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

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.