DNA-based tracking for salmon farming

Nr. 1 / May 2014

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Escape of farmed salmon is an undesirable event which has negative financial consequences for the farmer, and can lead to genetic impacts on wild salmon stocks. With an increased focus on a sustainable industry throughout the whole value chain, the introduction of a tracking system is a key element of the strategy for documenting the provenance of farmed salmon, and at the same time accountability for salmon producers.

DNA – the salmon’s identityCRW_5738_1-m-DNA-2

A salmon’s DNA – its genetic material – tells a story which stretches back to the very origin of the species, and can provide information about the unique history of the species’ development. Since the structure of DNA was first described by the pioneering molecular biologists Watson and Crick in 1953, we have little by little learned to decipher the genetic code. Advances in technology mean that we can retrieve ever more information from DNA from each individual salmon, and use this information for various practical purposes. The salmon inherits half of its genetic material from each of its parents, and has a great deal in common with its siblings. Each individual salmon nonetheless has its own unique DNA variant which makes it possible to determine kinship between different individuals.

Using DNA to track the provenance of salmon

The method of utilising DNA as a tracking tool was developed within the field of forensic science as early as the 1980s and today there are many examples of how genetic technology can be used to follow biological tracks. Since 1992 all paternity testing in Norway is carried out exclusively by means of DNA analysis. The mass media has popularised the concept of biological tracks in the context of solving crimes. This tracking system is made possible by the fact that a register has been established containing DNA profiles of persons who have been sentenced to serious criminal acts. Not so long ago a major food scandal arose in Europe after DNA testing uncovered the use of horse meat in products declared as beef. In a similar case, it was revealed that there were traces of pork in halal meat served in British prisons, with much media coverage as a result. DNA is used at present to verify the correct labelling of food with respect to species, provenance and country of origin. This is of ever greater significance in the global marketplace. In particular in the case of food manufacturers it is invaluable to be able to demonstrate responsible practice at all points of the chain of production, and traceability is a precondition for a sound strategy connected to food safety and sustainability.

What can the DNA of salmon tell us?

There are over 400 rivers in Norway containing local salmon populations, and salmon are on the whole in the habit of returning to the river they originated from in order to spawn. This inherited behaviour means that different populations of salmon are genetically distinct from one another, and DNA tracking can be used to determine a given salmon’s genetic provenance and thereby the provenance of a given wild salmon. Genetic markers have long been used in the management of Pacific salmon to regulate the fishing of particularly vulnerable salmon stocks. At the coast of Western Greenland a gathering of salmon of diverse origins is seen, both Canadian and European Atlantic salmon, and the use of DNA analyses is regulated in a transatlantic cooperation in order to determine which stocks that are fished in this region. Correspondingly, DNA analyses are used to monitor the development of different stocks of Baltic salmon. It is also expected that DNA analyses will become an important tool for the management of wild salmon in Norway as well. A research group consisting of participants from Nofima, NINA, CIGENE and AquaGen published in 2011 a study which shows that by means of a panel of 60 genetic markers it is possible to distinguish farmed salmon from wild salmon with a high degree of probability. With the project Quant Escape, NINA together with its partners wishes to characterise and quantify the flow of escaped farmed salmon back to wild salmon, amongst other things through the use of powerful genotyping tools developed by researchers at AquaGen and CIGENE. This will form an important knowledge base for following the genetic identity of wild salmon in the future.

Figure 1. The illustration shows one defined section of a DNA strand from two different fish. The letters indicate the four bases, A (Adenine), C (Cytosine), G (Guanine) and T (Thymine) which are elements of the nucleotides which the DNA strand is made up of. The sequence and which bases are found at specific points are decisive with respect to which types of proteins are coded for and which functions these receive in the fish. This can, once again, contribute to differences in characteristics in the fish. DNA analyses to establish the variations in the bases can be carried out by means of SNP (single nucleotide polymorphism) genetic markers and/or microsatellite genetic markers.

On the east coast of Canada and the USA, DNA tracking of farmed salmon has already been implemented. The Canadian and American authorities stipulate that breeding companies must archive DNA profiles of all fish which are used to produce eggs. This makes it possible to identify and track escaped fish back to the company responsible. In Norway a number of studies have been launched, financed by the Norwegian Seafood Research Fund, which amongst other things has evaluated and optimised methods for use in DNA tracking of escaped farmed salmon. These studies have demonstrated that combining DNA analyses of spawning fish by controlling unique batches of eggs from the egg producer to the hatchery and fish farms provides a reliable tracking of escaped farmed fish.

