Salmonid Genomic Sequencing Initiative: The case for sequencing the genomes of Atlantic salmon (Salmo salar) and rainbow trout (Oncorynchus mykiss)

Transcription

1 Salmonid Genomic Sequencing Initiative: The case for sequencing the genomes of Atlantic salmon (Salmo salar) and rainbow trout (Oncorynchus mykiss) William S. Davidson*, Yann Guiguen, Caird E. Rexroad 3 rd$, Stig W. Omholt #1 Simon Fraser University, Canada; INRA, Station Commune de Recherche en Ichtyophysiologie, Biodiversite et Envrionment, France; $ USDA/ARS, National Center for Cool and Cold Water Aquaculture, USA; # Norwegian University of Life Sciences, Norway [*Corresponding author: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6. Tel: ; Fax: ; wdavidso@sfu.ca] 1 The authors constitute the interim steering committe of cgrasp (the Consortium for Genomics Research on All Salmonids Program), the emerging international body for establishment and maintenance of salmonid genomic resources. Appendix 1 lists the current members of cgrasp. 1

2 Contents Background Commercial, Environmental and Scientific Rationales Current Salmonid Genomic Resources Identification of Missing Critical Pre-sequencing Phase Genomic Resources History of Major Funding for Salmonid Genomic Studies Costs and Benefits Data Release Policy References Background The salmonid fishes comprise several species of substantial importance for aquaculture 2, wild stock fisheries and recreational sport fisheries (Fig. 1). Besides their great economic and societal importance, the salmonids are also of considerable scientific importance in such fields as evolutionary biology, ecology, physiology, genetics, immunology, toxicology, nutritional and environmental science. There is no other species group that receives such a comprehensive combined commercial and scientific human attention. More is known about the biology, life history, population dynamics, biogeography and evolution of salmonids than any other fish family. There is thus a large and diverse scientific community working and publishing in the fields of salmonid biology and genomics (Table 2). Fig. 1. Salmonids are members of the Salmonidae family and include the whitefishes and ciscos (subfamily Coregoninae), graylings (subfamily Thymallinae) and trout, salmon and charr (subfamily Salmoninae). These fish have been further classified into nine genera and roughly sixty-eight species (Nelson 1994). The increasing interest in making use of genomics tools for salmonid research is reflected by the development of genomic resources for Atlantic salmon and rainbow trout 2 Atlantic salmon is by far the most abundant cultured salmonid with lesser amounts of rainbow trout, chinook, coho, Arctic charr and brook charr. The main producers of Atlantic salmon are Norway, Chile, the UK, Canada, the USA, Australia and Ireland while Chile, Norway, France, Denmark, Ireland and the USA are the main producers of rainbow trout (see Table 1). 2

3 over the last five years. It was recognized by several groups that there was a need for an international body to coordinate the research effort and ensure that existing and upcoming resources were made accessible to the community as a whole. At a workshop held on October 25-26, 2005 at the Norwegian University for Life Sciences at Aas, Norway, the Consortium for Genomic Research on All Salmonids Program (cgrasp) was formed. cgrasp will be the international collaborative structure for establishing priorities and maintaining salmonid genomic resources. The meeting resolved very clearly the need for at least one high-quality, whole genome salmonid sequence for making optimal use of genomics tools within salmonid research and development (see below), that most of the needed pre-sequencing phase resources will be in place before the end of 2007 (see below), that the task is technically feasible, and that the community is strongly committed to build and maintain the necessary organizational apparatus for handling the pre-sequencing phase, the draft sequence phase and the post draft sequence phase in a professional way. More specifically, after considering the various options, the participants reached the conclusion that the Atlantic salmon genome should be targeted as the reference sequence (6-8 fold coverage), and that a low-survey coverage of the rainbow trout genome should be done as a proof of principle of how the reference sequence can be exploited in other salmonid species (see Appendix II for a report on the Aas meeting). Commercial, Environmental and Scientific Rationales Commercial importance The worldwide production of salmonids was approximately 1,700,000 metric tonnes (MT) in By far the most important farmed salmonid is Atlantic salmon, with production accounting for 1,190,000 MT. Rainbow trout, sometimes described as Atlantic salmon s little sister, is a cosmopolitan fish. With the exception of the Antarctic, it is produced in all of the world s continents. FAO statistics name 70 countries in which this fish is farmed. In contrast to Atlantic salmon, rainbow trout farming receives much less attention although the production data for trout are quite impressive. Total world production reached 500,000 MT for the first time in The production figures for farmed Pacific salmon (coho and chinook) in 2004 were 149,000 MT. This represents a relatively small proportion of the total consumption of Pacific salmon, and most is still from the wild catch. For example, the 336,565 MT Alaskan Pacific salmon fishery (chinook, sockeye, pink, chum and coho) is worth US$ 230 million on an annual basis ( An estimate of the importance of the salmonid aquaculture industry to the economy of various countries can be seen from the export values of salmonid products for Norway (US$ 1.6 billion) and (Chile US$ 1.44 billion). Even the rather modest rainbow trout industry in the USA was worth US$ 57 million in annual sales for foodfish in 2004, and this does not include the industry aimed at restoration, conservation and recreation that was valued at an additional US$ 64.8 million. Table 1 shows salmonid production in 2004 in countries with researchers affiliated with cgrasp. 3

