Grouping of Bradyrhizobium USDA strains by sequence analysis of 168 rDNA and 168-238 rDNA internal transcribed spacer region

In order to select appropriate Bradyrhizobium USDA reference strains for primary grouping of indigenous soybean bradyrhizobia, we systematically constructed phylogenetic trees of 20 USDA strains based on DNA sequence analysis and PCR-restriction fragment length polymorphism (RFLP) targeted to 16S rDNA and the internal transcribed spacer (ITS) region between 16S and 23S rDNAs. The phylogenetic trees of 16S rDNA showed 3 major groups, cluster USDA 110 (USDA 62, 110, 122, 125, and 129), cluster USDA 6 (USDA 4, 6^T, 38, 115, 123, 127, 135, and 3622^T) and cluster B. elkanii (USDA 31, 46, 61, 76^T, 94, and 130), as well as the phylogenetically independent strain USDA 124. The topology of the ITS trees was almost similar to that of 16S rDNA, although the positions of two extra-slow-growing strains, USDA 135 and USDA 3622^T were variable among the ITS sequences, PCR-RFLP of the ITS region and 16S rDNA. Only two strains, USDA 110 and USDA 122, harbored hup genes and they fell into the USDA 110 cluster. These results suggest that PCR-RFLP analysis of 16S rDNA and the 16S-23S rDNA ITS region may be useful for the grouping of bradyrhizobia and for the first screening of hup-positive strains. Based on the above results, we propose a minimum set of USDA strains reflecting Bradyrhizobium diversity that includes B. japonicum USDA 6^T, B. japonicum USDA 110, B. japonicum USDA 124, and B. elkanii USDA 76^T. In addition, an extra-slow-growing strain with the serotype USDA 135 might be necessary for genomic diversity analysis of bradyrhizobia, because their phylogenetic positions were variable.