RESUMEN
Horizontal transfer (HT) refers to the exchange of genetic material between divergent species by mechanisms other than reproduction. In recent years, several studies have demonstrated HTs in eukaryotes, particularly in the context of parasitic relationships and in model species. However, very little is known about HT in natural ecosystems, especially those involving non-parasitic wild species, and the nature of the ecological relationships that promote these HTs. In this work, we conducted a pilot study investigating HTs by sequencing the genomes of 17 wild non-model species from a natural ecosystem, the Massane forest, located in southern France. To this end, we developed a new computational pipeline called INTERCHANGE that is able to characterize HTs at the whole genome level without prior annotation and directly in the raw sequencing reads. Using this pipeline, we identified 12 HT events, half of which occurred between lianas and trees. We found that mainly low copy number LTR-retrotransposons from the Copia superfamily were transferred between these wild plant species, especially those of the Ivana and Ale lineages. This study revealed a possible new route for HTs between non-parasitic plants and provides new insights into the genomic characteristics of horizontally transferred DNA in plant genomes.
Asunto(s)
Ecosistema , Genoma de Planta , Proyectos Piloto , Genoma de Planta/genética , Genómica , Retroelementos , Filogenia , Evolución Molecular , Transferencia de Gen Horizontal/genéticaRESUMEN
Abundant extrachromosomal circular DNA (eccDNA) is associated with transposable element (TE) activity. However, how the eccDNA compartment is controlled by epigenetic regulations and what is its impact on the genome is understudied. Here, using long reads, we sequence both the eccDNA compartment and the genome of Arabidopsis thaliana mutant plants affected in DNA methylation and post-transcriptional gene silencing. We detect a high load of TE-derived eccDNA with truncated and chimeric forms. On the genomic side, on top of truncated and full length TE neo-insertions, we detect complex structural variations (SVs) notably at a disease resistance cluster being a natural hotspot of SV. Finally, we serendipitously identify large tandem duplications in hypomethylated plants, suggesting that SVs could have been overlooked in epigenetic mutants. We propose that a high eccDNA load may alter DNA repair pathways leading to genome instability and the accumulation of SVs, at least in plants.
Asunto(s)
Arabidopsis , Humanos , Arabidopsis/genética , Elementos Transponibles de ADN/genética , Inestabilidad Genómica/genética , Interferencia de ARN , ADN CircularRESUMEN
The keystone of ribosome biogenesis is the transcription of 45S rDNA. The Arabidopsis thaliana genome contains hundreds of 45S rDNA units; however, they are not all transcribed. Notably, 45S rDNA units contain insertions/deletions revealing the existence of heterogeneous rRNA genes and, likely, heterogeneous ribosomes for rRNAs. In order to obtain an overall picture of 45S rDNA diversity sustaining the synthesis of rRNAs and, subsequently, of ribosomes in natura, we took advantage of 320 new occurrences of Arabidopsis thaliana as a metapopulation named At66, sampled from 0 to 1900 m of altitude in the eastern Pyrenees in France. We found that the 45S rDNA copy number is very dynamic in natura and identified new genotypes for both 5' and 3' External Transcribed Spacers (ETS). Interestingly, the highest 5'ETS genotype diversity is found in altitude while the highest 3'ETS genotype diversity is found at sea level. Structural analysis of 45S rDNA also shows conservation in natura of specific 5'ETS and 3'ETS sequences/features required to control rDNA expression and the processing of rRNAs. In conclusion, At66 is a worthwhile natural laboratory, and unraveled 45S rDNA diversity represents an interesting starting material to select subsets for rDNA transcription and alter the rRNA composition of ribosomes both intra- and inter-site.
RESUMEN
Throughout the years, most plant genomic studies were focused on nuclear chromosomes. Extrachromosomal circular DNA (eccDNA) has largely been neglected for decades since its discovery in 1965. While initial research showed that eccDNAs can originate from highly repetitive sequences, recent findings show that many regions of the genome can contribute to the eccDNA pool. Currently, the biological functions of eccDNAs, if any, are a mystery but recent studies have indicated that they can be regulated by different genomic loci and contribute to stress response and adaptation. In this review, we outline current relevant technological developments facilitating eccDNA identification and the latest discoveries about eccDNAs in plants. Finally, we explore the probable functions and future research directions that could be undertaken with respect to different eccDNA sources.
Asunto(s)
ADN Circular , Ácidos Nucleicos , Cromosomas , ADN , ADN Circular/genéticaRESUMEN
Long terminal repeat retrotransposons (LTR-RTs) are mobilized via an RNA intermediate using a 'copy and paste' mechanism, and account for the majority of repetitive DNA in plant genomes. As a side effect of mobilization, the formation of LTR-RT-derived extrachromosomal circular DNAs (eccDNAs) occurs. Thus, high-throughput sequencing of eccDNA can be used to identify active LTR-RTs in plant genomes. Despite the release of a reference genome assembly, carrot LTR-RTs have not yet been thoroughly characterized. LTR-RTs are abundant and diverse in the carrot genome. We identified 5976 carrot LTR-RTs, 2053 and 1660 of which were attributed to Copia and Gypsy superfamilies, respectively. They were further classified into lineages, families and subfamilies. More diverse LTR-RT lineages, i.e. lineages comprising many low-copy-number subfamilies, were more frequently associated with genic regions. Certain LTR-RT lineages have been recently active in Daucus carota. In particular, low-copy-number LTR-RT subfamilies, e.g. those belonging to the DcAle lineage, have significantly contributed to carrot genome diversity as a result of continuing activity. We utilized eccDNA sequencing to identify and characterize two DcAle subfamilies, Alex1 and Alex3, active in carrot callus. We documented 14 and 32 de novo insertions of Alex1 and Alex3, respectively, which were positioned in non-repetitive regions.
Asunto(s)
Daucus carota , Retroelementos , Daucus carota/genética , Evolución Molecular , Genoma de Planta/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Retroelementos/genética , Secuencias Repetidas Terminales/genéticaRESUMEN
Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high-quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe non-coding RNAs, pseudogenes and transposable elements, and highlight tissue-specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome-wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.
Asunto(s)
Thlaspi , Cromosomas , Ecosistema , Genoma de Planta/genética , Anotación de Secuencia Molecular , Thlaspi/genética , Investigación Biomédica TraslacionalRESUMEN
Extrachromosomal circular DNA (eccDNA) has been observed in different species for decades, and more and more evidence shows that this specific type of DNA molecules may play an important role in rapid adaptation. Therefore, characterizing the full landscape of eccDNA has become critical, and there are several protocols for enriching eccDNAs and performing short-read or long-read sequencing. However, there is currently no available bioinformatic tool to identify eccDNAs from Nanopore reads. More importantly, the current tools based on Illumina short reads lack an efficient standardized pipeline notably to identify eccDNA originating from repeated loci and cannot be applied to very large genomes. Here, we introduce a comprehensive tool to solve both of these two issues. Applying ecc_finder to eccDNA-seq data (either mobilome-seq, Circle-Seq and CIDER-seq) from Arabidopsis, human, and wheat (with genome sizes ranging from 120Mb to 17 Gb), we document the improvement of computational time, sensitivity, and accuracy and demonstrate ecc_finder wide applicability and functionality.
RESUMEN
Together with local chromatin structure, gene accessibility, and the presence of transcription factors, gene positioning is implicated in gene expression regulation. Although the basic mechanisms are expected to be conserved in eukaryotes, less is known about the role of gene positioning in plant cells, mainly due to the lack of a highly resolutive approach. In this study, we adapted the use of the ANCHOR system to perform real-time single locus detection in planta. ANCHOR is a DNA-labeling tool derived from the chromosome partitioning system found in many bacterial species. We demonstrated its suitability to monitor a single locus in planta and used this approach to track chromatin mobility during cell differentiation in Arabidopsis thaliana root epidermal cells. Finally, we discussed the potential of this approach to investigate the role of gene positioning during transcription and DNA repair in plants.
RESUMEN
Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula × Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit. We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements.
Asunto(s)
Populus , Metilación de ADN/genética , Sequías , Regulación de la Expresión Génica de las Plantas , Meristema , Populus/genética , Interferencia de ARNRESUMEN
Active transposable elements (TEs) generate insertion polymorphisms that can be detected through genome resequencing strategies. However, these techniques may have limitations for organisms with large genomes or for somatic insertions. Here, we present a method that takes advantage of the extrachromosomal circular DNA (eccDNA) forms of actively transposing TEs in order to detect and characterize active TEs in any plant or animal tissue. Mobilome-seq consists in selectively amplifying and sequencing eccDNAs. It relies on linear digestion of genomic DNA followed by rolling circle amplification of circular DNA. Both active DNA transposons and retrotransposons can be identified using this technique.
