The gene scene

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Odd Grydeland odd@fishfarmingxpert.com

The workshop was coordinated by Genome British Columbia, with funding support also coming from the Research Council of Norway (RCN), the Norwegian Seafood Research Fund (FHF), The Chilean Economic Development Agency, CORFO and InnovaChile – the same agencies that provided most of the funding for the sequencing of the Atlantic salmon genome. Entitled “Genomics 101: Industry workshop on practical applications for the Atlantic Salmon Genome Sequence in aquaculture”, the meeting was limited to 40 participants from the approximately 430 registered delegates to the main conference. In its initial invitation to the meeting, Genome BC announced that: “The workshop will allow aquaculture industry stakeholders to understand how they can use the reference sequence Atlantic salmon and other genomic resources in their day-to-day operations. The workshop will provide a layman’s introduction to genomics and present real-world examples of how genomics is improving aquaculture production and sustainability. Importantly, the workshop will provide information on how companies can implement genomics into their operations covering topics such as: working with academics, resources required, research and ROI timelines and possible sources of funding.” In her welcoming statement, Dr Rachael Ritchie, Director of Business Development for genome BC mentioned that a separate workshop was held in Norway recently where an announcement was made that the sequencing of the Atlantic salmon genome is now complete, and that interested parties will be informed about how to use it. The fact that a fully mapped and openly accessible Atlantic salmon genome had been completed was announced a year ago at a meeting of the International Cooperation to Sequence the Atlantic Salmon Genome (ICSASG) in Vancouver.

Dr Ben Koop from the Centre for Biomedical Research at the University of Victoria is very familiar with the salmon aquaculture industry, and has been an active participant in the scientific organizations involved with the sequencing of the Atlantic salmon genome, a process which he described in his presentation to the workshop. It all started with an agreement signed in Oslo, Norway in 2003, Dr Koop explained. This was followed by workshops at the Norwegian University of Life Sciences at Ås in Norway in 2005, at the Simon Fraser University (SFU) in Vancouver in 2006, in Chile in 2007 and in Quebec City in 2008. While the main drivers of the sequencing project were the representatives of a Scientific Executive Committee from Canada, Chile and Norway, scientists from some 17 countries were involved with the initiative, particularly during the 2006 meeting at SFU. A formal agreement resulting in the formation of the International Cooperation to Sequence the Atlantic Salmon Genome (ICSASG) was signed in Santiago, Chile in April 2009. The ICSASG was able to confirm that it had “raised sufficient funds to cover the cost of sequencing, assembling and annotating the Atlantic salmon genome”. The stated purpose of the organization was: To produce a genome sequence that: 1. Identifies and physically maps all of the genes in the Atlantic salmon genome. 2. Can act as a Reference/ Guide sequence for the genomes of other salmonids and more distantly related fish. Dr Koop explained how the process of breeding livestock for increased efficiency has developed from using a system of Index Selection* up to the late 1970s, to a process called BLUP (Best Linear Unbiased Prediction), to today’s opportunity to use selection based on genomics. He also told the meeting that, of over 69,000 scientific papers published about salmonids since 1955, only 1,919 deal with genetics. The salmon genome is one of the hardest to sequence, Dr Koop explained, but we now have one of the best genomes available to scientists anywhere, and the genomes of other salmonids will be much easier to sequence. Some numbers contained in Dr Koop’s presentation include: • There are 29 chromosome pairs in each Atlantic salmon. • Each chromosome on the average controls about 1,500 characteristics. • There are approximately 45,000 genes in each Atlantic salmon. • The Atlantic salmon genome consists of about 3.2 billion base pairs, of which we can sequence up to 900 at a time.

With respect to the genetic traits that can be of value to a salmon farmer and that can be addressed using genomics, Dr Koop listed the following;

• Pathogen resistance. • Growth. • Age at sexual maturity. • Pigment uptake. • Upper temperature tolerance.

He also mentioned that, in theory, one could eventually develop salmon with few lice problems (although this is not a serious issue in British Columbia, he acknowledged) and minimise the amount of deformities. As with other methods of genetic selection, Dr Koop said that one of the challenges associated with using genomics is to try to get some of the good genes without disturbing the others. Dr Koop’s presentation at Aquaculture Canada 2015 was followed by a session entitled “Genomics 101 – What is the salmon sequence and what is it good for?” by Dr Patricia Schulte, zoology professor at the University of British Columbia. She raised the question “what is the (Atlantic salmon genome) sequence?, and explained that all cells in a body of an animal such as the Atlantic salmon contain two copies of the specie’s genome. The sequence is basically the order of the bases (the A’s, C’s, G’s and the T’s) in the DNA of the fish. The sequence provides the “Instruction Manual” for making the organism with its particular traits, explained Dr Schulte, as well as the building blocks of chromosomes. She said that one can get a complete copy of the fish’s genome from just a fin clip from a salmon, which makes it easy to identify individual fish, which again leads to opportunities in broodstock development and traceability that can be used to improve production efficiencies and environmental sustainability. The genome sequence can be used to identify Single Nucleotide Polymorphisms (SNPs), which are the most common type of genetic variation among organisms. Each SNP represents a difference in a single DNA building block, called a nucleotide. Genomic selection consists of finding the association between SNPs and such important production traits as those identified by Dr Koop. This has yet to be done in aquaculture, Dr Schulte claimed, but it is commonly applied to other livestock. She said that this work can lead to production efficiencies, but warned that different genes can also be turned on/off as a result of environmental conditions. The technology should also be able to provide benefits in the area of feed development and fish health.