4 Table 1. Salmonid production in Metric Tonnes in 2004 in countries with researchers affiliated with cgrasp Country Atlantic Salmon Rainbow Trout Pacific Salmon Norway 537,000 82,000 Chile 343, , ,000 UK 139,000 17,200 Canada 96,800 4,800 18,000 USA 13,300 25, ,500 (wild fishery) France 46,712 Ireland 12,100 30,000 Denmark 40,000 Australia 15,200 Most, if not all, of the major agricultural vertebrate species are in the process of having their genomes sequenced. There are many good economic reasons for these sequencing projects, including the ability to identify genes that control production traits such as growth and disease resistance, and the subsequent use of this information in broodstock selection programs. There are equally good scientific reasons for investigating the genomes of these organisms. Many mammals, such as the cow and pig, have undergone extreme selection pressures for a wide variety of phenotypes, and several of these are relevant for understanding human health. What is happening with agriculturally important species should be equally applicable to the primary aquaculture species. The values Table 1 show very clearly that the salmonid aquaculture industry is already substantial, and that the potential for growth is considerable. However, it is important to realize that these figures are based upon the industry s ability to bring to the market Atlantic salmon and rainbow trout with phenotypic characteristics at a price and at an environmental cost that meet the expectations of the consumers. More specifically, most phenotypic traits in salmon and rainbow trout associated with health, growth, meat quality and the capacity for utilizing new and ecologically sustainable food items, are complex traits strongly influenced by the environment. To ensure a continuous reduction in production costs while being able to deliver highly competitive quality products in broad as well as narrow market segments requires an understanding of the underlying mechanistic basis of these traits. A deep biological understanding of phenotypic patterns of commercial importance ranging from intracellular molecular signatures to whole individual physiological, morphological and behavioral characteristics is synonymous with an understanding of the functioning of the underlying gene regulatory networks and how these are molded by the environment. This calls for identification of genes and characterization of their functional repertoire in various systemic settings. One could imagine that the establishment of this knowledge base could be achieved by numerous small scale sequencing efforts based on quantitative trait loci (QTL) scans and candidate gene approaches. However, this would take many years to achieve. It would be relatively very costly because of small-scale sequencing and bioinformatics operations. It would drain the ordinary funding sources to a substantial degree, and yet it would not provide the base for extensive comparative genomics nor would it give us a real overview of the global structural changes of the genome as a function of various types of selection and 4

5 interventions. From a quality assurance point of view this is a suboptimal solution. It would lead to a fragmented international salmonid community, which in turn will hamper knowledge-generation of critical importance to the aquaculture industry. 3 Impact on conservation biology and environmental monitoring Farmed salmon impact on wild salmon stocks: Today, approximately 300 million fish are kept in farm cages on the Norwegian coast, outnumbering the wild population 1,000-fold. The farmed salmon come from a number of closed populations that have been artificially selected for growth rate and other traits for up to eight generations. Since escaped farmed fish make up a significant portion of the spawners in the rivers, possibly deteriorating the genetic makeup of the wild populations, the coexistence of large, highly selected populations with their wild populations of origin is looked upon with concern. In addition, the escapees as well the population kept in cages in the sea may transmit serious diseases to wild stocks of salmon as well as sea trout. This potentially strong negative impact of domesticated escapees on wild stocks through interbreeding and disease transmission is manifested through complex life history or fitness traits in the wild stocks. Currently, we have only a vague understanding of these processes. Access to whole genome sequence information will be of crucial help to understand, monitor and minimize intra- and inter-species ecological effects of a growing salmonid aquaculture industry in Norway, Canada, Ireland, the UK and other countries. Monitoring of the aquatic environment: Salmonid species are widely distributed in lakes and streams throughout several countries in Europe and North America. By being present in natural environments near many industrialized centers, they are ideal for monitoring the possible presence of environmental contaminants of concern to human health. An American Society for Testing and Materials (ASTM) standard protocol established rainbow trout as the standard coldwater species used for regulatory 96-hour acute lethality testing for chemical pollutants entering freshwater aquatic ecosystems. It has been used in the USA and Canada for over 20 years. Studies using a salmonid cdna microarray have already revealed that genomics-based procedures provide more sensitive and discriminating analytical tools than the standard protocol. For example, it is possible to obtain high-resolution gene expression signatures from fish subjected to waste from the pulp and paper industry or drugs and other chemicals released into the environment through municipal sewage systems. However, these signatures need to be properly understood before they can serve as a rational basis for governmental and industrial management decision making. It is hard to see how an understanding of these highdimensional data in a toxico-genomic context can be achieved without access to whole genome sequence information. Impact on epistemic biological research Salmonid research community: The salmonid research community comprises numerous academic research groups that are not focused on aquaculture issues, but study salmonids for knowledge-oriented or epistemic reasons in basic disciplinary areas such as evolutionary biology, ecology, physiology, neurobiology, and immunology. Indeed, it should be noted that rainbow trout can be considered the aquatic lab rat with respect to 3 The lack of a whole genome salmonid sequence is already considered to be a problem by the salmonid research community as the agencies funding basic science are beginning to show a preference for those organisms that have already been sequenced. As this problem is likely to become more pronounced in the next few years, it may force researchers to leave the field. 5

