Comparative studies of X chromosomes in Cervidae family.

The family Cervidae is the second most diverse in the infraorder Pecora and is characterized by variability in the diploid chromosome numbers among species. X chromosomes in Cervidae evolved through complex chromosomal rearrangements of conserved segments within the chromosome, changes in centromere position, heterochromatic variation, and X-autosomal translocations. The family Cervidae consists of two subfamilies: Cervinae and Capreolinae. Here we build a detailed X chromosome map with 29 cattle bacterial artificial chromosomes of representatives of both subfamilies: reindeer (Rangifer tarandus), gray brocket deer (Mazama gouazoubira), Chinese water deer (Hydropotes inermis) (Capreolinae); black muntjac (Muntiacus crinifrons), tufted deer (Elaphodus cephalophus), sika deer (Cervus nippon) and red deer (Cervus elaphus) (Cervinae). To track chromosomal rearrangements during Cervidae evolution, we summarized new data, and compared them with available X chromosomal maps and chromosome level assemblies of other species. We demonstrate the types of rearrangements that may have underlined the variability of Cervidae X chromosomes. We detected two types of cervine X chromosome-acrocentric and submetacentric. The acrocentric type is found in three independent deer lineages (subfamily Cervinae and in two Capreolinae tribes-Odocoileini and Capreolini). We show that chromosomal rearrangements on the X-chromosome in Cervidae occur at a higher frequency than in the entire Ruminantia lineage: the rate of rearrangements is 2 per 10 million years.

Maps of Constitutive-Heterochromatin Distribution for Four Martes Species (Mustelidae, Carnivora, Mammalia) Show the Formative Role of Macrosatellite Repeats in Interspecific Variation of Chromosome Structure.

Constitutive-heterochromatin placement in the genome affects chromosome structure by occupying centromeric areas and forming large blocks. To investigate the basis for heterochromatin variation in the genome, we chose a group of species with a conserved euchromatin part: the genus Martes [stone marten (M. foina, 2n = 38), sable (M. zibellina, 2n = 38), pine marten (M. martes, 2n = 38), and yellow-throated marten (M. flavigula, 2n = 40)]. We mined the stone marten genome for the most abundant tandem repeats and selected the top 11 macrosatellite repetitive sequences. Fluorescent in situ hybridization revealed distributions of the tandemly repeated sequences (macrosatellites, telomeric repeats, and ribosomal DNA). We next characterized the AT/GC content of constitutive heterochromatin by CDAG (Chromomycin A3-DAPI-after G-banding). The euchromatin conservatism was shown by comparative chromosome painting with stone marten probes in newly built maps of the sable and pine marten. Thus, for the four Martes species, we mapped three different types of tandemly repeated sequences critical for chromosome structure. Most macrosatellites are shared by the four species with individual patterns of amplification. Some macrosatellites are specific to a species, autosomes, or the X chromosome. The variation of core macrosatellites and their prevalence in a genome are responsible for the species-specific variation of the heterochromatic blocks.

Comparative Studies of Karyotypes in the Cervidae Family.

The family Cervidae is the second most diverse family in the infraorder Pecora and is characterized by a striking variability in the diploid chromosome numbers among species, ranging from 6 to 70. Chromosomal rearrangements in Cervidae have been studied in detail by chromosome painting. There are many comparative cytogenetic data for both subfamilies (Cervinae and Capreolinae) based on homologies with chromosomes of cattle and Chinese muntjac. Previously it was found that interchromosomal rearrangements are the major type of rearrangements occurring in the Cervidae family. Here, we build a detailed chromosome map of a female reindeer (Rangifer tarandus, 2n = 70, Capreolinae) and a female black muntjac (Muntiacus crinifrons, 2n = 8, Cervinae) with dromedary homologies to find out what other types of rearrangements may have underlined the variability of Cervidae karyotypes. To track chromosomal rearrangements and the distribution of nucleolus organizer regions not only during Cervidae but also Pecora evolution, we summarized new data and compared them with chromosomal maps of other already studied species. We discuss changes in the pecoran ancestral karyotype in the light of new painting data. We show that intrachromosomal rearrangements in autosomes of Cervidae are more frequent than previously thought: at least 13 inversions in evolutionary breakpoint regions were detected.

The Cytogenetic Map of the Nile Crocodile (Crocodylus niloticus, Crocodylidae, Reptilia) with Fluorescence In Situ Localization of Major Repetitive DNAs.

