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Fishery Bulletin 100(3) 



to amplification, one of the primers was kinase-labeled 

 with y^-P-ATP by T4 polynucleoti(ie kinase (30 min, 37°C). 

 PCR reactions contained approximately 5 ng of genomic 

 DNA, 0.1 units of Tag DNA polymerase, 0.5 ^M of each 

 primer, 800 ^M dNTPs, 1-2 niM MgCl.^, IX Tag buffer at 

 pH 9.0 (Promega, Corp., Madison, WI), and sterile deion- 

 ized water in a total volume of 10 ^L. Thermal cycling was 

 carried out in 96-well plates as follows: denaturation (4.5 

 sec, 95°C), annealing (30 sec, temperature as in Appendix 

 Table 1), and polymerization (30 sec, 72°C) for 30 cycles. 

 Aliquots (3 fih) of each PCR reaction were electrophoresed 

 in 6% denaturing polyacrylamide ("sequencing") gels. Gels 

 were dried and exposed to x-ray film. Alleles at individual 

 microsatellites were scored as number of repeats by com- 

 parison to the cloned (and sequenced) allele. Genotypes at 

 each microsatellite for each individual were scored and 

 entered into a database. 



Initial statistical analysis involved generation of allele 

 frequencies and (direct-count) heterozygosity values, and 

 significance testing of genotypic proportions in relation 



to those expected under conditions of Hardy-Weinberg 

 equilibrium. Significance testing of Hardy-Weinberg 

 equilibrium proportions involved exact tests performed 

 using Markov-chain randomization (Guo and Thompson, 

 1992); probability (P) values for tests at each microsatel- 

 lite within each sample were estimated by permutation 

 (bootstrapping) with 1000 resamplings (Manly, 1991). 

 Significance levels for simultaneous tests were adjusted 

 with the sequential Bonferroni approach (Rice, 1989). 

 Tests of genotypic equilibrium at pairs of microsatellites 

 were carried out as a surrogate to assess whether any 

 microsatellites were genetically linked. Probability values 

 for (exact) tests of genotypic equilibrium were generated 

 by 1000 resamplings, and significance levels for simulta- 

 neous tests were adjusted with the sequential Bonferroni 

 approach. Allele frequencies and heterozygosity values 

 were obtained by using biosys-1.7 (Swofford and Selander, 

 1981), and tests of Hardy-Weinberg and genotypic equilib- 

 rium employed the package genepop (Raymond and Rous- 

 set, 1995). Exact tests also were used to test independence 



