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A calibration of different molecular markers for use in discrimination and management of stocks of commercially important fish species

Objective

The major aim of this project is to seek to provide improved methodology for fish stock discrimination through the application of modern molecular genetic methods, using four gadoid species; Atlantic cod Gadus morhua, blue whiting Micromesistius poutassou, hake Merluccius merluccius, and poor-cod Trisopterus minutus. This will be achieved by studying situations where populations are known to be genetically isolated and then, those where different stocks are suspected but have not been demonstrated by previous genetic methods. It is envisaged that through the integration of research on state-of-the-art molecular genetics and mathematical modelling, a cost-effective means of stock identification may be achieved, which could improve the accuracy of stock assessment and thus assist management. The molecular methods include PCRable mini- and microsatellite single locus VNTRs, certain mtDNA sequences, and nuclear DNA sequences including MHC and anonymous cDNA loci. Particular emphasis will be placed on determining low level differences, and to apply the tools so obtained to specific management problems in commercially important fish species.

Personnel in University of East Anglia (United Kingdom) developed PCR methods for analysing mtDNA and also developed several micro satellite loci specifically for the target species. It was demonstrated that few micro satellite loci developed for cod and whiting, work in other commercially important fish species (e.g. hake). One genomic library has been constructed for Atlantic cod, enriched for eight different types of micro satellite sequences, and one non-enriched library for hake, from which several micro satellite loci have been isolated. Screening of macro geographic variation in hake is in progress. The development of mathematical models for the integration of molecular genetics with fisheries management is underway. Workers at National University of Ireland, Cork (Ireland), made progress with satisfying the sampling requirements of the project, and tested two PCR-amplifiable minisatellite loci developed for whiting, in all target species. While one locus worked in each of cod, blue whiting and poor cod, neither worked with hake. In addition, to investigate potential null allele problems, a second set of primers was designed for each minisatellite locus. Macrogeographic analysis of cod with minisatellite loci has commenced. MHC I primers developed for Atlantic salmon, work with all target species. Trondheim Biological Station (Norway) researchers have progressed with the sampling, literature review and gathering of biological information. Micro satellite and minisatellite analysis of blue whiting has commenced. Isozyme analysis is now in progress for all species with the exception of hake, as is the literature review for blue whiting and poor cod. Personnel from the Marine Research Institute (Iceland) have made considerable progress in sampling for the macrogeographic and microgeographic genetic analyses. Macrogeographic analysis of the locus SypI has commenced for cod. The roles of CEFAS (United Kingdom) personnel in the project are sourcing samples and providing background fisheries information. Almost all macrogeographic samples are now secured.
Sampling With regard to macrogeographic sampling, some collections are taking longer than expected, since we cannot direct sampling and have to fit in with regular cruises of the fisheries labs. Most samples are frozen, which are more difficult to obtain and more expensive to transport and distribute, but allow for enzyme analysis as well as DNA analysis. In some cases only alcohol fixed samples are possible. We are attempting to obtain 100 individuals for each location since recent analyses and statistical developments indicate that such numbers may be necessary. Microgeographic sampling is also progressing. Once again 100 individuals will be sampled, and frozen where possible.

When the full range of molecular markers are applied to all samples, it will make a considerable contribution to assessing the relative value of different markers in this context and generally. The combination of information from different markers on this sample set, with their different properties, should allow for more complete understanding and explanation of population processes.

Literature.

By designating one lab to search the literature extensively for each species, a comprehensive bibliography is now available, and the true extent of the genetic knowledge of these fishes can be assessed. This can be placed into the context of the species biology. When our genetic screening and analysis is completed, the results may be interpreted in the light of this fuller knowledge of the species. There is a large literature on cod, while less is available on the other three species.

