Detection of Intraspecific.DNA Sequence Variation in ...

Detection of ntraspecific DNA Sequence Variation in the

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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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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