Designing Probes That Will Be Used to Identifying Patagonian Toothfish Haplotypes - Term Paper Example

Designing Probes That Will be Used to Identify Patagonian Toothfish Haplotyes and Use Then to Develop a new Identification Device Submitted by: To: In Application For: Table of Contents Lay Description: 3 Project Synopsis: 4 Hypotheses: 4 Expected Outcomes and Significance: 4 Background and Research Plan: 7 Background: 7 Research Plan: 15 Environmental Sampling 16 Tissue Biopsy 16 DNA Extraction 17 PCR Sequencing of mtDNA 18   Determination of the Haplotype frequencies 18 Designing the Probes 19 Developing the Device 19 Proposed Timetable: 20 Title: Designing Probes That Will be Used to Identify Patagonian Toothfish Haplotyes and Use Then to Develop a new Identification Device Lay Description: The increasing consumption of fish and fish products over the last decade has led to an increase in the need to identify the fish for purposes of both regulatory enforcement and consumer protection (Fao/Who, 2009). In particular the increase in the numbers of mislabeled fish and their products has led to illegal, unreported and unregulated fishing. This is a serious problem in the world market and one of the main hindrances of attaining sustainable fisheries (Marko & Lee, 2004). As a result, there has been a decline and collapse of many of the world’s finest fisheries leading to the promotion of consumption of sustainably harvested seafood that arises from the implementation of social marketing. Genetic analysis of Patagonian Toothfish is carried out on retail fish with eco-labels marketed as ‘Chilean sea bass’ to ensure that they are Marine Stewardship Council (MSC) certified. However, this has been found to be inaccurate and misleading. In solving this problem, we propose to design probes that are unique to Patagonian Toothfish Haplotyes which then be used to develop an identification device (Wong & Hanner, 2008). This development will be useful in forensic identification of the real Patagonian Toothfish that are being sold in the market and differentiate them from the mislabeled products by the fraudsters. This information which is also a great leap forward in the forensic equipment technology will lead to commercial production of the device that can be used in Patagonian Toothfish identification in an easy and affordable but sure manner. Project Synopsis: Aims: The main aim of this project is to develop genetic probes that will be used to identify the Patagonian Toothfish Haplotyes. Specifically, this involves designing probes with enzymes that are specific to Patagonian Toothfish Haplotyes immobilized on the surface of the device that will be used for identification (Ludwig, 2008). The ultimate aim is to commercially produce the device that will make it easy to identify Patagonian Toothfish in the social market and help solve the problem mislabeled Patagonian Toothfish. Hypotheses: The hypothesis to be tested is that the designed Patagonian Toothfish Haplotyes will lead to the development of a device that will provide an appropriate method and accurate way of identifying Patagonian Toothfish in the market. Expected Outcomes and Significance: In August, an article appearing in the journal of current biology revealed that some of the Patagonian toothfish appearing in the market with MSC eco-labels are not from the sustainable fishery while others are not in any manner toothfish (Marko & Lee, 2004). More reports assert that no all fish with the MSC eco-label originated from the certified fisheries. Forensic investigation revealed that there were significant genetic differences between the certified stock population and the retail sample. The retail labeling of the MSC certified Patagonian Toothfish has been found to be inaccurate and the labeling of the country-or-origin is misleading. The expected outcomes of this study will be geared towards solving the problem outlined above. The first outcome expected from this project is to provide a wealth of information pertaining to the idea that different types of fish have a varied genetic make-up. In addition to this, different species have a genetic signature pertaining to their genetic structure (D. Rogers et al, 2008). The research will also share the information that species separated by geographical factors may also have different genetic make-up which makes it easy to identify fish with respect to the country of origin. This study will therefore allow the development of genetic probes specific to the Patagonian Toothfish Haplotyes of the fish commonly known as the ‘Chilean sea bass’ (Dissostichus eleginoides). Secondly, it is expected that the designed probes will lead to creation of a database as a result of the success of this study from which information concerning the specific toothfish will be fished from and analysis done on them during future investigation of the mislabeled Patagonian Toothfish. Thirdly, it is expected that the results of this research will not be just exclusive to the proposed study but the information and the databases that will be generated will pave way for further research into identification of other types of fish which are important to the Australian market. Fourthly, the results of this study will help in potential development of more identification devices that will be used as testing