Progress 10/01/00 to 09/30/06

Outputs
Resource limitations currently prohibit the individual rearing of multiple larval families for striped bass performance testing, necessitating a breeding program that is based on communal rearing of progeny groups with molecular markers as genetic tags for offspring identification. This project addressed fundamental questions relevant to selective breeding of the male parent of the HSB, the striped bass, including: (1) Evaluation of genetic variation within a captive striped bass broodstock population; (2) Examination of the feasibility of communal rearing protocols based on microsatellite markers for progeny identification during performance evaluations of striped bass; and (3) Assessment of paternal variation in performance traits of striped bass at both research and commercial scale throughout the HSB production cycle. Examination of three captive striped bass broodstock strains using three highly variable microsatellite markers revealed that the broodstock population contains moderately high genetic diversity, with an average allelic richness of 13.7 alleles per locus and an average observed heterozygosity of 0.84. Crosses among the three differentiated strains should provide a valuable starting point for establishing a highly variable base population for selective breeding. Twenty-four experimental families were produced from captive, genotyped broodstock for communal evaluations of progeny survival and performance. Parentage was determined by microsatellite genotyping at six loci and more than 99% of progeny were attributable to a single sire-dam pair at each production phase and in all rearing environments. Application of large-scale communal rearing trials based on microsatellite markers for progeny identification should be a viable approach in a selective breeding program for striped bass. There was limited evidence of family effects on early growth or survival to 35 days of age; however, significant paternal effects on growth performance, body shape, and carcass traits were detected at later culture stages and variation in antimicrobial peptide activity, a measure of innate disease resistance, differed by strain within the research ponds. In general, progeny of domesticated Santee:Chesapeake sires out-performed those of other strains both at research-scale and in the commercial tank. In research ponds, performance of fish as yearlings (Phase II) allowed prediction of performance at Phase III (18-20 months of age). Performance in research ponds also was predictive of Phase III performance in the commercial tank. Results from performance evaluations provide evidence of genetic variation in economically important traits, which may be exploited for selective breeding of striped bass. This research provides fundamental information needed to accelerate selective breeding and to increase production efficiency for the hybrid striped bass industry.

Impacts
Results of this project demonstrate that existing domesticated strains of striped bass developed by NCSU possess sufficient genetic variation to act as founder stocks for the National Program of Genetic Improvement and Selective Breeding for the Hybrid Striped Bass Industry and that select families of striped bass exhibit far superior performance in growth related traits in commercial and experimental production when compared to other families. Our results identify a clear genetic basis for traits such as growth rate that can be exploited in the national breeding program. This study was the first to pioneer use of DNA markers for pedigree tracking of striped bass and the first to employ "common garden" rearing of multiple families of striped bass in progeny performance trials, practices that will be incorporated into an accelerated program for selective breeding of superior striped bass. The fact that family performance in research ponds predicts performance in commercial settings and that performance of fish evaluated as Phase II (~9 month old) fingerlings predicts their performance assessed as (~18 month old) market sized fish offers obvious avenues to design rapid and cost effective breeding trials. Semen from scores of males of the family of striped bass that was top-performing in commercial tanks and research ponds has been cryopreserved and shipped for storage at the National Animal Germplasm Program in Ft. Collins Colorado in order to protect these valuable genetic resources.

Publications

• Couch, C.R. 2006. Microsatellite DNA marker-assisted selective breeding of striped bass, Morone saxatilis (Under the direction of Dr. Craig. V. Sullivan). Ph.D. Dissertation, Department of Zoology, North Carolina State Universty, Raleigh. 314 p.
• Couch, C.R., Garber, A.F., Rexroad III, C.E., Abrams, J.M., Stannard, J.A., Westerman, M.A., and C.V. Sullivan. 2006. Isolation and characterization of 149 novel microsatellite DNA markers for striped bass, Morone saxatilis, and cross-species amplification in white bass, M. chrysops, and their hybrid. Molecular Ecology Notes 6:667-669.
• Garber, A.F., and C.V. Sullivan. 2006. Selective breeding for the hybrid striped bass (Morone chrysops, Rafinesque X M. saxatilis, Walbaum) industry: Status and perspectives. Aquaculture Research 37:319-338.
• Rexroad III, C.E., Vallejo, R., Coulibaly, I., Westerman, M.E., and C.V. Sullivan. 2006. Identification and characterization of microsatellites for striped bass from repeat-enriched libraries. Conservation Genetics 7 (6): 971-982.
• Skalski, G.T., Couch, C.R., Garber, A.F., Weir, B.S., and C.V. Sullivan. 2006. Evaluation of DNA pooling for the estimation of microsatellite allele frequencies: a case study using striped bass (Morone saxatilis). Genetics 173 (2): 863-875.

