ABSTRACT
We report the draft genome of Rhizobium leguminosarum strain I10 (7,598,161 bp scaffolded genome length, 60.5% GC, 7,061 protein-coding genes) isolated from the root nodules of Tephrosia purpurea.
KEYWORDS: Rhizobium leguminosarum, Tephrosia purpurea, genome, sequencing
ANNOUNCEMENT
Rhizobium leguminosarum, a gram-negative bacterium, is renowned for its symbiotic association with legumes and nitrogen fixation within root nodules. Rhizobium leguminosarum strain I10 was isolated from Tephrosia purpurea root nodules collected on 17 November 2022 at Dr. M.S. Swaminathan Agriculture College and Research Institute, Eachangkottai, Thanjavur (10.6621° N, 79.1594° E). Nodules were surface sterilized with 70% ethanol for 5–10 seconds, followed by 2% sodium hypochlorite for 1 minute, and then rinsed three times with sterile water (1, 2). Sterilized nodules (1 g) were crushed with a sterile glass rod in a test tube filled with a large drop of sterile water. The suspensions were serially diluted up to 10⁻⁶ dilutions and pour-plated onto Congo Red Yeast Extract Mannitol agar (3) and incubated aerobically at 28°C for 3–5 days. Pure cultures were obtained by repeated streaking on YEMA under the same temperature and aerobic conditions (4). To elucidate the genetic makeup of the isolate I10, whole-genome sequencing was subsequently performed.
Genomic DNA was extracted from a single purified colony grown in TY broth (tryptone, 5 g/L; yeast extract, 2.5 g/L) at 30°C for 18–24 h under shaking conditions, using a QIAGEN Genomic-tip 20/G Kit, following the manufacturer’s guidelines (5, 6). DNA quantity and integrity were assessed using a Qubit 4 Fluorometer and agarose gel electrophoresis (7). The genomic DNA was sent to ONEOMICS Private Limited (Tiruchirappalli, India) for whole-genome sequencing. Illumina libraries were prepared using the TruSeq DNA PCR-Free Kit (Illumina, Inc., USA); the genomic DNA is fragmented, end repaired, A-tailed, size selected, and ligated with Illumina sequencing barcoded adapters. Library concentration was quantified using a QuantStudio real-time PCR system (Applied Biosystems, USA), and fragment size distribution was verified on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA). Sequencing was performed on the Illumina NovaSeq 6000 platform (2 × 150 bp) (Illumina, Inc.), generating 35,036,626 raw reads totaling 5,255,493,900 bp.
Raw reads were quality-checked with FastQC v0.10.1 (8) and trimmed using TrimGalore (v0.6.10) (9). Genome assembly was generated using SOAPdenovo (v.2.04) (10) and polished with Pilon (v1.19) (11). The draft genome assembly spans 7,597,499 bp with 60.5% GC content (Table 1). The draft assembly was scaffolded with RagTag (v2.1.0) using the reference genome CP071626.1, resulting in a final scaffolded genome length of 7,598,161 bp, and the genome quality was evaluated using QUAST (v5.3.0) (12). Genome completeness was 99.58%, assessed using the BUSCO v5 bacteria_odb10 lineage data set (13, 14). Gene prediction and functional annotation were performed with PGAP (v6.10) (15, 16). Strain I10 was classified as Rhizobium leguminosarum using GTDB-Tk (v1.7.0 reference set R202) (17, 18). The closest GTDB representative genome was R. leguminosarum USDA 2370 (GCF_002008365.1), with an average nucleotide identity of 98.09%. Default parameters were used for all tools unless otherwise specified.
TABLE 1.
Detailed information of the draft genome
| Feature | Value |
|---|---|
| Genome size (ungapped length) (bp) | 7,597,499 bp |
| Number of chromosomes | 1 |
| Number of contigs | 7 |
| Contig N50 (bp) | 2,311,306 bp |
| Contig L50 | 2 |
| GC content (%) | 60.5 |
| Genome coverage | 625× |
| BUSCO completeness (%) | 99.58 |
| SRA | SRS24631670 |
| GeneBank assembly | CP196748 |
ACKNOWLEDGMENTS
We acknowledge the Dr. M.S. Swaminathan Agriculture College and Research Institute, Thanjavur, India, for providing necessary support for this study.
The authors declare that no external funding was received to support this study.
