Spindle architecture constrains karyotype evolution.

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome numbers vary dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

Multiple Horizontal Mini-chromosome Transfers Drive Genome Evolution of Clonal Blast Fungus Lineages.

Crop disease pandemics are often driven by asexually reproducing clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb accessory mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

Complete genome of the Medicago anthracnose fungus, Colletotrichum destructivum, reveals a mini-chromosome-like region within a core chromosome.

Colletotrichum destructivum (Cd) is a phytopathogenic fungus causing significant economic losses on forage legume crops (Medicago and Trifolium species) worldwide. To gain insights into the genetic basis of fungal virulence and host specificity, we sequenced the genome of an isolate from Medicago sativa using long-read (PacBio) technology. The resulting genome assembly has a total length of 51.7 Mb and comprises ten core chromosomes and two accessory chromosomes, all of which were sequenced from telomere to telomere. A total of 15, 631 gene models were predicted, including genes encoding potentially pathogenicity-related proteins such as candidate-secreted effectors (484), secondary metabolism key enzymes (110) and carbohydrate-active enzymes (619). Synteny analysis revealed extensive structural rearrangements in the genome of Cd relative to the closely related Brassicaceae pathogen, Colletotrichum higginsianum. In addition, a 1.2 Mb species-specific region was detected within the largest core chromosome of Cd that has all the characteristics of fungal accessory chromosomes (transposon-rich, gene-poor, distinct codon usage), providing evidence for exchange between these two genomic compartments. This region was also unique in having undergone extensive intra-chromosomal segmental duplications. Our findings provide insights into the evolution of accessory regions and possible mechanisms for generating genetic diversity in this asexual fungal pathogen.

Chromosome-Level Assembly Reveals a Fifteen-Chromosome Aneuploid Genome and Environmental Adaptation Strategy of Chinese Traditional Medical Fungus Wolfiporia hoelen.

The sclerotia of Wolfiporia hoelen are one of the most important traditional Chinese medicines and foods commonly used in China, Japan, Korea, and other Asian countries. To provide a high-quality reference genome and deepen our understanding of the genome of W. hoelen to elucidate various biological phenomena. In this study, we assembled three genomes of W. hoelen using a combination of Nanopore and Illumina sequencing strategies. The fifteen-chromosome genome L7 of W. hoelen was assembled with two-sided telomere and rDNA sequences for the first time. The chromosome count was subsequently confirmed through collinearity analysis, correcting the previous belief that W. hoelen had only fourteen chromosomes. Moreover, the aneuploid genome was discovered in W. hoelen for the first time through sequencing depth analysis of different chromosomes, and only some strains of W. hoelen exhibit aneuploid genomes. According to the genome analysis of homokaryotic offspring and protoplast-isolated strains, a potential variation in chromosome allocation patterns was revealed. Moreover, the gene function enrichment analysis of genes on reduplicated chromosomes demonstrated that aneuploidy in the genome may be the result of environmental adaptation for W. hoelen. The discovery of an aneuploid genome also provides new ideas for genetic improvement of W. hoelen.

Dynamic Live Cell Imaging of Budding Yeast Meiosis.

For over a century, major advances in understanding meiosis have come from the use of microscopy-based methods. Studies using the budding yeast, Saccharomyces cerevisiae, have made important contributions to our understanding of meiosis because of the facility with which budding yeast can be manipulated as a genetic model organism. In contrast, imaging-based approaches with budding yeast have been constrained by the small size of its chromosomes. The advent of advances in fluorescent chromosome tagging techniques has made it possible to use yeast more effectively for imaging-based approaches as well. This protocol describes live cell imaging methods that can be used to monitor chromosome movements throughout meiosis in living yeast cells.

Hi-C2B: Optimized Detection of Chromosomal Contacts Within Synchronized Meiotic S. cerevisiae Cells.

Hi-C, a genome-wide chromosome conformation capture assay, is a powerful tool used to study three-dimensional genome organization by converting physical pairwise interactions into counts of pairwise interactions. To study the many temporally regulated facets of meiotic recombination in S. cerevisiae, the Hi-C assay must be robust such that fine- and wide-scale comparisons between genetic datasets can be made. Here we describe an updated protocol for Hi-C (Hi-C2B) that generates reproducible libraries of interaction data with low noise and for a relatively low cost.

Identifying Chromosome Movement Patterns During Meiosis Using ChroMo.

During meiosis, transient associations between the nuclear envelope and telomeres transmit nuclear movements to chromosomes, enabling their pairing and recombination. Recent advances in the field of quantitative cell biology allow a large volume of information about the kinetics of these chromosome movements to be extracted and analyzed with the aim of identifying biologically relevant movement patterns. To this end, we have developed ChroMo, a freely available application for the unsupervised study of chromosome movements in fission yeast meiosis. ChroMo contains a set of time series algorithms to identify chromosome movement motifs that are not easily observable by direct human visualization and to establish causal relationships between phenotypes. In this chapter, we present a detailed protocol for the processing of raw live imaging data from fission yeast and its subsequent analysis in ChroMo.

Chromosome biology: Too big to fail.

