© 2005 International Council for the Exploration of the Sea
Novel DNA markers for rapid, accurate, and cost-effective discrimination of the continental origin of Atlantic salmon (Salmo salar L.)
a Fisheries Research Services, Freshwater Fisheries Laboratory Faskally, Pitlochry PH16 5LB, UK
b Fisheries Research Services, Marine Laboratory PO Box 101, Victoria Road, Aberdeen AB11 9DB, Scotland, UK
*Correspondence to J. Gilbey: tel: +44 1224 294459; fax: +44 1796 472060. e-mail: gilbeyj{at}marlab.ac.uk.
Salmon from geographically representative rivers in North America and Europe were typed for variation at the microsatellite locus SS1 and the mitochondrial DNA ND-1 restriction site 3971, using PCR amplification and agarose-gel electrophoresis. North American salmon showed near-fixation for SS1 alleles between 129 and 135 bp in length and the N mtDNA restriction type, while European salmon near-fixation for SS1 alleles between 183 and 219 bp and the A/D mtDNA type. Based on the observed variant frequencies, using these two markers in combination is predicted to give correct assignment of >99.5% of salmon to continent-of-origin. As both these continental markers can be screened by agarose-gel electrophoresis, their use offers a more rapid, cheaper, and simpler method for accurate assignment of Atlantic salmon to continent-of-origin than do existing methods. These markers can be applied to the identification of salmon in North Atlantic high-seas fisheries, in aquaculture stocks, and in rivers to determine the continent-of-origin of fish-farm escapes.
Keywords: continental origin, microsatellite marker, mtDNA, stock discrimination
Received 1 April 2005; accepted 10 July 2005.
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The development of methods for assigning the continent-of-origin of Atlantic salmon (Salmo salar L.) has been the subject of a considerable research effort over the last four decades (Reddin and Friedland, 1999), much of it focused on their discrimination in high-seas fisheries. Though Atlantic salmon from Europe and North America home to their natal rivers with high fidelity (e.g. Stabell, 1984; Youngson et al., 1994), both groups migrate into the North Atlantic and are often caught together during their feeding migrations in areas such as the seas off West Greenland (e.g. Reddin et al., 1988; ICES, 2004). Salmon from one continent may also be found within the range of the other where intercontinental fish transfers have occurred historically as part of stocking programmes or, more recently, as part of fish farming operations (e.g. NRC, 2004). In these situations, it is of interest for effective management to be able to establish the continental origin of individual fish so that their relative proportions in potential mixtures can be ascertained and their impacts properly evaluated.
Early efforts to develop markers for continental discrimination focused on the use of allozymes (see review in Verspoor et al., 2005), and parasites (Nyman and Pippy, 1972), as well as on body meristics and shape (e.g. Claytor et al., 1991), scale and otolith morphology (e.g. Friedland and Reddin, 1994), the elemental composition of scales (Lapi and Mulligan, 1981) and vertebrae (Mulligan et al., 1983). However, discrimination with these methods has proved problematic for various reasons. These reasons include limited or temporally variable divergence in the characters targeted, practical difficulties in obtaining baseline data, and excessive cost. Many of the problems with these methods have been overcome by using recently developed DNA markers which show consistent and stable differentiation, and can be typed from preserved scales or tissues. Studies of mitochondrial (e.g. Bermingham et al., 1991), minisatellite (e.g. Taggart et al., 1995), and microsatellite (e.g. McConnell et al., 1995; King et al., 2001) DNA have all identified potentially useful markers for discriminating European and North American salmon. Of these, microsatellite markers have to date been most useful, and a system of four microsatellite markers, Ssa202 (Genbank: U43695 [GenBank] ), Ssa289 (McConnell et al., 1995), SSOSL438 (Z49134 [GenBank] ), and SSOSL311 (Z48597 [GenBank] ), is currently applied to determine the origin of salmon in the West Greenland fishery (ICES, 2004).
