Five life stage-specific transcriptome assemblies for the reniform nematode, Rotylenchulus reniformis Linford & Oliveira

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Journal of Nematology

Society of Nematologists

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VOLUME 50 , ISSUE 4 (December 2018) > List of articles

Five life stage-specific transcriptome assemblies for the reniform nematode, Rotylenchulus reniformis Linford & Oliveira

Kurt C. Showmaker / William S. Sanders / Sebastian Eves-van den Akker / Brigitte E. Martin / Franklin E. Callahan / Daniel G. Peterson / Martin J. Wubben *

Citation Information : Journal of Nematology. Volume 50, Issue 4, Pages 1-2, DOI: https://doi.org/10.21307/jofnem-2018-053

License : (PUBLISHER)

Published Online: 03-December-2018

ARTICLE

ABSTRACT

The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a semi-endoparasitic nematode that is a pathogen of numerous major crops such as cotton and soybean. Here, the authors present transcriptome assemblies of the egg, second-stage juvenile (J2), J3, vermiform adult, and sedentary female life stages of this important plant pathogen.

The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a sedentary semi-endoparasitic nematode that causes significant economic damage to many important crops such as cotton, soybean, sweet potato, and pineapple (Robinson et al., 1997). Evolutionary studies indicated that R. reniformis is most closely related to the cyst nematode genera and share common ancestry with Radopholus spp. (Holterman et al., 2009). While both R. reniformis and the cyst nematodes are sedentary in nature and form similar feeding sites within the host root, the life cycles of these genera differ significantly. While the cyst nematodes initiate host root infection immediately upon egg hatching as second-stage juveniles (J2), R. reniformis J2 become immobile soon after hatching (Robinson et al., 1997). Syncytia formation is induced by cyst nematode J2 and the nutrients provided by this feeding site allow the cyst nematode to develop through the J3 and J4 life stages until adulthood. In contrast, R. reniformis proceeds through all juvenile stages in the soil, without feeding, until reaching the adult male and female vermiform life stage (Robinson et al., 1997). It is the R. reniformis vermiform female that infects the host root and initiates syncytium formation (Robinson et al., 1997).

The R. reniformis life stages of egg, J2, J3, vermiform adult (VA), and sedentary female (SF) were isolated as previously described (Ganji et al., 2013). Total RNA was extracted from individual life stages using Trizol reagent (Life Technologies, Grand Island, NY, USA) (Wubben et al., 2010). RNA quantity and integrity were determined with the Qubit RNA HS assay kit (Life Technologies) and with an Agilent Bioanalyzer 2100 via the Agilent RNA 6000 Nano kit (Agilent Technologies, Palo Alto, CA, USA), respectively. For each life stage, three technically replicated Illumina Truseq V2 RNA (Illumina, San Diego, CA) libraries were prepared by Global Biologics (Columbia, MO). Libraries were pooled and paired-end sequenced (2×100bp) on five lanes of the Illumina Hiseq 2000 using TruSeq v3 chemistry (Illumina) at the USDA-ARS Bovine Functional Genomics Laboratory (Beltsville, MD). The resulting reads were deposited in the SRA archive under the BioProject PRJNA286314.

Sequences were trimmed and filtered for adapters and low-quality base calls with Trimmomatic (v0.32; Bolger et al., 2014), and trimmed reads for each life stage were aligned to the reniform nematode reference genome (RREN1.0, GCA_001026735.1) with the STAR aligner (v2.6.0c; Dobin et al., 2013), and subsequently assembled with Trinity (v2.6.5; Grabherr et al., 2011). The resulting assemblies for each life stage were deposited in the Transcriptome Shotgun Assembly (TSA) database. Completeness assessment by the programs CEGMA (v2.5; Parra et al., 2007) and BUSCO (v3.0.1; Simão et al., 2015) ranged from 81.45 to 83.06 (92.34-93.95 including partial alignments) and 60.0 to 67.2% complete, respectively. Coding regions were predicted with TransDecoder (v5.1.0; http://transdecoder.github.io) guided by Diamond (v0.9.16; Buchfink et al., 2015), Blastp alignments to Swiss-Prot (release 2018_07), and hmmscan (v3.2.1; Eddy, 2011) alignments to Pfam-A (vPfam31.0; Finn et al., 2016). Clustering of all the predicted proteins with CD-HIT (v4.6.6; Li and Godzik, 2006) resulted in 71,321 protein clusters at the 90% identity level.

