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Molecular identification of Cynomorii Herba using ITS2 DNA barcoding
HOU Dian-yun1，2， SONG Jing-yuan2， SHI Lin-chun2， YANG Pei2， CHEN Shi-lin2，3， YAO Hui2*
（1.Agricultural College， Henan University of Science and Technology， Luoyang ， China；
2.The National Engineering Laboratory for Breeding of Endangered Medicinal Materials，
Institute of Medicinal Plant Development， Chinese Academy of Medical Sciences & Peking Union Medical College，
Beijing ， China；
3.Institute of Chinese Materia Medica， China Academy of Chinese Medical Sciences， Beijing ， China）
[Abstract] Objective： To identify the Cynomorii Herba and its analogues species using DNA barcoding technique. Method： Total genomic DNA extracted from all materials using the DNA extraction kit. The internal transcribed spacer 2（ITS2） regions were amplified using polymerase chain reaction （PCR）， and purified PCR products were sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner 3.7.1. The Kimura 2-Parameter （K2P） distances and GC content were computed using MEGA 5. 0. Species identification analyses were conducted through the species identification system for traditional Chinese medicine and neighbor-joining （NJ） trees. Result： The ITS2 sequence lengths of Cynomorii Herba were 229 bp. The average intra-specific genetic distances of Cynomorii Herba were 0.003. The average inter-specific genetic distances between Cynomorii Herba and its adulterants species were 0.760. The results showed that the minimum inter-specific divergence is larger than the maximum intra-specific divergence. The species identification system for traditional Chinese medicine and NJ trees results indicated that Cynomorii Herba and its adulterants species can be easily identification. Conclusion： The ITS2 region is an efficient barcode for identification of Cynomorii Herba，which provide a new technique to ensure clinical safety in utilization of traditional Chinese medicine.
[Key words] Cynomor II Herba； DNA barcoding； ITS2； molecular identification
doi：10.4268/cjcmm