DIFFERENTIATION OF MELOIDOGYNE FLORIDENSIS FROM M. ARENARIA USING HIGH-FIDELITY PCR AMPLIFIED MITOCHONDRIAL AT-RICH SEQUENCES
Keywords:high-fidelity pcr, meloidogyne, mitochondrial at-rich region, molecular diagnosis, stem and loop structure
AbstractStandard polymerase chain reaction (PCR) and high-fidelity PCR procedures were compared for amplifying DNA sequences from five root-knot nematode species. High-fidelity PCR was found to be at least four orders of magnitude more efficient than standard PCR. High-fidelity PCR was used to amplify mitochondrial DNA sequences located between the cytochome oxidase subunit II (COII) and 16S rRNA genes from the following root-knot nematode species; Meloidogyne mayaguensis (0.7 kb), M. floridensis (1.1 kb), M. arenaria (1.1 kb), M. incognita (1.5 kb), and M. javanica (1.6 kb). This region includes the non-coding "control" region, which is also referred to as AT-rich region and tRNA-His gene. The high-fidelity PCR products were cloned and sequenced. The COII 3' partial, tRNA-His, and 16S rRNA 5' partial sequences were found to display few nucleotide differences, but the AT-rich region displayed extensive nucleotide and length variations in all five species; M. mayaguensis (167 bp), M. arenaria (573 bp), M. floridensis (603 bp), M. incognita (963 bp), and M. javanica (1110 bp). A stem and loop structure, which has been characterized from insects and other higher organisms, was detected in a conserved block of AT-rich sequences from M. floridensis, M. arenaria, M. incognita, and M. javanica. Similar stem and loop structures were also detected from the AT-rich sequences previously reported from Caenorhabditis elegans and Ascaris suum and compared to the M. floridensis stem and loop structure. Even though the high-fidelity PCR products amplified from M. floridensis and M. arenaria were about the same size (1.1 kb), their AT-rich sequences were found to contain nucleotide differences at several locations. Hence, digesting the 1.1 kb high-fidelity PCR products from these species with the restriction enzyme Ssp I produced two distinct banding patterns that distinguished M. floridensis from M. arenaria.