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Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by MEMORIAL UNIV OF NEWFOUNDLAND on 03/29/12 For personal use only. 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 Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by MEMORIAL UNIV OF NEWFOUNDLAND on 03/29/12 For personal use only. (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- Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by MEMORIAL UNIV OF NEWFOUNDLAND on 03/29/12 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 cta aca G gga 45 * gac I atc E* gag T aca A gcc F ttc 30 98 D gat V gta N aac Y tac G ggc W tga L cta 45 135 A gcc S t F ttc F ttt F tte I att C 60 tgt 188 L ctc Y Y tat tat G ggt S tce tat L ctt 75 225 V t V gtc L ctt F ttc L ctt L tta V gta 98 2'90 V gtc c tat T acc S tca I atc C tgt R cgt cat A gct N t ggt H cac I att A gcc R G cga ggt E gag T aca W tga N aac I atc g T S tct F ttc V gta G Y ggt tat L cta G ggc L ctt tgc L cta F ttt L eta A gcc M ata H sac S tca S tcc V gta V* gtc H cac I att R cgg N aat M ata L c Y tat M atg V gta M ata acc ** * H * C Y * * C G * T L ctt T act L ctt G! caa ate S tct M ata H L tta G ggc F 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 Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by MEMORIAL UNIV OF NEWFOUNDLAND on 03/29/12 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 t a . . . s . . C a e g g . m s a " c ' c e c a n * . " . . a g a - c . t . . t a " - - . g - . . 4 2 1 1 3 8 N 9 - 1 - - - - - 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 - Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by MEMORIAL UNIV OF NEWFOUNDLAND on 03/29/12 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. 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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