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Fishery Bulletin 95(4), 1 997 
sected from 100 specimens at three sites and from 
50 specimens at Waitaki (Fig. 1). Tissue samples were 
frozen in liquid nitrogen at sea and stored at -70°C 
in the laboratory. 
Allozyme electrophoresis 
Eight enzyme systems were tested in heart, liver, 
and muscle tissues of orange roughy with cellulose 
acetate and starch gel electrophoresis following the 
methods in Smith (1986), except that BDH (British 
Drug House Chemicals Ltd, Poole, England) starch 
was substituted for Electrostarch (Electrostarch 
Company, USA). 
DNA extraction 
DNA was extracted from liver tissue of 50 orange 
roughy from each site. For each sample, 0.5 g of tis- 
sue was homogenized with 750 pL 4M guanidinium 
isothiocyanate in 8M urea and 2% sodium dodecyl 
sulfate (SDS) (Turner et al., 1989). DNA was ex- 
tracted by mixing with an equal volume of phenol 
chloroform and centrifugation at 13,000 rpm for 5 
min. The phenol-chloroform extraction was repeated 
and the aqueous fraction mixed with an equal vol- 
ume of chloroform-isoamyl alcohol (24:1). Following 
centrifugation at 13,000 rpm, the aqueous fraction 
was mixed with two volumes of ethanol and the DNA 
allowed to precipitate at -20°C overnight. The DNA 
pellet was washed in 70% ethanol, air dried, and re- 
suspended in 40 pL of sterile deionised water. 
mtDNA amplification and 
restriction enzyme digestion 
Three primer pairs were used to amplify the mtDNA. 
Amplification reactions were performed in 50-pL 
volumes in a Perkin Elmer Cetus DNA thermocycler: 
protocols followed those of Palumbi et al. (1991) and 
Cronin et al. (1993). The nucleotide sequences of the 
primers were the following: 
D-loop 5’-ATAGTGGGGTATCTAATCCCA-3' 
5’-RCRCCCAAAGCTRRRRTTCTA-3' 
(Palumbi et al., 1991); 
cytochrome b 5'-CCCTCAGAATGATA- 
TTTGTCCTCA-3' 
5’-TGACCTGAARAACCA- 
YCGTTG-3' 
(Palumbi et al.,1991); and 
ND 5/6 5'-AATAGTTTATCCA- 
GTTGGTCTTAG-3' 
5 ' -TTAC AACGATGGTTTTTC A- 
TAGTCA-3' (Cronin et al., 1993) 
Twelve restriction endonucleases recognizing 4-base 
sites (Bfa I, BstU I, Cfo I, Hae III, Hpa II, Mse I, Msp 
I, Nla III, Rsa I, Sal I, Sau 3A, and Taq I) were used 
to digest the D-loop primer amplification products. 
Eleven restriction endonucleases recognizing 4-base 
sites (Alu I, Bfa I, Cfo I, Hpa II, Msp I, Nar I, Rsa I, 
Sal I, Sau 3A, Taq I, and Tru I) were used to digest 
the cytochrome b primer amplification products. The 
ND 5/6 primers produced between 1 and 3 amplifi- 
cation products in different specimens, therefore no 
restriction digests were undertaken with the PCR 
products. 
For each primer pair and restriction enzyme, 24 
fish were tested, 6 from each area. The restriction 
enzymes that showed polymorphisms were used to 
test 50 fish from each site. The amplified and digested 
DNA products were separated in 1.4% agarose gels 
and detected with ethidium bromide under a UV light 
(312 nm). 
RAPD amplification and separation 
Six individuals from each sample site were ampli- 
fied with 24 RAPD primers. Each sample was am- 
plified separately with a 10-base oligonucleotide 
primer from Operon (OperonTechnologies, Alameda, 
CA). These primers were randomly selected from 
Operon series A, D, E, and H primers, but all have a 
G+C content of 60-70%. Amplification reactions were 
performed in 50-pL volumes in a Perkin Elmer Ce- 
tus DNA thermocycler. Serial dilutions of DNA 
samples were tested initially to determine optimum 
DNA concentration for amplification (Fig. 2). The 
DNA concentration in each sample was estimated 
fluorometrically and appropriate volumes were used 
for amplification. Each reaction contained approxi- 
mately 50 ng DNA in 10 mM Tris HC1 (pH8.3), 30 ng 
single 10-base primer, 50 mM KC1, 2 mM MgCl 2 , 100 
mM each of dATP, dCTP, dGTP, and dTTP, and 1 
unit Taq DNA polymerase in Perkin Elmer PCR 
buffer. The reaction was overlaid with mineral oil 
and amplified. The thermocycler was programmed 
for 40 cycles of 1-min duration at 94°C, 1 min at 36°C, 
and 2 min at 72°C. Amplification products were sepa- 
rated in 1.4% agarose gels and detected with 
ethidium bromide staining under a UV light (312 
nm). A DNA size-ladder was included in each gel. 
Control samples were amplified without a DNA tem- 
plate. Those primers that yielded variable fragment 
patterns were retested in the same fish. Primers pro- 
ducing repeatable fragment patterns in the initial 
six fish from each site were tested in 50 fish from 
each site. Polymorphisms were scored by the pres- 
ence or absence of an amplification product at spe- 
cific positions in the gel. 
