Restriction Fragment Length Polymorphism Analysis of 16S rDNA and Low Molecular Weight RNA Profiling of Rhizobial Isolates from Shrubby Legumes Endemic to the Canary Islands

Abstract

Thirty-six strains of slow-growing rhizobia isolated from nodules of four woody legumes endemic to the Canary islands were characterised by 16S rDNA PCR-RFLP analyses (ARDRA) and LMW RNA profiling, and compared with reference strains representing Bradyrhizobium japonicum, B. elkanii, B. liaoningense, and two unclassified Bradyrhizobium sp. (Lupinus) strains. Both techniques showed similar results, indicating the existence of three genotypes among the Canarian isolates. Analysis of the combined RFLP patterns obtained with four endonucleases, showed the existence of predominant genotype comprising 75% of the Canarian isolates (BTA-1 group) and the Bradyrhizobium sp. (Lupinus) strains. A second genotype was shared by nine Canarian isolates (BGA-1 group) and the B. japonicum and B. liaoningense reference strains. The BES-5 strain formed an independent group, as also did the B. elkanii reference strains. LMW RNA profile analysis consistently resolved the same three genotypes detected by 16S ARDRA among the Canarian isolates, and suggested that all these isolates are genotypically more related to B. japonicum than to B. elkanii or B. liaoningense. Cluster analysis of the combined 16S ARDRA and LMW RNA profiles resolved the BTA-1 group with the Bradyrhizobium sp. (Lupinus) strains, and the BES-5 isolate, as a well separated sub-branch of the B. japonicum cluster. Thus, the two types of analyses indicated that the isolates related to BTA-1 conform a group of bradyrhizobial strains that can be clearly distinguishable from representatives of the tree currently described Bradyrhizobium species. No correlation between genotypes, host legumes, and geographic location was found.