Genomes of two type strains of the Rhizobium tropici group: R. calliandrae CCGE524T and R. mayense CCGE526T

ABSTRACT The genome sequences of two nitrogen-fixing type strains of the Rhizobium tropici group were obtained: Rhizobium calliandrae CCGE524T and R. mayense CCGE526T. Genomic analyses confirmed their taxonomic position and identified three complete sequences of the repABC genes, indicative of three plasmids, one of them carrying symbiotic genes.

T he Rhizobium tropici group encompasses species of nitrogen-fixing rhizobia that, in general, are very promiscuous in nodulating several legumes and may contribute with high rates of nitrogen fixation (1)(2)(3). Rhizobium calliandrae CCGE524 T and R. mayense CCGE526 T were isolated from an effective nodule of the medicinal legume Calliandra grandiflora in rainforests of Chiapas, Mexico (4). Genetic characterization based on 16S rRNA and housekeeping genes (atpD, recA, and rpoB) positioned the two species into the R. tropici clade (4).
Nowadays, the use of genomic parameters is highly encouraged in studies of taxonomy and phylogeny of bacteria, including rhizobia (5). As there were no genomes available for these two type strains, we performed next-generation sequencing to obtain high-quality genomes. The type strains were recovered from cryopreserved stocks at −80°C in modified yeast extract mannitol (YM) culture medium (6) with 30% glycerol (vol/vol) from the "Diazotrophic and Plant Growth Promoting Bacteria Culture Collection of Embrapa Soja, " in Londrina, Paraná, Brazil, grown in liquid modified YM medium, and incubated at 28°C for 3 days. Total DNAs were extracted with the DNeasy Blood and Tissue kit (Qiagen). Libraries were constructed with Nextera XT kit, and paired-end sequencing was performed on the NextSeq 1000 platform (Illumina) at Embrapa Soja. The reads were assembled with the A5-MiSeq pipeline (de novo) v.20140604 (7), with default quality control parameters, trimming sequence adapters, and low-quality regions with Trimmomatic (8), errors were corrected with String Graph Assembler's (SGA's) k-mer-based algorithm (9), and annotated with the NCBI Prokaryotic Genome Annota tion Pipeline (PGAP), version 6.4 (10). ANI (Average Nucleotide Identity) (11) and digital DNA-DNA hybridization (dDDH) (12,13) were calculated with the species of the R. tropici clade.
The two strains present genomes of similar sizes, estimated at 7,547,379 bp for R. calliandrae CCGE524 T and 7,367,519 bp for R. mayense CCGE526 T . The genome coverages ranged from 140-to 143-fold, and the G + C contents were also similar for the two strains. These and other genomic parameters are shown in Table 1.
Three complete sequences of the repABC genes (plasmid replication genes) were detected in the two strains, that may indicate three plasmids, one of which carrying the nodulation and nitrogen fixation genes. We may suppose that the three plasmids correspond to the same organization reported for R. jaguaris CCGE525 T , another close species of the R. tropici group that carries a chromid, a symbiotic plasmid, and an additional plasmid (14). In addition, the genomes of R. jaguaris and of the two type strains CCGE524 T and CCGE526 T share the same organization of the genes nodABCD and nifABEHNQSTUWXZ.
The genome comparison of the species of the R. tropici clade indicated ANI values of 80.08%-91.24% for R. calliandrae CCGE524 T and of 80.37%-91.24% to R. mayense CCGE526 T , while the dDDH values ranged from 21.8% to 45.6%, and 22.0% to 45.6%, respectively (Table 1). Therefore, the values of ANI and dDDH are below the species delineation cutoffs established in literature, of 70% and 95%-96%, respectively (5, 15), confirming the taxonomic status of the two strains.

DATA AVAILABILITY
The whole-genome shotgun project for Rhizobium calliandrae CCGE524 T has been deposited at DDBJ/EMBL/GenBank under the GenBank accession num ber JARFYN000000000 (accession numbers BioProject PRJNA940114, BioSample SAMN33553174). The version described in this paper is JARFYN000000000. The wholegenome shotgun project of Rhizobium mayense CCGE526 T has been deposited at DDBJ/ EMBL/GenBank under the GenBank accession number JARFYM000000000 (accession number BioProject PRJNA940111, BioSample SAMN33553147). The version described in this paper is JARFYM000000000.