BACKGROUND: The i-motif is a tetrameric DNA structure based on the formation of hemiprotonated cytosine-cytosine (C+.C) base pairs. i-motifs are widely used in nanotechnology. In biological systems, i-motifs are involved in gene regulation and in control of genome integrity. In vivo, the i-motif forming sequences are subjects of epigenetic modifications, particularly 5-cytosine methylation. In plants, natively occurring methylation patterns lead to a complex network of C+.C, 5mC+.C and 5mC+.5mC base-pairs in the i-motif stem. The impact of complex methylation patterns (CMPs) on i-motif formation propensity is currently unknown. METHODS: We employed CD and UV-absorption spectroscopies, native PAGE, thermal denaturation and quantum-chemical calculations to analyse the effects of native, native-like, and non-native CMPs in the i-motif stem on the i-motif stability and pKa. RESULTS: CMPs have strong influence on i-motif stability and pKa and influence these parameters in sequence-specific manner. In contrast to a general belief, i) CMPs do not invariably stabilize the i-motif, and ii) when the CMPs do stabilize the i-motif, the extent of the stabilization depends (in a complex manner) on the number and pattern of symmetric 5mC+.5mC or asymmetric 5mC+.C base pairs in the i-motif stem. CONCLUSIONS: CMPs can be effectively used to fine-tune i-motif properties. Our data support the notion of epigenetic modifications as a plausible control mechanism of i-motif formation in vivo. GENERAL SIGNIFICANCE: Our results have implications in epigenetic regulation of telomeric DNA in plants and highlight the potential and limitations of engineered patterning of cytosine methylations on the i-motif scaffold in nanotechnological applications.
- MeSH
- cytosin metabolismus MeSH
- DNA rostlinná chemie genetika MeSH
- epigeneze genetická * MeSH
- metylace DNA * MeSH
- molekulární modely MeSH
- nanotechnologie * MeSH
- nukleotidové motivy genetika MeSH
- sekvence nukleotidů MeSH
- telomery genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.
- MeSH
- chromozomy rostlin genetika MeSH
- délka genomu genetika MeSH
- DNA rostlinná chemie genetika MeSH
- fazol genetika MeSH
- genom rostlinný genetika MeSH
- mapování chromozomů MeSH
- retroelementy genetika MeSH
- rostlinné geny genetika MeSH
- sekvenční analýza DNA metody MeSH
- syntenie MeSH
- vigna genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The carnivorous plant genus Utricularia L. (bladderwort) comprises about 240 species distributed worldwide and is traditionally classified into two subgenera (Polypompholyx and Utricularia) and 35 sections, based mainly on general and trap morphology. It is one out of the largest carnivorous genera, representing ca. 30% of all carnivorous plant species, and is also the most widely distributed. According to previous phylogenetic studies, most infrageneric sections are monophyletic, but there are several incongruences considering their relationships and also the dissenting position of some species as a result of a too few (mostly one or two) molecular markers analyzed. Thus, here we present a multilocus phylogeny for Utricularia species with a wide taxonomic sampling (78 species and 115 accessions) based on six plastid (rbcL, matK, rpl20-rps12, rps16, trnL-F) and nuclear DNA (ITS region) sequences. The aim is to reconstruct a well-resolved tree to propose evolutionary and biogeographic hypotheses for the radiation of lineages with inferences about the divergence times of clades using a molecular clock approach.
