Genome-wide single-nucleotide polymorphism map for Candida albicans

Single-nucleotide polymorphisms (SNPs) are essential tools for studying a variety of organismal properties and processes, such as recombination, chromosomal dynamics, and genome rearrangement. This paper describes the development of a genome-wide SNP map for Candida albicans to study mitotic recombination and chromosome loss. C. albicans is a diploid yeast which propagates primarily by clonal mitotic division. It is the leading fungal pathogen that causes infections in humans, ranging from mild superficial lesions in healthy individuals to severe, life-threatening diseases in patients with suppressed immune systems. The SNP map contains 150 marker sequences comprising 561 SNPs and 9 insertions-deletions. Of the 561 SNPs, 437 were transition events while 126 were transversion events, yielding a transition-to-transversion ratio of 3:1, as expected for a neutral accumulation of mutations. The average SNP frequency for our data set was 1 SNP per 83 bp. The map has one marker placed every 111 kb, on average, across the 16-Mb genome. For marker sequences located partially or completely within coding regions, most contained one or more nonsynonymous substitutions. Using the SNP markers, we identified a loss of heterozygosity over large chromosomal fragments in strains of C. albicans that are frequently used for gene manipulation experiments. The SNP map will be useful for understanding the role of heterozygosity and genome rearrangement in the response of C. albicans to host environments.     

Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.     

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.     

Fragile Site Instability in Saccharomyces cerevisiae Causes Loss of Heterozygosity by Mitotic Crossovers and Break-Induced Replication

Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.     

Efficient and rapid identification of Candida albicans allelic status using SNP-RFLP

Candida albicans is the most prevalent opportunistic fungal pathogen in the clinical setting, causing a wide spectrum of diseases ranging from superficial mucosal lesions to life-threatening deep-tissue infections. Recent studies provide strong evidence that C. albicans possesses an arsenal of genetic mechanisms promoting genome plasticity and that it uses these mechanisms under conditions of nutritional or antifungal drug stress. Two microarray-based methods, single nucleotide polymorphism (SNP) and comparative genome hybridization arrays, have been developed to study genome changes in C. albicans . However, array technologies can be relatively expensive and are not available to every laboratory. In addition, they often generate more data than needed to analyze specific genomic loci or regions. Here, we have developed a set of SNP-restriction fragment length polymorphism (RFLP) (or PCR-RFLP) markers, two per chromosome arm, for C. albicans . These markers can be used to rapidly and accurately detect large-scale changes in the C. albicans genome including loss of heterozygosity (LOH) at single loci, across chromosome arms or across whole chromosomes. Furthermore, skewed SNP-RFLP allelic ratios are indicative of trisomy at heterozygous loci. While less comprehensive than array-based approaches, we propose SNP-RFLP as an inexpensive, rapid, and reliable method to screen strains of interest for possible genome changes.     

High-Resolution Mapping of Spontaneous Mitotic Recombination Hotspots on the 1.1 Mb Arm of Yeast Chromosome IV

Although homologous recombination is an important pathway for the repair of double-stranded DNA breaks in mitotically dividing eukaryotic cells, these events can also have negative consequences, such as loss of heterozygosity (LOH) of deleterious mutations. We mapped about 140 spontaneous reciprocal crossovers on the right arm of the yeast chromosome IV using single-nucleotide-polymorphism (SNP) microarrays. Our mapping and subsequent experiments demonstrate that inverted repeats of Ty retrotransposable elements are mitotic recombination hotspots. We found that the mitotic recombination maps on the two homologs were substantially different and were unrelated to meiotic recombination maps. Additionally, about 70% of the DNA lesions that result in LOH are likely generated during G1 of the cell cycle and repaired during S or G2. We also show that different genetic elements are associated with reciprocal crossover conversion tracts depending on the cell cycle timing of the initiating DSB.     

Effect of the major repeat sequence on mitotic recombination in Candida albicans

The major repeat sequence (MRS) is known to play a role in karyotypic variation in Candida albicans. The MRS affects karyotypic variation by expanding and contracting internal repeats, by altering the frequency of chromosome loss, and by serving as a hotspot for chromosome translocation. We proposed that the effects of the MRS on translocation could be better understood by examination of the effect of the MRS on a similar event, mitotic recombination between two chromosome homologs. We examined the frequency of mitotic recombination across an MRS of average size (approximately 50 kb) as well as the rate of recombination in a 325-kb stretch of DNA adjacent to the MRS. Our results indicate that mitotic recombination frequencies across the MRS were not enhanced compared to the frequencies measured across the 325-kb region adjacent to the MRS. Mitotic recombination events were found to occur throughout the 325-kb region analyzed as well as within the MRS itself. This analysis of mitotic recombination frequencies across a large portion of chromosome 5 is the first large-scale analysis of mitotic recombination done in C. albicans and indicates that mitotic recombination frequencies are similar to the rates found in Saccharomyces cerevisiae.     

