DRAFT. Selection and Breeding Program for Rainbow Trout in Canada: Phase II. March 28, Prepared for: ACRDP

Transcription

1 1 Selection and Breeding Program for Rainbow Trout in Canada: Phase II DRAFT Prepared for: ACRDP March 28, 2010

2 2 TABLE OF CONTENTS Table of Contents 2 Summary Review of Phase I... 5 Questionnaire and Results... 6 Questionnaire and Responses... 7 Selective Breeding Program Type of Breeding Programs.. 11 Challenges to Implementation.. 12 Development Continuum.. 14 First Steps.. 14 Framework. 16 Development of a National Breeding Program. 19 Database and Data Collection. 19 Genetic Material Assessment.. 19 Family Based Breeding. 19 DNA Analysis and Assessment.. 20 National Breeding Program. 20 Needs and Concerns Toward Implementation. 20 Questions Posed.. 24 Conclusions Next Steps.... Additional Breeding Details and Associated Literature... Genetic Diversity.... Approaches Based on Phenotype.. Approaches Based on Genotype.... Marker-Assisted Selection... Literature Cited

3 3 Figure 1 Use of a Development Continuum to describe the effort over time for rainbow trout research and aquaculture development.... Figure 2 Framework for the initiation and execution of a selective breeding program for rainbow trout... Figure 3 Example breeding matrix using a partial factorial mating design... Figure 4 Example timeline for the execution of the first generation of a selective breeding program for rainbow trout. Figure 5 Details as to how the first three years of a selective breeding program for rainbow trout would initiate and proceed to harvest followed by subsequent broodstock selection.... Table 1 Modified framework for further discussion.... Appendix 1 Questionnaire ed or mailed to industry and associated individuals. Appendix 2 or mail cover letter. Appendix 3 Questionnaire.. Appendix 4 Slides developed for Phase II Workshop

4 4 Summary As part of the Industry Action Plan commissioned by the Interprovincial Partnership for Sustainable Freshwater Aquaculture Development (IPSFAD), a workshop on the Selection and Breeding of Rainbow Trout in Canada was convened February 2009 ( _NSERC_SWP_Workshop_on_Rainbow_Trout_Genetics.pdf). Following the workshop in 2009, a steering committee formed with the desire to continue onto the next steps toward the development of a selective breeding program for rainbow trout in Canada. The steering committee, comprised representation from IPSFAD, Northern Ontario Aquaculture Association (NOAA), Alma Research Centre and the University of Guelph, engaged the services of the Huntsman Marine Science Centre to conduct a survey of the key players in the rainbow trout aquaculture sector. This questionnaire was meant to serve as a next step to determine what type of framework could be developed and specifically what resources were available for implementing a national breeding program. The summary of this information was presented at a second workshop on 23 March The efforts made to date were reviewed and a potential framework for a selection and breeding program was discussed and modified. Conclusions were drawn as to what the next steps should be toward structure and implementation of a Selection and Breeding Program for Rainbow Trout in Canada.

5 5 Review of Phase I On February 12-13, 2009, the Interprovincial Partnership for Sustainable Freshwater Aquaculture Development (IPSFAD) held a Workshop on Selection and Breeding Program for Rainbow Trout Aquaculture in Canada in Guelph, Ontario. Delegates to this workshop included a diverse group of Canadian trout industry stakeholders and several invited experts in developing national breeding programs. The objectives of this workshop were threefold: 1) to assemble a team of experts in all aspects of Canadian rainbow trout aquaculture to generate ideas and strategies regarding the scope and nature of a Canadian Rainbow Trout Broodstock Program, 2) to review the current status of available technologies and practices regarding the selection and breeding of fish, in an effort to target effective approaches for a Canadian Rainbow Trout Broodstock Program, and 3) to identify the research, development and commercialization components necessary to establish a successful program. The rationale behind this workshop was to initiate an effort to enhance the quality and quantity of Canadian trout broodstock as long-term reliance on the present external suppliers is a threat to the rainbow trout sector. Currently, more than half the rainbow trout grown in Central and Arctic Region are imported from the United States. This importation of eggs is considered a risk on the sector in the event of border closures and, in addition, the genetic characteristics of the imported strains are not ideally suited to the grow-out conditions here in Canada. Dr. Graham Gall, one of the keynote speakers at the workshop, suggested that Part I of a breeding program is Step-by-Step Design including ten steps: 1) Assess the production system, 2) Formulate breeding objectives, 3) Review performance of available stocks, choose select initial stocks, 4) Obtain estimates of genetic parameters, 5) Devise system for animal evaluation, 6) Determine selection method, 7) Develop mating system for selected animals, 8) Develop plans for facilities, management and staffing, 9) System for expansion and dissemination, 10) Continuous evaluation and modification. Part II of a breeding program ( Selection Method Examples ) includes researching different data sources and selection schemes. Selection schemes presented were based on mass selection, combined family and within family selection and animal-model estimated breeding value selection. In mass selection, the observed harvest weight of the fish is assumed to be a reliable estimate of the breeding value of the fish with accuracy of selection a function of the estimated heritability. Combined and within family selection uses an index that is calculated for each individual fish performance of individual fish and average performance of all fullsib family members of those individual fish. Lastly, selection using the animal model with estimated breeding values (EBVs) was discussed. To calculate EBVs, all knowledge is used on individuals, families, parents used to produce those families (dams, sires) and grandparents (any additional pedigree data if available). The EBV

6 6 model typically includes additional fixed effects such as year, sex, location fish sampled if variable (e.g., different test stations) and usage of different stocks. Lastly, additional information taken from Richard M. Bourdon s Understanding Animal Breeding was discussed. This includes that breeding programs lasting for long periods of time display the attributes of understanding, good information, deliberation (taking time to think), consistency, simplicity and patience. Stressing that genetic progress is slow. Also, it is possible that environmental variations or any number of other shifts/changes can result in a next generation of fish who do not markedly differ from the previous generations. Breeders know, however, that if one generation does not show markedly improved results, then the next generation most likely will show the desired results. The workshop concluded with a review of the pertinent points discussed. Reviewing the production systems, in general, that are used across Canada. The desired traits or breeding objectives were listed. A list was devised of all stocks that may be available the list was not specific to the stocks that producers attending the meeting presently maintain and also included potential stocks from other companies (outside of Canada). In addition, it was expressed that the a system must be devised to accurately and similarly measure the traits important to commercial entities, That plans had to be devised for the facilities that would be used including management and staff and a general minimal cost of a program with the desire for dissemination across Canada (including British Columbia). Our approach to building on the information from this first workshop was to carry out a series of successive multi-stakeholder engagements (Primary Research) as well as a comprehensive review of the structures of selection and breeding programs through use of trade literature, internet sources, previously produced reports, etc. (Secondary Research). The results were presented at a Directed Phase II Workshop. Questionnaire and Results Following the workshop in 2009, a steering committee formed with the desire to continue onto the next steps toward the development of a selective breeding program for rainbow trout in Canada. The steering committee comprised representation from IPSFAD, NOAA, Alma Research Centre and the University of Guelph and engaged the services of the Huntsman Marine Science Centre to conduct a survey of the key players in the rainbow trout aquaculture sector. This questionnaire was meant to serve as a next step to determine what type of framework could be developed and specifically what resources were available for implementing this program. The summary of this information was presented on 23 March 2010.