Genotyping tools for Atlantic salmon

Traditionally, so-called microsatellitebased genetic markers have been used to analyse the DNA of salmon, both in breeding and for the purposes of tracking. In recent years a new method has been developed, so-called SNP analyses, which are more precise and are simpler to carry out where there are large quantities of samples (see Figure 1, which illustrates the difference between SNP and microsatellite markers). The great advantage of the use of socalled SNP panels or SNP chips is that a large number of markers can be tested simultaneously, ideally several tens to hundreds of thousands of markers in the same analysis. So far this technology has seen only limited use outside of the sphere of human medicine, because each individual analysis has proved expensive. Costs have however now been reduced, and it is now possible to implement the establishment of a DNA-based data bank of all broodfish which produce eggs for aquaculture in Norway at an acceptable price

How can it be done?

Figur 2. DNA-sporing av foreldrene til hver enkelt rognleveranse utføres ved at vevsprøver av alle hunn- og hannfiskene blir tatt ved befruktningstidspunktet. Vevsprøvene registreres elektronisk slik at resultater fra genotypingen kan spores tilbake til riktig rognleveranse og oppdretter i etterkant.

Figure 2. DNA tracking of the parents of each individual egg delivery is carried out by taking tissue samples of all female and male fish at the time of fertilisation. Tissue samples are registered electronically such that the results of genotyping can subsequently be traced back to the correct egg delivery and farm.

Egg producers can take a single tissue sample of each broodfish at stripping (Figure 2). Here it is simply necessary to follow established routines since most of the fish have already been genotyped for other genetic markers in socalled QTL-based selection. Strict routines must furthermore be observed for electronic tracking of both broodfish and eggs, and a clear separation must be maintained at all times between the various batches of eggs so as to prevent eggs from the same parents being sent to more than one hatchery. This will reduce flexibility in egg production and thus it will be necessary to provide a larger buffer capacity, albeit within an acceptable range and without this bringing about a disproportionate increase in cost. The tissue samples are sent to a laboratory for DNA purification, and then sent for genotyping. The genotyping results for the mother and father are sent to a database for archiving. In the event of an escape incident the escaped fish will be genotyped and compared against the database. If the mother and father are in the database, the provenance of the escaped fish will be able to be established with 100% certainty. Alternatively it will be possible to rule out that a suspected escaped fish originates from a particular facility if the facility in question has fish whose parents have been genotyped and entered into the database. In order to achieve close to 100% certainty in the analyses it is necessary for the salmon to be analysed for a minimum of 1000 genetic markers. If the number is increased to 10,000, confidence is extremely high and can analyse fish which are closely-related with a large number of possible parents in the database.

High density genetic marker sets in use

AquaGen and CIGENE have worked together to develop two high-density SNP panels for salmon. This work is based on full genome sequencing of close to 30 salmon. This resulted in the identification of more than 5 million SNP markers, of which a selection of 930,000 markers was used in the first SNP panel (930k). Around 1000 fish have been genotyped for 930,000 SNPs, and the 220,000 SNPs which gave the largest amount of information were used for the other SNP panel (220k). This has once again been used for genotyping of almost 7000 salmon, 1000 of which are wild salmon from the Quant Escape project. Several breeding companies breeding both domestic animals and plants have found that a SNP panel of around 50,000 SNPs provides an ideal amount of information relative to the costs connected with genotyping.  CIGENE is in the process of upgrading its instrument bank so as to be able to run a larger number of tests in a shorter timescale. AquaGen will use 50,000 of the best SNP markers for use in breeding work. AquaGen has invested a lot of time and resources into developing good genotyping tools, and will be able to develop a specially-designed tracking panel which contains 10,000 SNPs. This panel will be made accessible so that broodfish from other breeding programmes can be genotyped on the platform and included in the database, and the panel will form a crucial tool for studying wild salmon in general and especially interactions between farmed salmon and wild salmon (Figure 3).

