DNA methylation in clonal duckweed (Lemna minor L.) lineages reflects current and historical environmental exposures.

Environmentally induced DNA methylation variants may mediate gene expression responses to environmental changes. If such induced variants are transgenerationally stable, there is potential for expression responses to persist over multiple generations. Our current knowledge in plants, however, is almost exclusively based on studies conducted in sexually reproducing species where the majority of DNA methylation changes are subject to resetting in germlines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may therefore be more conducive to long-term inheritance of epigenetic marks. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we hypothesize that long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulphite sequencing approach (epiGBS), we show that temperature stress induces DNA hypermethylation at many CG and CHG cytosine contexts but not CHH. Additionally, differential methylation in CHG context that was observed was still detected in a subset of cytosines, even after 3-12 generations of culturing in a common environment. This demonstrates a memory effect of stress reflected in the methylome and that persists over multiple clonal generations. Structural annotation revealed that this memory effect in CHG methylation was enriched in transposable elements. The observed epigenetic stress memory is probably caused by stable transgenerational persistence of temperature-induced DNA methylation variants across clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants.

Heavy metal tolerance in Scopelophila cataractae: Transcriptomic and epigenetic datasets.

Studying how different plant groups deal with heavy metal exposure is crucial to improve our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used RNA sequencing (RNA-seq) and epigenotyping by sequencing (epiGBS) to assess gene expression and DNA methylation changes respectively in plants from four populations of the metallophyte moss Scopelophila cataractae treated with Cd or Cu in the laboratory. We built RNA-seq and epiGBS sequencing libraries from control and treated samples from each population and sequenced them using Illumina HiSeq 3000 (PE-150 bp) and Illumina HiSeq X-Ten System (PE-150 bp) respectively. For the RNA-seq data, we performed a read quality filter, mapped the reads to the de novo transcriptome created with Trinity, and estimated transcript abundance for each sample. For the epiGBS data, we used a custom pipeline (https://doi.org/10.5281/zenodo.7040291) to map the reads to a de novo reference genome and performed strand-specific nucleotide (single nucleotide polymorphisms, SNPs) and methylation (single cytosine methylation polymorphisms, SMPs) variant calling. We filtered out SNPs and SMPs with low coverage within (positions with <10 sequencing reads per sample) and across samples (positions with poor representation on the full set of samples). Finally, we performed pairwise comparisons between control and treated samples from each population and identified differentially expressed genes and differentially methylated cytosines associated to heavy metal exposure. We payed particular attention to the different responses of the more and the less tolerant populations of S. cataractae. These datasets could contribute to future comparative studies of abiotic stress response across plant groups.

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).

Epigenetic effects of parasites and pesticides on captive and wild nestling birds.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ectoparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free-living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced-representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we conducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identified a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontarget effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost-effective way to characterize genome-wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects.

Epigenetic Approaches in Non-Model Plants.

Reduced representation bisulfite sequencing is an emerging methodology for evolutionary and ecological genomics and epigenomics research because it provides a cost-effective, high-resolution tool for exploration and comparative analysis of DNA methylation and genetic variation. Here we describe how digestion of genomic plant DNA with restriction enzymes, subsequent bisulfite conversion of unmethylated cytosines, and final DNA sequencing allow for the examination of genome-wide genetic and epigenetic variation in plants without the need for a reference genome. We explain how the use of several combinations of barcoded adapters for the creation of highly multiplexed libraries allows the inclusion of up to 144 different samples/individuals in only one sequencing lane.

Evidence for rapid evolution in a grassland biodiversity experiment.

