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Restriction site polymorphism in mitochondrial DNA of rainbow trout, Salmo gairdneri Richardson, stocks in Finland
Aquuculture, 67 (1987) 283-289
Elsevier Science Publishers B.V.. Amsterdam -
263
Printed in The Netherlands
Restriction Site Polymorphism in Mitochondrial DNA of Rainbow Trout, Salmo gairdneri Richardson, Stocks in Finland TUULA K. PALVA’.3.4and E. TAP10 PALVA2,3 ‘Department of Applied Zoology, University of Kuopio, P.O. Box 138, SF-70101 Kuopio (Finland) ‘Molecular Genetics Laboratory Department of Genetics, University of Helsinki, SF-00100 Helsinki (Finland)
3Present address: Department of Molecular Genetics, Swedish University of Agricultural Sciences, Box 7003, S-750 07 Uppsala (Sweden) ‘To whom correspondence should be addressed. (Accepted 25 dune 1987)
ABSTRACT Palva, T.K. and Palva, E.T., 1987. Restriction site polymorphism in mitochondrial DNA of rainbow trout, Salmo gairdneri Richardson, stocks in Finland. Aquaculture, 67: 283-289. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) from four breeding stocks of rainbow trout, Salmo gairdneri Richardson, revealed extensive polymorphism. Two different restriction morphs were observed by Hind111 digestion and three different morphs by BgfII. All of the variant mtDNA types (A-D) could be explained by restriction site gains or losses.
INTRODUCTION
Effective fish farming requires knowledge of the breeding populations and identification of the breeding stocks. The determination of intraspecific genetic heterogeneity by phenotypic parameters is not necessarily adequate ( Avise et al., 1979a,b). To obtain maximal information nucleotide sequence heterogeneity should be determined. A good estimation of the sequence differences can be obtained by restriction endonuclease analysis (Avise et al., 1979a.b). The maternally inherited mitochondrial DNA (mtDNA) appears to be especially suitable for this type of analysis (Wilson et al., 1985a). It appears to evolve approximately one order of magnitude faster than the nuclear genome (Brown et al., 1979). Consequently, high levels of inter- and intraspecific heterogeneity of mtDNA have been demonstrated in several organisms. Restric-
00448486/87/%03.50
0 1987 Elsevier Science Publishers B.V.
204
tion analysis of mtDNA provides a method of determining this variation and can help to identify breeding stocks and populations (Brown and Simpson, 1981; Ferris et al., 1981; King et al., 1981; Brown et al., 1982). Recently, mtDNA restriction analysis has been applied to fish species (Avise and Saunders, 1984; Berg and Ferris, 1984; Graves et al., 1984; Wilson et al., 1985b; Birt et al., 1986; Thomas et al., 1986). These studies also include mtDNA analysis of Salmo gairdneri Richardson. Berg and Ferris (1984 ) describe mtDNA cleavage patterns obtained by a set of 13 restriction endonucleases from one maternal line of S. gairdneri. Wilson et al. (1985b) andThomas et al. (1986) have analysed mtDNA polymorphism in American S. gairdneri populations. Their results suggest that at least some of the populations can be differentiated on the basis of the restriction polymorphism present within the species. Here we report the employment of this method to detect mtDNA polymorphism in breeding stocks of rainbow trout, Salmogairdneri Richardson, in Finland. MATERIALS
AND METHODS
Fish material
Specimens from four different breeding stocks of rainbow trout (Salmo gairdneri Richardson) were obtained from the Laukaa Fish Culture Research Station of Finnish Game and Fisheries Research Institute, Laukaa, Finland. These stocks were designated A, Am, D and K, The A population is a hybrid between two stocks from the U.S.A. which, by selection, has been developed to spawn during autumn. The Am stock also originated from the U.S.A. and is a hybrid of two populations, one of which is anadromous. Stock D consists of fish which have been imported to Finland from Denmark since the beginning of the nineteen-sixties. This is the main stock farmed commercially in Finland. Stock K (Kamloops) is a hybrid between Kamloops fish (originating from Canada) imported to Finland both from the U.S.A. and Denmark. Isolation and restriction analysis of mtDNA
Crude mitochondrial preparations were obtained from liver samples of freshly killed animals by differential centrifugation essentially as described by Lansman et al. (1981). The mtDNA was isolated by a rapid extraction method as described previously ( Palva and Palva, 1985). The mtDNA samples were digested with restriction endonucleases indicated, according to the instructions provided by the supplier (Boehringer Mannheim GmbH) . If the digested fragments were end-labelled with ( CU-~%-)~ATP ( Amersham International), the method of Drouin (1980) was employed. The fragments were separated by
