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.

Exploring Genetic and Epigenetic Changes in Lingonberry Using Molecular Markers: Implications for Clonal Propagation.

Lingonberry (Vaccinium vitis-idaea L.) is an important and valuable horticultural crop due to its high antioxidant properties. Plant tissue culture is an advanced propagation system employed in horticultural crops. However, the progeny derived using this technique may not be true-to-type. In order to obtain the maximum return of any agricultural enterprise, uniformity of planting materials is necessary, which sometimes is not achieved due to genetic and epigenetic instabilities under in vitro culture. Therefore, we analyzed morphological traits and genetic and epigenetic variations under tissue-culture and greenhouse conditions in lingonberry using molecular markers. Leaf length and leaf width under greenhouse conditions and shoot number per explant, shoot height and shoot vigor under in vitro conditions were higher in hybrid H1 compared to the cultivar Erntedank. Clonal fidelity study using one expressed sequence tag (EST)-polymerase chain reaction (PCR), five EST-simple sequence repeat (SSR) and six genomic (G)-SSR markers revealed monomorphic bands in micropropagated shoots and plants in lingonberry hybrid H1 and cultivar Erntedank conforming genetic integrity. Epigenetic variation was studied by quantifying cytosine methylation using a methylation-sensitive amplification polymorphism (MSAP) technique. DNA methylation ranged from 32% in greenhouse-grown hybrid H1 to 44% in cultivar Erntedank under a tissue culture system. Although total methylation was higher in in vitro grown shoots, fully methylated bands were observed more in the greenhouse-grown plants. On the contrary, hemimethylated DNA bands were more prominent in tissue culture conditions as compared to the greenhouse-grown plants. The study conclude that lingonberry maintains its genetic integrity but undergoes variable epigenetic changes during in vitro and ex vitro conditions.

Wild epigenetics: insights from epigenetic studies on natural populations.

Epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNAs are increasingly targeted in studies of natural populations. Here, I review some of the insights gained from this research, examine some of the methods currently in use and discuss some of the challenges that researchers working on natural populations are likely to face when probing epigenetic mechanisms. While studies supporting the involvement of epigenetic mechanisms in generating phenotypic variation in natural populations are amassing, many of these studies are currently correlative in nature. Thus, while empirical data point to widespread contributions of epigenetic mechanisms in generating phenotypic variation, there are still concerns as to whether epigenetic variation is instead ultimately controlled by genetic variation. Disentangling these two sources of variation will be a key to resolving the debate about the importance of epigenetic mechanisms, and studies on natural populations that partition the relative contribution of genetic and epigenetic factors to phenotypic variation can play an important role in this debate.

Signatures of polygenic adaptation align with genome-wide methylation patterns in wild strawberry plants.

Epigenetic inheritance can drive adaptive evolution independently of DNA sequence variation. However, to what extent epigenetic variation represents an autonomous evolutionary force remains largely elusive. Through gene ontology and comparative analyses of genomic and epigenomic variation of wild strawberry plants raised in distinct drought settings, we characterised genome-wide covariation between single nucleotide polymorphisms (SNPs) and differentially methylated cytosines (DMCs). Covariation between SNPs and DMCs was independent of genomic proximity, but instead associated with fitness-related processes such as stress responses, genome regulation and reproduction. We expected this functional SNP-DMC covariation to be driven by adaptive evolution canalising SNP and DMC variation, but instead observed significantly lower covariation with DMCs for adaptive rather than for neutral SNPs. Drought-induced DMCs frequently co-varied with tens of SNPs, suggesting high genomic redundancy as a broad potential basis for polygenic adaptation of gene expression. Our findings suggest that stress-responsive DMCs initially co-vary with many SNPs under increased environmental stress, and that natural selection acting upon several of these SNPs subsequently reduces standing covariation with stress-responsive DMCs. Our study supports DNA methylation profiles that represent complex quantitative traits rather than autonomous evolutionary forces. We provide a conceptual framework for polygenic regulation and adaptation shaping genome-wide methylation patterns in plants.

(Epi)genomic adaptation driven by fine geographical scale environmental heterogeneity after recent biological invasions.

