Dissection of quantitative traits by bulk segregant mapping in a protoploid yeast species.

Since more than a decade ago, Saccharomyces cerevisiae has been used as a model to dissect complex traits, revealing the genetic basis of a large number of traits in fine detail. However, to have a more global view of the genetic architecture of traits across species, the examination of the molecular basis of phenotypes within non-conventional species would undoubtedly be valuable. In this respect, the Saccharomycotina yeasts represent ideal and potential non-model organisms. Here we sought to assess the feasibility of genetic mapping by bulk segregant analysis in the protoploid Lachancea kluyveri (formerly S. kluyveri) yeast species, a distantly related species to S. cerevisiae For this purpose, we designed a fluorescent mating-type marker, compatible with any mating-competent strains representative of this species, to rapidly create a large population of haploid segregants (>10(5) cells). Quantitative trait loci can be mapped by selecting and sequencing an enriched pool of progeny with extreme phenotypic values. As a test bed, we applied this strategy and mapped the causal loci underlying halotolerance phenotypes in L. kluyveri Overall, this study demonstrates that bulk segregant mapping is a powerful way for investigating the genetic basis of natural variations in non-model yeast organisms and more precisely in L. kluyveri.

Comparative genomics of protoploid Saccharomycetaceae.

Our knowledge of yeast genomes remains largely dominated by the extensive studies on Saccharomyces cerevisiae and the consequences of its ancestral duplication, leaving the evolution of the entire class of hemiascomycetes only partly explored. We concentrate here on five species of Saccharomycetaceae, a large subdivision of hemiascomycetes, that we call "protoploid" because they diverged from the S. cerevisiae lineage prior to its genome duplication. We determined the complete genome sequences of three of these species: Kluyveromyces (Lachancea) thermotolerans and Saccharomyces (Lachancea) kluyveri (two members of the newly described Lachancea clade), and Zygosaccharomyces rouxii. We included in our comparisons the previously available sequences of Kluyveromyces lactis and Ashbya (Eremothecium) gossypii. Despite their broad evolutionary range and significant individual variations in each lineage, the five protoploid Saccharomycetaceae share a core repertoire of approximately 3300 protein families and a high degree of conserved synteny. Synteny blocks were used to define gene orthology and to infer ancestors. Far from representing minimal genomes without redundancy, the five protoploid yeasts contain numerous copies of paralogous genes, either dispersed or in tandem arrays, that, altogether, constitute a third of each genome. Ancient, conserved paralogs as well as novel, lineage-specific paralogs were identified.

Ploidy influences cellular responses to gross chromosomal rearrangements in Saccharomyces cerevisiae.

BACKGROUND: Gross chromosomal rearrangements (GCRs) such as aneuploidy are key factors in genome evolution as well as being common features of human cancer. Their role in tumour initiation and progression has not yet been completely elucidated and the effects of additional chromosomes in cancer cells are still unknown. Most previous studies in which Saccharomyces cerevisiae has been used as a model for cancer cells have been carried out in the haploid context. To obtain new insights on the role of ploidy, the cellular effects of GCRs were compared between the haploid and diploid contexts. RESULTS: A total number of 21 haploid and diploid S. cerevisiae strains carrying various types of GCRs (aneuploidies, nonreciprocal translocations, segmental duplications and deletions) were studied with a view to determining the effects of ploidy on the cellular responses. Differences in colony and cell morphology as well as in the growth rates were observed between mutant and parental strains. These results suggest that cells are impaired physiologically in both contexts. We also investigated the variation in genomic expression in all the mutants. We observed that gene expression was significantly altered. The data obtained here clearly show that genes involved in energy metabolism, especially in the tricarboxylic acid cycle, are up-regulated in all these mutants. However, the genes involved in the composition of the ribosome or in RNA processing are down-regulated in diploids but up-regulated in haploids. Over-expression of genes involved in the regulation of the proteasome was found to occur only in haploid mutants. CONCLUSION: The present comparisons between the cellular responses of strains carrying GCRs in different ploidy contexts bring to light two main findings. First, GCRs induce a general stress response in all studied mutants, regardless of their ploidy. Secondly, the ploidy context plays a crucial role in maintaining the stoichiometric balance of the proteins: the translation rates decrease in diploid strains, whereas the excess protein synthesized is degraded in haploids by proteasome activity.

