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Detection of ntraspecific DNA Sequence Variation in the
Mitochondria Cytochrome b Gene of At antic Cod
ymerase Chain Reaction'
Steven M. Carr and W. Dawn Marshall
Genetics, Evolution, and lWolecular Systematics Laboratory, Department of Biology, Memorial University of Newfoundland, St. john's, Nfld. A IBr3X9,
Canada
Carr, S . M., and H. D.Marshall. 1991. Detection of intsaspecific DNA sequence variation i n the rnitochondsial
cytochrome b gene of Atlantic cod (Cadus rnorhua) by the polymerase chain reaction. Can. ). Fish.
Aquat. Sci. 48: 48-52.
We determined the DNA sequence of a portion sf the rnitochondrial cytochrorne b gene for 55 Atlantic cod
(Cadus morhua) from Norway and from 10 locations within the Northern Cod complex and adjacent stocks off
Newfoundland. DNA was prepared for sequencing by the polymerase chain reaction (PCR). Eleven variable
nucleotide positions within a 298 base region defined 1 2 genotypes. Genotype proportions differed significantly
between Newfoundland and Norwegian populations: the majority genotype among NewfoundBand populations
was present in a minority of Norwegian cod. Newfoundland cod showed less genotypic diversity than those from
the eastern Atlantic: nine genotypes were found among all 10 Newfoundland popuiations, as compared with
seven genotypes within the single Norwegian population. An exception was an overwintering, inshore Newfoundland population that showed four genotypes among five fish. As in other vertebrates, third position synonymous transitions predominate over other types of nucleotide changes. However, two amino acid replacement
substitutions occur among cod, and the ratio of purine transitions to pyrimidine transitions is significantly higher
than in other species. The existence of DNA sequence polymorphism permits the various hypotheses of the
distribution and differentiation of Newfoundland cod stocks to be tested, and points to the utility of PCR technology in fishery genetics.
Nous avons determine la sequence de I'ADN d'une portion du gene rnitocheandrial codant Be cytochrorne h ckez
55 morues franches (Gadus morhua) de Norvege et de 1(B endroits se treauvant dans le secteur du cornplexe de
la morue du nor$ et de stocks adjacents dks large des cdtes de Terre-Neuve. Pour Bes fins de I'analyse sequentielle,
I'ADN a et6 prepare au moyen de la reaction en chaCne 2 la pslyrnkrase. Onze positions de nucl6stides diffkrentes
ont 6t6 reperees dans un segment de 298 bases, soit 12 genotypes. Les proportions des genotypes variaient de
fason significative entre les populations de Terre-Neuve et de NorvGge: le genotype le plus frequent dans les
populations de Terre-Neuve etait minoritaire parmi les populations norvbgiennes. La diversite genotypique des
rnorues de Terre-Neuve etait inferieure a celle des rnorues de I'est de I'htlantique: les 18 populations de TerreNeuve cornptaient neuf genotypes tawdis que I'unique population de Norvege en comptait sept. Une population
c8tih-e hivernante de Terre-Neuve faisant cependant exception avec quatre genotypes observes sur cinq psissons.
Comme chez les autres vert6bres, les transitions synonymes en troisi6me position etaient les modifications nuclestidiques les plus frkquentes. Cependant, des substitutions de deux acides arnines existent chez la rnorue, et la
proportion des transitions des nuclkotides pyrimidiques aux transitions des nucleotides puriques est significativement superieure 2 celle observee chez d'autres es@ces. k'existence d'un pslyrnorpkisrne dans les skquences
d'ADN de la rnorue permet de mettre 2 I'kpreuve Bes diffbrentes hypotheses sur la distribution et la differenciation
des stocks de morue de TerreNeuve eta de quoi susciter un int6ret pour I'utilisation de la technique de la reaction
en chaine 3 la polym6rase dans le dsrnaine de la genetique appliquee aux p@ehes.
Received December 7 8, 1989
Accepted luly 23, 1990
(JA409)
A
tlantic cod (Gadus msrhua) in the western North AtHmtic exist as several biologically distinct stocks
(Templeman 1962). God in Northwest Atlantic Fisheries
Organization (NAFO) divisions 2J3KL northeast of insular
Newfoundland, referred to as the Northern Cod complex, have
been managed as a single stock since 1973. The degree to which
cod from the various banks, inshore, and offshore ssubcornpswents within 2J3KE represent separate stocks is the subject of
'The nucBesdide sequence data reported in this paper have been submitted to GenBank and assigned the accession numbers M5765257662.