Despite recent advances, the routine and widespread application of genetic typing in management is still constrained by the cost, speed, and technical complexity of existing methods. This paper describes two molecular genetic markers that can be used in combination to accurately, rapidly, and more cheaply assign Atlantic salmon to their continent-of-origin. Variation at the dinucleotide microsatellite SS1 (Genbank: AJ243170 [GenBank] ) was identified, during the course of another study (Gilbey et al., 2004), to be potentially informative as regards the continent-of-origin of salmon as well as resolvable by simple agarose-gel electrophoresis. Informative genetic variation at the mtDNA locus was identified from published analyses (Verspoor et al., 1999, 2002; King et al., 2000; Nilsson et al., 2001). Agarose-gel electrophoresis of the two markers would allow the routine discrimination of European and North American salmon in high-seas fisheries, aquaculture stocks, and wild populations.
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Material and methods |
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Genetic samples
Archival samples of salmon from a representative set of European and North American rivers were used to investigate the geographical distribution of variation at the two target markers (Figure 1). Total genomic DNA was isolated from skin samples using either the chloroform/ethanol extraction of Mullenbach (1989) or the simpler and more rapid method of Knox et al. (2002).
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SS1 microsatellite
The SS1 locus (AJ243170 [GenBank] ) was amplified by PCR which was performed in a 12.5 µl reaction volume containing 5–100 ng DNA, 75 mM Tris–HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% v/v Tween 20, 1.5 mM MgCl2, 200 µM each dNTP, 0.5 µM each primer, and 0.25 units of ABgene Taq DNA polymerase (ABgene, Surrey, England). The primers used were: forward primer 5'-CAT ACA TGA CAT CAA TCC AGC-3' and reverse primer 5'-TGT AAA GGG TTC ATT GAG TG-3'. Cycling conditions consisted of a modified "touchdown" sequence (Don et al., 1991) comprising a denaturing step of 95°C for 2 min, followed by a series of cycles consisting of 95°C for 30 s, annealing temperature for 30 s, and 72°C for 30 s. Annealing temperatures consisted of single cycles from 65°C to 56°C, decreasing in 0.5°C steps, followed by 20 cycles at 56°C. PCR was completed with an extension step of 72°C for 10 min. Allele sizes were determined using an ABI Prism 377 DNA Sequencer (Applied Biosystems, Cheshire, England) using FAM-labelled forward primer (MWG Biotech UK Ltd, Milton Keynes, England).
Variation at the SS1 locus was characterized precisely for 88 Atlantic salmon from five rivers in Europe (Laxa i Dolum, Iceland; Narcea, Spain; Pecha, Spain; North Esk, Scotland; Tornejoki, Sweden/Finland) and six in North America (Highlands, Miramichi, Narraguagus, St. Croix, Saint John, Stewiake in Canada) (Figure 1), to make a preliminary assessment of the potential utility of this marker for determining their continent-of-origin and obtain the exact sizes of the allelic variants. A further 187 Atlantic salmon were screened by using 2% agarose gels containing ethidium bromide and run at 95 V for 1 h and alleles visualized by viewing under UV light. Allelic variation was classified as either small (below 150 bp) or large (above 150 bp). This screening included additional salmon from the same rivers, with the exception of the Saint John, as well as salmon from the Michael's River in Labrador to extend the geographical range of the North American sites (Figure 1).
FST (Weir and Cockerham, 1984) and unbiased genetic distance DA (Nei, 1978) were calculated to compare levels of divergence observed at the markers examined with other loci. These measures were calculated using TFPGA version 1.3 (Miller, 1997).
