Beacham et al : Microsatellite DNA variation and estimation of stock composition of Oncorhynchus neika 



15 



A preliminary survey of DNA variation 

 at microsatellite loci indicated that there 

 was some differentiation among the Bark- 

 ley Sound sockeye salmon stocks (Nelson 

 et al., 1998). Evaluation of alternative 

 methods of stock identification indicated 

 that mixture analysis based on micro- 

 satellite allele frequencies would likely 

 provide reliable estimates of stock compo- 

 sition (Beacham et al., 1998). In the pres- 

 ent study, we expanded the analysis of 

 variation at microsatellite loci of Barkley 

 Sound sockeye salmon to six polymorphic 

 loci, examined the differentiation among 

 and within stocks at each locus, evalu- 

 ated the precision of data and accuracy of 

 stock composition estimates for a range 

 of mixture sample sizes based on data 

 from three to six loci, and finally used the 

 microsatellite variation to estimate stock 

 compositions from 1997 fishery samples. 



Materials and methods 



Collection of DNA samples and 

 amplification by PCR 



Scales were collected ft'om sockeye salmon 

 returning to spawn in the Sproat Lake and 

 Great Central Lake drainages in 1987, 

 1990, and 1992. Scales were collected from 

 Henderson Lake sockeye salmon in 1988 

 and 1993, and liver samples preserved in 

 95% ethanol were collected in 1995. Scales 

 or operculum punches were collected from 

 sockeye salmon sampled in fisheries in 

 1997. DNA was extracted fi-om scales as 

 outlined by Nelson et al. ( 1998). For the operculum or 

 liver samples, approximately 0.3 g of tissue was placed 

 in each well of a 96-well plate containing 0.2 mL of 5% 

 chelex in TE buffer ( 10 mM Tris pH 7.4, 1 mM EDTA 

 pH 8.0, 0.10 mg/mL proteinase K, and 0.1% SDS) and 

 incubated for 15 min at 50°C, and then incubated for 

 an additional 15 min at 95°C. The supernatant from 

 each well was collected and placed in a fresh 96-well 

 plate and stored at -20°C. About 1 mL of this extract 

 was required for each amplification of the sample by 

 the polymerase chain reaction (PCR). 



Loci amplified by PCR were the dinucleotide repeats 

 Omy77 and Ots3 and the tetranucleotide repeats 

 OtslOO, Otsl03, Otsl07, and Otsl08 (Table 1). For all 

 primer sets used in this study, PCR was conducted 

 in 25-juL reactions containing 12 pmol (0.48 ^M) of 

 each primer, 80 ^M of each nucleotide, 20 mM Tris-pH 



^4a40 — 



Figure 1 



Location of Barkley Sound on Vancouver Island. Sockeye salmon are produced in 

 Great Central Lake and Sproat Lake, both part of the Somass River drainage, as 

 well in Henderson Lake. 



8.8, 2 mM MgS04, 10 mM KCl, 0.1% Triton X-100, 

 10 mM (NH4)S04, and 0.1 mg/mL of nuclease-free 

 bovine serum albumin. Each PCR reaction was pre- 

 ceded by an initial denaturation step of three min at 

 94°C. All cycle extension (30 cycles for all loci except 

 OtslOS which was 35 cycles) steps were for 60 sec 

 at 72°C and all cycle denaturation steps were for 20 

 sec at 94 C. PCR of Omy77, Ots3, OtslOO, Otsl03, 

 Otsl07, and OtslOS was accomphshed with anneal- 

 ing temperatures of 48°C, 50°C, 57°C, 55°C, 48°C, and 

 46°C, respectively. Annealing times were 30 sec for 

 Omy77 and OtslOO, and 60 sec for the other loci. 



Gel electrophoresis and band analysis 



PCR products were size fractionated on 16 cm x 17 cm 

 nondenaturing polyacrylamide gels and visualized by 