48
an ongoing debate with important fishery management implications (Lex 1984; Keats et aB. 1986; Lear et al. 1986; Harris
1990).
Extensive protein electrophoretic data on genetic variation
applicable to problems of stock discrimination in cod are available (Samieson 19'95;Cross and Payne 19788;Mork et a!. 1985;
Grant et al. 1987; Grant and Stihl 1988a, 1988b): such data
show little variation attributable to stock differences in the
western Atlantic. h alternate genetic system, rraltschondial
DNA (rntDNA), has in the Bast 10 yr found wide use in the
study of local populations of many species (Wilson et al. 19851,
including the definition of stocks in other fish species
Can. J . Fish. Aqesab. Sci., Vol. 48, I991
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(Gyllensten and Wilson 1987; Bickham et al. 1989). Conventionai analysis of mtDNA has typically relied on physical purification of the molecule from individuals, cleavage with a series
of Type II restriction endonucleases, and inspection of the
resulting DNA fragment patterns for characteristic restriction
fragment length polymorphisms (RFLPs). Smith et al. (1989)
used this approach and reported no variation among 14 cod
from the Grand Banks sf Newfoundland, and RFEP variants in
two fish from the North Sea, based on 21 mtDNA restriction
fragments representing less than 0.8% of the genome. Johansen
et al. (1 998) reported a single W E P among 2 B Norwegian cod
examined with eight restriction endonucleases.
The alternative to R E P analysis, direct sequence determination, has until quite recently required molecular cloning:
Johansen et al. (1998) recently determined the sequence of two
thirds of the cod mitochondria1 genome by this approach, and
Beckenbach et al. (1990) compared a 2214 base pair portion of
the same genome among six rainbow trout (Oneorhynchus
mykiss). Cloning is, however, a technically demanding, laborious process that has precluded analysis of the large numbers
of individuals required in population studies. This limitation
has now been overcome by the advent of a new biotechnolsgy,
gene amplification by means of the polymerase chain reaction
( K R ) (Saiki et al. 1988). Specific gene segments can be enzymatically amplified from a crude cell extract in sufficient qumtities for direct sequencing. By appropriate choice sf gene
segments, it is possible to study DNA sequence variation among
individuals, local populations, or species (Kocher et al. 1989;
Vigilant et al. 1989).
In our ongoing study of cod, we wish to find genetic markers
that can identify discrete components of the Newfoundland cod
fishev, with a view towards improved management of this
resource. Comparison of Newfoundland cod with those from
the eastern Atlantic allows us to gauge the extent of intraspecific genetic differentiation*We present here results of our
preliminary study of DNA sequence polymorphism in the mitochondrid cytochrome b region. This paper represents the first
application of K W technology to a fishery management
question.
Materials and Methods
Cod from the western Atlantic were collected by personnel
of the Department of Fisheries md Oceans, the Marine Sciences Research Laboratory, Memorial University, and local
fishermen. Norwegian cod were supplied by R. Barrett, University of Tromso. Localities, date of collection, and sample
sizes are listed in Table 1 .
Mitochondrid DNA was isolated or purified by either of two
methods. First, cleared SDS detergent lysates of mitochondria%
fractions from fresh or frozen ( - 20 or - 70°C) cod hearts were
subjected to ultracentrifugation in cesium chloridelpropidiurn
iodide gradients to obtain highly purified mtDNA (Can and
Griffith 1987). mtDNA was also amplified directly from the
cleared lysates (steps 1-9 in the above procedure) without ultracentrifugation, and with NaCl substituted for CsCl in the final
sdt precipitation step.
We used as amplification and sequencing primers the following oligonuc8eotides, which correspond to highly conserved
cytochrome b sequences identified by Kocher et al. (1489):
5'-ccatccaacatctcagcatgatgaaa-a' (heavy-strand primer)
5'-gcccctcagaatgatatttgtcctca-3 '
(light-strand primer).
Can. 3. Fish. Aquat. Sci., Vol. 48, I991
TABLE1. Origins of cod used in this study.