Elucidating processes and mechanisms involved in rapid local adaptation to varied environments is a poorly understood but crucial component in management of invasive species. Recent studies have proposed that genetic and epigenetic variation could both contribute to ecological adaptation, yet it remains unclear on the interplay between these two components underpinning rapid adaptation in wild animal populations. To assess their respective contributions to local adaptation, we explored epigenomic and genomic responses to environmental heterogeneity in eight recently colonized ascidian (Ciona intestinalis) populations at a relatively fine geographical scale. Based on MethylRADseq data, we detected strong patterns of local environment-driven DNA methylation divergence among populations, significant epigenetic isolation by environment (IBE), and a large number of local environment-associated epigenetic loci. Meanwhile, multiple genetic analyses based on single nucleotide polymorphisms (SNPs) showed genomic footprints of divergent selection. In addition, for five genetically similar populations, we detected significant methylation divergence and local environment-driven methylation patterns, indicating the strong effects of local environments on epigenetic variation. From a functional perspective, a majority of functional genes, Gene Ontology (GO) terms, and biological pathways were largely specific to one of these two types of variation, suggesting partial independence between epigenetic and genetic adaptation. The methylation quantitative trait loci (mQTL) analysis showed that the genetic variation explained only 18.67% of methylation variation, further confirming the autonomous relationship between these two types of variation. Altogether, we highlight the complementary interplay of genetic and epigenetic variation involved in local adaptation, which may jointly promote populations' rapid adaptive capacity and successful invasions in different environments. The findings here provide valuable insights into interactions between invaders and local environments to allow invasive species to rapidly spread, thus contributing to better prediction of invasion success and development of management strategies.

Harnessing the power of multi-omics data for predicting climate change response.

Predicting how species will respond to future climate change is of central importance in the midst of the global biodiversity crisis, and recent work has demonstrated the utility of population genomics for improving these predictions. Here, we suggest a broadening of the approach to include other types of genomic variants that play an important role in adaptation, like structural (e.g. copy number variants) and epigenetic variants (e.g. DNA methylation). These data could provide additional power for forecasting response, especially in weakly structured or panmictic species. Incorporating structural and epigenetic variation into estimates of climate change vulnerability, or maladaptation, may not only improve prediction power but also provide insight into the molecular mechanisms underpinning species' response to climate change.

Manipulation of cytosine methylation does not remove latitudinal clines in two invasive goldenrod species in Central Europe.

Invasive species frequently differentiate phenotypically in novel environments within a few generations, often even with limited genetic variation. For the invasive plants Solidago canadensis and S. gigantea, we tested whether such differentiation might have occurred through heritable epigenetic changes in cytosine methylation. In a 2-year common-garden experiment, we grew plants from seeds collected along a latitudinal gradient in their non-native Central European range to test for trait differentiation and whether differentiation disappeared when seeds were treated with the demethylation agent zebularine. Microsatellite markers revealed no population structure along the latitudinal gradient in S. canadensis, but three genetic clusters in S. gigantea. Solidago canadensis showed latitudinal clines in flowering phenology and growth. In S. gigantea, the number of clonal offspring decreased with latitude. Although zebularine had a significant effect on early growth, probably through effects on cytosine methylation, latitudinal clines remained (or even got stronger) in plants raised from seeds treated with zebularine. Thus, our experiment provides no evidence that epigenetic mechanisms by selective cytosine methylation contribute to the observed phenotypic differentiation in invasive goldenrods in Central Europe.

Epigenetic Flexibility Underlies Somaclonal Sex Conversions in Hexaploid Persimmon.

Epigenetic regulation adds a flexible layer to genetic variations, potentially enabling long-term, but reversible, changes to a trait, while maintaining genetic information. In the hexaploid Oriental persimmon (Diospyros kaki), genetically monoecious cultivars bearing male flowers require the Y-encoded small RNA (smRNA) gene, OGI. This gene represses the expression of its autosomal counterpart gene, MeGI, as part of the canonical male production system. However, a D. kaki cultivar, Saijo, which lacks the OGI gene and originally bears only female flowers, occasionally produces somaclonal mutant male and revertant female (RF) branches. In this study, we investigated the mechanisms underlying these somaclonal sex conversions in persimmon. Specifically, we aimed to unravel how a genetically female tree without the OGI gene can produce male flowers and RF flowers. Applying multi-omics approaches, we revealed that this noncanonical male production system is basically consistent with the canonical system, in which the accumulation of smRNA targeting MeGI and the considerable DNA methylation of MeGI are involved. The epigenetic status of MeGI on CGN and CHG was synchronized to the genome-wide methylation patterns, both in transition to and from the male production system. These results suggest that the somaclonal sex conversions in persimmon are driven by the genome-wide epigenetic regulatory activities. Moreover, flexibility in the epigenetic layers of long-lived plant species (e.g. trees) is important for overcoming genetic robustness.

Unprogrammed epigenetic variation mediated by stochastic formation of ectopic heterochromatin.

