Whole-genome duplication (WGD) is a powerful evolutionary force, yet the mechanisms by which neopolyploids achieve transcriptomic stability and phenotypic success remain poorly understood. This study investigated the phenotypic and transcriptomic consequences of ploidy changes in the flatworm Macrostomum lignano, a “successful” neopolyploid model. We exploited two established sublines derived from the inbred DV1 line: the euploid DV1_8 (hidden tetraploid, SSL1L2) and the aneuploid DV1_10 (hidden hexaploid, SSL1L1L2L2). By integrating whole-genome sequencing (WGS)-informed normalization with RNA-seq analysis, we differentiated true regulatory shifts from gene-dosage effects. We revealed that while most genes scale linearly with ploidy, 1308 genes exhibited a nonlinear aneuploidy-induced transcriptional response. The remarkable trans-acting effects were observed across subgenome S encoded by disomic small chromosomes. Differentially expressed genes (DEGs) were enriched in pathways essential for homeostasis and growth: mTOR signaling, ubiquitin-mediated proteolysis, and the Hippo/Wnt pathways. Phenotypes of the DV1_10 worms exhibited increased body size, enhanced cell proliferation, and higher viability in comparison to the DV1_8 worms (60.25% vs. 21.5%). These findings suggest that M. lignano possesses mechanisms for dosage compensation to mitigate the deleterious effects of aneuploidy. Ultimately, this study demonstrates how genomic plasticity and rewiring of the transcriptome may facilitate the evolutionary success of animal neopolyploids.

Chromosomal inversions and copy-number variants (CNVs) drive genomic and phenotypic diversification in birds by reshaping recombination, gene expression, and genome architecture. Here, we report a complex structural polymorphism on great tit (Parus major) chromosome 1A that occurs in the Siberian population with a 19% heterozygote frequency. Using cytogenetic and genomic approaches, we show that this rearrangement combines a ~55 Mb paracentric inversion in the long arm with a dramatic (>30 Mb) expansion of the short arm driven by extensive amplification of multiple genomic loci. These include a region homologous to the poorly characterized FAM118A gene, whose paralog FAM118B has been recently shown to play a pivotal role in innate immune activation. This region is missing from the current reference genome assembly while present in ~20 copies on wild-type 1A chromosome and nearly twentyfold amplified in the rearranged variant. It contains a nested 630 bp tandem repeat, encompassing the entire exon 3, which has burst to a total of ~50,000 copies in the rearranged chromosome. While functional analyses are required to uncover the biological effects of the genomic features linked to this rearrangement, our results offer a unique framework for studying how complex structural polymorphisms drive genome innovation and adaptive diversity.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth’s death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient’s cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9 Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of genome doubling through allo- or autopolyploidization could lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are undoubtedly important for understanding one of the crucial stages of genome evolution. However, assembling neopolyploid appears to be a challenging task because its genome consists of two homologous (or homeologous) chromosome sets and therefore contains the extended paralogous regions with a high homology level. Post-WGD evolution of polyploids includes rediploidization, first part of which is cytogenetic diploidization led to the formation of species, whose polyploid origin might be hidden by disomic inheritance and diploid-like meiosis. Earlier we uncovered the hidden polyploid origin of free-living flatworms of the genus Macrostomum ( Macrostomum lignano, M. janickei , and M. mirumnovem ). Despite the different mechanisms for their genome doubling, cytogenetic diploidization in these species accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we reported unusual subgenomic organization of M. lignano through generation and sequencing of two new laboratory sublines of DV1 that differ only by a copy number of the large chromosome MLI1. Using non-trivial assembly-free comparative analysis of their genomes, including adapted multivariate k-mer analysis, and self-homology within the published genome assembly of M. lignano , we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano , which consists in (1) presence of three subgenomes, emerged via formation of large fused chromosome and its variants, and (2) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.

Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of WGD, allo- or autopolyploidization, lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are important for understanding the early stages of genome evolution. However, assembling neopolyploid is a challenging task due to the presence of 2 homologous (or homeologous) chromosome sets and therefore the existence of the extended paralogous regions in its genome. Post-WGD evolution of polyploids includes cytogenetic diploidization leading to the formation of species, whose polyploid origin might be hidden by disomic inheritance. Earlier we uncovered the hidden polyploid origin of the free-living flatworms of the genus Macrostomum (Macrostomum lignano, M. janickei, and M. mirumnovem). Cytogenetic diploidization in these species is accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we unravel the M. lignano genome organization through generation and sequencing of 2 sublines of the commonly used inbred line of M. lignano (called DV1) differing only in a copy number of the largest chromosome (MLI1). Using nontrivial assembly free comparative analysis of their genomes, we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano, which consists of (i) presence of 3 subgenomes, which emerged via formation of large fused chromosomes and its variants, and (ii) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.

Detection and precise genomic mapping of balanced chromosomal abnormalities in patients with impaired fertility or a clinical phenotype represent a challenge for current cytogenomics owing to difficulties with precise breakpoint localization in the regions enriched for DNA repeats and high genomic variation in such regions. Here, we present a comprehensive cytogenomic approach to breakpoint mapping in a rare paracentric inversion on 10q (in a patient with oligoasthenoteratozoospermia and necrozoospermia) that does not affect other phenotype traits. Multicolor banding, chromosomal microarray analysis, chromosome microdissection with reverse painting, and single-copy sequencing of the rearranged chromosome were performed to determine the length and position of the inverted region as well as to rule out a genetic imbalance at the breakpoints. As a result, a paracentric 19.251 Mbp inversion at 10q22.2q23.3 was described. The most probable location of the breakpoints was predicted using the hg38 assembly. The problems of genetic counseling associated with enrichment for repeats and high DNA variability of usual breakpoint regions were discussed. Possible approaches for cytogenomic assessment of couples with balanced chromosome rearrangements and problems like reproductive failures were considered and suggested as useful part of effective genetic counseling.

B chromosomes (Bs) or supernumerary chromosomes are extra chromosomes in the species karyotype that can vary in its copy number. Bs are widespread in eukaryotes. Usually, the Bs of specimens collected from natural populations are the object of the B chromosome studies. We applied another approach analyzing the Bs in animals maintained under the laboratory conditions as lines and cultures. In this study, three species of the Macrostomum genus that underwent a recent whole-genome duplication (WGD) were involved. In laboratory lines of M. lignano and M. janickei, the frequency of Bs was less than 1%, while in the laboratory culture of M. mirumnovem, it was nearer 30%. Their number in specimens of the culture varied from 1 to 14. Mosaicism on Bs was discovered in parts of these animals. We analyzed the distribution of Bs among the worms of the laboratory cultures during long-term cultivation, the transmission rates of Bs in the progeny obtained from crosses of worms with different numbers of Bs, and from self-fertilized isolated worms. The DNA content of the Bs in M. mirumnovem was analyzed with the chromosomal in situ suppression (CISS) hybridization of microdissected DNA probes derived from A chromosomes (As). Bs mainly consisted of repetitive DNA. The cytogenetic analysis also revealed the divergence and high variation in large metacentric chromosomes (LMs) containing numerous regions enriched for repeats. The possible mechanisms of the appearance and evolution of Bs and LMs in species of the Macrostomum genus were also discussed.

