Natalya A. Lemskaya

Genomic Organization and Physical Mapping of Tandemly Arranged Repetitive DNAs in Sterlet (Acipenser ruthenus)

Acipenseriformes represent a phylogenetically basal clade of ray-finned fish characterized by unusual genomic traits, including paleopolyploid states of extant genomes with high chromosome numbers and slow rates of molecular evolution. Despite a high interest in this fish group, only a limited number of studies have been accomplished on the isolation and characterization of repetitive DNA, karyotype standardization is not yet complete, and sex chromosomes are still to be identified. Here, we applied next-generation sequencing and cluster analysis to characterize major fractions of sterlet (Acipenser ruthenus) repetitive DNA. Using FISH, we mapped 16 tandemly arranged sequences on sterlet chromosomes and found them to be unevenly distributed in the genome with a tendency to cluster in particular regions. Some of the satellite DNAs might be used as specific markers to identify individual chromosomes and their paralogs, resulting in the unequivocal identification of at least 18 chromosome pairs. Our results provide an insight into the characteristic genomic distribution of the most common sterlet repetitive sequences. Biased accumulation of repetitive DNAs in particular chromosomes makes them especially interesting for further search for cryptic sex chromosomes. Future studies of these sequences in other acipenserid species will provide new perspectives regarding the evolution of repetitive DNA within the genomes of this fish order.

X chromosome evolution in cetartiodactyla

The phenomenon of a remarkable conservation of the X chromosome in eutherian mammals has been first described by Susumu Ohno in 1964. A notable exception is the cetartiodactyl X chromosome, which varies widely in morphology and G-banding pattern between species. It is hypothesized that this sex chromosome has undergone multiple rearrangements that changed the centromere position and the order of syntenic segments over the last 80 million years of Cetartiodactyla speciation. To investigate its evolution we have selected 26 evolutionarily conserved bacterial artificial chromosome (BAC) clones from the cattle CHORI-240 library evenly distributed along the cattle X chromosome. High-resolution BAC maps of the X chromosome on a representative range of cetartiodactyl species from different branches: pig (Suidae), alpaca (Camelidae), gray whale (Cetacea), hippopotamus (Hippopotamidae), Java mouse-deer (Tragulidae), pronghorn (Antilocapridae), Siberian musk deer (Moschidae), and giraffe (Giraffidae) were obtained by fluorescent in situ hybridization. To trace the X chromosome evolution during fast radiation in specious families, we performed mapping in several cervids (moose, Siberian roe deer, fallow deer, and Pere David’s deer) and bovid (muskox, goat, sheep, sable antelope, and cattle) species. We have identified three major conserved synteny blocks and rearrangements in different cetartiodactyl lineages and found that the recently described phenomenon of the evolutionary new centromere emergence has taken place in the X chromosome evolution of Cetartiodactyla at least five times. We propose the structure of the putative ancestral cetartiodactyl X chromosome by reconstructing the order of syntenic segments and centromere position for key groups.

Rapid Karyotype Evolution in Lasiopodomys Involved at Least Two Autosome - Sex Chromosome Translocations.

The generic status of Lasiopodomys and its division into subgenera Lasiopodomys (L. mandarinus, L. brandtii) and Stenocranius (L. gregalis, L. raddei) are not generally accepted because of contradictions between the morphological and molecular data. To obtain cytogenetic evidence for the Lasiopodomys genus and its subgenera and to test the autosome to sex chromosome translocation hypothesis of sex chromosome complex origin in L. mandarinus proposed previously, we hybridized chromosome painting probes from the field vole (Microtus agrestis, MAG) and the Arctic lemming (Dicrostonyx torquatus, DTO) onto the metaphases of a female Mandarin vole (L. mandarinus, 2n = 47) and a male Brandts vole (L. brandtii, 2n = 34). In addition, we hybridized Arctic lemming painting probes onto chromosomes of a female narrow-headed vole (L. gregalis, 2n = 36). Cross-species painting revealed three cytogenetic signatures (MAG12/18, 17a/19, and 22/24) that could validate the genus Lasiopodomys and indicate the evolutionary affinity of L. gregalis to the genus. Moreover, all three species retained the associations MAG1bc/17b and 2/8a detected previously in karyotypes of all arvicolins studied. The associations MAG2a/8a/19b, 8b/21, 9b/23, 11/13b, 12b/18, 17a/19a, and 5 fissions of ancestral segments appear to be characteristic for the subgenus Lasiopodomys. We also validated the autosome to sex chromosome translocation hypothesis on the origin of complex sex chromosomes in L. mandarinus. Two translocations of autosomes onto the ancestral X chromosome in L. mandarinus led to a complex of neo-X1, neo-X2, and neo-X3 elements. Our results demonstrate that genus Lasiopodomys represents a striking example of rapid chromosome evolution involving both autosomes and sex chromosomes. Multiple reshuffling events including Robertsonian fusions, chromosomal fissions, inversions and heterochromatin expansion have led to the formation of modern species karyotypes in a very short time, about 2.4 MY.