equipment of fish in the market which will allow fast and accurate determination of the type of fish and country-of-origin on site without having to waste time of going to the laboratory first. This means that testing and identification can be done anywhere and at any time. Although the analysis of mitochondrial DNA (mtDNA) using all time techniques such as microarrays, PCR, BstN1 digestion, and nucleotide sequencing is a trustworthy and reliable method of identifying Patagonian Toothfish and its products, they can really be expensive in terms of time, cost, equipment and skilled man-power. This can be very costly, time consuming and frustrating which makes the body involved in the certification and identification to be reluctant to take up such procedures. As a result, the certifications they come up with become inaccurate and misleading making the consumers to be at risk of consuming products that are likely to be of harm to their health. This study is therefore very significant to combating such problems. First of all, being able to specifically and accurately design for probes that will be used for specific Haplotypes of the Patagonian toothfish is a major significance in making sure that the fish in the market is of the right species. With this kind of information, investigators can be able to identify the Patagonian toothfish that is required in the market and separate it from those that have been mislabeled as it. Another major significance is the fact that this study will provide a wealth of information that can help to identify the Patagonian toothfish with respect to origin. This will greatly assist in determining that the fish under investigation is actually Chilean sea bass and not from any other origin (Jurenas, 2010). The third significance of this study is the potential development of a device that will be used to accurately and rapidly identify Patagonian toothfish without having to actually go to the laboratory. The device will bring with it the potential of developing a method that will involve fish identification in the market for purposes of getting rid of mislabeled Patagonian toothfish for good and to ensure the conservation of the same. This will make sure that only fish from the sustainable fisheries enter the market and are allowed to be passed on to the consumer. The significance of the outcome of producing the device is that it will be an easy to use device that can be used by investigators with no scientific skills which removes the requirement on skilled personnel in order to carry out the investigation (Ludwig, 2008). The fact the device is to be produced commercials has great economic significance to the future Australia. Background and Research Plan: Background: The Patagonian toothfish (Dissostichus eleginoides) is commonly referred to as the Chilean sea bass. It is the most valuable, demersal fish species found in Antarctic or sub Antarctic waters. It is large and grows up to a length of 2 meters with a lifespan of approximately 50 years. From six to 10 years of age, the fish becomes sexually mature, at a time it measures about 70 to 90 cm. this species of fish is said to have a low fertility rate producing between 48 000 and 500 000 eggs per its spawning season. It has a very low resilience with the population taking about 5 to 15 years to double. Its habitat is in the temperate waters of the Antarctic. The Chilean sea bass is mainly found in the Exclusive Economic Zones (EEZ) of Argentina and Chile and the sub-Antarctic islands under the jurisdiction of Australia, South Africa, France, United Kingdom and New Zealand. The major fishing areas for the Patagonian toothfish are Pacific Southeast, Pacific Southwest, Atlantic Southeast, Atlantic Southwest, Indian Ocean Antarctic, Indian Ocean Western, Atlantic Antarctic and Atlantic Southeast. The main catch areas as shown in the figure below: Figure 1: Landings of Patagonian Toothfish by main catch area, 1977-2000 (Fishstat + data, 2000) The most important fishing location is the Atlantic Southwest which produced 11000 (Metric Tones) MT in the year 2001 closely followed by the Southeast Pacific which produced 10500 MT. Other productive catch areas are the Indian Ocean Antarctica producing 9000 MT and Atlantic Antarctic with 4 700 MT (Fishstat + data, 2000). It is known that the market name for the Patagonian toothfish is Chilean sea bass selling at a little over $10 per kilo in the main markets. Its large fleshy body with few bones coupled with the long lifespan and late sexual maturity has made this species of fish susceptible to over fishing. Large-scale illegal fishing of the Chilean se bass has increased over the last few years. There have been increasing attempts to poach the fish from the major catch areas with the first numerous vessels illegally fishing first spotted in the year 1996. The fishing of the Patagonian toothfish started in the early 1970s in the sub Antarctic waters in the southern hemisphere. Before then, the toothfish had been caught in minor quantities from the grey rock cod and marbled rock beds especially in Kerguelen and South Georgia Islands. It was not until the years 1986 that the toothfish was caught in commercial quantities after a great deal of them was discovered in Kerguelen. However, the Chilean coast had been recognized as a substantial fishery from