RESUMEN
Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.
Asunto(s)
Citosina , Metilación de ADN , ADN Ribosómico , Silenciador del Gen , Citosina/metabolismo , ADN Ribosómico/genética , Variaciones en el Número de Copia de ADN , Transcripción Genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Cromosomas de las Plantas/genéticaRESUMEN
While the phenomenon of uniparental silencing of 35S rDNA in interspecific hybrids and allopolyploids is well documented, there is a notable absence of information regarding whether such silencing extends to the 5S RNA component of ribosomes. To address this gap in knowledge, we analyzed the 5S and 35S rDNA expression in Cardamine (Brassicaceae) allopolyploids, namely C. × insueta (2n = 3x = 24, genome composition RRA), C. flexuosa (2n = 4x = 32, AAHH), and C. scutata (2n = 4x = 32, PPAA) which share a common diploid ancestor (AA). We employed high-throughput sequencing of transcriptomes and genomes and phylogenetic analyses of 5S rRNA variants. The genomic organization of rDNA was further scrutinized through clustering and fluorescence in situ hybridization. In the C. × insueta allotriploid, we observed uniparental dominant expression of 5S and 35S rDNA loci. In the C. flexuosa and C. scutata allotetraploids, the expression pattern differed, with the 35S rDNA being expressed from the A subgenome, whereas the 5S rDNA was expressed from the partner subgenome. Both C. flexuosa and C. scutata but not C. × insueta showed copy and locus number changes. We conclude that in stabilized allopolyploids, transcription of ribosomal RNA components occurs from different subgenomes. This phenomenon appears to result in the formation of chimeric ribosomes comprising rRNA molecules derived from distinct parental origins. We speculate that the interplay of epigenetic silencing and rDNA rearrangements introduces an additional layer of variation in multimolecule ribosomal complexes, potentially contributing to the evolutionary success of allopolyploids.
Asunto(s)
Cardamine , Silenciador del Gen , Filogenia , Poliploidía , ARN Ribosómico 5S , ARN Ribosómico 5S/genética , Cardamine/genética , Genoma de Planta/genética , ADN Ribosómico/genética , Hibridación Fluorescente in Situ , Regulación de la Expresión Génica de las PlantasRESUMEN
Accumulation and selection of nucleotides is one of the most challenging problems surrounding the origin of the first RNA molecules on our planet. In the current work we propose that guanosine 3',5' cyclic monophosphate could selectively crystallize upon evaporation of an acidic prebiotic pool containing various other nucleotides. The conditions of the evaporative crystallization are fully compatible with the subsequent acid catalyzed polymerization of this cyclic nucleotide reported in earlier studies and may be relevant in a broad range of possible prebiotic environments. Albeit cytidine 3',5' cyclic monophosphate has the ability to selectively accumulate under the same conditions, its crystal structure is not likely to support polymer formation.
RESUMEN
The assembly of ancient informational polymers from nucleotide precursors is the central challenge of life's origin on our planet. Among the possible solutions, dry polymerization of 3',5'-cyclic guanosine monophosphate (3',5'-cGMP) has been proposed as a candidate to create oligonucleotides of 15-20â units in length. However, the reported sensitivity of the reaction to the presence of cations raised questions of whether this chemistry could be relevant in a geological context. The experiments in this study show that the presence of cations is not restrictive as long as the reaction is conducted in an acidic environment, in contrast to previous reports that suggested optimal conditions at pHâ 9.
Asunto(s)
GMP Cíclico , ARN , Catálisis , Oligonucleótidos , PolimerizacionRESUMEN
We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively.The data analyses show the following: (i) A high variability in 5S and 45S loci numbers, with both showing 50-fold or higher variability. However, karyotypes with an extremely high number of loci were rare, and medians generally converged to two 5S sites and two 45S rDNA sites per diploid genome. No relationship was observed between the number of 5S and 45S loci. (ii) The position of 45S rDNA on sex chromosomes was relatively frequent in some groups, particularly in arthropods (14% of karyotypes). Furthermore, 45S rDNA was almost exclusively located in microchromosomes when these were present (in birds and reptiles). (iii) The proportion of active NORs (positively stained with silver staining methods) progressively decreased with an increasing number of 45S rDNA loci, and karyotypes with more than 12 loci showed, on average, less than 40% of active loci. In conclusion, the updated version of the database provides some new insights into the organization of rRNA genes in chromosomes. We expect that its updated content will be useful for taxonomists, comparative cytogeneticists, and evolutionary biologists. .
