Electrophoretic Karyotyping Research Articles

An improved Lodderomyces elongisporus NRRL YB-4239 genome assembly substantiated by its electrophoretic karyotype.

Pacific Biosciences long-read sequencing was used to improve the genome assembly for Lodderomyces elongisporus strain NRRL YB-4239 (ATCC 11503). The new assembly included eight chromosomes that were substantiated by the electrophoretic karyotype. The nuclear genome was 16.1 Mb (37.2% GC) with 5,740 genes predicted.

A Chromosome-Level Genome Assembly for Yamadazyma tenuis ATCC 10573.

Pacific Biosciences (PacBio) long-read sequencing was used to generate a chromosome-level genome assembly for Yamadazyma tenuis strain ATCC 10573. The assembly featured 7 chromosomes that matched the electrophoretic karyotype and a 26.5-kb circular mitochondrial genome. The nuclear genome was 10.8 Mb, with a GC content of 43%, and 5,340 predicted genes.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Directed Evolution Detects Supernumerary Centric Chromosomes Conferring Resistance to Azoles in Candida auris.

Candida auris exhibits resistance to multiple antifungal drug classes and sterilization agents, posing threats to the immunocompromised worldwide. Among the four major geographical clades, the East Asian clade 2 isolates of C. auris are mostly drug susceptible. In this study, we experimentally evolved one such drug-susceptible isolate for multiple generations in the presence of the antifungal compound fluconazole and analyzed changes in the karyotype, DNA sequence, and gene expression profiles in three evolved drug-resistant isolates. Next-generation sequencing and electrophoretic karyotyping confirm the presence of segmental aneuploidy as supernumerary chromosomes originating from centromere-inclusive chromosomal duplication events in two such cases. A 638-kb region and a 675-kb region, both of which originated from chromosome 5 and contained its centromere region, are instances of supernumerary chromosome formation identified in two evolved fluconazole-resistant isolates. Loss of the supernumerary chromosomes from the drug-resistant isolates results in a complete reversal of fluconazole susceptibility. Transcriptome analysis of the third isolate identified overexpression of drug efflux pumps as a possible non-aneuploidy-driven mechanism of drug resistance. Together, this study reveals how both aneuploidy-driven and aneuploidy-independent mechanisms may operate in parallel in an evolving population of C. auris in the presence of an antifungal drug, in spite of starting from the same strain grown under similar conditions, to attain various levels of fluconazole resistance. IMPORTANCE Fungal pathogens develop drug resistance through multiple pathways by acquiring gene mutations, increasing the copy number of genes, or altering gene expression. In this study, we attempt to understand the mechanisms of drug resistance in the recently emerged superbug, C. auris. One approach to studying this aspect is identifying various mechanisms operating in drug-resistant clinical isolates. An alternative approach is to evolve a drug-susceptible isolate in the presence of an antifungal compound and trace the changes that result in drug resistance. Here, we evolve a drug-susceptible isolate of C. auris in the laboratory in the presence of a widely used antifungal compound, fluconazole. In addition to the already known changes like overexpression of drug efflux pumps, this study identifies a novel mechanism of azole resistance by the emergence of additional chromosomes through segmental duplication of chromosomal regions, including centromeres. The centric supernumerary chromosome helps stable amplification of a set of genes with an extra copy to confer fluconazole resistance.

Trypanosoma cruzi Genomic Variability: Array Comparative Genomic Hybridization Analysis of Clone and Parental Strain.

