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Evidence and importance of genetic exchange among field populations of Trypanosoma cruzi
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Many eukaryotic pathogenic microorganisms that were previously assumed to propagate clonally have retained cryptic sexual cycles. The principal reproductive mode of Trypanosoma cruzi, the aetiological agent of Chagas disease, remains a controversial topic. Despite the existence of two recent natural hybrid lineages, a pervasive view is that recombination has been restrained at an evolutionary scale and is of little epidemiological relevance to contemporary parasite populations. This article reviews the growing number of field studies which indicate that natural hybridization in T. cruzi may be frequent, non-obligatory and idiosyncratic; potentially involving independent exchange of kinetoplast and nuclear genetic material as well as canonical meiotic mechanisms. Together these observations now challenge the traditional paradigm of preponderate clonal evolution in T. cruzi and highlight the need for additional, intensive and appropriately sampled field surveys, complemented by high resolution, combined nuclear and mitochondrial population genetics analyses.
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How clonal are bacteria?
J. M. Smith, N Smith, M. O'Rourke … (1993)
Data from multilocus enzyme electrophoresis of bacterial populations were analyzed using a statistical test designed to detect associations between genes at different loci. Some species (e.g., Salmonella) were found to be clonal at all levels of analysis. At the other extreme, Neisseria gonorrhoeae is panmictic, with random association between loci. Two intermediate types of population structure were also found. Neisseria meningitidis displays what we have called an "epidemic" structure. There is significant association between loci, but this arises only because of the recent, explosive, increase in particular electrophoretic types; when this effect is eliminated the population is found to be effectively panmictic. In contrast, linkage disequilibrium in a population of Rhizobium meliloti exists because the sample consisted of two genetically isolated divisions, often fixed for different alleles: within each division association between loci was almost random. The method of analysis is appropriate whenever there is doubt about the extent of genetic recombination between members of a population. To illustrate this we analyzed data on protozoan parasites and again found panmictic, epidemic, and clonal population structures.
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The ‘obligate diploid’ Candida albicans forms mating-competent haploids
Meleah A. Hickman, Guisheng Zeng, Anja Forche … (2013)
Candida albicans, the most prevalent human fungal pathogen, was considered an obligate diploid that carried recessive lethal mutations throughout the genome. Here, we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions and appears to arise via a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids including the yeast-hyphal transition, chlamydospore formation, and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization and both haploids and auto-diploids display a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, suggesting that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen. Background The opportunistic fungal pathogen C. albicans has been studied extensively since the 1800’s and has been considered a strictly diploid organism with no haploid state. The diploid nature of the organism has complicated genetic and genomic analyses of C. albicans biology and virulence. Studies in the 1960’s through the early 1980’s debated the ploidy of C. albicans, and proposed that a haploid state existed based on cell size heterogeneity 1 , parasexual genetics 2 and estimates of DNA content by fluorimetry 1,3 . Haploid clinical isolates were reported 2,3 , but were later found to be diploid in genome content or were shown to be non-albicans Candida species (T. Suzuki, personal comm. and Fig. S1). Research supporting the diploid nature of the organism included DNA content measurements that suggested a genome size similar to that of diploid Saccharomyces cerevisiae 4,5 , molecular studies that demonstrated the need to delete two gene copies to produce a null mutant 6,7 , genetic studies that demonstrated heterozygosity of specific alleles 8,9 , and ultimately the complete genome sequence, which revealed heterozygosity throughout much of the genome 10 . This, together with the predominantly clonal nature of C. albicans within an individual host 11,12 , was interpreted as evidence that the organism spent most, if not all, of its life cycle in a diploid state. The ‘obligate diploid’ nature of C. albicans was proposed to be due to recessive lethal mutations dispersed throughout the genome 13,14 . However, studies of C. albicans chromosome monosomy 15,16 , recombination 17 and haplotype mapping 18 demonstrated that homozygosis of certain chromosomes can occur, arguing against this balanced lethal mutation hypothesis. A diploid-tetraploid parasexual cycle was also discovered in C. albicans and shown to involve a switch to the ‘opaque’ physiological state that renders cells mating-competent 19,20 , conjugation between opaque diploids to form tetraploids 21,22 , and subsequent ploidy reduction resulting in diploid progeny that often carry multiple aneuploid chromosomes 18,23 . Importantly, specific aneuploid chromosomes can provide a selective advantage under stressful conditions such as exposure to antifungal drugs 24 . Parasexual ploidy reduction in tetraploids occurs via a non-meiotic process termed ‘concerted chromosome loss’. This process can facilitate the rapid generation of diversity through the production of homozygous and aneuploid progeny, under conditions where outcrossing in the host is unlikely 25 . If a haploid state for C. albicans were to exist, it could facilitate the elimination of lethal alleles from the population. Furthermore, mating between different haploids could promote adaptation to changing conditions within the mammalian host. With the advent of whole genome approaches that can distinguish ploidy states, it is now possible to ask if C. albicans exists, even transiently, in the tetraploid or the haploid state. Detection of haploid C. albicans cells Haploid C. albicans was serendipitously discovered during experiments to follow loss of heterozygosity (LOH) at multiple independent loci. Using a multiply marked derivative of SC5314, the laboratory reference strain 10 , we selected for an initial LOH event at the GAL1 locus by growth on 2-deoxygalactose (2-DOG) and subsequently screened for additional LOH events at four other heterozygous loci. Importantly, growth in 2-DOG does not affect LOH rates (Fig. S2) and as such, the selection for cells with an LOH event does not artificially induce LOH. Amongst the ~2500 Gal− isolates, 42 exhibited additional LOH events and intriguingly one strain, Haploid I, was homozygous for all of the markers tested, as well as for multiple SNPs on every chromosome. Analysis of DNA content in Haploid I by flow cytometry