Comparative cytogenetics of rodents

Here, we present analysis of data on comparative chromosome painting produced using various chromosome-specific libraries for members of different Glires groups. Based on the results of comparative cytogenetic and molecular studies, the modern rodents can be conventionally classified into two groups with sharply differing directions and tempos of karyotypic evolution. One group (suborders Sciuromorpha, Castorimorpha, and Anomaluromorpha) preserved conserved genomes, which are probably close in structure to the genome of the ancestor of all mammals. The genomes of the other group (suborder Myomorpha) underwent “catastrophic evolution,” which resulted in numerous breaks and fusions of the ancient chromosomes. The current data do not allow unambiguously assigning the order Hystricomorpha to any of these groups.

Genome-wide comparative chromosome maps of Arvicola amphibius, Dicrostonyx torquatus, and Myodes rutilus.

The subfamily Arvicolinae consists of a great number of species with highly diversified karyotypes. In spite of the wide use of arvicolines in biological and medicine studies, the data on their karyotype structures are limited. Here, we made a set of painting probes from flow-sorted chromosomes of a male Palearctic collared lemming (Dicrostonyx torquatus, DTO). Together with the sets of painting probes made previously from the field vole (Microtus agrestis, MAG) and golden hamster (Mesocricetus auratus, MAU), we carried out a reciprocal chromosome painting between these three species. The three sets of probes were further hybridized onto the chromosomes of the Eurasian water vole (Arvicola amphibius) and northern red-backed vole (Myodes rutilus). We defined the diploid chromosome number in D. torquatus karyotype as 2n = 45 + Bs and showed that the system of sex chromosomes is X1X2Y1. The probes developed here provide a genomic tool-kit, which will help to investigate the evolutionary biology of the Arvicolinae rodents. Our results show that the syntenic association MAG1/17 is present not only in Arvicolinae but also in some species of Cricetinae; and thus, should not be considered as a cytogenetic signature for Arvicolinae. Although cytogenetic signature markers for the genera have not yet been found, our data provides insight into the likely ancestral karyotype of Arvicolinae. We conclude that the karyotypes of modern voles could have evolved from a common ancestral arvicoline karyotype (AAK) with 2n = 56 mainly by centric fusions and fissions.

The Ancestral Carnivore Karyotype As Substantiated by Comparative Chromosome Painting of Three Pinnipeds, the Walrus, the Steller Sea Lion and the Baikal Seal (Pinnipedia, Carnivora).

Karyotype evolution in Carnivora is thoroughly studied by classical and molecular cytogenetics and supplemented by reconstructions of Ancestral Carnivora Karyotype (ACK). However chromosome painting information from two pinniped families (Odobenidae and Otariidae) is noticeably missing. We report on the construction of the comparative chromosome map for species from each of the three pinniped families: the walrus (Odobenus rosmarus, Odobenidae–monotypic family), near threatened Steller sea lion (Eumetopias jubatus, Otariidae) and the endemic Baikal seal (Pusa sibirica, Phocidae) using combination of human, domestic dog and stone marten whole-chromosome painting probes. The earliest karyological studies of Pinnipedia showed that pinnipeds were characterized by a pronounced karyological conservatism that is confirmed here with species from Phocidae, Otariidae and Odobenidae sharing same low number of conserved human autosomal segments (32). Chromosome painting in Pinnipedia and comparison with non-pinniped carnivore karyotypes provide strong support for refined structure of ACK with 2n = 38. Constructed comparative chromosome maps show that pinniped karyotype evolution was characterized by few tandem fusions, seemingly absent inversions and slow rate of genome rearrangements (less then one rearrangement per 10 million years). Integrative comparative analyses with published chromosome painting of Phoca vitulina revealed common cytogenetic signature for Phoca/Pusa branch and supports Phocidae and Otaroidea (Otariidae/Odobenidae) as sister groups. We revealed rearrangements specific for walrus karyotype and found the chromosomal signature linking together families Otariidae and Odobenidae. The Steller sea lion karyotype is the most conserved among three studied species and differs from the ACK by single fusion. The study underlined the strikingly slow karyotype evolution of the Pinnipedia in general and the Otariidae in particular.