7 7 This questionnaire was distributed to 37 producers/growers from 31 companies/organizations and 32 additional individuals (association, government, funding, research, and not-for-profit). This represented in excess of 90% of all the rainbow trout production in Canada today. Additional individuals who responded mainly confirmed their interest in receiving updates and/or being directly involved with a selective breeding program if/when possible. Responses were received from 16 producers/growers by, fax or verbally by phoning individuals. Additional calls were made and messages were left when possible. Responses were received from producers and/or growers in Alberta, British Columbia, New Brunswick, Ontario, Prince Edward Island, Quebec and Saskatchewan. Individuals receiving the questionnaire with contact information are listed in Appendix I. The cover /letter sent is included as Appendix II. The questionnaire is included as Appendix III. Questionnaire and Responses: 1) From the discussions at the last workshop, there are two leading options for the development a national framework. As a key influencer in the rainbow trout sector in Canada, which option would you prefer to see implemented (pick one)? a. Centralized National Breeding Program i. In this scenario the broodstock would be housed in a central location (for example, Alma Research Centre, Alma, Ontario). Eggs would be purchased from this location by producers and/or farmers. ii. Family testing would occur at the various grow out locations in the field ( test stations ). All of the trait and pedigree data would be stored in a centralized location. The program would be supported by a fee paid for when purchasing the eggs and or juveniles from the program. b. Dispersed National Breeding Program i. In this scenario, the broodstock are housed at individual farm locations. The eggs would be purchased from these locations by farmers. ii. Family testing would occur at various grow out locations in the field. All trait and pedigree data would be stored in a centralized location. Broodstock selection lists from data collected would be provided to those individuals having broodstock and performance data would be provided to farmers for future purchase decisions (Note: Some stocks/strains might perform better in freshwater versus saltwater conditions.). The program would be supported by a fee paid for when purchasing the eggs and or juveniles from the program. 57% of responders indicated that a dispersed program was preferable.

8 8 2) Would you be willing to participate in a national program? i. Participation for producers of eggs and juveniles may mean providing genetic material and space within their facilities. Participation for farmers may mean keeping certain production lots separate so that performance data can be collected and used for future selections. 52.9% of responders indicated that he/she would be willing to participate. 35.3% were unsure and said that participation would be based on how a national breeding program was structured. 11.8% of responders indicated that he/she would not be likely participate. However, one of the two individuals response that participation would be unlikely indicated it in a manner that participation could only occur as a producer and the individual was a grower. 3) If you are interested, could you provide a brief description of your facility including details such as: a. Annual production capacity (units eggs, juveniles, lbs harvested) b. Growing conditions (i.e. groundwater, surface water, etc.) c. Types of rearing units d. Heating or chilling capacity (if applicable) This question was posed to help better elucidate if locations may be suitable for a breeding program. In addition, details on growing conditions were included as surface water, for example, could be a biosecurity risk as potential for broodstock exposure to environmental pathogens or environmental shifts would be more likely. If a producer has access to surface water for a hatchery, then added a disinfectant such as an ultraviolet light source would be suggested. Usage of groundwater was the most likely response (54.5%; e.g., spring water). 27.3% of responders indicated usage of surface water (e.g., creek water). 18.2% of responders indicated that a combination of both ground water and surface water were used on site. Types of rearing units included raceways, various types and descriptions of tanks, pond and cages. Recirculating aquaculture systems may also be a possibility. The responses indicated that variable rearing facilities exist over a diverse geographic range. This information will be useful when locating test stations. Variability in test stations is beneficial as GxE testing in a breeding program. This is genotype by environment and will reveal whether top-performing rainbow trout families grown in one region in one system will be the same as the top-performing families grown in another region.

9 9 4) If you own your own broodstock: a. Are you able to export eyes eggs to other parts of the country? b. Where did your stock(s) originate? c. Do you photomanipulate your broodstock? d. Do you have some type of selection or selection program in place? If so, could you provide a brief description. Of the seven responding individuals having broodstock, one indicated that the appropriate health certification was not in place for shipping to other parts of the country. The remaining individuals noted that shipping is possible. Follow up will be necessary to ensure that an FHPR permit is in place and local Introductions and Transfers permits would be obtainable. Stock origin included Donaldson, Kamloop, Washington State and wild fish in British Columbia. The amount of genetic variation; however, cannot be quantified until the onset of a breeding program. Some producers indicated that fish came from a general location (not a specific stock) or might have been a mixture from several farms. Most facilities indicated that selection had been occurring for 30+ years with some introductions over time. Photomanipulation is practiced at three locations and was noted as possible at an additional location. The remaining three locations answered broodstock were not photomanipulated. It was noted that photomanipulation was not necessary as a large variation in spawning time was maintained. Of the seven individuals with broodstock responding, one indicated that a formal selection program was presently in place, but more to retain genetic diversity and prevent inbreeding. The remaining responders indicated that informal selection was based on faster growth, uniformity, late maturation, attempts to minimize inbreeding, to retain variation in timing of spawning and/or increase length of spawning season and selecting for a more streamlined body conformation. These questions are also helpful for the broodstock development portion of a breeding program as a diverse population with ample genetic variation is desired at the onset. As mentioned, the amount of genetic variation existing will be better quantified at the onset of a breeding program. 5) How is your data stored (paper, Excel files, farm control, database, etc.)?

10 % of responders indicated that production and grow-out data is stored on paper. 54.5% of responders store data on paper and transfer to Excel, enter data in Excel or enter data into a database. 6) At the Workshop in February 2009, the following traits were identified as having significant commercial importance. Please rank these according to your view of importance (1 as most important, 8 as least important). a. growth/weight b. survival c. delayed sexual maturation d. body conformation e. timing of spawning/egg production f. fillet yield g. lower visceral fat h. carcass colour The most important trait to those individuals responding was growth/weight. The second most important trait was survival. The remaining traits had three similar groupings for important, so were categorized as third, fourth and fifth. The traits identified as third most important were fillet yield and delayed sexual maturation. The traits identified as fourth most important were lower visceral fat and timing of spawning/egg production. The traits identified as fifth most important were carcass colour and body conformation. As might be expected, ranking of traits was somewhat a result of whether or not a producer or grower was responding to the questionnaire. In addition, geographic location also played a factor and resulted in responses that were more dependent on environmental location and specific conditions for growout. In general, the traits of survival, fillet yield and delayed sexual maturation were generally ranked as either very high or very low. 7) Are there other traits, not directly included in question 6, that you feel are commercially important? With regards to survival, may include resistance to a specific disease, salinity tolerance, or temperature tolerance. With regards to growth, may include weight at a specific age. Please also list any other challenge you face that has not been specifically included in question 6. Three additional traits were repeated as being important: 1) survival specifically in saltwater (salinity tolerance), 2) disease resistance (especially to bacterial

11 11 coldwater disease and bacterial gill disease), and 3) temperature tolerance to both warm water and cold water extremes. Additional traits were mentioned as important were external colouration (similar to a steelhead), ease of spawning and decreased spinal deformities. Published results exist for many of the traits listed as important to a selection breeding program for rainbow trout in Canada including traits such as growth (Iwamoto et al. 1986, Pante et al. 2002); visceral lipid weight (visceral fat), fillet weight and fillet percentage (Kause et al. 2007); reproduction (Gall 1975, Kause et al. 2003), and bacterial cold water disease (Silverstein et al. 2009, Leeds et al. 2010). Selective Breeding Programs Type of Breeding Program A Centralized Program is typical to salmonid breeding programmes such as the Atlantic salmon and rainbow trout breeding programmes in the national Norwegian breeding programme, response to selection is ~10% per generation for each of seven traits (body weight at slaughter, age of sexual maturation, survival in challenge tests with furunculosis and ISA, flesh colour, total fat content, and amount of fat tissues). The base population for the Atlantic salmon breeding program was formed from 1971 to The programme comprises two breeding centres and each breeding centre has four test stations (Gjøen and Bentsen 1997). This Norwegian programme is similar to the programme set up for Atlantic salmon at the Atlantic Salmon Federation in St. Andrews, New Brunswick, Canada (Friars et al. 1995). This Canadian breeding programme was transferred to industry in Dispersed breeding programs have been used in the livestock industry, but this type of program is not presently used for any fish species. However, one program for rainbow trout improvement in the United States has a somewhat similar concept. At the National Center for Cool and Coldwater Aquaculture (NCCCWA) in Leetown, West Virginia, the USDA lab began by using three strains from industry and university (Silverstein et al. 2004). While the stocks had undergone population bottlenecks that should have reduced detectable genetic variation within stocks, numbers of alleles at each locus (alleles per marker) remained somewhat high (Silverstein et al. 2004). Using these strains, the NCCCWA has undergone three generations of selective breeding producing a trout that is approximately 25 percent larger at one year of age (about 750 grams

