Eric M. Hallerman

Breeding new strains of tilapia : development of an artificial center of origin and linkage map based on AFLP and microsatellite loci

Based on ideas from plant breeding and the opportunities offered by molecular biology, a program was initiated in 1995 to derive genetically superior tilapia from a synthetic stock .artificial center of origin, ACO produced by inter-crossing five groups of fish: Oreochromis w . . x

Detection of a chromosomal region with two quantitative trait loci, affecting cold tolerance and fish size, in an F2 tilapia hybrid

Abstract We searched for genetic linkage between microsatellite DNA markers and quantitative trait loci (QTL) for cold tolerance and fish size (body weight and standard length) in two unrelated F 2 families of interspecific tilapia hybrids ( Oreochromis mossambicus × Oreochromis aureus ). The first experiment was based on a family of 60 fish scanned for 20 microsatellites. A second experiment was conducted with a family of 114 fish scanned for 6 microsatellites in one linkage group, in order to test for QTL found in the first experiment. This two-step experimental design was used in order to protect against “false positive” associations. In both families, significant associations were found for two loci within the same linkage group. The two QTL, near UNH879 for cold tolerance, and near UNH130 for body size, were estimated to be 22 cM distant from each other, with no interaction found between the two traits. One of these loci, UNH879 , was also associated with sex determination. Distortion from the expected Mendelian genotypic ratio was observed for three markers: UNH130 , UNH180 and UNH907 , suggesting linkage with a QTL affecting survival. These results identify a chromosomal region in the tilapia genome harboring several QTL affecting fitness traits.

Transgenic fish and public policy: anticipating environmental impacts of transgenic fish.

Abstract Transfer of novel genes into fishes introduces a number of contentious issues into public policy debate among fisheries scientists and regulatory authorities. In the context of the technical status of development of transgenic strains of fishes, we discuss anticipated ecological impacts of releasing such fishes into natural environments. The major determinant of ecological impacts of transgenic fishes will be the phenotypic effect of the inserted genes. Three conceptual classes of phenotypic changes are anticipated, including changes in physiological rates, behavior, or tolerance of physical factors. The complex interactions among organisms and abiotic resources in aquatic communities suggest that it will be very difficult to predict ecosystem impacts of transgenic fishes. Based on current understanding of community-level impacts of stocking non-transgenic piscivorous fish, the release of certain transgenic fishes, particularly those exhibiting substantially altered performance, could destabilize...

SNP discovery and development of genetic markers for mapping innate immune response genes in common carp (Cyprinus carpio)

Single nucleotide polymorphisms (SNPs) in immune response genes have been reported as markers for susceptibility to infectious diseases in human and livestock. A disease caused by cyprinid herpesvirus 3 (CyHV-3) is highly contagious and virulent in common carp (Cyprinus carpio). With the aim to develop molecular tools for breeding CyHV-3-resistant carp, we have amplified and sequenced 11 candidate genes for viral disease resistance including TLR2, TLR3, TLR4ba, TLR7, TLR9, TLR21, TLR22, MyD88, TRAF6, type I IFN and IL-1beta. For each gene, we initially cloned and sequenced PCR amplicons from 8 to 12 fish (2-3 fish per strain) from the SNP discovery panel. We then identified and evaluated putative SNPs for their polymorphisms in the SNP discovery panel and validated their usefulness for linkage analysis in a full-sib family using the SNaPshot method. Our sequencing results and phylogenetic analyses suggested that TLR3, TLR7 and MyD88 genes are duplicated in the common carp genome. We, therefore, developed locus-specific PCR primers and SNP genotyping assays for the duplicated loci. A total of 48 SNP markers were developed from PCR fragments of the 13 loci (7 single-locus and 3 duplicated genes). Thirty-nine markers were polymorphic with estimated minor allele frequencies of more than 0.1. The utility of the SNP markers was evaluated in one full-sib family and revealed that 20 markers from 9 loci segregated in a disomic and Mendelian pattern and would be useful for linkage analysis.

