Bradyrhizobium agreste sp. nov., Bradyrhizobium glycinis sp. nov. and Bradyrhizobium diversitatis sp. nov., isolated from a biodiversity hotspot of the genus Glycine in Western Australia

Strains of the genus Bradyrhizobium associated with agronomically important crops such as soybean (Glycine max) are increasingly studied; however, information about symbionts of wild Glycine species is scarce. Australia is a genetic centre of wild Glycine species and we performed a polyphasic analysis of three Bradyrhizobium strains—CNPSo 4010T, CNPSo 4016T, and CNPSo 4019T—trapped from Western Australian soils with Glycine clandestina, Glycine tabacina and Glycine max, respectively. The phylogenetic tree of the 16S rRNA gene clustered all strains into the Bradyrhizobium japonicum superclade; strains CNPSo 4010T and CNPSo 4016T had Bradyrhizobium yuanmingense CCBAU 10071T as the closest species, whereas strain CNPSo 4019T was closer to Bradyrhizobium liaoningense LMG 18230T. The multilocus sequence analysis (MLSA) with five housekeeping genes—dnaK, glnII, gyrB, recA and rpoB—confirmed the same clusters as the 16S rRNA phylogeny, but indicated low similarity to described species, with nucleotide identities ranging from 93.6 to 97.6% of similarity. Considering the genomes of the three strains, the average nucleotide identity and digital DNA–DNA hybridization values were lower than 94.97 and 59.80 %, respectively, with the closest species. In the nodC phylogeny, strains CNPSo 4010T and CNPSo 4019T grouped with Bradyrhizobium zhanjiangense and Bradyrhizobium ganzhouense , respectively, while strain CNPSo 4016T was positioned separately from the all symbiotic Bradyrhizobium species. Other genomic (BOX-PCR), phenotypic and symbiotic properties were evaluated and corroborated with the description of three new lineages of Bradyrhizobium . We propose the names of Bradyrhizobium agreste sp. nov. for CNPSo 4010T (=WSM 4802T=LMG 31645T) isolated from Glycine clandestina, Bradyrhizobium glycinis sp. nov. for CNPSo 4016T (=WSM 4801T=LMG 31649T) isolated from Glycine tabacina and Bradyrhizobium diversitatis sp. nov. for CNPSo 4019T (=WSM 4799T=LMG 31650T) isolated from G. max.

Putative diploid ancestors of 80-chromosome Glycine tabacina

The objective of this study was to discover the diploid progenitors of 80-chromosome Glycine tabacina with adventitious roots (WAR) and no adventitious roots (NAR). Three synthetic amphiploids were obtained by somatic chromosome doubling. These were (i) (G. latifolia, 2n = 40, genome B1B1,) × (G. microphylla, 2n = 40, genome BB) = F1(2n = 40, genome BB1) – 0.1% colchicine treatment (CT) – 2n = 80, genome BBB1B1; (ii) (G. canescens, 2n = 40, genome AA) × G. microphylla, 2n = 40, genome BB) = F1 (2n = 40, genome AB) – (CT) – 2n = 80, genome AABB; (iii) (G. latifolia, 2n = 40, B1B1) × G. canescens, 2n = 40, AA) = F1 (2n = 40, genome AB1) – (CT) – 2n = 80, genome AAB1B1. The segmental allotetraploid BBB1B1 was morphologically similar to the 80-chromosome G. tabacina (WAR), but meiotic pairing data in F1 hybrids did not support the complete genomic affinity. Despite normal diploid-like meiosis in allotetraploids AABB and AAB1B1, AABB was completely fertile, while pod set in AAB1B1 was very sparse. Morphologically, allotetraploid AABB was indistinguishable from the 80-chromosome G. tabacina (NAR) but in their F1 hybrids, the range of univalents at metaphase I was wide (4–44). The allotetraploid AAB1B1 did not morphologically resemble the 80-chromosome G. tabacina (NAR). However, the F1 hybrid of AABB × AAB1B1 showed normal meiosis with an average chromosome association (range) of 1.7 I (0–4) + 39.2 II (38–40). Based on this information, we cannot correctly deduce the diploid progenitor species of the 80-chromosome G. tabacina (NAR). The lack of exact genome homology may be attributed to the geographical isolation, natural mutation, and growing environmental conditions since the inception of 80-chromosome G. tabacina. Thus, it is logical to suggest that the 80-chromosome G. tabacina (NAR) is a complex, probably synthesized from A genome (G. canescens, G. clandestina, G. argyrea, G. tomentella D4 isozyme group) and B genome (G. latifolia, G. microphylla, G. tabacina) species, and the 80-chromosome G. tabacina (WAR) complex was evolved through segmental allopolyploidy from the B genome species.Key words: Glycine spp., allopolyploidy, colchicine, genome, intra- and inter-specific hybridization, polyploid complex.