Beyond Complementation. Map-Based Cloning in Chlamydomonas reinhardtii    

Abstract

Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.Sometimes called green yeast (Goodenough, 1992), the unicellular, eukaryotic green alga Chlamydomonas reinhardtii (hereafter called Chlamydomonas) is a venerable model system for plant biology as well as for cell motility. The tag green yeast refers to its haploid vegetative state, the existence of two mating types, and the general similarity in applicable genetic techniques. These aspects of Chlamydomonas biology have been previously reviewed (Rochaix, 1995).Like many microorganisms, screening of Chlamydomonas strains for rare mutations is straightforward, since large numbers of cells can be plated on an appropriate selective medium, or nonswimmers, for example, can be selected from large numbers of swimming cells. At the same time, the ease of nuclear transformation in Chlamydomonas, coupled with the plant-like nonhomologous integration of transforming DNA, facilitates the creation of insertional mutant collections. Taken together, the assortment of techniques useable for Chlamydomonas indulges both the amateur and experienced geneticist, yielding sometimes overwhelming collections of mutant strains. In this report, we focus on mutants affecting photosynthesis, in keeping with the thrust of this journal, and the emphasis of the newly renewed and National Science Foundation-supported Chlamydomonas genome project (http://www.chlamy.org/). However, the map-based cloning tools described here are generally applicable to Chlamydomonas biology.The use of Chlamydomonas to study the elaboration and regulation of the photosynthetic apparatus is long established and was recently review...

Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions inChlamydomonas reinhardtii

“…A dCAPS marker is made for each BAC gene to be tested in the transformant (Figure 1). Markers predicted by Rymarquis et al (2005) are tested. If no predictions are available, 39-UTR sequences are amplified and tested with 3-10 restriction enzymes (Bowers et al 2003).…”

Rapid genetic mapping in Neurospora crassa

“…We chose 40 individuals as an ideal bulk size because, as discussed in section 3.2, this is the minimum number required to establish linkage if 25% recombination is observed (95% confidence level, Chi square). Although we could have analyzed only the albino class, which would be the norm for bulked segregant analysis in diploid organisms (Martinez-Morales et al, 2004;Michelmore et al, 1991;Rawls et al, 2003;Rymarquis et al, 2005), we also included the wild-type class for confirmation, and a 1:1 mix of the two pooled DNA samples as a control. In retrospect, the value of these extra samples in terms of increased confidence in the scoring far outweighs the modest increase in workload.…”

The Hsp70 and Hsp40 Chaperones Influence Microtubule Stability inChlamydomonas

“…These results suggested that the APM1 and APM2 gene products may have physical interactions or that the genes may function in the same process or structure. With the completion of the Chlamydomonas molecular map (Kathir et al 2003;Rymarquis et al 2005) and the sequence of the nuclear genome (Merchant et al 2007), we were able to determine that apm1 mutations are caused by lesions in the DNAJ1 gene encoding the cytosolic Hsp40 type I protein, while the genetically interacting apm2 mutations are in the gene encoding cytosolic Hsp70A. The results indicate a previously unsuspected role of the Hsp70 chaperone system in regulation of microtubule stability.…”