Genetic Analysis

Results for individual markers are as follows: Microsatellites: from primer pairs tested from other species only two loci showed defined bands. Consequently size selected and enriched libraries were constructed for the hake and cod. Suitable loci have been developed for blue whiting and hake, and are currently being screened for macrogeographic varlation.
Minisatellites: two minisatellite loci from whiting have been examined in detail and applied to the four species. Only one locus works in each of cod, poor cod and blue whiting. Since the attempt to transfer between species has yielded so few markers, new species specific loci are being developed. Macrogeographic analysis of cod is underway with available loci.
MtDNA: primer pairs have been optimised for the target species for several mtDNA regions including D loop, Cyt b and several other sections.
cDNA RFI>P: the chemiluminescent detection method employed initially (DM) was unsatisfactory, so enhanced chemiluminescence (ECI) is now being developed. Macrogeographic screening of genetic variation in cod at SypI has commenced.
Transcribed sequences: primers from Atlantic salmon for MHCI and MHCII have been tested while the suitability of a range of additional primers for transcribed sequences are also being examined. Samples are being compared from the extremes of the species range to gauge potential for stock discrimination.
Isozymes: isozyme techniques already developed have been adapted by investigators in Trondheim for the species under examination, and being applied to cod, blue whiting and poor cod. Most of the macrogeographic analysis is now complete.
It was previously thought that microsatellite and minisatellite loci should be useful in other related species, since the flanking primers are conserved sequences. Indeed some microsatellite loci primers isolated from bony fishes, produce bands in dogfish and lampreys (Rico et al, 1996). However, our experience is that very few of such non-specific primer pairs prove to be useful, being difficult to optimise, producing no product, single bands or too many bands. This is probably due in part to high mutation rates in the loci. In any event it means that research should focus on the development of new loci, both in this project and generally. Many laboratories are now turning to enrichment procedures, to increase the cloning efficiency of microsatellite and minisatellite sequences from genomic DNA of the target species. A number of such methods have recently been published and can yield 60% positive clones as compared to <1% from conventional cloning methods. This is the strategy we are now following.
The other markers we are developing, in addition to their application to these four fish species, are also of considerable general interest, and hence the subject of technical advancement. MHC loci, because of their high variability are likely to reflect population's differences due to local selection. Anonymous cDNA markers, are also attracting general interest as measure of
genomic variation. Because of the vast body of literature on allozymes and mtDNA, these will provide a baseline against which to judge other methods.
Mathematical modelling.

Recent analytical work has enhanced the utility of population genetic models in estimating demographic parameters. Members of our group (van-Oppen et al., 1997) have developed further the use of expectation based estimators of parameters such as effective population size while others have derived analytically the equilibrium values of these parameters for the various sequence and microsatellite mutation models (e.g. Rousset, 1996).In addition, a number of other workers with whom a member of our group (K M Ibrahim3 has collaborative links are exploring the combined use of coalescent theory and likelihood methods to derive robust parameter estimates by making use of all available data relating to the population biology of experimental species. The data generated in this study will enable members of our group to evaluate and make best use of these advances in analytical and theoretical population genetics. We shall pursue this in order to achieve the aim stated in our proposal; namely, to enhance the reliability of parameter estimates and to deal with the physical features of the study area and the biological traits of the study organisms.

