REP-PCR MEDIATED GENOMIC FINGERPRINTING OF RHIZOBIA AND COMPUTER-ASSISTED PHYLOGENETIC PATTERN-ANALYSIS

A rapid and reproducible method has been developed for genomic fingerp rinting of rhizobia and other soil microbes interacting with plants. T he method is based on the use of oligonucleotide primers, correspondin g to conserved motifs in naturally occurring interspersed repetitive D NA elements in bacteria (rep-elements), and the polymerase chain react ion (rep-PCR). Rep-PCR results in the amplification of inter-element g enomic DNA fragments of characteristic lengths and thereby generates a genomic fingerprint. These fingerprints resemble UPC bar code pattern s, and can be used to identify bacteria at the sub-species and strain level, as well as for phylogenetic analyses. Here we show that highly characteristic and very reproducible rep-PCR generated genomic fingerp rints can be obtained not only from purified genomic DNA, but also dir ectly from rhizobial cells derived from liquid cultures or from coloni es on plates, as well as from nodule tissue. We examine the effect of growth phase of the bacterial cells, serial subculturing and other par ameters on the reproducibility of the rep-PCR fingerprinting protocol. Moreover, we describe the results of mixing experiments designed to d etermine if individual genomic fingerprints can be recognized in mixtu res of strains. Lastly, we review the use of computer-based fragment d etection and phylogenetic analysis packages to analyse rep-PCR generat ed genomic fingerprints of a collection of Rhizobium loti and Bradyrhi zobium strains nodulating different Lotus spp.