Molecular characterization of Canadian populations of potato cyst nematodes, Globodera rostochiensis and G. pallida using ribosomal nuclear RNA and cytochrome b genes

Abstract

The mitochondrial cytochrome b gene (cytb), the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene were amplified, sequenced and used to characterize several populations of potato cyst nematodes, Globodera pallida and G. rostochiensis, collected from different areas in Canada. Diagnostic PCR-ITS-RFLP profiles with three restriction enzymes are provided for identification of both species. Sequences of ITS rRNA and cytb genes were compared with those in Genbank of other potato cyst nematode populations originating from Europe, South America, USA, Australia and New Zealand. The ITS rRNA sequences of Canadian G. rostochiensis were similar to those of all previously sequenced populations of this species. Sequence divergence of ITS rRNA for G. rostochiensis varied from 0 to 1.6%, whereas for G. pallida sequence divergence among populations reached 1.95%. Sequence and phylogenetic analysis of cytb and ITS rRNA genes using Bayesian inference revealed that Canadian G. pallida is almost identical to European and USA populations and formed a large clade with all these populations on the phylogenetic trees. The present molecular result with cytb confirmed the hypothesis that there are possibly several sibling species within G. pallida. Our study also supports a previously proposed hypothesis regarding introduction of G. pallida from a restricted area in Peru, firstly into Europe with subsequent spread to other continents including North America. Our sequence analysis revealed that several newly obtained sequences cannot be translated into functional cytb protein, because point indels disrupt the reading frame. Poly(T) variation in mtDNA genes in G. pallida might be explained by post-transcriptional editing mechanisms in Globodera mitochondria as well as by errors during PCR amplification of mononucleotide repeats within these genes.