indicated that the genome had half the amount of DNA of that in a diploid control (Fig. 1a). Once we became aware that haploid C. albicans cells could be detected, we used flow cytometry to screen isolates from many sources, including in vitro stresses, 26 from which one haploid isolate (Haploid IV) was identified amongst small colonies growing in the presence of fluconazole (in the halo of an E-test strip), a commonly used antifungal drug. We also screened C. albicans cells isolated from in vivo mouse models of candidemia 27 and candidiasis 28 and discovered several additional isolates with 1N DNA content. The overall population of post-in vivo isolates screened for ploidy included ~300 isolates from YPD plates (Gal+), and ~740 isolates from the 2-DOG selection plates (Gal−) and only the DOG-resistant colonies contained isolates that were haploid. We found that 3.2% of DOG-resistant colonies from the OPC model were haploid (10 out of 312), while 1.2% of DOG-resistant colonies from the systemic model were haploid (5 out of 431). Since LOH frequencies in vivo are ~10−3, 29 , this indicates that detectable haploids appeared at a frequency of 1–3 per 100,000 cells. Loss of GAL1 is often, but not always associated with the haploid state (e.g. Haploid IV), especially in diploid progenitors that are heterozygous for this locus. In total, we characterized eleven strains with haploid (or close to haploid) flow cytometry profiles (Fig. 1a, S3 and Table S1) that were derived from multiple independent and varied sources. Since previous reports of haploid C. albicans 3 included misidentified species (Fig. S1), we determined if these newly identified isolates were bona fide C. albicans by PCR amplifying and sequencing the mating-type-like (MTL) locus. All isolates contained sequences identical to either the MTL a or MTLα alleles of SC5314 (Fig. S4 and data not shown). Next, we analyzed the haploid genomes by hybridization to SNP/CGH arrays 30 which measure allelic ratios and also infer relative chromosome copy numbers. Six isolates were completely euploid (Fig. 1b and S5); in the remaining five haploids, one or two chromosomes (Chr6 and Chr7) were disomic. These disomic chromosomes were heterozygous except in one isolate (Haploid IV) where the disomic chromosome (Chr6) was homozygous. Based upon the C. albicans haplotype map 30 , each haploid chromosome primarily contained alleles from only one parental homolog with few, if any, obvious crossovers detectable (Fig. 1b and S5). This implies that haploids did not arise through conventional meiosis, which usually requires at least one crossover per chromosome 31 . The few crossovers detected likely arose by mitotic recombination prior to selection for LOH events. For example, Haploid I was isolated following selection for loss of GAL1, which is within the crossover region on Chr1. Furthermore, in C. albicans haploids (Fig. 1b and S5), the majority of disomic chromosomes were heterozygous, which would appear following segregation of homologs in meiosis I. Such meiosis I segregation is not seen in Drosophila male meioses, which lack recombination 32 ; it is seen in Candida lusitaniae meiotic progeny, which undergo high levels of recombination 33 . The low level of genetic recombination together with the presence of heterozygous disomic chromosomes remains most consistent with random homolog segregation in C. albicans. Accordingly, we propose that haploids arise via a concerted chromosome loss mechanism akin to that described for the diploid-tetraploid C. albicans parasexual cycle 23 , although a non-conventional meiotic program cannot be completely discounted. The existence of haploids refutes the argument that recessive lethal alleles are present on each C. albicans homolog 13,14 . Nonetheless, the absence of some chromosomal homologs from the haploid progeny suggests that recessive lethal alleles may exist. Indeed, only the ‘b’ homologs for Chr3, Chr4, Chr6, Chr7 and most of Chr1 were detected in the haploids analyzed (Fig. 1b and S5). Interestingly, a similar homolog bias was seen in diploid parasexual progeny derived from SC5314 18 . In contrast, homologs of Chr5, which carries the MTL, appeared in equal numbers (six MTL a and five MTLα) and were entirely of one or the other parental haplotype. Similarly, both homologs of ChrR and Chr2 were observed. As such, homologs from more than half the chromosomes (Chr3, Chr4, Chr6, Chr7 and most of Chr1) potentially carry at least one recessive lethal mutation and may limit the frequency with which viable haploids appear. Accordingly, we propose that haploidization may provide an effective mechanism for eliminating recessive lethal mutations from the predominantly diploid populations of C. albicans. Auto-diploidization of haploids In the course of these studies, prolonged propagation of haploid isolates yielded cultures with a mixed population of haploid and diploid cells (Fig. 1c). Subsequent colony purification yielded distinct haploid and/or diploid populations (Fig. 1d). Surprisingly, some haploid isolates also diploidized during the process of strain shipping, which involved storage, transit, and revival from partial dehydration on a solid surface. For example, a potential haploid isolate identified in Taiwan was sent to Minnesota on sterile filter paper. Once revived and grown in liquid culture, the genome was diploid (Fig. 1e) and homozygous for all SNPs (Fig. 1b, ‘Auto-dip.’), suggesting auto-diploidization from a haploid phase. While homozygous diploids could arise through mitotic defects or by self-mating 34 , mating between cells of the same mating-type was not detectable, as discussed below. Thus, we suggest that auto-diploids arise through mitotic defects that may be analogous to the auto-diploidization events in vertebrate haploid stem cell cultures 35–37 . Haploid Morphogenesis and Mating Consistent with reduced ploidy content in other yeast species 38 , haploid C. albicans cells were smaller, on average, for both cell and nuclear size compared to diploid cells (Fig. 2a and S6). The bud site selection patterns of haploids, like diploid C. albicans were predominantly axial, with a small fraction of cells displaying a bipolar budding pattern (Fig. 2b) 39 . Significantly, under conditions that induce morphogenesis of diploid C. albicans to form true hyphae, pseudohyphae, and chlamydospores 40,41 , similar morphogenetic events occurred in haploids (Fig. 2c). C. albicans diploid cells that are homozygous at the MTL can undergo a transcriptionally induced switch to the opaque state that renders them competent for mating 19,42 . Similarly, haploids readily switched from the white (Wh) to the opaque (Op) state, as detected by colony sectoring (Fig. 2d). Haploid opaque cells had elongated opaque-like cell morphology 43 , yet were considerably smaller than diploid opaque cells (Fig. 2d). Importantly, haploid opaque cells of opposite mating type were mating competent. Mating between cells with complementary genetic markers resulted in the formation of heterozygous diploids (Fig. 2e, Op x Op). In contrast, neither opaque cells of the same mating type, nor white cells of opposite mating type, mated efficiently (Fig. 2e). This is consistent