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits extensive inter- and intrastrain genetic diversity. As we have previously described, there are some genetic differences between the parental G strain and its clone D11, which was isolated by the limiting dilution method and infection of cultured mammalian cells. Electrophoretic karyotyping and Southern blot hybridization of chromosomal bands with specific markers revealed chromosome length polymorphisms of small size with additional chromosomal bands in clone D11 and the maintenance of large syntenic groups. Both G strain and clone D11 belong to the T. cruzi lineage TcI. Here, we designed intraspecific array-based comparative genomic hybridization (aCGH) to identify chromosomal regions harboring copy-number variations between clone D11 and the G strain. DNA losses were more extensive than DNA gains in clone D11. Most alterations were flanked by repeated sequences from multigene families that could be involved in the duplication and deletion events. Several rearrangements were detected by chromoblot hybridization and confirmed by aCGH. We have integrated the information of genomic sequence data obtained by aCGH to the electrophoretic karyotype, allowing the reconstruction of possible recombination events that could have generated the karyotype of clone D11. These rearrangements may be explained by unequal crossing over between sister or homologous chromatids mediated by flanking repeated sequences and unequal homologous recombination via break-induced replication. The genomic changes detected by aCGH suggest the presence of a dynamic genome that responds to environmental stress by varying the number of gene copies and generating segmental aneuploidy.

Molecular Markers Reveal Epidemiological Patterns and Evolutionary Histories of the Human Pathogenic Cryptococcus.

The human pathogenic Cryptococcus species are the main agents of fungal meningitis in humans and the causes of other diseases collectively called cryptococcosis. There are at least eight evolutionary divergent lineages among these agents, with different lineages showing different geographic and/or ecological distributions. In this review, we describe the main strain typing methods that have been used to analyze the human pathogenic Cryptococcus and discuss how molecular markers derived from the various strain typing methods have impacted our understanding of not only cryptococcal epidemiology but also its evolutionary histories. These methods include serotyping, multilocus enzyme electrophoresis, electrophoretic karyotyping, random amplified polymorphic DNA, restriction fragment length polymorphism, PCR-fingerprinting, amplified fragment length polymorphism, multilocus microsatellite typing, single locus and multilocus sequence typing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and whole genome sequencing. The major findings and the advantages and disadvantages of each method are discussed. Together, while controversies remain, these strain typing methods have helped reveal (i) the broad phylogenetic pattern among these agents, (ii) the centers of origins for several lineages and their dispersal patterns, (iii) the distributions of genetic variation among geographic regions and ecological niches, (iv) recent hybridization among several lineages, and (v) specific mutations during infections within individual patients. However, significant challenges remain. Multilocus sequence typing and whole genome sequencing are emerging as the gold standards for continued strain typing and epidemiological investigations of cryptococcosis.

Taxonomy of the yeast genus Vanderwaltozyma and proposal of Vanderwaltozyma meishanica sp. nov., Vanderwaltozyma huisunica sp. nov., and Vanderwaltozyma molinica sp. nov.

Using electrophoretic karyotyping, RAPD fingerprinting and phylogenetic analysis of ribosomal RNA gene sequences, twenty-six Vanderwaltozyma strains were studied. Out of 19 strains isolated in mountainous areas of Taiwan, eighteen strains were isolated from soil and one strain was isolated from the fruiting body of mushroom, six were identified as V. polyspora and three as V. verrucispora. Based on the results of a multigene sequence analysis (D1/D2, ITS and mitochondrial COX II gene) and DNA-DNA reassociation, three new ascosporic members of the genus Vanderwaltozyma are formally described: V. huisunica sp. nov. (GA1S06T = CBS 12250T = BCRC 23260T), V. meishanica sp. nov. (EN4S02T = CBS 12249T = BCRC 23255T) and V. molinica sp. nov. (GJ8S05T = CBS 12251T = BCRC 23264T), and the holotypes of these novel species are assigned as BCRC 23260T, BCRC 23255T and BCRC 23264T, respectively.

Forward Genetics Approach Reveals Host Genotype-Dependent Importance of Accessory Chromosomes in the Fungal Wheat Pathogen Zymoseptoria tritici.