ND1/16sRNA mitochondrial region
Salmon from the same rivers as screened for SS1 variation were independently typed for variation at base position 3971 in ND1/16sRNA region of the mitochondrial DNA (AF133701
[GenBank]
). This variation was detected by PCR amplification of the region around this site, HaeIII digestion of the resulting 111 bp fragment, electrophoretic separation of the fragments on a 3% agarose gel, and visualization of fragments using ethidium bromide and UV light (Knox et al., 2002). Individuals were either typed as A/D or N (Knox et al., 2002). The two haplotypes, A/D or N (Knox et al., 2002), are characterized by the presence or absence of a HaeIII restriction site at base position 3971 in the ND1/16sRNA region of the mitochondrial DNA, which is at position 58 in the 111 bp amplified fragment of this region. The A/D haplotype has a GGCC HaeIII restriction site at this position within which the enzyme will cut, producing two fragments of 58 and 53 bp. In contrast, the N haplotype has a GACC motif at this position within which there is no restriction site, thus leaving the 111 bp fragment intact.
PCR amplifications were carried out in a volume of 25 µl, and contained 10–100 ng of template DNA, 2x Biotaq reaction buffer (10 mM Tris, pH 8.8; 50 mM KCl; 1.5 mM MgCl2; 0.1% Triton-X-100), 200 µM of each dNTP, 0.5 µM of each primer, and 0.5 units of ABgene Taq DNA polymerase (ABgene, Surrey, England). The primers used were set 5 from Knox et al. (2002): forward primer 5'-CGC TTT CCT CAC CTT ACT CGA ACG-3' and reverse primer 5'-TTT AGG CCG TCC GCG ATA GG-3'. PCR cycling conditions consisted of an initial denaturing step of 95°C for 5 min followed by 29 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 1.5 min. This was followed by a final extension step of 72°C for 10 min. Digestions of the 111 bp amplified fragment with the restriction enzyme HaeIII (Life Technologies) were carried out in a 10 µl volume reaction, using four units of enzyme and 5 µl of amplified DNA, in the reaction buffer recommended by the manufacturers, with an overnight incubation at 37°C. Fragments were separated on 3% agarose gels run at 95 V for 1.5 h, and visualized by ethidium bromide staining. Fragment lengths were estimated by comparison with a size standard (
X174 DNA digested with HaeIII, Advanced Biotechnologies Ltd., Surrey, England).
As the A/D and N haplotypes are identified by the presence or absence of a cutting site, a positive control was used to confirm the success of the enzyme digestions. A 975 bp fragment of Atlantic salmon cytochrome-B mitochondrial DNA (see AF133701 [GenBank] ) was amplified in the same PCR conditions as the microsatellite SS1 above, but at a fixed annealing temperature of 55°C. The primers used were: forward primer 5'-CAA CAG CTT TTT CCT CTG TTT-3' and reverse primer 5'-AGC TAC TAG GGC AGG TTC ATT-3'. This fragment contains a HaeIII restriction site near its centre, producing fragments of 535 and 440 bp when successfully digested. The positive control was either digested in a separate restriction reaction, using the same conditions as the samples, and run in its own lane on the gel (shown for clarity in Figure 3), or 1 µl of the product from the positive control PCR amplification was added to each sample's enzyme reaction before digestion, giving a positive control for each sample.
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Results |
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Previous studies using microsatellites, in which the two alleles possessed by an individual differ significantly in length, have reported differences in the efficiency of the PCR amplification of both of them (e.g. Wattier et al., 1998; Gardner et al., 2002; Morand et al., 2002). This is thought to be due to technical limitations in the PCR leading to preferential amplification of the short alleles over the long alleles (termed "short allele dominance" or "upper allele dropout"; Wattier et al., 1998). The PCR-amplification efficiency of the SS1 microsatellite was examined using hybrids produced from crossings of North American and Scottish salmon (Gilbey, 2003) visualized on a silver-stained, 6%, denaturing, polyacrylamide gel. In the families examined there was a small decrease in the band intensity of the long alleles compared with the short. However, the long alleles were still readily scorable in these families, suggesting that even if short allele dominance is affecting the amplification, it should not influence the genotyping of this loci (the same as found with the microsatellites used by Morand et al., 2002).
