Draft Genome Sequence of Rhizobium sp. Strain 32-5/1, Isolated from Vicia cracca L. Root Nodules in the Russian Arctic

ABSTRACT

This study reports the whole-genome sequence of an endosymbiotic bacterium, Rhizobium sp. strain 32-5/1, isolated from root nodules of the legume Vicia cracca L. in the Arctic region of Russia. The genome consists of two plasmids and one chromosome, with a total length of 5,621,108 bp and 59.5% GC content.

ANNOUNCEMENT

Global climate change is followed by the active movement of plant communities to the north, filling new ecological niches (1). In these areas, pasture phytocenoses can form, a significant part of which are legumes that enter into symbiotic relationships with nitrogen-fixing nodule bacteria. Such pasture phytocenoses are extremely important for herbivore nutrition in the Arctic, especially reindeer and muskoxen (2). The legume Vicia cracca is a promising fodder plant in Arctic phytocenoses and currently has an extensive circumpolar habitat (3). Arctic plant ecosystems provide unique opportunities to study the response of legume-rhizobial symbiosis to climate change.

Nodules of V. cracca were collected in the Tiksi settlement (1 August 2021; 71.38302°N, 128.52411°E), situated on the Arctic Ocean coast (Sakha Republic, Russia). Individual root nodules were surface sterilized with 70% ethanol for 1 min, washed thoroughly with sterile tap water, and homogenized. Rhizobium strain 32-5/1 was obtained by plating 100 μL of the homogenized nodules on a yeast extract-maltose-sucrose agar (YMSA) plate. After 6 days of incubation at 28°C, colonies of strain 32-5/1 appeared. Further restreaking of the individual colony was performed for isolation of a pure culture. The pure culture of strain 32-5/1 was then picked and grown overnight in Reasoner’s 2A (R2A) broth at 28°C. Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Germany) according to the manufacturer’s recommendations. Libraries of genomic DNA were prepared using the ligation sequencing kit (SQK-LSK109) and the native barcoding expansion 1-12 kit (EXP-NBD104; ONT, UK), skipping the DNA-shearing step, sequenced using a MinION sequencer with an R.9.4.1 flow cell (ONT), and then base called using Guppy v5.0.11 with the high-accuracy model (ONT). Quality control of the raw data was performed using NanoStat v1.6.0 and resulted in 117,729 reads, with an average read length of 10,507 bp and an average quality of 11.4 (4). The draft genome sequence was obtained using the Flye v2.9 assembler; default parameters were used for all software unless otherwise noted (5). A circular genome was obtained using Circlator v1.5.5, without a specific starting point for the assembled contigs (6). The genomic statistics were measured using QUAST v5.0.2 (7). Three circular contigs—one chromosome with a length of 4,269,414 bp and two plasmids of 1,086,621 bp and 265,073 bp—were assembled, with a total size of 5.6 Mbp, a GC content of 59.5%, and a mean coverage of ~219×. The genome sequence was annotated using NCBI’s PGAP v6.4 software (8). A total of 5,649 genes were predicted in the genome, including 4,489 protein coding genes and 62 RNA genes (9 rRNAs, 49 tRNAs, and 4 noncoding RNAs [ncRNAs]).

The taxonomic rank was established using a 16S rRNA (rrs) BLASTn comparison, and the average nucleotide identity (ANI) was calculated using OrthoANI in OAT v0.93.1 software (9, 10). Strain 32-5/1 was most closely related to the type strain Rhizobium giardinii H152 (GenBank accession number GCF_000379605.1), with 78.99% average nucleotide identity and 98.11% rrs identity (Fig. 1). Based on the OrthoANI and 16S rRNA BLASTn results, strain 32-5/1 was identified as Rhizobium sp., without species definition.

FIG 1.

Phylogenetic tree and heat map of orthologous average nucleotide identity (OrthoANI) between strain 32-5/1 and related type strains from the order Hyphomicrobiales (genera Rhizobium and Ensifer), calculated using OAT software, with Bartonella harrusi 117A (GenBank accession number NZ_CP101114.1) used as the outgroup.

Data availability.

All data are available at GenBank under assembly accession numbers CP120638 to CP120640 and BioProject accession number PRJNA881974. The BioSample accession number is SAMN32638802. The raw MinION data can be found under SRA accession number SRR23086701.