Comparative genomics of Dothideomycete fungi

Fungi are a diverse group of eukaryotic micro-organisms particularly suited for comparative genomics analyses. Fungi are important to industry, fundamental science and many of them are notorious pathogens of crops, thereby endangering global food supply. Dozens of fungi have been sequenced in the last decade and with the advances of the next generation sequencing, thousands of new genome sequences will become available in coming years. In this thesis I have used bioinformatics tools to study different biological and evolutionary processes in various genomes with a focus on the genomes of the Dothideomycetefungi Cladosporium fulvum, Dothistroma septosporumand Zymoseptoria tritici. Chapter 1introduces the scientific disciplines of mycology and bioinformatics from a historical perspective. It exemplifies a typical whole-genome sequence analysis of a fungal genome, and focusses in particular on structural gene annotation and detection of transposable elements. In addition it shortly reviews the microRNA pathway as known in animal and plants in the context of the putative existence of similar yet subtle different small RNA pathways in other branches of the eukaryotic tree of life. Chapter 2addresses the novel sequenced genomes of the closely related Dothideomyceteplant pathogenic fungi Cladosporium fulvumand Dothistroma septosporum. Remarkably, it revealed occurrence of a surprisingly high similarity at the protein level combined with striking differences at the DNA level, gene repertoire and gene expression. Most noticeably, the genome of C. fulvumappears to be at least twice as large, which is solely attributable to a much larger content in repetitive sequences. Chapter 3describes a novel alignment-based fungal gene prediction method (ABFGP) that is particularly suitable for plastic genomes like those of fungi. It shows excellent performance benchmarked on a dataset of 7,000 unigene-supported gene models from ten different fungi. Applicability of the method was shown by revisiting the annotations of C. fulvumand D. septosporumand of various other fungal genomes from the first-generation sequencing era. Thousands of gene models were revised in each of the gene catalogues, indeed revealing a correlation to the quality of the genome assembly, and to sequencing strategies used in the sequencing centres, highlighting different types of errors in different annotation pipelines. Chapter 4focusses on the unexpected high number of gene models that were identified by ABFGP that align nicely to informant genes, but only upon toleration of frame shifts and in-frame stop-codons. These discordances could represent sequence errors (SEs) and/or disruptive mutations (DMs) that caused these truncated and erroneous gene models. We revisited the same fungal gene catalogues as in chapter 3, confirmed SEs by resequencing and successively removed those, yielding a high-confidence and large dataset of nearly 1,000 pseudogenes caused by DMs. This dataset of fungal pseudogenes, containing genes listed as bona fide genes in current gene catalogues, does not correspond to various observations previously done on fungal pseudogenes. Moreover, the degree of pseudogenization showing up to a ten-fold variation for the lowest versus the highest affected species, is generally higher in species that reproduce asexually compared to those that in addition reproduce sexually. Chapter 5describes explorative genomics and comparative genomics analyses revealing the presence of introner-like elements (ILEs) in various Dothideomycetefungi including Zymoseptoria triticiin which they had not identified yet, although its genome sequence is already publicly available for several years. ILEs combine hallmark intron properties with the apparent capability of multiplying themselves as repetitive sequence. ILEs strongly associate with events of intron gain, thereby delivering in silico proof of their mobility. Phylogenetic analyses at the intra- and inter-species level showed that most ILEs are related and likely share common ancestry. Chapter 6provides additional evidence that ILE multiplication strongly dominates over other types of intron duplication in fungi. The observed high rate of ILE multiplication followed by rapid sequence degeneration led us to hypothesize that multiplication of ILEs has been the major cause and mechanism of intron gain in fungi, and we speculate that this could be generalized to all eukaryotes. Chapter 7describes a new strategy for miRNA hairpin prediction using statistical distributions of observed biological variation of properties (descriptors) of known miRNA hairpins. We show that the method outperforms miRNA prediction by previous, conventional methods that usually apply threshold filtering. Using this method, several novel candidate miRNAs were assigned in the genomes of Caenorhabditis elegansand two human viruses. Although this chapter is not applied on fungi, the study does provide a flexible method to find evidence for existence of a putative miRNA-like pathway in fungi. Chapter 8provides a general discussion on the advent of bioinformatics in mycological research and its implications. It highlights the necessity of a prioriplanning and integration of functional analysis and bioinformatics in order to achieve scientific excellence, and describes possible scenarios for the near future of fungal (comparative) genomics research. Moreover, it discusses the intrinsic error rate in large-scale, automatically inferred datasets and the implications of using and comparing those.</p

An early warning indicator for atmospheric blocking events using transfer operators