Location
Tromsg, Noaway
Grey Island Shelf
Fogo HslmcV
Conception Bay
Flatrock"
Gull Is1md
PBaeentia Bay
Forthern Gmnd Banks
Ile aux Morts"
St. Pierre Bank
47"08'N, 55OCB7'W
47"QO'N,%0°16'W
N
NAFB
Division
Date of
collection
July 1989
June 1988
December 1989
July 1988
June 1989
July 1988
July 1988
June 1988
January 1989
January 1989
January 1989
January 1989
"Inshore fishery.
These primers amplify a 359 base pair region, which represents
about 2% of the 16.5 kilobase cud mtDNA genome (Johansen
et a%.1990). The primers were prepared on a Milligen oligonucleotide synthesizer in the DNA analysis facility at Memorial
University.
Double-stranded (symmetric) PCR amplifications were carried out in 25-pL reactions containing 67 m8uZ Tris (pH 8.8
2-mercaptoethanol, 2 ITBR% MgC12(all Sigma),
200 p,M each of dATB, dCTP, dGTP, and dTTP (Phmacia
or Bmhiinger-Mmnheipn), 4-00 dd each of the heavy- and
light-strand primers (18 pmol each per reaction), and 1 unit sf
Amplitaq polymerase (Perkin-Elmer Cetus). To this mixture
was added 1 p& of the DNA preparation to be amplified, either
purified mtDNA or the cleared lysate. The DNA was amplified
in a Perkin-Elmer Cetus Thermal Cycler on the following stepcycle profile: strand denaturation at 92°C for 45 s, primer
annealing at 50°C for 45 s, and primer extension at 72'C for
90 s, repeated for 30 cycles. R e l i m i n q denaturation at 95'C
for 5 min before the first cycle improved product yield in some
cases.
Eleetrophoresis of a 18-pL portion of the amplification product was done for 1 h at 100 V in a 2% NuSieve gel (FMC) in
This-acetate buffer (pH 7.4) containing e thidiurn bromide
(1 p.glrnL). DNA fragments were examined with 302-nm UV
illumination. A small portion of each 359 base pair product was
removed, added to 108 pL of H,Q, and remelted at 65'C for
10 min. Single-stranded (asymmetric) amplification was carried out on 1-2 yL of the remelted materid under the same
conditions as above, except that one primer was diluted 1:BW
(find concentration 4 nM, 0.4 pmol added per reaction) md
the total reaction volume was increased to 100 pL. (We typically diluted the light-strand primer, so as to obtain the light
strand as the single-strand product; asymmetric amplification
of the heavy strand was not routinely successful.)
The single-stranded DNA was desalted on a Centricon-30
ultrafiltration unit (Amicon) or an Ultrafree UFC-3 cartridge
(Millipore). Single-stranded DNA sequencing reactions were
prepared with Sequenase kits (version 2.0: U.S . Biochemical)
A T P England Nuclear) on 7 yL of the filter
and C X - ~ ~ S - ~ (New
retentate, according to the manufacturer's directions; the label
mix was diluted 158, which permits the sequence to be read
immediately after the primer, Sequences were separated in
48-cm 6% polyacrgrlamide (19: 1 BIS), 7 ha urea gels. Electro-
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For personal use only.
phoresis was done at 30 W constant power (approximately
1600 V) for either 1-1.5 or 4-5 h to obtain the 5' a d 3' ends
of the sequence, respectively. The gels were fixed in 5% methagnov%%acetic acid, dried onto filter paper, and autoradiographed with Ksdak AR or RP film,
Sequences were analyzed and prepared for publication with
the help of the ESEE program (E. Cabot, Depatment sf Biological Sciences, Simon Fraser University, Bumaby , B .C.
V5A 1S6). All sequences are given as their coding strand
equivalents. Sequence variants were confirmed by resequencing of reamplified pmducts from the extracted DNA.
Results and Discussion
Figure 1 shows the sequence of a 298 base popeion of the
coding strand sf the most common cod cytochrome b sequence,
dong with the inferred amino acid sequence. Eleven nucleotide
positions in this region vary among cod: the variable sites define
12 distinct genotypes (Table 2).
