Centromeres in the legume genera Pisum and Lathyrus exhibit unique morphological characteristics, including extended primary constrictions and multiple separate domains of centromeric chromatin. These so-called metapolycentromeres resemble an intermediate form between monocentric and holocentric types, and therefore provide a great opportunity for studying the transitions between different types of centromere organizations. However, because of the exceedingly large and highly repetitive nature of metapolycentromeres, highly contiguous assemblies needed for these studies are lacking. Here, we report on the assembly and analysis of a 177.6 Mb region of pea (Pisum sativum) chromosome 6, including the 81.6 Mb centromere region (CEN6) and adjacent chromosome arms. Genes, DNA methylation profiles, and most of the repeats were uniformly distributed within the centromere, and their densities in CEN6 and chromosome arms were similar. The exception was an accumulation of satellite DNA in CEN6, where it formed multiple arrays up to 2 Mb in length. Centromeric chromatin, characterized by the presence of the CENH3 protein, was predominantly associated with arrays of three different satellite repeats; however, five other satellites present in CEN6 lacked CENH3. The presence of CENH3 chromatin was found to determine the spatial distribution of the respective satellites during the cell cycle. Finally, oligo-FISH painting experiments, performed using probes specifically designed to label the genomic regions corresponding to CEN6 in Pisum, Lathyrus, and Vicia species, revealed that metapolycentromeres evolved via the expansion of centromeric chromatin into neighboring chromosomal regions and the accumulation of novel satellite repeats. However, in some of these species, centromere evolution also involved chromosomal translocations and centromere repositioning.
- MeSH
- centromera genetika MeSH
- chromatin genetika MeSH
- hrách setý * genetika MeSH
- lidé MeSH
- lidské chromozomy, pár 6 * MeSH
- satelitní DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Repeat-rich regions of higher plant genomes are usually associated with constitutive heterochromatin, a specific type of chromatin that forms tightly packed nuclear chromocenters and chromosome bands. There is a large body of cytogenetic evidence that these chromosome regions are often composed of tandemly organized satellite DNA. However, comparatively little is known about the sequence arrangement within heterochromatic regions, which are difficult to assemble due to their repeated nature. Here, we explore long-range sequence organization of heterochromatin regions containing the major satellite repeat CUS-TR24 in the holocentric plant Cuscuta europaea. Using a combination of ultra-long read sequencing with assembly-free sequence analysis, we reveal the complex structure of these loci, which are composed of short arrays of CUS-TR24 interrupted frequently by emerging simple sequence repeats and targeted insertions of a specific lineage of LINE retrotransposons. These data suggest that the organization of satellite repeats constituting heterochromatic chromosome bands can be more complex than previously envisioned, and demonstrate that heterochromatin organization can be efficiently investigated without the need for genome assembly.
- Publikační typ
- časopisecké články MeSH
Amplification of monomer sequences into long contiguous arrays is the main feature distinguishing satellite DNA from other tandem repeats, yet it is also the main obstacle in its investigation because these arrays are in principle difficult to assemble. Here we explore an alternative, assembly-free approach that utilizes ultra-long Oxford Nanopore reads to infer the length distribution of satellite repeat arrays, their association with other repeats and the prevailing sequence periodicities. Using the satellite DNA-rich legume plant Lathyrus sativus as a model, we demonstrated this approach by analyzing 11 major satellite repeats using a set of nanopore reads ranging from 30 to over 200 kb in length and representing 0.73× genome coverage. We found surprising differences between the analyzed repeats because only two of them were predominantly organized in long arrays typical for satellite DNA. The remaining nine satellites were found to be derived from short tandem arrays located within LTR-retrotransposons that occasionally expanded in length. While the corresponding LTR-retrotransposons were dispersed across the genome, this array expansion occurred mainly in the primary constrictions of the L. sativus chromosomes, which suggests that these genome regions are favourable for satellite DNA accumulation.