All songbirds studied so far have a germline-restricted chromosome (GRC), which is present in the germ cells and absent in the somatic cells. It shows a wide variation in size, morphology, and genetic content between the songbird species. In this paper, we analyzed GRC behavior in female and male meiosis of the great tit, using immunolocalization of meiotic proteins and FISH with GRC-derived DNA probes. We found that, despite dozens of million years of independent evolution, the great tit GRC displays a striking similarity with the GRCs of two species of martins and two species of estrildid finches examined earlier. It was usually present in two copies in females forming recombining bivalent and in one copy in males forming a condensed heterochromatic body with dotted-like axial elements of the synaptonemal complex. We observed mosaicism for the GRC copy number in the female and male great tit. This indicates that one of the GRC copies might be passively lost during premeiotic germ cell divisions. After the meiotic prophase, the GRC was ejected from most male germ cells. The reverse and interspecies FISH with GRC-specific microdissected DNA probes indicates that GRCs of the great tit, pale martin, and zebra finch differ substantially in their genetic content despite similarities in the meiotic behavior.

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

We analyzed the synapsis and recombination between Z and W chromosomes in the oocytes of nine neognath species: domestic chicken Gallus gallus domesticus, grey goose Anser anser, black tern Chlidonias niger, common tern Sterna hirundo, pale martin Riparia diluta, barn swallow Hirundo rustica, European pied flycatcher Ficedula hypoleuca, great tit Parus major and white wagtail Motacilla alba using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex, and MLH1, the mismatch repair protein marking mature recombination nodules. In all species examined, homologous synapsis occurs in a short region of variable size at the ends of Z and W chromosomes, where a single recombination nodule is located. The remaining parts of the sex chromosomes undergo synaptic adjustment and synapse non-homologously. In 25% of ZW bivalents of white wagtail, synapsis and recombination also occur at the secondary pairing region, which probably resulted from autosome−sex chromosome translocation. Using FISH with a paint probe specific to the germline-restricted chromosome (GRC) of the pale martin on the oocytes of the pale martin, barn swallow and great tit, we showed that both maternally inherited songbird chromosomes (GRC and W) share common sequences.

Interpreting the clinical significance of small supernumerary marker chromosomes (sSMCs) in prenatal diagnosis is still an urgent problem in genetic counselling regarding the fate of a pregnancy. We present a case of prenatal diagnosis of mosaic sSMC(10) in a foetus with a normal phenotype. Comprehensive cytogenomic analyses by array-based comparative genomic hybridization (aCGH), sSMC microdissection with next-generation sequencing (NGS) of microdissected library, fluorescence in situ hybridization (FISH) with locus-specific and telomere-specific DNA probes and quantitative real-time PCR revealed that sSMC(10) had a ring structure and was derived from the pericentromeric region of chromosome 10 with involvement of the 10p11.21-p11.1 and 10q11.21-q11.23 at 1.243 Mb and 7.173 Mb in size, respectively. We observed a difference in the length of sSMC(10) between NGS data of the DNA library derived from a single copy of sSMC(10), and aCGH results that may indicate instability and structural mosaicism for ring chromosomes in foetal cells. The presence of a 9 Mb euchromatin region in the analysed sSMC(10) did not lead to clinical manifestations, and a healthy girl was born at term. We suggest that the ring structure of sSMCs could influence sSMC manifestations and should be taken into account in genetic counselling during prenatal diagnosis.

Application of microdissected DNA libraries and DNA probes in numerous and various modern molecular cytogenetic studies showed them as an efficient and reliable tool in the analysis of chromosome reorganization during karyotypic evolution and in the diagnosis of human chromosome pathology. An important advantage of DNA probe generation by metaphase chromosome microdissection followed by sequence-independent polymerase chain reaction in comparison with the method of DNA probe generation using chromosome sorting is the possibility of DNA probe preparation from chromosomes of an individual sample without cell line establishment for the production of a large number of metaphase chromosomes. One of the main requirements for successful application of this technique is a possibility for identification of the chromosome of interest during its dissection and collection of its material from metaphase plates spread on the coverslip. In the present study, we developed and applied a technique for generation of microdissected DNA probes in the case when chromosome identification during microdissection appeared to be impossible. The technique was used for generation of two sets of Whole Chromosome Paints (WCPs) from all chromosomes of two species of free-living flatworms in the genus Macrostomum, M. mirumnovem and M. cliftonensis. The single-copy chromosome technique including separate collection of all chromosomes from one metaphase plate allowed us to generate WCPs that painted specifically the original chromosome by Chromosome In Situ Suppression Hybridization (CISS-Hybridization). CISS-Hybridization allowed identifying the original chromosome(s) used for DNA probe generation. Pooled WCPs derived from homologous chromosomes increased the intensity and specificity of chromosome painting provided by CISS-Hybridization. In the result, the obtained DNA probes appeared to be good enough for application in our studies devoted to analysis of karyotypic evolution in the genus Macrostomum and for analysis of chromosome rearrangements among the worms of laboratory cultures of M. mirumnovem.

