Figure 1.
Analysis of the Parasexual Mating Cycle in C. albicans
(A) Overview of the mating cycle in C. albicans. White MTLa and MTLα cells must switch to the opaque state to undergo mating and formation of a mononuclear tetraploid a/α cell. A reduction in ploidy back to the diploid (or near diploid) can occur by random chromosome loss.
(B) A scheme for selection of diploid progeny strains from tetraploids. A tetraploid strain, RBY18, heterozygous for the GAL1 gene on Chromosome (Chr) 1 and for all four MTL alleles on Chr 5 was constructed by mating MTLa and MTLα diploid strains, as shown. After induction of chromosome instability, strains that had undergone a reduction in ploidy were selected for by growth on 2-deoxygalactose (2-DOG) medium, as only strains that have lost the GAL1 gene are able to grow on medium containing 2-DOG. Progeny strains were subsequently analyzed by PCR of the MTL locus and by flow cytometric analysis to confirm they were diploid strains. Strains were then analyzed by SNP and CGH microarrays to determine their genetic content.
Figure 2.
Analysis of Progeny Strains from the Parasexual Mating Cycle by Flow Cytometry
Progeny strains derived from the tetraploid RBY18 were grown in liquid YPD medium, as described in Materials and Methods. Strains P1 to P7 (C–I) were derived from growth of RBY18 on pre-spo medium, while strains S1 to S6 (J–O) were derived from growth of RBY18 on sorbose medium. In both cases, progeny strains were found to be diploid, or near diploid, by flow cytometric analysis. For comparison, a parental diploid strain (A) and tetraploid strain (B) were also analyzed by flow cytometry. The x-axis of each graph (Sytox) represents a linear scale of nuclear fluorescence, and the y-axis (Counts) represents a linear scale of cell number.
Figure 3.
Morphology of Progeny Strains from the Parasexual Mating Cycle
Progeny strains derived from the tetraploid RBY18 strain by growth on pre-spo medium (P1 to P7) or sorbose medium (S1 to S6) were analyzed on YPD medium. Strains were grown at 30 °C for 7 d and photographed. Many strains exhibited a mutant morphology, including increased surface wrinkling of the colonies indicative of increased hyphal cell formation. A control diploid strain (SC5314) and tetraploid strain (RBY18) are included for comparison.
Figure 4.
Schematic Summary of Genomic Profiles of Progeny Strains Derived from Tetraploid Strains via the Parasexual Cycle
Progeny strains were analyzed by SNP and CGH whole-genome microarrays to determine the copy number of each chromosome and the configuration of chromosome homologues. Chromosome homologues are indicated by blue and pink bars to represent “maternal” and “paternal” homologues, respectively. Genetic recombination events are indicated by loss of heterogeneity between chromosome homologues. In cases where the chromosome is trisomic, this is indicated by a bracket to the right of the trisomic chromosome. P1 to P7 progeny strains were derived from growth of the tetraploid RBY18 strain on pre-spo medium, while S1 to S6 strains were derived from tetraploid growth on sorbose medium. Detailed SNP and CGH array data is provided in Table S4 and Figure S4.
Figure 5.
Expression of C. albicans Spo11 Protein in Mitotic Cells
The expression of a Spo11–13 × myc-tagged protein was analyzed by western blotting. Lane 1 shows a control strain lacking the Spo11-13myc fusion construct, while lane 2 shows expression of a Kar3-13myc protein for comparison. Lanes 3–6 show extracts from four independently transformed diploid strains with the Spo11-13myc construct (see Materials and Methods).
Figure 6.
Morphology of Diploid Progeny Strains Derived from the Δspo11 Tetraploid Strain
Progeny strains were grown on YPD medium at 30 °C for 7 d and colonies photographed. Progeny strains Ps1 to Ps8 were derived from growth of the Δspo11 tetraploid strain on pre-spo medium, while strains Ss1 to Ss10 were derived from growth on sorbose medium. A control diploid strain (SC5314) and tetraploid strain (RBY176) are shown for comparison.
Figure 7.
Schematic Summary of Genomic Profiles of Progeny Diploid Strains Derived from the Δspo11 Tetraploid Strain via the Parasexual Cycle
Progeny strains were analyzed by SNP and CGH microarrays to determine the genetic content of each strain. As described in the legend to Figure 4, chromosome homologues are indicated by blue and pink bars to represent “maternal” and “paternal” homologues, respectively. In cases where a chromosome is trisomic, this is indicated by a bracket to the right of the chromosome. Ps1 to Ps8 progeny strains were derived from growth of the Δspo11 tetraploid (RBY176 or RBY177) on pre-spo medium, while Ss1 to Ss10 strains were derived from Δspo11 tetraploid growth on sorbose medium. Detailed SNP and CGH array data are provided in Tables S5 and S6 and Figure S4.
Table 1.
Summary of Genotypes in Progeny Strains Derived from the Parasexual Mating Cycle