Dr Ruth Withler works as a geneticist for the Department of Fisheries and Oceans at the Pacific Biological Station in Nanaimo, and she has many years’ experience working with the aquaculture industry in BC and elsewhere. Her talk about the potential applications for the use of the Atlantic salmon genome centered around the issue of fish family analysis in broodstock management. She said that, as each cell in the body of a fish contains the entire fish’s genome, the breeding value of a fish can be determined as soon as it can be sampled, without having to wait until the fish has grown up. This is because you presumably know which alleles you are selecting from. (According to Genetics Home Reference, “An allele is one of two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent. If the two alleles are the same, the individual is homozygous for that gene. If the alleles are different, the individual is heterozygous”). Dr Withler said that the genome sequence knowledge can be used in breeding programs to avoid rapid inbreeding and loss of variability due to genetic drift. And she said that this new knowledge can do much more – making it possible to provide “Phenotypic (appearance) Information” about the fish and their associated breeding value. But she suggested that this work of identifying phenotypes and their associated alleles might require another 3-6 years of work. And just like in other, more traditional, fish breeding activities, it is imperative to start with genetic material with a high variation, Dr Withler pointed out. She added that the new technology could also assist in bringing beneficial alleles to fixation very rapidly, and this could be combined with other technologies, such as cryopreservation. Odd Magne Rødseth, CEO of AquaGen in Norway, told the audience that more or less by fluke, European scientists have discovered that Chromosome 25 in Atlantic salmon has been found to determine 80% of the resistance to the Infectious Pancreatic Necrosis Virus (IPNV), and that subsequent broodstock selection has resulted in the reduction of mortalities in Norwegian salmon farms due to this virus from about 200 outbreaks per year, to 50 in 2013, to 35 in 2014. The initial work was based on the fact that already by the late ’90s it had been documented that there was a significant genetic variation in salmon susceptibility to IPN.

His presentation focused on breeding and broodstock development. He said that, generally, there is an association between traits and alleles but while we now have new, fundamental tools in order to find these alleles, it is still a bit like looking for a needle in a haystack. As starting tools for a breeding program for Atlantic salmon, Mr Rødseth said that AquaGen has focused on the following; 1) AquaGen is part owner of BioBank, which has developed a secure and robust storage system with related information on biological material. Using the latest in cryopreservation technology, genes from many generations of Atlantic salmon are kept for future use. 2) High density SNPs: AquaGen and others have developed a SNP that analyses more than 900,000 genetic markers per sample from salmon – a major advance from the previously used set of 6,000 genetic markers. 3) People and money: about NOK 1.5 billion (€170 million) was spent by the Research Council of Norway to set up the infrastructure for genetic research of Atlantic salmon. Dr Rødseth said that his company is now working on Pancreas Disease, improved pigmentation and resistance to sea lice. The identification of three significant SNPs has led to a reduction of mortality in Chilean salmon farms due to Salmonid Rickettsia Septicemia (SRS) from 55% to about 25%. He concluded his presentation by stating that genetics has been a major driver in the “green revolution”, and it is sure to be the same in the current “blue revolution”.

Dr Tony Manning, of the Research and Productivity Council (RPC) in New Brunswick, spoke of the use of genomics in the context of fish health, where types of gene expression could include: • Stress response (to extreme temperatures, for example). • Immune response. • Host/parasite interactions. • Tolerance to drugs/chemicals.

He described two studies conducted on Canada’s east coast looking into the potential for vaccine development by looking at the immune response by Atlantic salmon to the exposure to ISA (Infectious Salmon Anaemia) virus. The work was conducted at Nellie Gagné’s Fisheries and Oceans Canada laboratory in Moncton, New Brunswick. Dr Manning said that gene expression biomarkers from these ISAV studies have potential as: • Vaccine development tools • Test and select new vaccine formulations which best activate the protective immune pathways and responses revealed by these studies. • Candidate DNA markers of ISAV resistance • Further study may reveal that a gene expression biomarker can also serve as a DNA marker for high-throughput screening of families. • Tools for testing marker-assisted selection improvements • Ensuring that gene expression responses associated with disease resistance are being enhanced by genetic selection *Description of animal breeding methods – various sources: Index Selection is a method of artificial selection in which several useful traits are selected simultaneously. First, each trait that is going to be selected is assigned a weight – the importance of the trait – ie if you were selecting for both height and the coat darkness in dogs, if height were the more important of the two one would assign that a higher weighting. For instance, height's weighting could be ten and coat darkness could be one. This weighting value is then multiplied by the observed value in each individual animal and then the score for each of the characteristics is summed for each individual. This result is the index score and can be used to compare the worth of each organism being selected. Therefore, only those with the highest index score are selected for breeding via artificial selection. In animal breeding, Best Linear Unbiased Prediction, or BLUP, is a technique for estimating genetic merits. In general, it is a method of estimating random effects. “Under ideal conditions the animal (BLUP) model is the best way for the prediction of breeding values in animal populations but only if the data set is large. The structure of the data also matters, as it explains the relationship of animals in the pedigree”. In the breeding work, the Molecular Genetic Methods contribute in an increasing degree towards shifting the focus from individual genes to the entire genome (genomics). This contributes to developing methods that are time- and labour-saving and which make the selection more effective. Methods that make it possible to study thousands of genes simultaneously open for new possibilities to identify genes than can be used in pathogenic studies.