Changes in gene expression via chromatin-mediated mechanisms are important for reprogramming and differentiation, but uncontrolled changes can potentially lead to harmful or adaptive phenotypic alteration. Thus, diversification of the genome-wide chromatin state must be strictly limited, but the underlying mechanism of this regulation is largely unknown. In this review, we focused on distribution of heterochromatin, a tight chromatin structure that negatively regulates gene expression. Heterochromatin is characterized by methylation of histone H3 at lysine 9, and its formation and spreading are controlled by H3K9-specific methyltransferases and reversal factors such as histone demethylases. We summarize recent findings and discuss how variability in the heterochromatin distribution is controlled in the unicellular eukaryote fission yeast. In this context, we recently found that the anti-silencing factor Epe1 plays a key role in the formation of the individual-specific heterochromatin distribution. In conclusion, recent studies revealed that there are many potential heterochromatin formation sites in the fission yeast genome, and several proteins contribute to suppression of spreading and genome-wide dispersal of heterochromatin; knowledge from fission yeast studies may provide insights into the mechanisms regulating epigenetic diversification in multicellular eukaryotes.

Intrauterine programming of obesity and type 2 diabetes.

The type 2 diabetes epidemic and one of its predisposing factors, obesity, are major influences on global health and economic burden. It is accepted that genetics and the current environment contribute to this epidemic; however, in the last two decades, both human and animal studies have consolidated considerable evidence supporting the 'developmental programming' of these conditions, specifically by the intrauterine environment. Here, we review the various in utero exposures that are linked to offspring obesity and diabetes in later life, including epidemiological insights gained from natural historical events, such as the Dutch Hunger Winter, the Chinese famine and the more recent Quebec Ice Storm. We also describe the effects of gestational exposure to endocrine disruptors, maternal infection and smoking to the fetus in relation to metabolic programming. Causal evidence from animal studies, motivated by human observations, is also discussed, as well as some of the proposed underlying molecular mechanisms for developmental programming of obesity and type 2 diabetes, including epigenetics (e.g. DNA methylation and histone modifications) and microRNA interactions. Finally, we examine the effects of non-pharmacological interventions, such as improving maternal dietary habits and/or increasing physical activity, on the offspring epigenome and metabolic outcomes.

Structure, stability and ecological significance of natural epigenetic variation: a large-scale survey in Plantago lanceolata.

Within-species diversity is an important driver of ecological and evolutionary processes. Recent research has found that plants can harbour significant epigenetic diversity, but its extent, stability and ecological significance in natural populations is largely unexplored. We analysed genetic, epigenetic and phenotypic variation in a large number of natural grassland populations of Plantago lanceolata, covering a broad geographical and environmental range. Within-population diversity and among-population differentiation were calculated from genetic and epigenetic marker data and from measurements of phenotypic traits, both for plants in the field and for the F1 generation grown in a common environment. We found weak but significant epigenetic population structure. A large part of the epigenetic population differences observed in the field was maintained in a common environment. Epigenetic differences were consistently related to genetic and environmental variation, and to a lesser degree to phenotypic variation and land use, with more grazed populations harbouring greater epigenetic diversity. Our study demonstrates that epigenetic diversity exists in natural populations of a common grassland species, and that at least part of this epigenetic diversity is stable, nonrandom and related to environmental variation. Experimental and more detailed molecular studies are needed to elucidate the mechanistic basis of these observed patterns.

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.

The clinical and prognostic value of miR-9 gene expression in Tunisian patients with bladder cancer.

There is a major need for the identification of biomarkers, which are able to guide personalized therapy for bladder cancer, in particular after resection of the primary tumor. Specifically, miR-9 upregulation has been preliminarily associated with a more aggressive phenotype of bladder cancer, namely muscle-invasive bladder cancer (MIBC) or high-grade non-muscle-invasive bladder cancer (HG NMIBC). In order to explore the potential utility of miR-9 as a biomarker in bladder cancer, we have investigated its expression pattern in a sample of Tunisian patients who have undergone primary resection. This is a retrospective study performed on BCa samples from 90 patients (44 specimens of HG NMIBC, 23 specimens of LG NMIBC, and 23 specimens of MIBC). Ten samples from the non-tumoral zone of cystectomy specimens were used as controls. For each specimen, we measured miR-9 expression and correlated it with the clinical characteristics of the patients. Overall, miR-9 was overexpressed in MIBC compared to NMIBC specimens (median fold change [FC]: - 8.89 vs 1.41, p = 0.001). Similarly, miR-9 expression was significantly different in LG NMIBC, HG NMIBC and MIBC subgroups (median FC: 0.68, 2.14 and 8.89, respectively; p = 0.001). ROC analysis showed that miR-9 expression pattern could be used as potential biomarker for distinguishing NMIBC subgroups: indeed miR-9 expression is relatively low in LG NMIBC and high in HG NMIBC. The thresholds are estimated at 0.063 and 21.597, respectively. Moreover, miR-9 was associated with a higher risk of progression. This study suggests the clinical value of miR-9 as a prognostic factor in bladder cancer after tumor resection. Should the prognostic ability of miR-9 be confirmed in larger studies, also on different ethnic groups, it would be useful to investigate whether urine sampling-which is easier to perform, less invasive and less costly-can provide the same results as analysis on surgical specimens.

