Beacham et al : Population structure of Oncorhynchus gorbuscha in British Columbia and Washington, determined with microsatellites 
243 
ing population structure in pink salmon in British Co- 
lumbia (Beacham et ah, 1988; Shakiee et ah, 1991) 
and elsewhere in the Pacific Rim distribution of the 
species (Aspinwall, 1974; Varnavskaya and Beacham, 
1992; Shakiee and Varnavskaya, 1994; Noil et ah, 2001; 
Hawkins et ah, 2002). Analysis of allozyme variation 
provided the general pattern of marked differentiation 
between the broodlines, but identification of fine-scale 
regional structure was limited, although estimation of 
stock composition of samples from mixed-stock fisheries 
was conducted (Beacham et ah, 1985; Shakiee et ah, 
1991). Development of DNA markers has led to new 
avenues of research for using genetic variation in de- 
fining population structure. Mitochondrial DNA varia- 
tion again showed strong differentiation between the 
broodlines, but differentiation among regional groups 
of pink salmon was limited (Brykov et ah, 1996, 1999; 
Churikov and Gharrett, 2002). However, Golovanov et 
ah (2009) noted that differentiation among even-year 
populations was higher than among odd-year popula- 
tions in the northern Sea of Okhotsk region. 
Microsatellites are reported to be useful for evaluat- 
ing fine-scale population structure in salmonids (Banks 
et ah, 2000) and have been used to evaluate large- 
scale and regional variation in chum salmon (O. keta) 
(Beacham et ah, 2009). Initial applications of micro- 
satellite variation to evaluate individual identification 
and population structure were reported by Olsen et ah 
(1998, 2000a). However, surveys of population variation 
were quite limited in these studies, and no comprehen- 
sive evaluation of variation at microsatellites has been 
conducted for pink salmon. A survey of microsatellite 
variation over a broader geographic range of pink salm- 
on distribution would likely be valuable for evaluating 
population structure. 
In the current study, we outline the microsatellite- 
based population structure of pink salmon in British 
Columbia and Washington as an initial step in evalu- 
ating whether higher resolution in estimation of stock 
composition may be possible when compared with esti- 
mates previously derived with aliozymes. This objec- 
tive was accomplished by analyzing variation at 16 
microsatellite loci to evaluate relationships in popula- 
tion structure of pink salmon, as well as by analyzing 
regional differences in allelic variation. The distribu- 
tion of genetic diversity among broodlines, regions, and 
populations was estimated in the study, as well as the 
stability of population structure. 
Materials and methods 
Collection of DNA samples and laboratory analysis 
Tissue samples were collected from mature pink salmon. 
Samples were preserved in 95% ethanol, and sent to the 
Molecular Genetics Laboratory at the Pacific Biologi- 
cal Station of Fisheries and Oceans Canada. DNA was 
extracted from the tissue samples by using a variety 
of methods, including a chelex resin protocol outlined 
Table 1 
Microsatellite loci surveyed in pink salmon ( Oncorhyn- 
chus gorbuscha) and their associated annealing and 
extension temperatures and times (seconds), as well as 
the number of cycles used in polymerase chain reaction 
amplifications. 
Locus 
Annealing 
Extension 
Cycles 
OkilO 
53°C/30s 
70°C/30s 
39 
OkilOl 
53°C/45s 
68°C/30s 
40 
OnelOl 
50°C/30s 
70°C/30s 
39 
Onel02 
50°C/30s 
70°C/30s 
39 
Onel04 
50°C/30s 
70°C/30s 
36 
Onel09 
55°C/30s 
70°C/30s 
34 
Onelll 
55°C/30s 
70°C/30s 
34 
Onell4 
50°C/30s 
70°C/45s 
38 
Ots213 
52°C/45s 
72°C/60s 
38 
Ots7e 
51°C/30s 
72°C/30s 
35 
OtsG253b 
60°C/45s 
72°C/45s 
35 
OtsG311 
50°C/45s 
68°C/45s 
34 
OtsG68 
50°C/30s 
70°C/30s 
36 
Ssa407 
60°C/30s 
70°C/30s 
39 
Ssa408 
60°C/45s 
70°C/45s 
40 
Ssa419 
50°C/30s 
70°C/30s 
40 
by Small et al. (1998), a Qiagen 96-well DNeasy® 1 pro- 
cedure (Qiagen, Mississauga, Ontario), or a Promega 
Wizard SV96 Genomic DNA Purification system (Pro- 
mega, Madison, WI). Once extracted DNA was avail- 
able, surveys of variation at 16 microsatellite loci were 
conducted: OkilO (Smith et a!., 1998), OkilOl (Beacham 
et ah, 2011), OtsG68, OtsG253b, OtsG311 (Williamson 
et ah, 2002), Ots213 (Greig et ah, 2003), Ots7e (Wright 
et ah, 2008), OnelOl, Onel02, Onel04, Onel09 , Onelll , 
Onell4 (Olsen et ah, 2000b), Ssa407 , Ssa408, Ssa419 
(Cairney et ah, 2000) 
In general, polymerase chain reaction (PCR) DNA 
amplifications were conducted by using a DNA En- 
gine Cycler Tetrad2 (BioRad, Hercules, CA) in 6-pL 
volumes consisting of 0.15 units of Taq polymerase, 
1-pL of extracted DNA, lxPCR buffer (Qiagen), 60 pM 
each nucleotide, 0.40 pM of each primer, and deionized 
water. The thermal cycling profile involved one cycle 
of Taq activation for 15 minutes at 95°C, followed by a 
denaturation cycle of 30 seconds at 94°C, with anneal- 
ing and extension conditions for each locus as outlined 
in Table 1. PCR fragments were initially size fraction- 
ated in denaturing polyacrylamide gels with an ABI 377 
automated DNA sequencer, and genotypes were scored 
by Genotyper, vers. 2.5 software (Applied Biosystems, 
Foster City, CA) by using an internal lane sizing stan- 
dard. Later in the study, microsatellites were size frac- 
tionated in an ABI 3730 capillary DNA sequencer, and 
1 Mention of trade names or commercial companies is for 
identification purposes only and does not imply endorsement 
by the National Marine Fisheries Service, NOAA. 