2%
123456789 v----v--
-
-21227
--
9
-
Fig. 1. Restriction analyses of S. gairdneri mtDNA isolated from samples of Am (lanes 1,3.5 and 7) and D (lanes 2, 4,6 and 8) populations. The mtDNA was isolated from liver samples as described in Materials and Methods and subjected to restriction with the endonucleases BglII (lanes 1 and 2), EcoRI (lanes 3 and 4), Hind111 (I anes 5 and 6) and XbaI (lanes 7 and 8). The fragments were end-labelled with (a-“%)dATP (Drouin. I980) prior to electrophoretic analysis on 1.5% agarose gels. A DNA digested with EcoRl and Hind111 and similarly end-la&&d was used as a standard (lane 9). The sizes of the ,l fragments (arrows) are in basepairs. The arrowheads indicate those of the Hind111 fragments (b, g and the probable sum fragment of b and g) that exhibit polymorphism.
electrophoresis in 0.8% or 1.5% agarose gels and were visualized either by EtBr staining or, when radioactively labelled, by autoradiography of dried gels. Bacteriophage 11DNA digested with EcoRI and Hind111 or this in combination with HindIiI digest of 1. DNA was used as molecular size marker.
286 TABLE 1 Distribution Stock
of mtDXA restriction morphs in Salmo gairdneri Richardson breeding stocks mtDNA” type
No of individuals analysed
Restriction
morphs”
Hind111
BglII
Am
A
8
i
ii
K K
A B
3 2
i i
ii .. .
D D D
A B C
9 1 2
i i ii
ii .. . 111 i
A A
C D
11 2
ii i
i i
111
“Nomenclature according to Brown and Simpson (1981). Restriction morph is defined to mean a restriction fragment pattern produced by gel electrophoresis of mtDNA from an individual animal after digestion with a single given restriction endonuclease. Each restriction morph is assigned with’s lower-case Roman number. The term type indicates a particular spectrum of restriction morphs produced by a given mtDNA. The designations used (A-D and i-iii) are arbitrary. For abbreviations denoting populations, see Materials and Methods.
RESULTS
AND DISCI-JSSION
Restriction analysis of different rainbow trout populations revealed clear heterogeneity in restriction sites with two of the enzymes tested. Intraspecific variation in fragment patterns signifying nucleotide sequence difference was obtained firstly wit.h HindIII (Fig. 1). With this enzyme two restriction morphs (Brown and Simpson, 1981; see also Table 1) were detected among the individuals analysed. The morph i mtDNA produced seven Hind111 fragments of 6530,3520,2170, 1780, 1220,109O and 250 bp. This pattern appears to be similar to that detected in an American population (Berg and Ferris, 1984). The morph ii mtDNA digested with Hind111 lacked the 250 bp fragment and the 3520 bp fragment appeared larger in size (3780 bp) , probably representing the sum of fragments b and g. These individuals (Fig. 1) did not exhibit variation with the other enzymes tested, and the fragment patterns (Fig. 1) appeared to be similar to those obtained by Berg and Ferris (1984). B&II digestion resulted in three different fragment patterns (Fig. 2). The morph i mtDNA contains four E&l11 fragments (7500, 4620, 3530 and 1230 bpf . In the second morph, ii, the a and b fragments have been replaced by a larger fragment that is the sum of a and b. This morph seems to be the same as described by Berg and Ferris (1984). In the third variant, iii, the fragments
1
ab
2
3
4
-21227
-a
cd
.w -
b ==-. -_c_
“, ~i_d
-
-
947
831
c and d ohservccl in the other morphs were rttplacccl by n new l;lrgcr fr;lpment (47GO lq,) apparently composed of c and d ( Pig. 2 1. The results suggest that the variant restriction pttterns otJtained with both NindIII and &.$I1 can be explained by reslriction site losses or gains, iis in all cases t.he new larger fragments seem to represent sums of smaller fragments in other morphs. Cenernlly this is the most common t>l)e of hetcrogencity otberved ( Lansman et al., 198 1 ) . Table 1 summarizes the polymorphism observed in the different hreecling stocks. Altogether four different. mtDNA types designated A-D were detected in these populat.ions. No clear population specific mtL)NA types were ohserved hut heterogeneity was present in most of the stocks. The only exception seems to he the Am stock which in this analysis was monomorphic. Restriction patterns of this stock resemble those of an Atnerican populat.ion ( Berg and Ferris, 1984).