Tandemly arranged and dispersed repetitive DNA sequences are important structural and functional elements that make up a significant portion of vertebrate genomes. Using high throughput, low coverage whole genome sequencing followed by bioinformatics analysis, we have identified seven major tandem repetitive DNAs and two fragments of LTR retrotransposons in the genome of the Nile crocodile (Crocodylus niloticus, 2n = 32). The repeats showed great variability in structure, genomic organization, and chromosomal distribution as revealed by fluorescence in situ hybridization (FISH). We found that centromeric and pericentromeric heterochromatin of C. niloticus is composed of previously described in Crocodylus siamensis CSI-HindIII and CSI-DraI repetitive sequence families, a satellite revealed in Crocodylus porosus, and additionally contains at least three previously unannotated tandem repeats. Both LTR sequences identified here belong to the ERV1 family of endogenous retroviruses. Each pericentromeric region was characterized by a diverse set of repeats, with the exception of chromosome pair 4, in which we found only one type of satellite. Only a few repeats showed non-centromeric signals in addition to their centromeric localization. Mapping of 18S-28S ribosomal RNA genes and telomeric sequences (TTAGGG)n did not demonstrate any co-localization of these sequences with revealed centromeric and pericentromeric heterochromatic blocks.

Karyotype Evolution in 10 Pinniped Species: Variability of Heterochromatin versus High Conservatism of Euchromatin as Revealed by Comparative Molecular Cytogenetics.

Pinnipedia karyotype evolution was studied here using human, domestic dog, and stone marten whole-chromosome painting probes to obtain comparative chromosome maps among species of Odobenidae (Odobenus rosmarus), Phocidae (Phoca vitulina, Phoca largha, Phoca hispida, Pusa sibirica, Erignathus barbatus), and Otariidae (Eumetopias jubatus, Callorhinus ursinus, Phocarctos hookeri, and Arctocephalus forsteri). Structural and functional chromosomal features were assessed with telomere repeat and ribosomal-DNA probes and by CBG (C-bands revealed by barium hydroxide treatment followed by Giemsa staining) and CDAG (Chromomycin A3-DAPI after G-banding) methods. We demonstrated diversity of heterochromatin among pinniped karyotypes in terms of localization, size, and nucleotide composition. For the first time, an intrachromosomal rearrangement common for Otariidae and Odobenidae was revealed. We postulate that the order of evolutionarily conserved segments in the analyzed pinnipeds is the same as the order proposed for the ancestral Carnivora karyotype (2n = 38). The evolution of conserved genomes of pinnipeds has been accompanied by few fusion events (less than one rearrangement per 10 million years) and by novel intrachromosomal changes including the emergence of new centromeres and pericentric inversion/centromere repositioning. The observed interspecific diversity of pinniped karyotypes driven by constitutive heterochromatin variation likely has played an important role in karyotype evolution of pinnipeds, thereby contributing to the differences of pinnipeds' chromosome sets.

Evolution of the Human Chromosome 13 Synteny: Evolutionary Rearrangements, Plasticity, Human Disease Genes and Cancer Breakpoints.

The history of each human chromosome can be studied through comparative cytogenetic approaches in mammals which permit the identification of human chromosomal homologies and rearrangements between species. Comparative banding, chromosome painting, Bacterial Artificial Chromosome (BAC) mapping and genome data permit researchers to formulate hypotheses about ancestral chromosome forms. Human chromosome 13 has been previously shown to be conserved as a single syntenic element in the Ancestral Primate Karyotype; in this context, in order to study and verify the conservation of primate chromosomes homologous to human chromosome 13, we mapped a selected set of BAC probes in three platyrrhine species, characterised by a high level of rearrangements, using fluorescence in situ hybridisation (FISH). Our mapping data on Saguinus oedipus, Callithrix argentata and Alouatta belzebul provide insight into synteny of human chromosome 13 evolution in a comparative perspective among primate species, showing rearrangements across taxa. Furthermore, in a wider perspective, we have revised previous cytogenomic literature data on chromosome 13 evolution in eutherian mammals, showing a complex origin of the eutherian mammal ancestral karyotype which has still not been completely clarified. Moreover, we analysed biomedical aspects (the OMIM and Mitelman databases) regarding human chromosome 13, showing that this autosome is characterised by a certain level of plasticity that has been implicated in many human cancers and diseases.

Comparative Chromosome Mapping of Musk Ox and the X Chromosome among Some Bovidae Species.