- MeSH
- centromera MeSH
- chromozomy rostlin MeSH
- DNA rostlinná genetika MeSH
- frekvence genu * MeSH
- genom rostlinný MeSH
- heterochromatin MeSH
- Lathyrus genetika MeSH
- molekulární evoluce MeSH
- nanopóry * MeSH
- retroelementy * MeSH
- satelitní DNA * MeSH
- tandemové repetitivní sekvence * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RepeatExplorer2 is a novel version of a computational pipeline that uses graph-based clustering of next-generation sequencing reads for characterization of repetitive DNA in eukaryotes. The clustering algorithm facilitates repeat identification in any genome by using relatively small quantities of short sequence reads, and additional tools within the pipeline perform automatic annotation and quantification of the identified repeats. The pipeline is integrated into the Galaxy platform, which provides a user-friendly web interface for script execution and documentation of the results. Compared to the original version of the pipeline, RepeatExplorer2 provides automated annotation of transposable elements, identification of tandem repeats and enhanced visualization of analysis results. Here, we present an overview of the RepeatExplorer2 workflow and provide procedures for its application to (i) de novo repeat identification in a single species, (ii) comparative repeat analysis in a set of species, (iii) development of satellite DNA probes for cytogenetic experiments and (iv) identification of centromeric repeats based on ChIP-seq data. Each procedure takes approximately 2 d to complete. RepeatExplorer2 is available at https://repeatexplorer-elixir.cerit-sc.cz .
- MeSH
- DNA sondy chemie genetika MeSH
- DNA chemie genetika MeSH
- genomika metody MeSH
- lidé MeSH
- repetitivní sekvence nukleových kyselin MeSH
- sekvenční analýza DNA metody MeSH
- shluková analýza MeSH
- software MeSH
- transpozibilní elementy DNA MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Satellite repeats are major sequence constituents of centromeres in many plant and animal species. Within a species, a single family of satellite sequences typically occupies centromeres of all chromosomes and is absent from other parts of the genome. Due to their common origin, sequence similarities exist among the centromere-specific satellites in related species. Here, we report a remarkably different pattern of centromere evolution in the plant tribe Fabeae, which includes genera Pisum, Lathyrus, Vicia, and Lens. By immunoprecipitation of centromeric chromatin with CENH3 antibodies, we identified and characterized a large and diverse set of 64 families of centromeric satellites in 14 species. These families differed in their nucleotide sequence, monomer length (33-2,979 bp), and abundance in individual species. Most families were species-specific, and most species possessed multiple (2-12) satellites in their centromeres. Some of the repeats that were shared by several species exhibited promiscuous patterns of centromere association, being located within CENH3 chromatin in some species, but apart from the centromeres in others. Moreover, FISH experiments revealed that the same family could assume centromeric and noncentromeric positions even within a single species. Taken together, these findings suggest that Fabeae centromeres are not shaped by the coevolution of a single centromeric satellite with its interacting CENH3 proteins, as proposed by the centromere drive model. This conclusion is also supported by the absence of pervasive adaptive evolution of CENH3 sequences retrieved from Fabeae species.
Centromeres are essential for proper chromosome segregation to the daughter cells during mitosis and meiosis. Chromosomes of most eukaryotes studied so far have regional centromeres that form primary constrictions on metaphase chromosomes. These monocentric chromosomes vary from point centromeres to so-called "meta-polycentromeres", with multiple centromere domains in an extended primary constriction, as identified in Pisum and Lathyrus species. However, in various animal and plant lineages centromeres are distributed along almost the entire chromosome length. Therefore, they are called holocentromeres. In holocentric plants, centromere-specific proteins, at which spindle fibers usually attach, are arranged contiguously (line-like), in clusters along the chromosomes or in bands. Here, we summarize findings of ultrastructural investigations using immunolabeling with centromere-specific antibodies and super-resolution microscopy to demonstrate the structural diversity of plant centromeres. A classification of the different centromere types has been suggested based on the distribution of spindle attachment sites. Based on these findings we discuss the possible evolution and advantages of holocentricity, and potential strategies to segregate holocentric chromosomes correctly.
We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel's original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.
- MeSH
- chromozomy rostlin genetika MeSH
- Fabaceae klasifikace genetika MeSH
- fenotyp MeSH
- fylogeneze MeSH
- genetická variace MeSH
- genom rostlinný * MeSH
- genomika MeSH
- hrách setý genetika MeSH
- lokus kvantitativního znaku * MeSH
- mapování chromozomů MeSH
- molekulární evoluce * MeSH
- referenční standardy MeSH
- regulace genové exprese u rostlin MeSH
- repetitivní sekvence nukleových kyselin MeSH
- rostlinné proteiny genetika MeSH
- sekvenování celého genomu MeSH
- zásobní proteiny semen genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Satellite DNA, a class of repetitive sequences forming long arrays of tandemly repeated units, represents substantial portions of many plant genomes yet remains poorly characterized due to various methodological obstacles. Here we show that the genome of the field bean (Vicia faba, 2n = 12), a long-established model for cytogenetic studies in plants, contains a diverse set of satellite repeats, most of which remained concealed until their present investigation. Using next-generation sequencing combined with novel bioinformatics tools, we reconstructed consensus sequences of 23 novel satellite repeats representing 0.008-2.700% of the genome and mapped their distribution on chromosomes. We found that in addition to typical satellites with monomers hundreds of nucleotides long, V. faba contains a large number of satellite repeats with unusually long monomers (687-2033 bp), which are predominantly localized in pericentromeric regions. Using chromatin immunoprecipitation with CenH3 antibody, we revealed an extraordinary diversity of centromeric satellites, consisting of seven repeats with chromosome-specific distribution. We also found that in spite of their different nucleotide sequences, all centromeric repeats are replicated during mid-S phase, while most other satellites are replicated in the first part of late S phase, followed by a single family of FokI repeats representing the latest replicating chromatin.
- MeSH
- anotace sekvence MeSH
- centromera metabolismus MeSH
- chromatinová imunoprecipitace MeSH
- DNA rostlinná genetika metabolismus MeSH
- genom rostlinný genetika MeSH
- mapování chromozomů metody MeSH
- molekulární evoluce MeSH
- načasování replikace DNA genetika MeSH
- satelitní DNA genetika MeSH
- sekvenční analýza DNA MeSH
- Vicia faba genetika metabolismus MeSH
- výpočetní biologie MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cíl studie: Zjistit, kolik pacientů podstupujících předanestetické vyšetření (PAV) má chytrý telefon a jaký je podíl pacientů ochotných podstoupit PAV nebo jeho část s využitím telefonu nebo jiné formy elektronické komunikace. Typ studie: Observační prospektivní multicentrická cross-sectional. Typ pracoviště: Anesteziologické ambulance pěti fakultních/krajských nemocnic. Materiál a metoda: Do studie byli zařazováni pacienti podstupující PAV před plánovaným operačním výkonem s požadavkem anesteziologické péče. U každého pacienta byly dotazníkovou formou zaznamenány údaje: věk, pohlaví, nejvyšší dosažené vzdělání a odpověď na otázky – „používáte internet?“, „máte přístup na e-mail?“, „máte chytrý telefon?“, „pokud by byla možnost provedení PAV přes internet a nemusel jste jít kvůli tomu do zdravotnického zařízení, využil/a byste dané možnosti?“. Byly použity deskriptivní statistické metody. Výsledky: Byla analyzována data od 1685 pacientů. Vlastnictví chytrého telefonu udáválo 957 pacientů (57 %), dostupnost internetu 1330 pacientů (79 %), vlastnictví e-mailového účtu 1260 pacientů (75 %). Ochotu absolvovat PAV s využitím internetu udávalo 536 pacientů (32 %). Ochota k provedení PAV přes internet byla významně vyšší u pacientů s VŠ vzděláním (164 z 343, 48 %). Závěr: Vybavenost mobilními technologiemi a přístup k internetu byly zjištěny u více než poloviny dotazovaných pacientů. Ochota k PAV bez osobního kontaktu s lékařem byla vyšší u pacientů s vysokoškolským vzděláním.
Objective: The aim of our study was to find out whether patients scheduled for elective surgery owned a smartphone, had access to internet/e-mail and whether they would be willing to undergo preoperative assessment (POA) using internet/mobile devices. Design: Observational, prospective, multicentre, cross-sectional study. Setting: Preoperative clinics at tertiary care hospitals in the Czech Republic. Materials and methods: The study was based on a simple questionnaire which the patients had to answer at the end of their POA appointment. The recorded data included age, gender, level of education, ownership of a smartphone, access to internet/e-mail and the patients' willingness to undergo remote POA. The obtained data were analysed using descriptive statistical methods. Results: We analysed data from 1685 patients. Total 957 (57%) of them owned a smartphone, 1330 (79%) had access to the internet and 1260 (75%) used e-mail. Total 536 patients (32%) were willing to undergo remote POA. Patients with a university degree (164 of 343, 48%) were more likely to be willing to have remote POA. Conclusion: More than half of the patients undergoing POA owned a smartphone, had internet connection and used e-mail. Overall one third of the patients expressed their willingness to undergo remote POA; this proportion was higher in patents with a degree.
