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Submitted to: Marine Biotechnology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 6/30/2009 Publication Date: 2/1/2010 Citation: Waldbieser, G.C., Bosworth, B.G., Quiniou, S. 2010. Production of viable homozygous, doubled haploid channel catfish (Ictalurus punctatus). Marine Biotechnology. 12:380-385. Interpretive Summary: Inbred lines are useful tools for the identification of genetic and environmental factors that contribute to variation in performance for economically important traits. In order to produce viable, completely inbred channel catfish, we fertilized catfish eggs with sperm that had been exposed to ultraviolet light. Because the sperm genome was not viable, the resulting one-celled eggs were haploid. These haploid eggs were treated with pressure or high temperature to permit duplication of the genome but inhibit the first mitotic division, and the resulting offspring contained a doubled haploid genome. Molecular analysis of genetic loci verified the offspring were homozygous at all loci with no paternal genetic contribution. The homozygous, doubled haploid catfish are completely inbred and can be used to produce completely inbred populations for genetic analyses. The genomic DNA from doubled haploid catfish is also valuable for gene identification and sequencing of the channel catfish genome. Technical Abstract: Production of doubled haploids via mitotic gynogenesis is a useful tool for the creation of completely inbred fish. In order to produce viable doubled haploid channel catfish, we utilized hydrostatic pressure or thermal treatments on eggs fertilized with sperm that had been exposed to ultraviolet light. At 1.5 h post-fertilization, the embryos were exposed to either 590 kg/cm2 hydrostatic pressure for 3 min, 37°C for 5 min, or 41°C for 3 min. In the pressure-treated group, only 21 offspring hatched from five spawns with family sizes of 1, 2, 2, 4, and 12 offspring each, respectively. Eight embryos from the 37°C treatment and 32 embryos from the 41°C treatment groups survived to hatch. Genotype analysis using tri- and tetranucleotide microsatellite loci demonstrated all 21 offspring resulting from pressure treatment were homozygous at the 64 loci tested, and none contained alleles unique to the donor male. Eleven of 32 offspring from the 41°C treatment were homozygous at the 18 loci tested, while 21 offspring were heterozygous at 6 to 12 of these loci. Again, no offspring contained alleles unique to the donor male. However, all 8 offspring from the 37°C treatment were heterozygous at multiple loci, and one contained unambiguous paternal alleles. These experiments demonstrated our ability to produce viable homozygous, doubled haploid channel catfish. Doubled haploid catfish can be used to create completely inbred populations for genetic analyses, and homozygous genomic templates will be useful in gene identification and genome characterization. |