Asunto(s)
ADN Ribosómico/genética , ARN Ribosómico 5S/genética , ARN Ribosómico/genética , Animales , Bases de Datos Genéticas , Evolución Molecular , Cariotipo , Cariotipificación , Especificidad de la EspecieRESUMEN
The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major SARS-CoV-2 lineages in the GISAID database, including genotypes arising during COVID-19 therapy. Globally, the C>U transitions and G>U transversions were the most represented mutations, accounting for the majority of single-nucleotide variations. Mutational spectra were not influenced by the time the virus had been circulating in its host or medical treatment. At the amino acid level, we observed about a 2-fold excess of substitutions in favor of hydrophobic amino acids over the reverse. However, most mutations constituting variants of interests of the S-protein (spike) lead to hydrophilic amino acids, counteracting the global trend. The C>U and G>U substitutions altered codons towards increased amino acid hydrophobicity values in more than 80% of cases. The bias is explained by the existing differences in the codon composition for amino acids bearing contrasting biochemical properties. Mutation asymmetries apparently influence the biochemical features of SARS CoV-2 proteins, which may impact protein-protein interactions, fusion of viral and cellular membranes, and virion assembly.
Asunto(s)
COVID-19/virología , Genoma Viral , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , SARS-CoV-2/genética , Proteínas Virales/química , Proteínas Virales/genética , Desaminasas APOBEC , Alelos , Sustitución de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Evolución Molecular , Variación Genética , Genotipo , Interacciones Huésped-Patógeno , Humanos , Filogenia , Polimorfismo de Nucleótido Simple , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.
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Betacoronavirus/genética , Citosina/metabolismo , Evolución Molecular , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Uracilo/metabolismo , Animales , Secuencia de Bases , Betacoronavirus/clasificación , Betacoronavirus/aislamiento & purificación , Quirópteros/virología , Islas de CpG , Humanos , Filogenia , Polimorfismo de Nucleótido Simple , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/clasificación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Introduction: In plants, the multicopy genes encoding ribosomal RNA (rDNA) typically exhibit heterochromatic features and high level of DNA methylation. Here, we explored rDNA methylation in early diverging land plants from Bryophyta (15 species, 14 families) and Marchantiophyta (4 species, 4 families). DNA methylation was investigated by methylation-sensitive Southern blot hybridization in all species. We also carried out whole genomic bisulfite sequencing in Polytrichum formosum (Polytrichaceae) and Dicranum scoparium (Dicranaceae) and used available model plant methyloms (Physcomitrella patents and Marchantia polymorpha) to determine rDNA unit-wide methylation patterns. Chromatin structure was analyzed using fluorescence in situ hybridization (FISH) and immunoprecipitation (CHIP) assays. Results: In contrast to seed plants, bryophyte rDNAs were efficiently digested with methylation-sensitive enzymes indicating no or low levels of CG and CHG methylation in these loci. The rDNA methylom analyses revealed variation between species ranging from negligible (<3%, P. formosum, P. patens) to moderate (7 and 17% in M. polymorpha and D. scoparium, respectively) methylation levels. There were no differences between coding and noncoding parts of rDNA units and between gametophyte and sporophyte tissues. However, major satellite repeat and transposable elements were heavily methylated in P. formosum and D. scoparium. In P. formosum rDNA, the euchromatic H3K4m3 and heterochromatic H3K9m2 histone marks were nearly balanced contrasting the angiosperms data where H3K9m2 typically dominates rDNA chromatin. In moss interphase nuclei, rDNA was localized at the nucleolar periphery and its condensation level was high. Conclusions: Unlike seed plants, the rRNA genes seem to escape global methylation machinery in bryophytes. Distinct epigenetic features may be related to rDNA expression and the physiology of these early diverging plants that exist in haploid state for most of their life cycles.
RESUMEN
The intergenic spacer (IGS) of rDNA is frequently built of long blocks of tandem repeats. To estimate the intragenomic variability of such knotty regions, we employed PacBio sequencing of the Cucurbita moschata genome, in which thousands of rDNA copies are distributed across a number of loci. The rRNA coding regions are highly conserved, indicating intensive interlocus homogenization and/or high selection pressure. However, the IGS exhibits high intragenomic structural diversity. Two repeated blocks, R1 (300-1250 bp) and R2 (290-643 bp), account for most of the IGS variation. They exhibit minisatellite-like features built of multiple periodically spaced short GC-rich sequence motifs with the potential to adopt non-canonical DNA conformations, G-quadruplex-folded and left-handed Z-DNA. The mutual arrangement of these motifs can be used to classify IGS variants into five structural families. Subtle polymorphisms exist within each family due to a variable number of repeats, suggesting the coexistence of an enormous number of IGS variants. The substantial length and structural heterogeneity of IGS minisatellites suggests that the tempo of their divergence exceeds the tempo of the homogenization of rDNA arrays. As frequently occurring among plants, we hypothesize that their instability may influence transcription regulation and/or destabilize rDNA units, possibly spreading them across the genome.
