Lampbrush chromosome studies in the post-genomic era.

Extraordinary extended lampbrush chromosomes with thousands of transcription loops are favorable objects in chromosome biology. Chromosomes become lampbrushy due to unusually high rate of transcription during oogenesis. However, until recently, the information on the spectrum of transcribed sequences as well as genomic context of individual chromomeres was mainly limited to tandemly repetitive elements. Here we briefly outline novel findings and future directions in lampbrush chromosome studies in the post-genomic era. We emphasize the fruitfulness of combining genome-wide approaches with microscopy imaging techniques using lampbrush chromosomes as a remarkable model object. We believe that new data on the spectrum of sequences transcribed on the lateral loops of lampbrush chromosomes and their structural organization push the boundaries in the discussion of their biological role. Also see the video abstract here: https://youtu.be/zexoHfzX9rM.

Karyotypes of water frogs from the Pelophylax esculentus complex: results of cross-species chromosomal painting.

Amphibian species have the largest genome size enriched with repetitive sequences and relatively similar karyotypes. Moreover, many amphibian species frequently hybridize causing nuclear and mitochondrial genome introgressions. In addition, hybridization in some amphibian species may lead to clonality and polyploidization. All such events were found in water frogs from the genus Pelophylax. Among the species within the genus Pelophylax, P. esculentus complex is the most widely distributed and well-studied. This complex includes two parental species, P. ridibundus and P. lessonae, and their hybrids, P. esculentus, reproducing hemiclonally. Parental species and their hybrids have similar but slightly polymorphic karyotypes, so their precise identification is still required. Here, we have developed a complete set of 13 chromosome painting probes for two parental species allowing the precise identification of all chromosomes. Applying chromosomal painting, we identified homologous chromosomes in both parental species and orthologous chromosomes in their diploid hemiclonal hybrids. Comparative painting did not reveal interchromosomal exchanges between the studied water frog species and their hybrids. Using cross-specific chromosome painting, we detected unequal distribution of the signals along chromosomes suggesting the presence of species-specific tandem repeats. Application of chromosomal paints to the karyotypes of hybrids revealed differences in the intensity of staining for P. ridibundus and P. lessonae chromosomes. Thus, both parental genomes have a divergence in unique sequences. Obtained chromosome probes may serve as a powerful tool to unravel chromosomal evolution in phylogenetically related species, identify individual chromosomes in different cell types, and investigate the elimination of chromosomes in hybrid water frogs.

Comparison of the somatic TADs and lampbrush chromomere-loop complexes in transcriptionally active prophase I oocytes.

In diplotene oocyte nuclei of all vertebrate species, except mammals, chromosomes lack interchromosomal contacts and chromatin is linearly compartmentalized into distinct chromomere-loop complexes forming lampbrush chromosomes. However, the mechanisms underlying the formation of chromomere-loop complexes remain unexplored. Here we aimed to compare somatic topologically associating domains (TADs), recently identified in chicken embryonic fibroblasts, with chromomere-loop complexes in lampbrush meiotic chromosomes. By measuring 3D-distances and colocalization between linear equidistantly located genomic loci, positioned within one TAD or separated by a TAD border, we confirmed the presence of predicted TADs in chicken embryonic fibroblast nuclei. Using three-colored FISH with BAC probes, we mapped equidistant genomic regions included in several sequential somatic TADs on isolated chicken lampbrush chromosomes. Eight genomic regions, each comprising two or three somatic TADs, were mapped to non-overlapping neighboring lampbrush chromatin domains - lateral loops, chromomeres, or chromomere-loop complexes. Genomic loci from the neighboring somatic TADs could localize in one lampbrush chromomere-loop complex, while genomic loci belonging to the same somatic TAD could be localized in neighboring lampbrush chromomere-loop domains. In addition, FISH-mapping of BAC probes to the nascent transcripts on the lateral loops indicates transcription of at least 17 protein-coding genes and 2 non-coding RNA genes during the lampbrush stage of chicken oogenesis, including genes involved in oocyte maturation and early embryo development.

