Library Preparation for Whole Genome Bisulfite Sequencing of Plant Genomes

Epigenetic mechanisms are a key interface between the environment and the genotype. These mechanisms regulate gene expression in response to plant development and environmental stimuli, which ultimately affects the plant’s phenotype. DNA methylation, in particular cytosine methylation, is probably the best studied epigenetic modification in eukaryotes. It has been associated to the regulation of gene expression in response to cell/tissue differentiation, organism development and adaptation to changing environments. Whole genome bisulfite sequencing (WGBS) is considered the gold standard to study DNA methylation at a genome level. Here we present a protocol for the preparation of whole genome bisulfite sequencing libraries from plant samples (grapevine leaves) which includes detailed instructions for sample collection and DNA extraction, sequencing library preparation and bisulfite treatment

The Regulation of Plant Vegetative Phase Transition and Rejuvenation: miRNAs, a Key Regulator

In contrast to animals, adult organs in plants are not formed during embryogenesis but generated from meristematic cells as plants advance through development. Plant development involves a succession of different phenotypic stages and the transition between these stages is termed phase transition. Phase transitions need to be tightly regulated and coordinated to ensure they occur under optimal seasonal, environmental conditions. Polycarpic perennials transition through vegetative stages and the mature, reproductive stage many times during their lifecycles and, in both perennial and annual species, environmental factors and culturing methods can reverse the otherwise unidirectional vector of plant development. Epigenetic factors regulating gene expression in response to internal cues and external (environmental) stimuli influencing the plant’s phenotype and development have been shown to control phase transitions. How developmental and environmental cues interact to epigenetically alter gene expression and influence these transitions is not well understood, and understanding this interaction is important considering the current climate change scenarios, since epigenetic maladaptation could have catastrophic consequences for perennial plants in natural and agricultural ecosystems. Here, we review studies focusing on the epigenetic regulators of the vegetative phase change and highlight how these mechanisms might act in exogenously induced plant rejuvenation and regrowth following stress

Shotgun Metagenomic Sequencing Data of Sunflower Rhizosphere Microbial Community in South Africa

This dataset presents shotgun metagenomic sequencing of sunflower rhizosphere microbiome in Bloemhof, South Africa. Data were collected to decipher the structure and function in the sunflower microbial community. Illumina HiSeq platform using next generation sequencing of the DNA was carried out. The metagenome comprised 8,991,566 sequences totaling 1,607,022,279 bp size and 66% GC content. The metagenome was deposited into the NCBI database and can be accessed with the SRA accession number SRR10418054. An online metagenome server (MG RAST) using the subsystem database revealed bacteria had the highest taxonomical representation with 98.47%, eukaryote at 1.23%, and archaea at 0.20%. The most abundant genera were the Conexibacter (17%), Nocardioides (8%), Streptomyces (7%), Geodermatophilus (6%), Methylobacterium (5%), and Burkholderia (4%). MG-RAST assisted analysis also revealed functional annotation based on subsystem, carbohydrates sequence had 13.74%, clustering based subsystem 12.93%, amino acids and derivatives 10.30% coupled with other useful functional traits needed for plant growth and health

Greenhouse Spatial Effects Detected in the Barley (\u3cem\u3eHordeum vulgare\u3c/em\u3e L.) Epigenome Underlie Stochasticity of DNA Methylation

Environmental cues are known to alter the methylation profile of genomic DNA, and thereby change the expression of some genes. A proportion of such modifications may become adaptive by adjusting expression of stress response genes but others have been shown to be highly stochastic, even under controlled conditions. The influence of environmental flux on plants adds an additional layer of complexity that has potential to confound attempts to interpret interactions between environment, methylome, and plant form. We therefore adopt a positional and longitudinal approach to study progressive changes to barley DNA methylation patterns in response to salt exposure during development under greenhouse conditions. Methylation-sensitive amplified polymorphism (MSAP) and phenotypic analyses of nine diverse barley varieties were grown in a randomized plot design, under two salt treatments (0 and 75 mM NaCl). Combining environmental, phenotypic and epigenetic data analyses, we show that at least part of the epigenetic variability, previously described as stochastic, is linked to environmental micro-variations during plant growth. Additionally, we show that differences in methylation increase with time of exposure to micro-variations in environment. We propose that subsequent epigenetic studies take into account microclimate-induced epigenetic variability

Il-15 enhances the persistence and function of bcma-targeting car-t cells compared to il-2 or il-15/il-7 by limiting car-t cell dysfunction and differentiation

Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the treatment of B-lymphoid malignancies. For multiple myeloma (MM), B-cell maturation antigen (BCMA)-targeted CAR-T cells have achieved outstanding complete response rates, but unfortunately, patients often relapse within a year of receiving the therapy. Increased persistence and reduced dysfunction are crucial features that enhance the durability of CAR-T cell responses. One of the factors that influence CAR-T cell in vivo longevity and loss of function, but which has not yet been extensively studied for BCMA-directed CAR-T cells, are the cytokines used during their production. We here compared the impact of IL-2, IL-15 and a combination of IL-15/IL-7 on the phenotype and function of ARI2h, an academic BCMA-directed CAR-T cell that is currently being administered to MM patients. For this study, flow cytometry, in vitro cytotoxicity assays and analysis of cytokine release were performed. In addition, ARI2h cells expanded with IL-2, IL-15, or IL-15/IL-7 were injected into MM tumor-bearing mice to assess their in vivo efficacy. We demonstrated that each of the cytokine conditions was suitable for the expansion of ARI2h cells, with clear in vitro activity. Strikingly, however, IL-15-produced ARI2h cells had improved in vivo efficacy and persistence. When explored further, it was found that IL-15 drove a less-differentiated ARI2h phenotype, ameliorated parameters related to CAR-T cell dysfunction, and lowered the release of cytokines potentially involved in cytokine release syndrome and MM progression. Moreover, we observed that IL-15 was less potent in inducing T cell senescence and DNA damage accumulation, both of which may contribute to an unfavorable CAR-T cell phenotype. These findings show the superiority of IL-15 to IL-2 and IL-15/IL-7 in the quality of anti-BCMA CAR-T cells, particularly their efficacy and persistence, and as such, could improve the duration of responses if applied to the clinical production of CAR-T cells for patients

Salt Stress Induces Non-CG Methylation in Coding Regions of Barley Seedlings (\u3cem\u3eHordeum vulgare\u3c/em\u3e)

Salinity can negatively impact crop growth and yield. Changes in DNA methylation are known to occur when plants are challenged by stress and have been associated with the regulation of stress-response genes. However, the role of DNA-methylation in moderating gene expression in response to salt stress has been relatively poorly studied among crops such as barley. Here, we assessed the extent of salt-induced alterations of DNA methylation in barley and their putative role in perturbed gene expression. Using Next Generation Sequencing, we screened the leaf and root methylomes of five divergent barley varieties grown under control and three salt concentrations, to seek genotype independent salt-induced changes in DNA methylation. Salt stress caused increased methylation in leaves but diminished methylation in roots with a higher number of changes in leaves than in roots, indicating that salt induced changes to global methylation are organ specific. Differentially Methylated Markers (DMMs) were mostly located in close proximity to repeat elements, but also in 1094 genes, of which many possessed gene ontology (GO) terms associated with plant responses to stress. Identified markers have potential value as sentinels of salt stress and provide a starting point to allow understanding of the functional role of DNA methylation in facilitating barley’s response to this stressor

Epigenetic rather than genetic factors may explain phenotipic divergence between coastal populations of diploid and tetraploid Limonium spp. (Plumbaginaceae) in Portugal

Research ArticleBackground: The genus Limonium Miller comprises annual and perennial halophytes that can produce sexual and/or asexual seeds (apomixis). Genetic and epigenetic (DNA methylation) variation patterns were investigated in populations of three phenotypically similar putative sexual diploid species (L. nydeggeri, L. ovalifolium, L. lanceolatum), one sexual tetraploid species (L. vulgare) and two apomict tetraploid species thought to be related (L. dodartii, L. multiflorum). The extent of morphological differentiation between these species was assessed using ten diagnostic morphometric characters. Results: A discriminant analysis using the morphometric variables reliably assigns individuals into their respective species groups. We found that only modest genetic and epigenetic differentiation was revealed between species by Methylation Sensitive Amplification Polymorphism (MSAP). However, whilst there was little separation possible between ploidy levels on the basis of genetic profiles, there was clear and pronounced interploidy discrimination on the basis of epigenetic profiles. Here we investigate the relative contribution of genetic and epigenetic factors in explaining the complex phenotypic variability seen in problematic taxonomic groups such as Limonium that operate both apomixis and sexual modes of reproduction. Conclusions: Our results suggest that epigenetic variation might be one of the drivers of the phenotypic divergence between diploid and tetraploid taxa and discuss that intergenome silencing offers a plausible mechanistic explanation for the observed phenotypic divergence between these microspecies. These results also suggest that epigenetic profiling offer an additional tool to infer ploidy level in stored specimens and that stable epigenetic change may play an important role in apomict evolution and species recognitioninfo:eu-repo/semantics/publishedVersio

First sexual intercourse and subsequent regret in three developing countries

Purpose: Adolescents who engage in sex can be affected by a range of negative physical and psychological consequences. We intend to analyze the reasons behind first sex, regret, and the association between reasons and regret. Methods: A questionnaire was implemented to 8495 high schools students, aged 14-18, in the Philippines, El Salvador and Peru. Sexually active participants responded whether several circumstances were reasons involved in their first sexual relationship. They also responded whether they regretted having already had sexual relationships. Results: More than a third of respondents reported at least one external pressure leading to first sex, and about half reported at least one reason implying getting carried away by sexual arousal. More females affirmed they regret having already had sex. Logistic regression shows that reasons for first sex associated with regret were partner insistence, "uncontrolled situations" and seeing sexual images. These reasons were associated with regret even when love was also reported as related to first sex. Conclusions: Adolescent sexual experience is often motivated by pressure and circumstances that lower the control over their decisions concerning sex, such as external pressure (because most friends already had sex or because of partner insistence) or getting carried away by sexual arousal (through an "uncontrolled situation" or viewing sexual images) rather than by mature decisions, and this may result in later regret. Adolescents should be helped by parents, educators and policy makers to be aware of these characteristics of adolescent sex and empowered to make assertive and informed decisions concerning their sexuality

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems

Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones