# Complete and Circularized Genome Sequences of Three Xanthomonas Strains Pathogenic on Soybean and Alfalfa

Mylène Ruh, Martial Briand, Sophie Bonneau, Armelle Darrasse, Marie-Agnès Jacques, Nicolas W. G. Chen

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Mylène Ruh, Martial Briand, Sophie Bonneau, Armelle Darrasse, Marie-Agnès Jacques, et al. Complete and Circularized Genome Sequences of Three Xanthomonas Strains Pathogenic on Soybean and Alfalfa. Microbiology Resource Announcements, 2021, 10 (31), 10.1128/MRA.00537-21. hal-03349711

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# Complete and Circularized Genome Sequences of Three Xanthomonas Strains Pathogenic on Soybean and Alfalfa

Mylène Ruh,ª Martial Briand,ª Sophie Bonneau,ª Armelle Darrasse,ª Marie-Agnès Jacques,ª Nicolas W. G. Chenª

**ABSTRACT** We report the complete and circularized genome sequences of two strains of *Xanthomonas citri* pv. glycines causing bacterial pustule on soybean and one strain of *Xanthomonas euvesicatoria* pv. alfalfae causing bacterial leaf and stem spot on alfalfa. These assemblies provide high-quality material for functional and evolutionary studies of these legume pathogens.

Xanthomonas euvesicatoria pv. alfalfae and Xanthomonas citri pv. glycines are two phylogenetically distant Xanthomonas pathovars causing disease on different legume species, including the model plants *Medicago truncatula* and soybean (*Glycine max*) (1, 2). Both bacteria are seed transmitted and can cause severe losses under warm and moist conditions (3, 4). However, while the host range of X. citri pv. glycines seems restricted to soybean, X. euvesicatoria pv. alfalfae can infect different legume species, including its main host alfalfa (Medicago sativa), as well as soybean, clover (Trifolium spp.), vetch (Vicia spp.), and M. truncatula (3, 4). Previously, HiSeq Illumina genome sequences were released for X. euvesicatoria pv. alfalfae and X. citri pv. glycines pathotype strains CFBP 3836 and CFBP 2526, both isolated in Sudan, and X. citri pv. glycines strain CFBP 7119, isolated in Brazil (5, 6).

Here, we produced PacBio sequences for the same three strains and performed de novo assemblies using both PacBio and HiSeq reads. Bacteria were grown on Trypticase soy (TS) agar for 2 days at 28°C, and then 5 to 6 clones were scraped, pooled, and cultured overnight on 10% TS agar to obtain fresh cultures. A loop (~5 µl) of cells was then suspended in sterile distilled water and collected by centrifugation. Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega) according to the manufacturer's recommendations. DNA was sheared using g-TUBE columns (Covaris). PacBio SMRTbell libraries were prepared from ~10 µg of genomic DNA, and size selection to 15 to 20 kb was done using BluePippin cassettes (Sage Scientific).

Sequencing was done using the PacBio RS II technology with P5-C3 chemistry at the Icahn School of Medicine at Mount Sinai (New York, NY). One single-molecule real-time (SMRT) cell per strain was used, leading to ~100,000 reads with an N<sub>50</sub> value of ~20 kbp, which corresponded to 252× to 308× genome coverage (Table 1). The reads were filtered using PreAssembler Filter v1 of SMRT Portal v2.3 (Pacific Biosciences, Inc., CA) with a minimum subread length of 500, minimum polymerase read quality of 0.80, and minimum polymerase read length of 100 bp. Assembly was performed using Canu v1.5 (7) with the setting genomeSize = 5m. Circularization was done using Berokka v0.2.3 (https://github.com/tseemann/berokka). For each molecule, the sequence start was fixed using Circlator v1.5.1 (8). Polishing was performed using variantCaller v2.2.2 (https://github.com/PacificBiosciences/GenomicConsensus) with the setting --algorithm best. PacBio assemblies were then corrected with Pilon v1.23 (9) with the setting --mindepth 0.5, using the HiSeq Illumina reads produced previously (5, 6). Default parameters were used for all software unless otherwise specified.

The genomes consisted of a chromosome plus one plasmid for strains CFBP 3836 and CFBP 2526, while strain CFBP 7119 contained two plasmids. The total genome

**Citation** Ruh M, Briand M, Bonneau S, Darrasse A, Jacques M-A, Chen NWG. 2021. Complete and circularized genome sequences of three Xanthomonas strains pathogenic on soybean and alfalfa. Microbiol Resour Announc 10:e00537-21. https://doi.org/10.1128/MRA.00537-21.

**Editor** David A. Baltrus, University of Arizona

**Copyright** © 2021 Ruh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Nicolas W. G. Chen, nicolas.chen@agrocampus-ouest.fr.