Spindles are microtubule-based machines that segregate chromosomes during cell division. Spindle morphology and dynamics are malleable based on forces within the spindle, and a new study reveals the extreme plasticity of the Saccharomyces cerevisiae spindle to adapt and segregate engineered mega-chromosomes.

Evidence of 14-3-3 proteins contributing to kinetochore integrity and chromosome congression during mitosis.

The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions in the cell. Homo- and hetero-dimers of these proteins mainly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or cells survive with severe growth defects, including gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work, we investigated their centromere- and kinetochore-related functions of Bmh1 and Bmh2. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and non-shared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, Bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.

Plasticity of the mitotic spindle in response to karyotype variation.

Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two "mega chromosomes," to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.

Comparative genomics of the closely related fungal genera Cryptococcus and Kwoniella reveals karyotype dynamics and suggests evolutionary mechanisms of pathogenesis.

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

DNA methylation-based high-resolution mapping of long-distance chromosomal interactions in nucleosome-depleted regions.

3C-based methods have significantly advanced our understanding of 3D genome organization. However, it remains a formidable task to precisely capture long-range chromosomal interactions between individual loci, such as those between promoters and distal enhancers. Here, we present Methyltransferase Targeting-based chromosome Architecture Capture (MTAC), a method that maps the contacts between a target site (viewpoint) and the rest of the genome in budding yeast with high resolution and sensitivity. MTAC detects hundreds of intra- and inter-chromosomal interactions within nucleosome-depleted regions (NDRs) that cannot be captured by 4C, Hi-C, or Micro-C. By applying MTAC to various viewpoints, we find that (1) most long-distance chromosomal interactions detected by MTAC reflect tethering by the nuclear pore complexes (NPCs), (2) genes co-regulated by methionine assemble into inter-chromosomal clusters near NPCs upon activation, (3) mediated by condensin, the mating locus forms a highly specific interaction with the recombination enhancer (RE) in a mating-type specific manner, and (4) correlation of MTAC signals among NDRs reveal spatial mixing and segregation of the genome. Overall, these results demonstrate MTAC as a powerful tool to resolve fine-scale long-distance chromosomal interactions and provide insights into the 3D genome organization.

A fully haplotype-resolved and nearly gap-free genome assembly of wheat stripe rust fungus.

Stripe rust fungus Puccinia striiformis f. sp. tritici (Pst) is a destructive pathogen of wheat worldwide. Pst has a macrocyclic-heteroecious lifecycle, in which one-celled urediniospores are dikaryotic, each nucleus containing one haploid genome. We successfully generated the first fully haplotype-resolved and nearly gap-free chromosome-scale genome assembly of Pst by combining PacBio HiFi sequencing and trio-binning strategy. The genome size of the two haploid assemblies was 75.59 Mb and 75.91 Mb with contig N50 of 4.17 Mb and 4.60 Mb, and both had 18 pseudochromosomes. The high consensus quality values of 55.57 and 59.02 for both haplotypes confirmed the correctness of the assembly. Of the total 18 chromosomes, 15 and 16 were gapless while there were only five and two gaps for the remaining chromosomes of the two haplotypes, respectively. In total, 15,046 and 15,050 protein-coding genes were predicted for the two haplotypes, and the complete BUSCO scores achieved 97.7% and 97.9%, respectively. The genome will lay the foundation for further research on genetic variations and the evolution of rust fungi.

Agent-based modeling of nuclear chromosome ensembles identifies determinants of homolog pairing during meiosis.

During meiosis, pairing of homologous chromosomes (homologs) ensures the formation of haploid gametes from diploid precursor cells, a prerequisite for sexual reproduction. Pairing during meiotic prophase I facilitates crossover recombination and homolog segregation during the ensuing reductional cell division. Mechanisms that ensure stable homolog alignment in the presence of an excess of non-homologous chromosomes have remained elusive, but rapid chromosome movements appear to play a role in the process. Apart from homolog attraction, provided by early intermediates of homologous recombination, dissociation of non-homologous associations also appears to contribute to homolog pairing, as suggested by the detection of stable non-homologous chromosome associations in pairing-defective mutants. Here, we have developed an agent-based model for homolog pairing derived from the dynamics of a naturally occurring chromosome ensemble. The model simulates unidirectional chromosome movements, as well as collision dynamics determined by attractive and repulsive forces arising from close-range physical interactions. Chromosome number and size as well as movement velocity and repulsive forces are identified as key factors in the kinetics and efficiency of homologous pairing in addition to homolog attraction. Dissociation of interactions between non-homologous chromosomes may contribute to pairing by crowding homologs into a limited nuclear area thus creating preconditions for close-range homolog attraction. Incorporating natural chromosome lengths, the model accurately recapitulates efficiency and kinetics of homolog pairing observed for wild-type and mutant meiosis in budding yeast, and can be adapted to nuclear dimensions and chromosome sets of other organisms.

A chromosome-scale genome provides new insights into the typical carotenoid biosynthesis in the important red yeast Rhodotorula glutinis QYH-2023 with anti-inflammatory effects.