Rainbow trout has no established nat.ural populations in Finland but is kept mostly in fisheries. The original st.ocks used are from several different sources with certain mixing having taken place. Therefore, it is maybe not so surprising that no clearcut population specific patterns were observed and that heterogeneity was present in three of the four stocks studied, probably reflecting their mixed origin. ACKXOW’LEDGEMENTS
JVe thank -Jill Thorngren for typing the manuscript. ported by the Academy of Finland.
This research
was sup-
REFERENCES Avise. .J.C. and Saunders. N.C.. 1981. Hybridization and introgression among species of sunfish (Leponris) : analysis by mitochondrial DNA and allnzyme markers. Genetics. 108: 237-2.53. Avise. J.C., Giblin-Davidson. C.. l’ntton. J.C. and Lansman. R.A., 1979a. Xlitochondrial DNA clones and matriarchal phylogeny within and among geographic populations of the pocket gopher. Gromys pin&. Proc. Natl. Acad. Sci. U.S.A., 76: 6694-6698. Avise. .J.C.. Lansman. R.A. and Shade, R.O.. 1979b. The use of restriction endonucleases t.o measure mitochondrial DNA sequence relat.edness in naturnl populations. I. Population structure and evolution in the genus h~romyscu.~. Genetics, 92: 279-295. Berg. W..J. und Ferris. SD., l!l%L. Restriction endonuclease analysis of aalrtrckl mitochondrial DNA. Can. J. Fish Aquatic Sci.. 4 1: l+tI- 1017. Birt. T.P., Green. .J.M. and Duvidscm, W.S.. 1986. Analysis of mitochondrial DNA in allopatric anadromous and Ilonanatlrc,nrt,us Atlantic Salmon. Sulmo safer. Chn. .I. Zwl.. 64: 118 - 120. Brown. G.G. and Simpson, M.V., 1981. Intra- and interspecific variat.ion of the mitochcmdrial gcnome in hftrs norccgiccw and fhltus ~cllfus: restriction enzyme analysis of variunt mitochondrial DNA molecules and their evc)lutionery relationships. Genetics. 97: 125-145. Brown. W.M.. (;eorKe. M. and Wilxon. AX., 1979. Rapid evolutionofanimal mitochtmdrial DNA. Proc. Not.1. Acad. Sci. U.S.A.. 76: 1967- 1971. Brown, W.M., Prager, EM.. Wung, A. and Wilson, A.C.. 1982. Mitochondrinl DNA sequences of primates: tempo and mode of evolution. J. Mol. Evol., 18: 225-239. Drouin. 4., 1980. Cloning of human mitochondrial DNA in Eschrrichia coli. J. Mol. Viol., 11[): 15-3-1. Ferris, SD., Brown, W.M., Davidson. W.S. and Wilson, A.C., 1981. Extensive polymorphism in the mitochtmdrial DNA of apes. Proc. Natl. Acad. Sci. U.S.A., ‘78: 6319-6323. Graves. J.E., Ferris, S.D. and Dizun. A.E.. 1984. Close genetic similarity of Atlantic and Pacific skipjack tuna (Katsuwonus pclamis) demonstrated with restriction endonuclease analysis of mitochondrial DNA. Mar. Biol.. 79: 315419. King, B.O.. Shade, R.O. and Lansman, R.A., 1981. The use of restriction endctnucleases to compare mitochondrial DNA sequences in Mu.s musculus: a detailed restricticm map of mitochondrial DNA from mouse L cells. Plasmid, 5: 3113-328. Lansman, R.A.. Shade, R.U.. Shapira. J.F. and Avise. .J.C., 1981. The use of restriction endonucleasev to measure mitochondrial DNA sequence relatedness in natural populations. III. Techniques and potential applications. J. Mol. Evol.. 17: 214226. Palva. T.K. and Palva. E.T.. 1985. Rapid isolation of animnl mitochondrial DNA by alkaline extraction. FEBS Lett., 192: X7-276).