The Ty1 LTR-retrotransposon population in Saccharomyces cerevisiae genome: dynamics and sequence variations during mobility.

Transposable element (TE) evolution in genomes has mostly been deduced from comparative genome analyses. TEs often account for a large proportion of the eukaryotic nuclear genome (up to 50%, depending on the species). Among the many existing genomic copies, only a small fraction may contribute to the mobility of a TE family. We have identified here, using a genetic screening procedure to trap Ty1 long terminal repeat-retrotransposon insertions in Saccharomyces cerevisiae, which among the populations of resident Ty1 copies are responsible for Ty1 mobility. Although the newly inserted Ty1 copies resulting from a single round of transposition were found to originate from a limited subset of Ty1 resident copies, they showed a high degree of diversity at the nucleotide level, mainly due to the reverse transcription-mediated recombination. In this process, highly expressed and strikingly nonautonomous mutant Ty1 were found to be the most frequently used resident copies, which suggests that nonautonomous elements play a key role in the dynamics of the Ty1 family.

Complete mitochondrial genome sequence of the yeast Pichia farinosa and comparative analysis of closely related species.

Yeasts of the Pichia genus have been isolated from different natural environments. Phylogenies based on multigene sequence analysis have shown that the genus is polyphyletic. Some species of this genus are member of the CTG group. In order to have a better insight into the relationship among species assigned to the yeast genera Pichia into the CTG group, we first sequenced the mitochondrial genome of the osmotolerant yeast Pichia farinosa. We then compared this genome with mitochondrial genomes of yeasts of the CTG group. The P. farinosa mitochondrial DNA is a circular-mapping genome of 32,065 bp, which contains 43 genes transcribed from both strands. It contains a complete set of tRNAs, the small and the large rRNAs, as well as 14 protein-coding genes. Yeasts of the CTG group contain the same core of mitochondrial genes. Phylogenetic analysis based on mitochondrial sequences clearly shows that the CTG group is divided into two distinct clades: the first one contains diploid Candida species, whereas the second mainly contains haploid Pichia species. Moreover, this analysis provides clear evidence that Pichia farinosa and Pichia sorbitophila, which were known to be unique species, are two distinct species.

Genome evolution in yeasts.

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

Influence of genetic background on the occurrence of chromosomal rearrangements in Saccharomyces cerevisiae.

BACKGROUND: Chromosomal rearrangements such as duplications and deletions are key factors in evolutionary processes because they promote genomic plasticity. Although the genetic variations in the Saccharomyces cerevisiae species have been well documented, there is little known to date about the impact of the genetic background on the appearance of rearrangements. RESULTS: Using the same genetic screening, the type of rearrangements and the mutation rates observed in the S288c S. cerevisiae strain were compared to previous findings obtained in the FL100 background. Transposon-associated rearrangements, a major chromosomal rearrangement event selected in FL100, were not detected in S288c. The mechanisms involved in the occurrence of deletions and duplications in the S288c strain were also tackled, using strains deleted for genes implicated in homologous recombination (HR) or non-homologous end joining (NHEJ). Our results indicate that an Yku80p-independent NHEJ pathway is involved in the occurrence of these rearrangements in the S288c background. CONCLUSION: The comparison of two different S. cerevisiae strains, FL100 and S288c, allowed us to conclude that intra-species genomic variations have an important impact on the occurrence of chromosomal rearrangement and that this variability can partly be explained by differences in Ty1 retrotransposon activity.

The complete mitochondrial genome of the yeast Pichia sorbitophila.

Here, we report the complete nucleotide sequence of the 39 107-bp mitochondrial genome of the yeast Pichia sorbitophila. This genome is closely related to those of Candida parapsilosis and Debaryomyces hansenii, as judged from sequence similarities and synteny conservation. It encodes three subunits of cytochrome oxidase (COX1, COX2 and COX3), three subunits of ATP synthase (ATP6, ATP8 and ATP9), the seven subunits of NADH dehydrogenase (NAD1-6 and NAD4L), the apocytochrome b (COB), the large and small rRNAs and a complete set of tRNAs. Although the mitochondrial genome of P. sorbitophila contains the same core of mitochondrial genes observed in the ascomycetous yeasts, those coding for the RNAse P and the ribosomal protein VAR1p are missing. Moreover, the mtDNA of P. sorbitophila contains several introns in its genes and has the particularity of possessing an intron, which is not linked to any upstream exon.

Spontaneous duplications in diploid Saccharomyces cerevisiae cells.

The duplication of DNA sequences is a powerful determinant of genomic plasticity and is known to be one of the key factors responsible for evolution. Recent genomic sequence data demonstrate the abundance of duplicated genes in all surveyed organisms. Over the past years, experimental systems were adequately designed to explore the molecular mechanisms involved in their formation in haploid Saccharomyces cerevisiae strains. To obtain a more global and accurate view of the events leading to DNA sequence duplications, we have selected and characterized duplication occurrences in diploid S. cerevisiae cells. The molecular analysis showed that two other predominant ways lead to duplication in this context: formation of extra chimeric chromosomes and non-reciprocal translocation events. Moreover, we demonstrated that these two types of rearrangements are RAD52 independent and therefore that homologous recombination plays no part in their formation. Finally, our results show the multiplicity of mechanisms involved in duplication events and provide the first experimental evidence that these mechanisms might be ploidy dependent.

Duplication processes in Saccharomyces cerevisiae haploid strains.

Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.

Large-Scale Survey of Intraspecific Fitness and Cell Morphology Variation in a Protoploid Yeast Species.

It is now clear that the exploration of the genetic and phenotypic diversity of nonmodel species greatly improves our knowledge in biology. In this context, we recently launched a population genomic analysis of the protoploid yeast Lachancea kluyveri (formerly Saccharomyces kluyveri), highlighting a broad genetic diversity (π = 17 × 10(-3)) compared to the yeast model organism, S. cerevisiae (π = 4 × 10(-3)). Here, we sought to generate a comprehensive view of the phenotypic diversity in this species. In total, 27 natural L. kluyveri isolates were subjected to trait profiling using the following independent approaches: (i) analyzing growth in 55 growth conditions and (ii) investigating 501 morphological changes at the cellular level. Despite higher genetic diversity, the fitness variance observed in L. kluyveri is lower than that in S. cerevisiae However, morphological features show an opposite trend. In addition, there is no correlation between the origins (ecological or geographical) of the isolate and the phenotypic patterns, demonstrating that trait variation follows neither population history nor source environment in L. kluyveri Finally, pairwise comparisons between growth rate correlation and genetic diversity show a clear decrease in phenotypic variability linked to genome variation increase, whereas no such a trend was identified for morphological changes. Overall, this study reveals for the first time the phenotypic diversity of a distantly related species to S. cerevisiae Given its genetic properties, L. kluyveri might be useful in further linkage mapping analyses of complex traits, and could ultimately provide a better insight into the evolution of the genotype-phenotype relationship across yeast species.

The Hidden Complexity of Mendelian Traits across Natural Yeast Populations.

Mendelian traits are considered to be at the lower end of the complexity spectrum of heritable phenotypes. However, more than a century after the rediscovery of Mendel's law, the global landscape of monogenic variants, as well as their effects and inheritance patterns within natural populations, is still not well understood. Using the yeast Saccharomyces cerevisiae, we performed a species-wide survey of Mendelian traits across a large population of isolates. We generated offspring from 41 unique parental pairs and analyzed 1,105 cross/trait combinations. We found that 8.9% of the cases were Mendelian. Further tracing of causal variants revealed background-specific expressivity and modified inheritances, gradually transitioning from Mendelian to complex traits in 30% of the cases. In fact, when taking into account the natural population diversity, the hidden complexity of traits could be substantial, confounding phenotypic predictability even for simple Mendelian traits.