Asunto(s)
Elementos Transponibles de ADN , ADN Circular/aislamiento & purificación , Animales , ADN de Plantas/genética , Técnicas de Amplificación de Ácido Nucleico , Plantas/genética , Análisis de Secuencia de ADNRESUMEN
Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.
Asunto(s)
Arabidopsis/genética , Resistencia a la Enfermedad/genética , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas , Genes de ARNr , Expresión Génica , Genes de Plantas , Genoma de Planta , Inestabilidad GenómicaRESUMEN
MAIN CONCLUSION: Copia/Ale is the youngest lineage in both Solanum tuberosum and S. commersonii. Within it, we identified nightshade, a new LTR element active in the cultivated potato. From an evolutionary perspective, long-terminal repeat retrotransposons (LTR-RT) activity during stress may be viewed as a mean by which organisms can keep up rates of genetic adaptation to changing conditions. Potato is one of the most important crop consumed worldwide, but studies on LTR-RT characterization are still lacking. Here, we assessed the abundance, insertion time and activity of LTR-RTs in both cultivated Solanum tuberosum and its cold-tolerant wild relative S. commersonii genomes. Gypsy elements were more abundant than Copia ones, suggesting that the former was somehow more successful in colonizing potato genomes. However, Copia elements, and in particular, the Ale lineage, are younger than Gypsy ones, since their insertion time was in average ~ 2 Mya. Due to the ability of LTR-RTs to be circularized by the host DNA repair mechanisms, we identified via mobilome-seq a Copia/Ale element (called nightshade, informal name used for potato family) active in S. tuberosum genome. Our analyses represent a valuable resource for comparative genomics within the Solanaceae, transposon-tagging and for the design of cultivar-specific molecular markers in potato.
Asunto(s)
Genoma de Planta/genética , Genómica , Retroelementos/ética , Solanum/genética , Secuencias Repetidas Terminales/genética , Frío , Evolución Molecular , Marcadores Genéticos/genética , Solanum/fisiología , Solanum tuberosum/genética , Solanum tuberosum/fisiología , Estrés FisiológicoRESUMEN
Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.
Asunto(s)
Arachis/genética , Arachis/clasificación , Argentina , Cromosomas de las Plantas/genética , Productos Agrícolas/genética , Metilación de ADN , ADN de Plantas/genética , Domesticación , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Variación Genética , Genoma de Planta , Hibridación Genética , Fenotipo , Poliploidía , Recombinación Genética , Especificidad de la Especie , TetraploidíaRESUMEN
Transposable elements (TEs) are parasitic DNA sequences that threaten genome integrity by replicative transposition in host gonads. The Piwi-interacting RNAs (piRNAs) pathway is assumed to maintain Drosophila genome homeostasis by downregulating transcriptional and post-transcriptional TE expression in the ovary. However, the bursts of transposition that are expected to follow transposome derepression after piRNA pathway impairment have not yet been reported. Here, we show, at a genome-wide level, that piRNA loss in the ovarian somatic cells boosts several families of the endogenous retroviral subclass of TEs, at various steps of their replication cycle, from somatic transcription to germinal genome invasion. For some of these TEs, the derepression caused by the loss of piRNAs is backed up by another small RNA pathway (siRNAs) operating in somatic tissues at the post transcriptional level. Derepressed transposition during 70 successive generations of piRNA loss exponentially increases the genomic copy number by up to 10-fold.
