| With the completion of the C. reinhardtii genome, a cornucopia of new genes awaits functional and interactional analysis. However, unlike the photosynthetic and flagellar gene complement, mutations in many of these genes are likely to be lethal in haploids, meaning that the approach of choice would be to induce and maintain the mutations in diploids and then screen for their effects either in haploid meiotic progeny or in diploids amenable to genetic analysis. A major drawback of heterozygous diploids is that they are refractory to genetic analysis: when induced to undergo gametogenesis by nitrogen starvation, they invariably differentiate as minus gametes due to their possession of the dominant MID gene in the MT- locus, and hence can only mate with plus gametes. Techniques for generating diploid plus strains have been developed using polyethylene glycol (PEG)-mediated protoplast cell fusion, but the PEG method is inefficient and finicky, and the resultant tetraploid meioses generate diploid products requiring additional backcross to express recessive traits. During the course of a study elucidating the homeoprotein-based induction of haploid → diploid → meiosis transitions in C. reinhardtii, we have developed procedures that induce self-mating in diploids and that generate diploid cell lines with the capacity to undergo meiosis without mating. These protocols, described in our presentation, should facilitate the isolation, characterization, mapping, and maintenance of recessive lethal mutations, followed by one-step haploidization to analyze recessive phenotypes. A protocol for generating haploid spores is also described that may abet long-term storage of mutant strains.
|