RESUMEN
Seed maturation is the developmental process that prepares the embryo for the desiccated waiting period before germination. It is associated with a series of physiological changes leading to the establishment of seed dormancy, seed longevity, and desiccation tolerance. We studied translational changes during seed maturation and observed a gradual reduction in global translation during seed maturation. Transcriptome and translatome profiling revealed specific reduction in the translation of thousands of genes. By including previously published data on germination and seedling establishment, a regulatory network based on polysome occupancy data was constructed: SeedTransNet. Network analysis predicted translational regulatory pathways involving hundreds of genes with distinct functions. The network identified specific transcript sequence features suggesting separate translational regulatory circuits. The network revealed several seed maturation-associated genes as central nodes, and this was confirmed by specific seed phenotypes of the respective mutants. One of the regulators identified, an AWPM19 family protein, PM19-Like1 (PM19L1), was shown to regulate seed dormancy and longevity. This putative RNA-binding protein also affects the translational regulation of its target mRNA, as identified by SeedTransNet. Our data show the usefulness of SeedTransNet in identifying regulatory pathways during seed phase transitions.
Asunto(s)
Arabidopsis , Germinación , Germinación/genética , Arabidopsis/metabolismo , Transcriptoma , Plantones/metabolismo , Semillas/metabolismoRESUMEN
The Populus genus is one of the major plant model systems, but genomic resources have thus far primarily been available for poplar species, and primarily Populus trichocarpa (Torr. & Gray), which was the first tree with a whole-genome assembly. To further advance evolutionary and functional genomic analyses in Populus, we produced genome assemblies and population genetics resources of two aspen species, Populus tremula L. and Populus tremuloides Michx. The two aspen species have distributions spanning the Northern Hemisphere, where they are keystone species supporting a wide variety of dependent communities and produce a diverse array of secondary metabolites. Our analyses show that the two aspens share a similar genome structure and a highly conserved gene content with P. trichocarpa but display substantially higher levels of heterozygosity. Based on population resequencing data, we observed widespread positive and negative selection acting on both coding and noncoding regions. Furthermore, patterns of genetic diversity and molecular evolution in aspen are influenced by a number of features, such as expression level, coexpression network connectivity, and regulatory variation. To maximize the community utility of these resources, we have integrated all presented data within the PopGenIE web resource (PopGenIE.org).
Asunto(s)
Populus/genética , Evolución Biológica , ADN de Plantas/genética , Evolución Molecular , Variación Genética , Genética de Población/métodos , Genoma de Planta , Genómica , Desequilibrio de Ligamiento/genética , Filogenia , Selección Genética/genética , Análisis de Secuencia de ADN/métodos , Árboles/genéticaRESUMEN
Seasonal cues influence several aspects of the secondary growth of tree stems, including cambial activity, wood chemistry, and transition to latewood formation. We investigated seasonal changes in cambial activity, secondary cell wall formation, and tracheid cell death in woody tissues of Norway spruce (Picea abies) throughout one seasonal cycle. RNA sequencing was performed simultaneously in both the xylem and cambium/phloem tissues of the stem. Principal component analysis revealed gradual shifts in the transcriptomes that followed a chronological order throughout the season. A notable remodeling of the transcriptome was observed in the winter, with many genes having maximal expression during the coldest months of the year. A highly coexpressed set of monolignol biosynthesis genes showed high expression during the period of secondary cell wall formation as well as a second peak in midwinter. This midwinter peak in expression did not trigger lignin deposition, as determined by pyrolysis-gas chromatography/mass spectrometry. Coexpression consensus network analyses suggested the involvement of transcription factors belonging to the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES and MYELOBLASTOSIS-HOMEOBOX families in the seasonal control of secondary cell wall formation of tracheids. Interestingly, the lifetime of the latewood tracheids stretched beyond the winter dormancy period, correlating with a lack of cell death-related gene expression. Our transcriptomic analyses combined with phylogenetic and microscopic analyses also identified the cellulose and lignin biosynthetic genes and putative regulators for latewood formation and tracheid cell death in Norway spruce, providing a toolbox for further physiological and functional assays of these important phase transitions.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Picea/genética , Estaciones del Año , Madera/genética , Cámbium/genética , Cámbium/crecimiento & desarrollo , Cámbium/metabolismo , Celulosa/biosíntesis , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Lignina/biosíntesis , Noruega , Floema/genética , Floema/crecimiento & desarrollo , Floema/metabolismo , Picea/crecimiento & desarrollo , Picea/metabolismo , Análisis de Componente Principal , Madera/crecimiento & desarrollo , Madera/metabolismo , Xilema/genética , Xilema/crecimiento & desarrollo , Xilema/metabolismoRESUMEN
Plastid sequences are a cornerstone in plant systematic studies and key aspects of their evolution, such as uniparental inheritance and absent recombination, are often treated as axioms. While exceptions to these assumptions can profoundly influence evolutionary inference, detecting them can require extensive sampling, abundant sequence data, and detailed testing. Using advancements in high-throughput sequencing, we analyzed the whole plastomes of 65 accessions of Picea, a genus of â¼35 coniferous forest tree species, to test for deviations from canonical plastome evolution. Using complementary hypothesis and data-driven tests, we found evidence for chimeric plastomes generated by interspecific hybridization and recombination in the clade comprising Norway spruce (P. abies) and 10 other species. Support for interspecific recombination remained after controlling for sequence saturation, positive selection, and potential alignment artifacts. These results reconcile previous conflicting plastid-based phylogenies and strengthen the mounting evidence of reticulate evolution in Picea. Given the relatively high frequency of hybridization and biparental plastid inheritance in plants, we suggest interspecific plastome recombination may be more widespread than currently appreciated and could underlie reported cases of discordant plastid phylogenies.
