Identification of candidate DNA markers associated with IHN virus resistance in backcrosses of rainbow (Oncorhynchus mykiss) and cutthroat trout (O. clarki)
Introduction
Infectious hematopoietic necrosis (IHN) is a rhabdoviral disease of salmonid species. Under the intensive culture conditions of salmonid aquaculture, particularly for rainbow trout (Oncorhynchus mykiss), losses can approach 100% in fry and fingerlings (Wolf, 1988). The economic impact of the disease is substantial (Busch, 1983).
Genetic bases for resistance to infection with IHN virus (IHNV) in salmonid species have been examined by quantifying additive genetic variation for tolerance to IHNV (McIntyre and Amend, 1978). Both intra- and inter-specific variations in resistance could be useful in developing IHNV-resistant strains for aquaculture (Fjalestad et al., 1993). Interspecific differences in IHNV resistance have been demonstrated in the principal salmonid species (Pilcher and Fryer, 1980; Mulcahy and Wood, 1986; Bootland et al., 1994), and interspecific hybridization has been demonstrated to improve IHN resistance (Parsons et al., 1986; Dorson et al., 1991).
Reproductively viable interspecific hybrids expressing IHNV resistance could be useful in breeding programs by allowing the introgression of desired genes from resistant species into the more productive, but susceptible species. Backcrossing programs to introgress disease resistance genes have been used extensively in plants (Young et al., 1988). The gene introgression approach requires numerous backcrosses to minimize the proportion of the unwanted genome. Genetic markers tightly linked to resistance could increase selection efficiency and reduce the number of necessary backcrosses (Hillel et al., 1990; Hillel, 1992).
The high level of variability detected with multilocus RFLP fingerprinting enables a large number of informative loci to be rapidly analyzed (Spruell et al., 1994; Palti et al., 1997; Young et al., 1998). However, analysis of DNA fingerprints of individuals segregating for a trait is technically challenging (Dunnington et al., 1990; Spruell et al., 1994; Palti et al., 1997). Bulked segregant analysis (BSA) is a rapid method used in plant breeding to detect markers linked to economic trait loci in segregating populations (Michelmore et al., 1991). BSA is analogous to DNA mixing techniques used in poultry research (Dunnington et al., 1990) and recently in fish research (Spruell et al., 1994; Thorgaard et al., 1995; Palti et al., 1997), and it has been successfully used to identify DNA markers linked to disease resistance in a variety of plants (Barua et al., 1993; Kesseli et al., 1994; Thomas et al., 1995). Two important requirements for a successful application of BSA are that (1) the pooled DNA samples compared are from individuals originating from a single cross of parents that differ by a discrete trait, and (2) a large number of segregating individuals is used in each pool, with the objective of minimizing or eliminating variation not associated with the trait of interest (Michelmore et al., 1991; Hormoza et al., 1994).
Initial observations suggested that Yellowstone cutthroat trout (O. clarki bouvieri) fry from Henry's Lake, Idaho, and hybrids of female rainbow trout×Henry's Lake cutthroat trout male, are significantly less susceptible than typical rainbow trout to IHN virus infection in a waterborne exposure challenge (Parsons, J., 1995, unpublished data). Such hybrids are productive, viable and fertile (Rohrer and Thorgaard, 1986).
BSA has not been studied extensively in fish to identify DNA markers linked to economic loci and gene introgression assisted with DNA markers linked to the desired gene(s). In this study, we applied DNA mixing and subsequent multilocus RFLP fingerprinting to identify candidate DNA markers linked to IHNV resistance or susceptibility. The DNA fingerprints of first mortalities and survivors of IHNV challenge from 16 full-sib families of backcross progeny (four female rainbow×four male hybrid in a diallelic mating scheme) were screened for the presence and absence of RFLP markers, and the frequency of candidate markers among individuals first to die and survivors was compared. Candidate markers that appear to be linked to resistance or susceptibility could be used in the selection of parents for an introgression program.
Section snippets
Source of gametes and mating design
Eggs for the IHNV resistance study were obtained from nine randomly selected females from a commercial rainbow trout brood stock held by Pisces Investments (Pisces, PO Box 83332, Hagerman, ID, USA). Eggs were stripped into individual plastic bags and transported on ice to the research laboratory at Blue Lakes Trout Farm (BLTF), located near Twin Falls, ID. Semen was collected from seven rainbow trout×cutthroat trout hybrid males into individual plastic bags and transported on ice to the BLTF
Results
A significant interaction (P=0.001) was found between the sire and dam effects on IHNV resistance (Table 1). The sire and dam effects were also significant (Table 2).
An average of 35 distinct bands were detected with each of the 10 multilocus probes used, and 33 candidate markers were identified in the Southern analysis of the pooled DNAs from the 16 BC1 families (Table 3). The candidate markers identified were between 3 and 23 kilobase-pairs (kb) in size. One candidate marker was identified in
Discussion
We found evidence that DNA markers associated with susceptibility or resistance to IHN virus could be identified by bulked segregant analysis of multilocus RFLP markers. Two markers associated with susceptibility were identified in one backcross family of full-sibs, and one marker associated with resistance was identified in another full-sib family. The two markers appear to be linked to two different loci, which may be involved in protecting the fish cells from IHNV infection and/or clearance
Acknowledgements
This project was supported by the USDA/SBIR grant number 95-33610-1988. We thank Karen Adams and Shenoa Creer for their assistance with the preparation of the DNA samples and the Southern analysis, Marc Evans for his assistance with the statistical analysis, and Sandra Ristow for her review of the manuscript.
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