: Bovidae, the largest family in Pecora infraorder, are characterized by a striking variability in diploid number of chromosomes between species and among individuals within a species. The bovid X chromosome is also remarkably variable, with several morphological types in the family. Here we built a detailed chromosome map of musk ox (Ovibosmoschatus), a relic species originating from Pleistocene megafauna, with dromedary and human probes using chromosome painting. We trace chromosomal rearrangements during Bovidae evolution by comparing species already studied by chromosome painting. The musk ox karyotype differs from the ancestral pecoran karyotype by six fusions, one fission, and three inversions. We discuss changes in pecoran ancestral karyotype in the light of new painting data. Variations in the X chromosome structure of four bovid species nilgai bull (Boselaphustragocamelus), saola (Pseudoryxnghetinhensis), gaur (Bosgaurus), and Kirk's Dikdik (Madoquakirkii) were further analyzed using 26 cattle BAC-clones. We found the duplication on the X in saola. We show main rearrangements leading to the formation of four types of bovid X: Bovinae type with derived cattle subtype formed by centromere reposition and Antilopinae type with Caprini subtype formed by inversion in XSB3.

An integrated chromosome-scale genome assembly of the Masai giraffe (Giraffa camelopardalis tippelskirchi).

BACKGROUND: The Masai giraffe (Giraffa camelopardalis tippelskirchi) is the largest-bodied giraffe and the world's tallest terrestrial animal. With its extreme size and height, the giraffe's unique anatomical and physiological adaptations have long been of interest to diverse research fields. Giraffes are also critical to ecosystems of sub-Saharan Africa, with their long neck serving as a conduit to food sources not shared by other herbivores. Although the genome of a Masai giraffe has been sequenced, the assembly was highly fragmented and suboptimal for genome analysis. Herein we report an improved giraffe genome assembly to facilitate evolutionary analysis of the giraffe and other ruminant genomes. FINDINGS: Using SOAPdenovo2 and 170 Gbp of Illumina paired-end and mate-pair reads, we generated a 2.6-Gbp male Masai giraffe genome assembly, with a scaffold N50 of 3 Mbp. The incorporation of 114.6 Gbp of Chicago library sequencing data resulted in a HiRise SOAPdenovo + Chicago assembly with an N50 of 48 Mbp and containing 95% of expected genes according to BUSCO analysis. Using the Reference-Assisted Chromosome Assembly tool, we were able to order and orient scaffolds into 42 predicted chromosome fragments (PCFs). Using fluorescence in situ hybridization, we placed 153 cattle bacterial artificial chromosomes onto giraffe metaphase spreads to assess and assign the PCFs on 14 giraffe autosomes and the X chromosome resulting in the final assembly with an N50 of 177.94 Mbp. In this assembly, 21,621 protein-coding genes were identified using both de novo and homology-based predictions. CONCLUSIONS: We have produced the first chromosome-scale genome assembly for a Giraffidae species. This assembly provides a valuable resource for the study of artiodactyl evolution and for understanding the molecular basis of the unique adaptive traits of giraffes. In addition, the assembly will provide a powerful resource to assist conservation efforts of Masai giraffe, whose population size has declined by 52% in recent years.

Improving Illumina assemblies with Hi-C and long reads: An example with the North African dromedary.

Researchers have assembled thousands of eukaryotic genomes using Illumina reads, but traditional mate-pair libraries cannot span all repetitive elements, resulting in highly fragmented assemblies. However, both chromosome conformation capture techniques, such as Hi-C and Dovetail Genomics Chicago libraries and long-read sequencing, such as Pacific Biosciences and Oxford Nanopore, help span and resolve repetitive regions and therefore improve genome assemblies. One important livestock species of arid regions that does not have a high-quality contiguous reference genome is the dromedary (Camelus dromedarius). Draft genomes exist but are highly fragmented, and a high-quality reference genome is needed to understand adaptation to desert environments and artificial selection during domestication. Dromedaries are among the last livestock species to have been domesticated, and together with wild and domestic Bactrian camels, they are the only representatives of the Camelini tribe, which highlights their evolutionary significance. Here we describe our efforts to improve the North African dromedary genome. We used Chicago and Hi-C sequencing libraries from Dovetail Genomics to resolve the order of previously assembled contigs, producing almost chromosome-level scaffolds. Remaining gaps were filled with Pacific Biosciences long reads, and then scaffolds were comparatively mapped to chromosomes. Long reads added 99.32 Mbp to the total length of the new assembly. Dovetail Chicago and Hi-C libraries increased the longest scaffold over 12-fold, from 9.71 Mbp to 124.99 Mbp and the scaffold N50 over 50-fold, from 1.48 Mbp to 75.02 Mbp. We demonstrate that Illumina de novo assemblies can be substantially upgraded by combining chromosome conformation capture and long-read sequencing.

Evolution of gene regulation in ruminants differs between evolutionary breakpoint regions and homologous synteny blocks.

