The unicellular green alga Chlamydomonas reinhardtii is the model organism of choice for studying many fundamental aspects of eukaryotic cell biology, physiology, and biochemistry. As examples, the biogenesis and function of flagella/cilia, basal bodies, and plastids are most easily investigated using Chlamydomonas. A draft sequence of the Chlamydomonas nuclear genome at >9X coverage has been released by the DOE's Joint Genome Institute, but the lack of reverse genetic resources is currently a major limitation for functional genomics of Chlamydomonas. TILLING is a high-throughput reverse genetics approach that combines traditional mutagenesis with modern technology for detection of point mutations. TILLING can provide an allelic series of mutations in any gene of interest. The goal of this project is to establish a publicly available TILLING resource for the Chlamydomonas research community, in collaboration with the Seattle TILLING Project, which has successfully developed similar resources for the Arabidopsis, Drosophila, and zebrafish communities.
The specific aims of this proposal are (1) to optimize UV/chemical mutagenesis of haploid Chlamydomonas and generate mutant populations for TILLING, (2) to perform pilot TILLING projects to determine if the haploid mutation density is sufficient for cost-effective TILLING, (3) to optimize UV/chemical mutagenesis of diploid Chlamydomonas and generate mutant populations for TILLING, (4) to perform pilot TILLING projects using a mutagenized diploid population, and (5) to provide a TILLING service to the Chlamydomonas research community. There is broad community support for the development of a TILLING resource for Chlamydomonas. The mutant populations and data generated by this project will be publicly available from the Chlamydomonas stock center and database. Relevance: Chlamydomonas is an important model for basic biological mechanisms and for human disease processes such as polycystic kidney disease, primary cilia dyskinesia, and Rh null disease. This project will generate a resource that will enable the determination of gene function for many genes that have been discovered by sequencing the Chlamydomonas genome. ? ? ?