Molecular Map of theChlamydomonas reinhardtiiNuclear Genome

Abstract

We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.Studies using the unicellular eukaryotic alga Chlamydomonas reinhardtii have yielded important insights into many cellular processes including photosynthesis (45, 123), flagellar assembly and motility (24,28,86,112,114,124,137,140), basal body assembly and positioning (115), gametogenesis and fertilization (35,42,167), DNA repair (109), phototaxis (49, 139), cell wall assembly (1), circadian rhythms (91, 162), and the regulation of metabolic pathways (3,14,31,50).A major strength of C. reinhardtii as an experimental system is its usefulness for genetic experiments (45,47,74). Vegetative cells are haploid, facilitating the analysis of mutant phenotypes, but stable diploid strains can be easily produced for dominance and complementation tests. Gametes can be crossed to yield diploid zygotes that sporulate to produce four products of meiosis, allowing routine tetrad analysis. Over the past 50 years, hundreds of mutations have been isolated; more than 200 genetic loci have been mapped to 17 linkage groups (28,29,45,46,52). Mutations induced by chemical or UV mutagenesis have been supplemented recently by mutations induced by transposition of one of several transposable element families in the genome (17,34,130,133) or by insertional mutagenesis (13,101,149).Insertional mutagenesis has become the favored method for generating mutations since the development of procedures for efficient transformation of the nuclear genome (59, 132). Upon transformation, plasmid DNA inserts in random positions into the nuclear genome, facilitating cloning of affected genes by using the transforming plasmid as a hybridization probe. This method of gene tagging has led to the isolation of numerous genes identified by mutation over the past several years. Despite its usefulness, the insertional-mutagenesis approach has drawbacks, including the inability to clone essential genes, difficulty in analyzing the large deletions that occur in some cases, and a limitation in the types of phenotypes that can be found by using a method that genera...

Beyond Complementation. Map-Based Cloning in Chlamydomonas reinhardtii

“…To date, 4 laboratories (see ''Materials and Methods'') have generated at least 506 STS, insertion/deletion (InDel), CAPS, SNP, 6 (a PCR fragment amplifiable in one polymorphic strain but not the other), and RFLP markers, whose respective utilities are discussed below. The combined molecular maps shown in Figure 1 contain 385 markers, representing 266 loci arranged on 17 linkage groups (LG), and differ from previously published maps (Kathir et al, 2003) by including markers generated by multiple laboratories. Fifteen markers could be assigned to an LG but not a specific location, whereas 107 are not displayed on the map because they are within marker-dense gene clusters or because precise marker order could not be determined due to insufficient data.…”

“…CAPS markers, on the other hand, require an additional step, since polymorphisms are revealed upon restriction enzyme digestion of the PCR product. SNPs are easy to find since only a single nucleotide difference is required, and on average 2.7 base substitutions were found in every 100 bp of sequence when S1-D2 and the laboratory strain were compared (Kathir et al, 2003). A disadvantage is that the reagents and technology for SNP detection are relatively expensive and some methods require special equipment.…”

“…Approximate distances were calculated based on the combined data and, where available, the genome sequence. Markers whose recombination data were not available were placed by alignment with the nuclear genome sequence, and their distances were calculated based on the assumption by Kathir et al (2003) that 1 cM equals 100 kb. This estimate was based on the total number of cM in the genome and an approximate genome size of 10 8 bp.…”

“…Mutations generated in this species can be mapped by crossing to the interfertile strain known as Chlamydomonas grossii, S1-D2 or its culture collection designation of CC-2290, which has a suitable profusion of sequence tagged sites (STS), cleavable amplified polymorphic sequence (CAPS), single nucleotide polymorphism (SNP), and RFLP markers (Gross et al, 1988;Vysotskaia et al, 2001;Grossman et al, 2003). Beginning with laborious RFLP-based mapping (Gross et al, 1988), Chlamydomonas mapping has moved toward a PCR-based method (Kathir et al, 2003) and now is poised to incorporate more high-throughput methods. This, in concert with an increasingly complete nuclear genome sequence (Grossman et al, 2003), provides the necessary tools for studies of all classes of mutations.…”

The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

“…The smallest genomic DNA fragment able to rescue the phenotype of 31E3 cells was a 9.7-kb subclone. It was mapped to Linkage group IX within 3 centiMorgans (cM) of the PF16 locus (Kathir et al, 2003). This map location and the mutant phenotype were in common with the uni2-1 mutation described by Dutcher and Trabuco (1998).…”