Long-read sequencing and structural variant characterization in 1,019 samples from the 1000 Genomes Project.

Structural variants (SVs) contribute significantly to human genetic diversity and disease 1-4 . Previously, SVs have remained incompletely resolved by population genomics, with short-read sequencing facing limitations in capturing the whole spectrum of SVs at nucleotide resolution 5-7 . Here we leveraged nanopore sequencing 8 to construct an intermediate coverage resource of 1,019 long-read genomes sampled within 26 human populations from the 1000 Genomes Project. By integrating linear and graph-based approaches for SV analysis via pangenome graph-augmentation, we uncover 167,291 sequence-resolved SVs in these samples, considerably advancing SV characterization compared to population-wide short-read sequencing studies 3,4 . Our analysis details diverse SV classes-deletions, duplications, insertions, and inversions-at population-scale. LINE-1 and SVA retrotransposition activities frequently mediate transductions 9,10 of unique sequences, with both mobile element classes transducing sequences at either the 3'- or 5'-end, depending on the source element locus. Furthermore, analyses of SV breakpoint junctions suggest a continuum of homology-mediated rearrangement processes are integral to SV formation, and highlight evidence for SV recurrence involving repeat sequences. Our open-access dataset underscores the transformative impact of long-read sequencing in advancing the characterisation of polymorphic genomic architectures, and provides a resource for guiding variant prioritisation in future long-read sequencing-based disease studies.

Arabidopsis histone deacetylase HD2A and HD2B regulate seed dormancy by repressing DELAY OF GERMINATION 1.

Seed dormancy is a crucial developmental transition that affects the adaption and survival of plants. Arabidopsis DELAY OF GERMINATION 1 (DOG1) is known as a master regulator of seed dormancy. However, although several upstream factors of DOG1 have been reported, the exact regulation of DOG1 is not fully understood. Histone acetylation is an important regulatory layer, controlled by histone acetyltransferases and histone deacetylases. Histone acetylation strongly correlates with transcriptionally active chromatin, whereas heterochromatin is generally characterized by hypoacetylated histones. Here we describe that loss of function of two plant-specific histone deacetylases, HD2A and HD2B, resulted in enhanced seed dormancy in Arabidopsis. Interestingly, the silencing of HD2A and HD2B caused hyperacetylation of the DOG1 locus and promoted the expression of DOG1 during seed maturation and imbibition. Knockout of DOG1 could rescue the seed dormancy and partly rescue the disturbed development phenotype of hd2ahd2b. Transcriptomic analysis of the hd2ahd2b line shows that many genes involved in seed development were impaired. Moreover, we demonstrated that HSI2 and HSL1 interact with HD2A and HD2B. In sum, these results suggest that HSI2 and HSL1 might recruit HD2A and HD2B to DOG1 to negatively regulate DOG1 expression and to reduce seed dormancy, consequently, affecting seed development during seed maturation and promoting seed germination during imbibition.

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 .

GSNOR Contributes to Demethylation and Expression of Transposable Elements and Stress-Responsive Genes.

In the past, reactive nitrogen species (RNS) were supposed to be stress-induced by-products of disturbed metabolism that cause oxidative damage to biomolecules. However, emerging evidence demonstrates a substantial role of RNS as endogenous signals in eukaryotes. In plants, S-nitrosoglutathione (GSNO) is the dominant RNS and serves as the •NO donor for S-nitrosation of diverse effector proteins. Remarkably, the endogenous GSNO level is tightly controlled by S-nitrosoglutathione reductase (GSNOR) that irreversibly inactivates the glutathione-bound NO to ammonium. Exogenous feeding of diverse RNS, including GSNO, affected chromatin accessibility and transcription of stress-related genes, but the triggering function of RNS on these regulatory processes remained elusive. Here, we show that GSNO reductase-deficient plants (gsnor1-3) accumulate S-adenosylmethionine (SAM), the principal methyl donor for methylation of DNA and histones. This SAM accumulation triggered a substantial increase in the methylation index (MI = [SAM]/[S-adenosylhomocysteine]), indicating the transmethylation activity and histone methylation status in higher eukaryotes. Indeed, a mass spectrometry-based global histone profiling approach demonstrated a significant global increase in H3K9me2, which was independently verified by immunological detection using a selective antibody. Since H3K9me2-modified regions tightly correlate with methylated DNA regions, we also determined the DNA methylation status of gsnor1-3 plants by whole-genome bisulfite sequencing. DNA methylation in the CG, CHG, and CHH contexts in gsnor1-3 was significantly enhanced compared to the wild type. We propose that GSNOR1 activity affects chromatin accessibility by controlling the transmethylation activity (MI) required for maintaining DNA methylation and the level of the repressive chromatin mark H3K9me2.

Repression of CHROMOMETHYLASE 3 prevents epigenetic collateral damage in Arabidopsis.

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

Nitric oxide coordinates growth, development, and stress response via histone modification and gene expression.

Nitric oxide (NO) is a signaling molecule with multiple regulatory functions in plant physiology and stress response. In addition to direct effects on transcriptional machinery, NO executes its signaling function via epigenetic mechanisms. We report that light intensity-dependent changes in NO correspond to changes in global histone acetylation (H3, H3K9, and H3K9/K14) in Arabidopsis (Arabidopsis thaliana) wild-type leaves, and that this relationship depends on S-nitrosoglutathione reductase and histone deacetylase 6 (HDA6). The activity of HDA6 was sensitive to NO, demonstrating that NO participates in regulation of histone acetylation. Chromatin immunoprecipitation sequencing and RNA-seq analyses revealed that NO participates in the metabolic switch from growth and development to stress response. This coordinating function of NO might be particularly important in plant ability to adapt to a changing environment, and is therefore a promising foundation for mitigating the negative effects of climate change on plant productivity.

ARADEEPOPSIS, an Automated Workflow for Top-View Plant Phenomics using Semantic Segmentation of Leaf States.

Linking plant phenotype to genotype is a common goal to both plant breeders and geneticists. However, collecting phenotypic data for large numbers of plants remain a bottleneck. Plant phenotyping is mostly image based and therefore requires rapid and robust extraction of phenotypic measurements from image data. However, because segmentation tools usually rely on color information, they are sensitive to background or plant color deviations. We have developed a versatile, fully open-source pipeline to extract phenotypic measurements from plant images in an unsupervised manner. ARADEEPOPSIS (https://github.com/Gregor-Mendel-Institute/aradeepopsis) uses semantic segmentation of top-view images to classify leaf tissue into three categories: healthy, anthocyanin rich, and senescent. This makes it particularly powerful at quantitative phenotyping of different developmental stages, mutants with aberrant leaf color and/or phenotype, and plants growing in stressful conditions. On a panel of 210 natural Arabidopsis (Arabidopsis thaliana) accessions, we were able to not only accurately segment images of phenotypically diverse genotypes but also to identify known loci related to anthocyanin production and early necrosis in genome-wide association analyses. Our pipeline accurately processed images of diverse origin, quality, and background composition, and of a distantly related Brassicaceae. ARADEEPOPSIS is deployable on most operating systems and high-performance computing environments and can be used independently of bioinformatics expertise and resources.