Thomas. W.K.. Withler. R.E. and Beckenback. A.T., 1986. Sfitnthondrial DNA analysis of Pacific salmonid evolution. Can. d. Zool.. 6-t: l&58- 1064. \~Ylson. XC.. Cann. R.L., Carr. SM., George. M., Gyllensten. U.B., Helm-Bychowski. Kkf.. Higuchi. R.G., Palumbi, S.R., Prager. E.M.. Sage, R.D. and Stoneking, 51.. 19858. Mitochondrial DNA and two perspectives on evolutionary genetics. Biol. .J. Linn. Sot.. 26: 3;.i-.W. LVilson. G.hI., Thomas, !V’.K. and Beckenbach, A.T.. 1985b. Intra- and inter-specific mitochondrial DNA sequence divergence in Salon: rainbow, stealhead, and cutthroat trouts. Can. .J. Zool.. 63: 2088-2094.
Elsevier Science Publishers B.V.. Amsterdam -
263
Printed in The Netherlands
Restriction Site Polymorphism in Mitochondrial DNA of Rainbow Trout, Salmo gairdneri Richardson, Stocks in Finland TUULA K. PALVA’.3.4and E. TAP10 PALVA2,3 ‘Department of Applied Zoology, University of Kuopio, P.O. Box 138, SF-70101 Kuopio (Finland) ‘Molecular Genetics Laboratory Department of Genetics, University of Helsinki, SF-00100 Helsinki (Finland)
3Present address: Department of Molecular Genetics, Swedish University of Agricultural Sciences, Box 7003, S-750 07 Uppsala (Sweden) ‘To whom correspondence should be addressed. (Accepted 25 dune 1987)
ABSTRACT Palva, T.K. and Palva, E.T., 1987. Restriction site polymorphism in mitochondrial DNA of rainbow trout, Salmo gairdneri Richardson, stocks in Finland. Aquaculture, 67: 283-289. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) from four breeding stocks of rainbow trout, Salmo gairdneri Richardson, revealed extensive polymorphism. Two different restriction morphs were observed by Hind111 digestion and three different morphs by BgfII. All of the variant mtDNA types (A-D) could be explained by restriction site gains or losses.
INTRODUCTION
Effective fish farming requires knowledge of the breeding populations and identification of the breeding stocks. The determination of intraspecific genetic heterogeneity by phenotypic parameters is not necessarily adequate ( Avise et al., 1979a,b). To obtain maximal information nucleotide sequence heterogeneity should be determined. A good estimation of the sequence differences can be obtained by restriction endonuclease analysis (Avise et al., 1979a.b). The maternally inherited mitochondrial DNA (mtDNA) appears to be especially suitable for this type of analysis (Wilson et al., 1985a). It appears to evolve approximately one order of magnitude faster than the nuclear genome (Brown et al., 1979). Consequently, high levels of inter- and intraspecific heterogeneity of mtDNA have been demonstrated in several organisms. Restric-
00448486/87/%03.50
0 1987 Elsevier Science Publishers B.V.