Single-Cell DNA Methylation Analysis of Chicken Lampbrush Chromosomes.

DNA methylation is an essential epigenetic regulation mechanism implicated in transcription and replication control, developmental reprogramming, retroelements silencing and other genomic processes. During mammalian development, a specific DNA methylation pattern should be established in germ cells to allow embryonic development. Less is known about germ cell DNA methylation in other species. To close this gap, we performed a single-cell methylome analysis of chicken diplotene oocytes. We comprehensively characterized methylation patterns in these cells, obtained methylation-based chicken genome segmentation and identified oocyte-specific methylated gene promoters. Our data show that despite the formation of specific transcriptionally hyperactive genome architecture in chicken diplotene oocytes, methylation patterns in these cells closely resemble genomic distribution observed in somatic tissues.

3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes' chromatin.

How chromosomes are folded, spatially organized and regulated in three dimensions inside the cell nucleus are among the longest standing questions in cell biology. Genome-wide chromosome conformation capture (Hi-C) technique allowed identifying and characterizing spatial chromatin compartments in several mammalian species. Here, we present the first genome-wide analysis of chromatin interactions in chicken embryonic fibroblasts (CEF) and adult erythrocytes. We showed that genome of CEF is partitioned into topologically associated domains (TADs), distributed in accordance with gene density, transcriptional activity and CTCF-binding sites. In contrast to mammals, where all examined somatic cell types display relatively similar spatial organization of genome, chicken erythrocytes strongly differ from fibroblasts, showing pronounced A- and B- compartments, absence of typical TADs and formation of long-range chromatin interactions previously observed on mitotic chromosomes. Comparing mammalian and chicken genome architectures, we provide evidence highlighting evolutionary role of chicken TADs and their significance in genome activity and regulation.

Heterochromatic regions in Japanese quail chromosomes: comprehensive molecular-cytogenetic characterization and 3D mapping in interphase nucleus.

Chromosomes of Japanese quail (Coturnix coturnix japonica, 2n=78), a galliform domestic species closely related to chicken, possess multiple heterochromatic segments. Due to the difficulties in careful analysis of such heterochromatic regions, there is a lack of data on their DNA composition, epigenetic status, as well as spatial distribution in interphase nucleus. In the present study, we applied giant lampbrush chromosome (LBC) microdissection for high-resolution analysis of quail centromeric regions of macrochromosomes and polymorphic short arms of submetacentric microchromosomes. FISH with the dissected material on mitotic and meiotic chromosomes indicated that in contrast to centromeres of chicken macrochromosomes, which are known to harbor chromosome-specific and, in some cases, tandem repeat-free sequences, centromeres of quail macroautosomes (CCO1-CCO11) have canonical organization. CCO1-CCO11 centromeres possess massive blocks of common DNA repeats demonstrating transcriptional activity at LBC stage. These repeats seem to have been subjected to chromosome size-correlated homogenization previously described primarily for avian microchromosomes. In addition, comparative FISH on chicken chromosomes supported the previous data on centromere repositioning events during galliform karyotype evolution. In interphase nucleus of different cell types, repetitive elements specific for microchromosome short arms constitute the material of prominent centrally located chromocenters enriched with markers of constitutive heterochromatin and rimmed with clusters of microchromosomal centromeric BglII-repeat. Thus, clustering of such repeats is responsible for the peculiar architecture of quail interphase nucleus. In contrast, centromere repeats of the largest macrochromosomes (CCO1 and CCO2) are predominantly localized in perinuclear heterochromatin. The possible involvement of the isolated repeats in radial genome organization is discussed.

Mutual maintenance of di- and triploid Pelophylax esculentus hybrids in R-E systems: results from artificial crossings experiments.