the year 1972 and many markets had already started recognizing this species. From that time, the fisheries specifically for this species have continued to de extensively expanded and rapidly developed to different locations like the New Zealand and Islands under the Australian Sovereignty. In the early 1990’s, the trawl fishery for this species started in Australia at the Macquarie Island which was immediately followed by the establishment of the fishery at Heard Island in the late 1990’s. Even though the fishing of the toothfish started as trawl fishing, it has since developed into fishing using the longline except for the fisheries in Australia. During the 1999-2000 periods, more than 14,500 MT of toothfish fished in the sub Antarctic. The Patagonian toothfish now occurs throughout the entire southern hemisphere due to the cool temperate climate and the waters of the sub Antarctic that are favorable to their growth and development. They usually inhabit deep waters from 300 m to a little over 2100 m in depth. From their habitat of choice, it is clear that the Patagonian toothfish is found close to the bed when fishing. It is a large and active predatory fish that gets its food mostly from the columns of water. They feed on squid and other fish but their diet is quite varied including the spawns and crabs which organisms are living on the sea bed. The Patagonian toothfish is a very important species of fish that now represents a fishing industry that is described to be a multi-million dollar industry in many countries. This has largely contributed to the economies of such countries in various ways counting that such a great industry provides employment. Fishing for the Patagonian toothfish is a major source of livelihood for fishermen in Chile, Argentina and other countries found within the catch areas of this toothfish. Some countries like Chile export the fish in bulk. The sea food usually undergoes a processing procedure before being exported. The exported product is usually beheaded and gutted and frozen or sold as fresh fillet. The export of this fish leads to the exporting country getting foreign exchange which increases the firm stand of such countries in the world market. In addition to this, there is no doubt that exporting the seafood adds value to the relationships between the countries involved thereby promoting international peace. One of the greatest importance of the Patagonian toothfish is the fact that it food for human consumption. There is no more benefit that it can give to the world apart from being one of the most preferred sea foods across the nations of the world. Its nutritional value makes everyone want to have a bite of it. Its taste is famous among the lovers of sea food. Its nutritional value changes from one environment to the next. Its flesh is extremely delicious close to that of the shrimp or the lobster. It is almost boneless and it rich in omega 3 which is an essential fatty acid that helps in reducing the risk of being susceptible to cardiovascular and inflammatory diseases. Eating the fish also helps you to develop a glowing skin and strong health hair. Its nutritional contents also help in strengthening the immune system as well as improving the overall health of a human being. When the Patagonian toothfish gets to the market, it usually has the MSC eco-label. However, there have been numerous cases of the presence of the Patagonian toothfish in the market that has been mislabeled. Although they bear the MSC eco-label, they are said not to originate from the fisheries certified by the MSC while others are not at all the Chilean sea bass. The MSC has since directed. The MSC has directed different researchers such as Peter Marko (2009), a professor at Clemson University who lead a research on the mislabeled toothfish to sample the necessary information needed to establish the truth of the products under investigation are really mislabeled. For a long time, there has been lack of such information like, time and date of sampling, where the sampling takes place, chain-of-custody certificate from MSC and the sampled product. What there we have are just promises to the MSC that the information will be available in the future. Another important research into the identification of mislabeled Patagonian toothfish was done by Dodgers, et al (2006). The findings of their research raised different issues about how much the MSC ‘Chain of Custody’ can be trusted for retail fish believed to be from certified fisheries that are known to be sustainable fisheries. At some point a long the supply chain, staring from the certification of a location as sustainable fishery by the MSC to the market where the retail fish has an eco-label certificate showing that it comes from a sustainable fishery, a great number of impostors come up. So, how just does the identification of the haplotype of Patagonian toothfish happen? The analysis of the mDNA (mitochondrial DNA) the Patagonian toothfish takes place so that it is compared against those that are thought to be “fake.” This happens with the use of genetic markers which happens with the use of Restriction fragment length polymorphisms (RFLPs) which are analyzed on two mtDNA. This helps in examining the haplotype variation in the Dissostichus eleginoides as in the research done by Appleyard at al (2002). The first region consists of the fragment referred to as the variable control fragment which is the BLC with the D-loop (Gaffney, 2000). This is usually amplified using primers such as 12SAR-H. This fragment is 1.3 kb that is then digested with the BstNI. The second fragment of the mtDNA is the ND2 that has NADH dehydrogenase attached to it as a subunit 2 gene and is usually amplified with the use of t-met primer and the Mt-76 primer(Park at al, 1993). This second fragment is approximately 1.1 kb and is digested using NIaIII. Seven loci of DNA micro satellites are then used to examine the nuclear variations of genes within the retail collection of the Chilean sea bass. These seven DNA micro satellites are To5, To2, cnrDe2, cnrDe9, cnrDe30, anrDe13 and cnrDe4. PCR amplification is then done for both the micro satellites and the mtDNA using a PE-Applied thermocyclers. The digested mtDNAs are then electrophoresed in 2.6% agarose gel (Appleyard et al, 2000). For the results of the mtDNA analysis, each of the patterns from the fragment’s two regions resulted in the formation of a complete haplotype. The molecular diversity of the haplotypes and their frequencies are then done for the Patagonian toothfish which is them compared to that of the retail collection under investigation. The specific tests for the mtDNA for the haplotype frequencies is then used to determine the frequencies of the haplotype in the sample using a method called the Analysis of Molecular Variance (AMOVA) (Excoffier et al, 1992) and the f-statistics are used to assess the differences among the collections. When the BCL fragment is digested using the BstNi on different samples of Patagonian toothfish from various habitats, this results in five haplotypes A,B,D,E and F while the digestion of the ND2 fragment gives rise to two haplotypes C and J after it has been digested with NIaIII. A total of seven different composite haplotypes are identified. Each of the collection has a common haplotype A. Further research has also show that there are different lineages of haplotypes of the Chilean sea bass from different habitats. This is due to the strong latitudinal differences in the sea temperatures. This results if large differences in mtDNA due to different environmental conditions causing selective identification for different haplotypes (Crochet et al. 2003). This therefore makes it easy for the proposed development of the device that will assist in identifying the origin of the fish in the market because the haplotypes are specific to the habitat. The analysis of mtDNA for the Patagonian toothfish purchased form the retail collection have resulted in 8% of the total toothfish sampled to be of other species. 155 to 34% of these fish had variations in the mtDNA that are unknown but they have been certified to be from the Chilean sea bass fishery. Further investigations have shown that their origin is actually the South Ocean between the southern tip of south America and Antarctica. Therefore, improperly labeled fish continue to be sold in the market. This means that there is still a gap in the certification and labeling of the Patagonian toothfish. Where and how the mislabeled toothfish make its way to the market is still a big question (Marko, et al, 2011). The gap is widened by the fact that MSC published it study in 2009 on the integrity of the certification process and the Patagonian toothfish supply chain stating that there was no evidence of illegal fish in the market nor mislabeling. This then forms the basis of our research proposal. The design of the Pataogonian toothfish haplotypes and the design of a device based on the haplotypes will help a great deal in ensuring that the problem is reduced. The device will use probes that are specific to the Patagonian tootfish Haplotypes. The idea of using the haplotypes in conj suction with molecular markers is good in winning the fight against the poachers and the people selling the illegal toothfish. The device will have a significant focus on the use of genetic markers of the Patagonian toothfish for its identification in the market. It will be used in the detection of false Chilean se bass which will be an important step towards combating the problem. This device will replace the current method of mtDNA analysis and using it to compare with other stocks in the database. This device will measure the level of Haplotype frequency by measuring the amount of color for each specific haplotype that is tested for. It will also be very helpful in determining the origin of the fish because the studies show that the populations of Patagonian toothfish from one habitat are genetically distinct from those from other habitats. Therefore, the device will help in recognition of those Patagonian Toothfish that are from the sustainable fisheries and differentiate from those that have been mislabeled to come from the sustainable habitats (Dale, 1999). It still remains a fact that the great idea of investigating the location of where the fish came from is a viable step. The scientific work in this area of profiling the genetic makeup of the species with respect to their origin is ground breaking. This has however reached a plateau because the fish certified by the MSC to be from the sustainable fisheries are usually mixed with those from other locations. The greatest