Abstract

The applicability of the random amplified polymorphic DNA (RAPD) and single-strand conformation polymorphism (SSCP) techniques using M13 and 16S rRNA primers, respectively, for genotyping of the phytopathogenic bacterium Xanthomonas campestris pv. campestris was studied. RAPD provided a simple, rapid, and reliable method to identify genetic variation, and SSCP was used to screen for sequence polymorphisms. A 730-bp region amplified using 16S rRNA primers was subjected to restriction digestion followed by SSCP analysis (restriction fragment length polymorphism (RFLP)-SSCP) to identify polymorphisms, and the results were compared with the RAPD profile obtained using the phage M13 core sequence as a single primer. SSCP is a useful tool for the detection of mutations but large amplicon size can hinder secondary structure formation. Therefore, RAPD using an M13 primer is more efficient for phylogeny detection. The use of a novel polymerase chain reaction (PCR)-based DNA fingerprinting method using both RAPD and SSCP to detect DNA sequence diversity is reported. This approach consists of PCR amplification of the 16S ribosomal DNA with universal primers and analysis of the PCR product by SSCP. This is the first report wherein plant pathogens are subjected to RFLP-SSCP combined with M13 primer-based analysis. This report provides a rapid and reliable method for phylogenetic analysis.

Key words: Xanthomonas campestris pv. campestris,  polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), microbial phylogenetics, randomly amplified polymorphic DNA (RAPD). 

Abbreviation

Abbreviations: SSCP, Single-strand conformation polymorphism; ssDNAs, single stranded DNAs; RAPD, random amplified polymorphic DNA; PBS, phosphate buffered saline; EDTA, ethylene diamine tetra acetate; SDS, sodium dodecyl sulfate; RFLP, restriction fragment length polymorphism; YDCC, yeast dextrose calcium carbonate; HTS, high-throughput screening; ARDRA, amplified rDNA restriction analysis; DGGE, denaturing and temperature gradient gel electrophoresis.