6 research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition (Thorgaard et al. 2002a,b). However, several of these efforts also provide important knowledge elements instrumental for applied research and development. An extensive elaboration of the added value of whole genome sequences for this section of the salmonid community is beyond the scope of this paper, but an indication of the potential impact can be obtained from assessing the actual impact of the whole genome sequencing efforts on the research profiles of the fruit fly (Drosophila melanogaster), the nematode (Caenorhabditis elegans) and mammalian (especially human and mouse) research communities. It is worthwhile though to emphasize specific assets of the salmonid family genomes, which cannot be exploited so easily and comprehensively in the vertebrate genomes sequenced to date. Vertebrate genome evolution: Vertebrates provide some of the most dramatic examples of morphological and physiological change during evolution. It has been suggested that the diversity of vertebrate groups may depend upon features of the vertebrate genome that include diversification of gene function following successive rounds of either genome or chromosome segment duplication. It is widely believed that genome duplications not only provide the raw material for evolutionary change but also facilitate speciation and the production of new classes of organisms. Genome mapping and sequencing provide evidence for an ancient, whole genome duplication in the lineage leading to the teleost radiation (Jaillon et al. 2004; Taylor and Raes 2004). How an inherently unstable duplicated genome reverts to a stable diploid state (i.e., rediploidization) is poorly understood. For example, although it is thought to include large-scale deletions, gene silencing and chromosomal rearrangements, it is not known if these events occur randomly along different lineages or if there is a burst of activity immediately after the duplication followed by stability in the resulting genomes (Wolfe 2001). The common ancestor of extant species in the Salmonidae underwent a genome duplication Mya (Allendorf and Thorgaard 1984); therefore, comparisons of the Atlantic salmon and rainbow trout genomes will provide an opportunity to discover how these organisms responded to a common tetraploidization event and to follow the rediploidization process along different lineages within a family. Further, it will provide an opportunity to examine in detail the Duplication, Degeneration and Complementation hypothesis (Lynch and Force 2000) in a vertebrate family spanning a whole range of habitats and life history regimes. Understanding complex traits in natural populations: Recent experimental studies in invertebrates and plants suggest that adaptive evolution is likely to occur by a mixture of genetic changes, some of which account for a substantial fraction of the total variance in evolutionary traits. These results are very promising from the point of view of achieving a detailed understanding of evolutionary processes in vertebrates, and for constructing an evolutionary genetics theory based on how genes actually work and interact. However, to understand the mechanistic basis for the establishment and maintenance of natural phenotypic variation, the effects of various selection pressures on this variation, and the evolution of new traits in vertebrates, is a daunting undertaking that demands comprehensive studies of vertebrate families that have experienced both long term and short term exposures to a variety of biotic and abiotic environments. The Salmonidae family offers a unique biological opportunity for detailed study of the mechanistic basis of species differences in vertebrates by being present in a variety of fresh and saltwater environments possessing very different characteristics, and by 6

7 displaying a variety of morphological, physiological and behavioral traits defining numerous life history regimes. Some of these traits have originated after the last Ice Age. Widespread melting of glaciers led to dramatic changes in sea level and land elevation. As a result of this global climate change, tens of thousands of new freshwater lakes and streams were created in formerly ice-covered regions throughout the Northern hemisphere, creating thousands of evolutionary experiments. The populations inhabiting these habitats have thus diverged over the course of only 10,000-15,000 years in response to different ecological conditions, including large differences in water characteristics, food sources, predators and seasonal environmental stability. This has given rise to new populations with marked changes in body size, body shape, body coloration, feeding specializations, salinity tolerance, temperature preference, parasite resistance, lifespan and behavior. Further study of the adaptive radiation of this family may thus reveal mechanisms that are broadly relevant to the evolution of complex traits and physiological differences in other groups that have recently migrated and adapted to different environments, such as humans. However, to seek to answer some of the most longstanding and fundamental questions about the type of DNA sequence alterations that underlie the appearance of new characters in natural populations, implies access to whole genome sequence information. Current Salmonid Genomic Resources Salmonid genomes Much of the basic information concerning salmonid genomes is known. For example, the haploid C-value for Atlantic salmon (Salmo salar) has recently been estimated as 3.27 pg (Hardie and Hebert 2003), which translates into a genome size of approximately 3 x 10 9 bp, which is similar to the estimate of the size of the rainbow trout genome (2.4 x 10 9 bp) (Young et al. 1998). The G+C content of the Atlantic salmon genome is 44.4% (Bucciarelli et al. 2002) whereas for rainbow trout it is 42.9% (Bernardi and Bernardi 1990). Therefore, salmonid genomes are quite similar to those of warm-blooded vertebrates with respect to size and overall base composition, but like other cold-water fish genomes, they appear to be devoid of isochore structures (Bernardi 1993). This is reflected in the inability to obtain G banding patterns in Atlantic salmon chromosomes (Hartley and Horne 1984). Chromosomes The karyotypes of several salmonid species, including Atlantic salmon and rainbow trout, have been described in detail. These studies reveal that many gross chromosomal rearrangements have occurred along the different lineages since the ancestral genome duplication occurred. It has been suggested that the diploid ancestor of salmonids possessed a karyotype with 48 acrocentric chromosomes resulting in 96 acrocentrics after the genome duplication. Phillips and Rab (2001) have reviewed the evolutionary changes that occurred in the karyotypes of the three subfamilies of the salmonid. Linkage maps Linkage maps have been constructed for Atlantic salmon (Moen et al. 2004; Gilbey et al. 2004; SALMAP consortium, Danzmann and Hoyheim, unpublished results), rainbow trout (Sakamoto et al. 2000; Nichols et al. 2003; Rexroad et al. 2006), brown trout (Gharbi et al. 2005) and Arctic charr (Woram et al. 2004). These maps, and the associated genetic markers, have enabled the identification of quantitative trait loci (QTL) for growth 7