Progress 10/01/04 to 09/30/05

Outputs
Breeding experiments were conducted at the NCSU Pamlico Aquaculture Field Laboratory (PAFL) using captive striped bass broodstock from three lineages. Performance assessments were made on fish reared in outdoor research ponds at PAFL, in an outdoor commercial production pond at Keo Fish Farms (Keo, Arkansas) and in an outdoor intensive culture tank at Kent Sea Tech (Mecca, California). Molecular pedigree tracking of communally reared fish by microsatellite genotyping was performed on the NCSU main campus and at the NCSU CALS Genome Research Laboratory on Centennial Campus. Prior to producing experimental families, we genotyped more than 120 adult broodstock available at PAFL to enable subsequent parentage assignment of their offspring. These broodstock represented three primary broodstock groups available at PAFL: a captive, wild Roanoke River lineage, a domestic Chesapeake lineage, and a domestic Santee:Chesapeake cross. Broodstock were crossed to produce 24 half-sibling families that were reared communally and sampled at the end of Phase I. Eighteen families were reared through Phase III. More than 99% of the sampled progeny could be assigned to their original parental pair with the six available microsatellite markers. Statistical analyses of data revealed that progeny from the Santee:Chesapeake broodstock group (domesticated at PAFL) were significantly larger (length, body weight) than other families, both in research ponds and in the commercial tank. A group of these top-performing fish were retained and grown to maturity for use as broodstock in later breeding experiments.

Impacts
Males (or cryopreserved semen) from the top-performing families has been provided to several commercial farmers for use in breeding HSB for progeny comparison with the industry standard HSB, produced largely from wild broodstock. Results of our project have stimulated much interest by HSB growers and other researchers, leading to development of a National Breeding Initiative for the genetic improvement of HSB. Dr. Sullivan is the Coordinator of the new breeding program and the bulk of breeding activities occur at PAFL. Top-performing fish from this project were utilized as broodfish in creating a highly diverse (>180 families) founder stock which are being utilized in future selective breeding efforts. Results of the research have been presented at the International Marine Biotechnology Conference, the International Plant and Animal Genome meeting, at several national and local meetings, by an undergraduate student for Research in the Capital at the NC State Legislature Building, and by several undergraduates at various NCSU Undergraduate Research Symposia. Additionally, the project has formed the basis of a doctoral dissertation in the Department of Zoology and has involved more than a dozen undergraduates in independent study, honors projects and hands-on training in molecular biology.

Publications

• Couch, C.R., Garber, A.F., Rexroad III, C.E., Abrams, J.M., Stannard, J.A., Westerman, M.E., and Sullivan, C.V. Sullivan. 2006. Isolation and characterization of 149 novel microsatellite DNA markers for striped bass, Morone saxatilis, and cross-species amplification in white bass, M. chrysops, and their hybrid. Molecular Ecology Notes. accepted.
• Garber, A.F., and Sullivan, C.V. Sullivan. 2006. Selective breeding for the hybrid striped bass industry: status and perspectives. Aquaculture Research. in press.

Progress 10/01/03 to 09/30/04

Outputs
Production of hybrid striped bass (HSB) is one of the fastest growing segments of U.S. aquaculture, yet neither the hybrid nor its white bass or striped bass parents have been genetically improved. The HSB farming industry recognizes that continued expansion will require full domestication and genetic improvement of these fishes. To evaluate the genetic basis of several commercially important traits, we performed a series of experimental crosses using three dams mated with groups of six sires from several striped bass lineages. Even-aged, half-sibling larvae were pooled by dam for communal rearing in a 1.6 ha commercial pond or in 0.1 ha research ponds. Fingerlings were harvested after 30 days and evaluated for sire-based differences in weight and length. Fingerlings were then stocked into replicate research-scale 0.1 ha ponds and flow-through tanks and into a single 17,000 gal commercial production tank for growout. Six microsatellite markers were utilized as innate genetic tags for parentage assignment. Preliminary analyses of harvest data from progeny of one dam from a single pond (n=400 fish) and from the commercial tank (n=252 fish) suggested that average weight and length differed significantly by genetic background in each environment. Rank order of half-sib families varied between environments; however, the two top-performing families in the research scale pond also performed best in the commercial tank. Data from these and other ongoing progeny evaluations will provide estimates of heritability for production traits in striped bass and will generate fundamental information needed to accelerate selective breeding for the HSB industry.

Impacts
The project has generated scores of new microsatellite DNA markers for Morone species, markers that will facilitate selective breeding and contribute to a genetic map of the Morone genome. Preliminary results of the project indicate that the striped bass domesticated at NCSU may produce superior performing progeny. The project has brought together as collaborators NCSU researchers, commercial producers of hybrid striped bass (Kent Sea Tech Corporation, Carolina Fisheries. Keo Fish Farms) and government scientists (Caird Rexroad III, USDA ARS National Center for Cool and Cold Water Aquaculture) and fostered establishment of the new national Program of Selective Breeding for the Hybrid Striped Bass Industry.