Transgenerational epigenetics: Inheritance of global cytosine methylation and methylation-related epigenetic markers in the shrub Lavandula latifolia.

PREMISE OF THE STUDY: The ecological and evolutionary significance of natural epigenetic variation (i.e., not based on DNA sequence variants) variation will depend critically on whether epigenetic states are transmitted from parents to offspring, but little is known on epigenetic inheritance in nonmodel plants. METHODS: We present a quantitative analysis of transgenerational transmission of global DNA cytosine methylation (= proportion of all genomic cytosines that are methylated) and individual epigenetic markers (= methylation status of anonymous MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring correlations and parental variance component estimation were applied to epigenetic features of field-growing plants ('maternal parents') and greenhouse-grown progenies. Transmission of genetic markers (AFLP) was also assessed for reference. KEY RESULTS: Maternal parents differed significantly in global DNA cytosine methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed significantly in global methylation, and their differences were significantly related to maternal origin. Methylation-sensitive amplified polymorphism (MSAP) markers exhibited significant transgenerational transmission, as denoted by significant maternal variance component of marker scores in greenhouse families and significant mother-offspring correlations of marker scores. CONCLUSIONS: Although transmission-related measurements for global methylation and MSAP markers were quantitatively lower than those for AFLP markers taken as reference, this study has revealed extensive transgenerational transmission of genome-wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. Similarity of results for global cytosine methylation and epigenetic markers lends robustness to this conclusion, and stresses the value of considering both types of information in epigenetic studies of nonmodel plants.

Biological distance at the Ryan Mound site.

OBJECTIVES: The Ryan Mound site in California spans 2000 years and has been utilized in over 200 studies. The Ryan Mound has been assumed to be a culturally and, therefore, a biologically continuous population over time. This study attempts to determine whether adults at the Ryan Mound consisted of a continuous population over the span of three temporal periods by using nonmetric skeletal traits. MATERIALS AND METHODS: Thirty-eight nonmetric cranial traits and four nonmetric post-cranial traits were scored on adults. Trait correlations were assessed for sex and age using chi-square and Fisher's exact tests. For bilateral traits, data were recorded for both sides, but only results from the left side are reported. Most data were recorded as present or absent. Twelve traits had scores that had more than two nominal categories, which were converted to binary values to enable mean measure of divergence (MMD) statistical analyses. RESULTS: After data reduction, 36 traits remained. Using these traits, standardized MMD analyses revealed that the oldest temporal period and the most recent temporal period individuals were significantly different. CONCLUSION: This study illustrates the importance of testing for biological continuity. Nonmetric studies provide a way to examine relationships within a sample to determine biological continuity. For the Ryan Mound, new populations may have moved into the region early on as part of the Meganos intrusion, or new populations may have moved into the region later, between AD 1500 and AD 1800. Comparative data from nearby sites further support the Meganos intrusion theory.

Comparative epigenetic and genetic spatial structure of the perennial herb Helleborus foetidus: Isolation by environment, isolation by distance, and functional trait divergence.

PREMISE OF THE STUDY: Epigenetic variation can play a role in local adaptation; thus, there should be associations among epigenetic variation, environmental variation, and functional trait variation across populations. This study examines these relationships in the perennial herb Helleborus foetidus (Ranunculaceae). METHODS: Plants from 10 subpopulations were characterized genetically (AFLP, SSR markers), epigenetically (MSAP markers), and phenotypically (20 functional traits). Habitats were characterized using six environmental variables. Isolation-by-distance (IBD) and isolation-by-environment (IBE) patterns of genetic and epigenetic divergence were assessed, as was the comparative explanatory value of geographical and environmental distance as predictors of epigenetic, genetic, and functional differentiation. KEY RESULTS: Subpopulations were differentiated genetically, epigenetically, and phenotypically. Genetic differentiation was best explained by geographical distance, while epigenetic differentiation was best explained by environmental distance. Divergence in functional traits was correlated with environmental and epigenetic distances, but not with geographical and genetic distances. CONCLUSIONS: Results are compatible with the hypothesis that epigenetic IBE and functional divergence reflected responses to environmental variation. Spatial analyses simultaneously considering epigenetic, genetic, phenotypic and environmental information provide a useful tool to evaluate the role of environmental features as drivers of natural epigenetic variation between populations.