**Received** 24 May 2021

**Accepted** 9 July 2021

**Published** 5 August 2021

<footnote>ªUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, Angers, France</footnote>
<table><caption>TABLE 1 Statistics</caption><thead><tr><th>Species</th><th>Pathovar</th><th>Strain</th><th>SRA no. (HiSeq reads)</th><th>No. of HiSeq reads</th><th>Coverage (HiSeq) (×)</th><th>SRA no. (PacBio reads)</th><th>No. of PacBio reads</th><th>Coverage (PacBio) (×)</th><th>N<sub>50</sub> (bp)</th><th>GenBank assembly accession no.</th><th>Size (bp)</th><th>G+C (%)</th><th>No. of plasmids</th><th>No. of CDs</th></tr></thead><tbody><tr><td>X. euvesicatoria</td><td>alfalfae</td><td>3836</td><td>SRR14026743</td><td>37,799,928</td><td>1,500</td><td>SRR14028218</td><td>102,443</td><td>255</td><td>19,326</td><td>GCA_017724035.1</td><td>5,120,436</td><td>64.7</td><td>1</td><td>4,253</td></tr><tr><td>X. citri</td><td>glycines</td><td>2526</td><td>SRR14026742</td><td>75,568,393</td><td>3,000</td><td>SRR14028219</td><td>97,171</td><td>252</td><td>19,333</td><td>GCA_017723855.1</td><td>5,304,469</td><td>64.6</td><td>1</td><td>4,477</td></tr><tr><td>X. citri</td><td>glycines</td><td>7119</td><td>SRR14027991</td><td>47,138,978</td><td>1,885</td><td>SRR14028187</td><td>112,444</td><td>308</td><td>21,508</td><td>GCA_017723895.1</td><td>5,581,022</td><td>64.4</td><td>2</td><td>4,743</td></tr></tbody></table>
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sizes were 5,120,436 bp, 5,304,469 bp, and 5,581,022 bp for strains CFBP 3836, CFBP 2526, and CFBP 7119, respectively, with respective G+C contents of 64.7%, 64.6%, and 64.4% (Table 1). Using CheckM v1.0.7 (10), the genomes were estimated to be >99.63% complete and <0.73% contaminated.

Coding sequence (CDS) predictions were retrieved from the NCBI Prokaryotic Genome Annotation Pipeline (11). Strains CFBP 3836, CFBP 2526 and CFBP 7119 were predicted to contain 4,253, 4,477, and 4,743 CDSs, respectively (Table 1). The three genome sequences comprised the usual arsenal of genes involved in *Xanthomonas* pathogenicity, including type III effectors, among which were genes encoding transcription activator-like effectors (12). In all, these circularized genomes offer high-quality materials for further comparative studies that could help in understanding the genetic determinants of *Xanthomonas* specificity to different legume species.

**Data availability.** We deposited all HiSeq Illumina reads, PacBio RS II reads, and assemblies at GenBank under the accession numbers listed in Table 1.

## ACKNOWLEDGMENTS

The research leading to these results was supported by grants from the French National Research Agency (ANR-14-CE19-0002-04 and ANR-2010-GENM-013).

We thank the CIRM-CFBP (Angers, France) for strain preservation and supply. We thank L. Legrand for fruitful discussions and CATI BARIC for bioinformatics facilities.

## REFERENCES

1. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang X-C, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA. 2010. Genome sequence of the palaeopolyploid soybean. Nature 463:178-183. https://doi.org/10.1038/nature08670.

2. Young ND, Debellé F, Oldroyd GED, Geurts R, Cannon SB, Udvardi MK, Benedito VA, Mayer KFX, Gouzy J, Schoof H, Van de Peer Y, Proost S, Cook DR, Meyers BC, Spannagl M, Cheung F, De Mita S, Krishnakumar V, Gundlach H, Zhou S, Mudge J, Bharti AK, Murray JD, Naoumkina MA, Rosen B, Silverstein KAT, Tang H, Rombauts S, Zhao PX, Zhou P, Barbe V, Bardou P, Bechner M, Bellec A, Berger A, Bergès H, Bidwell S, Bisseling T, Choisne N, Couloux A, Denny R, Deshpande S, Dai X, Doyle JJ, Dudez A-M, Farmer AD, Fouteau S, Franken C, Gibelin C, Gish J, et al. 2011. The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480:520-524. https://doi.org/10.1038/nature10625.

3. Hokawat S, Rudolph K. 1993. *Xanthomonas campestris* pv. glycines: cause of bacterial pustule of soybean, p 44–48. In *Swings R, Civerolo E, Swings J* (ed), Xanthomonas. Chapman & Hall, London, United Kingdom.

4. Bradbury JF. 1981. *Xanthomonas campestris* pv. alfalfae. [Descriptions of fungi and bacteria]. IMI descriptions of fungi and bacteria, no. 70, sheet 698. https://www.cabi.org/ISC/abstract/20056400698.

5. Darrasse A, Bolot S, Serres-Giardi L, Charbit E, Boureau T, Fisher-Le Saux M, Briand M, Arlat M, Gagnevin L, Koebnik R, Noël LD, Carrère S, Jacques MA. 2013. High-quality draft genome sequences of *Xanthomonas axonopodis* pv. glycines strains CFBP 2526 and CFBP 7119. Genome Announc 1:e01036-13. https://doi.org/10.1128/genomeA.01036-13.

6. Jacques MA, Bolot S, Charbit E, Darrasse A, Briand M, Arlat M, Gagnevin L, Koebnik R, Noël LD, Portier P, Carrère S, Boureau T. 2013. High-quality draft genome sequence of *Xanthomonas alfalfae* subsp. *alfalfae* strain CFBP 3836. Genome Announc 1:e01035-13. https://doi.org/10.1128/genomeA.01035-13.

7. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722-736. https://doi.org/10.1101/gr.215087.116.

8. Hunt M, De Silva N, Otto TD, Parkhill J, Keane JA, Harris SR. 2015. Circulator: automated circularization of genome assemblies using long sequencing reads. Genome Biol 16:1-10. https://doi.org/10.1186/s13059-015-0849-0.

9. 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. https://doi.org/10.1371/journal.pone.0112963.

10. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043-1055. https://doi.org/10.1101/gr.186072.114.

11. 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. https://doi.org/10.1093/nar/gkw569.

12. Perez-Quintero AL, Szurek B. 2019. A decade decoded: spies and hackers in the history of TAL effectors research. Annu Rev Phytopathol 57:459-481. https://doi.org/10.1146/annurev-phyto-082718-100026.