Title: A new chorismate mutase gene identified from Globodera ellingtonae and its utility as a molecular diagnostic marker Authors
Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 2, 2014
Publication Date: January 21, 2014
Citation: Chronis, D.N., Chen, S., Skantar, A.M., Zasada, I.A., Wang, X. 2014. A new chorismate mutase gene identified from Globodera ellingtonae and its utility as a molecular diagnostic marker. European Journal of Plant Pathology. 139(2):239-246. DOI:10.1007/s10658-014-0385-x. Interpretive Summary: Globodera ellingtonae is a new potato pest that was recently detected in a research farm in Oregon. This new Globodera species shares obvious morphological similarities with the two species of potato cyst nematodes (PCNs, Globodera rostochiensis and G. pallida) that are under quarantine in the U.S. Currently no molecular method for a rapid and sensitive identification of G. ellingtonae is available. In this study, we cloned and characterized a new chorismate mutase gene (Ge-cm-1) from G. ellingtonae. By detailed sequence analysis and comparison with the CM genes from closely-related species of G. rostochiensis, G. pallida, and G. tabacum (tobacco cyst nematode), we identified regions in Ge-cm-1 that are unique to G. ellingtonae. We then designed PCR primers and the TaqMan probe based on the unique regions and developed a TaqMan qPCR assay for identifying G. ellingtonae. Our results showed that the developed TaqMan qPCR assay could provide a highly specific and sensitive identification of G. ellingtonae. Owing to the fact that multiple Globodera potato pests currently occur in the U.S., this molecular diagnostic method is valuable and should be included with other standard protocols for screening soil survey samples that may contain PCN pests.
Technical Abstract: Globodera ellingtonae, a new cyst nematode species recently detected in Oregon and confirmed of reproduction on potato, shares key morphological features with the two species of potato cyst nematode (PCN; G. rostochiensis and G. pallida) of quarantine concern. Currently no methods are available for a molecular diagnosis of this emerging Globodera species. In this study, we cloned a new chorismate mutase effector 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. The comparison with the obtained Ge-cm-1 genomic sequence 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 of G. tabacum. Based on the sequence variations we designed PCR primers and TaqMan probe specific for Ge-cm-1 and developed a TaqMan qPCR assay that confirmed to provide a 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 developed molecular diagnostic method is valuable and should be included with other standard diagnostic methods to achieve a rapid and more accurate diagnosis of the two quarantine PCN species.