Detection of polymorphic patterns of genomic DNA amplified by RAPD-PCR in sweet potato weevils, Cylas formicarius (Fabricius)(Coleoptera: Brentidae).

Abstract

The polymorphic patterns of genomic DNA amplified by RAPD-PCR were detected in laboratory-cultured populations of sweet potato weevils, Cylas formicarius collected from the Southwest islands (Nansei-Shoto) of Japan. When three sets of primers, T13/T13, T05/T05 and T01/T07 were used for PCR, the polymorphic patterns of the amplified DNA were classified into nine types. Of these types, A 1 B 1 C 3 was common in all populations tested. The present study determined the useful primer sets that will enable the successful classification of sweet potato weevils based upon the polymorphic patterns of genomic DNA amplified by RAPD-PCR.

A convenient estimation of the sources of sweet potato weevils, Cylas formicarius (Fabricius) (Coleoptera: Brentidae), in recently invaded areas in Japan, by random amplified polymorphic DNA technique

“…DNA was extracted from the whole body of each individual using a method reported previously (Kawamura et al, 2002) and dissolved in 200 ml of TE buffer. After the DNA concentration was estimated using a spectrophotometer (DU 640, Beckman, CA, USA), the solution was diluted to 10 ng/ml and stored at 4°C.…”

“…The sample used for PCR was frozen at Ϫ20°C. We used three primer sets, i.e., T13/T13, T05/T05, and T01/T07 (Operon Technologies, CA, USA), which amplify DNA from C. formicarius reproducibly and provide RAPD patterns (Kawamura et al, 2002). PCR amplification was carried out under the same conditions described in Kawamura et al (2002), and 10 ml of the PCR products was electrophoresed in 1.5% agarose gel in TAE buffer.…”

“…We used three primer sets, i.e., T13/T13, T05/T05, and T01/T07 (Operon Technologies, CA, USA), which amplify DNA from C. formicarius reproducibly and provide RAPD patterns (Kawamura et al, 2002). PCR amplification was carried out under the same conditions described in Kawamura et al (2002), and 10 ml of the PCR products was electrophoresed in 1.5% agarose gel in TAE buffer. The gel was stained with 10 mg/ml ethidium bromide solution for 10 min, visualized on a UV transilluminator at a wavelength of 312 nm (FV1-20M, Funakoshi, Tokyo, Japan), and imaged with a Kodak DC120 Zoom digital camera using the Electrophoresis Documentation and Analysis System 120, Limited Edition (Kodak Digital Science, NY, USA), according to the manufacturer's instructions.…”

“…Based upon reproducible DNA fragments, the RAPD patterns of wild C. formicarius amplified using the three primer sets T13/T13, T05/T05, and T01/T07 are referred to as types A, B, and C RAPD patterns, respectively. Because there were multiple RAPD patterns for each type, each individual was given a triplet notation by combining the RAPD patterns of the three types detected for that individual, following the method of Kawamura et al (2002).…”

“…Although RAPD-PCR is not recommended for phylogenetic studies based on genetic distance, it is a fast and inexpensive method for the identification of insect species (Black, 1993). Kawamura et al (2002) detected genomic DNA polymorphisms in laboratory-reared C. formicarius using RAPD-PCR, established a reproducible protocol, and proposed a convenient procedure for classifying the genetic variation of C. formicarius more minutely by combining RAPD patterns from three primer sets. We used this procedure to examine the genetic variation of C. formicarius collected from various areas of Japan, and then estimated the sources of C. formicarius that invaded Muroto City, Kochi Prefecture, in 1995and Yakushima Island, Kagoshima Prefecture, in 1997(Hiramatsu, 1996Maehara, 1998).…”