The distribution of these genotypes differs between the western and eastern Atlantic. Genotype proportions in the two areas
are statistically differentiable: the frequency of the most common genotype (A in Table 2) is significantly smaller in the Norwegian population (27%) than in Newfoundland cod (88%)
(p << 0.01, Fisher's exact test). Genotypic diversity is also
lower in the western Atlantic. Among the 15 fish from the single Norwegian population, seven genotypes were found, four
of these in at least two individuals each. In contrast, examination of the 40 fish from 10 Newfoundland populations
revealed a totd of nine genotypes; except for the common type,
none was found in more than a single individual. Genotypic
diversity can be quantified by the nucleon diversity index h of
Nei m d Tajima (1981): h = (I - Z(aZ))(n)/(n- I), where x
is the proportion of each genotype and n is the total number of
individuals. By this measure, the single Norwegian population
is substantially more variable ( h = 0.88) than d l Newfoundlmd populations combined (h = 0.36). An exception to this
L
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pattern is the Fog0 Island sample, an overwintering, inshore
component of the 2J3Kk complex, which, with four genotypes
among five fish ( h = O.90), was the most variable Newfound4md population.
The pattern of nucleotide substitution in the mitochondrial
eytochrome b gene in cod differs from that of other vertebrate
species studied. Nine of the B 1 observed substitutions are synonymous sub~titutimsat third positions, eight of which are
transitions. This is similar to the pattern among closely related
individuals of other vertebrate species, where transitions greatly
outnumber trmsversions (Kocher et al. 1989). The same is true
in a different portion of the mitochondria%genome of rainbow
trout (Beckenback et al. 1990). In cod, silent purine and pyrimidine transitions are equally frequent (4 versus 4). In rainbow
trout, Beckenbach et iBB. (19%) found an excess of purine trmsitions (8 versus 4). In deer of the genus B$ocsi%eus,however,
22 sf 24 synonymous substitutions m o n g individuals are
pyrimidine transitions (S. M. C m and 6. A. Hughes, submitted), Both mammal/fish comparisons are significantly different
BB 0.05, Fisher's exact test). The remaining two substitutions in cod result in amino acid replacements: an a + g second
position transition at nucleotide 80 replaces glutamic acid with
glycine in genotype F, and a g + a first position transition at
nucleotide 100 replaces valine with isoleucine in genotype L
(Fig. 1). Neither replacement is considered radical (Grantham
1974). Replacement substitutions in this gene are unusual in
closely related individuals of other vertebrate species (Kocher
et al. 1989): in deer, no replacement substitutions were observed
either within or between species (S. M. C m and G. A. Hughes,
submitted). (The difference is, however, not significant.)
Beckenbach d al. (1990) found one replacement substitution
among six trout. These differences may indicate varying gatterns of mutation, fixation, and/or selection between bony fish
and mammals.
Documemtation of genetic variation and rnodificati~nof management practices so as to preserve or restore genetic diversity
are recognized goals of successful fishery management (Nelson
D
*
**
Y
15
FIG. 1. Sequence sf a 298 base region of the mitochondrial eytochme b gene from cod (Gadus mopha). The sequence of genotype A in
Table 2 is given: variable nucimtides and m i n o acids are marked with ban asterisk (see text). The inferred mino acid sequence is indicated.
50
Can. J . Fish. Aquat. Sci.,
48, 4991
TABLE2. Distribution sf DNA sequence variation among populations of Gadus morhraa. Sequences
are identical to genotype A unless otherwise indicated. All 3K variants are found in the Pogo Island
sample, except genotype H which occurs in the Grey Island Shelf sample.
Distribution
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For personal use only.
NucIeotide position in Fig. 1
Genotype 66 72 80 81 100 I02 105 120 201 243 249 Nor 3K 3% 3 0 3Pn 3Ps
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factual sequence variation if replication errors occur in early
and Sou16 1987). The existence of identifiable mtDNA
rounds of mplification. Otherwise, products with induced
sequence polymorphism in Newfoundland cod makes the various hypotheses of stock differentiation and discrete inshore
mutations contribute relatively small proportions to the total
breeding populations testable. Even with the relatively smdl
product, so that direst sequencing yields a consensus pattern
samples sizes used here, it has been possible to differentiate
corresponding to the genomic sequence (Saiki et al. 1988).