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Transferencia de Gen Horizontal , Picea/genética , Análisis de Secuencia de ADN/métodos , Evolución Biológica , ADN de Plantas/genética , Evolución Molecular , Genes de Plantas , Genoma de Plastidios , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hibridación Genética , Filogenia , Plastidios/genéticaRESUMEN
Xylan is one of the main compounds determining wood properties in hardwood species. The xylan backbone is thought to be synthesized by a synthase complex comprising two members of the GT43 family. We downregulated all GT43 genes in hybrid aspen (Populus tremula × tremuloides) to understand their involvement in xylan biosynthesis. All three clades of the GT43 family were targeted for downregulation using RNA interference individually or in different combinations, either constitutively or specifically in developing wood. Simultaneous downregulation in developing wood of the B (IRX9) and C (IRX14) clades resulted in reduced xylan Xyl content relative to reducing end sequence, supporting their role in xylan backbone biosynthesis. This was accompanied by a higher lignocellulose saccharification efficiency. Unexpectedly, GT43 suppression in developing wood led to an overall growth stimulation, xylem cell wall thinning and a shift in cellulose orientation. Transcriptome profiling of these transgenic lines indicated that cell cycling was stimulated and secondary wall biosynthesis was repressed. We suggest that the reduced xylan elongation is sensed by the cell wall integrity surveying mechanism in developing wood. Our results show that wood-specific suppression of xylan-biosynthetic GT43 genes activates signaling responses, leading to increased growth and improved lignocellulose saccharification.
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Proteínas de Plantas/genética , Populus/genética , Madera/crecimiento & desarrollo , Xilanos/biosíntesis , Cámbium/genética , Cámbium/crecimiento & desarrollo , Pared Celular/química , Pared Celular/genética , Celulosa/genética , Celulosa/metabolismo , Quimera , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas , Lignina/genética , Lignina/metabolismo , Familia de Multigenes , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Populus/crecimiento & desarrollo , Regiones Promotoras Genéticas , Interferencia de ARN , Azúcares/metabolismo , Madera/química , Madera/genética , Xilanos/genéticaRESUMEN
Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H2O2-scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H2O2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism.
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Peróxido de Hidrógeno/metabolismo , Fenoles/metabolismo , Picea/metabolismo , Antioxidantes/metabolismo , Espacio Extracelular/metabolismo , Depuradores de Radicales Libres/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genes de Plantas , Lignina/metabolismo , Anotación de Secuencia Molecular , Estrés Oxidativo , Picea/genética , Análisis de Componente Principal , Transducción de Señal , Especificidad por Sustrato , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
The advancement of high-throughput sequencing (HTS) technologies and the rapid development of numerous analysis algorithms and pipelines in this field has resulted in an unprecedentedly high demand for training scientists in HTS data analysis. Embarking on developing new training materials is challenging for many reasons. Trainers often do not have prior experience in preparing or delivering such materials and struggle to keep them up to date. A repository of curated HTS training materials would support trainers in materials preparation, reduce the duplication of effort by increasing the usage of existing materials, and allow for the sharing of teaching experience among the HTS trainers' community. To achieve this, we have developed a strategy for materials' curation and dissemination. Standards for describing training materials have been proposed and applied to the curation of existing materials. A Git repository has been set up for sharing annotated materials that can now be reused, modified, or incorporated into new courses. This repository uses Git; hence, it is decentralized and self-managed by the community and can be forked/built-upon by all users. The repository is accessible at http://bioinformatics.upsc.se/htmr.
