Complete and circularized genome sequences of five
nitrogen-fixing Bradyrhizobium sp. strains isolated from
root nodules of peanut, Arachis hypogaea, cultivated in
Tunisia

Besma Bouznif, Amira Boukherissa, Yan Jaszczyszyn, Mohamed Mars, Tatiana Timchenko, Jacqui A Shykoff, Benoît Alunni

► To cite this version:

Besma Bouznif, Amira Boukherissa, Yan Jaszczyszyn, Mohamed Mars, Tatiana Timchenko, et al. Complete and circularized genome sequences of five nitrogen-fixing Bradyrhizobium sp. strains isolated from root nodules of peanut, Arachis hypogaea, cultivated in Tunisia. Microbiology Resource Announcements, 2024, 13 (6), 10.1128/mra.01078-23. hal-04798785

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Distributed under a Creative Commons Attribution 4.0 International License
# Complete and circularized genome sequences of five nitrogen-fixing *Bradyrhizobium* sp. strains isolated from root nodules of peanut, *Arachis hypogaea*, cultivated in Tunisia

Besma Bouznif,<sup>1,2,3,4</sup> Amira Boukherissa,<sup>1,2</sup> Yan Jaszczyszyn,<sup>1</sup> Mohamed Mars,<sup>3</sup> Tatiana Timchenko,<sup>1</sup> Jacqui A. Shykoff,<sup>2</sup> Benoît Alunni<sup>1,5</sup>

**AUTHOR AFFILIATIONS** See affiliation list on p. 3.

**ABSTRACT** This manuscript reports the complete and circularized Oxford Nanopore Technologies (ONT) long read-based genome sequences of five nitrogen-fixing symbionts belonging to the genus *Bradyrhizobium*, isolated from root nodules of peanut (*Arachis hypogaea*) grown on soil samples collected from Tunisia.

**KEYWORDS** symbiosis, nitrogen fixation, *Bradyrhizobium*, peanut, Tunisia

Legume plants have evolved the capacity to harbor symbiotic nitrogen-fixing soil bacteria within root nodules. This symbiosis provides these plants the ability to grow in diverse habitats and to become major players in agricultural sustainability (1, 2). Bacteria belonging to the genus *Bradyrhizobium* are known as predominant peanut (*Arachis hypogaea*) symbionts (3–6).

In this announcement, we report the complete genome sequence of five bacterial strains isolated from peanut root nodules and belonging to the genus *Bradyrhizobium* based on average nucleotide identity (ANI) value analysis (Table 1). The soils were sampled from five sites in Tunisia (Table 1). Then, Tunisian traditional varieties of peanuts were grown in pots containing the sampled soils for bacterial trapping. After 7 weeks of greenhouse cultivation (28°C, 16 h photoperiod, 160 µmol·m⁻²·S⁻¹), nodules were collected and surface-sterilized in 96% ethanol for 1 min, then in 3% sodium hypochlorite solution for 3 min before being rinsed three times with sterile distilled water and individually crushed, plated on yeast extract mannitol (YM) agar medium and grown for 5 to 10 days at 28°C (7, 8). The strains were purified by successive streaking and single-colony picking and then stored in YM medium with 20% (vol/vol) glycerol at -80°C.

Total genomic DNA was extracted from the five strains grown in the YM medium for 4 days at 28°C using the MasterPure complete DNA and RNA Purification Kit (Epicentre). DNA libraries were prepared from intact genomic DNA with the Native Barcoding Kit 24 V14 (SQK-NBD114.24), and sequencing was performed on a MinION flow cell (R10.4.1). Basecalling was performed with the Super Accuracy model in Guppy 5.3.1. Nanopore library adaptors were trimmed using Porechop v2.0.4 (https://github.com/rrwick/Porechop). The read quality was assessed using NanoPlot v1.41.0 (9). The long reads were then assembled using Flye 2.8.3 (10), which generated a single contig assembly for four strains and two contigs for the strain BWA-3-5 (https://doi.org/10.6084/m9.figshare.24547348). The presence of plasmids was determined with plASgraph2 v1.0.0 (11). The assemblies were polished with Medaka v1.7.2 (model r1041_e82_260bps_sup_g632) (https://github.com/nanoporetech/medaka) using the Oxford Nanopore Technology (ONT) reads to create a consensus sequence. The assemblies of the chromosomes were then rotated with Circlator all at the DnaA gene (12).

**Editor** Leighton Pritchard, University of Strathclyde, Glasgow, United Kingdom

Address correspondence to Benoît Alunni, benoit.alunni@inrae.fr.

The authors declare no conflict of interest.

See the funding table on p. 3.

