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

Abstract

Bradyrhizobium japonicum USDA 110 has been shown to contain several genetically similar naturally occurring colony morphology variants. These variants differ in symbiotic nitrogen fixation ability and in the utilization of various carbon substrates. They have been shown to share extensive DNA homology and appear to be derived from a common ancestor. Despite these similarities certain B. japonicum USDA 110 variants have been shown to be devoid of symbiotic nitrogen fixation. One of these variants (L2-1 lo), however, was recently shown to possess significant levels of ex planta nitrogen fixation and to synthesize functional dinitrogenase enzyme within bacteroids. In an effort to identify genetic markers which could explain differences in symbiotic nitrogen fixation between B. juponicum variants, DNA fingerprints were generated by PCR using arbitrary primers. Two of these primers with GC rich sequences were able to differentiate between B. japonicum USDA 110 variants I-l 10, L2-110, and MN-110. Unique markers have now been identified which could be examined further to determine if they explain the differences in symbiotic nitrogen fixation between USDA 110 variants.

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“…Reported sequences of primers 18,19 RPO1 and CRL-7 were as follows: RPO1, 5 -AATTTTCAAGCGTCGTGCCA-3 ; CRL-7, 5 -GCCCGCCGCC-3 . PCR program was as follows: 95°C 15 s, 55°C 30 s, 72°C 90 s for 5 cycles, 95°C 15 s, 60°C 30 s, 72°C 90 s for 25 cycles, followed by 72°C 10 min.…”

The Diversity of Root Nodule Bacteria Associated with Lebeckia Species in South Africa

“…Nevertheless, phenotypic traits are useful when used in combination with genotypic DNA analysis in polyphasic classification approaches described below in section 1.5.1 (Rossum et al, 1995). Mathis & McMillin, 1996;. The main reason for this shift in focus is associated with the fact that DNA-based approaches are relatively easy to apply, robust and resolve even closely related species .…”

“…This method involves PCR amplification of genomic DNA using short arbitrary primers with 9-10 bases (Doyle et al, 1993;Micheli & Bova, 1996). According to Mathis & McMillin, (1996), variations in DNA structure of Bradyrizobium strains are observed when using RAPD-PCR with GC-rich arbitrary primers (e.g. CRL7) (Mathis & McMillin, 1996;Law et al, 2007).…”

“…According to Mathis & McMillin, (1996), variations in DNA structure of Bradyrizobium strains are observed when using RAPD-PCR with GC-rich arbitrary primers (e.g. CRL7) (Mathis & McMillin, 1996;Law et al, 2007). This approach is routinely used for the authentication of inoculant strains, identification of predominant nodulating strains from particular field sites, assessment of genetic diversity and relatedness amongst field populations of rhizobia Schneider, & de Bruijn, 1996;Law et al, 2007).…”

“…The nif gene derived primer RPO1 and arbitrary sequence primer CRL7 (Mathis and McMillin, 1996) (Table 3.2) were used to fingerprint the 76 Lebeckia rhizobia and the type strains (Tables 3.4). All PCR amplifications were conducted in a total reaction volume of 10 µl containing 5 ng/µl DNA, 1.5 mM MgCl 2 , 800 µM dNTPs, 1 µM primer, and 0.025 U/µl Taq polymerase and its reaction buffers (Promega, Madison, USA) .…”