Asunto(s)
Cucurbita/genética , ADN Espaciador Ribosómico/genética , Variación Genética , Repeticiones de Minisatélite , Conformación de Ácido Nucleico , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/metabolismo , Análisis de Secuencia de ADNRESUMEN
Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d-rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p-rDNA dominant progenitor were meiotically unstable, frequently switching to co-dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d-rDNA dominance, indicating immediate suppression of p-homeologs in F1 hybrids. Original p-rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p-rDNA and d-rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co-dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids.
Asunto(s)
ADN Ribosómico/genética , Epigenómica , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Genoma de Planta/genética , Tragopogon/genética , Metilación de ADN , ADN de Plantas/genética , Silenciador del Gen , Genotipo , Hibridación Genética , Fenotipo , PoliploidíaRESUMEN
In plants, genome duplication followed by genome diversification and selection is recognized as a major evolutionary process. Rapid epigenetic and genetic changes that affect the transcription of parental genes are frequently observed after polyploidization. The pattern of alternative splicing is also frequently altered, yet the related molecular processes remain largely unresolved. Here, we study the inheritance and expression of parental variants of three floral organ identity genes in allotetraploid tobacco. DEFICIENS and GLOBOSA are B-class genes, and AGAMOUS is a C-class gene. Parental variants of these genes were found to be maintained in the tobacco genome, and the respective mRNAs were present in flower buds in comparable amounts. However, among five tobacco cultivars, we identified two in which the majority of paternal GLOBOSA pre-mRNA transcripts undergo exon 3 skipping, producing an mRNA with a premature termination codon. At the DNA level, we identified a G-A transition at the very last position of exon 3 in both cultivars. Although alternative splicing resulted in a dramatic decrease in full-length paternal GLOBOSA mRNA, no phenotypic effect was observed. Our finding likely serves as an example of the initiation of homoeolog diversification in a relatively young polyploid genome.
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Proteínas de Homeodominio/genética , Proteínas de Plantas/genética , Mutación Puntual/genética , Precursores del ARN/genética , Transcripción Genética , Empalme Alternativo/genética , Exones/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/biosíntesis , Nucleótidos/genética , Proteínas de Plantas/biosíntesis , Poliploidía , Nicotiana/genéticaRESUMEN
DNA containing a sequence that generates a local curvature exhibits a pronounced retardation in electrophoretic mobility. Various theoretical models have been proposed to explain relationship between DNA structural features and migration anomaly. Here, we studied the capacity of 15 static wedge-bending models to predict electrophoretic behavior of 69 satellite monomers derived from four divergent families. All monomers exhibited retarded mobility in PAGE corresponding to retardation factors ranging 1.02-1.54. The curvature varied both within and across the groups and correlated with the number, position, and lengths of A-tracts. Two dinucleotide models provided strong correlation between gel mobility and curvature prediction; two trinucleotide models were satisfactory while remaining dinucleotide models provided intermediate results with reliable prediction for subsets of sequences only. In some cases, similarly shaped molecules exhibited relatively large differences in mobility and vice versa. Generally less accurate predictions were obtained in groups containing less homogeneous sequences possessing distinct structural features. In conclusion, relatively universal theoretical models were identified suitable for the analysis of natural sequences known to harbor relatively moderate curvature. These models could be potentially applied to genome wide studies. However, in silico predictions should be viewed in context of experimental measurement of intrinsic DNA curvature.
Asunto(s)
ADN Satélite/química , Ensayo de Cambio de Movilidad Electroforética/métodos , Electroforesis en Gel de Poliacrilamida Nativa/métodos , Secuencia de Bases , Simulación por Computador , ADN de Plantas/química , Ensayo de Cambio de Movilidad Electroforética/normas , Modelos Químicos , Datos de Secuencia Molecular , Electroforesis en Gel de Poliacrilamida Nativa/normas , Reproducibilidad de los Resultados , Alineación de Secuencia , Nicotiana/químicaRESUMEN
BACKGROUND: Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units. METHODS: We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci. RESULTS AND CONCLUSIONS: In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations.
