Genetic and environmental drivers of large-scale epigenetic variation in Thlaspi arvense.

Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being lower in colder regions and in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylated regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.

DNA methylation in the wild: epigenetic transgenerational inheritance can mediate adaptation in clones of wild strawberry (Fragaria vesca).

Due to the accelerating climate change, it is crucial to understand how plants adapt to rapid environmental changes. Such adaptation may be mediated by epigenetic mechanisms like DNA methylation, which could heritably alter phenotypes without changing the DNA sequence, especially across clonal generations. However, we are still missing robust evidence of the adaptive potential of DNA methylation in wild clonal populations. Here, we studied genetic, epigenetic and transcriptomic variation of Fragaria vesca, a predominantly clonally reproducing herb. We examined samples from 21 natural populations across three climatically distinct geographic regions, as well as clones of the same individuals grown in a common garden. We found that epigenetic variation was partly associated with climate of origin, particularly in non-CG contexts. Importantly, a large proportion of this variation was heritable across clonal generations. Additionally, a subset of these epigenetic changes affected the expression of genes mainly involved in plant growth and responses to pathogen and abiotic stress. These findings highlight the potential influence of epigenetic changes on phenotypic traits. Our findings indicate that variation in DNA methylation, which can be environmentally inducible and heritable, may enable clonal plant populations to adjust to their environmental conditions even in the absence of genetic adaptation.

Comprehensive benchmarking of software for mapping whole genome bisulfite data: from read alignment to DNA methylation analysis.

Whole genome bisulfite sequencing is currently at the forefront of epigenetic analysis, facilitating the nucleotide-level resolution of 5-methylcytosine (5mC) on a genome-wide scale. Specialized software have been developed to accommodate the unique difficulties in aligning such sequencing reads to a given reference, building on the knowledge acquired from model organisms such as human, or Arabidopsis thaliana. As the field of epigenetics expands its purview to non-model plant species, new challenges arise which bring into question the suitability of previously established tools. Herein, nine short-read aligners are evaluated: Bismark, BS-Seeker2, BSMAP, BWA-meth, ERNE-BS5, GEM3, GSNAP, Last and segemehl. Precision-recall of simulated alignments, in comparison to real sequencing data obtained from three natural accessions, reveals on-balance that BWA-meth and BSMAP are able to make the best use of the data during mapping. The influence of difficult-to-map regions, characterized by deviations in sequencing depth over repeat annotations, is evaluated in terms of the mean absolute deviation of the resulting methylation calls in comparison to a realistic methylome. Downstream methylation analysis is responsive to the handling of multi-mapping reads relative to mapping quality (MAPQ), and potentially susceptible to bias arising from the increased sequence complexity of densely methylated reads.

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional bayesian approaches.

BACKGROUND: Calling germline SNP variants from bisulfite-converted sequencing data poses a challenge for conventional software, which have no inherent capability to dissociate true polymorphisms from artificial mutations induced by the chemical treatment. Nevertheless, SNP data is desirable both for genotyping and to understand the DNA methylome in the context of the genetic background. The confounding effect of bisulfite conversion however can be conceptually resolved by observing differences in allele counts on a per-strand basis, whereby artificial mutations are reflected by non-complementary base pairs. RESULTS: Herein, we present a computational pre-processing approach for adapting sequence alignment data, thus indirectly enabling downstream analysis on a per-strand basis using conventional variant calling software such as GATK or Freebayes. In comparison to specialised tools, the method represents a marked improvement in precision-sensitivity based on high-quality, published benchmark datasets for both human and model plant variants. CONCLUSION: The presented "double-masking" procedure represents an open source, easy-to-use method to facilitate accurate variant calling using conventional software, thus negating any dependency on specialised tools and mitigating the need to generate additional, conventional sequencing libraries alongside bisulfite sequencing experiments. The method is available at https://github.com/bio15anu/revelio and an implementation with Freebayes is available at https://github.com/EpiDiverse/SNP.

Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates.

Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high-quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe non-coding RNAs, pseudogenes and transposable elements, and highlight tissue-specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome-wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

Extended lumbar drainage in idiopathic normal pressure hydrocephalus: a systematic review and meta-analysis of diagnostic test accuracy.

