Paris Relatives And The DNA Bar Code Identification Of Commercial Drugs

Objective:On4different single sequence fragment (matK, trnL-trnF, rpoC1, ITS) and its two series combined fragments and three sequences were screened for DNA bar code fragments, or group to be able to fast, efficient, and reliable identification of genuine Rhizoma Paridis and common relatives and Commercial Rhizoma Paridis combined. On using DNA barcoding Paris and its adulterants provide the method reference.Methods:Universal primers using chloroplast trnL-trnF, matK, rpoC1sequence and nuclear ITS sequences,1Paris control medicine and collected from8Sichuan Rhizoma Paridis Yunnan, closely related species (26samples) and market to buy10Rhizoma Paridis, fake bistort rhizome, white monkshood (a total of3samples) for PCR amplification and sequencing, amplification efficiency and sequencing comparison of the sequence of success rate; Clustal software for multiple sequence alignment of the sequence, MEGA5.0software after cutting, calculation of GC content, in combination with PAUP4B10software, mutation of conserved sites, information site; distribution frequency of Taxon DNA software statistics of intraspecific and interspecific genetic distance, barcoding gap analysis, combined with Wilconxon rank sum test to compare the different fragments between intraspecific and interspecific differences, screened sequences and sequences the ideal combination; by constructing phylogenetic tree (MP tree) analysis and evaluation of the sequence of authentic Paris and sibling identification efficiency of herbs and commercial medicinal materials.Results:(1) Amplification efficiency of ITS, matK, rpoC1, trnL-trnF sequence fragments of PCR, ITS amplification efficiency was98%, followed by rpoC193%, trnL-trnF91%, matK Amplification efficiency was87%; ITS sequencing success rate reached97%, the success rate of rpoC1sequencing, trnL-trnF sequence was96.7%,95%, the success rate of sequencing matK sequence for up to83.6%.(2) Barcoding gap analysis showed that, single sequence fragments, side ITS genetic variation within species distribution in numerical smaller (left), interspecific variation in side value of the larger (right), spacer intraspecific and interspecific than other sequence fragment is more obvious, its gap position in4%-5%, followed by trnL-trnF sequence, the gap position in between2%-3%, matK, rpoC1series of intraspecific and interspecific overlap more, no obvious area gap. Series two fragments, ITS+trnF-trnL gap area than other sequences, and is better than the barcoding gap diagram of the two single segment. Series three fragments of barcoding gap figure and not based on two fragment combination has obvious change.(3) Wilcoxon test showed that, variability within and between species based on single sequences, fragments, ITS are greater than other sequence, and P<0.01, the difference was significant, followed by trnF-trnL. Two series of fragments, ITS+trnF-trnL interspecific variability than other sequences, intraspecific variability than other combinatorial sequences. The three series combined fragments of intraspecific and interspecific differences are low.(4) Based on the system of ITS, ITS+trnL-trnF in the constructed tree, close relative relationship are gathered one branch respectively, and purchased10Rhizoma Paridis samples of commercial medicinal materials,3and seven chinensis gathered together first, suggested that the seven chinensis have close relationship, and commercial medicinal materials and Yunnan4Paris closer genetic relationship, the close relationship of commercial medicinal materials1,5,7and mosaic of Paris, phylogenetic relationship of commercial medicinal materials10and Paris fargesii near commercial medicinal materials,9,8and gross floor has a close genetic relationship of commercial medicinal materials,2,6and merge together at the periphery of Paris species, the commodity identification2,6is false. Conclusion:DNA barcoding ITS sequences can be used as identification of Rhizoma Paridis and common relatives and commercial medicinal materials, at the same time, we recommend ITS+trnL-trnF sequence as its complement sequence. And, in order to be able to direct effects on the production and application, the follow-up work should also study content into chemical fingerprint, reflect the difference in chemical composition contained in the type and quantity of goods between Paris and relatives, Rhizoma Paridis, excellent combination seek to identify the authentic Paris and relatives, Rhizoma Paridis genotype best and chemical type.