with opposite mating types and the opaque state being prerequisites for conventional mating 19,42 . Thus, we propose that haploids participate in a non-meiotic haploid-diploid parasexual cycle. Reduced growth and virulence of haploids The products from haploid a and α mating were heterozygous for MTL (Fig. S7), near-diploid in genome content (Fig. S7), and likely inherited the extra aneuploid copies of Chr6 and/or Chr7 from their MTLα parent. All the haploid isolates grew significantly slower than SC5314 (Fig. 3a and S8, p < 0.001 for all haploids). However, the mating products grew significantly faster than either of their haploid parents (Fig. 3a), as expected if heterozygosity restores fitness through the complementation of recessive alleles 44 . Consistent with this, auto-diploids exhibited growth rates indistinguishable from their haploid progenitors (Fig. 3a and S8), with the single exception of Haploid VIII (p = 0.02). We propose that the low fitness of haploids, as well as their corresponding auto-diploids, is a consequence of unmasking recessive alleles that reduce growth potential. Furthermore, the variability in fitness between haploids is likely due to the specific combination of alleles inherited by each haploid isolate. Significantly, the diploid mating products from haploid crosses grew more slowly than the highly heterozygous diploid SC5314. The generation of robust and diverse diploid progeny from haploid mating would require outcrossing, which is predicted to occur infrequently 11,12 , as many humans are colonized with a single strain of C. albicans. As it appears that some lethal mutations have accumulated on one of the two homologs for more than half the chromosomes (Fig. 1 and S5), mating between related haploids would not reestablish complete genome heterozygosity. Thus, even though the formation of haploids potentially eliminates lethal mutations, continued inbreeding between related haploids would perpetuate homozygosity of most chromosomes and consequently would reduce overall fitness. Consistent with reduced growth rates in vitro, Haploid II was avirulent in a mouse model of systemic candidiasis (Fig. 3b) and was cleared from the mouse after ten days. However, after 48 hours of infection with a haploid strain, cells could be recovered (Fig. S9a) and the majority of these isolates remained haploid. Subsequent in vivo experiments compared the colony forming units (CFUs) of haploids, auto-diploids and SC5314 isolated from mouse kidneys 48 hours post-inoculation in individual and direct competition experiments (Fig. 3c and S9b). The number of CFUs recovered from mice inoculated with haploids and auto-diploids was several orders of magnitude lower than the number of CFUs recovered from mice inoculated with SC5314 (Fig. 3c). Furthermore, competition experiments indicated that haploids and auto-diploids exhibited similar fitness in vivo, whereas both of these forms were outcompeted by heterozygous diploids (Fig. S9b). Similar to growth rates in vitro, the low fitness of haploids and their corresponding auto-diploids in vivo, indicates that it is not the diploid state, per se, that is beneficial to growth but rather extensive allelic heterozygosity, as evidenced with SC5314. From these data, we hypothesize that haploid formation may not be rare, but that haploids likely represent a very small fraction of the overall population due to their low competitive fitness relative to heterozygous diploids. Haploid strains as genetic tools The haploid state greatly facilitates experimental approaches such as classical genetic screens for recessive alleles and a single-round of gene-knockout phenotyping. To illustrate this potential, a set of auxotrophic C. albicans strains was derived from a relatively stable haploid, GZY792 (Fig. 4a and S10) and genes important for morphogenesis in diploids (HGC1, RVS167, SLA1, SEC3 and ACE2) were deleted in a single step. The resulting mutants exhibited morphogenesis defects closely resembling those of the corresponding diploid null mutants (Fig. 4c) 45–48 . Furthermore, because the deleted genes map to all eight chromosomes (Fig. 4b), the ability to delete each gene in a single step confirms that all chromosomes were monosomic in the parental haploid. Concluding remarks In summary, C. albicans can no longer be considered to be an obligate diploid. Rather, it has the ability to form haploids that subsequently mate to form diploids or undergo auto-diploidization. The lower fitness of haploids and auto-diploids suggests they will not persist in the population and thus should be detected only rarely. Nonetheless, efficient mating between viable haploids can produce heterozygous diploids that have increased fitness, presumably due to the complementation of detrimental recessive alleles. Furthermore, the reduction to a haploid state can serve as a vehicle to eliminate recessive lethal alleles from a heterozygous diploid population. We propose that C. albicans rapidly generates genetic diversity by producing a broad range of different ploidy states, including haploid, diploid, tetraploid, and aneuploid 25 . Each of these ploidy states are mating competent and genetic outcrossing can introduce, albeit infrequently, further genetic diversity into the population. While the ploidy reduction mechanism(s) used by C. albicans remain an enigma, the discovery of a haploid form and the potential for a haploid-diploid parasexual cycle significantly expands our ability to manipulate, and thereby better understand, this opportunistic pathogen. Furthermore, this study reveals how whole-genome analyses can lead to a re-evaluation of common assumptions about genomic structure in microbial organisms. Supplemental Methods Haploid screening from the mouse models of infection Bloodstream infections were performed by injecting 106 cells of parent strain AF7 (gal1Δ::URA3/GAL1), into the tail vein of 13 outbred ICR male mice (22–25 g, Harlan, Indianapolis) 27 . When moribund (at 5–7 days), mice were anesthetized using isofluorane, euthanized, and both kidneys were removed. Kidneys were combined, homogenized with 1 mL of water. 1:1000 dilutions of kidney homogenates were plated for total cell count onto YPD, and 1:10 dilutions of the same homogenate were plated onto 2-DOG medium to obtain Gal− colony counts at 3 days. For the oropharyngeal model of infection, Balb/C mice were immunosuppressed with cortisone on days −1, 1 and 3 of infection. Calcium alginate swabs were saturated with a suspension of 106 cells/ml of YJB9318 (gal1Δ::URA3/GAL1) under their tongues for 75 min. Mice were sacrificed at 1, 2, 3, and 5 days post-infection. 1:1000 dilutions of tongue tissue homogenates were plated for total cell count onto YPD, and 1:10 dilutions of the same homogenate were plated onto 2-DOG medium to obtain Gal− colony counts at 3 days. Importantly, to confirm that 2-DOG resistant cells only arose during in vivo passage and were not selected for by plating on 2-DOG medium, a gal1/gal1 strain and a gal1/GAL1 heterozygote strain were plated onto 2-DOG medium and observed for growth. 