Pinniped Karyotype Evolution Substantiated by Comparative Chromosome Painting of 10 Pinniped Species (Pinnipedia, Carnivora)

In memory of Ingemar Gustavsson 23rd International Colloquium on Animal Cytogenetics and Genomics (23 ICACG) took place in June 9–12, 2018 in Saint-Petersburg, Russia. Organized biennially, the Colloquium runs from 1970. From its very start this meeting is associated with the name of Ingemar Gustavsson to whom we dedicated the Colloquium 2018. The long and productive career of Ingemar Gustavsson had focused on chromosomes and their fundamental role in animal physiology, fertility, health and production in the context of agriculture and veterinary medicine. His meticulous analysis of breeding data performed back in 1964–69 resulted in the unequivocal identification of an association between heterozygosity for the 1/29 translocation in Swedish cattle and reduction in the fertility of the breed. Eventually, the argument in favor of selective elimination of bulls carrying the translocation from the breeding programs prevailed and the field of modern veterinary cytogenetics was established. Participants from fourteen different countries attended the 23 ICACG in Russia, the country having long lasting traditions in cytogenetics and the Scientific schools of N.K. Koltzov, S.S. Chetverikov and A.S. Serebrovsky, geneticists who made important conceptual contributions to studies of chromosomes and genes, population genetics and evolutionary theory as early as in the beginning of the XX-th century. All the abstracts received were subdivided between plenary and seven scientific sessions covering the issues in evolutionary and comparative cytogenetics, cytogenetics and genomes of domestic animals, meiosis studies, particular chromosome analyses, clinical cytogenetics, karyotypes and genomes of vertebrate and invertebrate animals, chromatin studies. In the abstract text below each presentation is marked with a capital letter: „L” stands for lectures, „O” for oral presentations and „P” for poster presentations. We gratefully acknowledge the support from the Saint-Petersburg Association of Scientists and Scholars (SPbSU), Veterinary Genetics Center ZOOGEN, Russian Foundation for Basic Research (RFBR), VEUK, Helicon, Axioma BIO, BioVitrum, Sartorius, DIA-M companies. The current collected abstracts comprise written contributions of the presentations during the 23 ICACG and were edited by Svetlana Galkina and Maria Vishnevskaya. The next Colloquium – 24 ICACG – will be held at the University of Kent in Canterbury (UK) in 2020. Please, cite abstracts as follows: Gall JG (2018) Giant chromosomes and deep sequences: what the amphibian egg tells us about transcription. In: Galkina SA, Vishnevskaya MS, Mikhailova EI (Eds) 23rd Inernational Colloquium on Animal Cytogenetics and Genomics (23rdICACG), June 9–12, 2018, St Petersburg, Russia. Comparative Cytogenetics 12(3): p–p. https://doi.org/10.3897/CompCytogen.v12i3.27748The ultimate aim of a genome assembly is to create a contiguous length of sequence from the p- to q- terminus of each chromosome. Most assemblies are however highly fragmented, limiting their use in studies of gene mapping, phylogenomics and genomic organisation. To overcome these limitations, we developed a novel scaffold-to-chromosome anchoring method combining reference-assisted chromosome assembly (RACA) and fluorescence in situ hybridisation (FISH) to position scaffolds from de novo genomes onto chromosomes. Using RACA, scaffolds were ordered and orientated into ‘predicted chromosome fragments’ (PCFs) against a reference and outgroup genome. PCFs were verified using PCR prior to FISH mapping. A universal set of FISH probes developed through the selection of conserved regions were then used to map PCFs of peregrine falcon (Falco peregrinus Tunstall, 1771), pigeon (Columba livia Gmelin, 1789), ostrich (Struthio camelus Linnaeus, 1758), saker falcon (Falco cherrug Gray, 1834) the budgerigar (Melopsittacus undulatus Shaw, 1805). Using this approach, we were able to improve the N50 of genomes seven-fold. Results revealed that Interchromosomal breakpoint regions are limited to regions with low sequence conservation, shedding light on why most avian species have very stable karyotypes. Our combined FISH and bioinformatics approach represents a step-change in the mapping of genome assemblies, allowing comparative genomic research at a higher resolution than was previously possible. The universal probe set facilitates research into avian karyotype evolution and the role of chromosome rearrangements in adaptation and phenotypic diversity in birds. Indeed, they have been used on over 20 avian species plus non-avian reptiles (including turtles), shedding light into the evolution of dinosaur species. Non-avian dinosaurs remain subjects of intense biological enquiry while pervading popular culture and the creative arts. While organismal studies focus primarily on their morphology, relationships, likely behaviour, and ecology there have been few academic studies that have made extensive extrapolations about the nature of non-avian dinosaur genome structure prior to the emergence of modern birds. We have used multiple avian whole genome sequences assembled at a chromosomal level, to reconstruct the most likely gross genome organization of the overall genome structure of the diapsid ancestor and reconstruct the sequence of inter and intrachromosomal events that most likely occurred along the Archosauromorpha-Archosauria-Avemetatarsalia-Dinosauria-Theropoda-Maniraptora-Avialae lineage from the lepidosauromorph-archosauromorph divergence ~275 million years ago through to extant neornithine birds.