In long-term grassland experiments, positive biodiversity effects on plant productivity commonly increase with time. Subsequent glasshouse experiments showed that these strengthened positive biodiversity effects persist not only in the local environment but also when plants are transferred into a common environment. Thus, we hypothesized that community diversity had acted as a selective agent, resulting in the emergence of plant monoculture and mixture types with differing genetic composition. To test our hypothesis, we grew offspring from plants that were grown for eleven years in monoculture or mixture environments in a biodiversity experiment (Jena Experiment) under controlled glasshouse conditions in monocultures or two-species mixtures. We used epiGBS, a genotyping-by-sequencing approach combined with bisulphite conversion, to provide integrative genetic and epigenetic (i.e., DNA methylation) data. We observed significant divergence in genetic and DNA methylation data according to selection history in three out of five perennial grassland species, namely Galium mollugo, Prunella vulgaris and Veronica chamaedrys, with DNA methylation differences mostly reflecting the genetic differences. In addition, current diversity levels in the glasshouse had weak effects on epigenetic variation. However, given the limited genome coverage of the reference-free bisulphite method epiGBS, it remains unclear how much of the differences in DNA methylation was independent of underlying genetic differences. Our results thus suggest that selection of genetic variants, and possibly epigenetic variants, caused the rapid emergence of monoculture and mixture types within plant species in the Jena Experiment.

Epigenetic population differentiation in field- and common garden-grown Scabiosa columbaria plants.

Populations often differ in phenotype and these differences can be caused by adaptation by natural selection, random neutral processes, and environmental responses. The most straightforward way to divide mechanisms that influence phenotypic variation is heritable variation and environmental-induced variation (e.g., plasticity). While genetic variation is responsible for most heritable phenotypic variation, part of this is also caused by nongenetic inheritance. Epigenetic processes may be one of the underlying mechanisms of plasticity and nongenetic inheritance and can therefore possibly contribute to heritable differences through drift and selection. Epigenetic variation may be influenced directly by the environment, and part of this variation can be transmitted to next generations. Field screenings combined with common garden experiments will add valuable insights into epigenetic differentiation, epigenetic memory and can help to reveal part of the relative importance of epigenetics in explaining trait variation. We explored both genetic and epigenetic diversity, structure and differentiation in the field and a common garden for five British and five French Scabiosa columbaria populations. Genetic and epigenetic variation was subsequently correlated with trait variation. Populations showed significant epigenetic differentiation between populations and countries in the field, but also when grown in a common garden. By comparing the epigenetic variation between field and common garden-grown plants, we showed that a considerable part of the epigenetic memory differed from the field-grown plants and was presumably environmentally induced. The memory component can consist of heritable variation in methylation that is not sensitive to environments and possibly genetically based, or environmentally induced variation that is heritable, or a combination of both. Additionally, random epimutations might be responsible for some differences as well. By comparing epigenetic variation in both the field and common environment, our study provides useful insight into the environmental and genetic components of epigenetic variation.

Recent and dynamic transposable elements contribute to genomic divergence under asexuality.

BACKGROUND: Transposable elements (TEs) are mobile pieces of genetic information with high mutagenic potential for the host genome. Transposition is often neutral or deleterious but may also generate potentially adaptive genetic variation. This additional source of variation could be especially relevant in non-recombining species reproducing asexually. However, evidence is lacking to determine the relevance of TEs in plant asexual genome evolution and their associated effects. Here, we characterize the repetitive fraction of the genome of the common dandelion, Taraxacum officinale and compare it between five accessions from the same apomictic lineage. The main objective of this study is to evaluate the extent of within-lineage divergence attributed to TE content and activity. We examined the repetitive genomic contribution, diversity, transcription and methylation changes to characterize accession-specific TEs. RESULTS: Using low-coverage genomic sequencing, we report a highly heterogeneous TE compartment in the triploid apomict T. officinale representing up to 38.6 % of the homoploid genome. The repetitive compartment is dominated by LTR retrotransposon families accompanied by few non-LTR retrotransposons and DNA transposons. Up to half of the repeat clusters are biased towards very high read identity, indicating recent and potentially ongoing activity of these TE families. Interestingly, the five accessions are divided into two main clades based on their TE composition. Clade 2 is more dynamic than clade 1 with higher abundance of Gypsy Chromovirus sequences and transposons. Furthermore, a few low-abundant genomic TE clusters exhibit high level of transcription in two of the accessions analysed. Using reduced representation bisulfite sequencing, we detected 18.9 % of loci differentially methylated, of which 25.4 and 40.7 % are annotated as TEs or functional genes, respectively. Additionally, we show clear evidence for accession-specific TE families that are differentially transcribed and differentially methylated within the apomictic lineage, including one Copia Ale II LTR element and a PIF-Harbinger DNA transposon. CONCLUSION: We report here a very young and dynamic repetitive compartment that enhances divergence within one asexual lineage of T. officinale. We speculate that accession-specific TE families that are both transcriptionally and epigenetically variable are more prone to trigger changes in expression on nearby coding sequences. These findings emphasize the potential of TE-induced mutations on functional genes during asexual genome evolution.

epiGBS: reference-free reduced representation bisulfite sequencing.