Macrostomum lignano is a free-living flatworm that is emerging as an attractive experimental animal for research on a broad range of biological questions. One feature setting it apart from other flatworms is the successful establishment of transgenesis methods, facilitated by a steady supply of eggs in the form of single-cell zygotes that can be readily manipulated. This, in combination with the transparency of the animal and its small size, creates practical advantages for imaging and fluorescence-activated cell sorting in studies related to stem cell biology and regeneration. M. lignano can regenerate most of its body parts, including the germline, thanks to the neoblasts, which represent the flatworm stem cell system. Interestingly, neoblasts seem to have a high capacity of cellular maintenance, as M. lignano can survive up to 210 Gy of γ-irradiation, and partially offset the negative consequence of ageing. As a non-self-fertilizing simultaneous hermaphrodite that reproduces in a sexual manner, M. lignano is also used to study sexual selection and other evolutionary aspects of sexual reproduction. Work over the past several years has led to the development of molecular resources and tools, including high-quality genome and transcriptome assemblies, transcriptional profiling of the germline and somatic neoblasts, gene knockdown, and in situ hybridization. The increasingly detailed characterization of this animal has also resulted in novel research questions, such as bio-adhesion based on its adhesion-release glands and genome evolution due to its recent whole-genome duplication.

The genus Macrostomum represents a diverse group of rhabditophoran flatworms with >200 species occurring around the world. Earlier we uncovered karyotype instability linked to hidden polyploidy in both M. lignano (2n = 8) and its sibling species M. janickei (2n = 10), prompting interest in the karyotype organization of close relatives. In this study, we investigated chromosome organization in two recently described and closely related Macrostomum species, M. mirumnovem and M. cliftonensis, and explored karyotype instability in laboratory lines and cultures of M. lignano (DV1/10, 2n = 10) and M. janickei in more detail. We revealed that three of the four studied species are characterized by karyotype instability, while M. cliftonensis showed a stable 2n = 6 karyotype. Next, we performed comparative cytogenetics of these species using fluorescent in situ hybridization (FISH) with a set of DNA probes (including microdissected DNA probes generated from M. lignano chromosomes, rDNA, and telomeric DNA). To explore the chromosome organization of the unusual 2n = 9 karyotype discovered in M. mirumnovem, we then generated chromosome-specific DNA probes for all chromosomes of this species. Similar to M. lignano and M. janickei, our findings suggest that M. mirumnovem arose via whole genome duplication (WGD) followed by considerable chromosome reshuffling. We discuss possible evolutionary scenarios for the emergence and reorganization of the karyotypes of these Macrostomum species and consider their suitability as promising animal models for studying the mechanisms and regularities of karyotype and genome evolution after a recent WGD.