transatlantic stocks. NAFO divisions 3h,3Ps, and 3K, which
Amplification and sequencing of both complementq strands
contain banks that are believed to correspond to separate
can corroborate the fidelity sf reciprocal asymmetric arnplifispawning areas (Templeman 2962; May 6966; Lea- I984), all
cations, but cannot detect e m r s introduced in the initial symcontain variant genotypes at low frequency; one inshore popmetric amplification, The authenticity of sequence variants can
ulation within 3K contains several such genotypes. The putaonly be verified by reamplifying and resequencing the same
tive genetic and/or ecological distinctness of such stocks,
strand from the original DNA source, as was done here. TherparticularppIy those sf the inshore, is the subject of ongoing manmal effects cam also be minimized by reducing the time of the
agement interest (Pinhorn 1984; Keats et al. 1986; Eear et al.
denaturation step, and reducing the number of thema1 cycles.
1986; Harris 1998).More extensive variation is expected within
The K R procedure offers several practical advantages over
more rapidly evolving portions of the rntDNA genome such as
conventional restriction endsnuclease methods for the analysis
the D-loop control region (Brown B983), which may be the
of intraspecific variation. The quantity of tissue and the quality
region of choice for studies of intraspecific polymorphism
of its preservation are much less problematic. Less than 100 rng
(Vigilant et al. 11989). It remains to be seen whether these or
of tissue was routinely used. Equally good double- and singleother genetic makers can, with larger sample sizes, be used to
stranded DNA amplifications and sequencing results were
differentiate spawning populations with statistical confidence
obtained horn purified or crude mtIlNA preparations, from
(S . M. C m md H. D. Marshall, work in progress).
material poorly or not quickly frozen, from tissue held at
The low genotypic diversity of Northern Cod populations is
- 20°C for up to 2 yr, and from tissue that had been repeatedly
reminiscent of the pattern seen with allozymes (Cross and Bayne
frozen and thawed. These considerations are especially impor1998; Mork et al. 198%).Whether this pattern is general among
tant when material is collected in the field. The results precod stocks in the western Atlantic, and if so its causes, are
sented here also indicate that the method is more sensitive to
questions for further investigation. The long-term consesubtle
variation within populations. This study analyzed about
quences of absence of genetic diversity in fishery stocks are
2%
of
the
cod miteschondrial genome, more than twice as much
well known (Nelson and Soul6 1987). Reduced effective popas
previous
RFLP analyses of the s m e genome (Smith et al.
ulation size and consequent loss of heterogeneity in cod could
1996), and has revealed a degree of intra1989;
Johanzsen
et
result from historical factors such as Pleistocene glaciations
specific
polymorphism
that the previous studies, on populations
(Cross and Payne 1978), bottlenecks at the &meof population
of similar origin, did not* Technically, our experience is that
origin (Grant and Stihl IBS8a, 6988b), homogenizing effects
of gene Wow (Smith et id. 1989), and/or fishing patterns (ha- direct sequence analysis of DNA amplified by K R is simpler
and more rapid than csnventional restriction analysis of p ~
et al. 1986), among other possibilities.
fied mtBNA. The limiting factor in this genetic assay is the
Sequence variation observed in PCW studies of natural populations may be authentic polymorphism, or may be an ar%ifact need for collection, alignment, and detailed comparison of individual sequences by hand and eye. Automation of sequence
introduced by errors in the PCR process itself. The Taq polyacquisition and data handling (Landegren et al. 1988; Wilson
merase used in PCR amplification is known to have a singleet al. 1990) may be especially useful where homologous regions
base substitution error rate of about 1 in 9000, due in part to
of a few hundred base pairs are to be compared repeatedly
the absence of 3 -+ 5' exonuclease proofreading activity; heat
amongst many individuals. Such technology, in conjunction
damage to the template DNA and degradation of the Taq
with K R amplification of DNA as performed in this study,
enzyme may also occur during repeated cycling to high temperature (Tindall and Kunkel 2988). These may result in m i may be well suited for the genetic analysis of fish stocks.
Caa. J . Fish. ihqasat. Sci., Vol. 48, 1998
-
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For personal use only.
This research is supported by the Canadian Centre for Fisheries
Innovation, Department sf Fisheries anad Oceans Science Subvention
awards asad Natural Sciences and Engineering Research Council of
Canada (NSERC) Operating md President's grants to S.M.C., rn
NSERG Equipment grant to 1. It. Ball, S.M.C., D. J. I m e s , and P,
G . Hempstead, an NSERC Equipment grant to W. S. Bavidson md
S.M.C., md an NSERG summer student fellowship to H . B . M . We
t h d W. S. Davidson, G. A. Hughes, D. 9. Innes, A. Meyer, P.
