Telomere-to-telomere assemblies of 142 strains characterize the genome structural landscape in Saccharomyces cerevisiae.

Pangenomes provide access to an accurate representation of the genetic diversity of species, both in terms of sequence polymorphisms and structural variants (SVs). Here we generated the Saccharomyces cerevisiae Reference Assembly Panel (ScRAP) comprising reference-quality genomes for 142 strains representing the species' phylogenetic and ecological diversity. The ScRAP includes phased haplotype assemblies for several heterozygous diploid and polyploid isolates. We identified circa (ca.) 4,800 nonredundant SVs that provide a broad view of the genomic diversity, including the dynamics of telomere length and transposable elements. We uncovered frequent cases of complex aneuploidies where large chromosomes underwent large deletions and translocations. We found that SVs can impact gene expression near the breakpoints and substantially contribute to gene repertoire evolution. We also discovered that horizontally acquired regions insert at chromosome ends and can generate new telomeres. Overall, the ScRAP demonstrates the benefit of a pangenome in understanding genome evolution at population scale.

Impact of the acquired subgenome on the transcriptional landscape in Brettanomyces bruxellensis allopolyploids.

Gene expression variation can provide an overview of the changes in regulatory networks that underlie phenotypic diversity. Certain evolutionary trajectories such as polyploidization events can have an impact on the transcriptional landscape. Interestingly, the evolution of the yeast species Brettanomyces bruxellensis has been punctuated by diverse allopolyploidization events leading to the coexistence of a primary diploid genome associated with various haploid acquired genomes. To assess the impact of these events on gene expression, we generated and compared the transcriptomes of a set of 87 B. bruxellensis isolates, selected as being representative of the genomic diversity of this species. Our analysis revealed that acquired subgenomes strongly impact the transcriptional patterns and allow discrimination of allopolyploid populations. In addition, clear transcriptional signatures related to specific populations have been revealed. The transcriptional variations observed are related to some specific biological processes such as transmembrane transport and amino acids metabolism. Moreover, we also found that the acquired subgenome causes the overexpression of some genes involved in the production of flavor-impacting secondary metabolites, especially in isolates of the beer population.

Towards accurate, contiguous and complete alignment-based polyploid phasing algorithms.

Phasing, and in particular polyploid phasing, have been challenging problems held back by the limited read length of high-throughput short read sequencing methods which can't overcome the distance between heterozygous sites and labor high cost of alternative methods such as the physical separation of chromosomes for example. Recently developed single molecule long-read sequencing methods provide much longer reads which overcome this previous limitation. Here we review the alignment-based methods of polyploid phasing that rely on four main strategies: population inference methods, which leverage the genetic information of several individuals to phase a sample; objective function minimization methods, which minimize a function such as the Minimum Error Correction (MEC); graph partitioning methods, which represent the read data as a graph and split it into k haplotype subgraphs; cluster building methods, which iteratively grow clusters of similar reads into a final set of clusters that represent the haplotypes. We discuss the advantages and limitations of these methods and the metrics used to assess their performance, proposing that accuracy and contiguity are the most meaningful metrics. Finally, we propose the field of alignment-based polyploid phasing would greatly benefit from the use of a well-designed benchmarking dataset with appropriate evaluation metrics. We consider that there are still significant improvements which can be achieved to obtain more accurate and contiguous polyploid phasing results which reflect the complexity of polyploid genome architectures.

Phased polyploid genomes provide deeper insight into the multiple origins of domesticated Saccharomyces cerevisiae beer yeasts.

Yeasts, and in particular Saccharomyces cerevisiae, have been used for brewing beer for thousands of years. Population genomic surveys highlighted that beer yeasts are polyphyletic, with the emergence of different domesticated subpopulations characterized by high genetic diversity and ploidy level. However, the different origins of these subpopulations are still unclear as reconstruction of polyploid genomes is required. To gain better insight into the differential evolutionary trajectories, we sequenced the genomes of 35 Saccharomyces cerevisiae isolates coming from different beer-brewing clades, using a long-read sequencing strategy. By phasing the genomes and using a windowed approach, we identified three main beer subpopulations based on allelic content (European dominant, Asian dominant, and African beer). They were derived from different admixtures between populations and are characterized by distinctive genomic patterns. By comparing the fully phased genes, the most diverse in our dataset are enriched for functions relevant to the brewing environment such as carbon metabolism, oxidoreduction, and cell wall organization activity. Finally, independent domestication, evolution, and adaptation events across subpopulations were also highlighted by investigating specific genes previously linked to the brewing process. Altogether, our analysis based on phased polyploid genomes has led to new insight into the contrasting evolutionary history of beer isolates.

