Identification Of Crude Drugs Come From Smilax Glabra And Its Closely Related Species Using The DNA Barcoding Technique

Aims:DNA barcoding has become one of hotspots of Traditional Chinese Medicine molecular identification research in recent years. It is a new biological method with which species identification is performed rapidly and accurately by using short and universal segments of DNA. Now the adulterants of the Smilax glabra were full of the market which mainly come from the related species, and the authentic and its adulterants appearance are similar, In this study,comparing Smilax glabra and its related species by DNA barcoding candidate sequences: ITS,ITS2,psbA-trnH,matK,rbcL. In order to establish the DNA barcoding method for identification of Smilax glabra and its related species method, provide reference for the DNA barcoding technology in traditional Chinese medicine authentication system. Methods:This study chose 34 Smilax glabra and its related species from different sources. And then the experiment selected 5 candidate sequence and amplified then in all of the nuclear genome DNA sequences of ITS and ITS2,psbA-trnH,matK,rbcL chloroplast genome DNA sequences. DNA was extracted from 3 medicinal materials by using the method of kit and PCR amplification reaction condition was optimized. The reaction product after bidirectional sequencing:The sequences and related data were analyzed using the software Codon Code Aligner V 3.7.2 and MEGA 6.0 The intra-and-inter Kimura-2-Parameter distances were calculated and the phylogenetic tree constructed using the Neighbor-joining method. psbA-trnH,matK,rbcL was by using SPSS 17.0 software to complete the wilcoxon signed rank test and the “barcoding Gap”test also be completed, As a measure between the different sequences of potential species identification capability. The sequence and Genbank registered sequence were analyzed by used the method of BLAST. Results:From the extraction of DNA, PCR amplification and electrophoresis conditions of the view, most of the medicinal materials of DNA can be extracted. In addition to ITS sequence,ITS2,psbA-trnH,matK,rbcL sequence are effective,And electrophoresis test strip bright, single. In addition to the ITS2 sequences appear a large number of hybrid or combined. psbA-trnH, matK,rbcL sequence can obtain higher quality of sequencing peak figure. The sequencing of success rate is 92%, 100%, 100% respectively. From the genetic distances, phylogenetic tree construction and investigation results of the view,the Smilax glabra of intraspecific variation is not obvious,psbA-trnH, matK,rbcL sequence of intraspecific genetic distance are 0.0018,0.0015,0.0000,respectively.But the Smilax glabra and its related species of interspecific difference is bigger, psbA-trnH, matK,rbcL sequence of interspecific genetic distance are: 0.3151,0.1450,0.0061,respectively.In the cluster dendrogram,the plants of Smilax glabra Roxb.come from various sources showed the monophyletic and simultaneously distinguished from the close relative species. Therefore, three sequences can identify Smilax glabra and its related species. Based on the Wilcoxon rank sum test and “ barcoding Gap ” test, the results show that: psbAtrnH,matK, rbcL sequences are accord with the basic requirements as a DNA barcode. This study by comparing gene sequences in the NCBI database,to determine the homology between sequences, Results indicate that the identification of species are in more than 95% success rate. Conclusions:DNA barcoding can be used for the identification of traditional Chinese medicine. And psbA-trnH, matK,rbcL sequence can be used for identification of Smilax glabra and its related species effectively.