Pepin, G. Rose, A. C Wilson, and two monymous reviewers for discussion and critical comment. We thank personnel of the Department
of Fisheries and Oceans, St. John's, Nfld., R. B m e t t , D. Clark, R.
Pagme, J. Wroblewski, and m n y Newfoundland fishermen far eosperation in sample collection. The courtesy of members sf the A. C.
Wilson laboratory for assistance with early experiments and T. D.
Kocher for providing primer sequences in advance af publication is
also gratefully acknowledged.
There are to OW knowledge no reported cases sf artifactual
sequence plymophism in natural populations produced by the
K W process; K R has, however, identified such artifacts in
sequences obtained by cloning (PHiibo, S ., and A. C . Wilson.
1988. Polymerase chain reaction reveals cloning artifacts.
Nature (Lond.) 334: 3 87-3 88).
References
BECKENBACH,
A. T.,W. H(. S OM AS, AND H. SQHRABI.
1998. In&aspcific
sequence variation in the mitocho~rialgenome of rainbow trout (Oncorhynchus mykiss). Genome 33: 13-15.
BICKHAM,
B. W . , S. M. CARW,B. G . HANKS,D. BURTON,AND B. L.
GALLOWAY.
1989. Genetic analysis of population variation in the arctic
cisco using electrophoretic, Wow cytometric, sand mitochondrid DNA
restriction analysis. Biol. Pap. Univ. Alaska 24: 112-122.
BROW, W. M. 1983. Evolution of animal rnitcsehondrid DNA, p. 62-88.h
M. Nei and R. Koehn [ed.] Evolution of genes and proteins. Sinauer,
Sunderlad, MA.
CARR,S. M., AND 0.M. G R I ~ T H1987.
.
Rapid isolalion of animal mitochondrial DNA in a small fixed-angle rotor at ultrahigh speed. Biochem.
Genet. 25: 385-398.
CROSS,T. P., AND R. H. PAYNE.1978. Geographic variation in Atlantic cod,
Gadus morhua, off eastern North America: a biochemical systematics
approach. J. Fish. Res. Board Can. 35: 117-123.
GRANT,W. S., AND G . STAHL.1988a. Evolution of Atlantic and Pacific cod:
loss of genetic variation and gene expression in Pxific cod. Evolution 42:
138-146.
1988b. Description of electrophoretic loci in Atlantic cod, Gadus
morhua, md c o m p ~ s o nwith Pacific cod, Gadus ma~rocephalus.Hereditas 108: 27-36.
GRANT,
W. S . , C. I. ZHANG,
AND T.KQBAYAHSI.
1987. Lack of genetic stock
discretion in Pacific cod (Gadus rnocrocephlus). Caw. J. Fish. Aquat.
Sci, 44: 4 9 W 9 8 .
GK~WAM
R., 1974. Amiw acid difference fornula to help explain protein
evolution. Science (Wash., BC) 185: 8 6 2 8 6 4 .
GYELENS~
U.,
N .AND A. C. WILSON.1987. Mitochsndrid DNA of salmonids,
p. 381-317.1~2N. Rymm and F. Utter led.] Population genetics and fishery management. University of Washington hess, Seattle, WA.
HARRIS,
L. 1930. Hndependent review of the state of the northern cod stock.
Final report. Prepared for the Honourable Thomas Siddon, February 1990.
Csrnmutaications Directorate, Department of Fisheries and Oceans,
Ottawa, Ont.
JMESON, A. 1975. Enzyme types sf Atlantic cod stocks on the North American banks, p. 491-515. In C. L. Markert [ed.] Isozymes. IV. Genetics
and Evolution. Academic Press, New York7NNY.
JOHANSEN,
S., P. H.GUDDAL,
AND T. JOHANSEN.
1990. Organization of the
mitochondria1 genome of Atlantic cod, Gabus morhua. Nucleic Acids
Res. 18: 411419.
KEATS,D.,D. H. STEELE,
AND J. M. GREEN.1986. A review of the recent
status of the northern cod stock (NAP0 Divisions 2J, 3K, and 3E) and
the declining inshore fishery. A report to the Newfoundland Inshore Pisheries Association on scientific problems in the worthern cod controversy.