2-DOG resistant colonies grew up from the gal1/gal1 mutant but not from the GAL1/gal1 heterozygote by day 3 after plating 27 . Therefore, we used a cut-off of day 3 for picking 2-DOG resistant colonies after plating. Approximately 300 colonies were transferred from YPD plates and ~740 colonies from 2-DOG plates (312 from OPC and 431 BSI isolates) into 96-well plates containing 50% glycerol and stored at −80°. All C. albicans cells isolated following in vivo passaging were analyzed by flow cytometry to determine cell ploidy. Flow cytometry preparation and analysis Mid-log phase cells were harvested, washed and resuspended in 50:50 TE (50mM Tris pH8 : 50mM EDTA) and fixed with 95% ethanol. Cells were washed with 50:50 TE and treated with 1 mg/ml RNAse A and then 5 mg/ml Proteinase K. Cells were washed with 50:50 TE and resuspended in SybrGreenI (1:85 dilution in 50:50 TE) incubated overnight at 4°. Stained cells were collected and resuspended in 50:50 TE and analyzed using a FACScaliber. Whole genome ploidy was estimated by fitting DNA content data with a multi-Gaussian cell cycle model that assumes the G2 peak has twice the fluorescence of the G1 peak and that minimizes S-phase cell contribution to the error function. Ploidy values were calculated by comparing the ratio of peak locations in experimental samples to those of diploid and tetraploid controls. Mating Assays Opaque or white cells were mixed together in equal cell numbers and incubated on Spider media 34 for 18 hours prior to replica-plating onto SDC −Ade + Nat (to select for mating products), as well as YPAD, YPAD + Nat, SDC −Ade and SDC −Ade + Nat to detect parental auxotrophies and then photographed 24 hours later. In vitro growth assays Strains were grown in YPD media supplemented with adenine, uridine and histidine in a 96-well microtiter plate and optical density was measured every 15 minutes with a plate reader (Tecan Sunrise) for 24 hours. Doubling times were calculated as previously described 30 . In vivo assays For virulence assays, eight mice per C. albicans strain were inoculated with ~6.0 × 105 CFUs by tail vein injection 49 . For survival assays, three mice per strain were inoculated by tail vein injection, both kidneys were harvested at 48h and CFUs were determined by plating on YPAD. Ploidy of randomly selected isolates was determined by flow cytometry. 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The Parasexual Cycle in Candida albicans Provides an Alternative Pathway to Meiosis for the Formation of Recombinant Strains
Anja Forche, Kevin Alby, Dana Schaefer … (2008)
Introduction In most sexually reproducing eukaryotes, meiosis is used to precisely halve the DNA content in the cell, often for the formation of haploid gametes from diploid precursor cells. This specialized form of cell division involves one round of DNA replication followed by two successive rounds of DNA division. Each round of DNA division is unique. During the first meiotic division (meiosis I) extensive DNA recombination takes place between maternal and paternal homologous chromosomes, which then are segregated from one another. The second round of DNA division (meiosis II) more closely resembles normal mitotic DNA division, in which sister chromatids are segregated to opposite poles. In the case of spores in fungi and spermatozoa in animals, all four haploid nuclei form four different haploid cells, while in the female meioses of animals only one haploid nucleus survives and forms the mature oocyte. The meiotic process has been studied extensively in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. In S. cerevisiae, mating of haploid MATa and MATα cells normally generates a stable diploid a/α cell that replicates mitotically until subsequently induced to undergo meiosis under conditions of limiting nitrogen availability and the presence of a non-fermentable carbon source [1]. In S. pombe, mating also occurs between haploid cells but the diploid state is often transient, immediately undergoing meiosis to regenerate the haploid form. The sexual program in S. pombe is again controlled by nutritional cues, as mating and meiosis normally occur only under starvation conditions [2]. In both S. cerevisiae and S. pombe, meiosis generates four recombinant haploid spores held together in an ascus. While S. cerevisiae and S. pombe are rarely pathogenic in humans, the related ascomycete C. albicans is an opportunistic pathogen capable of causing both debilitating mucosal infections and potentially life-threatening systemic infections [3]. C. albicans is normally a harmless commensal fungus, existing in the gastrointestinal tract of at least 70% of the healthy population [4]. However, C. albicans is also the most commonly isolated fungal pathogen, particularly targeting individuals with compromised immune systems and leading to death in up to 50% of patients with bloodstream infections [5–7]. Until recently, C. albicans was thought to be asexual, existing only as an obligate diploid organism and thus classified amongst the Fungi imperfecti [8]. However, a robust mating system has now been uncovered in this organism, in which mating occurs between diploid mating type-like (MTL) a and α strains to generate an a/α tetraploid strain. Mating occurs both under laboratory conditions and in different in vivo niches in a mammalian host [9–12]. Population studies of clinical isolates are also consistent with C. albicans strains undergoing genetic exchange in their natural environment, albeit at a limited rate [13]. While an efficient mating apparatus has now been identified in C. albicans, the mating cycle differs in several important respects from that of S. cerevisiae and other fungi. For example, mating in C. albicans is regulated by phenotypic switching; MTL homozygous C. albicans cells can reversibly switch between two heritable states termed white and opaque, and only the opaque form is competent for efficient mating [14]. This unusual mode of mating regulation is so far unique to C. albicans (and the very closely related yeast, Candida dubliniensis [15]) making it likely that this adaptation has evolved to regulate mating of C. albicans strains in their natural environment—that of a warm-blooded host. Completion of the mating cycle in C. albicans also seems to occur in an atypical manner. Although reductional DNA divisions by a meiotic program have not been observed, tetraploid strains of C. albicans have been shown to return to the diploid state via a parasexual mechanism. During this process, tetraploid cells exposed to certain laboratory media were induced to lose chromosomes in an apparently random, but concerted, fashion, thereby forming cells with a diploid, or very close to diploid, DNA content [16]. The genetic locus responsible for determining C. albicans mating type (the MTL locus) segregated randomly in these experiments so that many of the progeny cells were a and α diploid cells that were themselves mating competent. Mating of diploid cells to form tetraploid cells, followed by random chromosome loss to generate diploid progeny cells, thereby constitutes a parasexual mating cycle in C. albicans. In this study, we examined the genetic profile of strains formed by the parasexual mating process in C. albicans using SNP and comparative genome hybridization (CGH) techniques. We observed extensive shuffling of the