BACKGROUND: When appropriately selected, a high proportion of patients with suspected idiopathic normal pressure hydrocephalus (iNPH) will respond to cerebrospinal fluid diversion with a shunt. Extended lumbar drainage (ELD) is regarded as the most accurate test for this condition, however, varying estimates of its accuracy are found in the current literature. Here, we review the literature in order to provide summary estimates of sensitivity, specificity, positive- and negative predictive value for this test through meta-analysis of suitably rigorous studies. METHODS: Studies involving a population of NPH patients with predominantly idiopathic aetiology (>80%) in which the intention of the study was to shunt patients regardless of the outcome of ELD were included in the review. Various literature databases were searched to identify diagnostic test accuracy studies addressing ELD in the diagnosis of iNPH. Those studies passing screening and eligibility were assessed using the QUADAS-2 tool and data extracted for bivariate random effects meta-analysis. RESULTS: Four small studies were identified. They showed disparate results concerning diagnostic test accuracy. The summary estimates for sensitivity and specificity were 94% (CI 41-100%) and 85% (CI 33-100%), respectively. The summary estimates of positive and negative predictive value were both 90% (CIs 65-100% and 48-100%, respectively). CONCLUSION: Large, rigorous studies addressing the diagnostic accuracy of ELD are lacking, and little robust evidence exists to support the use of ELD in diagnostic algorithms for iNPH. Therefore, a large cohort study, or ideally an RCT, is needed to determine best practice in selecting patients for shunt surgery.

MethylScore, a pipeline for accurate and context-aware identification of differentially methylated regions from population-scale plant whole-genome bisulfite sequencing data.

Whole-genome bisulfite sequencing (WGBS) is the standard method for profiling DNA methylation at single-nucleotide resolution. Different tools have been developed to extract differentially methylated regions (DMRs), often built upon assumptions from mammalian data. Here, we present MethylScore, a pipeline to analyse WGBS data and to account for the substantially more complex and variable nature of plant DNA methylation. MethylScore uses an unsupervised machine learning approach to segment the genome by classification into states of high and low methylation. It processes data from genomic alignments to DMR output and is designed to be usable by novice and expert users alike. We show how MethylScore can identify DMRs from hundreds of samples and how its data-driven approach can stratify associated samples without prior information. We identify DMRs in the A. thaliana 1,001 Genomes dataset to unveil known and unknown genotype-epigenotype associations .

epiGBS2: Improvements and evaluation of highly multiplexed, epiGBS-based reduced representation bisulfite sequencing.

Several reduced-representation bisulfite sequencing methods have been developed in recent years to determine cytosine methylation de novo in nonmodel species. Here, we present epiGBS2, a laboratory protocol based on epiGBS with a revised and user-friendly bioinformatics pipeline for a wide range of species with or without a reference genome. epiGBS2 is cost- and time-efficient and the computational workflow is designed in a user-friendly and reproducible manner. The library protocol allows a flexible choice of restriction enzymes and a double digest. The bioinformatics pipeline was integrated in the Snakemake workflow management system, which makes the pipeline easy to execute and modular, and parameter settings for important computational steps flexible. We implemented bismark for alignment and methylation analysis and we preprocessed alignment files by double masking to enable single nucleotide polymorphism calling with Freebayes (epiFreebayes). The performance of several critical steps in epiGBS2 was evaluated against baseline data sets from Arabidopsis thaliana and great tit (Parus major), which confirmed its overall good performance. We provide a detailed description of the laboratory protocol and an extensive manual of the bioinformatics pipeline, which is publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.4764652).

The EpiDiverse Plant Epigenome-Wide Association Studies (EWAS) Pipeline.

Bisulfite sequencing is a widely used technique for determining DNA methylation and its relationship with epigenetics, genetics, and environmental parameters. Various techniques were implemented for epigenome-wide association studies (EWAS) to reveal meaningful associations; however, there are only very few plant studies available to date. Here, we developed the EpiDiverse EWAS pipeline and tested it using two plant datasets, from P. abies (Norway spruce) and Q. lobata (valley oak). Hence, we present an EWAS implementation tested for non-model plant species and describe its use.