8 (O Malley et al. 2003; Perry et al. 2005), upper temperature tolerance (Perry et al. 2001; Somorjai et al. 2003; Perry et al. 2005), body weight and condition factor (Reid et al. 2005) and spawning time (O Malley et al. 2003). As many of the microsatellite markers derived from one salmonid species amplify the DNA from other salmonid species, it has been relatively straightforward to carry out comparative analyses of the rainbow trout, Arctic charr and Atlantic salmon genomes (Danzmann et al. 2005), and to compare in detail the sex-determining regions of salmonid genomes (Woram et al. 2003). Sex-determination Salmonid fishes possess a strictly genetic XX/XY system of sex-determination (Johnstone et al. 1979), but the sex-determining master gene (SEX) has not been identified. Indeed, it is only recently that the chromosome containing the sex-determining region has been identified in Atlantic salmon using a procedure that integrated data from linkage mapping, physical mapping and FISH analysis (Artieri et al. 2005). Comparisons of the linkage groups containing the sex-determining factor in four salmonid species suggest that either the SEX has been translocated in different lineages or else there is a different master gene in each species (Woram et al. 2003). BAC libraries and physical maps Through a collaborative effort involving the Genomics Research on Atlantic Salmon Project funded by Genome Canada, the Norwegian Salmon Genome Project and Pieter de Jong s group at the Children s Hospital Oakland Research Institute (CHORI), a publicly available Atlantic salmon BAC (bacterial artificial chromosome) library (CHORI-214) was constructed and arrayed on to nylon membranes ( (Thorsen et al. 2005). The Atlantic salmon BAC library was fingerprinted using HindIII at the Genome Sciences Centre in Vancouver and arranged into contigs via FPC to create the first physical map of a salmonid genome (Ng et al. 2005a). This BAC fingerprint map is publicly available through a database server (icebox.bcgsc.ca) using the internet Contig Explorer (ice) version 3.4 (available at BAC libraries have also been constructed for rainbow trout (Katagiri et al. 2001; Palti et al. 2004) and chinook salmon (R. Devlin, unpublished results). cdna libraries and ESTs Approximately 200 cdna libraries have been constructed from many different tissues and developmental stages of several salmonid species (Davey et al. 2001; Martin et al. 2002; Tsoi et al. 2004; Rise et al. 2004a; Rexroad et al. 2004; Hagen-Larsen et al. 2005). As of May 12, 2006, more than 250,000 sequences had been obtained for Atlantic salmon and a further 246,000 for rainbow trout. These large EST databases are publicly available (web.uvic.ca/cbr/grasp) and they compliment other databases for Atlantic salmon ( ) and rainbow trout ( The EST databases are providing a rich source of material for identifying microsatellites (Ng et al. 2005b; Vasemagi et al. 2005; Rexroad et al. 2005; Coulibaly et al. 2005) and SNPs (Smith et al. 2005; Hayes et al., submitted), which can be used to compare the genomes of salmonids not only with one another but also with other vertebrate groups. Microarrays The availability of the ESTs and the cdna libraries has enabled the production of cdna microarrays that can be used to examine gene expression patterns in all salmonids tested to 8

9 date, and even in smelt, a representative of the diploid ancestor of the salmonids (Rise et al. 2004a; von Schalburg et al. 2005a; Koskinen et al. 2004; Tilton et al. 2005; Ewart et al. 2005). The microarrays have been used to investigate the response to a bacterial pathogen (Rise et al. 2004b; Martin et al. 2006) and a viral pathogen (Purcell et al. 2006), to survey the genes involved in the maturation and development of the rainbow trout ovarian and testicular tissues (von Schalburg et al. 2005b, 2006), to examine brain gene expression profiles in male salmon with different life history strategies (Aubin-Horth et al. 2005a, 2005b), to compare transcription profiles in wild and farmed Atlantic salmon (Roberge et al. 2006), to carry out toxicogenomic profiling of hepatic tumor promoters in rainbow trout (Tilton et al. 2005), to investigate the response of the rainbow trout transcriptome to model chemical contaminants (Koskinen et al. 2004) and to study gene expression in atrophying muscle (Salem et al. 2006). More than fifty groups around the world are using these microarrays, indicating that there is a large salmonid research community specifically interested in functional genomics. Identification of Missing Critical Pre-sequencing Phase Genomic Resources Although important genomic resources have been developed for salmonids, there are some significant gaps that should be addressed in order to allow the sequencing, assembly and annotation of the Atlantic salmon genome and its subsequent use as a platform for other salmonid genomes to proceed efficiently (see Gantt chart at end of document). BAC libraries and physical maps Two Atlantic salmon BAC libraries have been fingerprinted and used to produce a physical map. However, there are still ~4,000 contigs. A goal should be to have an Atlantic salmon physical map comprising ~500 contigs. Similar work has been initiated on one of the rainbow trout BAC libraries, and approximately a 1 x equivalent of the genome has been fingerprinted (Rexroad, unpublished results). This library should be fingerprinted to a much deeper coverage, and a physical map constructed for this species. Integration of linkage and physical maps One of the prerequisites for an efficient strategy for sequencing a vertebrate genome is to have a robust physical map that is integrated with the linkage map. By data-mining BAC end-sequences for microsatellites (Ng et al., unpublished results) and SNPs (Lien et al., unpublished results) and then placing these markers on the linkage map, a very highdensity linkage map that is tied to the physical map will be produced. A goal should be a SalHapMap with a minimum of 4,500 SNPs and microsatellite markers anchored to specific BACs. In addition to providing information for the assembly of the genome, this resource will enable quantitative trait loci to be mapped more effectively and so provide tools for marker assisted selection and allelic introgression in breeding programs. Similarly, there needs to be an integration of the rainbow trout high-density linkage map with a physical map. ESTs and full-length coding sequences As indicated above, there are 251,725 ESTs for Atlantic salmon and these have been placed into 72,101 contigs. Similarly, the 246,704 rainbow trout ESTs have been put into 68,200 contigs (web.uvic.ca/cbr/grasp). However, the success in annotating these contigs is rather poor, with only 13,645 annotations for Atlantic salmon and 15,381 for rainbow trout. In order to improve the annotation, it will be necessary to get the 5 and 3 ends of 9

