Androgenetic zebrafish (Danio rerio): generation, confirmation and genetic utility (fluorescent RAPD, FRAPD)

To help investigate the evolutionary origin of the imprinting (parent of origin mono-allelic expression) of paternal genes observed in mammals, I developed methods to produce haploid and diploid androgenetic zebrafish (Danio rerio). Androgenotes receive their genomic DNA solely from their male parent, just as gynogenotes receive their genomic DNA from their female parent. Haploid androgenotes were produced by fertilizing eggs that had been x-ray irradiated to eliminate the maternal genome. Diploid androgenotes were produced by inhibition of the first mitotic division by heat shock. Analysis of parentally polymorphic DNA markers (Random Amplified Polymorphic DNA and Simple Sequence Repeats) confirmed the lack of significant maternal transmission to the androgenotes. Haploid androgenotes completed embryonic development but arrested as larvae, showing defects typical of the "haploid syndrome". Diploid androgenotes developed normally and have been bred. The survival of androgenetic zebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development. Production of haploid androgenotes is being used to determine the meiotic recombination rate in male zebrafish. Androgenesis has other useful genetic applications which are discussed. To produce androgenotes and to provide genetic evidence that androgenotes had been produced, two techniques were developed. Zebrafish eggs must normally be inseminated within a few minutes of being expressed from the female, allowing insufficient time to irradiate eggs for production of androgenotes. I developed the use of ovarian fluid from coho salmon (Oncorhynchus kisutch) for delayed in vitro fertilization, allowing manipulations of the eggs prior to fertilization. I also developed the Fluorescent Random Amplified Polymorphic DNA (FRAPD) technique for use on an automated DNA sequencer. This technique allowed the efficient production of strong genetic evidence to confirm the paternal origin of the genomes of the androgenotes produced. None of the 157 maternal-specific DNA markers analyzed using FRAPD, some of which were apparently homozygous, were passed on to any of the 18 putative androgenotes analyzed.