The USDA/ARS National Center for Cool and Cold Water Aquaculture (NCCCWA) in Leetown, West Virginia, is working to integrate molecular genetic technologies into a selective breeding program aimed at the genetic improvement of rainbow trout for aquaculture production efficien- cy. Our multidisciplinary approach aims to incorporate genome information with respect to dis- ease resistance and stress, growth, and reproductive physiology into our selective breeding pro- gram. Selective breeding is based on the hypothesis that sufficient genetic variation exists in broodstock to realize genetic improvement through contributions of superior performing germplasm to future generations. Our associated goal is to develop and transfer improved germplasm and technologies to the aquaculture industry. Our approach to implementing molec- ular information into our breeding program is outlined under three objectives: (a) development of a genetic map to reveal chromosomal locations affecting traits associated with aquaculture pro- duction, (b) use of a candidate gene approach to identify and characterize genes affecting impor- tant aquaculture production traits through functional genomic technologies, and (c) development of bioinformatic strategies to implement genetic mapping and functional genomic information into a selective breeding program

Palti, Yniv

Rexroad, Caird E. III

Silverstein, Jeffery T.

Vallejo, Roger L.

eVols at University of Hawaii at Manoa

The Open Access Israeli Journal of Aquaculture – BamidgehAs from January 2010 The Israeli Journal of Aquaculture - Bamidgeh (IJA) will be published  exclusively  as  an  on-line  Open  Access  (OA) quarterly  accessible  by  all AquacultureHub  (http://www.aquaculturehub.org)  members  and  registered  individuals and institutions.  Please visit our website (http://siamb.org.il) for free registration form, further information and instructions.  This transformation from a subscription printed version to an on-line OA journal, aims at supporting the concept that scientific peer-reviewed publications should be made available to all, including those with limited resources. The OA IJA does not enforce author or subscription fees and will endeavor to obtain alternative sources of income to support this policy for as long as possible.Editor-in-ChiefDan Mires Editorial BoardSheenan Harpaz Agricultural Research OrganizationBeit Dagan, IsraelZvi Yaron Dept. of ZoologyTel Aviv UniversityTel Aviv, IsraelAngelo Colorni National Center for Mariculture, IOLREilat, IsraelRina Chakrabarti Aqua Research LabDept. of ZoologyUniversity of DelhiIngrid Lupatsch Swansea UniversitySingleton Park, Swansea, UKJaap van Rijn The Hebrew University Faculty of AgricultureIsraelSpencer Malecha Dept. of Human Nutrition, Food and Animal SciencesUniversity of HawaiiDaniel Golani The Hebrew University of JerusalemJerusalem, IsraelEmilio Tibaldi Udine UniversityUdine, ItalyCopy Editor Ellen Rosenberg Published under auspices ofThe Society of Israeli Aquaculture and Marine Biotechnology (SIAMB), University of Hawaii at Manoa Library andUniversity of Hawaii Aquaculture Program in association withAquacultureHub http://www.aquaculturehub.org              ISSN 0792 - 156X Israeli Journal of Aquaculture - BAMIGDEH.PUBLISHER: Israeli Journal of Aquaculture - BAMIGDEH -Kibbutz Ein Hamifratz, Mobile Post 25210, ISRAELPhone: + 972 52 3965809http://siamb.org.il BARD Workshop: Aquaculture Genetics - Status and Prospects 323Integration of Molecular Genetic Information into theNCCCWA Selective Breeding Program for Rainbow TroutCaird E. Rexroad III*, Yniv Palti, Roger L. Vallejo and Jeffrey T. SilversteinUSDA/ARS National Center for Cool and Cold Water Aquaculture, Leetown, West Virginia 25430, USAKey words: rainbow trout, microsatellite, genetic map, expressed sequence tagAbstractThe USDA/ARS National Center for Cool and Cold Water Aquaculture (NCCCWA) in Leetown,West Virginia, is working to integrate molecular genetic technologies into a selective breedingprogram aimed at the genetic improvement of rainbow trout for aquaculture production efficien-cy. Our multidisciplinary approach aims to incorporate genome information with respect to dis-ease resistance and stress, growth, and reproductive physiology into our selective breeding pro-gram. Selective breeding is based on the hypothesis that sufficient genetic variation exists inbroodstock to realize genetic improvement through contributions of superior performinggermplasm to future generations. Our associated goal is to develop and transfer improvedgermplasm and technologies to the aquaculture industry. Our approach to implementing molec-ular information into our breeding program is outlined under three objectives: (a) development ofa genetic map to reveal chromosomal locations affecting traits associated with aquaculture pro-duction, (b) use of a