Genetica

c Indian Academy of Sciences

ONLINE RESOURCES

Development of single locus DNA microsatellite markers in Oryctes rhinoceros (Linnaeus) using 5 anchored RAMs-PCR method
G. MANJERI1 , R. MUHAMAD1 ∗ , Q. Z. FARIDAH2 and S. G. TAN3
1

Department of Plant Protection, Faculty of Agriculture, 2 Department of Biology, Faculty of Science and 3 Department of Cell and Molecular Biology, Faculty ofBiotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
[Manjeri G., Muhamad R., Faridah Q. Z. and Tan S. G. 2012 Development of single locus DNA microsatellite markers in Oryctes rhinoceros (Linnaeus) using 5 anchored RAMs-PCR method. J. Genet. 91, e92–e96. Online only: http://www.ias.ac.in/jgenet/OnlineResources/91/ e92.pdf]

IntroductionOryctes rhinoceros, commonly known as rhinoceros beetle, is an important pest in oil palm plantations. The presence of this pest in replanting sites as early as six months after replanting has alarmed planters due to the possibility of increased crop damage (Samsuddin et al. 1993; Kamarudin and Wahid 1997). Being a nocturnal animal with a destructive feeding habit, it is difficult to eliminate thispest (Young 1986). Pheromone trapping using a speciesspecific-aggregation pheromone is commonly used to trap O. rhinoceros in replanting sites (Hallet et al. 1995). However, not all population of O. rhinoceros in the field were observed to be attracted by it. This suggests the possibility of a cryptic species complex occurrence in this insect. To investigate this notion, a thorough study utilizingmolecular markers is necessary. An example of a powerful marker with proven capability for resolving such issues is the single-locus DNA microsatellite marker. Being codominant, multiallelic and highly polymorphic, microsatellites are a powerful and promising genetic marker, suitable for precise discrimination of closely related individuals (Smouse and Chevillon 1998). A previous study on thegenetic variation of O. rhinoceros had highlighted the necessity of further studying this pest species using single-locus microsatellite DNA markers as O. rhinoceros were reported to exhibit possible occurrence in two groups (Manjeri et al. 2011). Therefore, with interest to further study the population genetic structure of O. rhinoceros; this study was carried out to isolate sufficiently novelsingle-locus microsatellite markers for the insect. The isolation was carried out based on the 5 -anchored polymerase chain reaction (PCR) technique (Fisher et al. 1996)
∗ For correspondence. E-mail: ritamuhamad@yahoo.com.

using anchored randomly amplified microsatellite (RAM) primers (Kumar et al. 2002; Hoh et al. 2008).

Materials and method
Genomic DNA was extracted from O. rhinoceros beetle usingthe Wizard Genomic DNA Purification kit (Promega, Madison, USA) according to the manufacturer’s protocol for animal tissue genomic DNA isolation with minor modifications. Tissues from the thorax and head of the beetle were used to avoid contamination. Extracted DNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). Fifteen RAMs primer were testedusing the extracted DNA. However, only nine primers with clear banding profile were chosen. PCR was performed in a total volume of 10 μL per reaction containing 25 ng of genomic DNA, 2.5 mM MgCl2 , 1× PCR Buffer (10 mM Tris-HCl, 50 mM KCl and 0.1 Triton R_X-100), 0.4 mM dNTPs mix, optimized specific concentration of primer, 3 U Taq DNA polymerase (Promega, Madison, USA) and topped with deionizeddistilled water up to 10 μL. Amplifications were performed in a Techne TC-412 thermal cycler (Burlington, USA) with an initial 3 min of predenaturation at 96◦ C, followed by 40 cycles of 20 s denaturation at 95◦ C, 20 s annealing at optimized temperature for each primer and 35 s extension at 68◦ C. A final extension step of 68◦ C for 5 min was included. PCR product, 5 μL was electrophoresed on...