The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms. The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms

Bartlett, Benjamin D

Eves-van den Akker, Sebastian

Hsu, Chuan-Yu

Martin, Brigitte E

Peterson, Daniel G

Platt, Roy N

Sanders, William S

Showmaker, Kurt C

Stokes, John V

Wubben, Martin J

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A genomic resource for the sedentary semi-endoparasitic reniform nematode, Rotylenchulus reniformis Linford & Oliveira

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1JOURNAL OF NEMATOLOGYe2019-13 | Vol. 51Article | DOI: 10.21307/jofnem-2019-013© 2019 Authors. This is an Open Access article licensed under the Creative Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/A genomic resource for the sedentary semi-endoparasitic reni-form nematode, Rotylenchulus reniformis Linford & OliveiraAbstractThe reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.Kurt C. Showmaker1,2,3,‡, William S. Sanders2,3,4,‡, Sebastian Eves-van den Akker5, Brigitte E. Martin6, Roy N. Platt II7, John V. Stokes8, Chuan-Yu Hsu3, Benjamin D. Bartlett9, Daniel G. Peterson3,9 and Martin J. Wubben2,10,*1Department of Crop Science,  University of Illinois at Urbana- Champaign, Urbana, IL.2Department of Biochemistry,  Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Starkville, MS 39759.3Institute for Genomics, Biocomputing, and Biotechnology, Mississippi State University, Starkville, MS 39759.4The Jackson Laboratory,  Farmington, CT.5Department of Plant Sciences, Uni-versity of Cambridge, Cambridge, UK.6Department of Molecular and  Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL.7Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX.8Department of Basic Sciences,  College of Veterinary Medicine,  Mississippi State University, Starkville, MS 39759.9Department of Plant and Soil Sciences, Mississippi State  University, Starkville, MS 39759.10USDA–ARS, Crop Science  Research Laboratory, Mississippi State, Starkville, MS 39759.*Email: Martin.Wubben@ars.usda.govThis paper was edited by  Erik J. Ragsdale‡These authors contributed equally.Received for publication August 17, 2018The reniform nematode (Rotylenchulus reniformis Linford & Oliveira) is a sedentary semi-endoparasitic species that infects 77 plant families, including cotton and soybean, and other high-value crops such as sweet potato and pineapple (Robinson et al., 1997). Here, we report the genome sequencing and assembly of R. reniformis.Biological material for sequencing was a R. reniformis stock culture maintained on cotton. Eggs of R. reniformis were collected from infected cotton roots (Hussey and Barker, 1973), purified (Jenkins, 1964), surface-sterilized (Baum et al., 2000), pelleted by centrifugation, flash-frozen with liquid nitrogen, and stored at −80°C until DNA extrac-tion. Genomic DNA extraction was performed as described by Blin and Stafford (1976).Three short-read small-insert libraries (250, 350, and 550 bp) and four large-insert Nextera mate-pair libraries (2-3.5-, 4-6-, 8-10-, and 2-10-kb) were constructed from a pooled population of R. reniformis eggs using standard protocols provided by Illumina (San Diego, CA). Additionally, a single R. reniformis egg was isolated, its DNA was amplified by whole genome amplification (WGA) using the Qiagen Rep-li-G single cell kit (Valencia, CA) and short-read small-insert libraries (400- and 600-bp) were constructed and sequenced using standard protocols provided by Illumina (San Diego, CA). Paired-end sequenc-ing was conducted on Illumina GAIIx and Illumina MiSeq systems. We obtained 19.17 Gb and 29.85 Gb from the pooled population short- and long-read libraries, respectively, and 22.29 Gb from the WGA librar-ies. Pooled population and WGA sequence data was combined for genome assembly.Mate-pair reads were processed with NextClip (v1.3; Richard et al., 2014) with default parameters to identify fragments containing linker sequence indicating proper circulation and removal of duplicate reads. All libraries were trimmed for adapters and low-quality base calls with Trimmomatic (v0.32; Bolger et al., 2014) and then assembled with ABySS (v1.5.2; Simpson et al., 2009) with a k-mer value of 175. The mate-pair libraries were used for scaffolding the assembly with 2A genomic resource for the sedentary semi-endoparasitic reniform nematode, Rotylenchulus reniformis Linford & OliveiraSSPACE (v3.0; Boetzer et al., 2011) followed by gap filling of all libraries with GapFiller (v1.10; Nadalin et al., 2012).The resultant R. reniformis genome assembly (RREN1.0, GCA_001026735.1) contained 314 Mb distributed among 100,525 scaffolds with scaf-fold and contig