NUCLEOTIDE POLYMORPHISMS AND AN IMPROVED PCR-BASED MTDNA DIAGNOSTIC FOR PARTHENOGENETIC ROOT-KNOT NEMATODES (MELOIDOGYNE SPP)

Sequence analysis of 2212 bp of six closely related mitochondrial DNA (mtDNA) haplotypes that dominate Australian populations of Meloidogyne (Hugall et at, 1994) revealed twelve polymorphic nucleotide sites and one deletion. Despite this low diversity, there are enough variable r estriction enzyme sites among these sequences to provide diagnostic te sts. 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 o r MnII. The diagnostic test identifies the haplotypes, even in mixture s, found in M. arenaria, M. incognita, M, javanica, and M. hispanica a nd also M. hapla and M. chirwoodi. This is an improvement over previou s mtDNA PCR tests for Meloidogyne in that it discriminates between mor e species and races (e.g., M. arenaria races from M. javanica) and, be cause smaller products are amplified, it should be more robust. We als o developed primers to amplify a region of 63 bp variable number tande m repeats. The resulting DNA banding pattern may differentiate isolate s within restriction enzyme haplotypes and, potentially, can be used t o verify the identity of nematode isolates maintained in culture.