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Description
Microsatellites are codominantly inherited nuclear-DNA markers (Wright and Bentzen, 1994) that are now commonly used to assess both stock structure and the effective population size of exploited fishes (Turner et al., 2002; Chistiakov et al., 2006; Saillant and Gold, 2006). Multiplexing is the combination of polymerase chain reaction (PCR) amplification products from multiple loci into a single lane of an electrophoretic gel (Olsen et al., 1996; Neff et al., 2000) and is accomplished either by co-amplification of multiple loci in a single reaction (Chamberlain et al., 1988) or by combination of products from multiple single-locus PCR amplifications (Olsen et al., 1996). The advantage of multiplexing microsatellites lies in the significant reduction in both personnel time (labor) and consumable supplies generally required for large genotyping projects (Neff et al., 2000; Renshaw et al., 2006).
In this note, we report the development of multiplex panels of microsatellites that will facilitate population-level genetic studies of both gray (Lutjanus griseus) and lane (L. synagris) snappers. The overexploitation of Gulf red snapper (L. campechanus) in U.S. waters and the increasing restrictions on both commercial and recreational red snapper catches (Gillig et al., 2001) have led to increased fishing pressure on other snapper species (Fischer et al., 2005), including both gray (Burton, 2001; Fischer et al., 2005) and lane snappers (GMFMC (1)). Although neither species has yet been classified as "overfished" or subject to "overfishing," the increased exploitation of the two species could jeopardize these snapper resources in the future. In this study, we optimized multiplex panels for gray and lane snappers from among microsatellite markers designed originally for red snapper by Gold et al. (2001) and vermillion snapper (Rhomboplites aurorubens) by Bagley and Geller (1998).
Materials and methods
Samples of gray and lane snappers were obtained off the west coast of Florida during April of 2004. Fin clips and pieces of liver were preserved in 95% ethanol, brought to the laboratory, and stored at room temperature. Genomic DNA was extracted by using an alkaline-lysis method (Saillant et al., 2002) and stored at -20[degrees]C.
Microsatellites were first evaluated in single-locus (simplex) reactions in order to determine the size range and ease of scoring of PCR products in each species. PCR amplifications were performed in ll.5-[micro]L volumes comprising 1.5 [micro]L of DNA... |
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