GenBank accession numbers: the raw sequence data and transcriptome assemblies were deposited at GenBank under BioProject no. PRJNA286314. The Transcriptome Shotgun Archive (TSA) projects has been deposited at DDBJ/ENA/GenBank and the versions described in this paper are the first versions; GGVV01000000 (egg), GGVO01000000 (J2), GGVP01000000 (J3), GGVQ01000000 (VA), and GGVR01000000 (SF). Disclaimer: Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture.

Acknowledgements

The authors would like to thank Dr John Brooks and Dr Jack McCarty (USDA-ARS) for their critical review of the manuscript. Kurt C. Showmaker and William S. Sanders contributed equally to this work.

References


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  9. Ganji, S., Wubben, M. J., and Jenkins, J. N.. 2013. Two simple methods for the collection of individual life stages of reniform nematode, Rotylenchulus reniformis. Journal of Nematology 45: 87-91.
  10. Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B. W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., and Regev, A.. 2011. Full-length transcriptome assembly from RNA-seq data without a reference genome. Nature Biotechnology 29: 644-652.
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  11. Li, W., and Godzik, A.. 2006. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22 (13): 1658-1659.
    [CROSSREF]
  12. Parra, G., Bradnam, K., and Korf, I.. 2007. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23: 1061-1067.
    [CROSSREF]
  13. Robinson, A. F., Inserra, R. N., Caswell-Chen, E. P., Vovlas, N., and Troccoli, A.. 1997. Rotylenchulus species: identification, distribution, host ranges, and crop plant resistance. Nematropica 27: 127-180.
  14. Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., and Zdobnov, E. M.. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31 (19): 3210-3212.
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REFERENCES

  1. Baum, 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 Heterodera schachtii. Journal of Nematology 32: 166-173.
  2. Blin, N., and Stafford, D. W.. 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Research 3: 2303-2308.
    [CROSSREF]
  3. Bolger, A. M., Lohse, M., and Usadel, B.. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114-2120.
    [CROSSREF]
  4. Buchfink, B., Xie, C., and Huson, D. H.. 2015. Fast and sensitive protein alignment using DIAMOND. Nature Methods 12 (1): 59-60.
    [CROSSREF]
  5. Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T. R.. 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29 (1): 15-21.
    [CROSSREF]
  6. Eddy, S. R.. 2011. Accelerated Profile HMM Searches. PLoS Computational Biology 7 (10): e1002195.
    [CROSSREF]
  7. Eves-van den Akker, S., et al. 2016. The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence. Genome Biology 17: 124.
    [CROSSREF]
  8. Finn, R. D., Coggill, P., Eberhardt, R. Y., Eddy, S. R., Mistry, J., Mitchell, A. L., Potter, S. C., Punta, M., Qureshi, M., Sangrador-Vegas, A., Salazar, G. A., Tate, J., and Bateman, A.. 2016. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Research 44 D1: D279-D285.
    [CROSSREF]
  9. Ganji, S., Wubben, M. J., and Jenkins, J. N.. 2013. Two simple methods for the collection of individual life stages of reniform nematode, Rotylenchulus reniformis. Journal of Nematology 45: 87-91.
  10. Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B. W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., and Regev, A.. 2011. Full-length transcriptome assembly from RNA-seq data without a reference genome. Nature Biotechnology 29: 644-652.
    [CROSSREF]
  11. Li, W., and Godzik, A.. 2006. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22 (13): 1658-1659.
    [CROSSREF]
  12. Parra, G., Bradnam, K., and Korf, I.. 2007. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23: 1061-1067.
    [CROSSREF]
  13. Robinson, A. F., Inserra, R. N., Caswell-Chen, E. P., Vovlas, N., and Troccoli, A.. 1997. Rotylenchulus species: identification, distribution, host ranges, and crop plant resistance. Nematropica 27: 127-180.
  14. Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., and Zdobnov, E. M.. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31 (19): 3210-3212.
    [CROSSREF]

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