Reverse genetics of Chlamydomonas using TILLING
Krishna K. Niyogi1, Marilyn C. Kobayashi1, William Inwood1, Marina N. Sharifi1, Christine Codomo2, Jennifer L. Cooper2, Bradley J. Till2, Margaret C. Darlow2, Elizabeth A. Greene2, Steven Henikoff2, and Sydney Kustu1
1) Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102 USA
2) Fred Hutchinson Cancer Research Center, Seattle, WA 98107, USA
Despite the development of insertional mutagenesis and RNAi tools, a lack of reverse genetic resources remains a limitation for functional genomics of Chlamydomonas. Targeting Induced Local Lesions IN Genomes (TILLING) is a high-throughput reverse genetics approach that combines traditional mutagenesis with modern technology for detection of point mutations using a Li-Cor DNA Analyzer. In principle, TILLING can provide an allelic series of mutations in any gene of interest. To establish a publicly available TILLING resource for the Chlamydomonas research community, we generated a UV-mutagenized population of ~5,000 haploid colonies and assessed mutagenesis phenotypically using loss-of-function mutations in AMT4. Genomic DNA was prepared from each mutant in the population, and two-dimensional 16-fold pool plates were arrayed for high-throughput screening for point mutations. Beta test screening of 4,024 mutants discovered 23 mutations in four targets, and we calculated an average mutation density of 1 UV-induced mutation per 910 kb. Including earlier pilot screening of nine additional genes, we found a total of 78 mutations, of which 4% were truncations, 33% were missense, and 63% were silent. The number of mutations discovered per target gene ranged from 2 to 19. Several examples will be presented that illustrate how point mutations discovered through TILLING can provide new insight into gene function in Chlamydomonas. This work was supported by the NIH.
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