITS2 secondary structures of the samples were analyzed. Results: Oxytropis medicinal materials ITS2 sequence was shorter, with sequence length of 216 ~ 218 bp, which was in favor of DNA extraction, PCR amplification and sequencing. Genetic distances of Oxytropis myriophylla, Oxytropis fetissovii and Oxytropis grandiflora were much larger than the genetic distances of themselves. In the NJ tree, Oxytropis medicinal materials and counterfeits could be distinguished, and Oxytropis medicinal materials could be distinguished from Astragalus membranaceus. Conclusion: The DNA barcode based on ITS2 sequence is a powerful and efficient tool for identification of Oxytropis medicinal materials.

[DNA Barcoding of Mongolian Medicinal Plant Scutellaria scordifolia].

Objective: To authenticate Scutellaria scordifolia and its closely related species using ITS2 sequence. Methods: Total genomic DNA was isolated from Scutellaria scordifolia and its related species. Nuclear DNA ITS2 sequences were amplified and sequenced,and ITS2 sequences of the other species of plants were obtained in Gen Bank,The Kimura 2-parameter( K2P) distances were calculated. Identification analyses were performed using Neighbor-joining( NJ) and secondary structure of ITS2 sequence methods. Results: The genetic distances of ITS2 between Scutellaria scordifolia and its closely related species were 0. 014 ~ 0. 141,which were obviously higher than those in the intra-species of Scutellaria scordifolia. The NJ tree and secondary structure of ITS2 sequence can distinguish Scutellaria scordifolia and its closely related species. Conclusion: ITS2 sequence can effectively and accurately identify the Mongolian medicinal plant Scutellaria scordifolia and its closely relatives.