Asunto(s)
ADN de Plantas/genética , ADN Ribosómico/genética , Diploidia , Sitios Genéticos/genética , Variación Genética/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Nicotiana/genética , Southern Blotting , ADN Espaciador Ribosómico/genética , Dosificación de Gen/genética , Genes de Plantas/genética , Análisis de Secuencia de ADNRESUMEN
⢠Allopolyploidy, a driving force in plant evolution, can induce rapid structural changes in parental subgenomes. Here, we examined the fate of homologous subtelomeric satellites in intrasection allotetraploid Nicotiana arentsii formed from N. undulata and N. wigandioides progenitors < 200,000 yr ago. ⢠We cloned and sequenced a number of monomers from progenitors and the allotetraploid. Structural features of both cloned and genomic monomers were studied using double-strand conformation polymorphism analysis. ⢠Two homologous satellites were isolated from N. undulata (called NUNSSP) and N. wigandioides (NWISSP). While the NUNSSP monomers were highly homogeneous in nucleotide sequences, the NWISSP monomers formed two separate clades. Likewise, the genomic NUNSSP monomers showed less DNA conformation heterogeneity than NWISSP monomers, with distinct conformations. While both satellites predominantly occupy subtelomeric positions, a fraction of the NWISSP repeats was found in an intercalary location, supporting the hypothesis that dispersion prevents the repeats becoming homogeneous. Sequence, structural and chromosomal features of the parental satellites were faithfully inherited by N. arentsii. ⢠Our study revealed that intergenomic homogenization of subtelomeric satellite repeats does not occur in N. arentsii allotetraploid. We propose that the sequence and structural divergence of subtelomeric satellites may render allopolyploid chromosomes less vulnerable to intergenomic exchanges.
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ADN Satélite/genética , Nicotiana/genética , Conformación de Ácido Nucleico , Polimorfismo Genético , Poliploidía , Análisis de Secuencia de ADN/métodos , Southern Blotting , Cromosomas de las Plantas/genética , ADN Satélite/aislamiento & purificación , Diploidia , Genoma de Planta/genética , Cariotipificación , Datos de Secuencia Molecular , Filogenia , Secuencias Repetitivas de Ácidos Nucleicos/genética , Especificidad de la EspecieRESUMEN
Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.
Asunto(s)
Adenosilhomocisteinasa/metabolismo , Epigénesis Genética/fisiología , Flores/anatomía & histología , Regulación de la Expresión Génica de las Plantas/fisiología , Germinación/fisiología , Nicotiana/fisiología , Adenina/análogos & derivados , Adenina/toxicidad , Adenosilhomocisteinasa/antagonistas & inhibidores , Southern Blotting , Metilación de ADN , Cartilla de ADN/genética , ADN Complementario/genética , Epigénesis Genética/efectos de los fármacos , Flores/fisiología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Germinación/efectos de los fármacos , Proteínas de Plantas/metabolismo , Polen/fisiología , Estadísticas no Paramétricas , Nicotiana/enzimologíaRESUMEN
BACKGROUND: Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids. RESULTS: Using Southern blot hybridization and fluorescent in situ hybridization (FISH), we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals) and four lines of synthetic T. miscellus (71 individuals). Variation among diploid parents accounted for most of the observed gene imbalances detected in F1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids. CONCLUSIONS: Uniparental reductions of homeologous rRNA gene copies occurred in both synthetic and natural populations of Tragopogon allopolyploids. The extent of these rDNA changes was generally higher in natural populations than in the synthetic lines. We hypothesize that locus-specific and chromosomal changes in early generations of allopolyploids may influence patterns of rDNA evolution in later generations.
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Asteraceae/genética , ADN Ribosómico/genética , Evolución Molecular , Southern Blotting , Diploidia , Hibridación Genética/genética , Hibridación Fluorescente in Situ , TetraploidíaRESUMEN
Allopolyploids represent natural experiments in which DNA sequences from different species are combined into a single nucleus and then coevolve, enabling us to follow the parental genomes, their interactions and evolution over time. Here, we examine the fate of satellite DNA over 5 million yr of divergence in plant genus Nicotiana (family Solanaceae). We isolated subtelomeric, tandemly repeated satellite DNA from Nicotiana diploid and allopolyploid species and analysed patterns of inheritance and divergence by sequence analysis, Southern blot hybridization and fluorescent in situ hybridization (FISH). We observed that parental satellite sequences redistribute around the genome in allopolyploids of Nicotiana section Polydicliae, formed c. 1 million yr ago (Mya), and that new satellite repeats evolved and amplified in section Repandae, which was formed c. 5 Mya. In some cases that process involved the complete replacement of parental satellite sequences. The rate of satellite repeat replacement is faster than theoretical predictions assuming the mechanism involved is unequal recombination and crossing-over. Instead we propose that this mechanism occurs with the deletion of large chromatin blocks and reamplification, perhaps via rolling circle replication.