Asunto(s)
Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Células Germinativas/metabolismo , Ovario/metabolismo , ARN Interferente Pequeño/genética , Aneuploidia , Animales , Drosophila melanogaster/citología , Femenino , Silenciador del Gen , Genoma de los Insectos/genética , Células Germinativas/citología , Ovario/citología , Transducción de Señal/genéticaRESUMEN
MAIN CONCLUSION: We provide advances in DCL and RDR gene diversity in Solanaceae. We also shed light on DCL and RDR gene expression in response to cold stress. DICER-like (DCL) and RNA-dependent RNA polymerase (RDR) genes form the core components to trigger small non-coding RNA (ncRNA) production. In spite of this, little is known about the two gene families in non-model plant species. As their genome sequences are now available, the cultivated potato (Solanum tuberosum) and its cold-tolerant wild relative Solanum commersonii offer a valuable opportunity to advance our understanding of the above genes. To determine the extent of diversification and evolution of DCLs and RDRs in these species, we performed a comparative analysis. Seven DCLs were identified in the two species, whereas seven and six RDR genes were found in S. tuberosum and S. commersonii, respectively. Based on phylogenetic analysis with DCLs and RDRs from several species, we provide evidence for an increase in their number in both potato species. We also disclosed that tandem duplications played a major role in the evolution of these gene families in Solanaceae. DCL and RDR expression was investigated in different tissues and under cold and virus stresses, with divergent profiles of the tandem duplicated genes being found in different tissues. DCL paralogs showed a contrasting expression in S. tuberosum and S. commersonii following cold stress and virus infection. By contrast, no change in RDR transcript activity was detected following both stresses. Overall, this study provides the first comparative genomic analysis of the core components of the RNAi machinery in Solanaceae and offers a scaffold for future functional analysis of these gene families.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Genes de Plantas/genética , ARN Polimerasa Dependiente del ARN/genética , Ribonucleasa III/genética , Solanum tuberosum/genética , Solanum/genética , Duplicación de Gen/genética , Regulación de la Expresión Génica de las Plantas/genética , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Solanum/enzimología , Solanum tuberosum/enzimología , Estrés Fisiológico/genéticaRESUMEN
Transposable elements (TEs) were first identified through the polymorphisms they induced in plants and animals. Genomic studies have later revealed that TEs were highly abundant in eukaryotic genomes. Recently, more precise single individual genomic analyses have unravelled the huge diversity of TE insertions in many plant and animal species. In most cases the stress conditions behind this diversity are not known and neither is the adaptive capacity of these natural TE-induced variants. Here, we review some of the most recent examples of TE-related impacts on gene expression at the locus or the genome level and discuss the rich diversity of the TE repertoire and its potential role in adaptive evolution.
Asunto(s)
Elementos Transponibles de ADN/genética , Eucariontes/genética , Evolución Molecular , Genoma/genética , Animales , Expresión Génica/genética , GenómicaRESUMEN
The RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. We find that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. We suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.
Asunto(s)
Antivirales/inmunología , ADN Viral/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/inmunología , ARN Helicasas/metabolismo , Virus ARN/inmunología , Ribonucleasa III/metabolismo , Animales , Arbovirus/inmunología , Arbovirus/patogenicidad , Culicidae/inmunología , ARN Helicasas DEAD-box/metabolismo , Proteínas de Drosophila/genética , Genes Virales/genética , Genoma Viral , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Mutación Puntual , ARN Helicasas/genética , Interferencia de ARN/inmunología , Infecciones por Virus ARN , Virus ARN/genética , Virus ARN/patogenicidad , ARN Interferente Pequeño/genética , ARN Viral/metabolismo , Ribonucleasa III/genética , Carga Viral , Replicación ViralRESUMEN
Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes.
Asunto(s)
Ecología , Epigénesis Genética , Plantas , Metilación de ADN , EcosistemaRESUMEN
BACKGROUND: Transposables elements (TEs) contribute to both structural and functional dynamics of most eukaryotic genomes. Because of their propensity to densely populate plant and animal genomes, the precise estimation of the impact of transposition on genomic diversity has been considered as one of the main challenges of today's genomics. The recent development of NGS (next generation sequencing) technologies has open new perspectives in population genomics by providing new methods for high throughput detection of Transposable Elements-associated Structural Variants (TEASV). However, these have relied on Illumina platform that generates short reads (up to 350 nucleotides). This limitation in size of sequence reads can cause high false discovery rate (FDR) and therefore limit the power of detection of TEASVs, especially in the case of large, complex genomes. The newest sequencing technologies, such as Oxford Nanopore Technologies (ONT) can generate kilobases-long reads thus representing a promising tool for TEASV detection in plant and animals. RESULTS: We present the results of a pilot experiment for TEASV detection on the model plant species Arabidopsis thaliana using ONT sequencing and show that it can be used efficiently to detect TE movements. We generated a ~0.8X genome coverage of a met1-derived epigenetic recombinant inbred line (epiRIL) using a MinIon device with R7 chemistry. We were able to detect nine new copies of the LTR-retrotransposon Evadé (EVD). We also evidenced the activity of the DNA transposon CACTA, CAC1. CONCLUSIONS: Even at a low sequence coverage (0.8X), ONT sequencing allowed us to reliably detect several TE insertions in Arabidopsis thaliana genome. The long read length allowed a precise and un-ambiguous mapping of the structural variations caused by the activity of TEs. This suggests that the trade-off between read length and genome coverage for TEASV detection may be in favor of the former. Should the technology be further improved both in terms of lower error rate and operation costs, it could be efficiently used in diversity studies at population level.