Saprolegnia parasitica is a pathogenic oomycete that cause saprolegniosis. Freshwater fish like salmon and trout species are particularly vulnerable to infection, which is characterized by cotton-like grayish mycelial growth on the surface of the fish. Currently, an effective treatment for this disease is not available. This pathogen has a great impact on freshwater fish species world-wide. An initial step to keep this devastating disease at bay is the ability to detect the responsible pathogen, so that appropriate actions could be taken before it becomes widespread. The development of molecular tools that will accurately and rapidly detect S. parasitica is the main goal of this project. This project is divided into two main sections. The first section describes initial marker design efforts that were focused on the internal transcribed spacer (ITS) regions. Efforts were also made for the collection of field samples and the generation of our own ITS data that includes a number of Saprolegnia spp. Compiled sequence data allowed the identification of unknown samples and the adoption of the clade taxonomic system that other researchers had established for species designations. The accumulated sequence data helped to clarify the taxonomy within the genus Saprolegnia and complemented previous studies. The design of broad specificity PCR primers also allowed a quick initial detection of Saprolegnia spp., which could then be identified to species either by determining ITS nucleotide sequence or by a subsequent step of RFLP marker. Isolates sequence data in the compiled sample collection could be used for validation purposes in further marker development. The second section of the project described the development of higher specificity molecular markers for the detection of S. parasitica. These were based on the study of three different gene loci as potential markers. These included the Pumilio RNA-binding protein (Puf), Glutathionylspermidine synthetase (Gsp) and the thiazole biosynthetic enzyme (Thi4). The nucleotide sequence of each locus was studied to develop suitable PCR primers that were then refined through testing against our isolate collection to improve their specificity for the target species. Saprolegnia parasitica-specific markers were developed for the Puf and Gsp loci and these were further evaluated using our field collected samples. / Graduate

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