Asunto(s)
Biología Computacional/educación , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Algoritmos , Interpretación Estadística de Datos , Educación , Humanos , EnseñanzaRESUMEN
BACKGROUND: Evolutionary theory suggests that males and females may evolve sexually dimorphic phenotypic and biochemical traits concordant with each sex having different optimal strategies of resource investment to maximise reproductive success and fitness. Such sexual dimorphism would result in sex biased gene expression patterns in non-floral organs for autosomal genes associated with the control and development of such phenotypic traits. RESULTS: We examined morphological, biochemical and herbivory traits to test for sexually dimorphic resource allocation strategies within collections of sexually mature and immature Populus tremula (European aspen) trees. In addition we profiled gene expression in mature leaves of sexually mature wild trees using whole-genome oligonucleotide microarrays and RNA-Sequencing. CONCLUSIONS: We found no evidence of sexual dimorphism or differential resource investment strategies between males and females in either sexually immature or mature trees. Similarly, single-gene differential expression and machine learning approaches revealed no evidence of large-scale sex biased gene expression. However, two significantly differentially expressed genes were identified from the RNA-Seq data, one of which is a robust diagnostic marker of sex in P. tremula.
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Populus/genética , Regulación de la Expresión Génica de las Plantas , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/genética , Populus/fisiología , Análisis de Secuencia de ARNRESUMEN
Gene regulatory and gene co-expression networks are powerful research tools for identifying biological signal within high-dimensional gene expression data. In recent years, research has focused on addressing shortcomings of these techniques with regard to the low signal-to-noise ratio, non-linear interactions and dataset dependent biases of published methods. Furthermore, it has been shown that aggregating networks from multiple methods provides improved results. Despite this, few useable and scalable software tools have been implemented to perform such best-practice analyses. Here, we present Seidr (stylized Seiðr), a software toolkit designed to assist scientists in gene regulatory and gene co-expression network inference. Seidr creates community networks to reduce algorithmic bias and utilizes noise corrected network backboning to prune noisy edges in the networks. Using benchmarks in real-world conditions across three eukaryotic model organisms, Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana, we show that individual algorithms are biased toward functional evidence for certain gene-gene interactions. We further demonstrate that the community network is less biased, providing robust performance across different standards and comparisons for the model organisms. Finally, we apply Seidr to a network of drought stress in Norway spruce (Picea abies (L.) H. Krast) as an example application in a non-model species. We demonstrate the use of a network inferred using Seidr for identifying key components, communities and suggesting gene function for non-annotated genes.
RESUMEN
Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (PolÏ´). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Proteínas Quinasas/genética , Reparación del ADN/genética , ADN/genéticaRESUMEN
The rate of meiotic recombination is one of the central factors determining genome-wide levels of linkage disequilibrium which has important consequences for the efficiency of natural selection and for the dissection of quantitative traits. Here we present a new, high-resolution linkage map for Populus tremula that we use to anchor approximately two thirds of the P. tremula draft genome assembly on to the expected 19 chromosomes, providing us with the first chromosome-scale assembly for P. tremula (Table 2). We then use this resource to estimate variation in recombination rates across the P. tremula genome and compare these results to recombination rates based on linkage disequilibrium in a large number of unrelated individuals. We also assess how variation in recombination rates is associated with a number of genomic features, such as gene density, repeat density and methylation levels. We find that recombination rates obtained from the two methods largely agree, although the LD-based method identifies a number of genomic regions with very high recombination rates that the map-based method fails to detect. Linkage map and LD-based estimates of recombination rates are positively correlated and show similar correlations with other genomic features, showing that both methods can accurately infer recombination rate variation across the genome. Recombination rates are positively correlated with gene density and negatively correlated with repeat density and methylation levels, suggesting that recombination is largely directed toward gene regions in P. tremula.
Asunto(s)
Variación Genética , Populus/genética , Recombinación Genética , Cromosomas de las Plantas/genética , Metilación de ADN , Desequilibrio de LigamientoRESUMEN
Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pregenomic, qualitative methods. To help address these limitations, we combined machine learning and in silico enrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae: Picea abies). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats in P. abies, we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: Recombination dynamics were heterogeneous across vascular plants and highly active small repeats (ca. 200 bp) were present in about one-third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.