204
tion analysis of mtDNA provides a method of determining this variation and can help to identify breeding stocks and populations (Brown and Simpson, 1981; Ferris et al., 1981; King et al., 1981; Brown et al., 1982). Recently, mtDNA restriction analysis has been applied to fish species (Avise and Saunders, 1984; Berg and Ferris, 1984; Graves et al., 1984; Wilson et al., 1985b; Birt et al., 1986; Thomas et al., 1986). These studies also include mtDNA analysis of Salmo gairdneri Richardson. Berg and Ferris (1984 ) describe mtDNA cleavage patterns obtained by a set of 13 restriction endonucleases from one maternal line of S. gairdneri. Wilson et al. (1985b) andThomas et al. (1986) have analysed mtDNA polymorphism in American S. gairdneri populations. Their results suggest that at least some of the populations can be differentiated on the basis of the restriction polymorphism present within the species. Here we report the employment of this method to detect mtDNA polymorphism in breeding stocks of rainbow trout, Salmogairdneri Richardson, in Finland. MATERIALS
AND METHODS
Fish material
Specimens from four different breeding stocks of rainbow trout (Salmo gairdneri Richardson) were obtained from the Laukaa Fish Culture Research Station of Finnish Game and Fisheries Research Institute, Laukaa, Finland. These stocks were designated A, Am, D and K, The A population is a hybrid between two stocks from the U.S.A. which, by selection, has been developed to spawn during autumn. The Am stock also originated from the U.S.A. and is a hybrid of two populations, one of which is anadromous. Stock D consists of fish which have been imported to Finland from Denmark since the beginning of the nineteen-sixties. This is the main stock farmed commercially in Finland. Stock K (Kamloops) is a hybrid between Kamloops fish (originating from Canada) imported to Finland both from the U.S.A. and Denmark. Isolation and restriction analysis of mtDNA
Crude mitochondrial preparations were obtained from liver samples of freshly killed animals by differential centrifugation essentially as described by Lansman et al. (1981). The mtDNA was isolated by a rapid extraction method as described previously ( Palva and Palva, 1985). The mtDNA samples were digested with restriction endonucleases indicated, according to the instructions provided by the supplier (Boehringer Mannheim GmbH) . If the digested fragments were end-labelled with ( CU-~%-)~ATP ( Amersham International), the method of Drouin (1980) was employed. The fragments were separated by
2%
123456789 v----v--
-
-21227
--
9
-
Fig. 1. Restriction analyses of S. gairdneri mtDNA isolated from samples of Am (lanes 1,3.5 and 7) and D (lanes 2, 4,6 and 8) populations. The mtDNA was isolated from liver samples as described in Materials and Methods and subjected to restriction with the endonucleases BglII (lanes 1 and 2), EcoRI (lanes 3 and 4), Hind111 (I anes 5 and 6) and XbaI (lanes 7 and 8). The fragments were end-labelled with (a-“%)dATP (Drouin. I980) prior to electrophoretic analysis on 1.5% agarose gels. A DNA digested with EcoRl and Hind111 and similarly end-la&&d was used as a standard (lane 9). The sizes of the ,l fragments (arrows) are in basepairs. The arrowheads indicate those of the Hind111 fragments (b, g and the probable sum fragment of b and g) that exhibit polymorphism.
electrophoresis in 0.8% or 1.5% agarose gels and were visualized either by EtBr staining or, when radioactively labelled, by autoradiography of dried gels. Bacteriophage 11DNA digested with EcoRI and Hind111 or this in combination with HindIiI digest of 1. DNA was used as molecular size marker.
286 TABLE 1 Distribution Stock
of mtDXA restriction morphs in Salmo gairdneri Richardson breeding stocks mtDNA” type
No of individuals analysed
Restriction
morphs”
Hind111
BglII
Am
A
8
i
ii
K K
A B
3 2
i i
ii .. .
