Identification of Tibetan Medicine Jiawa and Easily Mixed Species Based on ITS2 Barcode
OBJECTIVE To establish an effective identification method for Jiawa and easily mixed species by using DNA barcod-ing ITS2 sequence.METHODS The total DNA of 72 Jiawa and its similar species samples were extracted by using the plant genomic DNA Extraction kit,PCR amplification and bidirectional sequencing were carried out for the ITS2 fragments,another 30 items of the genus were downloaded from GenBank.MEGA6.0 software was applied to analyze 102 ITS2 sequences,calculate the intraspecific and interspecific K2-P distance,construct neighber-jioning(N-J)phylogenetic tree,and predict the secondary structure of ITS2.RESULTS K2-P genetic distance analysis showed that,in addition to Pleurospermum franchetianum Hemsl.and Pleurospermum wrightianum de Boiss.,Pleurospermum hookeri var.thomsonii C.B.Clarke.,Sphallerocarpus gracilis(Bess.)K.-Pol.,Vicatia thibetica de Boiss.,Carum carvi L.,Heracleum millefolium Diels.,Ligusticum daucoides(Franch.)Franch.and Anthriscus sylvestris subsp.Nemorosa have obvious barcode spacing.N-J tree cluster analysis showed that each species could be clustered into one branch with a bootstrap support rate of＞50％,and the monophyleticity was good.The secondary structure analysis of ITS2 showed that the number,size and position of stem-loops in the four helical regions of Jiawa and its miscible were different.CONCLUSION The ITS2 sequence can be used to identify Jiawa and its easily mixed species,which is beneficial to the rational development and utilization of Jiawa resources.
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