12 12 versus 940 grams). A separate line of fish shows greater resistance to bacterial cold water disease with survival increases over a similar duration of approximately 45 percent (results from USDA US Program Aquaculture Accomplishments, 18 February 2010). In addition to selective breeding efforts, NCCCWA scientists are also researching production of tetraploids and triploids, cryopreservation, stress response, feed efficiency, production of all female lines, feed intake, time to first cleavage and diet by genotype interactions. While those contributing germplasm have access to the improved fish, much of the program is one of technology transfer. As the scientists make improvements to various techniques, this information is transferred to industry. In addition, the scientists also work directly with industry in their own breeding programs at a collaborative level. In this capacity, the NCCCWA is able to help a company improve production without directly contributing germplasm that could be later used to the benefit of another producer (pers. comm. Dr. Caird E. Rexroad III). The latter part of this model is being implemented, to some extent, with many companies who work directly with Universities, government or not-for-profit facilities that are helping industry to answer particular questions. However, in these cases, it may take longer to see improvements for particular traits as research may be tied to funding cycles that may not be continuous and/or may be tied to the schedule of a graduate student or other individual who cannot be fully dedicated to the effort. Challenges to Implementation Both breeding methods above inherently include a set of pros and cons most of which can be avoided by viewing as challenges and implementing the program accordingly. One challenge to both programs is the long term commitment from industry (producers and growers) and government. The only way the breeding program can become a success is to continue on a yearly basis (or twice yearly basis if production occurs in fall and spring). Those involved must realize that appreciable gains in traits such as growth may not be appreciable as (with a dispersed program) broodstock that produced fast growing progeny may not still be available and sisters or brothers to those broodstock may not yet be readily identifiable. In addition to a long term commitment, the program needs to be structured like a business. Several individuals feared that the program may benefit a particular scientist s research program first rather than directly benefiting industry. Strict biosecurity is a requirement and can be a challenge for either type of breeding program. Biosecurity is necessary to aid in the prevention of spreading pathogens from

13 13 one tank to another, within a facility or between facilities. If a facility is using surface water, then one method of increasing biosecurity is to use ultraviolet light on all incoming water. Other methods of biosecurity include foot baths, hand wash stations, booties and possibly gloves. This may become especially important if traffic of individuals increases between facilities. In addition, those individuals included within the breeding program will need to retain or obtain FHPR health certification. This certification will allow for transfer of eyed eggs and fingerling. When implementing a program with diploid progeny, those progeny can be selected directly and later used as broodstock. Therefore, both individual and family selection is implemented. With the rainbow trout industry, it may be necessary to create all female progeny for later use as broodstock and this may require an additional line or fish or tracking additional fish for use as future broodstock. In a dispersed program, production of triploid progeny would mean that each facility would need to have pressure shocking equipment as transferring this equipment between sites would introduce an additional biosecurity risk. In addition, if rainbow trout families that perform best in fresh water vary from those families performing best in salt water, then it might be necessary to produce two lines of progeny (or a producer could focus one of the two environments). In addition, if real time verification of progress is desired then a control line or essentially randomly mated synthetic line is needed. However, in Norway, wild fish were compared to selectively bred fish after a few generations to generally assess progress. Interprovincial transfer is a potential challenge to either type of breeding program (e.g., eggs sent to British Columbia have to be quarantined for three months). Alternative methods of gamete storage and transfer such as cryopreservation may help to alleviate this issue; however, transfer of gametes may not fit with a dispersed breeding program.

14 14 Development Continuum To more easily describe efforts that have been made over time toward the initiation of a National Breeding Program for rainbow trout, a Development Continuum was constructed. This continuum starts with Time Zero and works toward the Initiation of a Selective Breeding Program. Within this Development Continuum, a separate Framework for a selective breeding program is detailed. This framework was discussed on 23 March 2010 at a second workshop ( Workshop on a Selection and Breeding Program for Rainbow Trout Aquaculture in Canada: Phase II ) and, based on those discussions, modifications have been made to allow for further comments and changes. Figure 1. Use of a Development Continuum to describe the effort over time for rainbow trout research and aquaculture development. First Steps In the Development Continuum Time Zero was noted as the discovery or rather naming of rainbow trout (Oncorhynchus mykiss, Walbaum). This was followed by culture for sport fishing and also for aquaculture. Significant time and effort was placed into these endeavours; however, focus was placed on those efforts that have occurred most recently toward the development of a Selection and Breeding Program for Rainbow Trout Aquaculture in Canada.

15 15 In 2006, the Initiative became a registered not-for-profit organization the Inter- Provincial Partnership for Sustainable Freshwater Aquaculture Development (IPSFAD) with a mission to promote sustainable development of freshwater aquaculture in Canada. As part of the planning process and third industry action plan (2007/2009), 49 issues were identified in regional meetings and these were re-classified into 16 applied research, development and commercialization efforts within six thematic groups. The third thematic groups was identified as Broodstock Management. Key Issues within Broodstock Management were the desire to achieve economic gains via genetic selection to enhance quality and performance of rainbow trout populations. This might include the introduction of commercial strains from other jurisdictions and introduction of new genetic material from captive or feral populations both of these introductions with the target of improving performance. These desires led to the initiation of Project 9 within the overall action plan. Project 9 defined as the development of a national broodstock program to develop enhanced performance in rainbow trout targeting improved fillet yield, enhanced growth rate, and greater tolerance to warm-water conditions. These traits enhancements would be elucidated after evaluation of genetic characteristics (performance) of the target strains and their disease profiles. The first workshop Workshop on a Selection and Breeding Program for Rainbow Trout Aquaculture in Canada was a step toward the development of such a program. Please see above for a summary of the Phase I workshop. Within this workshop various methodologies were discussed as to how a selective breeding program might take shape. A questionnaire was sent to producers and growers (along with many supporting individuals researchers, government, etc.) to better determine what kind of framework could be developed. To do this, it was necessary to poll industry as to which type of breeding program (centralized or dispersed) was most attractive to them, their interest in involvement and the resources that they have and may be able to make available for implementation into the decided upon program. The results from the questionnaire have been detailed above and allowed for Option Development for a National Breeding Program with the most popular option being a Dispersed National Breeding Program. This program would allow each producer to retain his/her broodstock and performance testing would occur on those broodstock.

16 16 Framework Figure 2. Framework for the initiation and execution of a selective breeding program for rainbow trout. From the results of the questionnaire, it was thought that five producers would be both interested and would most likely be able to be involved at the onset of a selection and breeding program. The results also revealed that it may be possible to rear (grow-out) rainbow trout in five separate rearing locations. However, as an actual program unfolds, there may be a desire to either increase or decrease these numbers. Figure 3. Example breeding matrix using a partial factorial mating design. To obtain initial numbers of families and total numbers that might made sense from a production and rearing perspective, 40 families from each site were proposed for initiation of the program. It was proposed that these 40 families originate from 20 male and 20 female broodstock following the Berg and Henryon (1998) partial factorial mating

17 17 design (Figure 3). This type of mating design allows for genetic analysis of both paternal and maternal effects and also maximizes potential genetic diversity at the onset by using each male or female broodstock individual a minimum number of times. A mating design that produces both full sibling and half sibling (related) families is desirable. The half sibling relatedness allows the quantitative geneticist to tease apart the, for example, amount of increased growth that is a result of genetics (inheritance) versus the amount of increased growth that is a result of the environment. Fin clips would be collected and labeled from each of the broodstock used at the time of crossing. These fin clips would allow for the creation of a multiplex (group) of microsatellite DNA markers that could be used for the later identification of progeny at harvest. Coordination between the producers would be necessary to set aside a specific day(s) to make the crosses for the program and ensure that as little variation existed between sites possibly by agreeing upon a standard protocol if subtle variation in production exists. It was proposed, then, that 40 families from each of five producers of 200 families be produced and at least 1250 green eggs be shipped to a central hatchery. The central hatchery would then mix ~1250 eyed eggs from each family just prior to hatching. This would hopefully allow for later representation from all families or a larger number of families. The eggs would then be mixed and reared in the hatchery until they were a specified weight. The weight proposed was grams for two reasons 1) rainbow trout need to be grams prior to salt water entry and 2) questionnaire comments that a larger rainbow trout at entry into fresh water might be preferable. In addition, all rearing sites were marked to receive the same number of fish per site and this may vary as different locations may prefer different numbers of fish. Fish (progeny) would be reared at each site until harvest. At harvest, data on desired traits would be recorded from 2000 random progeny in the processing facility. From these progeny, a fin clip would be collected and preserved in ethanol. Progeny would then be genotyped using the pre-determined multiplex of microsatellite DNA markers and assigned to specific parents (that had been genotyped previously). The quantitative geneticist would then analyze this data and provide the results to the producers for selection of the next generation of broodstock. The cost of each fingerling progeny was listed as $3.00. Of the $3.00, $1.50 would return to the hatchery to pay for eggs and grow-out from green eggs to fingerlings and the remaining $1.50 would be used to fuel the breeding program. Several issues being inherent with this proposal including: 1) the quantitative geneticist must start prior to production and therefore no money would yet be generated, 2) initiation of the database might have some cost if purchased or if constructed and would need to be housed somewhere (probably at the University of Guelph), 3) $10.00 per genotyping sample is a bare minimum with a cost that may be closer to $25.00 per sample, and 4) data collection would incur additional costs for equipment, personnel and possible rental of space within a processing facility. In addition, parents of progeny would also need to be genotyped. At this time or potentially prior to this time, each producer may want to tag each fish (Passive Integrated Transponder) that may be used as a parent and

18 18 potentially initial fish as well. At minimum, the parents used for the program would need to be genotyped at a cost that is not presently included in the framework. Figure 4. Example timeline for the execution of the first generation of a selective breeding program for rainbow trout. Figure 5 Details as to how the first three years of a selective breeding program for rainbow trout would initiate and proceed to harvest followed by subsequent broodstock selection. Figures 4 and 5 give examples of timing for the framework described in Figure 2. Most importantly, the quantitative geneticist would need to start prior to the initiation of the

19 19 selective breeding program. In addition, production or families generated in 2010 would not inform broodstock selection until Development of a National Breeding Program Database and Data Collection Prior to data collection, a database should be created. This database could either be constructed by the quantitative geneticist hired to oversee the breeding program or purchased and set-up to fit the specific needs of a program for rainbow trout (e.g., Gen- Database from AquaInnovo). When the quantitative geneticist meets with those individuals interested in being involved with the breeding program, he/she will get a better feel for the structure of a database. This might include historical data from producers (and potentially growers). The historical data would be the first data collected for the database and might include information collected when broodstock were PIT tagged for later identification such as successive spawning events over a several year period. Subsequent data would be collected during spawning and continue to be collected until and including harvest each year. Entering data in a single location allows for efficient retrieval for analysis one query can link and output parentage data from spawning with progeny data at harvest for input into an analysis program such as ASReml (used to estimate breeding values). The database would include both private and shared parts. Privately, the quantitative geneticist can work with each producer to structure a program at each farm site that will allow them to track broodstock performance and develop a pedigree (family tree). These producers would have access to this data for their particular farm, but unless agreed upon, that data would not be accessible to other producers or growers. The shared data would be in the form of harvest results which families from which producers performed the best in which environments as some families may grow best in fresh versus salt water. Other families may have better survival. Genetic Material Assessment The next step is to assess genetic material that is available. From the results of the questionnaire, the dispersed breeding program was selected over a centralized approach. With this approach, at minimum, broodstock used to produce progeny for the program would be fin clipped and DNA would be isolated from these fish. This would allow for the selection of the best microsatellite markers available. These would be markers that are the most diverse (number of different alleles) within and between broodstock group(s) from each producer. Family Based Breeding

20 20 This would follow the traditional quantitative genetic approach previously discussed. Not discussed in any detail, but depending how the breeding program is set up, the collection of fin clip or other tissues may aid in the future implementation of a Quantitative Trait Loci (QTL) approach to selection of the best performing individuals. This type of selection, also termed Marker-Assisted Selection (MAS) allows for selection of the best performing individuals as soon as those individuals are large enough to be fin clipped as only a minimal amount of tissue is required for DNA isolation and subsequent amplification and testing. DNA Analysis and Assessment As previously mentioned, fin clip or other tissue samples would be collected during a harvest assessment along with the associated data. These tissue samples would allow for the genotyping of progeny. Progeny would be assigned parents. This data individual progeny, parents, dam, sire and trait measurements would allow for estimation of breeding values. These breeding values would allow the quantitative geneticist to provide selection lists to each producer. National Breeding Program The steps from Database to Data Analysis and Assessment and back to Database would create the loop that would serve as the Selection and Breeding Program for Rainbow Trout Aquaculture in Canada. Needs and Concerns Toward Implementation At the Phase II workshop held on 23 March 2010 concerns to implementation of the framework presented and initiation of a selective breeding program were discussed by the organizing committee, producers and growers. It was agreed that a quantitative geneticist should start prior to the initiation of a selective breeding program. The start date of this individual might be dependent on the ability to obtain external funding to cover this position for at least one year, but more likely several years of funding would be required. This funding period would allow the quantitative geneticist to have a secure position while working directly with producers and growers. Meeting and working with producers would allow the individual to help customize broodstock programs at each site and provide the technology transfer necessary to make these programs successful. Meeting and working with growers would allow the individual to ensure any necessary or important data be recorded along the way data that might help with subsequent production and/or may allow for the elucidation of variations in production that could cause variations in results seen during the analysis phase.

21 21 Genotyping rather than tagging progeny with Passive Integrated Transponder (PIT) tags is one method of tracking progeny. While genotyping does not allow for multiple measurements of progeny over time as PIT tagging does, genotyping does allow for testing of progeny at a commercial scale. It was agreed that testing at a commercial scale is important, so a breeding program should initiate using genotyping for progeny identification. It was agreed that a central hatchery should be used so that equal amounts of eggs from each family can be mixed. Also, if any variations in eggs between families or sites exist, then these variations can be noted. Differences in egg quality, for example, could result in differences in initial survival that would not be apparent after mixing but would become apparent at harvest. However, after mixing, sending eyed eggs to various sites for hatch and grow-out may be necessary as a result of transportation costs for fingerlings. For example, if Alma Research Station or another facility in Ontario is used for the central hatchery, then shipping eyed eggs to the east coast, west coast and/or prairies might be more economically feasible than shipping fingerlings. This might be best for a central hatchery as the hatchery may not have the ability to grow the number of fingerlings proposed to the desired size. It was noted that shipping fingerlings from PEI to Ontario cost $12-15,000. Several individuals inquired as to whether or not initial rearing in different hatcheries might cause problems for later analysis as initial rearing may affect performance. Specifically for analysis, this would not be an issue as the performance of every fish is compared to every other fish at each grow-out location (testing station), in addition to looking at individual and family performance over all sites. If one hatchery affect specific families, then presumably another hatchery might affect those families differently. Also, one method proposed to better research a variation in hatchery conditions would be to send the same family mixtures reared in two different hatcheries to one grow-out location with two cages at the same site. [Note: Variations could still exist between cages at the same site, but would be reduced if considering two cages at two different sites.] In the proposed framework, spawning and creation of families was only proposed to occur at one time point each year. There was a concern that one facility may have better fall spawners while another facility may have better spring spawners or the time of year decided to create families may be several weeks from the best time of year for each producer. In addition, some producers or the central hatchery may not have the needed fish health certification. To become FHPR certified, it takes approximately two years. For these reasons, it was discussed that the best option may be to start a quantitative geneticist sometime in 2010, but delay initiation of a selective breeding program until 2011 or The quantitative geneticist along with additional interested researchers could then assess the stocks that would be used for production and, with the help of producers, agree upon the best time to initiate the program by making family crosses. This delay would also allow producers to obtain the needed fish health