candidate gene approach to identify and characterize genes affecting impor-tant aquaculture production traits through functional genomic technologies, and (c) developmentof bioinformatic strategies to implement genetic mapping and functional genomic information intoa selective breeding program.Genetic Markers and MappingTo date, much of our efforts have focused ondeveloping the diverse arsenal of species-specific genome tools and reagents that willbe required to exploit molecular genetic datain selective breeding. To this end, a suite ofDNA genetic markers called microsatelliteshas been developed to enhance our breedingprogram through a number of applications. Microsatellites are DNA repeats of 1-6nucleotides that occur naturally in thegenome. The number of times a repeat occursin tandem (alleles) can differ between individ-uals; fish that share common alleles are moreclosely related to each other than fish with dif-ferent sets of alleles (Weber and May, 1989).The most basic application of microsatel-*  Corresponding author. Tel.: +1-304-724-8340, ext. 2129; fax: +1-304-725-0351; e-mail: caird.rexroadiii@ars.usda.gov324lites is to determine parentage, allowing theevaluation of families in a common garden.For instance, fry that are too young to mark byother technologies can be combined to con-duct an experiment; parents of superior per-formers can be identified after performancetraits are measured (Palti et al., 2006). A sec-ond application is to characterize the geneticstructure of a population to avoid inbreedingthroughout generations of selection (Silver-stein et al., 2004). Although one result ofselection is increased inbreeding, the use ofgenetic markers can estimate the degree ofinbreeding due to selection and enable moni-toring to maintain an acceptable level ofgenetic diversity within broodstock acrossgenerations.The most complex use of markers is thegenetic mapping of genes affecting productiontraits. Genetic maps are constructed byobserving the inheritance of genetic markersacross generations of families. Traits of inter-est (e.g., disease resistance) can be mappedto a specific chromosomal region (or locus) byanalyzing co-segregation of marker alleleswith the phenotype in the same families thatwere used to construct genetic maps. Thisallows for the identification of a chromosomeregion (and perhaps a gene and its allelic vari-ants) that affect a trait. Identification of suchgenes allows for selection based on markergenotypes, referred to as marker or gene-assisted selection, where phenotypic perfor-mance has been shown to be correlated withmarkers or gene allelic variants. Genetic map-ping approaches target (a) traits that areexpensive or difficult to measure or requiresacrificing fish, (b) traits that are to be intro-gressed from an outside population into high-ly performing broodstock, (c) selection pro-grams where multiple traits can not be evalu-ated in the same fish, or (d) traits estimated tohave low heritability.Genetic markers and maps have beendeveloped for rainbow trout and othersalmonids at Washington State University, theUniversity of Guelph, INRA, and NCCCWA(Young et al., 1998; Sakamoto et al., 2000;Nichols et al., 2003a; Gilbey et al., 2004;Moen et al., 2004; Woram et al., 2004). Thesemaps have been used to identify loci influenc-ing natural killer cell-like activity (Zimmermanet al., 2004), temperature tolerance (Jacksonet al., 1998), spawning date (Sakamoto et al.,1999), body weight (O’Malley et al., 2003),resistance to infectious pancreatic necrosisvirus (Ozaki et al., 2001), resistance toCeratomyxa shasta (Nichols et al., 2003),embryonic development rate (Robison et al.,2001), and albinism (Nakamura et al., 2001).We elected to target three traits due to theirrelevance to rainbow trout aquaculture pro-duction efficiency: stress tolerance (crowdingstress measured by cortisol response), resis-tance to the bacterial pathogen Flavobacteri-um psychrophilum, and feed efficiency. Genetic mapping aims to identify DNAsequence variation associated with a genethat is responsible for variation in a trait. Oncea set of markers have been associated with atrait of interest, the next step is to identify thegene(s) responsible for the change in the trait.For this, molecular biologists and geneticistsconduct a series of fine mapping experiments.This includes genotyping more markers,development of more markers targeting thatspecific region of the genome, investigatingadditional crosses or populations with linkagedisequilibrium mapping, and identification ofcandidate genes from gene function orsequence information. Often candidate genesare selected based on comparative informa-tion from another species, whether it is func-tional or mapping information. In salmonids,we have the benefit of closely related specieshaving comparative maps (Danzmann et al.,2005). We recently began to develop