A great number of interspersed repetitive sequences in chromosomes make it difficult to identify chromosomal material via fluorescence in situ hybridization (FISH). The traditional approach to solve this problem is chromosome in situ suppression hybridization (CISS-hybridization). Unfortunately, it is impossible to be performed or fails with chromosomes of many eukaryote species. The aim of this study was to consider the image enhance procedure [1] and the in silico method of chromosome specific signal visualization (method VISSIS) [2] as alternatives to CISS-hybridization. The effectiveness of approaches for identification of specific signals was estimated by signal-to-background ratio (SNR). The computer methods were applied to images of human chromosomes, obtained with FISH of the whole chromosome painting DNA probes. Results showed that effectiveness of image processing methods depends on ratio of short and line interspersed elements (SINEs/LINEs) in DNA probes. The closer chromosomes in ratio of SINEs/LINEs, the higher specific signal intensities and signal-to-background ratios could be achieved. This suggests that computer methods can be efficient only with application of DNA probes derived from chromosomes characterized with similar ratio of SINE and LINE contents.

Despite the rapid development of new methods of molecular and molecular cytogenetic diagnostics, the small supernumerary marker chromosomes (sSMCs), described for the first time in 1961, are still one of the most difficult cases in the diagnosis of human chromosomal pathologies. In this article, new approaches, methods of analysis and description of sSMC, as well as the existing problems of assessing their possible clinical significance are considered. There are the prospects of improving and developing new methods and approaches to solving problems in the field of molecular cytogenetic diagnosis of human chromosomal pathologies analyzed.

All songbirds studied to date have an additional Germline Restricted Chromosome (GRC), which is not present in somatic cells. GRCs show a wide variation in genetic content and little homology between species. To check how this divergence affected the meiotic behavior of the GRC, we examined synapsis, recombination and copy number variation for GRCs in the closely related sand and pale martins ( Riparia riparia and R. diluta ) in comparison with distantly related estrildid finches. Using immunolocalization of meiotic proteins and FISH with GRC-specific DNA probes, we found a striking similarity in the meiotic behavior of GRCs between martins and estrildid finches despite the millions of years of independent evolution. GRCs are usually present in two copies in female and in one copy in male pachytene cells. However, we detected polymorphism in female and mosaicism in male martins for the number of GRCs. In martin and zebra finch females, two GRCs synapse along their whole length, but recombine predominately at their ends. We suggest that the shared features of the meiotic behavior of GRCs have been supported by natural selection in favor of a preferential segregation of GRCs to the eggs.

The free-living flatworm Macrostomum mirumnovem is a neopolyploid species whose genome underwent a recent Whole Genome Duplication (WGD). In the result of chromosome fusions of the ancient haploid chromosome set, large metacentric chromosomes were formed. In addition to three pairs of small metacentrics, the current karyotype of M. mirumnovem contains two pairs of large metacentric chromosomes, MMI1 and MMI2. The generation of microdissected DNA libraries enriched for DNA repeats followed by DNA probe preparation and fluorescent in situ hybridization (FISH) were performed. The DNA probes obtained marked chromosome regions enriched for different DNA repeats in the M. mirumnovem chromosomes. The size and localization of these regions varied in different copies of large chromosomes. They varied even in homologous chromosomes, suggesting their divergence due to genome re-diploidization after a WGD. Besides the newly formed chromosome regions enriched for DNA repeats, B chromosomes were found in the karyotypes of the studied specimens of M. mirumnovem. These B chromosomes varied in size and morphology. FISH with microdissected DNA probes revealed that some Bs had a distinct DNA content. FISH could paint differently B chromosomes in different worms and even in the same sample. B chromosomes could carry a bright specific fluorescent signal or could show no fluorescent signal at all. In latter cases, the specific FISH signal could be absent even in the pericentromeric region of the B chromosome. Possible mechanisms of B chromosome formation and their further evolution are discussed. The results obtained indicate an important role that repetitive DNAs play in genome re-diploidization initiating a rapid differentiation of large chromosome copies. Taking together, karyotype peculiarities (a high level of intraspecific karyotypic diversity associated with chromosome number variation, structural chromosomal rearrangements, and the formation of new regions enriched for DNA repeats) and some phenotypic features of M. mirumnovem (small body size, short lifecycle, easy maintenance in the laboratory) make this species a perspective model in the studies of genomic and karyotypic evolution in species passed through a recent WGD event.