- MeSH
- Bayesova věta MeSH
- biologická evoluce MeSH
- buněčné jádro genetika MeSH
- DNA rostlinná chemie izolace a purifikace metabolismus MeSH
- fylogeneze MeSH
- hluchavkotvaré klasifikace genetika MeSH
- plastidy genetika MeSH
- ribulosa-1,5-bisfosfát-karboxylasa klasifikace genetika metabolismus MeSH
- rostlinné proteiny klasifikace genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční seřazení MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: S. latifolia is a model organism for the study of sex chromosome evolution in plants. Its sex chromosomes include large regions in which recombination became gradually suppressed. The regions tend to expand over time resulting in the formation of evolutionary strata. Non-recombination and later accumulation of repetitive sequences is a putative cause of the size increase in the Y chromosome. Gene decay and accumulation of repetitive DNA are identified as key evolutionary events. Transposons in the X and Y chromosomes are distributed differently and there is a regulation of transposon insertion by DNA methylation of the target sequences, this points to an important role of DNA methylation during sex chromosome evolution in Silene latifolia. The aim of this study was to elucidate whether the reduced expression of the Y allele in S. latifolia is caused by genetic degeneration or if the cause is methylation triggered by transposons and repetitive sequences. RESULTS: Gene expression analysis in S. latifolia males has shown expression bias in both X and Y alleles. To determine whether these differences are caused by genetic degeneration or methylation spread by transposons and repetitive sequences, we selected several sex-linked genes with varying degrees of degeneration and from different evolutionary strata. Immunoprecipitation of methylated DNA (MeDIP) from promoter, exon and intron regions was used and validated through bisulfite sequencing. We found DNA methylation in males, and only in the promoter of genes of stratum I (older). The Y alleles in genes of stratum I were methylation enriched compared to X alleles. There was also abundant and high percentage methylation in the CHH context in most sequences, indicating de novo methylation through the RdDM pathway. CONCLUSIONS: We speculate that TE accumulation and not gene decay is the cause of DNA methylation in the S. latifolia Y sex chromosome with influence on the process of heterochromatinization.
Two new genera (Streptosarcina and Streptofilum) and three new species (Streptosarcina arenaria, S. costaricana and Streptofilum capillatum) of streptophyte algae were detected in cultures isolated from terrestrial habitats of Europe and Central America and described using an integrative approach. Additionally, a strain isolated from soil in North America was identified as Hormidiella parvula and proposed as an epitype of this species. The molecular phylogeny based on 18S rRNA and rbcL genes, secondary structure of ITS-2, as well as the morphology of vegetative and reproductive stages, cell ultrastructure, ecology and distribution of the investigated strains were assessed. The new genus Streptosarcina forms a sister lineage to the genus Hormidiella (Klebsormidiophyceae). Streptosarcina is characterized by packet-like (sarcinoid) and filamentous thalli with true branching and a cell organization typical for Klebsormidiophyceae. Streptofilum forms a separate lineage within Streptophyta. This genus represents an easily disintegrating filamentous alga which exhibits a cell coverage of unique structure: layers of submicroscopic scales of piliform shape covering the plasmalemma and exfoliate inside the mucilage envelope surrounding cells. The implications of the discovery of the new taxa for understanding evolutionary tendencies in the Streptophyta, a group of great evolutionary interest, are discussed.
- MeSH
- DNA rostlinná chemie genetika MeSH
- ekosystém * MeSH
- fylogeneze * MeSH
- konformace nukleové kyseliny MeSH
- mezerníky ribozomální DNA chemie genetika MeSH
- mikroskopie MeSH
- půdní mikrobiologie MeSH
- ribozomální DNA chemie genetika MeSH
- ribulosa-1,5-bisfosfát-karboxylasa genetika MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční analýza DNA MeSH
- shluková analýza MeSH
- Streptophyta klasifikace genetika ultrastruktura MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Severní Amerika MeSH
- Střední Amerika MeSH
The movement of nuclear DNA from one vascular plant species to another in the absence of fertilization is thought to be rare. Here, nonnative rRNA gene [ribosomal DNA (rDNA)] copies were identified in a set of 16 diploid barley (Hordeum) species; their origin was traceable via their internal transcribed spacer (ITS) sequence to five distinct Panicoideae genera, a lineage that split from the Pooideae about 60 Mya. Phylogenetic, cytogenetic, and genomic analyses implied that the nonnative sequences were acquired between 1 and 5 Mya after a series of multiple events, with the result that some current Hordeum sp. individuals harbor up to five different panicoid rDNA units in addition to the native Hordeum rDNA copies. There was no evidence that any of the nonnative rDNA units were transcribed; some showed indications of having been silenced via pseudogenization. A single copy of a Panicum sp. rDNA unit present in H. bogdanii had been interrupted by a native transposable element and was surrounded by about 70 kbp of mostly noncoding sequence of panicoid origin. The data suggest that horizontal gene transfer between vascular plants is not a rare event, that it is not necessarily restricted to one or a few genes only, and that it can be selectively neutral.