KWMW, T. D., W. K. THOMAS,
A. MBYER,
S. V. EDWARDS,
S. PAABO,IF. X.
VILLABLANCA,
AND A. C. WILSON.
1989. Dynamics of mitochondria1DNA
evolution in animals: amplification and sequencing with conserved
primers. Roc. NatI. A c d . Sci. USA 86: 619M2W.
LANBEGREN,
U.,R. KAISER:
C. T. CASKEY,
AND E. HOOD.1988. DNA diagnostics - molecular techniques and automation. Science (Wash., DC) 24%:
229-237.
LEAR,W. H. 1984. Discrimination of the stock complex of Atlantic cod (Gadus
morhua) off southern Labrador md eastern Newfoundland, as inferred
from tagging studies. J. Northwest Atl. Fish. Sci. 5: 143-159.
LMR,W. H . , J. W. BAIRD,J. C.RICE.J. E. CARSCADDEN,
G. R. LILLY,AND
S. A. A ~ N H E A1986.
D . An examination of factors affecting catch in the
inshore fishery of Labrador and eastern Newfound%and.Can. Tech. Rep.
Fish. Aquat. Sci. 1449: iv + 71 p.
MAY,A. W. 1946. Biology and fishery of Atlantic cod (Gadas morhua L.)
from Labrador. Ph.B. thesis, McGill University, Montreal, Que.
MORK,J., N. RYMAN,
G.STAHL,F. M. UTTER,AND G. SUDNES.
1985. Genetic
variation in Atlantic cod (Gadas morhuce) throughout its range. Can. J.
Fish. Aquat. Sci. 42: 1586B-1587.
NEB,M., AND F. TBJIMA.1981. DNA polymorphism detectable by restriction
endsnucleases. Genetics 97: 145-163.
NELSON,
K., AND M. SOULB.Genetic consewation of exploited fishes, 345368. In N. Ryman and F. Utter [ed.] Population genetics md fishery mmagement. University sf Washington Press, Seattle, WA.
PINHORN,
A. T. 9984. Inshore exploitation of Atlantic cod, Gadus mrhua, in
Labrador and eastern Newfoundland waters. J. Northwest Atl. Fish. Sci.
5: 79-84.
SAIKI,R. K., D.H.GELWAN,
S. STOFFEL,S. J. SCHARF,
R. HIGUCHI,
G. T.
HORN,K. B. MULLIS,AND H.A. EARLBCH.
1988. Primer-directed enzymatic amplification of DNA with a themostable DNA polymerase. Science (Wash., DC) 239: 487491.
SMITH,P. J., A. J. BIRLEY,A. BAMIESON,
AND C. A. BISHOP.1989. Mitochondrid DNA in the Atlantic cod, Gadus morhw: lack of genetic divergence between eastern and western populations. J. Fish. Biol. 34: 369373.
TEMPLEMAN,
W. 1962. Division of cod stocks in the Northwest Atlrantic. ICNAF
Redkook 1962(HIH): 79-1 29.
TBNDALL,
K. R . , AND T.A. KUNKEL.
1988. Fidelity of DNA synthesis by the
Thermw aquaticus DNA polymerase. Biochemistry 27: 6WM113.
VIGILANT,
L., R. ~ N N I N G T O N , W. MARPENDING,
'k. D. KOCHER,
AND A. C.
WILSON.1989. Mitochowdrial DNA sequences in single hairs from a
southern African population. Roc. Natl. Acad. Sci. USA 84: 9350-9354.
WILSON,A. C., R. L. CANN,S. M.C m , M.J. GEORGE
JR., U. B. GYLLENSEN, K. HELM-BYCHOWSKI,
R. G . H~csvc~r,S. R. PALUMBI,
E. M.
PRAGER,
R. D.SAGE,AND M.SFONERING.
1985. Mitochondria1 DNA and
two perspectives on evolutionary genetics. Biol. J. Linm. S x . 26: 375404).
W r w o ~ R.
, K., C. CHEN,AND L. HOOD. 1998. Optimization of asymmetric
polymerase c h i n reaction for rapid fluorescent DNA sequencing. BioTechniques 8: 184-1 89.
Can. J. Fish. Aqleaf. Sci., V01. 48, 1991