Toward detection of quantitative trait loci and marker‐assisted selection in fish

Abstract Genes affecting aquaculture performance, or quantitative trait loci (QTLs), can be mapped in relation to naturally occurring genetic markers. Knowledge of linkages between marker and QTL alleles can be used for marker‐assisted selection (MAS), increasing the rate of genetic progress above that for selective breeding alone. Although fish may provide an attractive system for detecting QTLs and executing MAS, QTL detection and MAS have not yet been practiced on an aquaculture species. We review the technical literature on QTL detection and MAS in other species in order to advance critical discussion of how best to pursue QTL detection and MAS in fish. Over 100 highly polymorphic markers would be needed for complete genome coverage, although a subset of QTLs might be detected with fewer markers. The need for cost‐effectively screening markers suggests polymerase chain reaction‐based screening of a collection of microsatellite loci or RAPDs (random amplified polymorphic DNAs). Experimental power calcu...

Incorporating risk assessment and risk management into public policies on genetically modified finfish and shellfish

Abstract Genetically modified finfish and shellfish pose economic benefits to aquaculture, but also pose ecological and genetic risks to ecosystems receiving such organisms. Realization of benefits with minimization of risks posed by a new technology can be addressed through the processes of risk assessment and risk management. Public policies adopted by individual countries will reflect differences in the outcome of risk assessment and risk management processes resulting from differences among the receiving ecosystems and sets of human values at issue. A number of countries and international institutions have begun development of policies for oversight of genetically modified aquatic organisms. In the United States, a working group commissioned by the U.S. Department of Agriculture incorporated risk assessment and risk management principles into draft performance standards for safely conducting research with genetically modified finfish and shellfish. The performance standards address research with a broad range of aquatic GMOs, and compliance is intended to be voluntary. In contrast, the Canadian policy mandates adherence to specified guidelines for experiments with transgenic aquatic organisms; establishment as national policy is expected soon. Based upon the recently-adopted Gene Technology Act, Norwegian policy does not preclude use of genetically engineered aquatic organisms in the aquaculture industry, but the Norwegian government seems unlikely to support such use. Policies on aquatic GMOs have been adopted by leading international institutions concerned with fisheries management or aquaculture. The philosophy and technical content of oversight policies have important implications for scientists involved in research with aquatic GMOs.

Molecular analysis and growth evaluation of northern pike (Esox Iucius) microinjected with growth hormone genes

Gross, M.L., Schneider, J.F., Moav, N., Moav, B., Alvarez, C., Myster, S.H., Liu, Z., Hallerman, E.M., Hackett, P.B., Guise, K.S., Faras, A.J. and Kapuscinski, A.R., 1992. Molecular analysis and growth evaluation of northern pike (Esox lucius) microinjected with growth hormone genes. Aquaculture, 103: 253-273. Bovine (bGH) or chinook salmon (Oncorhynchus tshawytscha) growth hormone (csGH) cDNA genes were transferred by microinjection into newly fertilized northern pike (&OX lucks) eggs. Nonlethal screening of fin tissue showed genomic integration of transgenes in 88 of 1398 putative transgenie fish. Expression of bGH transgenes under transcriptional control of the Rous sarcoma virus long terminal repeat was detected in 36 of 1218 putative transgenic fish examined by radioimmunoassay of blood serum, Bovine growth hormone was also detected in mesodermal tissue of fins from microinjected fish using thin slice immunohistochemistry. Southern hybridizations of six tissues from a sample of 40 microinjected individuals revealed a high degree of mosaicism, with 30% of the fish containing detectable transgenic DNA in one or more tissues and only 41% of these containing detectable transgenes in fins. Growth of microinjected fish was quantitatively evaluated in three experiments. Average weight of microinjected fish was greater than that of controls of the same sex in four out of six groups. Significant growth enhancement (PC 0.05) was detected only for microinjected males in one experiment. Comparisons among molecular assays and individual fish growth in the founder generation indicated that the high degree of mosaicism prevented non-lethal indentification of all transgenic individuals and influences detection of growth enhancement.

Suggestive association of major histocompatibility IB genetic markers with resistance to bacterial cold water disease in rainbow trout (Oncorhynchus mykiss).

