Periodic Reporting for period 2 - EPIDIVERSE (Epigenetic Diversity in Ecology) Reporting period: 2019-09-01 to 2022-02-28 Summary of the context and overall objectives of the project Plants are the foundation of all ecosystems and they provide humans with essential services. Global environmental changes are now putting plants under increased risk, and it is important to understand the capacity of plants to adapt to this rapid changes. EpiDiverse has taken a cross-disciplinary approach to investigate the role that epigenetic mechanisms, specifically DNA methylation, play in plant adaptation. Understanding the epigenetic contribution to adaptation will help to predict species responses to global warming and can open new directions for sustainable agriculture and crop breeding. EpiDiverse brought together a consortium of academic and entrepreneurial groups to train 15 early-stage researchers (ESRs) to become a first generation of expert eco-epigeneticists, equipping them with the interdisciplinary skills - molecular, (epi)genomic, ecological and bioinformatics - to successfully tackle this new research area. Objectives of EpiDiverse were: (1) Optimize genomic and bioinformatic tools to enable high-resolution DNA methylation analysis in non-model plant species; (2) determine natural epigenetic diversity, and its relationship with environmental factors, climate, and phenotypes, in three common wild plant species across Europe; and (3) characterize the molecular basis and functional consequences of epigenetic responses to specific environmental factors. This approach is innovative and timely because it integrates several research areas that have not been optimally connected thus far. And it transfers analytical methods from laboratory research to the real world.The action was successful in developing a range of tools that facilitate epigenomic analysis, including plant reference genomes and novel software and best-practices analysis pipelines. Applying these tools to three focal plant species (Black poplar, Woodland strawberry and Field pennycress) generated novel insights in the nature of the epigenetic response to biotic and abiotic environments. The EpiDiverse research showed that this response differs between environments and plant species. It unraveled genetic versus environmental determinants of epigenetic variation in natural plant populations; and it revealed that the stability and dynamics of epigenetic variation differ between plant species with different reproduction strategies. Importantly, EpiDiverse trained a cohort of young researchers in a truly cross-disciplinary way. The EpiDiverse approach to let ESRs from different research domains (bioinformatics, ecology, molecular biology) collaborate on individual case studies generated much interaction between domains. It became easy for the ESRs to cross between these domains. This gave the ESRs an interdisciplinary research perspective on the field. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The ‘Genomes and Bioinformatics’ work package laid the methodological foundation for epigenomic analysis in the EpiDiverse study species. This work package generated an annotated reference genome for Black poplar and contributed to a reference genome for Field pennycress. Novel bioinformatic tools were generated for: (1) inferring genetic information from bisulfite-converted DNA samples (which are typically used for DNA methylation analysis), (2) the annotation of small RNAs and active transposable elements, (3) improving reference genomes by reconciling different assemblies, (4) optimizing DNA methylation analysis when no reference genome is available. Best-practices bioinformatic pipelines were created. These combined existing and novel tools for (1) the DNA methylation analysis using whole-genome bisulfite sequencing data (WGBS pipeline), (2) identifying genomic regions that are differentially methylated between experimental groups (DMR pipeline), and (3) finding associations between DNA methylation, climatic variation, and underlying genetic variation in samples from natural plant populations (EWAS pipeline). The 'Natural Patterns' work package applied the best-practices pipelines to natural populations of the three focal plant species, and to plant species exposed to specific ecological conditions. Experiments in this work package were based on extensive field sampling throughout Europe, subsequent common garden experiments, and large-scale DNA methylation screening. This work was supplemented in some species with genetic variation analysis and gene expression analysis. Results show in all three focus species that environmental and genetic factors impact different components of DNA methylation in plant genomes. Asexually reproducing species showed a multi-generation epigenetic memory of stress exposure. Furthermore, experimental manipulation of DNA methylation in living plants indicated that DNA methylation plays a functional role in local adaptation and in plant herbivory responses.The ‘Molecular Mechanisms’ work package characterized in detail the epigenetic response of plants when exposed to different experimental stress conditions. Many stress environments triggered only modest epigenetic responses, but some stresses (specifically drought and heat) triggered very strong responses. Some of these epigenetic changes were clonally propagated for three generations, but the functional consequences of this transgenerational epigenetic memory were relatively weak. EpiDiverse organized four summer schools as the basis of the ESRs’ training programme, on plant ecological epigenetics; bioinformatic skill development; data analysis for linking epigenetic to environmental and genetic data; and translating and communicating science. The network hosted 37 extended research visits (2-3 per ESR) in which each ESR gained training experience both in academic and non-academic environments. For dissemination of scientific results, in addition to scientific publications and conference presentations by the ESRs, EpiDiverse organized one open scientific conference that highlighted project results for a broader scientific community. A key output of the network’s dissemination and utilization efforts has been the development of an open, online learning portal for plant epigenetics. This portal is meant for end users as a resource for learning and teaching on plant ecological epigenetics. It includes online lectures, unlocks the bioinformatic analysis pipelines, and contains an online text book on plant ecological epigenetics with chapters that are written by the ESRs. Public outreach was achieved via an EpiDiverse YouTube channel (containing for instance 15 short movies in which the ESRs present their own research) and via participation in the European Researchers Night 2020. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) EpiDiverse delivered a systematic analysis of the involvement of epigenetic mechanisms in ecological adaptation of different plant species. The scientific impact of this is that high-resolution epigenomic analysis approaches are translated from model species in controlled laboratory environments to different wild species and natural populations. Insight in these underlying mechanisms of ecological adaptation is important for better understanding, and possibly managing, plant adaptability to environmental challenges. Specific resources that were generated by EpiDiverse will be useful for training purposes (EpiDiverse online learning portal) and research purposes (EpiDiverse genomic and bioinformatic analysis tools), and can be used by companies involved in analysis of epigenomic data. EpiDiverse group in Leipzig EpiDiverse ESRs at the EpiDiv21 conference logo of the EPidiverse ITN EpiDiverse group picture 2018