First cytogenetic analysis of lesser gymnures (Mammalia, Galericidae, Hylomys) from Vietnam

Abstract Gymnures are an ancient group of small insectivorous mammals and are characterized by a controversial taxonomic status and the lack of a description of karyotypes for certain species. In this study, conventional cytogenetic techniques (Giemsa, CBG- and GTG-banding, Ag-NOR), CMA3-DAPI staining, and fluorescent in situ hybridization (FISH) with telomeric DNA probes were used to examine for the first time the karyotypes of lesser gymnures of group Hylomyssuillus Müller, 1840 from northern and southern Vietnam. All studied specimens had karyotypes with 2n=48, NFa=64. C-positive heterochromatic blocks existed in centromeric regions of 7 bi-armed autosomes and the submetacentric X chromosome. The Y chromosome is a C-positive and dot-like. The nucleolus organizer regions resided terminally on the short arms of 2 small bi-armed pairs. Positive signals at the telomeres of all chromosomes were revealed by FISH. CMA3-positive blocks were localized on the telomeric and pericentric regions of most bi-armed and acrocentric chromosomes. Despite the large genetic distances between Hylomys Müller, 1840, lesser gymnures from H.suillus-group from northern and southern Vietnam have similar karyotypic characteristics.

A Female Patient with FMR1 Premutation and Mosaic X Chromosome Aneuploidy and Two Sons with Intellectual Disability

In this report, we describe a molecular cytogenetic study of a family burdened with intellectual disability (ID) and suicide. Our study revealed that the mother has a heterozygous premutation in the FMR1 gene and supernumerary X chromosomes as well as X-derived marker chromosomes. Both of her sons have ID and a normal chromosome number. One of the sons has fragile X syndrome, and the other has ID of an unclear nature.

X-derived marker chromosome in patient with mosaic Turner syndrome and Dandy-Walker syndrome: a case report

BackgroundSmall supernumerary marker chromosomes can be derived from autosomes and sex chromosomes and can accompany chromosome pathologies, such as Turner syndrome.Case presentationHere, we present a case report of a patient with mosaic Turner syndrome and Dandy-Walker syndrome carrying a marker chromosome. We showed the presence of the marker chromosome in 33.8% of blood cells. FISH of the probe derived from the marker chromosome by microdissection revealed that it originated from the centromeric region of chromosome X. Additionally, we showed no telomeric sequences and no XIST sequence in the marker chromosome. This is the first report of these two syndromes accompanied by the presence of a marker chromosome.ConclusionMarker chromosome was X-derived and originated from centromeric region. Patient has mild symptoms but there is no XIST gene in marker chromosome.Trial registrationCPG137. Registered 03 March 2017.