Contributor Information
Marimuthu Subramanian, Email: sm20@tnau.ac.in.
Vidya de Gannes, Email: vidya.degannes@uwi.edu.
Leighton Pritchard, University of Strathclyde, Glasgow, United Kingdom.
DATA AVAILABILITY
The whole-genome sequence project for Rhizobium leguminosarum I10 has been deposited at GenBank under accession number CP196748. The raw reads are available under BioProject accession number PRJNA1247883, and the BioSample accession number is SAMN47832398. The sequence data obtained in this work have been deposited in the NCBI Sequence Read Archive under accession number SRS24631670. Additionally, the assembled genome sequence is available in GenBank under accession number GCA_051904995.1.
REFERENCES
- 1. Jorrin B, Palacios JM, Peix Á, Imperial J. 2020. Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L. Syst Appl Microbiol 43:126090. doi: 10.1016/j.syapm.2020.126090 [DOI] [PubMed] [Google Scholar]
- 2. Varun A, Patel A, Banjare U, Kumar S, Kumar Singh R. 2017. Standardization of isolation technique of rhizobium from root nodules of lentil. IntJCurrMicrobiolAppSci 6:132–134. doi: 10.20546/ijcmas.2017.611.017 [DOI] [Google Scholar]
- 3. Perera S, Dineesha B. 2023. Isolation and identification of rhizobium species from different legume plant roots. GARI International Journal of Multidisciplinary Research 09. doi: 10.63572/gari4435 [DOI] [Google Scholar]
- 4. Ojo-Omoniyi OA, Oseni OS, Animashaun MA, Mohammed AO. 2025. Molecular characterization of root-nodule bacteria from soybean (Glycine Max L. Merrill) rhizosphere in three agro-ecological systems in Nigeria. SJB:11–23. doi: 10.47752/sjb.8.1.11.23 [DOI] [Google Scholar]
- 5. Bearzatto B, Durant J-F, Ambroise J, Gala J-L. 2025. Rapid, user-friendly, cost-effective DNA and library preparation methods for whole-genome sequencing of bacteria with varying cell wall composition and GC content using minimal DNA on the illumina platform. BMC Genomics 26:396. doi: 10.1186/s12864-025-11598-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Haendiges J, Timme R, Kastanis G, Balkey M. 2020. Manual DNA extraction using Qiagen DNeasy blood and tissue Kit v1. doi: 10.17504/protocols.io.bi4dkgs6 [DOI]
- 7. Zhou L, Ng HK, Drautz-Moses DI, Schuster SC, Beck S, Kim C, Chambers JC, Loh M. 2019. Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing. Sci Rep 9:10383. doi: 10.1038/s41598-019-46875-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Andrews S, Krueger F, Segonds-Pichon A, Biggins L, Krueger C, Wingett S. 2010. FastQC. A quality control tool for high throughput sequence data 370
- 9. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, et al. 2012. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1:18. doi: 10.1186/2047-217X-1-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. doi: 10.1371/journal.pone.0112963 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Manni M, Berkeley MR, Seppey M, Zdobnov EM. 2021. BUSCO: assessing genomic data quality and beyond. Curr Protoc 1:e323. doi: 10.1002/cpz1.323 [DOI] [PubMed] [Google Scholar]
- 14. Seppey M, Manni M, Zdobnov EM. 2019. BUSCO: assessing genome assembly and annotation completeness. Methods Mol Biol 1962:227–245. doi: 10.1007/978-1-4939-9173-0_14 [DOI] [PubMed] [Google Scholar]
- 15. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Ciufo S, Li W. 2013. Prokaryotic genome annotation pipeline, p 1–23. National Center for Biotechnology Information, Bethesda, MD. [Google Scholar]
- 16. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. 2022. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics 38:5315–5316. doi: 10.1093/bioinformatics/btac672 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. 2019. GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics 36:1925–1927. doi: 10.1093/bioinformatics/btz848 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The whole-genome sequence project for Rhizobium leguminosarum I10 has been deposited at GenBank under accession number CP196748. The raw reads are available under BioProject accession number PRJNA1247883, and the BioSample accession number is SAMN47832398. The sequence data obtained in this work have been deposited in the NCBI Sequence Read Archive under accession number SRS24631670. Additionally, the assembled genome sequence is available in GenBank under accession number GCA_051904995.1.