ABSTRACTThe fungal wheat pathogen Zymoseptoria tritici possesses a large complement of accessory chromosomes showing presence/absence polymorphism among isolates. These chromosomes encode hundreds of genes; however, their functional role and why the chromosomes have been maintained over long evolutionary times are so far not known. In this study, we addressed the functional relevance of eight accessory chromosomes in reference isolate IPO323. We induced chromosome losses by inhibiting the β-tubulin assembly during mitosis using carbendazim and generated several independent isogenic strains, each lacking one of the accessory chromosomes. We confirmed chromosome losses by electrophoretic karyotyping and whole-genome sequencing. To assess the importance of the individual chromosomes during host infection, we performed in planta assays comparing disease development results in wild-type and chromosome mutant strains. Loss of the accessory chromosomes 14, 16, 18, 19, and 21 resulted in increased virulence on wheat cultivar Runal but not on cultivars Obelisk, Titlis, and Riband. Moreover, some accessory chromosomes affected the switch from biotrophy to necrotrophy as strains lacking accessory chromosomes 14, 18, 19, and 21 showed a significantly earlier onset of necrosis than the wild type on the Runal cultivar. In general, we observed that the timing of the lifestyle switch affects the fitness of Z. tritici. Taking the results together, this study was the first to use a forward-genetics approach to demonstrate a cultivar-dependent functional relevance of the accessory chromosomes of Z. tritici during host infection.

The Value of Long-Chain Fatty Acid Analysis, Randomly Amplified Polymorphic DNA and Electrophoretic Karyotyping for the Characterization of Wine Yeast Strains

Wine yeast strains of Saccharomyces had previously been classified into several different species or varieties. This classification system was based mainly on sugar fermentation and assimilation patterns. Subsequently, most of these species were reclassified as Saccharomyces cerevisiae. The assignment of the majority of wine yeast strains to a single species does, however, not imply that all stains of S. cerevisiae are equally suitable for wine fermentation. These physiological strains of S. cerevisiae differ significantly in their fermentation performance and their ability to contribute to the final bouquet and quality of the various types of wine and distillates. Therefore, to ensure strain authenticity, security and proper strain management, it is of cardinal importance to have reliable taxonomic techniques available to identify and characterize individual strains of commercial cultures. In this study, 18 commercial wine yeast strains were characterized in order to evaluate and compare three taxonomic techniques, namely long-chain fatty acid analysis, randomly amplified polymorphic DNA (RAPD) and electrophoretic karyotyping. As a single identification technique, electrophoretic karyotyping seems to be the most useful method for routine fingerprinting of wine yeast strains. However, we propose that the combined use of these three techniques provides the most reliable means of differentiating amongst commercial wine yeast strains.

Genetic and biochemical characterization of yeasts isolated from Antarctic soil samples

The Polish Arctowski Station is situated in the maritime Antarctic on the western shore of Admiralty Bay and encompasses terrestrial habitats which are not permanently covered by ice, in contrast to more than 90% of the island’s surface area. Over the past several decades, studies exploring the soils of those habitats have revealed a considerable diversity of bacteria, filamentous fungi, and, to a lesser extent, yeasts; however, characterization of this complex microbiome, especially at the molecular level, is still far from satisfactory. The isolates were assigned to their respective genera and species based on genetic analysis of the D1/D2 and ITS1-5.8S-ITS2 regions of rDNA. In the studied soil samples, the most abundant microorganisms belonged to the genera Cryptococcus, Rhodotorula, and Debaryomyces. Physiological and biochemical analysis of Cryptococcus gilvescens (pro tempore Goffeauzyma gilvescens) and Rhodotorula mucilaginosa showed only a limited level of intraspecies diversity. Cellular DNA content and karyotypes were determined using flow cytometry and pulsed-field gel electrophoresis for several selected strains. For the first time, genome size and electrophoretic karyotypes were investigated in C. gilvescens (pro tem G. gilvescens), Cryptococcus saitoi (pro tem Naganishia globosa), Cryptococcus gastricus (pro tem Goffeauzyma gastrica), and Cryptococcus albidus (pro tem Naganishia albida). In addition, plate tests showed Antarctic yeasts to be a potential source of biotechnologically important enzymes. This study in biodiversity, presenting physiological and molecular characterization of psychrotolerant yeast strains isolated from the soils of western Admiralty Bay, contributes to a better understanding of the microbial ecology of this unique ecosystem.

