Application of flow cytometry for genome size determination in <i>Geosmithia</i> fungi: A comparison of methods

Veselská, Tereza; Svoboda, Jan; Růžičková, Žaneta; Kolařík, Miroslav

doi:10.1002/cyto.a.22500

Cited by 12 publications

(10 citation statements)

References 62 publications

(78 reference statements)

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“…Flow cytometry is one of the most effective methods for the estimation of the genome size of eukaryotes and has largely replaced cytological methods. However, the adaptation of this technique to the analysis of fungal genome size is in some cases a challenging task [ 27 , 28 ]. So far, we were unsuccessful to apply flow cytometry to D .…”

Section: Resultsmentioning

confidence: 99%

A draft genome sequence of the rose black spot fungus Diplocarpon rosae reveals a high degree of genome duplication

et al. 2017

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BackgroundBlack spot is one of the most severe and damaging diseases of garden roses. We present the draft genome sequence of its causative agent Diplocarpon rosae as a working tool to generate molecular markers and to analyze functional and structural characteristics of this fungus.ResultsThe isolate DortE4 was sequenced with 191x coverage of different read types which were assembled into 2457 scaffolds. By evidence supported genome annotation with the MAKER pipeline 14,004 gene models were predicted and transcriptomic data indicated that 88.5% of them are expressed during the early stages of infection. Analyses of k-mer distributions resulted in unexpectedly large genome size estimations between 72.5 and 91.4 Mb, which cannot be attributed to its repeat structure and content of transposable elements alone, factors explaining such differences in other fungal genomes. In contrast, different lines of evidences demonstrate that a huge proportion (approximately 80%) of genes are duplicated, which might indicate a whole genome duplication event. By PCR-RFLP analysis of six paralogous gene pairs of BUSCO orthologs, which are expected to be single copy genes, we could show experimentally that the duplication is not due to technical error and that not all isolates tested possess all of the paralogs.ConclusionsThe presented genome sequence is still a fragmented draft but contains almost the complete gene space. Therefore, it provides a useful working tool to study the interaction of D. rosae with the host and the influence of a genome duplication outside of the model yeast in the background of a phytopathogen.

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Section: Resultsmentioning

confidence: 99%

A draft genome sequence of the rose black spot fungus Diplocarpon rosae reveals a high degree of genome duplication

et al. 2017

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“…Flow cytometry was carried out on isolated nuclei by the method described in Veselská et al () with a few modifications, using Aspergillus fumigatus CEA10 with a GS of 29.2 Mb (Fedorova et al, ) as an external standard for GS calculations. Lichen apothecia or sterile mycelium of A. fumigatus were fixed in methanol: acetic acid (3: 1 v/v), 10% DMSO, 0.1% Triton X‐100 for one hour at 4 °C and then washed with 0.1% Triton X‐100.…”

Section: Methodsmentioning

confidence: 99%

Substrate switches, phenotypic innovations and allopatric speciation formed taxonomic diversity within the lichen genus Blastenia

Vondrák

Frolov

Košnar

et al. 2019

J of Sytematics Evolution

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Blastenia is a widely distributed lichen genus in Teloschistaceae. We reconstructed its phylogeny in order to test species delimitation and to find evolutionary drivers forming recent Blastenia diversity. The origin of Blastenia is dated to the early Tertiary period, but later diversification events are distinctly younger. We recognized 24 species (plus 2 subspecies) within 6 infrageneric groups. Each species strongly prefers a single type of substrate (17 species occur on organic substrates, 7 on siliceous rock), and most infrageneric groups also show a clear substrate preference. All infrageneric groups tend to have the Mediterranean and Macaronesian distribution, but some epiphytic species have much larger geographic ranges and some evolved after a long-distance dispersal outside the region. Chlorinated and nonchlorinated anthraquinone chemosyndromes co-occur in apothecia of most species, but the chemotype has been secondarily reduced in some lineages. One infrageneric group has a marked reduction in apothecial size, associated with a substrate shift to twigs. Only seven species have vegetative diaspores; they also produce apothecia but have smaller ascospores. Genome sizes (22-35 Mb in Blastenia) are significantly higher in epilithic species. Within-species genetic variation is low in widely distributed species but high in some epilithic species with small geographical ranges. New taxa are: B. afroalpina, B. anatolica, B. caucasica, B. gennargentuae, B. herbidella subsp. acidophila, B. lauri, B. monticola, B. palmae, B. psychrophila, B. purpurea, B. relicta, B. remota, B. xerothermica, and B. xerothermica subsp. macaronesica. New combinations are: B. festivella and B. subathallina; both names and B. catalinae are lectotypified.

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“…As the nuclear content is doubled in diploid isolates compared to haploid isolates, staining the nuclear content of spores with propidium iodide (PI) and measurement by using Fluorescence-Activated Cell Sorting (FACS) technology can differentiate the ploidy level as already shown by de Lucas et al in 1998 [18]. The same selection of isolates for benomyl susceptibility testing were also subjected to FACS analysis as previously described by Veselská et al [19] with minor adaptations. Finally, as a proof of principle, a diploid isolate was haploidized resulting in reduced cell size while this is not possible for a haploid isolate.…”

Section: Confirmation Of Diploidymentioning

confidence: 99%

Parasexual recombination enablesAspergillus fumigatusto persist in cystic fibrosis

et al. 2020

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Aspergillus fumigatus is a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical for A. fumigatus to adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply in A. fumigatus due to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis of A. fumigatus isolates cultured from different origins.As diploids are the hallmark of parasex, we screened 799 A. fumigatus isolates obtained from patients with CF, chronic pulmonary lung disease, acute invasive aspergillosis and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid associated genetic variation.We identified 11 diploids in isolates recovered from six of 11 (55%) CF-patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from acute infection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate.Parasexual recombination allows A. fumigatus to adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology of A. fumigatus in the human lung.

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Application of flow cytometry for genome size determination in Geosmithia fungi: A comparison of methods

Cited by 12 publications

References 62 publications

A draft genome sequence of the rose black spot fungus Diplocarpon rosae reveals a high degree of genome duplication

A draft genome sequence of the rose black spot fungus Diplocarpon rosae reveals a high degree of genome duplication

Substrate switches, phenotypic innovations and allopatric speciation formed taxonomic diversity within the lichen genus Blastenia

Parasexual recombination enablesAspergillus fumigatusto persist in cystic fibrosis

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