D D D
A B C
9 1 2
i i ii
ii .. . 111 i
A A
C D
11 2
ii i
i i
111
“Nomenclature according to Brown and Simpson (1981). Restriction morph is defined to mean a restriction fragment pattern produced by gel electrophoresis of mtDNA from an individual animal after digestion with a single given restriction endonuclease. Each restriction morph is assigned with’s lower-case Roman number. The term type indicates a particular spectrum of restriction morphs produced by a given mtDNA. The designations used (A-D and i-iii) are arbitrary. For abbreviations denoting populations, see Materials and Methods.
RESULTS
AND DISCI-JSSION
Restriction analysis of different rainbow trout populations revealed clear heterogeneity in restriction sites with two of the enzymes tested. Intraspecific variation in fragment patterns signifying nucleotide sequence difference was obtained firstly wit.h HindIII (Fig. 1). With this enzyme two restriction morphs (Brown and Simpson, 1981; see also Table 1) were detected among the individuals analysed. The morph i mtDNA produced seven Hind111 fragments of 6530,3520,2170, 1780, 1220,109O and 250 bp. This pattern appears to be similar to that detected in an American population (Berg and Ferris, 1984). The morph ii mtDNA digested with Hind111 lacked the 250 bp fragment and the 3520 bp fragment appeared larger in size (3780 bp) , probably representing the sum of fragments b and g. These individuals (Fig. 1) did not exhibit variation with the other enzymes tested, and the fragment patterns (Fig. 1) appeared to be similar to those obtained by Berg and Ferris (1984). B&II digestion resulted in three different fragment patterns (Fig. 2). The morph i mtDNA contains four E&l11 fragments (7500, 4620, 3530 and 1230 bpf . In the second morph, ii, the a and b fragments have been replaced by a larger fragment that is the sum of a and b. This morph seems to be the same as described by Berg and Ferris (1984). In the third variant, iii, the fragments
1
ab
2
3
4
-21227
-a
cd
.w -
b ==-. -_c_
“, ~i_d
-
-
947
831
c and d ohservccl in the other morphs were rttplacccl by n new l;lrgcr fr;lpment (47GO lq,) apparently composed of c and d ( Pig. 2 1. The results suggest that the variant restriction pttterns otJtained with both NindIII and &.$I1 can be explained by reslriction site losses or gains, iis in all cases t.he new larger fragments seem to represent sums of smaller fragments in other morphs. Cenernlly this is the most common t>l)e of hetcrogencity otberved ( Lansman et al., 198 1 ) . Table 1 summarizes the polymorphism observed in the different hreecling stocks. Altogether four different. mtDNA types designated A-D were detected in these populat.ions. No clear population specific mtL)NA types were ohserved hut heterogeneity was present in most of the stocks. The only exception seems to he the Am stock which in this analysis was monomorphic. Restriction patterns of this stock resemble those of an Atnerican populat.ion ( Berg and Ferris, 1984).
Rainbow trout has no established nat.ural populations in Finland but is kept mostly in fisheries. The original st.ocks used are from several different sources with certain mixing having taken place. Therefore, it is maybe not so surprising that no clearcut population specific patterns were observed and that heterogeneity was present in three of the four stocks studied, probably reflecting their mixed origin. ACKXOW’LEDGEMENTS
JVe thank -Jill Thorngren for typing the manuscript. ported by the Academy of Finland.