BACKGROUND: Interspecies animal hybrids can employ clonal or hemiclonal reproduction modes where one or all parental genomes are transmitted to the progeny without recombination. Nevertheless, some interspecies hybrids retain strong connection with the parental species needed for successful reproduction. Appearance of polyploid hybrid animals may play an important role in the substitution of parental species and in the speciation process. RESULTS: To establish the mechanisms that enable parental species, diploid and polyploid hybrids coexist we have performed artificial crossing experiments of water frogs of Pelophylax esculentus complex. We identified tadpole karyotypes and oocyte genome composition in all females involved in the crossings. The majority of diploid and triploid hybrid frogs produced oocytes with 13 bivalents leading to haploid gametes with the same genome as parental species hybrids usually coexist with. After fertilization of such gametes only diploid animals appeared. Oocytes with 26 bivalents produced by some diploid hybrid frogs lead to diploid gametes, which give rise to triploid hybrids after fertilization. In gonads of all diploid and triploid hybrid tadpoles we found DAPI-positive micronuclei (nucleus-like bodies) involved in selective genome elimination. Hybrid male and female individuals produced tadpoles with variable karyotype and ploidy even in one crossing owing to gametes with various genome composition. CONCLUSIONS: We propose a model of diploid and triploid hybrid frog reproduction in R-E population systems. Triploid Pelophylax esculentus hybrids can transmit genome of parental species they coexist with by producing haploid gametes with the same genome composition. Triploid hybrids cannot produce triploid individuals after crossings with each other and depend on diploid hybrid females producing diploid eggs. In contrast to other population systems, the majority of diploid and triploid hybrid females unexpectedly produced gametes with the same genome as parental species hybrids coexist with.

Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing.

BACKGROUND: Over the past two decades, chromosome microdissection has been widely used in diagnostics and research enabling analysis of chromosomes and their regions through probe generation and establishing of chromosome- and chromosome region-specific DNA libraries. However, relatively small physical size of mitotic chromosomes limited the use of the conventional chromosome microdissection for investigation of tiny chromosomal regions. RESULTS: In the present study, we developed a workflow for mechanical microdissection of giant transcriptionally active lampbrush chromosomes followed by the preparation of whole-chromosome and locus-specific fluorescent in situ hybridization (FISH)-probes and high-throughput sequencing. In particular, chicken (Gallus g. domesticus) lampbrush chromosome regions as small as single chromomeres, individual lateral loops and marker structures were successfully microdissected. The dissected fragments were mapped with high resolution to target regions of the corresponding lampbrush chromosomes. For investigation of RNA-content of lampbrush chromosome structures, samples retrieved by microdissection were subjected to reverse transcription. Using high-throughput sequencing, the isolated regions were successfully assigned to chicken genome coordinates. As a result, we defined precisely the loci for marker structures formation on chicken lampbrush chromosomes 2 and 3. Additionally, our data suggest that large DAPI-positive chromomeres of chicken lampbrush chromosome arms are characterized by low gene density and high repeat content. CONCLUSIONS: The developed technical approach allows to obtain DNA and RNA samples from particular lampbrush chromosome loci, to define precisely the genomic position, extent and sequence content of the dissected regions. The data obtained demonstrate that lampbrush chromosome microdissection provides a unique opportunity to correlate a particular transcriptional domain or a cytological structure with a known DNA sequence. This approach offers great prospects for detailed exploration of functionally significant chromosomal regions.

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts.

Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription.

Transcription of subtelomere tandemly repetitive DNA in chicken embryogenesis.

Transcription of tandemly repetitive DNA in embryogenesis seems to be of special interest due to a crucial role of non-coding RNAs in many aspects of development. However, only a few data are available on tandem repeats transcription at subtelomere regions of chromosomes during vertebrate embryogenesis. To reduce this gap, we examined stage and tissue-specific pattern of subtelomeric PO41 (pattern of 41 bp) tandem repeat transcription during embryogenesis of chicken (Gallus gallus domesticus). Using whole-mount RNA fluorescent in situ hybridization and reverse transcription PCR with specific primers, we demonstrated that both strands of PO41 repeat are transcribed at each of the studied stages of chicken embryo development: from 7-8 HH to 20 HH stages. Subtelomere-derived transcripts localize in the nuclei of all cell types and throughout the all embryonic bodies: head, somites, tail, wings and buds. In embryo-dividing cells and cultured embryonic fibroblasts, PO41 RNAs envelop terminal regions of chromosomes. PO41-containing RNAs are predominantly single-stranded and can be polyadenylated, indicating appearance of non-nascent form of subtelomeric transcripts. PO41 repeat RNAs represent a rare example of ubiquitously transcribed non-coding RNAs, such as Xist/XIST RNA or telomere repeat-containing RNA. Distribution of PO41 repeat transcripts at different stages of embryo development and among cell types has extremely uniform pattern, indicating on possible universal functions of PO41 non-coding RNAs.