parental configurations of chromosomes by the parasexual cycle, giving rise to many types of recombinant C. albicans progeny. Many of the progeny strains are not true (euploid) diploids; rather, they are aneuploid strains that are often trisomic for one or more chromosomes. In addition, we provide the first evidence that tetraploid strains experiencing chromosome instability and subsequent chromosome loss also undergo genetic recombination between homologous chromosomes. We also report that genetic recombination in C. albicans tetraploids was dependent on the presence of Spo11p, a conserved protein that in other eukaryotes initiates meiotic recombination by the introduction of double-strand breaks (DSBs) into the DNA [17]. These results suggest that the parasexual pathway in C. albicans has evolved as an alternative pathway to meiosis for promoting a reduction in cell ploidy, and furthermore, that at least one gene that normally functions in meiotic recombination has been co-opted for use in the parasexual mating cycle. Results A Strain for Studying the Parasexual Mating Cycle in C. albicans The parasexual cycle of C. albicans, as currently envisaged, is shown in Figure 1A. Note that no meiotic program has been observed in C. albicans, despite the presence of many genes in the genome whose homologues function specifically in meiosis in other fungi [18]. However, C. albicans strains have been found to undergo a parasexual cycle; tetraploid strains become genetically unstable when incubated on certain laboratory media, losing chromosomes and generating diploid (and aneuploid) progeny strains that are themselves mating competent. The chromosome loss process is concerted, with loss of one or more chromosomes predisposing the cell to lose additional chromosomes, and the diploid state being the final product [16]. While tetraploids are stable when grown on YPD medium at different temperatures, two culture conditions were identified that induced genetic instability in C. albicans: (i) growth of tetraploid strains on S. cerevisiae “pre-sporulation” (pre-spo) medium at 37 °C, and (ii) growth of tetraploid strains on medium containing L-sorbose at 30 °C. The latter condition was previously shown to also induce chromosome loss in diploid C. albicans strains [19]. More specifically, diploid strains were unable to grow on L-sorbose medium unless they first underwent loss of one copy of Chromosome (Chr) 5, becoming monosomic for this chromosome. In contrast, diploid strains were relatively stable when grown on pre-spo medium, indicating that diploid and tetraploid strains exhibit very different selective pressures when cultured on this medium. 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. To monitor changes in ploidy in tetraploid strains of C. albicans, we exploited a genetically marked tetraploid strain, RBY18, containing markers on Chr 1 and 5. The strain was constructed by mating a/Δα and Δa/α cell types, as shown in Figure 1B [16]. Strain RBY18 is heterozygous for the GAL1 gene 1 on Chr 1, which is counterselectable. Strains carrying wild-type GAL1 are unable to grow on medium containing 2-deoxygalactose (2-DOG) as the carbon source, while derivative strains that have lost both copies of the GAL1 gene are able to grow on 2-DOG medium [20]. In most cases, it is expected that loss of GAL1 function in RBY18 will occur by loss of both chromosomes carrying the GAL1 allele, although GAL1 function also can be lost by mutation or genetic recombination. The RBY18 tetraploid strain is also heterozygous for all four MTL alleles on Chr 5: WTa, WTα, Δ a 1/a2, and Δα1/α2, which are easily distinguishable using whole cell PCR and oligonucleotides specific to each MTL allele [14]. Selection of Diploid Progeny after Parasexual Chromosome Reduction To generate progeny strains that have undergone the parasexual mating cycle, the marked tetraploid strain RBY18 was induced to undergo chromosome loss on either pre-spo or sorbose medium and gal1 − strains were selected by growth on 2-DOG medium. These 2-DOG resistant (DOGR) strains were subsequently analyzed by PCR to confirm that loss of MTL alleles on Chr 5 had accompanied loss of GAL1 alleles on Chr 1, an indication that cells had undergone a reduction in overall cell ploidy (unpublished data). PCR of the MTL loci was also used to detect possible jackpot effects, where several gal1 − progeny might have been derived from a single cell having undergone a chromosome loss event. Where possible, progeny cells with different combinations of MTL alleles were used for subsequent analysis. Selected progeny strains were grown in YPD medium at 30 °C and analyzed by flow cytometry to determine the overall ploidy of each strain, as shown in Figure 2. Flow cytometric analyses confirmed that each strain was diploid, or close to diploid, in DNA content, as judged by staining of the DNA with sytox green [9]. Seven strains (P1 to P7) were derived from RBY18 by growth on pre-spo medium, and six strains (S1 to S6) were derived from RBY18 by growth on sorbose medium (Figure 2). Subtle differences were observed in the flow cytometry DNA profiles between isolates, where distinct peaks were evident representing non-replicated (G1 phase) and replicated (G2 phase) DNA. In some strains the majority of the cells contained replicated DNA (e.g., S5 and S6, Figure 2, panels N and O), while others had an almost equal distribution of cells with unreplicated and replicated DNA (e.g., P4, panel F). However, there was no obvious correlation between DNA profiles analyzed by flow cytometry and cell growth rates. 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. To further characterize the strains generated by parasexual chromosome reduction, progeny were plated for single colonies on rich (YPD) medium to examine colony growth. After incubation at 30 °C for 7 d, colonies were compared for overall size and morphology (Figure 3). A wide range of phenotypes was observed, including smaller colony sizes relative to diploid and tetraploid parental strains and altered colony morphologies. Some of the isolates produced hyper-filamentous morphologies, as evidenced by increased surface wrinkling of the colonies (e.g., progeny strains P3, P4, and P6; Figure 3, panels E, F, and H). Normally, C. albicans cells grow as budding yeast, pseudohyphal, or true hyphal cells. Examination of cells from the wrinkled colonies by microscopy confirmed that these colonies contained many filamentous (pseudohyphal and true hyphal) cells, while the unwrinkled colonies (including control strains) contained very few filamentous cells (unpublished data). Some progeny strains also exhibited reduced filamentation on medium that normally induces hyphae formation (Spider medium and serum-containing medium, KA and RJB, unpublished data). 