Nucleotide polymorphisms and an improved PCR-based mtDNA diagnostic for parthenogenetic root-knot nematodes (Meloidogyne spp.)

Abstract

Sequence analysis of 2212 bp of six closely related mitochondrial DNA (mtDNA) haplotypes that dominate Australian populations of Meloidogyne (Hugall et al., 1994) revealed twelve polymorphic nucleotide sites and one deletion. Despite this low diversity, there are enough variable restriction enzyme sites among these sequences to provide diagnostic tests. Using a selection of these sites, we have developed a multiplexed PCR-based diagnostic that simultaneously amplifies two small regions of the mitochondrial genome, and then digests the product with HinfI or MnII. The diagnostic test identifies the haplotypes, even in mixtures, found in M. arenaria, M. incognita, M. javanica, and M. hispanica and also M. hapla and M. chitwoodi. This is an improvement over previous mtDNA PCR tests for Meloidogyne in that it discriminates between more species and races (e.g., M. arenaria races from M. javanica) and, because smaller products are amplified, it should be more robust. We also developed primers to amplify a region of 63 bp variable number tandem repeals. The resulting DNA banding pattern may differentiate isolates within restriction enzyme haplotypes and, potentially, can be used to verify the identify of nematode isolates maintained in culture.