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Isolation and inheritance of microsatellite loci in the Dungeness crab (Brachyura: Cancridae: Cancer magister) Pamela C. Jensen and Paul Bentzen
Abstract: The isolation, PCR amplification, and descriptive statistics of six microsatellite loci are described for the Dungeness crab, Cancer magister. Also reported is the inheritance of these loci in two families obtained from artificial crosses in the laboratory. All six loci conform to expectations under Mendelian inheritance and there is no evidence for linkage between any of the loci. Allelic size ranges for three of the loci are relatively large, ranging from 135–357 bp between the smallest and largest allele detected at that locus. At two of these loci upper allelic drop out (nonamplification of the larger allele in a heterozygous individual) can be problematic for scoring. Results from crossspecies amplification in nine congeners are summarized. These loci will be valuable in studies requiring high-resolution genetic markers in Dungeness crabs and related species. Key words: Cancer magister, microsatellite, Mendelian Inheritance, cross species amplification, Brachyura. Résumé : Le clonage, l’amplification PCR et les statistiques descriptives de six locus microsatellites chez le crabe dormeur, Cancer magister, sont décrits. Les auteurs rapportent également l’hérédité de ces locus chez deux familles obtenues suite à des croisements réalisés en laboratoire. Les six locus ont montré une ségrégation mendélienne et aucune évidence de liaison entre les locus n’a été obtenue. La gamme d’allèles était relativement grande pour trois des locus, la différence entre le plus petit et le plus grand allèle variant entre 135 et 137 pb. Chez deux de ces locus, l’absence d’amplification d’un allèle (l’impossibilité d’amplifier l’allèle de plus grande taille au sein d’un hétérozygote) peut s’avérer problématique lors du génotypage. Les résultats d’amplifications interspécifiques, chez neuf espèces apparentées, sont résumés. Ces locus seront utiles dans le cadre d’études nécessitant des marqueurs génétiques à haute résolution chez le crabe dormeur et des espèces apparentées. Mots clés : Cancer magister, microsatellite, hérédité mendélienne, amplification interspécifique, Brachyura. [Traduit par la Rédaction]
Jensen and Bentzen
Introduction The Cancridae comprise a well-known and generally readily accessible family of brachyuran crabs. One of the more familiar cancrids, the Dungeness crab, Cancer magister, has been regularly fished for approximately 150 years, is often locally abundant, and is both an important predator and prey organism. However, most aspects of its life history, including mating system, larval dispersal, and population Received 24 March 2003. Accepted 5 November 2003. Published on the NRC Research Press Web site at http://genome.nrc.ca on 23 March 2004. Corresponding Editor: J.B. Bell. P.C. Jensen1,2 and P. Bentzen.3 Marine Molecular Biotechnology Laboratory, School of Aquatic and Fishery Sciences, University of Washington, 3707 Brooklyn Ave. NE, Suite No. 175, Seattle, WA 98105, U.S.A. 1
Present address: National Marine Fisheries Service, Alaska Fisheries Science Centre – Resource Assessment and Conservation Engineering (AFSC–RACE), 7600 Sand Point Way NE, Seattle, WA 98115, U.S.A. 2 Corresponding author (e-mail:
[email protected]). 3 Present address: Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada. Genome 47: 325–331 (2004)
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structure, remain poorly understood. The use of molecular markers to examine aspects of these processes should provide information that might otherwise be difficult to obtain. Dungeness crabs are distributed along the North American Pacific coast from Santa Barbara, Calif., to the Pribilof Islands, Alaska (Hart 1982; Jensen 1995). In considering only the movements of tagged adults as documented in the literature (Cleaver 1949; Butler 1957; Snow and Wagner 1965; Gotshall 1978), the data suggests that separate subpopulations could exist. However, Dungeness crab larval stages are planktonic for 3–4 months (Poole 1966; Reilly 1983) and are found in swift-flowing currents that potentially could distribute them over great distances. Most population studies of C. magister have inferred structure based upon measurements of life history stages, behavior, knowledge of oceanographic currents, or models of larval drift (e.g., MacKay and Weymouth 1935; Weymouth and MacKay 1936; PFMC 1979; Tasto 1983; Stevens and Armstrong 1984; Orensanz and Gallucci 1988; Jamieson and Armstrong 1991; Dinnel et al. 1993; Jamieson and Phillips 1993); it remains unknown whether these differences between populations are genetically based or whether they reflect latitudinal gradients. Similarly, the literature investigating mating systems of brachyuran crabs is fragmentary and knowledge of even ba-
doi: 10.1139/G03-135
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sic processes involved in reproduction is incomplete for all but perhaps a handful of species (see reviews by Hartnoll 1969; Hazlett 1975; Ridley 1983; Salmon 1983; Christy 1987; Diesel 1991). Several reports describe components of various cancrid mating systems, but relatively little is known about any one species, and attributes of the mating system of one species are not necessarily applicable to the others (e.g., Orensanz and Gallucci 1988; Jensen et al. 1996). Because of their lack of variability in Dungeness crabs, allozymes, one of the most commonly used molecular markers, lack sufficient resolving power to elucidate population structure or kinship in this species (Soule and Tasto 1983). In contrast, microsatellite markers have revealed extensive genetic variability in a number of species that show little allozyme variation (e.g., grey seals (Allen et al. 1995); Atlantic cod (Bentzen at al. 1996; Ruzzante et al. 1996); marine turtles (FitzSimmons et al. 1995); snow crab (Urbani et al. 1998); and crayfish (Baker et al. 2000; Belfiore and May 2000; Gouin et al. 2000)). Few decapod microsatellites have been described, but those that are known are variable and useful as high-resolution markers (Penaeus spp. (Garcia et al. 1994, 1996; Vonau et al. 1999; Xu et al. 1999; Sugaya et al. 2002); Homarus spp. (Tam and Kornfield 1996; Jones et al. 2003); Chionoecetes spp. (Urbani et al. 1998); crayfish (Baker et al. 2000; Belfiore and May 2000; Gouin et al. 2000)). This article describes the isolation and characterization of six novel microsatellite loci for use in kinship and population studies in C. magister. Also reported are tests confirming Mendelian inheritance of these six loci in C. magister, and their conservation in nine other species of Cancer.
Materials and methods Collections and families Juvenile and adult Dungeness crabs (N = 120) were collected from Puget Sound at Mukilteo, Wash., by SCUBA and one of their third walking legs removed and preserved in 100% v/v ethanol for DNA extraction. Two virgin (
