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RSα is a repeated DNA sequence found within the nitrogen-fixation gene cluster of Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium that nodulates soybean. Bradyrhizobium japonicum strain 110spc4 contains 12 repeats, each located on a separate XhoI DNA restriction fragment between 1.2 and 14 kb in length. Although Fix+ and Fix− derivatives of B. japonicum USDA 110 were first reported more than two decades ago, genotypic differentiation, on the basis of RSα hybridization pattern, was reported only recently. Bradyrhizobium japonicum strain USDA 110 had only single copies of the RSα-hybridizing bands, but a particular Fix− derivative, MSDJG1, carried doublets of two distinct XhoI fragments that carry RSα3 and RSα4. In this study, RSα hybridization patterns were analysed further in both Fix+ and Fix− derivatives of strain 110 to test for duplication of these particular genomic regions. It was concluded that the duplication, or not, of genetic regions carrying RSα3 and RSα4 in strain USDA 110 derivatives is unrelated to symbiotic nitrogen-fixation ability. Like Fix− MSDJG1, Fix+ strain 110 derivatives I-110 and MN-110 had duplications of the XhoI DNA restriction fragments carrying RSα3 and RSα4, but Fix− strain 110 derivative L2-110 lacked these duplications. Thus, it is now clear that Fix− derivatives MSDJG1 and L2-110 arose via distinct genetic mechanisms. Interestingly, Fix+ derivatives of strain 110 from the laboratories of Elkan and Hennecke differed in RSα hybridization profile.