DNA Extraction And Identification Of Zanthoxylum Using DNA Barcoding

There are 39 different Zanthoxylum Rutaceae plant species in China. DNA barcoding is a new method for identifying species, and it is a Molecular technique which use a relatively short fragment of the genome to identify species. The study discussed the DNA extration methods of Zanthoxylum Rutaceae plants and improve the extraction method of different plant materials plant on Zanthoxylum plants. This paper identified nine species of Zanthoxylum using DNA barcoding. Aim at using the molecular identification means to identify Zanthoxylum more conveniently and fastly, and trying to identify the different varieties of the Z. Bungeanum and the Z. armatum by using ITS2 sequence.The main conclusions of this study are as follows:(1) Two kinds of commonly used methods, i.e. CTAB method and Reagent Kit method were used to extract the collected specimens of Zanthoxylum. The results showed that the genomic DNA of leveas dried by silica gelcould which be successfully extracted by both CTAB and Reagent Kit method. For the samples of pericarp of Zanthoxylum, the nuclear isolation buffer was added into the powdered pericarp samples and mixed thoroughly before splitting to improve the success rate of extraction. In addition, the extraction efficiency wasn’t affected when the water bath time was 1h. The ITS2 sequences were successfully amplified from 121 samples, which is informative for the extraction of materia medica from Zanthoxylum.(2) In this study, the ITS2 sequence was used to distinguish the Zanthoxylum and the results indicated that ITS2 was an ideal barcode to identify the collected nine species of Zanthoxylum. Apart from Z. Bungeanum, the intra-specific K2P genetic distances of the remaining eight species were smaller than the intre-ones, which could be used for species discrimination. In the multiple sequence alignment, all nine species possess stable variation sites which were unique to each species, making ITS2 sequence appropriate to identify the nine species of Zanthoxylum. Furthermore, the NJ tree indicated that the clades of nine species showed great monophyly, which could be used for the identifacation of Zanthoxylum.(3) Among the four varieties of Z. bungeanum, ITS2 sequences showed no feasibility for the identification of Hanyuan Z. bungeanum、Yuexi Z. bungeanum and Lingshan Z. bungeanum, because their ITS2 sequence were identical. Morever, the ITS2 sequence of Z. bungeanum from Sonpan, Maoxian, Wenchuan in Sichuan province had three parallel sites toones from Lixian, Linxia, Tianshui in Gansu province, which didn’t seen in the rest of producing area.Furthermore, no stable and obvious variations were detected in three varieties of Z. armatum.