Chromosomal rearrangements as a major mechanism in the onset of reproductive isolation in Saccharomyces cerevisiae.

Understanding the molecular basis of how reproductive isolation evolves between individuals from the same species offers valuable insight into patterns of genetic differentiation as well as the onset of speciation [1, 2]. The yeast Saccharomyces cerevisiae constitutes an ideal model partly due to its vast ecological range, high level of genetic diversity [3-6], and laboratory-amendable sexual reproduction. Between S. cerevisiae and its sibling species in the Saccharomyces sensu stricto complex, reproductive isolation acts postzygotically and could be attributed to chromosomal rearrangements [7], cytonuclear incompatibility [8, 9], and antirecombination [10, 11], although the implication of these mechanisms at the incipient stage of speciation remains unclear due to further divergence in the nascent species. Recently, several studies assessed the onset of intraspecific reproductive isolation in S. cerevisiae by evaluating the effect of the mismatch repair system [12-14] or by fostering incipient speciation using the same initial genetic background [15-18]. Nevertheless, the overall genetic diversity within this species was largely overlooked, and no systematic evaluation has been performed. Here, we carried out the first species-wide survey for postzygotic reproductive isolation in S. cerevisiae. We crossed 60 natural isolates sampled from diverse niches with the reference strain S288c and identified 16 cases of reproductive isolation with reduced offspring viabilities ranging from 44% to 86%. Using different mapping strategies, we identified reciprocal translocations in a large fraction of all isolates surveyed, indicating that large-scale chromosomal rearrangements might play a major role in the onset of reproductive isolation in this species.

Pichia sorbitophila, an Interspecies Yeast Hybrid, Reveals Early Steps of Genome Resolution After Polyploidization.

Polyploidization is an important process in the evolution of eukaryotic genomes, but ensuing molecular mechanisms remain to be clarified. Autopolyploidization or whole-genome duplication events frequently are resolved in resulting lineages by the loss of single genes from most duplicated pairs, causing transient gene dosage imbalance and accelerating speciation through meiotic infertility. Allopolyploidization or formation of interspecies hybrids raises the problem of genetic incompatibility (Bateson-Dobzhansky-Muller effect) and may be resolved by the accumulation of mutational changes in resulting lineages. In this article, we show that an osmotolerant yeast species, Pichia sorbitophila, recently isolated in a concentrated sorbitol solution in industry, illustrates this last situation. Its genome is a mosaic of homologous and homeologous chromosomes, or parts thereof, that corresponds to a recently formed hybrid in the process of evolution. The respective parental contributions to this genome were characterized using existing variations in GC content. The genomic changes that occurred during the short period since hybrid formation were identified (e.g., loss of heterozygosity, unilateral loss of rDNA, reciprocal exchange) and distinguished from those undergone by the two parental genomes after separation from their common ancestor (i.e., NUMT (NUclear sequences of MiTochondrial origin) insertions, gene acquisitions, gene location movements, reciprocal translocation). We found that the physiological characteristics of this new yeast species are determined by specific but unequal contributions of its two parents, one of which could be identified as very closely related to an extant Pichia farinosa strain.

Osmoresistant yeast Zygosaccharomyces rouxii: the two most studied wild-type strains (ATCC 2623 and ATCC 42981) differ in osmotolerance and glycerol metabolism.