Different trajectories of polyploidization shape the genomic landscape of the Brettanomyces bruxellensis yeast species.

Polyploidization events are observed across the tree of life and occur in many fungi, plant, and animal species. During evolution, polyploidy is thought to be an important source of speciation and tumorigenesis. However, the origin of polyploid populations is not always clear, and little is known about the precise nature and structure of their complex genome. Using a long-read sequencing strategy, we sequenced 71 strains from the Brettanomyces bruxellensis yeast species, which is found in anthropized environments (e.g., beer, contaminant of wine, kombucha, and ethanol production) and characterized by several polyploid subpopulations. To reconstruct the polyploid genomes, we phased them by using different strategies and found that each subpopulation had a unique polyploidization history with distinct trajectories. The polyploid genomes contain either genetically closely related (with a genetic divergence <1%) or diverged copies (>3%), indicating auto- as well as allopolyploidization events. These latest events have occurred independently with a specific and unique donor in each of the polyploid subpopulations and exclude the known Brettanomyces sister species as possible donors. Finally, loss of heterozygosity events has shaped the structure of these polyploid genomes and underline their dynamics. Overall, our study highlights the multiplicity of the trajectories leading to polyploid genomes within the same species.

nPhase: an accurate and contiguous phasing method for polyploids.

While genome sequencing and assembly are now routine, we do not have a full, precise picture of polyploid genomes. No existing polyploid phasing method provides accurate and contiguous haplotype predictions. We developed nPhase, a ploidy agnostic tool that leverages long reads and accurate short reads to solve alignment-based phasing for samples of unspecified ploidy ( https://github.com/OmarOakheart/nPhase ). nPhase is validated by tests on simulated and real polyploids. nPhase obtains on average over 95% accuracy and a contiguous 1.25 haplotigs per haplotype to cover more than 90% of each chromosome (heterozygosity rate ≥ 0.5%). nPhase allows population genomics and hybrid studies of polyploids.

Evidence for Two Main Domestication Trajectories in Saccharomyces cerevisiae Linked to Distinct Bread-Making Processes.

Production of leavened bread dates to the second millennium BCE. Since then, the art of bread making has developed, yet the evolution of bread-associated microbial species remains largely unknown. Nowadays, leavened bread is made either by using a pure commercial culture of the yeast Saccharomyces cerevisiae or by propagating a sourdough-a mix of flour and water spontaneously fermented by yeasts and bacteria. We studied the domestication of S. cerevisiae originating from industrial sources and artisanal sourdoughs and tested whether different bread-making processes led to population divergence. We found that S. cerevisiae bakery strains are polyphyletic with 67% of strains clustering into two main clades: most industrial strains were tetraploid and clustered with strains having diverse origins, including beer. By contrast, most sourdough strains were diploid and grouped in a second clade of strains having mosaic genomes and diverse origins, including fruits and natural environments. They harbored a higher copy number of genes involved in maltose utilization, and a high level of gene flow from multiple contributors was detected. Bakery strains displayed higher CO2 production than do strains from other domesticated lineages (such as beer and wine), revealing a specific phenotypic signature of domestication. Interestingly, industrial strains had a shorter fermentation onset than sourdough strains, which were better adapted to a sourdough-like environment, suggesting divergent selection by industrial and artisanal processes. Our results reveal that the domestication of bakery yeast has been accompanied by dispersion, hybridization, and divergent selection through industrial and artisanal processes.

The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose.

Anti-cancer strategies that target the glycolytic metabolism of tumors have been proposed. The glucose analog 2-deoxyglucose (2DG) is imported into cells and, after phosphorylation, becomes 2DG-6-phosphate, a toxic by-product that inhibits glycolysis. Using yeast as a model, we performed an unbiased mass spectrometry-based approach to probe the cellular effects of 2DG on the proteome and study resistance mechanisms to 2DG. We found that two phosphatases that target 2DG-6-phosphate were induced upon exposure to 2DG and participated in 2DG detoxification. Dog1 and Dog2 are HAD (haloacid dehalogenase)-like phosphatases, which are evolutionarily conserved. 2DG induced Dog2 by activating several signaling pathways, such as the stress response pathway mediated by the p38 MAPK ortholog Hog1, the unfolded protein response (UPR) triggered by 2DG-induced ER stress, and the cell wall integrity (CWI) pathway mediated by the MAPK Slt2. Loss of the UPR or CWI pathways led to 2DG hypersensitivity. In contrast, mutants impaired in the glucose-mediated repression of genes were 2DG resistant because glucose availability transcriptionally repressed DOG2 by inhibiting signaling mediated by the AMPK ortholog Snf1. The characterization and genome resequencing of spontaneous 2DG-resistant mutants revealed that DOG2 overexpression was a common strategy underlying 2DG resistance. The human Dog2 homolog HDHD1 displayed phosphatase activity toward 2DG-6-phosphate in vitro and its overexpression conferred 2DG resistance in HeLa cells, suggesting that this 2DG phosphatase could interfere with 2DG-based chemotherapies. These results show that HAD-like phosphatases are evolutionarily conserved regulators of 2DG resistance.

