Polymorphic DNA microsatellites identified in the yellow dung fly ( Scathophaga stercoraria )

Sperm competition in yellow dung flies (Scathophaga stercoraria) has been extensively investigated since Parker's (1970a) seminal work (e. These flies serve as a model system for understanding the mechanisms and outcomes of sperm competition in internal fertilizers. Invariably however, these investigations have been laboratory based, and typically involved competition between only two males. How the results of such studies relates to free-living flies is unknown, but it is unlikely that the experimental conditions employed exist in nature, and therefore outcomes may not reflect true female sperm utilization patterns (Eady & Tubman 1996). This is exemplified by a study of sperm competition in pseudoscorpions, which showed that second-male mating advantage breaks down when females mate with more than two males (Zeh & Zeh 1994). In addition, Ward (2000) has shown that females are able to subtly alter paternity patterns under conditions that are likely to be common in the field. With this in mind, our aim was to develop appropriate genetic markers to allow paternity to be accurately assigned in clutches laid by free-living female yellow dung flies. A subgenomic library enriched for CA repeat microsatellites was constructed following standard protocols outlined in Tenzer et al. (1999), with slight modifications. Genomic DNA isolated from a single S. stercoraria male using standard phenol– chloroform extraction and ethanol precipitation (Sambrook et al. 1989) was digested using Tsp509I (New England Biolabs). A 500 –1000 bp size fraction was isolated from a LM-MP agarose (Boehringer Mannheim) gel by first excising the appropriate size range from the gel. The gel fragment was melted in a 65 °C water bath and volume was increased to 500 µL using double distilled water. An equal volume of equilibrated phenol (pH 8.0) was added, the solution vortexed briefly and then put at – 80 °C for 30 min. The sample was then thawed and extraction was completed following standard phenol – chloroform extraction methods (Sambrook et al. 1989). This isolate was used for ligation with TSPADSHORT/ TSPADLONG linkers (Tenzer et al. 1999) and then amplified via the polymerase chain reaction (PCR), using TSPADSHORT as a primer. PCR was performed using the following conditions: Total reaction volume was 25 µL included 100 ng DNA, 1 U Taq DNA polymerase (Quantum-Appligene), 10 mm Tris-HCl, pH 9.0, 50 mm KCl, 1.5 mm MgCl 2 , 0.01% TritonX100, 0.2 mg BSA (Quantum-Appligene), 100 µm of each dNTP (Promega), and 1 µm of TSPADSHORT. PCR …