The yeast Zygosaccharomyces rouxii is known for its high tolerance to osmotic stress, which is thought to be caused by sets of specific genes. Relatively few Z. rouxii genes have been identified so far, all of them having homologues in Saccharomyces cerevisiae; none of them was Z. rouxii-specific. Most of the known Z. rouxii genes were isolated from two wild-type strains, ATCC 2623 and ATCC 42981. In this study, we compared these two strains with regard to some of their morphological, physiological and genomic properties. Important differences were found in their salt tolerance and assimilation of glycerol and karyotype; slight differences were also present in their cell morphology. The ATCC 42981 strain showed a higher resistance to salts, higher glycerol production and, unlike ATCC 2623, was able to assimilate glycerol. Under conditions of osmotic stress, the glycerol production in both Z. rouxii strains was much lower than in a S. cerevisiae S288c culture, which suggested the presence of a system that efficiently retains glycerol inside Z. rouxii cells. The karyotype analysis revealed that ATCC 42981 cells contain more chromosomes and have a bigger genome size than those of ATCC 2623.

Tools for the genetic manipulation of Zygosaccharomyces rouxii.

A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP-kanMX-loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase.

Characterisation of Zygosaccharomyces rouxii centromeres and construction of first Z. rouxii centromeric vectors.

Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its high osmotolerance, the basis of which still remains unknown. By exploring the Génolevures I database, four Z. rouxii fragments homologous to Saccharomyces cerevisiae centromeres were identified. Two of them were subjected to further analysis. Their function as centromeres in Z. rouxii was proved, and they were localized to Z. rouxii chromosomes II and VII, respectively. The species-specificity of centromeres was observed; plasmids with a Z. rouxii centromere were not recognized as centromeric in S. cerevisiae, and a S. cerevisiae centromere did not function as a centromere in Z. rouxii. Constructed plasmids bearing Z. rouxii centromeres serve as the first specific centromeric plasmids, and thus contribute to the so-far limited set of genetic tools needed to study the Z. rouxii specific features.

Spontaneous deletions and reciprocal translocations in Saccharomyces cerevisiae: influence of ploidy.

Studying spontaneous chromosomal rearrangements throws light on the rules underlying the genome reshaping events occurring in eukaryotic cells, which are part of the evolutionary process. In Saccharomyces cerevisiae, translocation and deletion processes have been frequently described in haploids, but little is known so far about these processes at the diploid level. Here we investigated the nature and the frequency of the chromosomal rearrangements occurring at this ploidy level. Using a positive selection screen based on a particular mutated allele of the URA2 gene, spontaneous diploid revertants were selected and analysed. Surprisingly, the diploid state was found to be correlated with a decrease in chromosome rearrangement frequency, along with an increase in the complexity of the rearrangements occurring in the target gene. The presence of short DNA tandem repeat sequences seems to be a key requirement for deletion and reciprocal translocation processes to occur in diploids. After discussing the differences between the haploid and diploid levels, some mechanisms possibly involved in chromosome shortening and arm exchange are suggested.

Recovery of a function involving gene duplication by retroposition in Saccharomyces cerevisiae.

The duplication of DNA sequences contributes to genomic plasticity and is known to be one of the key factors responsible for evolution. The mechanisms underlying these rare events, which have been frequently mentioned by authors performing genomic analysis, have not yet been completely elucidated. These mechanisms were approached here in the yeast Saccharomyces cerevisiae, using a positive selection screen based on a particular mutated allele of the URA2 gene. Spontaneous revertants containing a duplication of the terminal part of the URA2 gene were selected and analyzed. Some important features of the duplicated regions, such as their chromosome location, size, and insertion sites, were characterized. The events selected correspond to a single inter- or intrachromosomal gene duplication process. The duplicated ATCase sequence is generally punctuated by a poly(A) tract and is always located in Ty1 sequences. In addition, the activation of a Ty1 transcription process increased the frequency of the duplication events. All in all, these data suggest that the duplication mechanism involves the reverse transcription of mRNA and the subsequent integration of the cDNA into a Ty1 area. The Ty1 elements and the retrotransposon-encoded function are key factors contributing to chromosomal reshaping. The genomic rearrangements described constitute experimental evidence for the recovery of a function involving duplication by retroposition.