Genotyping for the G4946E site of ryanodine receptor gene in Plutella xylostella (Lepidoptera: Yponomeutidae) considering gene duplication

Genotyping for the previously reported G4946E mutation site of the ryanodine receptor (RyR) gene was conducted individually in Plutella xylostella Linnaeus strains with different sensitivities to diamide insecticides. Results obtained by direct sequencing using primers previously reported for the mutation site were not indicative for the resistance status of the strains examined, possibly because of gene duplication. A typical example is that the RyR gene sequences with the G4946E mutation were found in the laboratory-susceptible strain. However, all clones with the G4946E mutation lacked introns, suggesting that the mutation encoded in intronless sequences does not function as a resistance factor. Primers exclusively amplifying the RyR gene sequences with introns were designed based on data obtained by cloning and nucleotide sequencing of RyR gene sequences amplified from 28 insects belonging to six strains. Using the primers, genotyping for the mutation site was conducted individually. Results showed that resistant strains contain more insets of resistant homozygote.