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. Thus, the parasexual cycle of C. albicans can generate variant strains with diverse colony morphologies. Changes in the ability to undergo the yeast-hyphal transition have been closely linked with the pathogenic potential of C. albicans strains [21–24]. It is therefore likely that many of these variant strains will exhibit reduced virulence in models of candidiasis; but it is also possible that some of these isolates could have increased fitness under particular selective conditions, leading to improved colonization of defined in vivo niches in the host. Genomic Profiling of Progeny Cells from the Parasexual Cycle SNP and CGH microarrays are powerful approaches for examining genetic recombination and genome structure in C. albicans [25–28]. SNP arrays were designed to exploit the sequence diversity between chromosome homologues in the diploid C. albicans genome. The genome-wide SNP arrays used here included 152 SNPs, distributed across all eight chromosomes of C. albicans. As each SNP is specific for one of the parental chromosome homologues, each homologue can be distinguished in progeny from the parasexual mating cycle. In addition, loss of heterozygosity (LOH) at SNPs on otherwise heterozygous chromosomes can be used as a marker for genetic recombination. Quantitative SNP analysis can also be used to determine the relative copy number of each homologue in a sample (see Materials and Methods). CGH analysis provides a complementary approach to SNP arrays for the determination of the copy number of each gene on each chromosome in the sample. Labeled genomic DNA from experimental samples (Cy3 labeled) and labeled DNA from a reference diploid SC5314 strain (Cy5 labeled) were hybridized to whole genome arrays containing >6,000 C. albicans ORFs [27,28]. CGH data provides information on the copy number of every chromosome, as well as indicating large-scale aneuploidies. In this study, we used both CGH and SNP approaches to obtain a detailed picture of the products of the C. albicans parasexual cycle following concerted chromosome loss. SNP and CGH arrays were first used to analyze RBY18 and the diploid parental strains that had been used to construct this tetraploid strain. SNP analysis confirmed that MTLa and MTLα parental diploid strains were heterozygous for most of the SNPs on the array, although in the parental MTLα strain Chr 2 was homozygous for all markers (Table S4). CGH array data confirmed that the parental strains were euploid diploids and RBY18 was a euploid tetraploid, as they contained two and four copies of each of the eight C. albicans chromosomes, respectively. We then analyzed 13 progeny strains produced by concerted chromosome loss from RBY18 using SNP and CGH arrays (see Figures 4 and S4, and Tables S1 and S4). Only three of the 13 strains were true diploids (P2, P5, and P6). The majority (10/13) of the progeny strains contained at least one extra chromosome: four of the seven strains derived from growth of the tetraploid on pre-spo medium were trisomic for one to three chromosomes and all six strains derived from growth on sorbose were also trisomic for up to three of the eight C. albicans chromosomes (Figure 4). Thus, concerted chromosome loss was often incomplete and did not immediately result in true diploid strains. 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. Curiously, there was a strong bias towards trisomy of Chr 4 in the progeny strains; all strains carrying at least one trisomic chromosome (four pre-spo-selected strains and all sorbose-selected strains) were trisomic for Chr 4. Trisomies of Chr R, 2, 5, 6, or 7 were also detected in at least one of the progeny. As expected, Chr 1 was always present in the disomic parental configuration (one copy of each homologue) because selection of DOGR progeny requires that the strains lose both Chr 1 homologues from the MTLα mating parent (Figure 1B). Genetic Recombination in the Parasexual Cycle of C. albicans The most striking feature of the progeny genetic profiles was that three strains contained a number of short LOH tracts (six or seven LOH tracts were observed in each strain), evidence of multiple recombination events between homologous chromosomes. Isolates P1, S3, and S4 exhibited recombination events that included LOH at SNPs on multiple chromosomes (including Chr R, 1, 2, 4, 5, 6, and 7, see Figure 4). While selection on 2-DOG required inheritance of the gal1Δ alleles on Chr 1, the LOH events detected here are independent of the GAL1 locus. Moreover, these events did not involve homozygosis of all of Chr 5, which might be expected to occur in response to sorbose selection. Instead, the recombination events we observed appear to be selection independent. Overall, the appearance of multiple gene conversion tracts within several strains, and the general absence of gene conversion tracts in other strains, suggests that some cells become generally competent for recombination at more than one locus, while other strains do not undergo such recombination events at all. In at least one example (Chr 2 in strain P1) one complete chromosome arm (Chr 2L) became homozygous (Figure 4). This recombination event may have arisen in one of two ways: (i) A cross-over between chromosomes led to reciprocal recombination between homologues, as commonly occurs during meiosis in other fungi. In this case, the partner DNA involved in the reciprocal exchange was lost during the process of concerted chromosome loss. (ii) A break-induced replication event occurred. In this case, a DSB in one chromosome was repaired by DNA replication that copied the template strand from the break near the centromere all the way to the telomere in the homologous chromosome. Break-induced replication is a non-reciprocal recombination event and in S. cerevisiae is often restricted to repair of DNA DSBs where only one end of the break shares homology with the template [29]. Potential hotspots for recombination were identified in the three strains that had undergone inter-homologue recombination. For example, SNPs HST3 and 2340/2493 on Chr 5 underwent LOH in P1, S3, and S4 recombinant strains. Additional experiments are necessary to fully document hotspots for recombination. However, our results indicate that recombination events are not uniform across the C. albicans genome during the parasexual cycle. Does the C. albicans Genome Harbor Recessive Lethal Alleles? Natural isolates of C. albicans are diploid, and it has been proposed that haploid forms cannot exist because of the presence of recessive lethal alleles in the genome. Evidence supporting this idea came from classical mitotic recombination studies [30,31]; however, no systematic investigation of possible recessive lethal alleles in the C. albicans genome has been reported. Using the present dataset, we can rule out the presence of recessive lethal alleles on some chromosomes. For example, it was already known that Chr 5 does not harbor recessive lethal alleles: loss of either homologue can be induced in diploid cells by growth on sorbose medium [19]. The SNP data presented here supports this finding, as both AA and BB configurations of Chr 5 homologues were observed in the progeny strains P2 and P6, respectively (this nomenclature assigns the parental configuration of chromosome homologues as AB). Similarly, several other chromosomes did not carry recessive lethal alleles, as their homologues could be lost during the parasexual cycle. Chr R, 2, 3, 5, 6, and 7 were all found to be homozygous in at least one independent isolate. However, only one homozygous configuration was observed for each chromosome (either AA or BB), leaving open the possibility that the other chromosome homologue carries recessive lethal alleles. We will revisit the issue of recessive lethal alleles below. The Role of Meiosis Genes in C. albicans The C. albicans parasexual cycle provides an alternative mechanism to meiosis for a reduction in cell ploidy. Although no experimental evidence for a meiotic pathway in C. albicans currently exists, the genome contains homologues of many genes that function specifically in meiosis in the related yeast S. cerevisiae [18]. Some of the meiosis genes from C. albicans even complement for meiotic function in S. cerevisiae, demonstrating they encode a conserved protein activity [32]. It seems likely that either (i) C. albicans has a cryptic meiotic program still to be discovered, or (ii) meiotic genes have been adapted to other processes in C. albicans, perhaps some in the parasexual pathway. To address the latter possibility, we investigated the potential role of the Spo11 protein in genetic recombination during the parasexual cycle. In fungi such as S. cerevisiae and S. pombe and in higher eukaryotes, Spo11p makes meiosis-specific DSBs in DNA via a topoisomerase-like mechanism of DNA cleavage [33,34]. C. albicans ORF19.11071 on Chr 2 encodes a potential homolog of S. cerevisiae SPO11 (http://www.candidagenome.org). An alignment of this ORF with SPO11 genes from diverse species including S. pombe, S. cerevisiae, Kluyveromyces lactis, and Drosophila reveals that several of the critical conserved residues identified for DNA strand cleavage are present in the C. albicans sequence (Figure S1). In particular, the conserved active site tyrosine residue, required for breakage of the DNA and formation of a phosphotyrosine bond, is present in the C. albicans protein. Similarly, Glu-233 and Asp-288 residues that are required in S. cerevisiae Spo11p for meiotic recombination [35] are conserved in the C. albicans protein. ORF19.11071 is a homologue of the Spo11 family and will therefore be referred to as C. albicans Spo11p in the rest of this study. Attempts to complement S. cerevisiae Spo11 function with C. albicans Spo11p, as measured by rates of meiotic recombination in return-to-growth experiments, were unsuccessful (Table S2). This result is perhaps not surprising as SPO11 sequences from diverged species are poorly conserved outside of the core catalytic residues [36] (Figure S1). It is also worth noting that meiotic proteins in general are faster evolving than most cellular proteins [37,38], an issue that is taken up again in the Discussion. To investigate whether C. albicans Spo11p is expressed in mitotically dividing cells, a Spo11-13myc fusion protein was constructed in diploid C. albicans strains. Western blots show that the Spo11-13myc protein was detectable in mitotic extracts of diploid cells grown in YPD medium, although the level of expression was relatively low (see comparison of protein levels with that of the mitotic spindle protein Kar3-13myc) (Figure 5). Thus, in C. albicans, the Spo11 protein is expressed in mitotically dividing cells. 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). Genetic Recombination in the C. albicans Parasexual Mating Cycle Is Dependent on Spo11 Function The observation that C. albicans Spo11p is expressed during mitotic growth is consistent with it having a function outside of meiosis. To examine if C. albicans Spo11p is required for genetic recombination in the parasexual mating cycle, we deleted all four copies of the SPO11 gene in genetically marked tetraploid strains (RBY176/RBY177) that were heterozygous for GAL1 on Chr 1. The strains were induced to undergo concerted chromosome loss on pre-spo or sorbose medium and were then exposed to 2-DOG to select for strains that had lost both copies of GAL1. Eighteen DOGR colonies were selected from tetraploid growth on pre-spo (eight colonies) or sorbose (ten colonies) and subsequently analyzed by flow cytometry to determine if they were diploid, or near diploid, strains (Figure S2). Indeed, we detected diploid Δspo11 progeny strains, indicating that Spo11p is not necessary for the process of concerted chromosome loss in tetraploid C. albicans strains. We next analyzed the colony morphologies of the Δspo11 diploid progeny. As was seen with progeny from wild-type tetraploids (Figure 4), many of the Δspo11 progeny strains exhibited altered colony morphologies on YPD medium (Figure 6). 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. Genomic profiles of the Δspo11 diploid progeny (along with the parental diploid and tetraploid strains) were generated using SNP and CGH microarrays (see Figure 7, as well as Tables S5 and S6, and Figures S1 and S4). One of the diploid parents (RBY79, MTLα parent) was initially homozygous for Chr 2, and the other parent (RBY77, MTL a parent) carried a long tract of LOH on Chr 2 (Figure 7). This is reflected in the patterns of Chr 2 inheritance in the diploid progeny which either received only one type of Chr 2 homologue (Ps2, Ps3, Ps4, Ps5, Ps6, Ss1, Ss2, Ss3, Ss4, Ss8, and Ss10) or received two homologues that only differ near the Chr 2R telomere (Ps1, Ps7, Ps8, Ss5, Ss6, Ss7, and Ss9). Similarly, one of the gal1Δ Chr 1 homologues in the parental MTLa strain had undergone LOH of a single SNP near the telomere of Chr 1L and this LOH tract was retained in all of the progeny. 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. As in the wild-type (SPO11+ ) progeny that were close to diploid, a majority (11/18) of the strains carried at least one and up to three trisomies, and Chr 4 was often one of the trisomic chromosomes (5/11 strains). Other chromosomes that became trisomic were Chr R, Chr 1, Chr 2, Chr 5, Chr 6, and Chr 7. The only chromosome that did not become trisomic in these strains or in the wild-type diploid progeny strains was Chr 3. Concerted chromosome loss did result in homozygosis of Chr R in nine strains (and trisomy in one strain) with the same homologue always being retained (the blue-colored “A” homologue in Figure 7). Interestingly, while no trisomies of Chr 3 were found, Chr 3 underwent LOH in ten strains, with seven of them retaining homologue B (colored pink) and three retaining the A homologue (Figure 7). The most striking feature of the Δspo11 progeny strains was that they did not undergo any detectable genetic recombination events. No single LOH events (gene conversion events) or chromosome crossing over events (long-range LOH) events were observed (although we note that if reciprocal recombination events occurred, in which both recombinant chromosomes were retained, these would not be detected by SNP analysis). In contrast, progeny derived from SPO11+ strains exhibited multiple recombination events in three out of 13 strains (Figure 4), a difference that is statistically significant (p = 0.05 were insignificant. Western blotting. Cultures of strains CAY126 (Spo11-13myc), RSY84 (Kar3-13myc), and the untagged RBY1118 strain were grown to logarithmic phase in YPD medium at 30 °C and cells harvested. Whole-cell extracts from these strains were prepared by resuspending cell pellets in lysis buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM dithiothreitol) containing