754 



Fishery Bulletin 100(4) 



In total, nine samples were collected from 1977 through 

 1999: six from each of the major spawning aggregations 

 shown in Figure 1, and three samples that were interan- 

 nual replicates from Prince William Sound (PWS), Shelikof 

 Strait (SHEL), and Bogoslof Island (BOG). Other samples 

 included Unimak Pass (UNI) and Middleton Island (MID) 

 in 1998 and Kronotsky Bay (KRON) in 1999. Tissue sam- 

 ples from heart, liver, muscle, and eye were taken from 100 

 individuals per population with the exception of Unimak 

 Pass (n=40) and Kronotsky Bay (7?=96). Only muscle tissue 

 was sampled from Bogoslof Island walleye pollock in 1997. 

 Samples were stored at -80°C until analyzed. 



Allozyme analysis 



AUozyme alleles were resolved by using horizontal starch- 

 gel electrophoresis and enzyme-specific histochemical 

 staining procedures described by Aebersold et al. (1987). 

 Thirty six loci were screened for polymorphism (Table 2): 

 sAAT-l*,sAAT-2*,mAAT-l*.ADA-l*\ADA-2*.AH-l*.AH- 

 2*,ALAT*, CK-A'\ FH*, GAPDHl*. G3PDH-1*, G3PDH- 

 2*. G3PDH-3', G3PDH-4\ GPIl*. GPL2\ IDHPl*. 

 IDHP-2*. IDHP-3\ LDH-2'\ LDH-3'. MDH-A*. MEP-l*, 

 MEP-2*. MPI'. PEPA\ PEPB*, PEPD*. PEPLT*, PGDN*, 

 PGM-P, PGM-2*, SOD-1*. SOD-2*. and TPI*. Twenty- 

 four loci were polymorphic in at least one of nine popula- 

 tions, and these loci were used in the tests of spatial and 

 temporal genetic variation (Table 3). 



DNA analysis 



Total genomic DNA was isolated from 20-30 mg of heart 

 tissue by using a Centra Systems^'^ (Minneapolis, MN) 

 Puregene DNA isolation kit. Precipitated DNA was 

 hydrated in 50-100 pL tris-ethylenediaminetetraacetic 

 acid (EDTA) buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) 

 and heated at 55°C for approximately 12 h. Approximately 

 1 pL of hydrated DNA was used from each sample for poly- 



merase chain reaction (PCR) amplification of mtDNA and 

 microsatellite fragments. 



mtDNA For an initial screen, six segments of the walleye 

 pollock mitochondrial genome were examined for restric- 

 tion fragment length polymorphism in a sample of 12 wall- 

 eye pollock. The mtDNA segments examined were ND5/6 

 (-2400 base pairs /bp/; Cronin et al., 1993), cytochrome b 

 1-1200 bp and~800 bp; Bickham et al., 1995), cytochrome 

 oxidase I (-700 bp; PowersM, D-loop (-1400 bp; Cronin et 

 al., 1993), and 16S (-600 bp; Palumbi et al., 1991). We used 

 the following restriction enzymes: A/m I.Apa I, Ase l,Ava 



I, Ava II, BamH I, Bel I, Bgl 1, Bgl II. BstE II. BstU I, Dpn 



II, EcoR I, EcoR V. Hae II, Hae III, Hha I, Hinfl, Hind III, 

 Kpn I, Mse I, Msp I, Nci I, Pst I, Rsa I, Sac I, Sac II, Sau96 

 I. Sea I, Stii I. Tag I. Xba I. and Xho I. 



PCRs were performed in 25-100 pL volumes containing 

 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0-3.5 niM MgCl.^, 

 0.8 mM dNTPs, 0.05 units/pL Tag polymerase, 0.3-1.2 pM 

 primer, and about 250 ng DNA template. The PCR profile 

 was 92°C (2 min) -^ 30-40 cycles of (92°C (30 sec) -hX°C (30 

 sec) -I- 72°C (140 sec)) -t- 72°C (5 min). where the anneal- 

 ing temperature X varied among primer pairs. Restriction 

 digests followed manufacturer's specifications (New Eng- 

 land Biolabs. Beverly, Maine). 



The mtDNA fragment and enzyme combinations ND5/ 

 6 (-2400 bp) — Ase I, Ava I, Msp I, Rsa I; cytochrome b 

 (-1200 bp) — Alu I. Hae III, Mse I; and cytochrome oxidase 

 (-700 bp) — Alu I revealed polymorphism and were used 

 to test for spatial and temporal genetic variation. The 

 PCR annealing temperatures for these fragments were 

 the following: (for ND5/6) 50°C; (for cytochrome b) 54°C; 

 and (for cytochrome oxidase) 50°C. Restriction fragments 



' Powers. D. A. 1997. The use of molecular techniques to dis- 

 sect the genetic architecture of pollock populations. Unpubl. 

 rep. to the National Marine Fisheries Service, lip. Hopkins 

 Marine Station. Stanford University, Pacific Grove, CA, 93950. 