Genes within the major histocompatibility complex (MHC) are important for both innate and adaptive immune responses in mammals; however, much less is known regarding their contribution in teleost fishes. We examined the involvement of four major histocompatibility (MH) genomic regions in rainbow trout in resistance to the causative agent of bacterial coldwater disease (BCWD), Flavobacterium psychrophilum. Fish from the 2005 NCCCWA brood-year (71 full-sib families) were challenged with F. psychrophilum strain CSF 259–93. The overall mortality rate was 70%, with large variation in mortality between families. Disease resistance was quantified as post-challenge days to death. Phenotypic variation and additive genetic variation were estimated using mixed models of survival analysis. To examine association, eight microsatellite markers were isolated from MH gene-containing BAC clones and mapped onto the rainbow trout genetic linkage map. The parents and grandparents of the 2005 brood-year families were genotyped with these eight markers and another two markers tightly linked to the MH-IB region to assess the extent of linkage disequilibrium (LD) of MH genomic regions MH-IA, MH-IB, TAP1, and MH-II with survival post-challenge. MH-IB and MH-II markers were linked to BCWD survivability when data were analyzed by family. Tests for disease association at the population level substantiated the involvement of MH-IB, but not MH-II, with disease resistance. The impact of selective breeding for disease resistance on MH sequence variation is discussed in the context of aquaculture production.

GENETIC MANAGEMENT GUIDELINES FOR CAPTIVE PROPAGATION OF FRESHWATER MUSSELS (UNIONOIDEA)

Abstract Although the greatest global diversity of freshwater mussels (~300 species) resides in the United States, the superfamily Unionoidea is also the most imperiled taxon of animals in the nation. Thirty-five species are considered extinct, 70 species are listed as endangered or threatened, and approximately 100 more are species of conservation concern. To prevent additional species losses, biologists have developed methods for propagating juvenile mussels for release into the wild to restore or augment populations. Since 1997, mussel propagation facilities in the United States have released over 1 million juveniles of more than a dozen imperiled species, and survival of these juveniles in the wild has been documented. With the expectation of continued growth of these programs, agencies and facilities involved with mussel propagation must seriously consider the genetic implications of releasing captive-reared progeny. We propose 10 guidelines to help maintain the genetic resources of cultured and wild populations. Preservation of genetic diversity will require robust genetic analysis of source populations to define conservation units for valid species, subspecies, and unique populations. Hatchery protocols must be implemented that minimize risks of artificial selection and other genetic hazards affecting adaptive traits of progeny subsequently released to the wild. We advocate a pragmatic, adaptive approach to species recovery that incorporates the principles of conservation genetics into breeding programs, and prioritizes the immediate demographic needs of critically endangered mussel species.

Detection of Alternaria fungal contamination in cereal grains by a polymerase chain reaction-based assay.

Alternaria sp. are important fungal contaminants of grain products; they secrete four structural classes of compounds that are toxic or carcinogenic to plants and animals and cause considerable economic losses to growers and the food-processing industry. Alternaria toxins have been detected by high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay, and other techniques. Here, we report the development of a polymerase chain reaction (PCR)-based method for the detection of Alternaria DNA. PCR primers were designed to anneal to the ITS1 and ITS2 regions of the 5.8S rDNA gene of Alternaria alternata or Alternaria solani but not to other microbial or plant DNA. We compared the sensitivity of PCR in detecting Alternaria DNA, that of the HPLC method in detecting Alternaria alternariol and alternariol methyl ether toxins, and that of the morphological examination of mycelia and conidia in experimentally infested corn samples. The sensitivity of toxin detection for HPLC was above the level of contamination in a set of commercially obtained grain samples, resulting in negative scores for all samples, while the PCR-based method and mold growth plating followed by morphological identification of Alternaria gave parallel, positive results for 8 of 10 samples. The PCR assay required just 8 h, enabling the rapid and simultaneous testing of many samples at a low cost. PCR-based evidence for the presence of Alternaria DNA followed by positive assay results for Alternaria toxins would support the rejection of a shipment of grain.