The preliminary geographic survey showed North American fish with alleles ranging in size from 129 to 135 bp, while all but two of the European fish had larger alleles of 183–219 bp in size (Table 1); two salmon from the River Narcea had an allele in the lower-size class. Size differences between the larger, typically European, and smaller, typically North American, alleles were resolved after an hour of electrophoresis on a 2% agarose gel, as shown in Figure 2, and show a clear separation of the two allele size classes. In the additional typing of 187 fish, based on the two size classes (Figure 2; Table 2), all but three (1.5%) of the North American fish showed alleles in the smaller-size class, while all but nine (5.3%) of the European fish had alleles in the larger-size class. Taking all fish into account (n = 275), DA between continents for SS1 is 0.898 and the FST is 0.948.
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Mitochondrial DNA typing of salmon from the same rivers (Figure 3), including those typed for SS1, shows even less overlap between rivers on the two continents (Table 3). Of the 309 North American salmon analysed, only one (0.3%), from Michael's River in Labrador, had the A/D type. In Europe, 7 of 285 (2.5%), again all from one river – the Pecha in northern Russia, had the N type. Taking all fish into account (n = 594), DA between continents based on mtDNA differences is 0.945 while FST is 0.973.
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Discussion |
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Differences between salmon from European and North American rivers at microsatellite loci were first reported by McConnell et al. (1995). They found three microsatellite loci which showed an overall genetic distance (DA) of 0.32 between the stocks they examined. Using a much larger set of rivers and a larger set of microsatellite loci, King et al. (2001) found DA values to range between 0.46 and 0.75, and an FST of 0.274. These values are considerably less than those observed for SS1, with DA = 0.898 and FST = 0.948, and similar estimates have only previously been observed for Ssa-A45/2/2, a nuclear minisatellite locus studied by Taggart et al. (1995). They found values of DA and FST were 0.914 and 0.990, respectively. Differentiation at SS1 is also of a similar magnitude to EST-D*, another nuclear locus, though population screening for this allozyme locus has been more limited (Bourke et al., 1997; Makhrov et al., 1998; Skaala et al., 1998). mtDNA, which behaves as a single haploid locus, showed similar high levels of differentiation in the present study, with DA and FST values of 0.945 and 0.973, respectively.
More extensive investigations of variation at allozyme (Verspoor et al., 2005), minisatellite (Taggart et al., 1995) and microsatellite loci (King et al., 2001), suggest that the level of differentiation observed at SS1 in the samples analysed here is likely to be representative of the populations on the two continents. At more extensively studied loci, conclusions drawn from more limited sets of rivers have generally been found to reflect patterns of differentiation on a broader scale, provided the rivers included are geographically representative of the species range. In general, populations of anadromous salmon rivers in broad geographic regions tend to be similar (e.g. King et al., 2001; Verspoor et al., 2005). With regard to mtDNA, the differentiation reported here is in agreement with previously published studies of other populations in North America and Europe (Verspoor et al., 1999, 2002; King et al., 2000; Nilsson et al., 2001; Knox et al., 2002).
Despite the high differentiation observed between salmon from North America and Europe, these two markers were not completely diagnostic for continent-of-origin. Neither the allele class (129–135 bp) which typifies North America, nor that found most commonly for European salmon (185–215 bp) is fixed in populations on either continent. Using SS1 alone as a marker, 0.7% of the European salmon typed would have been classified as North American, though no North American salmon would have been classified as European. Additionally, 6.8% of the European salmon and 2.1% of the North American salmon, which had an allele in each size category, would not have been assigned, giving an overall classification success of 95.2%. This compares with current methods, using four microsatellite markers, which achieve an assignment success rate in excess of 99%. Assignment success for mtDNA is 99.7% and 97.5% for the North American and European salmon, respectively. However, unlike the "nuclear genes" situation where salmon can be heterozygous and have one of each variant type, all fish are either one mtDNA variant type or the other. Using SS1 in combination with mtDNA, over 99.99% of North American and 99.8% of European fish analysed would be expected to be classified successfully, assuming the frequencies of the North American- and European-type alleles observed in the locations sampled are representative of their respective continents. This equates to fewer than 1 fish in 10 000 being misclassified, at least as good as the assignment success achievable using the existing suite of four microsatellite markers (ICES, 2004).
