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.

Figure 1.

Molecular map of C. reinhardtii. A total of 506 molecular markers have been arranged on the 17 LGs; 385 are shown here. Markers are color coded as to their type: CAPS markers are in black, RFLP markers are in blue, STS and InDel makers are in red, and ± markers are in purple. All SNP markers are underlined; those that can be assayed using an alternative method are indicated by a color other than orange. Broken lines indicate gene clusters, where only representative markers are shown; the remaining markers are in the supplemental data (Supplemental Table I).

Figure 2.

Dhc4 is linked to mcd4. DNAs isolated from the mapping population described in the text were amplified with the Dhc4 CAPS primers (supplemental data) and digested with PstI. Digests were analyzed in a 3% agarose gel. The U indicates the undigested PCR product of 235 bp. Upon digestion, the mcd4 allele yields 191- and 44-bp products, whereas the S1-D2 (Mcd4) allele yields approximately 170-, 44-, and approximately 30-bp products. Forty-five out of 53 progeny tested are shown here, and the parental controls are at the left of the top row.

Figure 3.

DNA of up to six progeny can be combined for BSA. The indicated ratios of mcd5:S1-D2 DNA were used to simulate bulked progeny. PCR products for the markers indicated at right were analyzed in 1% agarose gels, and product sizes in basepairs are shown at the left.

Figure 4.

BSA affects mapping resolution. DNAs from four randomly selected mcd4 progeny were bulked per reaction and analyzed for the markers indicated on the left, with separation of PCR products in 1% agarose gels. Products were scored for number of progeny that contained the mcd4 allele. Actual mcd4 allele frequencies were determined by scoring individual unbulked samples (data not shown). Bulks from 48 out of the 53 progeny tested are shown here.

Figure 5.

Markers can be duplexed and combined with BSA. A, PCR products from 3 duplexed sets are shown, analyzed in 1% agarose gels. B, The Ald/Vfl2 duplexed primers were used in combination with BSA. PCR products were analyzed in a 1% agarose gel. The table at right shows expected product sizes for each marker. The symbols denoting the respective products from amplification of Ald or Vfl2 from mcd5 or S1-D2 are used to mark product positions on the relevant gels. S1-D2 generates a second, artifactual band with Ald primers that migrates at approximately 75 bp (below 122 bp S1-D2 product).

Figure 6.

Summary of mapping data showing the region of LG II surrounding mcd4. Available sequence from scaffolds 2 and 66 (genome version 2) is represented by the black bars, with red representing gaps in the sequence. The large gap is the estimated distance between scaffolds 2 (left) and 66 (right). Markers are color coded as shown in the box at top left. The potential positions of mcd4 are indicated by dotted lines. Distances between selected markers and mcd4 are given in centimorgans below the map. A BAC contig developed for the CNA45 region is shown to illustrate typical coverage where such contigs have been anchored.

Figure 7.

Flow chart for map-based cloning in Chlamydomonas. The scheme begins with a cross between the strain carrying the mutation of interest and the appropriate mating type of S1-D2. In the second step, one clone showing the mutant phenotype is selected, although in principle the S1-D2 (nonmutant) allele could be mapped by selecting one wild-type clone per tetrad. The initial mapping phase identifies a linkage group and chromosome arm, and depending on location, phenotype, and linkage, several subsequent steps may ensue.