EpiDiverse Toolkit: a pipeline suite for the analysis of bisulfite sequencing data in ecological plant epigenetics.

The expanding scope and scale of next generation sequencing experiments in ecological plant epigenetics brings new challenges for computational analysis. Existing tools built for model data may not address the needs of users looking to apply these techniques to non-model species, particularly on a population or community level. Here we present a toolkit suitable for plant ecologists working with whole genome bisulfite sequencing; it includes pipelines for mapping, the calling of methylation values and differential methylation between groups, epigenome-wide association studies, and a novel implementation for both variant calling and discriminating between genetic and epigenetic variation.

Improving Efficacy of Beauveria bassiana against Stored Grain Beetles with a Synergistic Co-Formulant.

The potential of a dry powder co-formulant, kaolin, to improve the control of storage beetles by the entomopathogenic fungus Beauveria bassiana, isolate IMI389521, was investigated. The response of Oryzaephilus surinamensis adults to the fungus when applied to wheat at 1 × 10(10) conidia per kg with and without kaolin at 1.74 g per kg wheat was assessed. Addition of kaolin increased control from 46% to 88% at day 7 and from 81% to 99% at day 14 post-treatment. Following this the dose response of O. surinamensis and Tribolium confusum to both kaolin and the fungus was investigated. Synergistic effects were evident against O. surinamensis at ≥0.96 g of kaolin per kg of wheat when combined with the fungus at all concentrations tested. For T. confusum, adult mortality did not exceed 55%, however, the larvae were extremely susceptible with almost complete suppression of adult emergence at the lowest fungal rate tested even without the addition of kaolin. Finally, the dose response of Sitophilus granarius to the fungus at 15 and 25 °C, with and without kaolin at 1 g per kg of wheat, was examined. Improvements in efficacy were achieved by including kaolin at every fungal rate tested and by increasing the temperature. Kaolin by itself was not effective, only when combined with the fungus was an effect observed, indicating that kaolin was having a synergistic effect on the fungus.

Dupuytren's contracture: emerging insight into a Viking disease.

Dupuytren's disease is a fibroproliferative condition of the palm, with a predilection for men, which has affected Northern Europeans since the Viking conquests. Although strongly heritable, clear evidence exists for environmental factors that modify the underlying genetic risk, such as diabetes, heavy drinking, and smoking. Evidence also exists for epilepsy (probably due to treatment with certain anti-epileptic drugs), and Human Immunodeficiency Virus infection. Recent large studies have shown no relationship with manual labour or vibrating tools. Two theories have emerged regarding the pathogenic mechanism: the first attributes the aberrant healing process that characterises Dupuytren's to free radicals, generated as a result of microangiopathy, whereas the second cites a genetic tendency toward apoptosis-resistant myofibroblasts. Despite only one study demonstrating linkage, emerging data from genome-wide association studies highlight a series of single nucleotide polymorphisms near members of the Wnt signalling pathway, and transcriptional profiling studies have consistently identified certain components of the extracellular matrix.

Not always asthma: clinical and legal consequences of delayed diagnosis of laryngotracheal stenosis.

Laryngotracheal stenosis (LTS) is a rare condition that occurs most commonly as a result of instrumentation of the airway but may also occur as a result of inflammatory conditions or idiopathically. Here, we present the case of a patient who developed LTS as a complication of granulomatosis with polyangiitis (GPA), which was misdiagnosed as asthma for 6 years. After an admission with respiratory symptoms that worsened to the extent that she required intubation, a previously well 14-year-old girl was diagnosed with GPA. Following immunosuppressive therapy, she made a good recovery and was discharged after 22 days. Over subsequent years, she developed dyspnoea and "wheeze" and a diagnosis of asthma was made. When she became pregnant, she was admitted to hospital with worsening respiratory symptoms, whereupon her "wheeze" was correctly identified as "stridor," and subsequent investigations revealed a significant subglottic stenosis. The delay in diagnosis precluded the use of minimally invasive therapies, with the result that intermittent laser resection and open laryngotracheal reconstructive surgery were the only available treatment options. There were numerous points at which the correct diagnosis might have been made, either by proper interpretation of flow-volume loops or by calculation of the Empey or Expiratory Disproportion Indices from spirometry data.