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Genoma Mitocondrial , Picea/genética , Recombinación Genética , Simulación por Computador , Cycadopsida/genética , ADN de Plantas/química , Genes de Plantas , Variación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Máquina de Vectores de SoporteRESUMEN
Leaf shape is a defining feature of how we recognize and classify plant species. Although there is extensive variation in leaf shape within many species, few studies have disentangled the underlying genetic architecture. We characterized the genetic architecture of leaf shape variation in Eurasian aspen (Populus tremula L.) by performing genome-wide association study (GWAS) for physiognomy traits. To ascertain the roles of identified GWAS candidate genes within the leaf development transcriptional program, we generated RNA-Seq data that we used to perform gene co-expression network analyses from a developmental series, which is publicly available within the PlantGenIE resource. We additionally used existing gene expression measurements across the population to analyze GWAS candidate genes in the context of a population-wide co-expression network and to identify genes that were differentially expressed between groups of individuals with contrasting leaf shapes. These data were integrated with expression GWAS (eQTL) results to define a set of candidate genes associated with leaf shape variation. Our results identified no clear adaptive link to leaf shape variation and indicate that leaf shape traits are genetically complex, likely determined by numerous small-effect variations in gene expression. Genes associated with shape variation were peripheral within the population-wide co-expression network, were not highly connected within the leaf development co-expression network, and exhibited signatures of relaxed selection. As such, our results are consistent with the omnigenic model.
RESUMEN
Current extinction rates are comparable to five prior mass extinctions in the earth's history, and are strongly affected by human activities that have modified more than half of the earth's terrestrial surface. Increasing human activity restricts animal movements and isolates formerly connected populations, a particular concern for the conservation of large carnivores, but no prior research has used high throughput sequencing in a standardized manner to examine genetic connectivity for multiple species of large carnivores and multiple ecosystems. Here, we used RAD SNP genotypes to test for differences in connectivity between multiple ecosystems for African wild dogs (Lycaon pictus) and lions (Panthera leo), and to test correlations between genetic distance, geographic distance and landscape resistance due to human activity. We found weaker connectivity, a stronger correlation between genetic distance and geographic distance, and a stronger correlation between genetic distance and landscape resistance for lions than for wild dogs, and propose a new hypothesis that adaptations to interspecific competition may help to explain differences in vulnerability to isolation by humans.
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Evolución Biológica , Canidae/genética , Carnivoría , Leones/genética , Animales , Canidae/fisiología , Genotipo , Leones/fisiología , Filogenia , Polimorfismo de Nucleótido SimpleRESUMEN
Norway spruce (Picea abies (L.) Karst.) is a conifer species of substanital economic and ecological importance. In common with most conifers, the P. abies genome is very large (â¼20 Gbp) and contains a high fraction of repetitive DNA. The current P. abies genome assembly (v1.0) covers approximately 60% of the total genome size but is highly fragmented, consisting of >10 million scaffolds. The genome annotation contains 66,632 gene models that are at least partially validated (www.congenie.org), however, the fragmented nature of the assembly means that there is currently little information available on how these genes are physically distributed over the 12 P. abies chromosomes. By creating an ultra-dense genetic linkage map, we anchored and ordered scaffolds into linkage groups, which complements the fine-scale information available in assembly contigs. Our ultra-dense haploid consensus genetic map consists of 21,056 markers derived from 14,336 scaffolds that contain 17,079 gene models (25.6% of the validated gene models) that we have anchored to the 12 linkage groups. We used data from three independent component maps, as well as comparisons with previously published Picea maps to evaluate the accuracy and marker ordering of the linkage groups. We demonstrate that approximately 3.8% of the anchored scaffolds and 1.6% of the gene models covered by the consensus map have likely assembly errors as they contain genetic markers that map to different regions within or between linkage groups. We further evaluate the utility of the genetic map for the conifer research community by using an independent data set of unrelated individuals to assess genome-wide variation in genetic diversity using the genomic regions anchored to linkage groups. The results show that our map is sufficiently dense to enable detailed evolutionary analyses across the P. abies genome.
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Mapeo Cromosómico , Genoma de Planta , Genómica , Haploidia , Picea/genética , Ligamiento Genético , Marcadores Genéticos , Genética de Población , Genómica/métodos , Noruega , Polimorfismo de Nucleótido SimpleRESUMEN
With the rapid advancement of high throughput sequencing, large numbers of genetic markers can be readily and cheaply acquired, but most current software packages for genetic map construction cannot handle such dense input. Modern computer architectures and server farms represent untapped resources that can be used to enable higher marker densities to be processed in tractable time. Here we present a pipeline using a modified version of OneMap that parallelizes over bottleneck functions and achieves substantial speedups for producing a high density linkage map (N = 20,000). Using simulated data we show that the outcome is as accurate as the traditional pipeline. We further demonstrate that there is a direct relationship between the number of markers used and the level of deviation between true and estimated order, which in turn impacts the final size of a genetic map.