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Mapping of recombination breakpoints in a chytrid fungus Russell Poulter and Margi Butler Russell Poulter Department of Biochemistry Otago University [email protected] Batrachochytrium dendrobatidis (Bd) is the only chytrid known to infect vertebrates -causes chytridiomycosis in frogs and other amphibians -chytridiomycosis is a potentially fatal epidermal infection of amphibians -has caused mass mortality, population declines and extinctions -mechanistic/proximate cause of mortality is unknown, but may relate to osmotic imbalance/loss of electrolytes -symptoms include include abnormal posture, lethargy, loss of righting reflex Archey’s Frog Leiopelma archeyi Photo: Phil Bishop, Zoology Chytrids have been isolated from NZ frogs Maximum-likehood tree of B.atrachochytrium dendrobatidis among the eukaryotes Phylogeny of B. d. based on rDNA operon. (James et al., 2006) Southern Corroboree frog, Pseudophryne corroboree, one of the endangered Australian amphibian species. Bd has caused the species to become extinct in the wild Litoria ewingii (Brown Tree frog) infected with Batrachochytrium B. dendrobatidis occurs on all continents except Antarctica 1961 1938 Effect of Bd on amphibians, varies with host species and climate -Bd is a generalist pathogen known to infect over 90 amphibian species on five continents -a continuum exists in the host response to Bd -several species are known to carry infection without any recognised pathogenic effects -other species suffer up to 100 % mortality, rapid species declines and in some cases, extinction First described by Joyce Longcore in 1997 who was sent samples from dendrobatid (poison dart) frogs kept at the National Zoo (Washington) Zoospore of Bd is ~ 5 µm dia Discharge papillae form and contents cleave into zoospores. Only very low levels of genetic variation have been found in global surveys of Batrachochytrium isolates Morehouse et al., . Mol Ecol. 12:395-403 (2003) Only five variable nucleotide positions were detected among 10 loci (5918 bp) This suggests that Bd might be a recently emerged clone that has spread rapidly across the world. The alternative is that Bd has a very stable genome, that there are very few new mutations and that the heterozygosities are ancient and stable. http://www.broad.mit.edu/annotation/genome/batrachochytrium_dendrobatidis Strain sequenced = JEL423 http://genome.jgi-psf.org/Batde5/Batde5.home.html BLASTN search using part of a Broad contig as the query Query= supercont1.1 of Batrachochytrium dendrobatidis [DNA] 16000001610000 Searching not the assembled data but the raw(ish) sequence reads (the ‘traces’) Region with homozygous sites in both strains Region with heterozygous sites in both strains Region with sites polymorphic between two strains Supercontig 5: Haplotype data generated from examination of the Trace Data from the two whole Bd genome projects. Supercontig 5 is 1.7Mb. JEL423 JAM081 Haplotype A Haplotype B Repetitious Elements Supercontig 5: recombination breakpoints occur where there is a boundary between heterozygous and homozygous sequence. Six strains share a common breakpoint and must have a shared, common ancestor. original ancestral heterozygous supercontig 5 JEL229 JEL197 JAM081 * JEL423, JEL225, JEL308, RTP1-4 * JEL253 * P16 Supercontig 1: Five strains share a common breakpoint and must have a shared, common ancestor. These strains were also clustered in the supercontig 5 analysis, together with JEL 253. original ancestral heterozygous supercontig 1 JEL404 JEL229 JEL197 JAM081 JEL423 JEL225, P16 * * * RTP1-4 JEL253 * JEL308 Sequence chromatograms showing heterozygous sites supercontig 5 (position~ 600,000) JEL 197 JEL225 JEL253 JEL308 P16 Data: Cynthia Huang, MSc student Acknowledgments: University of Otago Research Committee Joyce Longcore, University of Maine The Broad Institute, MIT/Harvard Joint Genome Institute (JGI), University of California/Dept. of Energy, USA University of Otago New Zealand