REFERENCES

Arnason, E., Polsson, S. and Arason, A. 1992a. Gene flow and lack of population differentiation in Atlantic cod, Gadus morhua L., from Iceland, and comparison of cod from Norway and Newfoundland. J. Fish Biol. 40:751-770.
Bucklin, A., La Jennesse, T.C., Curry, E., Wallinga, J. and Garrison, K. 1996. Molecular diversity of the cope pod, Nannocalanus minor Genetic evidence of species and population structure in the North Atlandtic Ocean. J. Marine Res. 54: 285-310.
Bussman, B. 1984. investigation on stock separation of bluewhiting (Micromesistius poutassou, Risso 1810) in Northeast Atlantic. Meereforschung Rep. Mar. Res. 1984 (30): 196-208.
Carr, S.M., Snellen, K.A., Howse, K.A., Wroblewski, J.S. 1995. Mitochondrial DNA sequence variation and genetic stock structure of Atlantic cod (Gadus morhua) from bay and offshore locations on the Newfoundland continental shel£ Mol. Ecol. 4:79-88.
Carvalho, G.R. and Hauser, L. 1995. Molecular genetics and the stock concept in fisheries. In "Molecular genetics in fisheries'' G.R. Carvalho and T.J. Pitcher (eds),New York: Chapman and Hall. 55-79pp.
Carvalho, G.R. and Pitcher T.J. 1995. Molecular genetics in fisheries (eds), New York: Chapman and Hall. 141pp.
Cross, T. F. and Payne, R. H. 1978. Geographic variation in Atlantic cod, Gadus morhua, off Eastern North America: A biochemical systematics approach. J. Fish. Res. Board Can. 35: 117-123.
Dahle, G. 1991. Cod, Gadus morhua L., populations identified by mitochondrial DNA. J. Fish Biol. 38:295-303.
Gyllensten, U. 1985. The genetic structure of fish: differences in the intraspecific distribution of biochemical genetic variation between marine, anadromous and freshwater species. J. Fish. Biol. 26: 691-699.
Hislop, J.R.G. 1984. A comparison of reproductive tactics and strategies of cod, haddock, whiting and Norway pout in the North Sea. In " Fish reproduction: strategies and tactics. G.W. Potts and R.J. Wootton (eds) Academic Press, London:
Isaev, N.A. and Seliverstov, A.S. 1991. Population structure of the Hebridean- Norwegian school of blue whiting, Micromesistius poutassou, . Ichthyol. (31): 45-58.
Mork, J., Ryman, N., Stahl, G., Utter, F., & Sundnes, G. 1985. Genetic variation in Atlantic cod (Gadus morhua) throughout its range. Can. J. Fish. Aquat. Sci. 42 (10): 1580-1587.
Pogson, G.H., Mesa, K.A., Boutilier, R.G. 1995. Genetic population-structure and gene flow in the Atlantic cod
Gadus-morhua - a comparison of allozyme and nuclear RFLP loci. Genetics 139: 375-385
Rico, C., Rico, I. & Hewitt, G. 1996. 470 million years of conservation of microsatellite loci among fish species. Proc. Roy. Soc. B. 263: 549-557.
Rousset, F (1996). Equilibrium values of measures of measures of population subdivision for stepwise mutation process. Genetics 142: 1357-1362.
Smith, P. J., Birley, A. J., Jamieson, A. and Bishop, C. A. 1989. Mitochondrial DNA in the Atlantic cod, Gadus morhua: lack of genetic divergence between Eastern and Western populations. J. Fish Biol. 34:369-373.
Utter, F.M. 1991. Biochemical genetics and fishery management: an historical perspective. J. Fish Biol. 39 (Suppl.A): 1-20.
van-Oppen, MJH, Turner, GF, Rico, C, Deutsch, JC, Ibrahim KM, Robinson, RL and Hewitt, GM (1997). Unusually fine-scale genetic structuring found in rapidly speciating Malawi cichlid fishes. Proc. Roy. Soc. B 264:1803-1812.
INTRODUCTION

The effective management of marine fisheries requires the ability to discriminate among stocks. One needs to identify non-interbreeding populations, to assess the gene flow between different genetic stocks, and to monitor temporal changes in the gene pools (Carvalho & Hauser, 1995 and references therein). Many freshwater and anadromous fish stocks were readily identifiable by protein electrophoresis (Utter, 1991), but until the mid 1980s, little difference was detectable between adjacent marine fish groupings (Gyllensten, 1985; Ward et al, 1994).