Effects of sub-culturing on genetic and physiological parameters in different Beauveria bassiana isolates

Beauveria bassiana is a fungus which is widely used as a biological insecticide to control a number of economically important insect pests. Knowledge of the genetic diversity of the isolates, understanding the underlying nature of these evolutionary phenomena and finding appropriate and simple screening tools play an important role in developing effective biocontrol agents. Here, we monitored changes of electrophoretic karyotype of small molecules of extrachromosomal DNAs, presumably mitochondrial DNA or plasmids in several individual isolates of B. bassiana during the forced in vitro evolution by continual subculture on artificial media and then we evaluated these changes on the virulence of the isolates. Through agarose gel electrophoresis of the small extrachromosomal DNAs molecules, we found that mutations accumulate quickly and obvious changes take place in extrachromosomal DNAs of some isolates, although this did not always occur. This plasticity in response to culturing pressure suggests that buffering capacity of fungal genome against mutations is isolate dependent. Following the forced evolution by sub-culturing, five discriminable electrophoretic karyotype of extrachromosomal DNAs was observed among isolates. The results showed that some isolates are prone to deep mutations, but during enforced sub-culturing some others have efficiently conserved genome. These differences are influensive in screening appropriate isolates for mass production as a keystone in biocontrol program. To determine the effects of these changes on isolate traits, virulence, germination rate and spore-bound Pr1 activity were assessed parallel to sub-culturing. The results clearly revealed that parallel to sub-culturing and in correlation with karyotypic changes, isolates significantly suffered from virulence, germination rate and spore-bound Pr1 activity deficiencies.

Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

Seasonal Variation of Indigenous Saccharomyces cerevisiae Strains Isolated from Vineyards of the Western Cape in South Africa

There is strong support for the use of naturally-occurring Saccharomyces cerevisiae strains that improve the sensory quality of wines and reflect the characteristics of a given region. Contrary to popular belief, S. cerevisiae is found at very low numbers on healthy, undamaged grapes and is rarely isolated from intact berries. The majority of studies on the population kinetics and geographic distribution of indigenous S. cerevisiae strains have not adequately focused on the variation in their numbers over a longer period of time. This paper discusses the results obtained in the first phase of a comprehensive research programme aimed at assessing how the natural population dynamics of S. cerevisiae are affected over the long term by abiotic factors. Indigenous strains of S. cerevisiae were aseptically isolated from eight sites in four areas in the coastal regions of the Western Cape, South Africa, during 1995 through 1998. Thirty colonies per site were isolated and the S. cerevisiae strains were characterised by electrophoretic karyotyping. Strain numbers per site varied over the four-year study period. Weather conditions resulting in severe fungal infestations and heavy applications of chemical sprays dramatically reduced the numbers of S. cerevisiae strains recovered during 1997. A return to normal weather patterns in 1998 resulted in a gradual recovery of the indigenous population. Indications are that some of the strains isolated are widespread in the study area and may represent yeasts typical of the area. Commercial wine yeast strains were recovered in only a few instances and the likelihood that commercial yeasts will eventually replace the natural yeast microflora in vineyards seems remote.

Lachancea quebecensis sp. nov., a yeast species consistently isolated from tree bark in the Canadian province of Québec.

A thorough sampling of maple, oak, birch, and apple tree bark in North America yielded a set of isolates that represent a yeast species not yet formally described. The strains obtained were all isolated from the Canadian province of Québec. These four isolates have identical electrophoretic karyotypes, distinct from other species of the genus Lachancea, and are most closely related to the formally recognized species Lachancea thermotolerans according to the D1/D2 domain of the LSU rDNA gene and 5.8S–ITS region. Previous studies revealed the existence of a population of strains closely related to L. thermotolerans, with unique D1/D2 sequences and the ability to grow on melibiose, which is also true for these isolates. The sequences obtained here (for the D1/D2, and 5.8S–ITS region) are identical among the four strains, and in a phylogenetic analysis of the D1/D2 region, the strains form a distinct clade with the previously described population closely related to L. thermotolerans, composed of isolates from Japan, as well as from the provinces of Ontario and Québec in Canada. On the basis of select physiological and phylogenetic characteristics, a novel ascosporogenous yeast species, Lachancea quebecensis sp. nov., is proposed. The type strain LL11_022T ( = CBS 14138T = CLIB 1763T = UCDFST 15-106T) was isolated from maple tree bark in the Station Duchesnay, QC region of Québec, Canada. The MycoBank number is MB811749.

Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts.

Industrial yeasts, economically important microorganisms, are widely used in diverse biotechnological processes including brewing, winemaking and distilling. In contrast to a well-established genome of brewer's and wine yeast strains, the comprehensive evaluation of genomic features of distillery strains is lacking. In the present study, twenty two distillery yeast strains were subjected to electrophoretic karyotyping and array-based comparative genomic hybridization (array-CGH). The strains analyzed were assigned to the Saccharomyces sensu stricto complex and grouped into four species categories: S. bayanus, S. paradoxus, S. cerevisiae and S. kudriavzevii. The genomic diversity was mainly revealed within subtelomeric regions and the losses and/or gains of fragments of chromosomes I, III, VI and IX were the most frequently observed. Statistically significant differences in the gene copy number were documented in six functional gene categories: 1) telomere maintenance via recombination, DNA helicase activity or DNA binding, 2) maltose metabolism process, glucose transmembrane transporter activity; 3) asparagine catabolism, cellular response to nitrogen starvation, localized in cell wall-bounded periplasmic space, 4) siderophore transport, 5) response to copper ion, cadmium ion binding and 6) L-iditol 2- dehydrogenase activity. The losses of YRF1 genes (Y' element ATP-dependent helicase) were accompanied by decreased level of Y' sequences and an increase in DNA double and single strand breaks, and oxidative DNA damage in the S. paradoxus group compared to the S. bayanus group. We postulate that naturally occurring diversity in the YRF1 gene copy number may promote genetic stability in the S. bayanus group of distillery yeast strains.

Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris.

BackgroundCandida auris is a multidrug resistant, emerging agent of fungemia in humans. Its actual global distribution remains obscure as the current commercial methods of clinical diagnosis misidentify it as C. haemulonii. Here we report the first draft genome of C. auris to explore the genomic basis of virulence and unique differences that could be employed for differential diagnosis.ResultsMore than 99.5 % of the C. auris genomic reads did not align to the current whole (or draft) genome sequences of Candida albicans, Candida lusitaniae, Candida glabrata and Saccharomyces cerevisiae; thereby indicating its divergence from the active Candida clade. The genome spans around 12.49 Mb with 8527 predicted genes. Functional annotation revealed that among the sequenced Candida species, it is closest to the hemiascomycete species Clavispora lusitaniae. Comparison with the well-studied species Candida albicans showed that it shares significant virulence attributes with other pathogenic Candida species such as oligopeptide transporters, mannosyl transfersases, secreted proteases and genes involved in biofilm formation. We also identified a plethora of transporters belonging to the ABC and major facilitator superfamily along with known MDR transcription factors which explained its high tolerance to antifungal drugs.ConclusionsOur study emphasizes an urgent need for accurate fungal screening methods such as PCR and electrophoretic karyotyping to ensure proper management of fungemia. Our work highlights the potential genetic mechanisms involved in virulence and pathogenicity of an important emerging human pathogen namely C. auris. Owing to its diversity at the genomic scale; we expect the genome sequence to be a useful resource to map species specific differences that will help develop accurate diagnostic markers and better drug targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1863-z) contains supplementary material, which is available to authorized users.

Electrophoretic karyotyping of Hypsizygus marmoreus and evaluation of variation among its basidiospores.