N50s of 22,705 bp and 5,991 bp, respectively. The genome assembly contained 86.29% of the core eukaryotic proteins utilized by CEGMA (v2.5; Parra et al., 2007). Flow cytometry of propidium iodide stained R. reniformis nuclei indicated a genome size of ~190 Mb. The larger ge-nome size (310 Mb) represented by the assembly is likely the result of unresolved haplotypes stemming from heterogeneity within the R. reniformis popu-lation used for DNA extraction. Repeat prediction with RepeatModeler (Smit and Hubley, 2015) and repeat quantification with RepeatMasker (Smit et al., 2015) identified 245 repeat families within 35.1 Mb of the assembled genome; most elements were DNA transposons or LTR retroelements (20.6 Mb and 11.3 Mb, respectively), while low com-plexity repeats accounted for 7 Mb. Homologs of known plant-parasitic nematode effector molecules were identified via tBLASTn and included beta-1–4 endogluconase, chitinase, CLE, CEP, chorismate mutase, invertase, ubiquitin extension protein, and venom allergen-like protein as well as cyst nematode pioneers 4D06, GLAND1, GLAND11, GLAND14, GLAND15, 10C02, 20E03, 33A09, 4G05, 7E05, and 10A06 (reviewed by Mitchum et al., 2013). A variant of the DOG (DOrsal Gland) box DNA motif, an enhancer element associated with dorsal gland effectors (Eves-van den Akker et al., 2016), was identified in the assembly.GenBank accession numbers: The raw DNA sequence data and genome assembly were de-posited at GenBank under BioProject No. PRJ-NA214681.AcknowledgmentsMention of trade names or commercial products in this paper is solely for the purpose of providing spe-cific information and does not imply recommendation or endorsement by the US Department of Agriculture. The authors would like to thank Dr. Satish Ganji for his assistance with flow cytometry and whole genome amplification. The authors would also like to thank Drs. William Rutter and Osman Gutierrez (USDA–ARS) for their critical review of the manuscript. This project was funded, in part, by USDA ARS Non-As-sistance Cooperative Agreements 58-6066-6-046 and 58-6066-6-059.ReferencesBaum, T. J., Wubben, M. J. E., Hardy, K. A., Su, H. and Rodermel, S. R. 2000. A screen for Arabidopsis thaliana mutants with altered susceptibility to Heterod-era schachtii. Journal of Nematology 32:166–73.Blin, N. and Stafford, D. W. 1976. A general method for isolation of high molecular weight DNA from eukar-yotes. Nucleic Acids Research 3:2303–8.Boetzer, M., Christiaan, V. H., Hans, J. J., Butler, D. and Pirovano, W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–9.Bolger, A. M., Lohse, M. and Usadel, B. 2014. Trim-momatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–20.Eves-van den Akker, S., et al. 2016. The genome of the yellow potato cyst nematode, Globodera ros-tochiensis, reveals insights into the basis of parasitism and virulence. Genome Biology 17:124.Hussey, R. S. and Barker, K. R. 1973. A comparison of methods of collecting inocula of Meloidogyne spp., includ-ing a new technique. Plant Disease Reporter 57:1025–8.Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48:692.Mitchum, M. G., Hussey, R. S., Baum, T. J., Wang, X., Elling, A. A., Wubben, M. and Davis, E. L. 2013. Nematode effector proteins: an emerging paradigm of parasitism. New Phytologist 199:879–94.Nadalin, F., Vezzi, F. and Policriti, A. 2012. Gapfiller: a de novo assembly approach to fill the gap within paired reads. BMC Bioinformatics 13 No. S14:S8.Parra, G., Bradnam, K. and Korf, I. 2007. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23:1061–7.Richard, M. L., Bernardo, J. C., Leah, C., Matthew, D. C. and Mario, C. 2014. Nextclip: an analysis and read preparation tool for Nextera long mate pair librar-ies. Bioinformatics 30:566–8.Robinson, A. F., Inserra, R. N., Caswell-Chen, E. P., Vovlas, N. and Troccoli, A. 1997. Rotylenchulus spe-cies: Identification, distribution, host ranges, and crop plant resistance. Nematropica 27:127–80.Simpson, J. T., Wong, K., Jackman, S. D., Schein, J. E., Jones, S. J. M. and Birol, İ. 2009. ABySS: A par-allel assembler for short read sequence data. Genome Research 19:1117–23.Smit, A. F. A. and Hubley, R. 2015. Repeatmodeler open-1.0, 2008–2015, http://www.reeatmasker.org/faq.html#faq3.Smit, A. F. A. Hubley, R. and Green, P. 2015. Repeatmasker open-4.0, 2013–2015.

A genomic resource for the sedentary semi-endoparasitic reniform nematode, Rotylenchulus reniformis Linford & Oliveira.

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A genomic resource for the sedentary semi-endoparasitic reniform nematode, Rotylenchulus reniformis Linford & Oliveira.

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