compar-ative mapping information between trout andmodel research species (Rexroad et al.,2005).Fine mapping is the process of “zoomingin” on a smaller and more specific chromoso-mal region and narrowing down the list ofgenes in the locus affecting the trait. An exam-ple of the need for fine mapping is the chro-mosomal region that controls natural killercell-like activity in rainbow trout (Zimmermanet al., 2004). This locus is responsible for63.4% of the genetic variation and spans agenetic distance of 25.7 centiMorgans. ThisThe Israeli Journal of Aquaculture - Bamidgeh 58(4), 2006corresponds to roughly 21 million base pairsof DNA and includes roughly 200 genes.Fine mapping is greatly facilitated by bac-terial artificial chromosome (BAC) libraries.The rainbow trout genome has been estimat-ed to be 2.4 x 109 base pairs. To facilitategenome research, molecular biologists workwith small fragments of the genome retainedin bacterial cells. These BAC libraries aremanageable and conducive towards DNAsequencing and other laboratory manipula-tion. Libraries for rainbow trout have beenconstructed at the Tokyo University of MarineScience and Technology (2; Katagiri et al.,2001), NCCCWA (2; Palti et al., 2004), andWashington State University (Phillips et al.,2003). The BAC libraries can be used to con-struct a physical map of the genome based onoverlapping restriction enzyme digest frag-ments, a method also referred to as DNA fin-gerprinting (e.g., Ng et al., 2005). Once a setof clones representing a chromosome regionof interest has been obtained, they can besequenced to identify candidate genes. Thesegenes and the surrounding regions are theninvestigated to identify differences in DNAsequences between individuals that result indifferent phenotypes. Candidate Gene ApproachThe candidate gene approach uses informa-tion about known genes to investigate potentialeffects on traits of interest. For instance, theMajor Histocompatibility Complex in humansand many other vertebrates has been shown tomediate immune response to pathogens,therefore it is reasonable to expect that the cor-responding Major Histocompatibility Regions introut would serve the same function. The can-didate gene approach is greatly facilitated by alarge volume of gene sequence data fromwhich to identify candidates. This is obtainedby partially sequencing genes from differenttissues, periods in the life cycle, or experimen-tal treatments. The goal of obtaining these rep-resentative sequences is to identify as manyunique genes as possible and associate themwith a function.GenBank, a genome database hosted bythe National Center for BiotechnologyInformation (www.ncbi.nlm.nih.gov), currentlyhas 238,460 gene sequences from rainbowtrout, about 50% of which were submitted byNCCCWA. Use of this large amount of data isfacilitated by bioinformatic analyses such asthose conducted by The Institute for GenomeResearch (TIGR). The TIGR gene indices area user friendly and open access database toretrieve information (Rexroad et al., 2003).This includes potential or predicted proteinsequences, potential mutations, functionalannotation, and comparative informationobtained by comparing with sequences fromother species. The large volumes of genesequence information have enabled the useof microarray technology for salmonids (Riseet al., 2004; Krasnov et al., 2005; Tilton et al.,2005; Von Schalburg et al., 2005). This tech-nology allows investigators to examine theexpression of thousands of genes simultane-ously, whereas traditional protocols addresseach gene individually. ConclusionsSignificant progress has been made throughinternational collaborations in developingtools for rainbow trout genome researchincluding genetic maps, expressed sequencetags, and bacterial artificial chromosomelibraries (Thorgaard et al., 2002). The additionof these resources enhances our researchprograms and facilitates the development ofnew selective breeding strategies whichemploy molecular genetic data.ReferencesDanzmann R.G., Cairney M., DavidsonW.S., Ferguson M.M., Gharbi K., GuyomardR., Holm L.E., Leder E., Okamoto N., OzakiA., Rexroad C.E. 3rd, Sakamoto T., TaggartJ.B. and R.A. Woram, 2005. A comparativeanalysis of the rainbow trout genome with 2other species of fish (Arctic charr and Atlanticsalmon) within the tetraploid derivativeSalmonidae family (subfamily: Salmoninae).Genome, 48:1037-1051.Gilbey J., Verspoor E., McLay A. and D.Houlihan, 2004. A microsatellite linkage mapfor Atlantic salmon (Salmo salar). 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Integration of molecular genetic information into the NCCCWA selective breeding program for rainbow trout

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Integration of molecular genetic information into the NCCCWA selective breeding program for rainbow trout

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