gap is in the lack of practical application and follow-up research into the cost and efficiency of the instruments used. This research proposal will be geared towards inventing the mechanism that will identify, beyond any reasonable doubt the real identity and origin of the fish that is present in the market. Research Plan: The method employed in this study is geared towards provision of the most effective and reliable way of designing the genetic probes for the Patagonian fish haplotypes and coming up with a device that will be used to identify the real Chilean sea bass in the market and differentiate it from the mislabeled ones. The technique to be used in this study will be aimed at providing the most effective and reliable way to identify the origin of Patagonian toothfish for each sample under study This technical workflow follows a slightly modified version of that described in an comprehensive review of the process and a similar workflow described other researchers working on similar studies (Taggart et al, 1992). Wherever it was possible kits have been integrated into the workflow. These manufactured kits have already been broadly optimized and this will increase analysis efficiency. The techniques utilized in this pilot study have been chosen because they allow analysis of each species of Patagonian toothfish and their haplotypes in a sample and given the advances in technology these methods are now efficient and relatively cheap. In accordance with Workplace Health and Safety, Standard PC2 Laboratory Workplace Health and Safety (WHS) Protocol will be followed and this protocol may be found in the Laboratory Quality Manual (LABDOC#1528). In field methods will follow the agreed WHS protocol between the laboratory and the Brisbane City Council (WHSRFPDOC#1277). Environmental Sampling The waters surrounding the sub-Antarctic island of South Georgia and the nearby plateau at Shag Rocks are the only sustainable fisheries certified by the MSC. All the Patagonian fish habitats in the southern hemisphere including the Heard Island and McDonald Islands fisheries in Australia have been chosen for this study. The Heard Island and McDonald Islands fisheries are ecologically viable for the growth of the Patagonian toothfish and have also been licensed by the Australian Fisheries Management Authority (AFMA) for Patagonian toothfish catching. They are also ecologically similar to the fisheries in Chile where the majority of the Patagonian toothfish are found and the fish grown in them are then assumed to be genetically similar to the ones in the Chilean fisheries. All the Patagonian toohfish fisheries are also easily accessible. These factors are important, since this is a pilot study that will lead to great progresses in the identification of the Chilean sea bass, we must use environments that harbor the right type of fish to be studied. As both chosen environments harbor the type of Chilean sea bass under study, it is easy to decide on a number of replicates and area to sample. The best course of action determined is to divide an area of each habitats into a grid patterns to enable easy sampling of the fish. Tissue Biopsy The Patagonia toothfish caught from the environment of choice will be have their scale removed and get to the location between the adipose fins and the dorsal fins. A longitudinal slice will be removed using a surgical blade from around the dorsal region. Collection of the skeletal tissue will take place and the collected tissue will be stored at room temperatures in 95% ethanol until the right time comes for their use. DNA Extraction Total DNA will be extracted from the extracted tissues that have been stored in ethanol according to the protocol fronted by Taggart et al (1992). The muscle will be cut into tiny pieces and put in 500 ml STE buffer which in essence consists of 0.05 M Trish, 0.1 M NaCl and 0.01 M Na2 EDTA, pH 7.5), 30 ml proteinase K (10 mg/ml, and 10 ml SDS 10%. The solution will then be left to digest for two hours at 500C. The digestion will be accelerated by inverting the tubes several times during the process. After the conclusion of the digestion process, 3 ml of DNAse free-RNAse will be added and the mixture will be incubated at 370C for 300minutes. The final mixture of digestion will then be extracted once with the use of 500 ml of buffer saturated in phenol at pH 8.0, it will also be extracted once with chloroform-isoamyl alcohol and once with pheno-chloroform-isoamyl alcohol. The DNA will then be precipitated in sodium acetate (3 M, NaOAc, pH 5.3) and 1 ml cold ethanol (Maniatis et al., 1982). The DNA pellets will then be dried and dissolved in 100 ml TE buffer (1 mM Tris-HCl, 7.6 and 0.1 mM EDTA, pH 8.0). the mtDNA will be extracted using 2 ml of the total DNA extract and then digested with the endonucleases ApaI (GGGCC/C) and HpaI (GTT/AAC restriction enzyme. The digested DNA samples will then be analysed using the agarose gel at 0.8% for a period of 1 hour. The gell will then be denatured using 0.5 M NaOH and 1.5 M NaCl and then neutralized with the use of 1.0 M Tris-HCl in 1.5 M NaCl at pH 7.2. the DNA fragments will then be immobilized on a membrane (nylon) using the method called the southern transfer. The detection of mtDNA will be done by hybridization using homologous probes of Dissostichus eleginoides. The