10 cdnas, and then to fill in the intervening gaps as necessary. A goal should be to have a set of 24,000 full-length coding sequences for mrnas for both species. In addition to providing invaluable information for microarray experiments, this dataset will form the basis for annotating salmonid genomic sequences as they become available. Moreover, these data will enable the identification of homeologs, and so provide information concerning the fate of duplicated genes. Microarrays It is obvious from the publications and the number of groups around the world who are using the salmonid microarrays that the research community has embraced this technology. A goal is to expand the most recent 16,000 cdna microarray to 24,000 cdnas by the middle of This upgraded array will benefit from the full-length coding sequences as each of the cdnas will be well-characterized and annotated. In turn, the microarray results will feed into the annotation of the genome by providing direct evidence that a transcript is actually expressed in a particular tissue and under what conditions. Snapshot of genome Approximately twenty Atlantic salmon BACs have been sequenced to date, and this provides a snapshot of the genome organization (Hunt et al. 2005; Koop et al., unpublished results). It appears that repetitive DNA accounts for approximately thirty to thirty five percent of the genome, with a Tc1-like element making up six to ten percent. This means that it will be very important to have a good physical map with a well-defined minimum tiling path for an efficient assembly of the sequence of the Atlantic salmon genome. Regions that resulted from the genome duplication have been investigated, and it was revealed that these are readily distinguishable at the sequence level (Mitchell et al., in prep.). Therefore, the genome duplication should not cause any difficulty for assembling the genomic sequence. Another approach to getting a snapshot of the genome is to examine BAC end sequences. A goal is to have sequences available from both ends of 100,000 Atlantic salmon BACs by the middle of A similar initiative should be undertaken for rainbow trout. The BAC sequences and the BAC-end sequences have been used to create a repeat database (Ng et al., unpublished results available at grasp.mbb.sfu.ca), and this can be used to mask sequences when probes are being designed. In addition, this repeat database will be expanded as more sequence information becomes available, and it will prove a valuable resource for the annotation of the genome sequence. Double haploid fish One of the problems that plagued the initial sequencing of the zebrafish genome was the extent of polymorphism within and between the individuals of the pooled group that was used as the source of the DNA for the project. This problem has subsequently been overcome by using a single, double haploid fish that was produced by androgenesis. Double haploid clonal lines of rainbow trout have been produced (Parsons and Thorgaard 1985), and these have been extensively characterized (Scheerer et al. 1991; Young et al. 1996). Indeed, one of the rainbow trout BAC libraries was produced from such a fish. The availability of DNA from a totally homozygous individual will greatly facilitate the assembly of whole genome shotgun sequences. A goal should be to produce double haploid Atlantic salmon. 10