We describe epiGBS, a reduced representation bisulfite sequencing method for cost-effective exploration and comparative analysis of DNA methylation and genetic variation in hundreds of samples de novo. This method uses genotyping by sequencing of bisulfite-converted DNA followed by reliable de novo reference construction, mapping, variant calling, and distinction of single-nucleotide polymorphisms (SNPs) versus methylation variation (software is available at https://github.com/thomasvangurp/epiGBS). The output can be loaded directly into a genome browser for visualization and into RnBeads for analysis of differential methylation.

Evidence for an epigenetic role in inbreeding depression.

Inbreeding depression (i.e. negative fitness effects of inbreeding) is central in evolutionary biology, affecting numerous aspects of population dynamics and demography, such as the evolution of mating systems, dispersal behaviour and the genetics of quantitative traits. Inbreeding depression is commonly observed in animals and plants. Here, we demonstrate that, in addition to genetic processes, epigenetic processes may play an important role in causing inbreeding effects. We compared epigenetic markers of outbred and inbred offspring of the perennial plant Scabiosa columbaria and found that inbreeding increases DNA methylation. Moreover, we found that inbreeding depression disappears when epigenetic variation is modified by treatment with a demethylation agent, linking inbreeding depression firmly to epigenetic variation. Our results suggest an as yet unknown mechanism for inbreeding effects and demonstrate the importance of evaluating the role of epigenetic processes in inbreeding depression.

Genomic toolboxes for conservation biologists.

Conservation genetics is expanding its research horizon with a genomic approach, by incorporating the modern techniques of next-generation sequencing (NGS). Application of NGS overcomes many limitations of conservation genetics. First, NGS allows for genome-wide screening of markers, which may lead to a more representative estimation of genetic variation within and between populations. Second, NGS allows for distinction between neutral and non-neutral markers. By screening populations on thousands of single nucleotide polymorphism markers, signals of selection can be found for some markers. Variation in these markers will give insight into functional rather than neutral genetic variation. Third, NGS facilitates the study of gene expression. Conservation genomics will increase our insight in how the environment and genes interact to affect phenotype and fitness. In addition, the NGS approach opens a way to study processes such as inbreeding depression and local adaptation mechanistically. Conservation genetics programs are directed to a fundamental understanding of the processes involved in conservation genetics and should preferably be started in species for which large databases on ecology, demography and genetics are available. Here, we describe and illustrate the connection between the application of NGS technologies and the research questions in conservation. The perspectives of conservation genomics programs are also discussed.

Long-term submergence-induced elongation in Rumex palustris requires abscisic acid-dependent biosynthesis of gibberellin1.

Rumex palustris (polygonceae) responds to complete submergence with enhanced elongation of its youngest petioles. This process requires the presence of gibberellin (GA) and is associated with an increase in the concentration of GA1 in elongating petioles. We have examined how GA biosynthesis was regulated in submerged plants. Therefore, cDNAs encoding GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, and the GA-deactivating enzyme GA 2-oxidase were cloned from R. palustris and the kinetics of transcription of the corresponding genes was determined during a 24 h submergence period. The submergence-induced elongation response could be separated into several phases: (1) during the first phase of 4 h, petiole elongation was insensitive to GA; (2) from 4 to 6 h onward growth was limited by GA; and (3) from 15 h onward underwater elongation was dependent, but not limited by GA. Submergence induced an increase of GA1 concentration, as well as enhanced transcript levels of RpGA3ox1. Exogenous abscisic acid repressed the transcript levels of RpGA20ox1 and RpGA3ox1 and thus inhibited the submergence-induced increase in GA1. Abscisic acid had no effect on the tissue responsiveness to GA.