22 22 certification. It was noted that if the central hatchery was private, then a private company would be apprehensive in transporting in eyed eggs from another site and especially any site lacking an FHPR permit (if transfer would even be possible without this permit). It was also noted that if the central hatchery was located at Alma Research Centre then additional health certification would be necessary but would be achievable. The FHPR permits would also be necessary for those individuals receiving eyed eggs and/or fingerlings. It was also noted that even if broodstock and subsequent eggs are certified, this does not indicate that those eggs are resistant to a particular disease such as coldwater disease. If a hatchery has an issue with coldwater disease, then eggs from a non-resistant site might later look to be poor performers. This would be less of an issue if rainbow trout were grown at several hatcheries. Also, it would be standard protocol to note any disease problems, low oxygen events, temperature fluctuations and etc. which may allow for a correlation later. It was noted that both size of fingerlings and number of fingerlings desired for grow out varies between companies. An gram fingerling would need to be grown to a 3-4 pound fish to be profitable whereas a 25 gram fingerling would need to be grown to pounds to be profitable. In addition, some fingerlings are vaccinated prior to stocking. A program would need to be flexible in accommodating the needs of each company growing fish. For example, the east coast might require either 22,000 or 53,000 fish per sea cage and Ontario may require 70,000 fish to later be split into two 35,000 fresh water cages as fish grow. The cost of each fingerling may have to decrease, at least initially, as these rainbow trout will not yet be enhanced as a result of the broodstock program and will cost quite a bit more than those fingerlings presently purchased at $3.00 per fingerling. It was noted that one company could purchase a 100 gram vaccinated fingerling for $1.00 plus delivery. This is $2.00 less than proposed in the framework. However, $1.00 per fingerling is less than what other individuals may be paying as a per fingerling cost (expressed during a phone interview). There appeared to be a general agreement that, initially, the rainbow trout fingerlings would need to be sold for a competitive or possibly reduced amount as performance would be unknown in comparison to present fingerlings. This is a necessity as the fish would not be marketable for a premium. If the consume is not willing to pay more, then the company growing the fish cannot pay more. It was also noted that a company may not be able to justify taking fingerlings that have not be tested in salt water. Huntsman Marine Science Centre was noted as having facilities that would allow for the hatching of eyed eggs and production of fingerlings on the east coast. This facility could also do salt water testing in tanks at the onset or possibly a pay-for-survival insurance could be agreed upon with a salt water company in case mass mortality was experienced when fish were transferred to salt water. As variations exist in amount each producer sells an egg and in the amount each grower purchases a fingerling, those individuals otherwise interested in being involved in the breeding program will need to agree upon equivalent sale and purchase prices.

23 23 Further discussion must occur on the rainbow trout produced. At present, companies grow diploid rainbow trout, all female rainbow trout or triploid all female rainbow trout. Sometimes, the type of rainbow trout is the result of a personal preference and other times it is a provincial requirement. Detailed discussion did not occur as to the specific type of rainbow trout that would be produced. This may be partially resultant on the ability of the producer to produce a specific type of brood fish and it may also be partially dependent on the location and desire of the producers. It was noted that right now there is an ability to expand on the east coast and different provinces have different requirements (e.g., Nova Scotia requires that rainbow trout be all female). Some discussion of traits occurred. It was noted that some traits specific and important to producers may be discussed with the quantitative geneticist and that individual would be able to provide details on how best to incorporate that into a selection program for each producer. It should also be noted that initial harvest assessment should include several or all of the associated traits if it is deemed feasible from a time and economic perspective. Measuring multiple traits will allow the quantitative geneticist to look at the relationship between those traits as body weight and fillet yield, for example, might be so tightly genetically correlated that selection for body weight might result in better fillet yields. Measuring traits on rainbow trout pre-harvest was also mentioned as one method of shortening the production cycle and ensuring that some of the best broodstock producing one generation can be used in the next generation. To continue to make improvements, producers will need to discuss setting up a pedigree for their particular location with the quantitative geneticist as the same good broodstock can only be used so many times and using the same broodstock will not help in making genetic improvements. It was agreed that a framework can vary over time. Some individuals may stay with the program while others might enter and exit over time. In addition, changes can be made if certain pieces are found to work more efficiently in a different way. All appear to agree, though, that a selective breeding program must have some type of infrastructure that will allow it to continue. It was noted that there NEEDS to be a selective breeding program even if the program starts small and grows over time. Companies outside Canada are benefiting from breeding programs while producers in Canada lack the formal and support. Therefore, fish in Canada are not improving and will eventually lose any competitive edge they may presently have (at this point they may be lacking the competitive edge those companies have).

24 24 Table 1. Modified framework for further discussion. Note: Assumed price of eggs included in hatchery cost for production. Genotyping cost has been increased as a commercial scale of genotyping is not available at $10 per sample (presently looking into lower cost genotyping facility in Ontario). Data collection is an estimation and may be lower or higher. The overall cost of data collection will be somewhat dependent on the processing facility and whether or not any equipment can be provided in kind for measurements Item Estimated Expense Hatchery (~$1.50 per fish*175,000) 262,500 Quantitative Geneticist 70,000 Genotyping ($25 per fish*10,200) 255,000 Data Collection 20,000 Database/Support (Univ. of Guelph) 3,000 Total 610,500 Total Generated ($1.00 per fish) 175,000 Total Needed for Initiation 435,500 Questions Posed 1. Who is going to do this? It was noted that Alma Research Centre would be one possibility as having existing infrastructure that might be able to be modified with an initial infusion to work for this purpose (funding for reconfiguration and construction). Also, it was asked as to whether or not a private company may decide to serve as the central hatchery. In this case, a mechanism to finance start-up that would be repayable may be needed. 2. Will there be an external funding mechanism? It was agreed that this would be necessary to fuel the infrastructure noted above. Also, a quantitative geneticist would need to be hired prior to any profits from fingerling sales. Lastly, as discussed, fingerling sales may need to be decreased to increase the ability of growers in justifying grow-out of essentially untested individuals (some growers are already purchasing eggs and fingerlings from some producers, but not all producers that may be interested). It was discussed that several funding agencies may be able to provide the dollars needed for start-up. In addition, it may be beneficial to hire the quantitative geneticist prior to initiation of a program as this individual may be able to help put the network of funding together to cover such a program. i. IRAP, for instance, if working with a for-profit company or a collaboration of several companies (possibly under NOAA) could cover the salary of a quantitative geneticist.

25 25 ii. OMAFRA may be an option and the next funding intiative would be this fall. It was noted that matching funding for OMAFRA can be any type of funding (including funding from a government agency). iii. Each producer, for example, setting up a breeding program to their specifications, should look into SR&ED tax credits if he/she is not already benefiting from those. SR&ED tax credits can help with purchase of equipment. iv. AIMAP has announced a second round of funding with a deadline of 30 April The next round of proposals will be due in December 2010 for v. NSERC money can be used for long term research and development. vi. Program, once underway, will generate funds to cover staff and personnel. 3. Who will decide? Will one organization such as IPSFAD be charged with the decision as to the location and further details or should this be decided by a committee of individuals. If a committee, then will this committee be effective in coming to this decision?

26 26 Conclusions Next Steps Five conclusions or next steps were identified as a result of the Workshop on Selection and Breeding Program for Rainbow Trout Aquaculture in Canada: Phase II held on 23 March ) There is interest from all involved parties industry, government, not-for-profit, and researchers in initiating a selective breeding program. 2) A Coalition of the Willing will be formed primarily comprised of stakeholders (producers and growers). However, associated organizations should continue to be involved to see this to fruition. The steering committee for the workshop will convene the new group and hand off the action item. 3) A funding mechanism needs to be established. This funding mechanism may include various funding bodies. This is the first priority. 4) A decision as where this will be located and who will decide must be made. 5) Staff need to be hired (e.g., quantitative geneticist).