Stress-induced memory alters growth of clonal offspring of white clover (Trifolium repens).

PREMISE OF THE STUDY: The phenotype of an individual can be modified by the environment experienced by its predecessors, a phenomenon called transgenerational or maternal effects. These effects are studied mostly across sexual generations and are thought to be mediated also by epigenetic variation. However, we do not know how important transgenerational effects are across asexual generations of clonal plants. METHODS: We investigated the role of different drought intensities and durations experienced by parental plants of Trifolium repens on the growth of offspring ramets after transplantation of clonal cuttings to control conditions. We also treated half of the plants with 5-azacytidine, which is a demethylating agent, to test the potential role of DNA methylation on transgenerational effects. KEY RESULTS: Transgenerational effects were manifested as increased biomass of offspring ramets if parental plants experienced medium drought applied for a short period and decreased biomass of offspring ramets if parental plants experienced intense drought for a short period. These transgenerational effects were not observed for offspring of parents from the same treatments if these were treated with 5-azacytidine, whose application significantly decreased the amount of 5-methyl-2'-deoxycytidine in plants. CONCLUSIONS: Transgenerational effects might play an important role in the clonal plant Trifolium repens and are probably mediated by epigenetic variation. The growth and behavior of clonal plants might be affected not only by the ambient environment but also by environments that are no longer present at the time of clonal reproduction. This phenomenon can have yet unacknowledged ecological and evolutionary implications for clonal plants.

Characterization and machine learning prediction of allele-specific DNA methylation.

A large collection of Single Nucleotide Polymorphisms (SNPs) has been identified in the human genome. Currently, the epigenetic influences of SNPs on their neighboring CpG sites remain elusive. A growing body of evidence suggests that locus-specific information, including genomic features and local epigenetic state, may play important roles in the epigenetic readout of SNPs. In this study, we made use of mouse methylomes with known SNPs to develop statistical models for the prediction of SNP associated allele-specific DNA methylation (ASM). ASM has been classified into parent-of-origin dependent ASM (P-ASM) and sequence-dependent ASM (S-ASM), which comprises scattered-S-ASM (sS-ASM) and clustered-S-ASM (cS-ASM). We found that P-ASM and cS-ASM CpG sites are both enriched in CpG rich regions, promoters and exons, while sS-ASM CpG sites are enriched in simple repeat and regions with high frequent SNP occurrence. Using Lasso-grouped Logistic Regression (LGLR), we selected 21 out of 282 genomic and methylation related features that are powerful in distinguishing cS-ASM CpG sites and trained the classifiers with machine learning techniques. Based on 5-fold cross-validation, the logistic regression classifier was found to be the best for cS-ASM prediction with an ACC of 0.77, an AUC of 0.84 and an MCC of 0.54. Lastly, we applied the logistic regression classifier on human brain methylome and predicted 608 genes associated with cS-ASM. Gene ontology term enrichment analysis indicated that these cS-ASM associated genes are significantly enriched in the category coding for transcripts with alternative splicing forms. In summary, this study provided an analytical procedure for cS-ASM prediction and shed new light on the understanding of different types of ASM events.

Epigenetic variation in maize agronomical traits for breeding and trait improvement.

Epigenetics-mediated breeding (Epibreeding) involves engineering crop traits and stress responses through the targeted manipulation of key epigenetic features to enhance agricultural productivity. While conventional breeding methods raise concerns about reduced genetic diversity, epibreeding propels crop improvement through epigenetic variations that regulate gene expression, ultimately impacting crop yield. Epigenetic regulation in crops encompasses various modes, including histone modification, DNA modification, RNA modification, non-coding RNA, and chromatin remodeling. This review summarizes the epigenetic mechanisms underlying major agronomic traits in maize and identifies candidate epigenetic landmarks in the maize breeding process. We propose a valuable strategy for improving maize yield through epibreeding, combining CRISPR/Cas-based epigenome editing technology and Synthetic Epigenetics (SynEpi). Finally, we discuss the challenges and opportunities associated with maize trait improvement through epibreeding.