Rhodotorula spp. has been studied as one powerful source for a novel cell factory with fast growth and its high added-value biomolecules. However, its inadequate genome and genomic annotation have hindered its widespread use in cosmetics and food industries. Rhodotorula glutinis QYH-2023, was isolated from rice rhizosphere soil, and the highest quality of the genome of the strain was obtained at chromosome level (18 chromosomes) than ever before in red yeast in this study. Comparative genomics analysis revealed that there are more key gene copies of carotenoids biosynthesis in R. glutinis QYH-2023 than other species of Rhodotorula spp. Integrated transcriptome and metabolome analysis revealed that lipids and carotenoids biosynthesis was significantly enriched during fermentation. Subsequent investigation revealed that the over-expression of the strain three genes related to carotenoids biosynthesis in Komagataella phaffii significantly promoted the carotenoid production. Furthermore, in vitro tests initially confirmed that the longer the fermentation period, the synthesized metabolites controlled by R. glutinis QYH-2023 genome had the stronger anti-inflammatory properties. All of the findings revealed a high-quality reference genome which highlight the potential of R. glutinis strains to be employed as chassis cells for biosynthesizing carotenoids and other active chemicals.

Chromosome-level genome assembly of the yeast Lodderomyces beijingensis reveals the genetic nature of metabolic adaptations and identifies subtelomeres as hotspots for amplification of mating type loci.

Lodderomyces beijingensis is an ascosporic ascomycetous yeast. In contrast to related species Lodderomyces elongisporus, which is a recently emerging human pathogen, L. beijingensis is associated with insects. To provide an insight into its genetic makeup, we investigated the genome of its type strain, CBS 14171. We demonstrate that this yeast is diploid and describe the high contiguity nuclear genome assembly consisting of eight chromosome-sized contigs with a total size of about 15.1 Mbp. We find that the genome sequence contains multiple copies of the mating type loci and codes for essential components of the mating pheromone response pathway, however, the missing orthologs of several genes involved in the meiotic program raise questions about the mode of sexual reproduction. We also show that L. beijingensis genome codes for the 3-oxoadipate pathway enzymes, which allow the assimilation of protocatechuate. In contrast, the GAL gene cluster underwent a decay resulting in an inability of L. beijingensis to utilize galactose. Moreover, we find that the 56.5 kbp long mitochondrial DNA is structurally similar to known linear mitochondrial genomes terminating on both sides with covalently closed single-stranded hairpins. Finally, we discovered a new double-stranded RNA mycovirus from the Totiviridae family and characterized its genome sequence.

Centromere pairing enables correct segregation of meiotic chromosomes.

Proper chromosome segregation in meiosis I relies on the formation of connections between homologous chromosomes. Crossovers between homologs provide a connection that allows them to attach correctly to the meiosis I spindle. Tension is transmitted across the crossover when the partners attach to microtubules from opposing poles of the spindle. Tension stabilizes microtubule attachments that will pull the partners toward opposite poles at anaphase. Paradoxically, in many organisms, non-crossover partners segregate correctly. The mechanism by which non-crossover partners become bioriented on the meiotic spindle is unknown. Both crossover and non-crossover partners pair their centromeres early in meiosis (prophase). In budding yeast, centromere pairing is correlated with subsequent correct segregation of the partners. The mechanism by which centromere pairing, in prophase, promotes later correct attachment of the partners to the metaphase spindle is unknown. We used live cell imaging to track the biorientation process of non-crossover chromosomes. We find that centromere pairing allows the establishment of connections between the partners that allows their later interdependent attachment to the meiotic spindle using tension-sensing biorientation machinery. Because all chromosome pairs experience centromere pairing, our findings suggest that crossover chromosomes also utilize this mechanism to achieve maximal segregation fidelity.

The landscape and predicted roles of structural variants in Fusarium graminearum genomes.

Structural rearrangements, such as inversions, translocations, duplications, and large insertions and deletions, are large-scale genomic variants that can play an important role in shaping phenotypic variation and in genome adaptation and evolution. We used chromosomal-level assemblies from eight Fusarium graminearum isolates to study structural variants and their role in fungal evolution. We generated the assemblies of four of these genomes after Oxford Nanopore sequencing. A total of 87 inversions, 159 translocations, 245 duplications, 58,489 insertions, and 34,102 deletions were detected. Regions of high recombination rate are associated with structural rearrangements, and a significant proportion of inversions, translocations, and duplications overlap with the repeat content of the genome, suggesting recombination and repeat elements are major factors in the origin of structural rearrangements in F. graminearum. Large insertions and deletions introduce presence-absence polymorphisms for many genes, including secondary metabolite biosynthesis cluster genes and predicted effectors genes. Translocation events were found to be shuffling predicted effector-rich regions of the genomes and are likely contributing to the gain and loss of effectors facilitated by recombination. Breakpoints of some structural rearrangements fall within coding sequences and are likely altering the protein products. Structural rearrangements in F. graminearum thus have an important role to play in shaping pathogen-host interactions and broader evolution through genome reorganization, the introduction of presence-absence polymorphisms, and changing protein products and gene regulation.