Three-dimensional architecture of tandem repeats in chicken interphase nucleus.

Tandem repeats belong to a class of genomic repetitive elements that form arrays of head-to-tail monomers. Due to technical difficulties in sequencing and assembly of large tandem repeat arrays, it remains largely unknown by which mechanisms tandem-repeat-containing regions aid in maintenance of ordered radial genome organization during interphase. Here we analyzed spatial distribution of several types of tandem repeats in interphase nuclei of chicken MDCC-MSB1 cells and somatic tissues relative to heterochromatin compartments and nuclear center. We showed that telomere and subtelomere repeats generally localize at the nuclear or chromocenters periphery. A tandem repeat known as CNM, typical for centromere regions of gene-dense microchromosomes, forms interchromosome clusters and occupies DAPI-positive chromocenters that appear predominantly within the nuclear interior. In contrast, centromere-specific tandem repeats of the majority of gene-poor macrochromosomes are embedded into the peripheral layer of heterochromatin. Chicken chromocenters rarely comprise centromere sequences of both macro- and microchromosomes, whose territories localize in different radial nuclear zones. Possible mechanisms of observed tandem repeats positioning and its implication in highly ordered arrangement of chromosome territories in chicken interphase nucleus are discussed.

Cytological maps of lampbrush chromosomes of European water frogs (Pelophylax esculentus complex) from the Eastern Ukraine.

BACKGROUND: Hybridogenesis (hemiclonal inheritance) is a kind of clonal reproduction in which hybrids between parental species are reproduced by crossing with one of the parental species. European water frogs (Pelophylax esculentus complex) represent an appropriate model for studying interspecies hybridization, processes of hemiclonal inheritance and polyploidization. P. esculentus complex consists of two parental species, P. ridibundus (the lake frog) and P. lessonae (the pool frog), and their hybridogenetic hybrid - P. esculentus (the edible frog). Parental and hybrid frogs can reproduce syntopically and form hemiclonal population systems. For studying mechanisms underlying the maintenance of water frog population systems it is required to characterize the karyotypes transmitted in gametes of parental and different hybrid animals of both sexes. RESULTS: In order to obtain an instrument for characterization of oocyte karyotypes in hybrid female frogs, we constructed cytological maps of lampbrush chromosomes from oocytes of both parental species originating in Eastern Ukraine. We further identified certain molecular components of chromosomal marker structures and mapped coilin-rich spheres and granules, chromosome associated nucleoli and special loops accumulating splicing factors. We recorded the dissimilarities between P. ridibundus and P. lessonae lampbrush chromosomes in the length of orthologous chromosomes, number and location of marker structures and interstitial (TTAGGG)n-repeat sites as well as activity of nucleolus organizer. Satellite repeat RrS1 was mapped in centromere regions of lampbrush chromosomes of the both species. Additionally, we discovered transcripts of RrS1 repeat in oocytes of P. ridibundus and P. lessonae. Moreover, G-rich transcripts of telomere repeat were revealed in association with terminal regions of P. ridibundus and P. lessonae lampbrush chromosomes. CONCLUSIONS: The constructed cytological maps of lampbrush chromosomes of P. ridibundus and P. lessonae provide basis to define the type of genome transmitted within individual oocytes of P. esculentus females with different ploidy and from various population systems.

High-resolution mapping and transcriptional activity analysis of chicken centromere sequences on giant lampbrush chromosomes.