mitochondrial DNA will be extracted using a method fronted by Chapman and Powers (1984) and then digested with BamHI restriction enzyme. This will result in two fragments of 1.3 kb and 1.1 kb. PCR Sequencing of mtDNA After the mtDNA has been detected, the two fragments will each sequence using the PCR so that more of them will be ready for the design of the probes. The two fragments were amplified with standard methods for the PCR and those of the MJ PTC-225 thermocycler using the 25-µL volumes using 2 µL of DNA extracts as the templates. The primers to be used will be MT16498H and L19 (Jerry & Baverstock, 1998) under the stanadard conditions: initial process of denaturing for 5 minutes at 94 °C followed by 35 cycles with denaturing at 94 °C for 1 minute each, annealing at 51 degree Celsius for 1 minute and finally extension at 72 ° C for one minute. This will be followed by a final step of extension at 72 °C for 5 minutes. The products of PCR will be purified using the QIAGEN MinElute 96 PCR purification plates.   Determination of the Haplotype frequencies For the determination of the haplotype frequencies from the analysis of mtDNA analysis, each of the patterns from the fragment’s two regions will be investigated in the formation of a complete haplotype. The molecular diversity of the haplotypes and their frequencies will then be done on each of the two fragments. The specific tests for the mtDNA for the haplotype frequencies will then used to determine the frequencies of the haplotype in the sample using a method called the Analysis of Molecular Variance (AMOVA) (Excoffier et al, 1992) and the f-statistics will be used to assess the differences among the collections. When the first fragment is digested using the BstNi, this should results in five haplotypes A,B,D,E and F while the digestion of the ND2 fragment should give rise to two haplotypes C and J after it has been digested with NIaIII. A total of seven different composite haplotypes will be identified. Each of the seven haplotyes are specific to different environmental habitats and they will be used in the device for the identification of the origin of the fish. Designing the Probes The designing of probes that have complementary sequence to the Haplotypes will be done using a method known as hybridization. First, fixation of the mtDNA fragments containing the haplotypes will take place. The process of fixation will be geared towards stabilizing the macromolecules and the mtDNA molecule so as to prevent the denaturing of the mtDNAt during the hybridization process. The fixed mtDNA will then be incubated (hybridized) in a buffer solution at a specific temperature. This allows the probes to be specifically bound to the target when they will be used in the device. This means that the probes are also bound to an enzyme that is specific to each Patagonian fish halpotype. Ideally, only those probe/haplotype pairs will form with no mismatches in the hybrid. As result, only target cells with contain the probes specific to the haplotypes will show the color signature when using the device. This is then followed by a step to wash and remove all the unbound probe molecules. Finally, the hybridized probes will be labeled with a letter to represent each of the seven haplotypes specific to the Patagonian toothfish by flow cytometry or epifluorescence microscopy. Developing the Device The device will be developed by using a tube made of a capillary membrane. This membrane will have on its sides a coating of immobilized enzyme’s substrates and Probes that specific for each of the Patagonian toothfish haplotype. At one end is the opening where a liquidated DNA sample is fed is moves through the membrane by capillary action. Near the opening where the DNA is fed is a layer containing free Probes that are common in all Patagonian toothfish haplotypes , each conjugated with enzymes. The probes that the column is coated wil be the ones developed during the study. As the DNA flows through, the free Probes-enzymes complexes on the first layer will attaché to the DNA and the DNA-probes-enzymes complexes will continue to flow. When DNA-probes-enzymes complexes arrive to the layer that contain immobilised specific probes for the haplotypes and substrates for enzymes, DNA-probes-enzymes complexes will not continue to flow, as they are attached to the immobilised probes. Enzymes will interact with substrates and make colour lines to show that the DNA fed to the device really belongs to the Patagonian Toothfish and the specific haplotype that gives a specific color will help identify the origin of the Patagonian toothfish. The remaining probes-enzymes complexes will attached to control layer(last layer) that have immobilised complementary probes for the probes in probes-enzymes complexes and will make another colour layer. If the DNA does not come from the Patagonian toothfish, the probes-enzymes complexes will flow to the last membrane and there will be no color lines in the capillary membrane. Proposed Timetable: Oct - Dec: Environmental sampling and DNA Extractionm Jan – Feb: mtDNA sequencing and probe designing Marc – April: Developing the device Reference: Appleyard, S.A., Ward R.D. and R. Williams, 2002, Population structure of the Patagonian toothfish. Ant. Sci. 14: 364-373. 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