27 27 Additional Breeding Details and Associated Literature Genetic Diversity Typically, prior to the initiation of a selective breeding program, prior knowledge of the endemic range and genetic structure of the populations would be used so that genetically diverse founder broodstock with sufficient additive genetic variation (V A, breeding value, or measure of the amount that an individual s genetic makeup contributes additively to progeny phenotypes) be present for those traits targeted for improvement (e.g., Gjedrem 2000, Fjalestad et al. 2003). Collection of multiple potential breeders from genetically discrete populations would be important to evaluate variation in the performance of individuals from various geographic locations so that selective breeding protocols can be rationally designed. However, there is a need to assess genetic variation underlying fish performance in direct experimental trials rather than predicting it a priori based on indirect measures such as microsatellite DNA diversity as marker diversity does not predict variation in performance (Knibb 2000). In the framework presented, microsatellite marker variation is needed to identify broodstock to some extent and later assign parentage of progeny at harvest. The amount of microsatellite marker diversity within and between broodstock could be limited as a result, in part, of the selection that has occurred on many farms for 30+ generations. It is this selection, though, that could have also increased the overall genetic variation. When genetic variation was assessed using nine microsatellite markers for three strains of domesticated rainbow trout, Silverstein et al. (2004) found that adequate variation existed within and between stocks. Using allozymes and mitochondrial DNA, Ferguson et al. (1993) found that the mixed stock at Lyndon Fish Hatcheries selected for external appearance and growth was genetically diverse. McDonald et al. (2004) evaluated three strains of rainbow trout. One strain was from Ontario and having undergone many generations of selection for faster growth had reduced genetic variation at microsatellite loci (3-7 alleles per marker). This level of variation was less than that reported in Silverstein et al. (2004). No recent project has assessed the microsatellite genetic variation of the broodstock populations maintained by a rainbow trout producers in Canada. Approaches Based on Phenotype As has been discussed, it is essential that clear breeding goals be defined a priori and that breeding protocols that are practical for use within the existing industry infrastructure be developed. These protocols will, by necessity, be based on traditional application of quantitative genetics using selection based on phenotype, although it is likely that they will eventually include use of DNA markers and genetic maps to accelerate breeding gains.

28 28 Traditionally, selective breeding of organisms has been based on phenotypic or observable characteristics which develop as a result of the genotype (genetic makeup), the environment, and interactions between the two. The majority of traits likely to be exploited in a selective breeding program are quantitative traits, metric characters thought to result from the cumulative expression of multiple genes, each having a small effect on the phenotype. The collective expression of all combinations of alleles for the many genes influencing a quantitative trait produces a continuous spectrum of possible phenotypes approximating a normal frequency distribution (Falconer & Mackay 1996). Therefore, any of the many traits having a measurable and continuous probability distribution could be a potential target for improvement through selective breeding. In evaluating such traits, fairly large numbers of individuals must be measured in order to draw precise genetic conclusions. This requirement limits the types and numbers of traits that can be selected to those that can be practically and economically assessed in the laboratory or on the farm. The traditional quantitative genetic approach utilizes statistical parameters for traits of interest (e.g., heritabilities, genetic variances, genetic correlations) based on recorded pedigree data often in the absence of knowledge about the number, location, and effect of genes whose expression contributes to a trait, or the frequencies of favorable alleles at each relevant gene locus (Dekkers & Hospital 2002; Pagnacco & Carta 2003). Therefore, the expected increases in mean population performance per generation are proportional to the accuracy of estimates of breeding values (value of an individual estimated by the mean value of its offspring), intensity of selection (proportion of population selected for breeding), and population genetic variation (Dekkers & Hospital 2002). These phenotype-based selection methods have allowed for major achievements in cattle and pig breeding over the past 50 years (Pagnacco & Carta 2003) and they also have been employed successfully with Atlantic salmon in Norway (Gjøen & Bentsen 1997). Through selective breeding, a 10-15% increase in values of certain production traits per generation (~4 years) has been achieved for salmonids, and these carnivorous fish are now two to three times more efficient than pigs or broiler chickens in the conversion of energy and protein into edible food for humans (Gjedrem 2000). Four generations (~10 years) of selective breeding of coho salmon Oncorhynchus kisutch (Walbaum) has resulted in an increase in body weight greater than 60% of fish reared for 8 months in marine net pens (Hershberger et al. 1990). A study of tilapia Oreochromis niloticus L., which lasted 10 years and included 12 generations of fish subjected to fairly high selection intensities, resulted in genetic gains for 16-week body weight of over 10% per year (Bolivar & Newkirk 2002). For channel catfish Ictalurus punctatus (Rafinesque), domestication alone has resulted in growth rates increasing 2-6% per generation (~3 years) on average, with mass selection increasing body weight up to 55% after only four generations (Dunham & Liu 2003; mass selection identifies a percentage of the top performing fish and uses them as broodstock for production of the next generation). These observations illustrate the importance of domestication and selective breeding for

29 29 the aquaculture industry to meet market demands that can no longer be satisfied by the wild fishery (Bentsen & Olesen 2002). As mentioned previously, the salmonid breeding programs in Norway make approximately 10% gains on improvement of traits per generation (Gjøen and Bentsen 1997). The NCCCWA program has made an increase of ~25% in growth at one year of age (750 grams versus 940 grams) which decreases the time to market and ~45% in resistance to bacterial cold water disease which increases overall survival. These increase were made over three generations (results from USDA US Program Aquaculture Accomplishments, 18 February 2010). Knibb (2000) found that a marginal aquaculture company (approximately 5% profit on cost) achieving modest genetic gains of approximately 10% on important production traits could double its profits. Approaches Based on Genotype It is inherently difficult to effectively select individuals for breeding on the basis of values for traits that are very time consuming to measure (e.g., disease resistance), sex limited (e.g., egg quality), age limited (e.g., time to maturity), or recorded post mortem (e.g., flesh quality or fat content of fillets). These traits often have a low heritability and measurements must be made on siblings or other close relatives of candidate breeders. Using phenotypic measures alone, the accuracy of selection may be low for such traits or the cost of measurement may be too high to be economically feasible. Because samples of a candidate breeder s DNA can be obtained non-lethally, even from juveniles (e.g., blood samples, fin clips), genetic markers can be utilized in some of these cases to allow the direct selection of breeding candidates with regard to such traits, as well as to improve selection for traits that usually have high heritability and are easy to measure (e.g., growth). A genetic marker is a readily identifiable physical or molecular characteristic that is polymorphic (exists in two or more forms) and may be inherited in association with a particular gene or trait of interest. Traditional genetic markers for breeding, those that give rise to a visible phenotype, are rare since the effect of individual alleles at many loci combine to control quantitative traits. However, the advent of modern molecular biology has led to the development of many categories of genetic markers that can be mined to discover those linked to particular traits. Such markers are linked to quantitative trait loci (QTL) and may include the sequence of genes or proteins directly involved in expression of a quantitative trait (Type I markers) or anonymous segments of genomic DNA with no coding function whose inheritance can be tracked and correlated with a particular quantitative trait of interest (Type II markers). Type I DNA markers that have been discussed with rainbow trout include microsatellite DNA markers, expressed sequence tags (ESTs), and single nucleotide polymorphisms (SNPs). Most DNA markers employed in animal breeding programs are Type II

30 30 markers, but the demand for Type I markers is increasing as mapping of QTL and marker-assisted selection gain importance (Liu & Cordes 2004). At present, DNA markers also are being used to track animal pedigrees, assess the relatedness of individuals, and manage the rate of inbreeding. Marker-Assisted Selection Marker-assisted selection (MAS) involves the utilization of genetic markers closely linked to QTL to identify animals that carry genes for desirable (or undesirable) traits so that they can be selected or avoided as future broodstock. In MAS, progeny are selected based on whether or not they bear a particular marker allele at a locus that is presumed to flank a particular gene or suite of genes influencing a trait of interest. While the benefits of marker usage have been well documented, difficulties that arise in most cases include, but are not limited to, the cost of adding markers to a selective breeding program, alteration of a breeding program to accommodate markers, and lack of a genetic linkage map with sufficient marker resolution for the species of interest. In aquaculture, only a few species are subjects of a selective breeding program and selection based on genetic markers remains in its infancy. Nevertheless, several mapping projects (Young et al. 1998, Nichols et al. 2003) and an expressed sequence tag identification project (Rexroad et al. 2003) have been completed for rainbow trout. In addition, various projects have made strides in the identification of QTL for thermotolerance and length (Perry et al. 2005), spawning time (Sakamoto et al. 1999), spawning time and body weight (O Malley et al. 2002), resistance/susceptibility to infectious pancreatic necrosis virus (Ozaki et al. 2001). These projects are important as this information will decrease the time to utility of QTL in a MAS program, so markers be chosen as a method for selection of rainbow trout in the future. In fact, rainbow trout are one of the most widely studied model fish species with reference to genomics (Thorgaard et al. 2002). However, manuscripts exist on cryopreservation (Salte et al. 2004), nutrition (Gomes et al. 1995, Chaiyapechara et al. 2003), inbreeding, use of clove oil as an anaesthetic (Keene et al. 1998, Holloway et al. 2004) and salinity tolerance (Johnsson and Clarke 1988, Bystriansky et al. 2006), just to name a few studies.