Subsequently, DNA methods (Carvalho & Pitcher, 1995) have shown inconsistencies when different types of genetic markers were used (Carr et al, 1995; Pogson et al, 1995 and references therein). Low levels of differentiation in marine fish species have been attributed to the large size of most marine fish populations (i.e. little genetic drift), to limited migration between marine populations, or to the recent origin of populations, possibly involving a bottleneck followed by a radiation event (Smith et al, 1989; Arnason et al, 1992). The level and significance of gene flow in the ocean remains controversial (e.g. Pogson et al, 1995 Bucklin et al, 1996).

Most gadoid and merlucid species produce large numbers of pelagic eggs and their life histories are highly conducive to dispersal, perhaps over intercontinental distances (Hislop, 1984). Many of them support major commercial fisheries in the Northeast Atlantic and Mediterranean sea, and are major components of the ecosystems of these waters (Arnason et al, 1992; Hislop, 1984). The genetic structure of gadoid populations has been assessed utilising allozyme and mtDNA analyses, especially for Atlantic cod, Gadus morhua (L.), (Cross & Payne, 1978; Mork et al, 1985; Smith et al, 1989; Dahle, 1991; Arnason et al, 1992) and blue whiting, Micromesistius poutassou (Risso) (Bussmann, 1984; Isaev & Seliverstov, 1991).

The major aim of the present study is to seek to provide improved methodology for fish stock discrimination through the application of modern molecular genetic methods. This will be achieved by studying situations where populations are known to be genetically isolated and then, those where different stocks are suspected but have not been demonstrated by previous genetic methods. This project will specifically attempt to:

i) calibrate the most discriminatory genetic methods (i.e. PCRable mini- and microsatellites single locus VNTRs, certain mtDNA transcribed sequences, and nuclear DNA sequences and RFLP loci including MHC and cDNA's) for use in the detection and characterisation of marine fish stocks at different levels of genetic differentiation using four gadoid species; Atlantic cod Gadus morhua , blue whiting Micromes istius po utassou , hake Me rluccius me rluccius , and poor-cod Trisopterus minutus.

ii) develop mathematical models to estimate effective population sizes for stock assessment based on the genetic data obtained using the proposed molecular techniques.

MATERIALS AND METHODS

We set out to study the population genetic structure of Atlantic cod, blue whiting, hake, and poor-cod, throughout their range using mini-, microsatellite DNA, mtDNA control region and transcribed sequences, and nDNA, including anonymous and/or characterised cDNAs and MHC genes, and protein electrophoresis as genetic markers. Significant inter- and intra-specific differences in genetic differentiation are predicted for stocks whose biology and/or distribution is characterised by large differences.

Samples of 50 fish or more are being collected, preferably during the spawning season, from at least six distant geographical location throughout the range of each species, and then analysed using these molecular markers. Where different sub-species or closely related species occur these will be sampled in some cases as an out-group control. This will elucidate the macro geographic genetic structure of the resource. Then, samples of 50 fish will be collected simultaneously and for two consecutive years, from two or three adjacent geographical locations. One sample will be taken during the spawning season and one sample, six months later during the aggregations on the feeding grounds. Only the former sample will be taken for two years to investigate inter-year variation. Samples will be then analysed with the proposed molecular markers to establish the micro geographic genetic structure of the species. Extensive literature reviews are being carried out through computerised databases, to gather all relevant biological information of the species studied. Specific environmental aspects such as predominant oceanic and tidal currents and presence of presumed geographical barriers relevant to species studied are also the subject of extensive literature reviews.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

University of East Anglia
Address
University Plain
NR4 7TJ Norwich
United Kingdom

Participants (4)

Marine Research Institute of Iceland
Iceland
Address
4,Keldnaholt
112 Reykjavik
THE CENTRE FOR ENVIRONMENT, FISHERIES AND AQUACULTURE SCIENCE
United Kingdom
Address
Pakefield Road
Lowestoft, Suffolk
UNIVERSITY COLLEGE CORK
Ireland
Address
Lee Maltings Prospect Row
Cork
University of Trondheim
Norway
Address
Bynesveien 46
7018 Trondheim