This research
was sup-
REFERENCES Avise. .J.C. and Saunders. N.C.. 1981. Hybridization and introgression among species of sunfish (Leponris) : analysis by mitochondrial DNA and allnzyme markers. Genetics. 108: 237-2.53. Avise. J.C., Giblin-Davidson. C.. l’ntton. J.C. and Lansman. R.A., 1979a. Xlitochondrial DNA clones and matriarchal phylogeny within and among geographic populations of the pocket gopher. Gromys pin&. Proc. Natl. Acad. Sci. U.S.A., 76: 6694-6698. Avise. .J.C.. Lansman. R.A. and Shade, R.O.. 1979b. The use of restriction endonucleases t.o measure mitochondrial DNA sequence relat.edness in naturnl populations. I. Population structure and evolution in the genus h~romyscu.~. Genetics, 92: 279-295. Berg. W..J. und Ferris. SD., l!l%L. Restriction endonuclease analysis of aalrtrckl mitochondrial DNA. Can. J. Fish Aquatic Sci.. 4 1: l+tI- 1017. Birt. T.P., Green. .J.M. and Duvidscm, W.S.. 1986. Analysis of mitochondrial DNA in allopatric anadromous and Ilonanatlrc,nrt,us Atlantic Salmon. Sulmo safer. Chn. .I. Zwl.. 64: 118 - 120. Brown. G.G. and Simpson, M.V., 1981. Intra- and interspecific variat.ion of the mitochcmdrial gcnome in hftrs norccgiccw and fhltus ~cllfus: restriction enzyme analysis of variunt mitochondrial DNA molecules and their evc)lutionery relationships. Genetics. 97: 125-145. Brown. W.M.. (;eorKe. M. and Wilxon. AX., 1979. Rapid evolutionofanimal mitochtmdrial DNA. Proc. Not.1. Acad. Sci. U.S.A.. 76: 1967- 1971. Brown, W.M., Prager, EM.. Wung, A. and Wilson, A.C.. 1982. Mitochondrinl DNA sequences of primates: tempo and mode of evolution. J. Mol. Evol., 18: 225-239. Drouin. 4., 1980. Cloning of human mitochondrial DNA in Eschrrichia coli. J. Mol. Viol., 11[): 15-3-1. Ferris, SD., Brown, W.M., Davidson. W.S. and Wilson, A.C., 1981. Extensive polymorphism in the mitochtmdrial DNA of apes. Proc. Natl. Acad. Sci. U.S.A., ‘78: 6319-6323. Graves. J.E., Ferris, S.D. and Dizun. A.E.. 1984. Close genetic similarity of Atlantic and Pacific skipjack tuna (Katsuwonus pclamis) demonstrated with restriction endonuclease analysis of mitochondrial DNA. Mar. Biol.. 79: 315419. King, B.O.. Shade, R.O. and Lansman, R.A., 1981. The use of restriction endctnucleases to compare mitochondrial DNA sequences in Mu.s musculus: a detailed restricticm map of mitochondrial DNA from mouse L cells. Plasmid, 5: 3113-328. Lansman, R.A.. Shade, R.U.. Shapira. J.F. and Avise. .J.C., 1981. The use of restriction endonucleasev to measure mitochondrial DNA sequence relatedness in natural populations. III. Techniques and potential applications. J. Mol. Evol.. 17: 214226. Palva. T.K. and Palva. E.T.. 1985. Rapid isolation of animnl mitochondrial DNA by alkaline extraction. FEBS Lett., 192: X7-276).
Thomas. W.K.. Withler. R.E. and Beckenback. A.T., 1986. Sfitnthondrial DNA analysis of Pacific salmonid evolution. Can. d. Zool.. 6-t: l&58- 1064. \~Ylson. XC.. Cann. R.L., Carr. SM., George. M., Gyllensten. U.B., Helm-Bychowski. Kkf.. Higuchi. R.G., Palumbi, S.R., Prager. E.M.. Sage, R.D. and Stoneking, 51.. 19858. Mitochondrial DNA and two perspectives on evolutionary genetics. Biol. .J. Linn. Sot.. 26: 3;.i-.W. LVilson. G.hI., Thomas, !V’.K. and Beckenbach, A.T.. 1985b. Intra- and inter-specific mitochondrial DNA sequence divergence in Salon: rainbow, stealhead, and cutthroat trouts. Can. .J. Zool.. 63: 2088-2094.