DNA-based genotyping techniques for the detection of point mutations associated with insecticide resistance in Colorado potato beetle Leptinotarsa decemlineata

Three DNA-based genotyping techniques, bi-directional PCR amplification of specific allele (bi-PASA), single-stranded conformational. polymorphism (SS CP) and minisequencing, have been developed and compared for the detection of the S291G (insensitive acetylcholinesterase) and L1014F (insensitive sod ium channel), mutations associated with azinphos-methyl and permethrin resi stance, respectively, in the Colorado potato beetle (Leptinotarsa decemline ata). Extraction of genomic DNA from individual neonates that were hatched from previously collected egg masses is the most efficient and reliable mea ns to obtain suitable templates, in terms, of convenience, economy, speed a nd DNA quality. Bi-PASA, employing two allele-specific primers, appears to be the most efficient and rapid genotyping method for the simultaneous, det ection of both resistant/susceptible homozygous (SS, RR) and heterozygous ( SR) alleles. Its resolution, however, is strongly dependent on the quality of template genomic DNA. SSCP also: allows unambiguous genotyping, includin g the detection of heterozygous alleles, and is less dependent on template DNA quality, but requires a longer processing time. Minisequencing. is amen able to a 96-well microtiter plate format for the processing of a large num ber of samples and allows direct detection of resistant/susceptible homozyg ous alleles but is not as efficient as the PASA and SSCP in detecting heter ozygous alleles. In considering the advantages and disadvantages of each te chnique, DNA-based genotyping is best employed in combinations, with the bi -PASA as the primary method and the SSCP and minisequencing as the secondar y validating methods. These methods are rugged, rapid, cost-effective and c apable of resolving SS, RR and SR individuals. The availability of such DNA -based genotyping techniques, using neonate genomic DNA as templates,, will enable the precise monitoring of the resistant and susceptible allele freq uencies, including. those of heterozygote individuals, in field populations of L decemlineata. (C) 2001 Society of Chemical Industry.