Differentiation of Diaprepes abbreviatus and Pachnaeus litus (Coleoptera: Curculionidae) egg masses: PCR-restriction fragment-length polymorphism and species-specific PCR amplification of 18S rDNA products
2003
Weathersbee, A.A III | Bullock, R.C. | Panchal, T.D. | Dang, P.M.
The root weevils Diaprepes abbreviatus (L.) and Pachnaeus litus (Germar) are both pests of Florida horticulture, but D. abbreviatus is a regulated exotic species that causes severe damage whereas P. litus is considered a minor pest. Egg masses of these two weevil species are indistinguishable when they are detected on host plants. Two approaches to differentiating the egg masses are described herein. Total genomic DNAs extracted from D. abbreviatus and P. litus were used for polymerase chain reaction (PCR) amplification and the PCR products were sequenced to obtain the 2014-bp sequence of the complete 18S rRNA gene for each species. A 446-bp region amplified from the 5' end of the 18S rDNA of P. litus contained a restriction fragment-length polymorphism marker, an extra BstU I recognition site that was not present in D. abbreviatus. Agarose gel electrophoresis of the restriction enzyme-digested PCR products produced a restriction pattern that enabled differentiation of the egg masses. Additionally, two species-specific reverse primers were designed to exploit a single nucleotide polymorphism (SNP) that occurred at the restriction fragment-length polymorphism marker site. These reverse primers differed only by a single nucleotide at the 3' end. When used in concert with a standard 18S rDNA forward primer, each species-specific reverse primer distinctly amplified a 256-bp product only when the correct genomic DNA was present.
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