**Received** 13 November 2023

**Accepted** 15 April 2024

**Published** 15 May 2024

Copyright © 2024 Besma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<table><caption>TABLE 1 Genome features of the five *Bradyrhizobium* sp. strains</caption><thead><tr><th rowspan="2">Strain characteristic(s)</th><th colspan="5">Data for:</th></tr><tr><th>Bradyrhizobium sp. BEA-2-5</th><th>Bradyrhizobium sp. BWA-3-5</th><th>Bradyrhizobium sp. BWC-3-1</th><th>Bradyrhizobium sp. NDS-1</th><th>Bradyrhizobium sp. sBnM-33</th></tr></thead><tbody><tr><td>Sampling site</td><td>Béja, Elmarja</td><td>Béja, Wechtata</td><td>Béjà, Wechtata</td><td>Nabeul, Dar Allouch</td><td>Ben Gardane</td></tr><tr><td>Peanut variety</td><td>Arbi</td><td>Arbi</td><td>Chounfakhi</td><td>Siniya</td><td>Massriya</td></tr><tr><td>No. of reads (post QC)</td><td>91,274</td><td>118,365</td><td>141,671</td><td>99,886</td><td>80,650</td></tr><tr><td>No. of contigs</td><td>1</td><td>2</td><td>1</td><td>1</td><td>1</td></tr><tr><td>No. of genes</td><td>7,837</td><td>7,729</td><td>8,367</td><td>7,134</td><td>8,642</td></tr><tr><td>Genome length (bp)</td><td>8,406,336</td><td>8,041,125<br>(chromosome: 7,897,608;<br>plasmid: 143,517)</td><td>8,767,305</td><td>7,645,890</td><td>9,184,954</td></tr><tr><td>Coverage (x)</td><td>148</td><td>188</td><td>161</td><td>190</td><td>146</td></tr><tr><td>N50 (bp)</td><td>8,406,336</td><td>7,897,608</td><td>8,767,305</td><td>7,645,890</td><td>9,184,954</td></tr><tr><td>GC content (%)</td><td>63.92</td><td>62.51</td><td>62.92</td><td>64.01</td><td>61.62</td></tr><tr><td>BUSCO score (%)</td><td>99.1</td><td>99.9</td><td>99.5</td><td>99.4</td><td>99.2</td></tr><tr><td>Closest taxonomic assignment—accession<br>(ANI value to closest species [%])</td><td>Bradyrhizobium pachyrhizi—<br>GCF_029714545.1<br>(95.89)</td><td>Bradyrhizobium hereditatis—<br>GCF_020329435.1<br>(89.14)</td><td>Bradyrhizobium canariense—<br>GCF_019402665.1<br>(94.70)</td><td>Bradyrhizobium frederickii—<br>GCF_004570865.1<br>(90.13)</td><td>Bradyrhizobium retamae—<br>GCF_001440415.1<br>(92.53)</td></tr><tr><td>BioSample ID</td><td>SAMN37684786</td><td>SAMN37684780</td><td>SAMN37683990</td><td>SAMN37684784</td><td>SAMN37684830</td></tr><tr><td>SRA ID</td><td>SRR26337671</td><td>SRR26335727</td><td>SRR26335726</td><td>SRR26337672</td><td>SRR26336054</td></tr><tr><td>GenBank ID</td><td>CP136629</td><td>CP136626-<br>CP136627</td><td>CP136625</td><td>CP136628</td><td>CP136624</td></tr></tbody></table>

The plasmid of the strain BWA-3-5 was rotated with Prodigal v2.6.2 at position 73241 (13). The statistics of the assembly were obtained with QUAST v5.2.0 (https://github.com/ablab/quast), and the completeness of the genomes was assessed using BUSCO v5.6.1 (14) (Table 1). Functional annotation of the genomes was performed using the NCBI

<figure><img src="image_2.png" /><figcaption>FIG 1 Heatmap of ANI values between the five *Bradyrhizobium* sp. strains. ANI values for pairwise comparisons of these five strains are in the range between 81.2% and 87.7%, below the species threshold of 95%, and therefore appear to belong to five distinct *Bradyrhizobium* species.</figcaption></figure>
Prokaryotic Genome Annotation Pipeline v6.3 (15, 16). ANI calculation was carried out using ANIclustermap v1.2.o (Fig. 1)(17).

## ACKNOWLEDGMENTS

B.B. benefited from support from the Programme Hubert Curien UTIQUE Project 17G0918, the Government of Tunisia, for a mobility grant (Bourse d'Alternance) and a doctoral partner scholarship from the French Ministry of Foreign and European Affairs. A.B. benefited from a Ph.D. contract in the frame of the CNRS 80|PRIME—2021 program. We acknowledge the sequencing and bioinformatics expertise of the I2BC High-throughput sequencing facility, supported by France Génomique (funded by the French National Program "Investissement d'Avenir" ANR-10-INBS-09). This work has benefited from a French State grant (Saclay Plant Sciences, reference no. ANR-17-EUR-0007, EUR SPS-GSR) under a France 2030 program (reference no. ANR-11-IDEX-0003).

## AUTHOR AFFILIATIONS

¹Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France

²IDEEV—Laboratoire Ecologie, Systématique et Evolution, CNRS, AgroParisTech Université Paris-Saclay, Gif-sur-Yvette, France

³Research Unit Biodiversity and Valorization of Arid Areas Bioresources (BVBAA), Faculty of Sciences, Gabès, Tunisia

⁴Université de Picardie Jules Verne, UMRt BioEcoAgro 1158-INRAE, BIOPI, Amiens, France

⁵Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB), Versailles, France

## AUTHOR ORCIDs

Benoît Alunni http://orcid.org/0000-0002-3616-0669

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Programme Hubert Curien</td><td>UTIQUE Project 17G0918</td><td>Bouznif Besma Mars Mohamed Shykoff Jacqui</td></tr><tr><td>Centre National de la Recherche Scientifique (CNRS)</td><td>80 Prime</td><td>Boukherissa Amira Benoît Alunni</td></tr><tr><td>Agence Nationale de la Recherche (ANR)</td><td>ANR-10-INBS-09</td><td>Jaszczyszyn Yan Benoît Alunni</td></tr><tr><td>Agence Nationale de la Recherche (ANR)</td><td>ANR-17-EUR-0007</td><td>Timchenko Tatiana Benoît Alunni</td></tr><tr><td>Agence Nationale de la Recherche (ANR)</td><td>ANR-11-IDEX-0003</td><td>Timchenko Tatiana Benoît Alunni</td></tr></tbody></table>

## DATA AVAILABILITY

The raw reads and complete genomes of the five *Bradyrhizobium* sp. strains were deposited at GenBank under the BioProject no. PRJNA1023532. The BioSample numbers, raw reads, and assembly GenBank accession numbers are given in Table 1.
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