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| Molecular Identification of Ilicis Chinensis Folium from Its Closely Related Species and Adulterants Based on Ribosomal Internal Transcribed Spacer 2 (ITS2) Sequence |
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Abstract Ilicis Chinensis Folium is a traditional Chinese herbal medicine, which derived from one kind of ornamental plants named Ilex chinensis. In order to identify adulterants emerged in the market and closely related species in Ilex rapidly and reliably by DNA barcoding, ribosomal internal transcribed spacer 2 (ITS2) from 37 materials of I. chinensis, Ilex rotunda, Ilex asprella, Ilex cornuta and Ligustrum lucium was amplified and sequenced, respectively. Assembled by CodonCode Aligner 4.2.7, all of the 37 ITS2 sequences ampilified and 28 GenBank sequences of Ilex, L. lucium, Symplocos setchuensis were annotated based on hidden Markov model (HMM) to cut off 5.8S and 28S so that complete ITS2 region could be gained. Kimura 2-parameter (K2P) genetic distance analysis of intraspecific and interspecific accompanied by neighbor-joining tree (NJ tree) construction was performed by molecular evolutionary genetics analysis (MEGA6.0). Results indicated that unique bands were obtained from gel electrophoresis demonstrating the successful amplification of ITS2 sequences by the universal primer. Sequencing results showed that the aligned sequence length of I. chinensis was 239 bp and the interspecific one was 269 bp. Four variable sites of intraspecies were discovered, which divided the I. chinensis into 6 haplotypes. In contrast, there were 143 interspecific variable sites which were much more than those of I. chinensis. Sequence characteristics analysis of intraspecific and interspecific by MEGA6.0 showed the maximum K2P genetic distance of intraspecies was 0.013, which was much shorter than the minimum K2P genetic distance of interspecies of 0.094. L. lucium and S. setchuensis clustered into separate clade with bootstrap scores both of 100%. I. chinensis distributed to a single cluster that belonged to the cluster of the Ilex. Species in the same subgenus of Ilex could also cluster together. Therefore, ITS2, as one of the universal DNA barcodes, could differentiate I. chinensis from its adulterants and closely related species effectively. As a molecular biological method for discrimination, DNA barcoding possessed huge potential to authenticate the Ilex, meanwhile, it provides scientific foundation for clinical medication safety, exploration and application.
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Received: 15 November 2014
Published: 17 March 2015
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