Diversity of root-nodule bacteria has been revealed by many studies and almost all of the data reported previously indicate that there is a high level of genetic diversity in these bacteria. An assessment of the genetic diversity and genetic relationships among strains could provide valuable information about bacterial genotypes that are well adapted to a certain environment. The aim of this study was to characterize very effective putative rhizobia strains isolated from different Iranian regions using a polyphasic approach that includes phenotypic properties and genetic analysis of ribosomal and symbiotic genes. 80 strains of rhizobia were isolated from alfalfa (Medicago sativa L.) nodules grown in six different regions of East Azarbayjan and Hamedan provinces in Iran. Colony morphology (size, color, mucosity, transparency, borders and elevation) and acid/alkaline reaction in vitro were evaluated. PCR amplification of repetitive regions of the DNA (rep- PCR) was carried out with BOX-A1R, enterobacterial repetitive intergenic consensus (ERIC) primers. The DNA of each strain was amplified with the universal primers of 16S rDNA gene (rD1/fD1). PCR products were then digested separately with each of the following restriction enzymes: MspI, HhaI and HaeIII, as recommended by the manufacturers. For the nifH region, the DNA of each strain was amplified with primers nifHF and nifHI resulting in a product of approximately 780 bp. Amplification of the nodC region was achieved using nodCF and nodCI primers and resulting in a fragment of about 930 bp. After amplification, PCR products were digested separately with the following restriction enzymes: HpaII, HaeIII and RsaI. The fragments obtained were analyzed by electrophoresis in a 3% agarose gel at 80V for 3 h. The sizes of the fragments in each analysis were normalized according to the MW of the DNA markers. The fingerprintings obtained in the RFLP-PCR and in the rep-PCR analyses were analyzed using Ntsys 2.02 software. All
