RESUMO
BACKGROUND: DNA barcoding uses a 650 bp segment of the mitochondrial cytochrome c oxidase I (COI) gene as the basis for an identification system for members of the animal kingdom and some other groups of eukaryotes. PCR amplification of the barcode region is a key step in the analytical chain, but it sometimes fails because of a lack of homology between the standard primer sets and target DNA. RESULTS: Two forward PCR primers were developed following analysis of all known arthropod mitochondrial genome arrangements and sequence alignment of the tRNA-W gene which was usually located within 200 bp upstream of the COI gene. These two primers were combined with a standard reverse primer (LepR1) to produce a cocktail which generated a barcode amplicon from 125 of 141 species that included representatives of 121 different families of Hexapoda. High quality sequences were recovered from 79% of the species including groups, such as scale insects, that invariably fail to amplify with standard primers. CONCLUSIONS: A cocktail of two tRNA-W forward primers coupled with a standard reverse primer amplifies COI for most hexapods, allowing characterization of the standard barcode primer binding region in COI 5' as well as the barcode segment. The current results show that primers designed to bind to highly conserved gene regions upstream of COI will aid the amplification of this gene region in species where standard primers fail and provide valuable information to design a primer for problem groups.
Assuntos
Artrópodes/genética , Impressões Digitais de DNA/métodos , Primers do DNA/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , RNA de Transferência/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Genoma Mitocondrial/genética , Dados de Sequência MolecularRESUMO
Mealybugs are under a strict regulation at foreign trades of agricultural products because they are one of the most economically damaging groups of insects on food crops and ornamental plants. However, the absence of morphological characteristics enabling the discrimination of early life stages often cause a significant delay or rejection of a shipment when infested fruit is discovered, causing significant economic loss. A polymerase chain reaction-based method for species identification was developed for six mealybug species known to infest Korean pears including two regulated insects, Planococcus kraunhiae (Kuwana) and Crisicoccus matsumotoi (Siraiwa). Six sets of species-specific primers were designed based on the sequence comparison of the internal transcribed spacer 1 and 2 regions. Efficiency tests against 29 mealybug samples showed that this method could effectively discriminate different mealybug species regardless of their developmental stages. Blind tests against 11 field collected mealybug nymph samples indicated that a single polymerase chain reaction is enough to discriminate unidentified mealybugs collected on Korean pears. This new method will facilitate trade and export requirements, as well as identify the species at any stage of mealybug intercepted.