The combined use of SS1 and mtDNA for determination of the continent-of-origin of salmon offers a number of advantages. First, only two markers are required to be screened to achieve the same accuracy, given the higher differentiation shown at each locus. Second, unlike existing microsatellite markers, the two largely diagnostic allele size classes of SS1 (129–135 bp vs. 185–215 bp) can be resolved using simple and expedient agarose-gel electrophoresis, as can the two ND1/16sRNA mtDNA haplotypes (Figures 2 and 3). A preliminary assessment suggests that typing to continent-of-origin using these two markers resolved by agarose gels is likely to be at least twice as fast and one quarter the cost of screening for the four microsatellites currently employed as markers.
The overall assignment accuracy estimated in the present study should reflect assignment rates achieved where large numbers of populations from each continent mix, such as in the West Greenland fishery. However, in more local situations, this will vary somewhat. In areas such as Newfoundland and Labrador, where both "European" type variants for both SS1 and mtDNA occur, slightly more fish are likely to be incorrectly assigned, though in most cases the proportions will still be very small. For example, in the case of Michael's River, in which the highest frequencies of "European" SS1 and mtDNA variants were observed (Tables 1 and 2), the estimated rate of successful assignment would still be 99.5%. A similar, slightly lowered assignment rate would also be achieved in the Pecha in northern Russia, where both "North American" SS1 and mtDNA variants occur at low frequency (Tables 1 and 2). In Europe, in a river such as the North Esk only 72.7% of salmon are expected to be assigned correctly using SS1 alone. However, 100% are expected to be assigned correctly when this marker is used with mtDNA as, similar to most rivers across Europe (Verspoor et al., 1999), no "North American" variant types are found there (Table 2). Accurate assignment of all fish can also be expected in regions such as the Inner Bay of Fundy and Gulf of Maine in North America, where studies with other microsatellite and mtDNA markers have found that European variants appear to be absent in wild, native populations (this paper; King et al., 2001; Verspoor et al., 2002).
Studies to date have failed to find any genetic marker where alleles of one type are fixed in North American salmon, and others fixed in European salmon, despite hundreds of loci having been surveyed. This suggests that finding a locus which shows complete fixation for alternative alleles on the two continents is highly unlikely. Yet, at some loci in relation to most populations, fixed differences occur. This suggests the overall populations on the two continents have been highly isolated reproductively for a considerable period of time and that gene flow between the two continents is generally absent. The distribution of "European" variant types in North America, and "North American" variant types in Europe, is most likely explained by low-level, post-glacial gene flow between the two continents during the early period of colonization of parts of the species' modern range following the retreat of the Pleistocene ice sheets (Knox et al., 2002; Verspoor et al., 2005). If so, this would explain the failure to find molecular markers that are fully diagnostic as regards continent-of-origin. However, despite this, it is clear that using two unlinked and highly differentiated markers, such as SS1 and mtDNA, can effectively provide the same discrimination as a single fully diagnostic locus. Nuclear loci such as SS1 also allow for the identification of first generation hybrids, where it can be established that the wild populations under study are fixed for one or other diagnostic variant class. This may allow insight to be gained into possible interbreeding between escaped farm or stocked salmon and wild salmon, where stocked fish or farm escapees entering rivers have a different continent-of-origin than the wild native salmon.
Further screening of samples from rivers throughout the species range for both loci will be carried out over the next few years. This will provide a more robust baseline data set for using the markers for continental discrimination. This information as it becomes available can be obtained via the corresponding author or the fourth author.
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Acknowledgements |
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The authors wish to thank Tim King, Dave Reddin, Tom Cross, and numerous others over the years for providing the samples of tissue used, and two anonymous referees for their very helpful comments during the preparation of this manuscript.
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References |
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