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A new chorismate mutase gene identified from Globodera ellingtonae and its utility as a molecular diagnostic marker

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Abstract

Globodera ellingtonae, a new cyst nematode species recently detected in Oregon and confirmed to reproduce on potato, shares key morphological features with the two species of potato cyst nematode (PCN; G. rostochiensis and G. pallida) which are of quarantine concern. Currently no methods are available for the molecular diagnosis of this new Globodera species. In this study, we cloned a chorismate mutase gene (Ge-cm-1) from G. ellingtonae. Our detailed sequence analysis identified two different Ge-cm-1 mRNA transcripts, named as Ge-cm-1 and Ge-cm-1-IRII, of which Ge-cm-1-IRII differs from Ge-cm-1 by a 93-base pair (bp) insertion. Comparison of the Ge-cm-1-IRII transcript with the genomic sequence of Ge-cm-1 revealed that Ge-cm-1-IRII was an alternatively spliced transcript generated by intron retention, further confirming a previous discovery that alternative splicing of CM genes is conserved among Globodera species. The genomic sequence of Ge-cm-1 contains three introns with intron 1 showing significant divergence compared to those of CM genes from the two PCN species as well as a related species G. tabacum. Based on the sequence variations, we designed PCR primers and a TaqMan probe specific for Ge-cm-1 and developed a TaqMan qPCR assay that provides reliable and sensitive identification of G. ellingtonae. Due to the fact that multiple Globodera species that infect potato currently occur in the U.S., this new molecular diagnostic method is valuable and should be included with other standard diagnostic methods to achieve a rapid and accurate differentiation of Globodera species.

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Abbreviations

AS:

Alternative splicing

bp:

Base pair

CM :

Chorismate mutase

Ge-cm-1 :

Globodera ellingtonae chorismate mutase gene

Gr-cm-1 :

Globodera rostochiensis chorismate mutase gene

Gp-cm-1 :

Globodera pallida chorismate mutase gene

Gt-cm-1 :

Globodera tabacum chorismate mutase gene

ITS:

Internal transcribed spacer

PCN:

Potato cyst nematode

qPCR:

Quantitative real-time polymerase chain reaction

RFLP:

Restriction fragment length polymorphism

rRNA:

Ribosomal RNA

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Acknowledgments

We would like to thank Eric Grenier for providing DNA from Globodera populations from South Peru, Chile and UK, Geert Smant for providing DNA from PCN populations from the Netherlands, Solke De Boer for providing DNA from a G. pallida population from Newfoundland, Canada, Melissa Mitchum for providing G. tabacum, and Eric Davis for providing Heterodera glycines. This study was supported by funding from USDA-ARS and USDA-APHIS.

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Authors and Affiliations

  1. USDA-ARS, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, 14853, USA

    Demosthenis Chronis & Xiaohong Wang

  2. Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY, 14853, USA

    Shiyan Chen & Xiaohong Wang

  3. USDA-ARS, Nematology Laboratory, Building 010A, Beltsville, MD, 20705, USA

    Andrea M. Skantar

  4. USDA-ARS, Horticultural Crops Research Laboratory, 3420 NW Orchard Avenue, Corvallis, OR, 97330, USA

    Inga A. Zasada

Authors
  1. Demosthenis Chronis
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  2. Shiyan Chen
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  3. Andrea M. Skantar
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  4. Inga A. Zasada
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  5. Xiaohong Wang
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Corresponding author

Correspondence to Xiaohong Wang.

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Supplementary Fig. 1

Alignment of chorismate mutase protein sequences from different Globodera species. Included protein sequences are from G. rostochiensis (GenBank accession number ABR19889), G. pallida (CAD29887), G. tabacum (AEA07495), and G. ellingtonae (KF360243). The sequence alignment was made using Clustal W (Larkin et al. 2007) and BOXSHADE (Subramaniam 1998). The signal peptide predicted by SignalP (Nielsen et al. 1997) and the chorimate mutase domain were boxed and labelled as indicated. Identical and similar amino acid residues are shaded in black and gray, respectively. (JPEG 143 kb)

High resolution image (TIFF 11973 kb)

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Chronis, D., Chen, S., Skantar, A.M. et al. A new chorismate mutase gene identified from Globodera ellingtonae and its utility as a molecular diagnostic marker. Eur J Plant Pathol 139, 245–252 (2014). https://doi.org/10.1007/s10658-014-0385-x

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  • DOI: https://doi.org/10.1007/s10658-014-0385-x

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