The role of chromosome rearrangements in driving evolution has been a long-standing question of evolutionary biology. Here we focused on ruminants as a model to assess how rearrangements may have contributed to the evolution of gene regulation. Using reconstructed ancestral karyotypes of Cetartiodactyls, Ruminants, Pecorans, and Bovids, we traced patterns of gross chromosome changes. We found that the lineage leading to the ruminant ancestor after the split from other cetartiodactyls was characterized by mostly intrachromosomal changes, whereas the lineage leading to the pecoran ancestor (including all livestock ruminants) included multiple interchromosomal changes. We observed that the liver cell putative enhancers in the ruminant evolutionary breakpoint regions are highly enriched for DNA sequences under selective constraint acting on lineage-specific transposable elements (TEs) and a set of 25 specific transcription factor (TF) binding motifs associated with recently active TEs. Coupled with gene expression data, we found that genes near ruminant breakpoint regions exhibit more divergent expression profiles among species, particularly in cattle, which is consistent with the phylogenetic origin of these breakpoint regions. This divergence was significantly greater in genes with enhancers that contain at least one of the 25 specific TF binding motifs and located near bovidae-to-cattle lineage breakpoint regions. Taken together, by combining ancestral karyotype reconstructions with analysis of cis regulatory element and gene expression evolution, our work demonstrated that lineage-specific regulatory elements colocalized with gross chromosome rearrangements may have provided valuable functional modifications that helped to shape ruminant evolution.

A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin.

Сonstitutive heterochromatin areas are revealed by differential staining as C-positive chromosomal regions. These C-positive bands may greatly vary by location, size, and nucleotide composition. CBG-banding is the most commonly used method to detect structural heterochromatin in animals. The difficulty in identification of individual chromosomes represents an unresolved problem of this method as the body of the chromosome is stained uniformly and does not have banding pattern beyond C-bands. Here, we present the method that we called CDAG for sequential heterochromatin staining after differential GTG-banding. The method uses G-banding followed by heat denaturation in the presence of formamide with consecutive fluorochrome staining. The new technique is valid for the concurrent revealing of heterochromatin position due to differential banding of chromosomes and heterochromatin composition (AT-/GC-rich) in animal karyotyping.

The Case of X and Y Localization of Nucleolus Organizer Regions (NORs) in Tragulus javanicus (Cetartiodactyla, Mammalia).

There are differences in number and localization of nucleolus organizer regions (NORs) in genomes. In mammalian genomes, NORs are located on autosomes, which are often situated on short arms of acrocentric chromosomes and more rarely in telomeric, pericentromeric, or interstitial regions. In this work, we report the unique case of active NORs located on gonоsomes of a eutherian mammal, the Javan mouse-deer (Tragulus javanicus). We have investigated the position of NORs by FISH experiments with ribosomal DNA (rDNA) sequences (18S, 5.8S, and 28S) and show the presence of a single NOR site on the X and Y chromosomes. The NOR is localized interstitially on the p-arm of the X chromosome in close proximity with prominent C-positive heterochromatin blocks and in the pericentromeric area of mostly heterochromatic Y. The NOR sites are active on both the X and Y chromosomes in the studied individual and surrounded by GC enriched heterochromatin. We hypothesize that the surrounding heterochromatin might have played a role in the transfer of NORs from autosomes to sex chromosomes during the karyotype evolution of the Javan mouse-deer.

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.

Comparative Chromosome Map and Heterochromatin Features of the Gray Whale Karyotype (Cetacea).

Cetacean karyotypes possess exceptionally stable diploid numbers and highly conserved chromosomes. To date, only toothed whales (Odontoceti) have been analyzed by comparative chromosome painting. Here, we studied the karyotype of a representative of baleen whales, the gray whale (Eschrichtius robustus, Mysticeti), by Zoo-FISH with dromedary camel and human chromosome-specific probes. We confirmed a high degree of karyotype conservation and found an identical order of syntenic segments in both branches of cetaceans. Yet, whale chromosomes harbor variable heterochromatic regions constituting up to a third of the genome due to the presence of several types of repeats. To investigate the cause of this variability, several classes of repeated DNA sequences were mapped onto chromosomes of whale species from both Mysticeti and Odontoceti. We uncovered extensive intrapopulation variability in the size of heterochromatic blocks present in homologous chromosomes among 3 individuals of the gray whale by 2-step differential chromosome staining. We show that some of the heteromorphisms observed in the gray whale karyotype are due to distinct amplification of a complex of common cetacean repeat and heavy satellite repeat on homologous autosomes. Furthermore, we demonstrate localization of the telomeric repeat in the heterochromatin of both gray and pilot whale (Globicephala melas, Odontoceti). Heterochromatic blocks in the pilot whale represent a composite of telomeric and common repeats, while heavy satellite repeat is lacking in the toothed whale consistent with previous studies.

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.