Microsatellite Multiplexing in Fish

—Microsatellite multiplexing is a powerful technique that can increase the productivity of genetic studies in fisheries biology. We review multiplexing methods and present an optimized and detailed protocol for microsatellite multiplexing that is specifically tailored for use with radioisotopes. The protocol can significantly reduce the cost associated with microsatellites and provides high polymerase chain reaction (PCR) fidelity and band resolution. Comparing three radioisotopes, we find that end labeling with 33P provides the highest resolution. We also present a quick and inexpensive DNA isolation protocol that is successful with fish larvae. Finally, we find that PCR fidelity depends on the quality of the DNA template, and we therefore review preservation and isolation methods specific to various fish tissue types. Together, these microsatellite multiplexing and DNA isolation protocols can significantly reduce the time and expense associated with genetic analyses in fish. Microsatellites are versatile genetic markers that are finding applications in many studies of ecology, evolution, and conservation (for reviews, see Wirgin and Waldman 1994; O’Reilly and Wright 1995; Jarne and Lagoda 1996; O’Connell and Wright 1997). In fisheries biology, these markers are widely used to assess the following: effective population size of stocks (Reilly et al. 1999), stock identification (Shaklee and Bentzen 1998), levels of inbreeding (Tessier et al. 1997), population structure and gene flow (DeLeon et al. 1997; Arnegard et al. 1999), parentage (Knight et al. 1998), and quantitative traits (Jackson et al. 1998). Polymerase chain reaction (PCR) multiplexing, the coamplification of two or more loci in a single PCR reaction (Chamberlain et al. 1988), is an innovative technique that considerably reduces the time and costs associated with microsatellite genetic analyses. However, many fisheries laboratories are not multiplexing because of the lack of effective and detailed protocols and because of the general apprehension that multiplexing considerably increases the complexity of using microsatellites. * Corresponding author: [email protected] Received November 12, 1998; accepted May 14, 1999 Although several multiplexing protocols exist for the fluorescent tags and automated detection systems (e.g., Edwards et al. 1991; Kimpton et al. 1993; Oetting et al. 1995; Paetkau et al. 1995; Ricciardone et al. 1997; Fishback et al. 1999), these systems are expensive and are unavailable to most fish laboratories. Instead, these laboratories use radioisotopes, polyacrylamide gel electrophoresis, and autoradiography to visualize microsatellites. The use of radioisotopes presents several additional complexities to multiplexing that the protocols tailored to fluorescent tags do not address. For example, microsatellite loci with alleles that overlap in size cannot be differentiated with radioisotopes. Further, the lack of internal size standards in individual multiplex reactions impedes accurate scoring of radioisotope-labeled banding patterns, thereby placing emphasis on resolution and visualization. Finally, existing radioisotope-based protocols (e.g., Huang et al. 1992; O’Reilly et al. 1996) are generally costly for large sample sizes, and they may produce inconsistent resolution, particularly when researchers are amplifying dinucleotide microsatellites. This paper (1) reviews the key papers that discuss multiplexing; (2) presents a step-by-step protocol for the design and optimization of multiplexes specifically tailored for use with radioisotopes; (3) evaluates the performance of three radioisotopes; (4) evaluates the effect of DNA purity on multiplex fidelity; and (5) summarizes methods of preservation and isolation of DNA for different types of fish tissue. The step-by-step protocol provides a comprehensive set of procedures, including primer design, PCR coamplification, multiplex optimization, and electrophoresis and visualization.