protease inhibitors (pepstatin A, leupeptin, phenylmethyl sulfonyl chloride, and aprotinin) and lysis achieved by bead beating for 12–15 cycles (30 s vortexing following by 30–60 s on ice). An aliquot from each sample was separated by SDS-PAGE and analyzed by western blotting. The myc-tagged proteins were detected using an anti-myc antibody at 1/2,000 dilution (4a6 antibody; Millipore) followed by an anti-mouse HRP (horseradish peroxidase)-conjugated antibody at 1/1,000 dilution (Jackson Laboratories). Antibody binding was visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposure to autoradiography film. Supporting Information Figure S1 Alignment of C. albicans Spo11 with Conserved Spo11 Proteins from Other Species C. albicans Spo11 (product of ORF19.11071) was aligned with other fungal Spo11 sequences from S. pombe, S. cerevisiae, and K. lactis, as well as with the Drosophila Spo11 protein. The active site tyrosine residue is highlighted in yellow, with other highly conserved regions highlighted in blue. (53 KB PPT) Click here for additional data file. Figure S2 Flow Cytometric Analysis of Progeny Diploid Strains Derived from Δspo11 Tetraploids via the Parasexual Cycle As a control, a parental diploid strain (A) and the tetraploid RBY18 strain (B) were analyzed by flow cytometry for comparison. 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. (32.9 MB TIF) Click here for additional data file. Figure S3 Growth Rates of Progeny Strains from Wild-Type (SPO11 +) and Δspo11 Tetraploid Strains (A) Doubling times (DT) of each of the progeny strains during exponential growth in YPD medium at 30 °C (min). (B) Graph indicates the correlation between increased numbers of aneuploid (trisomic) chromosomes and increased cell doubling times. (C) Increased cell doubling times of progeny strains is dependent on the number of extra chromosomes they contain (over the normal diploid complement). The progeny strains were compared to SC5314, an a/α diploid control strain. The standard error is shown for the averaged data. Growth rates for progeny containing two or three trisomic chromosomes (+2 Chr or +3 Chr, respectively) differed significantly from euploid diploid strains (p < 0.005), using a t-test (two sample assuming equal variances). (70 KB DOC) Click here for additional data file. Figure S4 CGH Analysis of Progeny Strains Derived from Wild-Type (SPO11 +) and Δspo11 Tetraploid Strains Plots of each chromosome are shown with the y-axes representing gene copy number calculated from log 2 values. Abbreviations are CSE4, centromeric DNA; MRS, major repeat sequence; transp., transposon; CARE-2, the CARE-2 repetitive element; telom., telomeric sequences. (3.86 MB PPT) Click here for additional data file. Figure S5 Oligonucleotides Used to Amplify the SPO11 Gene Disruption Construct (12 KB PDF) Click here for additional data file. Table S1 Comparison of CGH and SNP Data Table shows large chromosomal changes indicated by CGH and SNP analysis. These changes include chromosomal aneuploidies (trisomic chromosomes; 3×) and LOH events (whole chromosomes or partial chromosomes). This table shows the high correlation between identification of trisomic chromosomes by CGH and SNP techniques. (23 KB XLS) Click here for additional data file. Table S2 Complementation of Spo11 Function in S. cerevisiae Meiotic Recombination SKY10 (Δspo11) was transformed with ARS/CEN plasmids carrying the indicated SPO11 alleles. Intragenic recombination was measured as the HIS+ prototroph frequency (frequency × 103 per viable cell) after 8 h at 30 °C in SPM medium [35]. Each value is the mean ± standard deviation of at least four experimental samples. The premeiotic prototroph frequencies have not been subtracted. (25 KB DOC) Click here for additional data file. Table S3 Additional SNPs Used in This Study 29 additional SNPs were included in this study as listed. Each SNP has a unique identifier number (151–179) and a descriptive name (e.g., ERG7/1). The chromosomal location of each SNP in the Contig 19 database (http://www.candidagenome.org) is listed. The chromosome position is indicated by both the chromosome number and the Sfi fragment on which the SNP is found (e.g., 2U indicates the Sfi fragment U on Chromosome 2). For more details on the physical map of C. albicans go to http://albicansmap.ahc.umn.edu. Note that some SNPs are located on the same PCR product (e.g., SNPs 152, 153, and 154 are all located on the same PCR product on Chromosome 2). (26 KB XLS) Click here for additional data file. Table S4 SNP Data for Progeny Strains Derived from the Wild-Type (SPO11 +) Tetraploid Strain (RBY18) Table shows SNP data for parental diploids (DIP), tetraploid RBY18 strain (TET), and 13 progeny (PR) strains. Strains P1 to P7 were derived from pre-spo medium, while strains S1 to S6 were derived from sorbose medium. Homozygous loci are indicated by red text (AA configuration) or pink text (BB configuration). Trisomic loci are indicated by green text (AAB configuration) or blue text (ABB configuration). Black text indicates the locus is heterozygous (AB configuration). NaN; data not applicable. (67 KB XLS) Click here for additional data file. Table S5 SNP Data for Progeny Strains Derived from Δspo11 Tetraploids Grown on Pre-Spo Medium Table shows SNP data for parental diploids (DIP), tetraploid strains (RBY176/177; TET), and eight progeny (PR) strains. Strains Ps1 to Ps8 were derived from Δspo11 tetraploids grown on pre-spo medium. Homozygous loci are indicated by red text (AA configuration) or pink text (BB configuration). Trisomic loci are indicated by green text (AAB configuration) or blue text (ABB configuration). Black text indicates the locus is heterozygous. NaN; data not applicable. (222 KB XLS) Click here for additional data file. Table S6 SNP Data for Progeny Strains Derived from Δspo11 Tetraploids Grown on Sorbose Medium Table shows SNP data for parental diploids (DIP), tetraploid strains (RBY176/177; TET), and ten progeny (PR) strains. Strains Ss1 to Ss10 were derived from Δspo11 tetraploids grown on sorbose medium. Homozygous loci are indicated by red text (AA configuration) or pink text (BB configuration). Trisomic loci are indicated by green text (AAB configuration) or blue text (ABB configuration). Black text indicates the locus is heterozygous. NaN; data not applicable. (66 KB XLS) Click here for additional data file.
Author and article information
Contributors
Louisa A. Messenger
Journal
Journal ID (nlm-ta): Acta Trop
Journal ID (iso-abbrev): Acta Trop
Title:
Acta Tropica
Publisher:
Elsevier
ISSN
(Print):
0001-706X
ISSN
(Electronic):
1873-6254
Publication date PMC-release: 1
November
2015
Publication date
(Print):
November
2015
Volume: 151
Pages: 150-155
Affiliations
Author notes
Article
Publisher ID: S0001-706X(15)00141-2DOI: 10.1016/j.actatropica.2015.05.007
PMC ID: 4644990
PubMed ID: 26188331
SO-VID: 7330edec-130e-4f61-9bb7-89455feb2349
Copyright © © 2015 The Authors
License:
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
History
Date
received
: 18
March
2015
Date
revision received
: 25
April
2015
Date
accepted
: 6
May
2015
Categories
Subject:
Article
ScienceOpen disciplines: Ecology
Data availability:
ScienceOpen disciplines: Ecology