CONSTRUCTION OF A GENETIC LINKAGE MAP OF ZEBRAFISH

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This grant application is submitted in response to the RFA for Genomic Resources for the Zebrafish, requesting applications for funds to construct a 0.3 cM map in zebrafish. Our goal in responding to this RFA is to generate a dense microsatellite linkage map of the zebrafish Danio rerio, one that will have a dramatic effect on the ease of cloning genes by position, and to do so in a manner of ready utility for the entire community of scientists using this organism. More than 600 genes of developmental importance have been defined in zebrafish by chemical mutagenesis screens. Our institute is one major site for such screens and the cloning of the disrupted genes, especially those which are relevant to organ development. The mutations speak to important human diseases, including heart failure and arrhythmias, cancer, hematopoietic deficiency, retinal degeneration, polycystic kidney disease, and neural degeneration. Isolation of these genes will require a dense and robust genetic map, composed of reliable, easy-to- use markers that can be used in crosses between many strains of fish. Microsatellite markers based on simple sequence length polymorphisms (SSLPs) repeats throughout the genome are now the standard starting point for positional cloning and anchoring of other maps in most genetic organisms. They are abundant and well distributed through the genome; they are highly polymorphic et between outbred strains; unlike RAPDs, their high incidence of codominance makes them especially useful in diploids and non-inbred strains; and they are single locus amplification products, making them an essential tool for the anchoring of a radiation hybrid (RH)-based expressed sequence tag (EST) map to the genetic map, which is the other component goal of this RFA. During the initial years of our mapping project, we developed a low- density complete map with sufficient markers (600-700) to cover all 25 chromosomes at an average interval of 5 centiMorgans (cM). Many investigators around the world have used our current low-density microsatellite map to initiate positional cloning of important genes, but agree that we, as a field, must increase the number of microsatellite markers on our map and make them rapidly available. Our laboratory, building on the experience gained while making the low density map, has developed a streamlined method to do so.