How clonal are clones? A quest for loss of heterozygosity during asexual reproduction in Daphnia magna

Due to the lack of recombination, asexual organisms are predicted to accumulate mutations and show high levels of within‐individual allelic divergence (heterozygosity); however, empirical evidence for this prediction is largely missing. Instead, evidence of genome homogenization during asexual reproduction is accumulating. Ameiotic crossover recombination is a mechanism that could lead to long genomic stretches of loss of heterozygosity (LOH) and unmasking of mutations that have little or no effect in heterozygous state. Therefore, LOH might be an important force for inducing variation among asexual offspring and may contribute to the limited longevity of asexual lineages. To investigate the genetic consequences of asexuality, here we used high‐throughput sequencing of Daphnia magna for assessing the rate of LOH over a single generation of asexual reproduction. Comparing parthenogenetic daughters with their mothers at several thousand genetic markers generated by restriction site‐associated DNA (RAD) sequencing resulted in high LOH rate estimation that largely overlapped with our estimates for the error rate. To distinguish these two, we Sanger re‐sequenced the top 17 candidate RAD‐loci for LOH, and all of them proved to be false positives. Hence, even though we cannot exclude the possibility that short stretches of LOH occur in genomic regions not covered by our markers, we conclude that LOH does not occur frequently during asexual reproduction in D. magna and ameiotic crossovers are very rare or absent. This finding suggests that clonal lineages of D. magna will remain genetically homogeneous at least over time periods typically relevant for experimental work.     

Extensive loss of heterozygosity is suppressed during homologous repair of chromosomal breaks

Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 x 10(-5); however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.     

A system for studying genetic changes in Candida albicans during infection

Candida albicans is a diploid yeast with a dimorphic life history. It exists commensally in many healthy humans but becomes a potent pathogen in immunocompromised hosts. The underlying genetic mechanisms by which C. albicans switches from a commensal to a pathogenic form in the host are not well understood. To study the evolution of virulence in mammalian hosts, we used GAL1 as selectable marker system that allows for both positive and negative selection in selective media. We show that the deletion of one or both copies of GAL1 in the C. albicans genome does not change virulence in a systemic mouse model. We obtained estimates for the frequency of mitotic recombination at the GAL1 locus during systemic infection. Our observations suggest that genetic changes such as mitotic recombination and gene conversion occur at a high enough frequency to be important in the transition of C. albicans from a commensal to a pathogenic organism.     

Haplotype mapping of a diploid non-meiotic organism using existing and induced aneuploidies

Haplotype maps (HapMaps) reveal underlying sequence variation and facilitate the study of recombination and genetic diversity. In general, HapMaps are produced by analysis of Single-Nucleotide Polymorphism (SNP) segregation in large numbers of meiotic progeny. Candida albicans, the most common human fungal pathogen, is an obligate diploid that does not appear to undergo meiosis. Thus, standard methods for haplotype mapping cannot be used. We exploited naturally occurring aneuploid strains to determine the haplotypes of the eight chromosome pairs in the C. albicans laboratory strain SC5314 and in a clinical isolate. Comparison of the maps revealed that the clinical strain had undergone a significant amount of genome rearrangement, consisting primarily of crossover or gene conversion recombination events. SNP map haplotyping revealed that insertion and activation of the UAU1 cassette in essential and non-essential genes can result in whole chromosome aneuploidy. UAU1 is often used to construct homozygous deletions of targeted genes in C. albicans; the exact mechanism (trisomy followed by chromosome loss versus gene conversion) has not been determined. UAU1 insertion into the essential ORC1 gene resulted in a large proportion of trisomic strains, while gene conversion events predominated when UAU1 was inserted into the non-essential LRO1 gene. Therefore, induced aneuploidies can be used to generate HapMaps, which are essential for analyzing genome alterations and mitotic recombination events in this clonal organism.     

Infrequent genetic exchange and recombination in the mitochondrial genome of Candida albicans

Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast Candida albicans. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of C. albicans from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of C. albicans, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.     

Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome.

Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated. Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome. Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element. Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination. Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.     

Rad52 function prevents chromosome loss and truncation in Candida albicans

RAD52 is required for almost all recombination events in Saccharomyces cerevisiae. We took advantage of the heterozygosity of HIS4 in the Candida albicans SC5314 lineage to study the role of Rad52 in the genomic stability of this important fungal pathogen. The rate of loss of heterozygosity (LOH) at HIS4 in rad52-ΔΔ strains was ～10(-3) , at least 100-fold higher than in Rad52(+) strains. LOH of whole chromosome 4 or truncation of the homologue that carries the functional HIS4 allele was detected in all 80 rad52-ΔΔ His auxotrophs (GLH -GL lab His(-)) obtained from six independent experiments. Isolates that had undergone whole chromosome LOH, presumably due to loss of chromosome, carried two copies of the remaining homologue. Isolates with truncations carried centric fragments of broken chromosomes healed by de novo telomere addition. GLH strains exhibited variable degrees of LOH across the genome, including two strains that became homozygous for all the heterozygous markers tested. In addition, GLH strains exhibited increased chromosomal instability (CIN), which was abolished by reintroduction of RAD52. CIN of GLH isolates is reminiscent of genomic alterations leading to cancer in human cells, and support the mutator hypothesis in which a mutator mutation or CIN phenotype facilitate more mutations/aneuploidies.     

A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae

Loss of heterozygosity (LOH) can be a driving force in the evolution of mitotic/somatic diploid cells, and cellular changes that increase the rate of LOH have been proposed to facilitate this process. In the yeast Saccharomyces cerevisiae, spontaneous LOH occurs by a number of mechanisms including chromosome loss and reciprocal and nonreciprocal recombination. We performed a screen in diploid yeast to identify mutants with increased rates of LOH using the collection of homozygous deletion alleles of nonessential genes. Increased LOH was quantified at three loci (MET15, SAM2, and MAT) on three different chromosomes, and the LOH events were analyzed as to whether they were reciprocal or nonreciprocal in nature. Nonreciprocal LOH was further characterized as chromosome loss or truncation, a local mutational event (gene conversion or point mutation), or break-induced replication (BIR). The 61 mutants identified could be divided into several groups, including ones that had locus-specific effects. Mutations in genes involved in DNA replication and chromatin assembly led to LOH predominantly via reciprocal recombination. In contrast, nonreciprocal LOH events with increased chromosome loss largely resulted from mutations in genes implicated in kinetochore function, sister chromatid cohesion, or relatively late steps of DNA recombination. Mutants of genes normally involved in early steps of DNA damage repair and signaling produced nonreciprocal LOH without an increased proportion of chromosome loss. Altogether, this study defines a genetic landscape for the basis of increased LOH and the processes by which it occurs.     

High-resolution genome-wide analysis of irradiated (UV and γ-rays) diploid yeast cells reveals a high frequency of genomic loss of heterozygosity (LOH) events

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.     

Mating is rare within as well as between clades of the human pathogen Candida albicans

Candida albicans is a diploid yeast that can undergo mating and a parasexual cycle, but is apparently unable to undergo meiosis. Characterization of the population structure of C. albicans has shown that reproduction is largely clonal and that mating, if it occurs, is rare or limited to genetically related isolates. Because molecular typing has delineated distinct clades in C. albicans, we have tested whether recombination was common within clades, but rare between clades. Two hundred and three C. albicans isolates have been subjected to multilocus sequence typing (MLST) and the haplotypes at heterozygous MLST genotypes characterized. The C. albicans isolates were distributed among nine clades, of which five corresponded to those previously identified by Ca3 fingerprinting. In each of these clades with more than 10 isolates, polymorphic nucleotide positions located on between 3 and 4 of the six loci were in Hardy-Weinberg disequilibrium. Moreover, each of these polymorphic sites contained excess heterozygotes. This was confirmed by an expanded analysis performed on a recently published MLST dataset for 1044 isolates. On average, 66% of polymorphic positions in the individual clades were in significant excess of heterozygotes over the five clades. These data indicate that mating within clades as well as self-fertilization are both limited and that C. albicans clades do not represent a collection of cryptic species. The study of haplotypes at heterozygous loci performed on our dataset indicates that loss of heterozygosity events due to mitotic recombination is moderately common in natural populations of C. albicans. The maintenance of substantial heterozygosity despite relatively frequent loss of heterozygosity could result from a selective advantage conferred by heterozygosity.