Exploration into morphofunctional organisation of centromere DNA sequences is important for understanding the mechanisms of kinetochore specification and assembly. In-depth epigenetic analysis of DNA fragments associated with centromeric nucleosome proteins has demonstrated unique features of centromere organisation in chicken karyotype: there are both mature centromeres, which comprise chromosome-specific homogeneous arrays of tandem repeats, and recently evolved primitive centromeres, which consist of non-tandemly organised DNA sequences. In this work, we describe the arrangement and transcriptional activity of chicken centromere repeats for Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 and non-repetitive centromere sequences of chromosomes 5, 27, and Z using highly elongated lampbrush chromosomes, which are characteristic of the diplotene stage of oogenesis. The degree of chromatin packaging and fine spatial organisations of tandemly repetitive and non-tandemly repetitive centromeric sequences significantly differ at the lampbrush stage. Using DNA/RNA FISH, we have demonstrated that during the lampbrush stage, DNA sequences are transcribed within the centromere regions of chromosomes that lack centromere-specific tandem repeats. In contrast, chromosome-specific centromeric repeats Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 do not demonstrate any transcriptional activity during the lampbrush stage. In addition, we found that CNM repeat cluster localises adjacent to non-repetitive centromeric sequences in chicken microchromosome 27 indicating that centromere region in this chromosome is repeat-rich. Cross-species FISH allowed localisation of the sequences homologous to centromeric DNA of chicken chromosomes 5 and 27 in centromere regions of quail orthologous chromosomes.

Three-dimensional organisation of RNA-processing machinery in avian growing oocyte nucleus.

Chromosome architecture and assorted nuclear compartments play an essential role in RNA transcription and processing. Growing vertebrate oocytes represent an advantageous model to study the principles of nuclear structure and function. In this article, the data on three-dimensional (3D) organisation of intact and non-deformed oocyte nuclei (germinal vesicles) in four species of birds (domestic chicken, Japanese quail, rock pigeon and chaffinch) obtained by confocal laser scanning microscopy are presented. The nucleus of the growing avian oocyte has an unusual structure of RNA processing machinery. Germinal vesicles from any of the avian species studied, with the exception of the rock pigeon, are characterized by absence of extrachromosomal RNA-enriched nuclear bodies including Cajal bodies, histone locus bodies and interchromatin granule clusters. The absence of Cajal bodies and histone locus bodies in chicken oocytes correlated with the inactivation of nucleolus organizer and clustered histone genes. Splicing factors such as SR-protein SC35 accumulated in chromosome-associated domains that were classified as complex loops (terminal giant and lumpy loops). Formation of such depot in avian oocyte nuclei is supposed to be nucleated by transcripts of non-coding tandem repeats. The results obtained strongly support a model of RNA-mediated nuclear domains formation.

Centromere positions in chicken and Japanese quail chromosomes: de novo centromere formation versus pericentric inversions.

Chicken (Gallus gallus domesticus, GGA) and Japanese quail (Coturnix coturnix japonica, CCO) karyotypes are very similar. They have identical chromosome number (2n = 78) and show a high degree of synteny. Centromere positions on the majority of orthologous chromosomes are different in these two species. To explore the nature of this divergence, we used high-resolution comparative fluorescent in situ hybridization mapping on giant lampbrush chromosomes (LBCs) from growing oocytes. We applied 41 BAC clones specific for GGA1, 2, 3, 11, 12, 13, 14, and 15 to chicken and quail LBCs. This approach allowed us to rule out a pericentric inversion earlier proposed to explain the difference between GGA1 and CCO1. In addition to a well-established large-scale pericentric inversion that discriminates GGA2 and CCO2, we identified another, smaller one in the large inverted region. For the first time, we described in detail inversions that distinguish GGA3 from CCO3 and GGA11 from CCO11. Despite the newly identified and confirmed inversions, our data suggest that, in chicken and Japanese quail, the difference in centromere positions is not mainly caused by pericentric inversions but is instead due to centromere repositioning events and the formation of new centromeres. We also consider the formation of short arms of quail microchromosomes by heterochromatin accumulation as a third scenario that could explain the discrepancy in centromeric indexes.