Asunto(s)
ADN Satélite/genética , Nicotiana/genética , Poliploidía , Secuencias Repetitivas de Ácidos Nucleicos/genética , Southern Blotting , Clonación Molecular , Diploidia , Hibridación Fluorescente in Situ , Patrón de Herencia/genética , Filogenia , Reacción en Cadena de la Polimerasa , Especificidad de la Especie , Factores de Tiempo , Nicotiana/citologíaRESUMEN
The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Genes de Plantas , Familia de Multigenes , Nicotiana/enzimología , Nicotiana/genética , Secuencia de Bases , Clonación Molecular , ADN (Citosina-5-)-Metiltransferasas/clasificación , Metilación de ADN/genética , ADN de Plantas/genética , Diploidia , Epigénesis Genética , Expresión Génica , Genoma de Planta , Datos de Secuencia Molecular , Filogenia , Poliploidía , Secuencias Repetitivas de Ácidos Nucleicos , Selección Genética , Distribución TisularRESUMEN
BACKGROUND: Polyploidy, frequently termed "whole genome duplication", is a major force in the evolution of many eukaryotes. Indeed, most angiosperm species have undergone at least one round of polyploidy in their evolutionary history. Despite enormous progress in our understanding of many aspects of polyploidy, we essentially have no information about the role of chromosome divergence in the establishment of young polyploid populations. Here we investigate synthetic lines and natural populations of two recently and recurrently formed allotetraploids Tragopogon mirus and T. miscellus (formed within the past 80 years) to assess the role of aberrant meiosis in generating chromosomal/genomic diversity. That diversity is likely important in the formation, establishment and survival of polyploid populations and species. METHODOLOGY/PRINCIPAL FINDINGS: Applications of fluorescence in situ hybridisation (FISH) to natural populations of T. mirus and T. miscellus suggest that chromosomal rearrangements and other chromosomal changes are common in both allotetraploids. We detected extensive chromosomal polymorphism between individuals and populations, including (i) plants monosomic and trisomic for particular chromosomes (perhaps indicating compensatory trisomy), (ii) intergenomic translocations and (iii) variable sizes and expression patterns of individual ribosomal DNA (rDNA) loci. We even observed karyotypic variation among sibling plants. Significantly, translocations, chromosome loss, and meiotic irregularities, including quadrivalent formation, were observed in synthetic (S(0) and S(1) generations) polyploid lines. Our results not only provide a mechanism for chromosomal variation in natural populations, but also indicate that chromosomal changes occur rapidly following polyploidisation. CONCLUSIONS/SIGNIFICANCE: These data shed new light on previous analyses of genome and transcriptome structures in de novo and establishing polyploid species. Crucially our results highlight the necessity of studying karyotypes in young (<150 years old) polyploid species and synthetic polyploids that resemble natural species. The data also provide insight into the mechanisms that perturb inheritance patterns of genetic markers in synthetic polyploids and populations of young natural polyploid species.
Asunto(s)
Cromosomas de las Plantas , Evolución Molecular , Poliploidía , Tragopogon/genética , ADN Ribosómico/genética , Diploidia , Genoma de Planta , Hibridación Fluorescente in Situ , Cariotipificación , Meiosis/fisiología , Mitosis/fisiología , Tragopogon/anatomía & histologíaRESUMEN
Analyses of selected bacterial artificial chromosomes (BACs) clones suggest that the retrotransposon component of angiosperm genomes can be amplified or deleted, leading to genome turnover. Here, Nicotiana allopolyploids were used to characterize the nature of sequence turnover across the whole genome in allopolyploids known to be of different ages. Using molecular-clock analyses, the likely age of Nicotiana allopolyploids was estimated. Genomic in situ hybridization (GISH) and tandem repeat characterization were used to determine how the parental genomic compartments of these allopolyploids have diverged over time. Paternal genome sequence losses, retroelement activity and intergenomic translocation have been reported in early Nicotiana tabacum evolution (up to 200,000 yr divergence). Here it is shown that within 1 million years of allopolyploid divergence there is considerable exchange of repeats between parental chromosome sets. After c. 5 million years of divergence GISH fails. This GISH failure may represent near-complete genome turnover, probably involving the replacement of nongenic sequences with new, or previously rare sequence types, all occurring within a conserved karyotype structure. This mode of evolution may influence or be influenced by long-term diploidization processes that characterize angiosperm polyploidy-diploid evolutionary cycles.