Figure 3. DNA from a possible escaped farmed salmon can be genotyped and checked against the database of farmed salmon parents. If the DNA analysis shows a positive match between the salmon and the parents in the database, the specific information regarding the egg delivery can be traced back to the correct hatchery with 100% accuracy. Alternatively, the analysis can also reveal whether the salmon belongs to one of various wild salmon stocks or mixed salmon stocks consisting of a mixture of wild salmon and farmed salmon.

 

Genotyping, operation of the database and assignment of escaped fish can be carried out by CIGENE, with reporting back to the breeding company responsible for ongoing tracking of the delivery together with the farmer. As we are at present seeing an ever increasing share of integrated operations, it will in most cases be possible to identify the company responsible. In the case of independent smolt producers, tracing the company responsible can present more of a challenge, but in this area production can be organised so that each smolt customer receives fish from separate egg batches.

How quickly can this be implemented?

The three north-Norwegian fish farmers Cermaq, Nordlaks and Nova Sea are implementing tracking for all egg batches delivered from October 2014 onwards. Following this, it will be possible for incidents of escape in these companies’ areas of production to be checked against the parentage database, and thereby for the involvement of these companies to be confirmed or rule out. The investigation will also reveal any possible wild fish which have mistakenly been suspected of being farmed fish. This system can be easily expanded if other fish farms or breeding companies wish to introduce genetic tracking, and ultimately the establishment of a national platform will be possible. AquaGen has made the genotyping tool (10,000 SNPs) accessible for tracking purposes. CIGENE, in the capacity of an independent third party, carries out genotyping, operates the database and assigns broodfish parents. The breeding companies own the results of the genotyping of their own stock, but make the results accessible for the purposes of tracking.

DNA tracking gives secure answers

The greatest advantage of DNA tracking is that it is based upon an underlying technology which has over the course of the past 20 years been developed, optimised, approved and implemented for various purposes of benefit to society. The method has achieved a high degree of reliability and general acceptance both in the legal system and in society as a whole. Implementing this method for use in tracking escaped farmed salmon does not entail any physical marking which could cause the fish pain or discomfort. Nor does it entail any extra sorting or handling of the fish, nor special diets. The marking is also very secure since it cannot fall off or be altered in the course of the fish’s lifetime. The cost is low compared with alternative methods, notwithstanding the fact that it is applied to both broodstock and the egg stage. On a national level this means that the cost of genotyping and operation of the database for an annual quantity of 50,000 broodfish will be divided between the 350 million eggs produced. In addition comes costs associated with adapting production both on egg, hatching and farming phases, but notwithstanding this the costs remain low compared to many of the proposed alternatives. DNA tracking is used at present to a limited extent in research and mapping of wild fish stocks. The method can reliably and efficiently distinguish farmed salmon from wild salmon and also distinguish different wild salmon stocks from one another. By expanding use of this method of tracking to encompass tracking of escaped farmed salmon, it will simultaneously become possible to build up a knowledge base relating to the central challenges within wild salmon management, such as the extent of accidental migration of different wild salmon stocks and the occurrence of hybridisation between farmed and wild salmon.

A combination of several methods probably gives the best results

All of the possible tracking methods evaluated have both weaknesses and disadvantages. It may therefore be appropriate to utilise a combination of methods, such as DNA tracking in combination with mineral analysis of fish scales. These two approaches are in many respects complimentary, and can supplement one another should there be any doubt.

The DNA method can be implemented immediately and will provide reliable tracking back to the hatchery. In instances of mixing of smolt groups at the hatchery, and possible distribution of the same smolt group to several on-growing sites, DNA tracking will reduce the number of potential sources of escaped fish to just a handful of facilities. The mineral element method has the potential to provide more specific information that reflects water source in freshwater and seawater.

Facts about genetic markers

A SNP (single nucleotide polymorphism) genetic marker reveals a single base at a specific point on the DNA strand. By using a combination of a number of different SNPs spread over the genome, a high level of certainty can be achieved in the determination the genetic characteristics of an individual.
A microsatellite genetic marker locates areas where there are many consecutive recurring bases on the DNA strand. The bases located close to the microsatellite will as a rule also be conserved at reproduction, and some key genetic traits have been linked with such microsatellites. This type of marker is most used in kinship tests, but SNP analyses have become more relevant due to increased precision, effectiveness and lower costs compared with microsatellite analyses.