Characterisation of Rhizobium isolates by amplification of DNA polymorphisms using random primers

Abstract

The use of single random primers, selected in the absence of target sequence information, has been shown to be effective in producing DNA amplifications that provide fingerprints which are unique to individual organisms. DNA amplification by random priming was applied to the DNA from isolates of Rhizobium leguminosarum biovar trifolii. Amplification products were produced using a number of primers, and the resulting fingerprints allowed strain differentiation. However, the effectiveness of primers was dependent upon length and GC content. It was also possible to amplify DNA directly from cells in culture and in nodule tissue. Lysis of these cells was achieved simply through heat applied in the initial DNA denaturation stage of the thermal reaction. The ability to produce varied amplification patterns from different Rhizobium isolates, especially directly from nodules, gives this method potential for use in examining genetic structures and relationships in Rhizobium populations.

Genetic characterization of naturally occurringRhizobium melilotipopulations and their potential to form effective symbioses with lucerne

“…Following further centrifugation, the washed pellet was resuspended in SDW and further diluted to achieve a concentration of 10 4 -10 5 cells 50 ml −1 reaction. Amplification conditions and agarose gel electrophoresis were as described by Harrison et al (1992).…”

“…Harrison et al (1992), Dooley et al (1993) and Dye et al (1995) used arbitrary primers of varying length to produce Randomly Amplified Polymorphic DNA (RAPD) fingerprints for rhizobial identification.…”

Detection of genetic variation in Bradyrhizobium japonicum USDA 110 variants using DNA fingerprints generated with GC rich arbitrary PCR primers

“…In order to determine which primers might be useful in detecting polymorphisms between USDA 110 colony morphology variants, 28 primers were screened using representative DNA samples from USDA 110 and USDA 122. The following primers were screened, OPE-01 -OPEl0 and OPO-1 -OPO-10 (Operon Company, Alameda California, USA), ARP-2, ARP-5, ARP-6 (McMillin and Muldrow, 1992), ARP-7 (GTACGTGGCG), SPH-1 (Harrison et al~ 1992), MICROSAT, [(T or A)(T or A)GAGGGTGG], CRL-7 and CRL-13 (Kubelik and Szabo, 1995). Each of the primers which amplified and detected polymorphisms between USDA 110 and USDA 122 were used to amplify DNA from B. juponicum USDA 110 variants.…”

Biocontrol Strain Pseudomonas sp. W34: Specific Detection and Quantification in the Rhizosphere of Cucumis sativus with a DNA Probe and Genotypic Characterization by DNA Fingerprinting

“…This method proved to be suitable for antagonistic pseudomonads in our study. At the same time, this method allows insights into the phylogenetic relationships of the organisms studied on the strain and isolate level (Harrison et al, 1992). In our study, RA PD results were extended and supported by repetitive sequence-based PCR (rep-PCR, Versalovic et al, 1994) using B O X and enterobacterial repetitive intergeneric con sensus (E R IC ) primers, a technique that also yields strain-specific genomic fingerprints (de Bruijn, 1992;Rademaker et al, 1997).…”