The molecular karyotype of Hypsizygus marmoreus was explored by contour-clamped homogeneous electric field gel electrophoresis. Eleven chromosomal bands were separated from the dikaryotic mycelia of H.marmoreus (strain Hm 3-10), and the chromosomes ranged in size from 1.9 to 5.8Mb. The total genome size of the strain was estimated to be 36.3Mb. The chromosome numbers were also confirmed by telomere fingerprinting, and 22 telomeric bands were identified. This result suggests that 11 chromosomes exist in Hm 3-10. The marker sequences for each chromosome were determined and were applied to identify each chromosome. Karyotyping and Southern blot analysis revealed that the size of chromosomes in the basidiospores were greatly different from those of parental dikaryon Hm 3-10 cells.

English

Candida is a medically important fungi because of its high frequency as a commensal and pathogenic microorganism causing superficial as well as invasive infections. Because the accurate diagnosis of candidiasis remains difficult, a fast and reliable assay for characterization of fungal pathogens is critical for the early initiation of adequate antifungal therapy and/or for introduction of preventive measures. As novel molecular genetic techniques are continuously introduced, their role in the management of infectious diseases has also been growing .Strain typing and delineation of the species are essential for understanding its biology, epidemiology and population structure. A wide range of molecular techniques have been used for this purpose including non-DNA-based methods (multi-locus enzyme electrophoresis), conventional DNA-based methods (electrophoretic karyotyping, random amplified polymorphic DNA, amplified fragment length polymorphism, restriction enzyme analysis with and without hybridization, rep-PCR) and DNA-based methods called exact typing (PCR based methods, microstallite length polymorphism, multilocus sequence typing, DNA microarray ) because they generate unambiguous and highly reproducible typing data. Today, molecular strategies complement conventional methods and provide more accurate and detailed insight. It can be expected that future technical development will improve their potential furthermore.In this article, we provide a critical review on the vigorously fermenting field of molecular approaches to Candida identification and typing and summarized their advantages and limitations with regard to their discriminatory power, reproducibility, cost and ease of performance.

Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting

BackgroundMeyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes.ResultsWe targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping.ConclusionsWe herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used as a reliable tool for rapid and accurate identification of closely related species of M. guilliermondii complex and for differentiating emerging infectious yeasts of the Saccharomycotina CTG clade.

Breakage-fusion-bridge Cycles and Large Insertions Contribute to the Rapid Evolution of Accessory Chromosomes in a Fungal Pathogen

Chromosomal rearrangements are a major driver of eukaryotic genome evolution, affecting speciation, pathogenicity and cancer progression. Changes in chromosome structure are often initiated by mis-repair of double-strand breaks in the DNA. Mis-repair is particularly likely when telomeres are lost or when dispersed repeats misalign during crossing-over. Fungi carry highly polymorphic chromosomal complements showing substantial variation in chromosome length and number. The mechanisms driving chromosome polymorphism in fungi are poorly understood. We aimed to identify mechanisms of chromosomal rearrangements in the fungal wheat pathogen Zymoseptoria tritici. We combined population genomic resequencing and chromosomal segment PCR assays with electrophoretic karyotyping and resequencing of parents and offspring from experimental crosses to show that this pathogen harbors a highly diverse complement of accessory chromosomes that exhibits strong global geographic differentiation in numbers and lengths of chromosomes. Homologous chromosomes carried highly differentiated gene contents due to numerous insertions and deletions. The largest accessory chromosome recently doubled in length through insertions totaling 380 kb. Based on comparative genomics, we identified the precise breakpoint locations of these insertions. Nondisjunction during meiosis led to chromosome losses in progeny of three different crosses. We showed that a new accessory chromosome emerged in two viable offspring through a fusion between sister chromatids. Such chromosome fusion is likely to initiate a breakage-fusion-bridge (BFB) cycle that can rapidly degenerate chromosomal structure. We suggest that the accessory chromosomes of Z. tritici originated mainly from ancient core chromosomes through a degeneration process that included BFB cycles, nondisjunction and mutational decay of duplicated sequences. The rapidly evolving accessory chromosome complement may serve as a cradle for adaptive evolution in this and other fungal pathogens.