Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2–6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36–49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.

Illumina sequencing has revolutionized yeast genomics, with prices for commercial draft genome sequencing now below $200. The popular SPAdes assembler makes it simple to generate a de novo genome assembly for any yeast species. However, whereas making genome assemblies has become routine, understanding what they contain is still challenging. Here, we show how graphing the information that SPAdes provides about the length and coverage of each scaffold can be used to investigate the nature of an assembly, and to diagnose possible problems. Scaffolds derived from mitochondrial DNA, ribosomal DNA, and yeast plasmids can be identified by their high coverage. Contaminating data, such as cross-contamination from other samples in a multiplex sequencing run, can be identified by its low coverage. Scaffolds derived from the bacteriophage PhiX174 and Lambda DNAs that are frequently used as molecular standards in Illumina protocols can also be detected. Assemblies of yeast genomes with high heterozygosity, such as interspecies hybrids, often contain two types of scaffold: regions of the genome where the two alleles assembled into two separate scaffolds and each has a coverage level C , and regions where the two alleles co-assembled (collapsed) into a single scaffold that has a coverage level 2 C . Visualizing the data with Coverage- vs. -Length (CVL) plots, which can be done using Microsoft Excel or Google Sheets, provides a simple method to understand the structure of a genome assembly and detect aberrant scaffolds or contigs. We provide a Python script that allows assemblies to be filtered to remove contaminants identified in CVL plots.

Metschnikowia strain UCD127 was isolated from soil in Ireland and sequenced. It is a highly heterozygous diploid strain with 385,000 single nucleotide polymorphisms (SNPs). Its ribosomal DNA has the highest similarity to that of M. chrysoperlae, but its ACT1 and TEF1 loci and mitochondrial genome show affinity to those of M. fructicola, whose genome is significantly larger.

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Centromeres pose an evolutionary paradox: strongly conserved in function but rapidly changing in sequence and structure. However, in the absence of damage, centromere locations are usually conserved within a species. We report here that isolates of the pathogenic yeast species Candida parapsilosis show within-species polymorphism for the location of centromeres on two of its eight chromosomes. Its old centromeres have an inverted-repeat (IR) structure, whereas its new centromeres have no obvious structural features but are located within 30 kb of the old site. Centromeres can therefore move naturally from one chromosomal site to another, apparently spontaneously and in the absence of any significant changes in DNA sequence. Our observations are consistent with a model in which all centromeres are genetically determined, such as by the presence of short or long IRs or by the ability to form cruciforms. We also find that centromeres have been hotspots for genomic rearrangements in the C. parapsilosis clade.

Diutina catenulata (Candida catenulata) is an ascomycetous yeast that has been isolated from humans, animals and environmental sources. The species is a contaminant of dairy products, and has been linked to superficial and invasive infections in both humans and animals. Previous phylogenetic analyses have assigned the species to the Saccharomycetales, but failed to identify its specific clade. Here, we report the genome sequence of an environmental isolate of D. catenulata. Examination of the tRNA repertoire and coding potential of this species shows that it translates the CUG codon as serine and not leucine. In addition, two phylogenetic analyses using 204 ubiquitous gene family alignments and 3,826 single-copy genes both confirm the placement of the species in the Debaryomycetaceae/Metschnikowiaceae, or CTG-Ser clade. The sequenced isolate contains an MTLα idiomorph. However, unlike most MTL loci in related species, poly (A) polymerase (PAP) is not adjacent to MTLα1.

50Aneuploidy is associated with drug resistance in fungal pathogens. In tropical 51 countries, Candida tropicalis is the most frequently isolated Candida species from 52 patients. To facilitate the study of genomic rearrangements in C. tropicalis, we 53 assembled its genome in seven gapless chromosomes by combining next-54 generation sequencing (NGS) technologies with chromosome conformation capture 55 sequencing (3C-seq). Our 3C-seq data revealed interchromosomal centromeric and 56 telomeric interactions in C. tropicalis, similar to a closely related fungal pathogen 57 Candida albicans. By performing a genome-wide synteny analysis between C. 58 tropicalis and C. albicans, we identified 39 interchromosomal synteny breakpoints 59 (ICSBs), which are relics of ancient translocations. Majority of ICSBs are mapped 60 within 100 kb of homogenized inverted repeat-associated (HIR) centromeres (17/39) 61 or telomere-proximal regions (7/39) in C. tropicalis. Further, we developed a genome 62 assembly of Candida sojae and used the available genome assembly of Candida 63 viswanathii, two closely related species of C. tropicalis, to identify the putative 64 centromeres. In both species, we identified the putative centromeres as HIR-65 associated loci, syntenic to the centromeres of C. tropicalis. Strikingly, a centromere-66 specific motif is conserved in these three species. Presence of similar HIR-67 associated putative centromeres in early-diverging Candida parapsilosis indicated 68 that the ancestral CUG-Ser1 clade species possessed HIR-associated centromeres. 69We propose that homology and spatial proximity-aided translocations among the 70 ancestral centromeres and loss of HIR-associated centromere DNA sequences led 71 to the emergence of evolutionary new centromeres (ENCs) on unique DNA 72 sequences. These events might have facilitated karyotype evolution and centromere-73 type transition in closely-related CUG-Ser1 clade species. 74 75 76 Significance Statement 77We assembled the genome of Candida tropicalis, a frequently isolated fungal 78 pathogen from patients in tropical countries, in seven complete chromosomes. 79 Comparative analysis of the CUG-Ser1 clade members suggests chromosomal 80 rearrangements are mediated by homogenized inverted repeat (HIR)-associated 81 centromeres present in close proximity in the nucleus as revealed by chromosome 82 conformation capture. These translocation events facilitated loss of ancestral HIR-83 associated centromeres and seeding of evolutionary new centromeres on unique 84 DNA sequences. Such karyotypic rearrangements can be a major source of genetic 85 variability in the otherwise majorly clonally propagated human fungal pathogens of 86 the CUG-Ser1 clade. The improved genome assembly will facilitate studies related to 87 aneuploidy-induced drug resistance in C. tropicalis. 88 89 Introduction 90 91 The efficient maintenance of the genetic material and its propagation to subsequent 92 generations determine the fitness of an organism. Genomic rearrangements are 93 often associat...