Primer Notes

The predominant mode of reproduction for most marine fishes is pelagic spawning; gametes are released into the water where sperm, often contributed by more than one male, fertilize eggs of a female. As a consequence of external fertilization males are frequently in sperm competition with each other. However, little is known about the actual effects of sperm competition on the fitness of a pelagic spawning male. We have isolated and characterized microsatellite loci from the bluehead wrasse, Thalassoma bifasciatum. This coralreef fish has been extensively used in studies of sexual selection, life-history traits (Warner & Schultz 1992) and recruitment ecology (Caselle & Warner 1996). For our purposes microsatellite markers were developed to determine paternity of larvae from spawns involving multiple males. We developed microsatellite primers for T. bifasciatum not only for their suitability in paternity studies because of potential high variability (Tautz 1989), but also for their codominant allelic patterns, critical for powerful paternityexclusion analysis. Moreover, microsatellites require small tissue amounts and are specific to target DNA. These are important attributes for our study, because minute larvae and sperm are collected from sea water contaminated with various DNA sources. Microsatellite loci were isolated and characterized using the construction of a partial genomic DNA library of short fragments of T. bifasciatum as per Strassmann et al. (1996). Genomic DNA was isolated in large quantities from gill filaments and digested to completion by the restriction enzyme Sau3A1. Restriction fragments were separated by agarose gel electrophoresis and the 200Ð600 bp fragments were isolated and ligated into the pUC-18 vector pBluescript (Stratagene). Following transformation of competent DH5α bacterial cells, plating onto selective agar media, and replica plating onto nylon filters, the library was screened using [αP]-dCTP end-labelled oligonucleotides corresponding to common microsatellite motifs: (AAT)10, (CAT)10, (TAG)10, (AAG)10 and (AAC)10. A total of 24 positive clones was sequenced on an automated sequencer (ABI/Perkin Elmer) of which 12 contained dinucleotide or trinucleotide repeats. Two of these microsatellite loci did not contain adequate flanking region for primer set design. Thus 10 primer sets have been designed of which six are variable (Table 1). Population screens were conducted to determine allelic variability at the six loci by using genomic DNA from seven to 43 presumably unrelated individuals collected in 1993 from different coral reefs of St Croix, US Virgin Islands. The final concentrations of the PCR reagents in a volume of 15 μL were as follows: ≈10 ng of genomic DNA, 1× PCR buffer Molecular Ecology (1998) 7, 1613Ð1621