31 31 Literature Cited Bentsen, H.B., Olesen, I., Designing aquaculture mass selection programs to avoid high inbreeding rates. Aquaculture 204: Berg, P., Henryon, M., A comparison of mating designs for inference on genetic parameters in fish. Proceedings of the Sixth World Congress on Genetics Applied to Livestock Production, Armidale, Australia, January, vol. 27, p Bolivar, R.B., Newkirk, G.F., Response to within family selection for body weight in Nile tilapia (Oreochromis niloticus) using a single-trait animal model. Aquaculture 204: Bystriansky, J.S., Richards, J.G., Schulte, P.M., Ballantyne, J.S., Reciprocal expression of gill Na+/K+-ATPase α-subunit isoforms α1a and α1b during seawater acclimation of three salmonid fishes that vary in their salinity tolerance. The Journal of Experimental Biology 209: Chaiyapechara, S., Casten, M.T., Hardy, R.W., Dong, F.M., Fish performance, fillet characteristics, and health assessment index of rainbow trout (Oncorhynchus mykiss) fed diets containing adequate and high concentrations of lipid and vitamin E. Aquaculture 219: Dekkers, J.C.M., Hospital, F., 2002.The use of molecular genetics in the improvement of agricultural populations. Nature Reviews Genetics 3: Dunham, R.A., Liu, Z., Gene mapping, isolation and genetic improvement in catfish. In: Aquatic genomics: steps toward a great future (ed. by Shimizu N., Aoki T., Hirono I. & Takashima F.), pp Springer-Verlag, New York. Falconer, D.S., Mackay, T.F.C., Introduction to quantitative genetics. Prentice Hall, England. pp Ferguson, M.M., Danzmann, R.G., Ska, A., Mitochondrial-DNA and allozyme variation in Ontario cultured rainbow-trout spawning in different seasons. Aquaculture 117: Fjalestad, K.T., Moen, T., Gomez-Raya, L., Prospects for genetic technology in salmon breeding programmes. Aquaculture Research 34: Friars, G.W., Bailey, J.K., O Flynn, F.M., Applications of selection for multiple traits in cage-reared Atlantic salmon (Salmo salar). Aquaculture 137:

32 32 Gall, G.A.E., Genetics of reproduction in domesticated rainbow trout. Journal of Animal Science 40: Gjedrem, T., Genetic improvement of cold-water fish species. Aquaculture Research 31: Gjøen, H.M., Bentsen, H.B., Past, present, and future genetic improvement in salmon aquaculture. ICES Journal of Marine Science 54: Gomes, E.F., Rema, P., Kaushik, S.J., Replacement of fish meal by plant proteins in the diet of rainbow trout (Oncorhynchus mykiss): digestibility and growth performance. Aquaculture 130: Hershberger, W.K., Myers, J.M., Iwamoto, R.N., McAuley, W.C., Saxton A.M., Genetic changes in the growth of coho salmon (Oncorhynchus kisutch) in marine net-pens, produced by ten years of selection. Aquaculture 85: Holloway, A.C., Keene, J.L., Noakes, D.G., Moccia R.D., Effects of clove oil and MS-222 on blood hormone profiles in rainbow trout Oncorhynchus mykiss, Walbaum. Aquaculture Research 35: Iwamoto, R.N., Myers, J.M., Hershberger, W.K., Genotype-environment interactions for growth of rainbow trout, Salmo gairdneri. Aquaculture 57: Johnsson, J., Clarke W.C., Development of seawater adaptation in juvenile steelhead trout (Salmo gairdneri) and domesticated rainbow trout (Salmo gairdneri) Effects of size, temperature and photoperiod. Aquaculture 71: Kause, A., Paananen, T., Ritola, O., Koskinen, H., Direct and indirect selection of visceral lipid weight, fillet weight, and fillet percentage in a rainbow trout breeding program. Journal of Animal Science 85: Kause, A., Ritola, O., Paananen, T., Mäntysaari, E., Eskelinen, U., Selection against early maturity in large rainbow trout Oncorhynchus mykiss: The quantitative genetics of sexual dimorphism and genotype-by-environment interactions. Aquaculture 228: Keene, J.L., Noakes, D.L.G., Moccia, R.D., Soto, C.G., The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture Research 29:

33 33 Knibb W., Genetic improvement of marine fish which method for industry? Aquaculture Research 31, Leeds, T.D., Silverstein, J.T., Weber, G.M., Vallejo, R.L., Palti, Y., Rexroad III, C.E., Evenhuis, J., Hadidi, S., Welch, T.J., Wiens, G.D., Response to selection for bacterial cold water disease resistance in rainbow trout. Journal of Animal Science online first. Liu, Z.J., Cordes, J.F., DNA marker technologies and their applications in aquaculture genetics. Aquaculture 238:1-37. McDonald, G.J., Danzmann, R.G., Ferguson, M.M., Relatedness determination in the absence of pedigree information in three cultured strains of rainbow trout (Oncorhynchus mykiss). Aquaculture 233: Nichols, K.M., Young, W.P., Danzmann, R.G., Robison, B.D., Rexroad, C., et al., A consolidated linkage map for rainbow trout (Oncorhynchus mykiss). Animal Genetics 34: O Malley, K.G., Sakamoto, T., Danzmann, R.G., Ferguson, M.M., Quantitative trait loci for spawning date and body weight in rainbow trout: testing for conserved effects across ancestrally duplicated chromosomes. Journal of Heredity 94: Ozaki, A., Sakamoto, T., Khoo, S., Nakamura, K., Coimbra, M.R.M., Akutsu, T., Okamoto, N., Quantitative trait loci (QTLs) associated with resistance/susceptibility to infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss). Molecular Genetics and Genomics 265: Pagnacco, G., Carta, A., Animal breeding from infinitesimal model to MAS: the case of a backcross design in dairy sheep (Sarda X Lacaune) and its possible impact on selection. In: Biotechnology in food and agriculture, Conference 10, Marker assisted selection: a fast track to increase genetic gain in plant and animal breeding? Session II: MAS in animals, pp Pante, M.J.R., Gjerde, B., McMillan, I., Misztal, I., Estimation of additive and dominance genetic variances for body weight at harvest in rainbow trout, Oncorhynchus mykiss. Aquaculture 204:

34 34 Perry, G.M.L., Ferguson, M.M., Sakamoto, T., Danzmann, R.G., Sex-linked quantitative trait loci for thermotolerance and length in the rainbow trout. Journal of Heredity 96: Rexroad III, C.E., Lee, Y., Keele, J.W., Karamycheva, S., Brown, G., Koop, B., Gahr, S.A., Pali, Y., Quackenbush, J., Sequence analysis of a rainbow trout cdna library and creation of a gene index. Cytogenetic Genome Research 102: Sakamoto, T., Danzmann, R.G., Okamoto, N., Ferguson, M.M., Ihssen, P.E., Linkage analysis of quantitative trait loci associated with spawning time in rainbow trout (Oncorhynchus mykiss). Aquaculture 173: Salte, R., Galli, A., Falaschi, U., Fjalestad, K.T., Aleandri, R., A protocol for the on-site use of frozen milt from rainbow trout (Oncorhynchus mykiss Walbaum) applied to the production of progeny groups: Comparing males from different populations. Aquaculture Silverstein, J.T., Rexroad III, C.E., King, T.L., Genetic variation measured by microsatellites among three strains of domesticated rainbow trout (Oncorhynchus mykiss, Walbaum). Aquaculture Research 35: Silverstein, J.T., Vallejo, R.L., Palti, Y., Leeds, T.D., Rexroad III, C.E., Welch, T.J., Wiens, G.D., Ducrocq, V., Rainbow trout resistance to bacterial cold-water disease is moderately heritable and is not adversely correlated with growth. Journal of Animal Science 87: Thorgaard, G.H., Bailey, G.S., Williams, D., Buhler, D.R., Kaattari, S.L., Ristow, S.S., Hansen, J.D., Winton, J.R., Bartholomew, J.L., Nagler, J.J., Walsh, P.J., Vijayan, M.M., Devlin, R.H., Hardy, R.W., Overturf, K.E., Young, W.P., Robison, B.D., Rexroad, C., Palti, Y., Status and opportunities for genomics research with rainbow trout. Comparative Biochemistry and Physiology Part B 133: Young, W.P., Wheeler, P.A., Coryell, V.H., Keim, P., Thorgaard, G.H., A detailed linkage map of rainbow trout produced using doubled haploids. Genetics 148:

35 35 Appendix I Questionnaire ed or mailed to industry and associated individuals. Method of Initial Contact Name/Details Phone British Columbia Alberta Manitoba mail mail mail mail mail Saskatchewan Ontario Hans Lehman, Miracle Springs Jim Powell, Freshwater Fisheries Society of BC Larry Albright, Freshwater Aquaculture Association BC Lourne Louden, Ackenberry Trout Farm Max Ménard, Smoky Trout Farm Clinton Boyd, Valley Fish Farm Ken Markosky, Clear Springs Aqua Farms Peter Palaschuk, Arctic Aquafarms Howard Plett, Two Fish Pond John Wityshyn, Manitoba RT Farmers Association Dean Foss, Wild West Steelhead Collin and Rachel Keet, Keet's Fish Farm Dale Jordison, Coldwater Fisheries Robert Devine, Coldwater Fisheries Tom Horne, Coldwater Fisheries Gord Cole, Aqua- Cage Fisheries Dan Glofcheskie, Northwind Fisheries info@miraclespringsinc.com jim.powell@gofishbc.com albright@sfu.ca ackenberry1@aol.com max@smokytroutfarm.com dfoss@wws-sk.ca info@keetsfishfarm.com djordison@coldwaterfisheries.com rdevine@coldwaterfisheries.com thomas.horne@sympatico.ca aquacage@vianet.on.ca norwin@cyberbeach.net (250) (604) (780) (403) (204) (204) (204) (204) (204) (306) (306) , (306) (705) (705) (705) (705)

36 36 Quebec Maritimes Ben Kanaswe, Buzwah Fisheries Pete Kanaswe, Buzwah Fisheries Mike Meeker, NOAA - MTM Aquaculture benkanasawe@yahoo.ca peterkanasawe@yahoo.ca mtmaqua@xplornet.com Karen Tracey, NOAA noaa@manitoulin.net Todd Gordon, NOAA todd.noaa@manitoulin.net mail mail Joanne Stevenson, Rainbow Springs Trout Hatchery Jim Taylor, Cedar Crest Trout Farm Lynn Rieck & Sean Pressey, Lyndon Fish Hatcheries Alvis Fogels, Springhills Trout Farm Jim Taylor, Linwood Acres Goossens Trout Farm, Brian Goossen Yves Boulanger, Pisciculture des Alléghanys Normand Roy, Ferme Piscicole des Bobines Dr. John Buchanan, Aqua Bounty Canada Dawn Runighan, Aqua Bounty Canada Sherman D'Entremont, Nova Fish Farms Inc. (CW NL) Jennifer Woodland, Cold Ocean Salmon & Nordland Aquaculture Ltd. Vernon Watkins, Long Island Resources Limited Jim_Taylor@everus.ca sean.lfhi@bellnet.ca alvis.fogels@sympatico.ca linwoodacres@sympatico.ca goose@oxford.net alleghan@globetrotter.net lesbobines@hotmail.com jbuchanan@aquabounty.com drunighan@aquabounty.com sdentremont@coldwaterfisheries.com jennifer.woodland@cookeaqua.com vernon.watkins@persona.ca Don Wolverton speckle@nb.sympatico.ca Harry Wolverton hwolverton@nb.sympatico.ca (705) (705) (705) (519) (519) (519) (519) (905) (519) (418) (819) (858) (902) (902) (709) (709) (506) (506)

37 37 Aaron Craig Chris Bridger, Aquaculture Engineering Group Other Sylvain Lareau, AAQ Bruce Swift, Swift Aquaculture Dr. Roy Danzmann, Integrative Biology, U. of Guelph Dr. Andy Robinson, Centre for Genetic Improvement of Livestock, U. of Guelph Prof. Richard Moccia, Dept. of Animal & Poultry Science, U. of Guelph Dr. Céline Audet, ISMER, UQAR Dr. Louis Bernatchez, Department of biology, U. Laval Dr. Brian Glebe, DFO, Aquaculture and Biological Interactions Dr. Bob Devlin, DFO, Centre for Aquaculture and Environmental Research Eric Gilbert, DFO Jay Parsons, DFO Steve Naylor, Ontario Ministry of Agriculture and Food Chris Wilson, Ontario Ministry of Natural Resources Richard Morin, MAPAQ (506) (506) (819) (604) (519) x58364 (519) x53679 (519) (418) x1744 (418) (506) (604) (613) (613) (519) (705) (418) x3374 Doug Geilling, DFO (705) Sylvain Langlois, (613) 996-

38 38 NSERC 7135 Tricia Gheorghe, (613) 998- DFO Science 2904 Guilaume Dagenais, (613) 990- DFO AMD 8745 Dan Stechey, (905) 377- Canadian 8501 Aquaculture Systems Luc Picard, Centre de transfert et de sélection des salmonidés Stephanie Houle, SORDAC Eric Boucher, IPSFAD Grant Vandenberg, IPSFAD David Bevan, AARS Michael Burke, AARS Ron Evans, IRAP Tim Jackson, IRAP Alistair Struthers, DFO Amber Garber, HMSC Bill Robertson, HMSC (418) (418) x3423 (418) x6580 (418) x6541 (519) x52689 (519) (519) (506) (613) (506) (506)

39 39 Appendix II or mail cover letter. Cover letter sent to individuals receiving questionnaire. Note: Last response included 19 March From: Amber Garber Sent: Monday, February 08, :48 AM To: Amber Garber Subject: Questionnaire - National Framework for a Selection and Breeding Program for Rainbow Trout As a follow up to the Workshop on a Selection and Breeding Program for Rainbow Trout in Canada held last spring in Guelph, Ontario and hosted by IPSFAD; we are seeking your input into the development of a national framework for the creation of such a program. Our key objective is to develop a plan that will be implemented to benefit the Canadian Rainbow Trout industry. There are two primary ways to participate: 1) fill out the questionnaire attached and return to us via by 16 February 2010, 2) respond to this with contact details so we can conduct a short phone interview and/or for additional explanation of any questions in the attached questionnaire. All of the responses will be pooled and the results will be presented at an upcoming second workshop in March Thanking you in advance, Amber Amber F. Garber, PhD Research Scientist The Huntsman Marine Science Centre 1 Lower Campus Road St. Andrews, NB E5B 2L7 T: F: E: agarber@huntsmanmarine.ca W: Please consider the environment before printing this.

40 40 Appendix III Questionnaire

41 41

42 42

43 43

44 44 Appendix IV Slides Developed for Phase II Workshop.

45 45

46 46

47 47

48 48