Incompatibilities in Mismatch Repair Genes MLH1-PMS1 Contribute to a Wide Range of Mutation Rates in Human Isolates of Baker's Yeast.

Laboratory baker's yeast strains bearing an incompatible combination of MLH1 and PMS1 mismatch repair alleles are mutators that can adapt more rapidly to stress, but do so at the cost of long-term fitness. We identified 18 baker's yeast isolates from 1011 surveyed that contain the incompatible MLH1-PMS1 genotype in a heterozygous state. Surprisingly, the incompatible combination from two human clinical heterozygous diploid isolates, YJS5845 and YJS5885, contain the exact MLH1 (S288c-derived) and PMS1 (SK1-derived) open reading frames originally shown to confer incompatibility. While these isolates were nonmutators, their meiotic spore clone progeny displayed mutation rates in a DNA slippage assay that varied over a 340-fold range. This range was 30-fold higher than observed between compatible and incompatible combinations of laboratory strains. Genotyping analysis indicated that MLH1-PMS1 incompatibility was the major driver of mutation rate in the isolates. The variation in the mutation rate of incompatible spore clones could be due to background suppressors and enhancers, as well as aneuploidy seen in the spore clones. Our data are consistent with the observed variance in mutation rate contributing to adaptation to stress conditions (e.g., in a human host) through the acquisition of beneficial mutations, with high mutation rates leading to long-term fitness costs that are buffered by mating or eliminated through natural selection.

Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

Variation of the meiotic recombination landscape and properties over a broad evolutionary distance in yeasts.

Meiotic recombination is a major factor of genome evolution, deeply characterized in only a few model species, notably the yeast Saccharomyces cerevisiae. Consequently, little is known about variations of its properties across species. In this respect, we explored the recombination landscape of Lachancea kluyveri, a protoploid yeast species that diverged from the Saccharomyces genus more than 100 million years ago and we found striking differences with S. cerevisiae. These variations include a lower recombination rate, a higher frequency of chromosomes segregating without any crossover and the absence of recombination on the chromosome arm containing the sex locus. In addition, although well conserved within the Saccharomyces clade, the S. cerevisiae recombination hotspots are not conserved over a broader evolutionary distance. Finally and strikingly, we found evidence of frequent reversal of commitment to meiosis, resulting in return to mitotic growth after allele shuffling. Identification of this major but underestimated evolutionary phenomenon illustrates the relevance of exploring non-model species.

Mismatch Repair Incompatibilities in Diverse Yeast Populations.

An elevated mutation rate can provide cells with a source of mutations to adapt to changing environments. We identified a negative epistatic interaction involving naturally occurring variants in the MLH1 and PMS1 mismatch repair (MMR) genes of Saccharomyces cerevisiae We hypothesized that this MMR incompatibility, created through mating between divergent S. cerevisiae, yields mutator progeny that can rapidly but transiently adapt to an environmental stress. Here we analyzed the MLH1 and PMS1 genes across 1010 S. cerevisiae natural isolates spanning a wide range of ecological sources (tree exudates, Drosophila, fruits, and various fermentation and clinical isolates) and geographical sources (Europe, America, Africa, and Asia). We identified one homozygous clinical isolate and 18 heterozygous isolates containing the incompatible MMR genotype. The MLH1-PMS1 gene combination isolated from the homozygous clinical isolate conferred a mutator phenotype when expressed in the S288c laboratory background. Using a novel reporter to measure mutation rates, we showed that the overall mutation rate in the homozygous incompatible background was similar to that seen in compatible strains, indicating the presence of suppressor mutations in the clinical isolate that lowered its mutation rate. This observation and the identification of 18 heterozygous isolates, which can lead to MMR incompatible genotypes in the offspring, are consistent with an elevated mutation rate rapidly but transiently facilitating adaptation. To avoid long-term fitness costs, the incompatibility is apparently buffered by mating or by acquiring suppressors. These observations highlight effective strategies in eukaryotes to avoid long-term fitness costs associated with elevated mutation rates.

PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells.

Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3'-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5'-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure.

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.

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.