Publications

• No publications reported this period

Progress 10/01/02 to 09/30/03

Outputs
We generated multiple genetically tagged full- and half-sib families of striped bass (Morone saxitalis) produced from wild and domesticated parents, reared the fish through all phases of commercial culture, and sampled them at the termination of each culture phase, as follows: Phase 1 (harvest after 30 days as Phase I fingerlings), Phase II (harvest after 9 months as Phase II fingerlings), and Phase III (harvest at market size after 18 months). The parents used to make these crosses were genotyped or are being genotyped at several microsatellite DNA loci so that progeny can later be genotyped and assigned to a specific cross. When sampled at the end of each culture phase, offspring were evaluated with respect to a number of performance traits including, as examples, survival, growth (length, weight), morphology (appearance, body conformation, abnormalities), gender, gonadosomatic index, viscerosomatic index, dress-out weight (phase III only), and production by gill tissue of antimicrobial peptides (surrogate measure of potential disease resistance). Progeny are currently being genotyped for assignment to parents. Statistical analyses of the data will identify production phases during which contributions of genetic variation to fish performance are evident and can be manipulated through selective breeding. They also will provide initial estimates of heritability for specific performance traits and indicate whether or not several generations of domestication of the striped bass at NCSU has produced superior broodstocks. Preliminary results from this project were reported in a paper given at Plant and Animal Genome XII: an International Conference on the Status of Plant and Animal Genome Research held in San Diego in January 2004.

Impacts
The project has generated scores of new microsatellite DNA markers for Morone species, markers that will facilitate selective breeding and contribute to a genetic map of the Morone genome. Preliminary results of the project indicate that the striped bass domesticated at NCSU may produce superior performing progeny. The project has brought together as collaborators NCSU researchers, commercial producers of hybrid striped bass (Kent Sea Tech Corporation, Carolina Fisheries. Keo Fish Farms) and government scientists (Caird Rexroad III, USDA ARS National Center for Cool and Cold Water Aquaculture) and fostered establishment of the new national Program of Selective Breeding for the Hybrid Striped Bass Industry.

Publications

• No publications reported this period

Progress 10/01/01 to 09/30/02

Outputs
Breeding experiments were carried out as proposed using striped bass broodstock that were previously genotyped at several microsatellite DNA loci. Production, sampling, and restocking of phase I striped bass and hybrid striped bass fingerlings was accomplished as proposed. The Phase II fingerlings were recovered from the pond at the Pamlico Aquaculture Field Laboratory, graded into size categories, measured and weighed, fin clipped for identification of parentage, and sampled for tissue and blood to measure the endocine growth parameters. The Phase II fingerlings were restocked into the 0.1 ha ponds as proposed and grown out to near market size. At the time of this writing, they are being recovered from the ponds, enumerated, graded, and sampled as described above. Several parameters not listed in the original proposal also are being measured. These included an index of morphology noting any abnormalities (e.g. missing fins or abbreviated opercles) as well as gill tissue sampled for evaluation of natural antimicrobial activity. In addition, each fish sampled is photographed using a digital camera for analyses of body shape by truss analyses. Extraction of DNA from the finclip samples has proceeded with thousands of extracted samples being archived for later analyses of microsatellite genotype. Microsatelite analysis of these samples has begun and has included evaluation of the utility of additional loci using primer sets developed by us or our collaborators at Kent Sea Tech Corporation. Procedures for detection of microsatellite genotypes, including PCR and subsequent separation of amplicons on an ABI 377 DNA sequencer, have been optimized and multiplex strategies have been developed.

Impacts
Availability of improived strains of hybrid striped bass will lower production costs by increasing production efficiency. This development will ultimately allow hybrid striped bass to penetrate traditional retail markets, from which they are now excluded because the price point for hybrid striped bass is too high. Hybrid striped bass are currently marketed almost exclusively to restuarants and asian ethnic markets. Penetration of retail markets will allow hybrid striped bass farming to again grow exponentially, as it did in this country through much of the 1980s.

Publications

• No publications reported this period

Progress 10/01/00 to 09/30/01

Outputs
We pioneered DNA marker-assisted selective breeding of striped bass (SB) and white bass (WB) broodstock for the hybrid striped bass (HSB; genus Morone) farming industry. Procedures were established for high through put genotyping of fish based on microsatellite DNA markers (MDM). Our broodstock were genoyped at several MDM loci, but more markers are needed to allow flexibility when making crosses. In collaboration with Kent SeaTech (San Diego), we finished developing highly enriched phagemid MDM libraries and are `mining' them for additional markers. The new markers will contribute toward a map of the Morone genome, which can be used to identify quantitative trait loci and accelerate selective breeding. We initiated experiments aimed at discovering which phases of the SB production cycle will be amenable to quantitative genetic analyses. We generated genetically-tagged, full- and half-sib SB families that were pooled in equal numbers for communal rearing in `common garden' performance trials. Results will identify production phases in which contributions of additive genetic variation to fish performance are evident and can be manipulated through selective breeding. They will provide the first estimates of heritability of specific SB performance traits. We initiated similar growout trials of genetically-tagged HSB produced from wild versus domesticated broodstock. Results will indicate whether or not several generations of domestication has produced superior broodstock for commercial HSB production.

Impacts
Superior performing fish are needed because the HSB industry faces severe competition from foreign producers of similar white-fleshed fish, either wild-caught or farmed. Improved efficiency of production, via genetic improvements to the fish, will ensure continued competitiveness of HSB farming in the USA.

Publications

• No publications reported this period

Progress 10/01/99 to 09/30/00

Outputs
N/A

Impacts
(N/A)

Publications

• No publications reported this period