- MeSH
- buněčné jádro genetika MeSH
- diploidie MeSH
- DNA rostlinná chemie genetika MeSH
- fylogeneze * MeSH
- ječmen (rod) klasifikace genetika MeSH
- lipnicovité klasifikace genetika MeSH
- mezerníky ribozomální DNA chemie genetika MeSH
- molekulární evoluce MeSH
- přenos genů horizontální * MeSH
- ribozomální DNA chemie genetika MeSH
- rostlinné geny genetika MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Telomeres and genes encoding 45S ribosomal RNA (rDNA) are frequently located adjacent to each other on eukaryotic chromosomes. Although their primary roles are different, they show striking similarities with respect to their features and additional functions. Both genome domains have remarkably dynamic chromatin structures. Both are hypersensitive to dysfunctional histone chaperones, responding at the genomic and epigenomic levels. Both generate non-coding transcripts that, in addition to their epigenetic roles, may induce gross chromosomal rearrangements. Both give rise to chromosomal fragile sites, as their replication is intrinsically problematic. However, at the same time, both are essential for maintenance of genomic stability and integrity. Here we discuss the structural and functional inter-connectivity of telomeres and rDNA, with a focus on recent results obtained in plants.
- MeSH
- chromatin chemie metabolismus MeSH
- DNA rostlinná chemie metabolismus MeSH
- epigeneze genetická MeSH
- histony genetika metabolismus MeSH
- nestabilita genomu MeSH
- replikace DNA MeSH
- ribozomální DNA chemie metabolismus MeSH
- rostliny genetika MeSH
- telomery genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
HTS-1 is a new kind of pistillody wheat. All or parts of its stamen are transformed into pistils or pistil-like structures, and it has more seed sets per floret than normal wheat under normal cultivation conditions. To investigate the expression divergence in this mutant, an annealing control primer system was used to identify differentially expressed genes (DEGs) in the young spikelets. As a result, three DEGs, including HDB2, HGF2, and HCG4, were detected, with variable expression in HTS-1 and the control. After further confirmation using real-time reverse transcription polymerase chain reaction analysis, these genes were overexpressed in HTS-1 wheat. NGF2 was identified in the double ridge to floret differentiation stages; HDB2 and HCG4 were identified in the stage of pistil and stamen-differentiating. Therefore, we inferred that the homeotic transformation of stamens into pistil-like structures occurred during the early stage of stamen development. Sequence alignment analysis revealed that HDB2 encodes a putative protein of 189 amino acids, with high homology to the DEAD-box ATP-dependent RNA helicase, and HCG4 was identical to the Chinese spring wheat cDNA clone predicted protein according to GenBank. However, NGF2 was not found to have significant similarity to any reported proteins, suggesting it is a new functional gene in wheat. The results suggest that HDB2, HCG4, and HGF2 are minor genes contributing to pistillody trait formation in HTS-1.
- MeSH
- cytoplazma metabolismus MeSH
- DNA primery genetika MeSH
- DNA rostlinná chemie genetika MeSH
- fenotyp MeSH
- komplementární DNA chemie genetika MeSH
- květy genetika růst a vývoj MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- pšenice genetika růst a vývoj MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné proteiny genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza DNA MeSH
- sekvenční seřazení MeSH
- semena rostlinná genetika růst a vývoj MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A protocol is described for production of micrograms of DNA from single copies of flow-sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome-derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single-chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome-specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole-genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.
- MeSH
- chromozomy rostlin genetika MeSH
- DNA rostlinná chemie genetika MeSH
- genom rostlinný genetika MeSH
- genomika metody MeSH
- kontigové mapování metody MeSH
- kořeny rostlin genetika MeSH
- průtoková cytometrie MeSH
- pšenice genetika MeSH
- reprodukovatelnost výsledků MeSH
- rostlinné geny genetika MeSH
- sekvenční analýza DNA MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
With the expansion of molecular techniques, the historical collections have become widely used. Studying plant DNA using modern molecular techniques such as DNA sequencing plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods or microsatellites, AFLP).
- MeSH
- cetrimoniové sloučeniny chemie MeSH
- chemická frakcionace metody MeSH
- DNA rostlinná chemie genetika izolace a purifikace MeSH
- druhová specificita MeSH
- filtrace MeSH
- kyselina dusičná chemie MeSH
- listy rostlin chemie MeSH
- mikrosatelitní repetice MeSH
- rostliny chemie MeSH
- semena rostlinná chemie MeSH
- uchovávání tkání * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