Assignment of the somatic A/B compartments to chromatin domains in giant transcriptionally active lampbrush chromosomes.

BACKGROUND: The three-dimensional configuration of the eukaryotic genome is an emerging area of research. Chromosome conformation capture outlined genome segregation into large scale A and B compartments corresponding mainly to transcriptionally active and repressive chromatin. It remains unknown how the compartmentalization of the genome changes in growing oocytes of animals with hypertranscriptional type of oogenesis. Such oocytes are characterized by highly elongated chromosomes, called lampbrush chromosomes, which acquire a typical chromomere-loop appearance, representing one of the classical model systems for exploring the structural and functional organization of chromatin domains. RESULTS: Here, we compared the distribution of A/B compartments in chicken somatic cells with chromatin domains in lampbrush chromosomes. We found that in lampbrush chromosomes, the extended chromatin domains, restricted by compartment boundaries in somatic cells, disintegrate into individual chromomeres. Next, we performed FISH-mapping of the genomic loci, which belong to A or B chromatin compartments as well as to A/B compartment transition regions in embryonic fibroblasts on isolated lampbrush chromosomes. We found, that in chicken lampbrush chromosomes, clusters of dense compact chromomeres bearing short lateral loops and enriched with repressive epigenetic modifications generally correspond to constitutive B compartments in somatic cells. A compartments align with lampbrush chromosome segments with smaller, less compact chromomeres, longer lateral loops, and a higher transcriptional status. Clusters of small loose chromomeres with relatively long lateral loops show no obvious correspondence with either A or B compartment identity. Some genes belonging to facultative B (sub-) compartments can be tissue-specifically transcribed during oogenesis, forming distinct lateral loops. CONCLUSIONS: Here, we established a correspondence between the A/B compartments in somatic interphase nucleus and chromatin segments in giant lampbrush chromosomes from diplotene stage oocytes. The chromomere-loop structure of the genomic regions corresponding to interphase A and B compartments reveals the difference in how they are organized at the level of chromatin domains. The results obtained also suggest that gene-poor regions tend to be packed into chromomeres.

Non-canonical Cajal bodies form in the nucleus of late stage avian oocytes lacking functional nucleolus.

In the somatic cell nucleus, there are several universal domains such as nucleolus, SC35-domains, Cajal bodies (CBs) and histone locus bodies (HLBs). Among them, CBs were described more than 100 years ago; however, we still do not have a final understanding of their nature and biological significance. The giant nucleus of avian and amphibian growing oocytes represents an advantageous model for analysis of functions and biogenesis of various nuclear domains. Nevertheless, in large-sized avian oocytes that contain transcriptionally active lampbrush chromosomes, CB-like organelles have not been identified yet. Here we demonstrate that in the pigeon (Columba livia) oocyte nucleus, characterized by absence of any functional nucleoli, extrachromosomal spherical bodies contain TMG-capped spliceosomal snRNAs, core proteins of Sm snRNPs and the protein coilin typical for CBs, but not splicing factor SC35 nor the histone pre-mRNA 3'-end processing factor symplekin. The results establish that coilin-rich nuclear organelles in pigeon late-stage oocyte are not the equivalents of HLBs but belong to a group of CBs. At the same time, they do not contain the snoRNP/scaRNP protein fibrillarin involved in 2'-O-methylation of snoRNAs and snRNAs. Thus, the nucleus of late-stage pigeon oocytes houses CB-like organelles that have an unusual molecular composition and are implicated in the snRNP biogenesis pathway. These data demonstrate that snRNP-rich non-canonical CBs can form in the absence of nucleolus. We argue that pigeon oocytes represent a new promising model to investigate CB modular organization, functions and formation mechanism.

Delete and survive: strategies of programmed genetic material elimination in eukaryotes.

