Screening And Applying Of DNA Barcodes For Clavibacter And Curtobacterium

DNA barcoding is one of the molecular diagnostic techniques that can identifying species by analyzing a standard DNA regions.DNA barcoding technology is a combination of molecular biology and bioinformatics.It provides us an easy,rapid and accurate method relying on mature PCR amplification and sequencing technology as well as developed network information technology.This technology has become a hot spot in the research of biological taxonomy in recent years,and it is an effective complement to the traditional identification methods.The main principle of DNA barcoding is that the variation of the interspecies is higher than intraspecies,so as to distinguishing the species.Clavibacter and Curtobacterium,two of the important plant pathogenic bacteria,are selected in this study to screening the best DNA barcode gene for these two genera.In order to choose a suitable barcoding gene for Clavibacter,4 candidate gene(16S rRNA,cpn60,gyrB,rpoD)were tested on 95 samples in Clavibacter and other closer genera.After the PCR amplification and sequencing,amplification rate,sequencing success rate and other information of the obtained sequences to each genes are calculated and arranged,we carry on some necessary analysis including Wilcoxon signed rank test, barcoding gap as well as NJ phylogenetic trees and get the conclusion that the gyrB gene is the best choice for DNA barcoding gene of Clavibacter.This gene can successfully distinguish five subspecies in Clavibacter.Cpn60 gene can also achieve the goal of identifying different subspecies,however,the distinguish effect of cpn60 is a bit lower than gyrB gene.At the same time,16 S rRNA gene has the advantage of distinguishing Clavibacter on the speices level,while the distinguishing between subspecies could be a weakness in this gene.The rpoD gene was excluded because of its low amplification rate.The same 4 candidate genes were tested on 68 stains in Curtobacterium and other closer genera.In order to screen the suitable barcode gene of Curtobacterium,3 candidate genes and four gene combination(16S+cpn60,16S+gyrB,cpn60+gyrB, 16S+cpn60+gyrB) are also prepared to be analysed.After all necessary analysis we get the conclusion that all the sequences we used are not able to differentiate the 4 pathovars of the Curtobacterium flaccumfaciens.Thus new DNA barcode genes should be screened to accomplish the goal of identifying different pathovars in Curtobacterium flaccumfaciens.The application of DNA barcoding is also tested in the study. DNA barcode technology were used to identify the bacteria isolated from oats seeds,the resulte proved that DNA barcode technology is feasible to identify and distinguish subspecific in genus Clavibacter. This study also screening the specific primers to detect Clavibacter michiganense subsp.michiganense and the result show that PSA8/PSAR are the best choice.