The existence of persistent midlatitude atmospheric flow regimes with time-scales larger than 5-10 days and indications of preferred transitions between them motivates to develop early warning indicators for such regime transitions. In this paper, we use a hemispheric barotropic model together with estimates of transfer operators on a reduced phase space to develop an early warning indicator of the zonal to blocked flow transition in this model. It is shown that, the spectrum of the transfer operators can be used to study the slow dynamics of the flow as well as the non-Markovian character of the reduction. The slowest motions are thereby found to have time scales of three to six weeks and to be associated with meta-stable regimes (and their transitions) which can be detected as almost-invariant sets of the transfer operator. From the energy budget of the model, we are able to explain the meta-stability of the regimes and the existence of preferred transition paths. Even though the model is highly simplified, the skill of the early warning indicator is promising, suggesting that the transfer operator approach can be used in parallel to an operational deterministic model for stochastic prediction or to assess forecast uncertainty

Novel Introner-Like Elements in fungi are involved in parallel gains of spliceosomal introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom

Wavelength dependent collective effects in the multiphoton ionization of atomic deuterium

This paper presents the results of an experimental investigation into collective effects in the transient plasma formed by multiphoton ionization of atomic deuterium with a pulsed laser. The laser wavelength is varied in a narrow range around 243 nm, so that the photoionization is resonant with the metastable 2S1/2 state. The ion yield, the ion time-of-flight spectra, and the yield of Lyman-a photons have been measured as a function of laser intensity ~from 1 to 340 MW/cm2! and laser detuning around the 1S1/2-2S1/2 two-photon resonance. During and shortly after the laser pulse, collective effects resulting from the mutual interaction of the photoelectrons and the ions affect the spatial and temporal distribution of the ions. Because of the near-degeneracy of the 2S1/2, 2P1/2 , and 2P3/2 states, the resonant multiphoton ionization is affected by the Stark mixing of these states in the collective field. As a result, the time-dependent yields of ions and of Lyman-a photons are modulated by the interplay of the multiphoton ionization of the atoms and the collective effects in the plasma. From the measurements it is deduced that collective effects are important above a critical charge density of 33108 ions/cm3. An asymmetry is observed in the line profile of the total ion yield as a function of laser detuning. This asymmetry is interpreted to be due to the effect of the collective field upon the intermediate resonant 2S1/2 state of the photoionization process

Wavelength dependent collective effects in the multiphoton ionization of atomic deuterium

This paper presents the results of an experimental investigation into collective effects in the transient plasma formed by multiphoton ionization of atomic deuterium with a pulsed laser. The laser wavelength is varied in a narrow range around 243 nm, so that the photoionization is resonant with the metastable 2S1/2 state. The ion yield, the ion time-of-flight spectra, and the yield of Lyman-a photons have been measured as a function of laser intensity ~from 1 to 340 MW/cm2! and laser detuning around the 1S1/2-2S1/2 two-photon resonance. During and shortly after the laser pulse, collective effects resulting from the mutual interaction of the photoelectrons and the ions affect the spatial and temporal distribution of the ions. Because of the near-degeneracy of the 2S1/2, 2P1/2 , and 2P3/2 states, the resonant multiphoton ionization is affected by the Stark mixing of these states in the collective field. As a result, the time-dependent yields of ions and of Lyman-a photons are modulated by the interplay of the multiphoton ionization of the atoms and the collective effects in the plasma. From the measurements it is deduced that collective effects are important above a critical charge density of 33108 ions/cm3. An asymmetry is observed in the line profile of the total ion yield as a function of laser detuning. This asymmetry is interpreted to be due to the effect of the collective field upon the intermediate resonant 2S1/2 state of the photoionization process

The principle of a virtual multi-channel lock-in amplifier and its application to magnetoelectric measurement system

This letter presents principles and applications of a virtual multi-channel lock-in amplifier that is a simple but effective method to recover small ac signal from noise with high presison. The fundamentals of this method are based on calculation of cross-correlation function. Via this method, we successfully built up a magnetoelectric measurement system which can perform precise and versatile measurements without any analog lock-in amplifier. Using the virtual multi-channel lock-in amplifier, the output of the magnetoelectric measurement system is extensively rich in magnetoelectric coupling behaviors, including coupling strength and phase lag, under various dc bias magnetic field and ac magnetic field.Comment: 11 pages, 6 figures. To be submitted to Rev. Sci. Instr

Polarization Correlation Measurements of Electron Impact Excitation of Hs2pd at 54.4 eV

First direct measurements are reported of the linear reduced Stokes parameters P1,P2 for H(2p) excited by electron impact at the benchmark energy of 54.4 eV. The results differ significantly from previous values deduced from angular correlation measurements which are in serious conflict with all sophisticated theoretical approaches. Our results support the trend of theoretical predictions for P2 and confirm that its value is negative at electron scattering angles above 100±, as predicted by theory