Genome stability is a crucial feature of eukaryotic organisms because its alteration drastically affects the normal development and survival of cells and the organism as a whole. Nevertheless, some organisms can selectively eliminate part of their genomes from certain cell types during specific stages of ontogenesis. This review aims to describe the phenomenon of programmed DNA elimination, which includes chromatin diminution (together with programmed genome rearrangement or DNA rearrangements), B and sex chromosome elimination, paternal genome elimination, parasitically induced genome elimination, and genome elimination in animal and plant hybrids. During programmed DNA elimination, individual chromosomal fragments, whole chromosomes, and even entire parental genomes can be selectively removed. Programmed DNA elimination occurs independently in different organisms, ranging from ciliate protozoa to mammals. Depending on the sequences destined for exclusion, programmed DNA elimination may serve as a radical mechanism of dosage compensation and inactivation of unnecessary or dangerous genetic entities. In hybrids, genome elimination results from competition between parental genomes. Despite the different consequences of DNA elimination, all genetic material destined for elimination must be first recognised, epigenetically marked, separated, and then removed and degraded.

Maintenance of pure hybridogenetic water frog populations: Genotypic variability in progeny of diploid and triploid parents.

An intriguing outcome of hybridisation is the emergence of clonally and hemiclonally reproducing hybrids, that can sustain, reproduce, and lead to the emergence of polyploid forms. However, the maintenance of diploid and polyploid hybrid complexes in natural populations remains unresolved. We selected water frogs from the Pelophylax esculentus complex to study how diploid and triploid hybrids, which reproduce hemiclonally via hybridogenesis, are maintained in natural populations. During gametogenesis in diploid hybrids, one of the parental genomes is eliminated, and the remaining genome is endoreplicated. In triploid hybrids, the single-copy genome is typically eliminated, while genome endoreplication does not occur. To investigate how diploid and triploid hybrid frogs reproduce in populations without parental species, we crossed these hybrid animals from two separate pure hybrid populations located in Poland. Using cytogenetic analysis of tadpoles that emerged from the crosses, we established which gametes were produced by parental hybrids. The majority of hybrid females and hybrid males produced one type of gamete with the P. ridibundus genome. However, in both studied populations, approximately half of the diploid and triploid hybrids simultaneously produced gametes with different genome compositions and ploidy levels, specifically, the P. ridibundus and P. lessonae genomes, as well as diploid gametes with genomes of both parental species. Triploid hybrid males and females mostly produced haploid gametes with the P. lessonae genome; however, gametes with the P. ridibundus genome have also been observed. These results suggest that not all hybrids follow the classical hybridogenetic reproduction program and reveal a significant level of alterations in the gametogenesis pathways. In addition, we found a variable survival rate of particular progeny genotypes when we crossed hybrid females with different males suggesting the important role of postzygotic barriers on the maintenance of pure hybrid systems. We suggest that the observed variability in produced gametes and the different survival rate of the progeny with certain genotypes is crucial for the existence of pure hybrid systems.

FISH Going Meso-Scale: A Microscopic Search for Chromatin Domains.

The intimate relationships between genome structure and function direct efforts toward deciphering three-dimensional chromatin organization within the interphase nuclei at different genomic length scales. For decades, major insights into chromatin structure at the level of large-scale euchromatin and heterochromatin compartments, chromosome territories, and subchromosomal regions resulted from the evolution of light microscopy and fluorescence in situ hybridization. Studies of nanoscale nucleosomal chromatin organization benefited from a variety of electron microscopy techniques. Recent breakthroughs in the investigation of mesoscale chromatin structures have emerged from chromatin conformation capture methods (C-methods). Chromatin has been found to form hierarchical domains with high frequency of local interactions from loop domains to topologically associating domains and compartments. During the last decade, advances in super-resolution light microscopy made these levels of chromatin folding amenable for microscopic examination. Here we are reviewing recent developments in FISH-based approaches for detection, quantitative measurements, and validation of contact chromatin domains deduced from C-based data. We specifically focus on the design and application of Oligopaint probes, which marked the latest progress in the imaging of chromatin domains. Vivid examples of chromatin domain FISH-visualization by means of conventional, super-resolution light and electron microscopy in different model organisms are provided.