11 History of Major Funding for Salmonid Genomic Studies Although individual researchers were receiving grants from national agencies during the 1980s and 1990s to carry out genetic studies on salmonids, the funding was not sufficient for any group to make significant headway on its own. It took a concerted, large-scale effort in the form of the SALMAP consortium to make the breakthrough. This venture, involving researchers from Norway, Scotland, Denmark, France and Canada, received Euro 1,431,000 through the EU FAIR Program from 1997 to 2000 to develop DNA markers and genetic maps for salmonid fishes (Atlantic salmon, rainbow trout and brown trout). The following year SALGENE, with researchers from Ireland, Norway, Denmark and Scotland, obtained Euro 1,179,000 over a three-year period ( ) to produce a genetic body map for Atlantic salmon by characterizing several hundred cdnas from each of a variety of tissues. These two projects paved the way for other large-scale genomics projects, and clearly demonstrated the need for collaboration among groups if major advances were to occur. The Norwegian Salmon Genome Project and the Genomics Research on Atlantic Salmon Project (GRASP), funded from 2001 until 2005 by Genome Canada, allowed great progress to be made, but it was evident to the participants that even more could be achieved if they pooled resources and worked together. This was the basis for forming cgrasp: the Consortium for Genomics Research on All Salmonids Project, which was successful in bringing together salmonid genomics teams from Canada, Norway, Scotland and the USA and was able to put together a project with funding from several sources that amounts to Cdn$15.2 million. cgrasp is set to run from January, 2006 until December During this period, the work carried out by cgrasp will provide a solid foundation and many of the resources that are considered essential for efficiently sequencing the Atlantic salmon and rainbow trout genomes. Costs and Benefits With the completion of the human genome sequence and several other mammalian genomes at an advanced stage, it is clear that there is a need to investigate and characterize non-mammalian genomes in order to gain an understanding of the complexity of vertebrate genomes and how they have evolved. Fish and mammals share a common ancestor that dates back to the very origin of the vertebrate lineage. There are approximately 25,000 species of fish, making them the most successful vertebrate group, and it is their diversity that makes them useful model systems in different disciplines of biology. For example, the zebrafish (Danio rerio) with its short generation time, easy maintenance and external development of a transparent embryo, provides the opportunity to investigate vertebrate developmental biology on a scale similar to Drosophila melanogaster and Caenorhabditis elegans. The small genomes of the marine pufferfish (Takifugu rubripes) and the spotted green pufferfish (Tetraodon nigrovridis) made then candidates for cataloguing the genes in a vertebrate at a fraction of the cost for a mammalian species. Other fish whose genomes are being sequenced include an aquarium species, the medaka (Oryzias latipes) that has been used as a model in genetics for about a century, and the threespine stickleback (Gasterosteus aculeatus), a species that has undergone one of the most recent and dramatic adaptive radiations of any vertebrate (Roest Crollius and Weissenbach 2005). Genomic resources are being amassed for several other fish species (Cossins and Crawford 2005), and the time has come to expand the number of fish genomes being sequenced to include representatives of the most important economic species, namely the salmonids. 11

12 As indicated in Table 2, there is a vibrant community working on salmonid fishes. Members of this community are already making use of the genomic resources that have been developed, and they are poised to take advantage of sequence information as it becomes available. Many teams have expressed an interest in being involved in the annotation of salmonid genomes, and given the diversity of research interests in these groups, it will be easy to find leaders who will be responsible for particular gene families and pathways. Table 2. PubMed references in salmonids versus fishes with an already sequenced genome (Search for salmonids = salmon and trout, May 10, 2006). Total references and in (last 5 years references) Salmonids Zebrafish Pufferfish Total publications 16,896 (4,072) 7,652 (4,650) 635 (511) Genome 1204 (529) 2,168 (1,249) 412 (324) Physiology 12,863 (3,096) 6,962 (4,160) 549 (439) Hormone 4,365 (759) 434 (280) 47 (40) Nutrition 495 (172) 17 (10) 0 Genetic 1,039 (396) 1,801 (1,117) 145 (124) Transgenic 88 (35) 478 (314) 37 (30) Biochemistry 869 (205) 495 (311) 15 (13) Evolution 932 (377) 1,073 (772) 331 (285) Behavior 730 (277) 329 (219) 7 (6) Cancer 841 (129) 516 (341) 13 (10) Carcinogenesis 121 (13) 73 (54) 0 Toxicology 55 (15) 39 (29) 0 Toxicity 1,356 (458) 301 (195) 23 (18) Immunology 1,457 (453) 174 (103) 35 (27) Antibody 1,177 (291) 246 (134) 17 (17) Environment 2,721 (914) 326 (190) 22 (18) Development 2,057 (718) 3,632 (2,204) 75 (64) It is estimated that the cost to obtain a 6-8 fold coverage of the Atlantic salmon genome and a low survey coverage of the rainbow trout genome with the associated annotations will be less than the US$ 50M required for ~15 fold coverage of the bovine or pig genome. In part this is because of advances in technology and expertise that have accumulated during the sequencing of mammalian genomes. However, it is also a result of the extensive work that has been carried out to date in the pre-sequencing phase of the salmonid genome initiative. The gannt chart (Fig. 2) reveals the genomic resources that have already been developed, for which funding has been obtained and are on-going, and for which new funding is being sought. The decision on which of several possible approaches will be adopted for sequencing the Atlantic salmon and rainbow trout genomes will depend on the sources of funding for the project. However it is achieved, the genomic information that will result 12

13 from the work of cgrasp will provide answers to fundamental scientific questions and in so doing will provide resources and novel applications in economically important and socially relevant sectors; namely, aquaculture, conservation and the environment. Data Release Policy cgrasp is committed to a Data Release Policy of open and rapid dissemination of fundamental genomic information and related resources. cgrasp will adhere to the Bermuda Principles as modified by the Ft. Lauderdale agreement ( References Allendorf FW and GH Thorgaard Tetraploidy and the evolution of salmonid fishes, pp in Evolutionary Genetics of Fishes, edited by BJ Turner. Plenum Press, New York. Artieri CG, LA Mitchell, SHS Ng, SE Parisotto, RG Danzmann, BJ Hoyheim, RB Phillips, M Morasch, BF Koop and WS Davidson Identification of the sexdetermining locus of Atlantic salmon (Salmo salar) on chromosome 2. Cytogenetic and Genome Research. 112: Aubin-Horth N, BH Letcher and HA Hofmann. 2005a. Interaction of rearing environment and reproductive tactic on gene expression profiles in Atlantic salmon. Journal of Heredity. 96: Aubin-Horth N, CR Landry, BH Letcher and HA Hofmann. 2005b. Alternative life histories shape brain gene expression profiles in males of the same population. Proceedings Biological Sciences. 272: Bernardi G The isochore organization of the human genome and its evolutionary history--a review. Gene. 135: Bernardi G, Bernardi G. Compositional patterns in the nuclear genome of cold-blooded vertebrates. Journal of Molecular Evololution : Bucciarelli G, G Bernardi and G Bernardi An ultracentrifugation analysis of two hundred fish genomes. Gene. 295: Cossins AR and DL Crawford Fish as models for environmental genomics. Nature Reviews Genetics. 6: Coulibaly I, K Gharbi, RG Danzmann, J Yao and CE Rexroad 3 rd Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags. Animal Genetics. 36: Danzmann RG, M Cairney, WS Davidson, MM Ferguson, K Gharbi, R Guyomard, LE Holm, N Okamoto, A Ozaki, CE Rexroad 3 rd, T Sakamoto, JB Taggart and RA Woram A comparative analysis of the rainbow trout genome with 2 other species of fish (Arctic charr and Atlantic salmon) within the tetraploid derivative Salmonidae family (subfamily: Salmoninae). Genome 48:

14 Davey GC, NC Caplice, SA Martin and R Powell A survey of genes in the Atlantic salmon (Salmo salar) as identified by expressed sequence tags. Gene. 263: Ewart KV, JC Belanger, J Williams, T Karakach, S Penny, SC Tsoi, RC Richards and SE Douglas. Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cdna microarray technology. Developmental and Comparative Immunology. 29: Gharbi K, A Gautier, RG Danzmann, S Gharbi, T Sakamoto, B Hoyheim, JB Taggart, M Cairney, R Powell, F Kreig, N Okamoto, MM Ferguson, LE Holm and R Guyomard A linkage map for brown trout (Salmo trutta): chromosome homeologies and comparative genome organization with other salmonid fish. Genetics. 172: Gilbey J, E Verspoor, A McLay and D Houlihan A microsatellite linkage map for Atlantic salmon (Salmo salar). Animal Genetics. 35: Hagen-Larsen H, JK Laerdahl, F Panitz, A Adzhubei and B Hoyheim An ESTbased approach for identifying genes expressed in the intestine and gills of presmolt Atlantic salmon (Salmo salar). BMC Genomics. 6: 171 Hardie DC and PD Hebert The nucleotype effects of cellular DNA content in cartilaginous and ray-finned fishes. Genome. 46: Hartley SE and MT Horne Chromosome relationships in the genus Salmo. Chromosoma. 90: Hunt PND, MD Wilson, KR von Schalburg, WS Davidson and BF Koop Expression and genomic organization of zonadhesin-like genes in three species of fish give insight into the evolutionary history of a mosaic protein. BMC Genomics. 6:165 Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, Bouneau L, Fischer C, Ozouf-Costaz C, Bernot A, Nicaud S, Jaffe D, Fisher S, Lutfalla G, Dossat C, Segurens B, Dasilva C, Salanoubat M, Levy M, Boudet N, Castellano S, Anthouard V, Jubin C, Castelli V, Katinka M, Vacherie B, Biemont C, Skalli Z, Cattolico L, Poulain J, De Berardinis V, Cruaud C, Duprat S, Brottier P, Coutanceau JP, Gouzy J, Parra G, Lardier G, Chapple C, McKernan KJ, McEwan P, Bosak S, Kellis M, Volff JN, Guigo R, Zody MC, Mesirov J, Lindblad-Toh K, Birren B, Nusbaum C, Kahn D, Robinson-Rechavi M, Laudet V, Schachter V, Quetier F, Saurin W, Scarpelli C, Wincker P, Lander ES, Weissenbach J, Roest Crollius H Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature. 431: Johnstone R, TH Simpsons, AF Youngson and C Whitehead Sex reversal in salmonid culture. Part II. The progeny of sex reversed rainbow trout. Aquaculture. 18: Katagiri T, S Asakawa, S Minagawa, N Shimizu, I Hirono and T Aoki Animal Genetics. 32:

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16 Perry GML, M Ferguson, T Sakamoto and RG Danzmann Sex-linked Quantitative Trait Loci for Thermotolerance and Length in the Rainbow Trout. Journal of Heredity. 96: Phillips R and P Rab Chromosome evolution in the Salmonidae (Pisces): an update. Biological Reviews Cambridge Philosophical Society. 76: Purcell MK, KM Nichols, JR Winton, G Kurath, GH Thorgaard, P Wheeler, JD Hansen. RP Herwig and LK Park Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic virus. Molecular Immunology. 43: Reid DP, Z Szanto, B Glebe, RG Danzmann and MM Ferguson QTL for body weight and condition factor in Atlantic salmon (Salmo salar); comparative analysis with rainbow trout (Oncorhynchus mykiss) and Arctic charr (Salvelinus aplinus). Heredity. 94: Rexroad CE 3 rd, Y Lee, JW Keele, S Karamycheva, G Brown, BF Koop, SA Gahr, Y Palti and J Quackenbush Sequence analysis of a rainbow trout cdna library and creation of a gene index. Cytogenetics Genome Research. 102: Rexroad CE 3 rd, Y Palti, G Thorgaard, J Hansen, A Benmansour, A Teale, A Krasnov, A Felip-Edo, A Cossins, B Robison, B Koop, B May, B Hershberger, B Shepherd, C Ostberg, C Secombes, C Michel, C Bayne, D Lee, D Williams, D Buhler, FM Rodriguez, F Allendorf, F Goetz, G Bailey, G Young, G Goss, G Warr, G Weber, G Wiens, H Mölsä, I Coulibaly, J Nagler, J Parsons, J Petersen, J Winton, JF Bernardet, J Hard, J Silverstein, J Bartholomew, J Dijkstra, J Taggart, J Bautista, J Brunelli, J Cloud, K Gharbi, K Blemings, K Cain, K Overturf, K Reed, K Nichols, L Holm, L Park, M Vijayan, M Rise, M Cairns, M Ford, M Skinner, M Dorson, N Bury, N Karrow, N Okamoto, P Iturra, P Prunet, P Walsh, P Cash, P Boudinot, P Rescan, R Guyomard, R Stet, R Bogden, R Devlin, R Ingermann, R Hardy, R Danzmann, R Phillips, S Ristow, S Gahr, S LaPatra, S Kaattari, S Winberg, T Sakamoto, T Nakanishi, T Chen and J L Ziu A Proposal Advocating Draft Sequencing the Genome of Rainbow Trout, Oncorhynchus mykiss. submitted to the DOE Joint Genome Institute Community Sequencing Program. Rexroad CE 3 rd, MF Rodriguez, I Coulibaly, K Gharbi, RG Danzmann, J Dekoning, R Phillips and Y Palti Comparative mapping of expressed sequence tags containing microsatellites in rainbow trout (Oncorhynchus mykiss). BMC Genomics. 6:54. Rexroad CE3 rd, R Danzmann, Y Palti and Vallejo 2006 The NCCCWA genetic map for rainbow trout. Plant and Animal Genome XIV. January 14-15, San Diego, CA Rise, M.L., KR von Schalburg, GD Brown, RH Devlin, MA Mawer, N Kuipers, M Busby, M Beetz-Sargent, R Alberto, AR Gibbs, P Hunt, R Shukin, JA Zeznik, C Nelson, SRM Jones, DE Smailus, SJM Jones, JE Schein, MA Marra, YSN Butterfield, JM Stott, SH Ng, WS Davidson and BF Koop Development and application of a salmonid EST database and cdna microarray: data mining and interspecific hybridization characteristics. Genome Research. 14:

17 Rise ML, SRM Jones, GD Brown, KR von Schalburg, WS Davidson and BF Koop Piscirickettsia salmonis infection consistently regulates expression of a suite of genes in cultured Atlantic salmon macrophages and in hematopoietic kidney. Physiological Genomics. 20: Roberge C, S Einum, H Guderley and L Bernatchez Rapid parallel evolutionary changes of gene transcription profiles in farmed Atlantic salmon. Molecular Ecology.15: Roest Crollius H and Weissenbach Fish genomics and biology. Genome Research. 15: Sakamoto T, RG Danzmann, K Gharbi, P Howard, A Ozaki, SK Khoo, RA Woram, N Okamoto, MM Ferguson, LE Holm, R Guymard and B Hoyheim A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates. Genetics. 155: Salem M, PB Kennedy, CE Rexroad and J Yao Microarray gene expression analysis in atrophying rainbow trout muscle: A unique non-mammalian muscle degradation model. Physiological Genomics. In press. Scheerer PD, GH Thorgaard and FW Allendorf Genetic analysis of androgenetic rainbow trout. Journal of Experimental Zoology. 260: Smith CT, CM Elfstrom, LW Seeb and JE Seeb Use of sequence data from rainbow trout and Atlantic salmon for SNP detection in Pacific salmon. Molecular Ecology. 14: Somorjai IM, RG Danzmann and MM Ferguson Distribution of temperature tolerance quantitative trait loci in Arctic charr (Salvelinus alpinus) and inferred homologies in rainbow trout (Oncorhynchus mykiss). Genetics. 165: Taylor JS and J Raes Duplication and divergence: the evolution of new genes and old ideas. Annual Review of Genetics. 38: Thorgaard GH, GS Bailey, D Williams, DR Buhler, SL Kaattari, SS Ristow, JD Hansen, JR Winton, JL Bartholomew, JJ Nagler, PJ Walsh, MM Vijayan, RH Devlin, RW Hardy, KE Overturf, WP Young, BD Robison, C Rexroad and Y Palti. 2002a. Status and opportunities for genomics research with rainbow trout. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 133: Thorgaard G, G Bailey, D Williams, D Buhler, S Kaattari, S Ristow, J Hansen, J Winton, J Bartholomew, J Nagler, P Walsh, M Vijayan, R Devlin, R Hardy, K Overturf, W Young, B Robison, CE3rd Rexroad, Y Palti, B May, S LaPatra, R Phillips, L Park, T Sakamoto, N Okamoto, R Danzmann, F Allendorf, L Holm, R Bogden, P Iturra, R Guyomard and Guiguen Y. 2002b. A White Paper Advocating Complete Sequencing of the Genome of the Rainbow Trout, Oncorhynchus mykiss. submitted to the NIH. Thorsen J, B Zhu, E Frengen, K Osoegawa, PJ de Jong, BF Koop, WS Davidson and B Hoyheim A highly redundant BAC library of Atlantic salmon (Salmo salar): an important tool for salmon projects. BMC Genomics. 6: