Chromosomal Evolution of the Talpinae.

In recent years, the number of mole species with species status confirmed by genetic methods has been continuously increasing. Unfortunately, cytogenetic data are not yet available for all species. Here, for the first time, a GTG-banded karyotype of the small-toothed mole from Vietnam, Euroscaptor parvidens, a representative of the Eastern clade of the genus Euroscaptor, has been described. Through comparative analysis of available Euroscaptor (Euroscaptor parvidens, Euroscaptor klossi, and Euroscaptor malayana) and Oreoscaptor (Oreoscaptor mizura) karyotypes, we found cytogenetic signatures for each of the studied species. Zoo-FISH with sorted chromosomes of the Siberian mole (Talpa altaica) on chromosome sets of the small-toothed mole (E. parvidens), the small Japanese mole (Mogera imaizumii) from the closely related genus, and the Japanese shrew mole (Urotrichus talpoides) from the tribe Urotrichini made it possible to identify syntenic regions between these species. We propose a possible ancestral karyotype of the tribe and, based on it, traced the features of chromosomal rearrangements accompanying the divergence of moles. The low rates of chromosomal evolution within the species of the genus Talpa-T. altaica and T. europaea-and the high rates of karyotypic reshuffling within the Asian genera of the tribe were confirmed. The karyotype of the Japanese mountain mole O. mizura seems to be the most conserved among the Asian moles. The most frequently occurring types of chromosomal rearrangements in moles are the pericentric inversions and amplification of heterochromatin. The pericentric inversions on four pairs of autosomes are shared between the closely related genera Euroscaptor, Oreoscaptor, and Mogera, while many more apomorphic rearrangements have occurred in each lineage additionally. The highest rate of chromosomal changes, with five rearrangements occurring over approximately 7 million years, was recorded in the lineage of the small-toothed mole.

Chromosome Distribution of Highly Conserved Tandemly Arranged Repetitive DNAs in the Siberian Sturgeon (Acipenser baerii).

Polyploid genomes present a challenge for cytogenetic and genomic studies, due to the high number of similar size chromosomes and the simultaneous presence of hardly distinguishable paralogous elements. The karyotype of the Siberian sturgeon (Acipenser baerii) contains around 250 chromosomes and is remarkable for the presence of paralogs from two rounds of whole-genome duplications (WGD). In this study, we applied the sterlet-derived acipenserid satDNA-based whole chromosome-specific probes to analyze the Siberian sturgeon karyotype. We demonstrate that the last genome duplication event in the Siberian sturgeon was accompanied by the simultaneous expansion of several repetitive DNA families. Some of the repetitive probes serve as good cytogenetic markers distinguishing paralogous chromosomes and detecting ancestral syntenic regions, which underwent fusions and fissions. The tendency of minisatellite specificity for chromosome size groups previously observed in the sterlet genome is also visible in the Siberian sturgeon. We provide an initial physical chromosome map of the Siberian sturgeon genome supported by molecular markers. The application of these data will facilitate genomic studies in other recent polyploid sturgeon species.

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.

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

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.

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.

Evolutionary plasticity of acipenseriform genomes.

Acipenseriformes is an order of ray-finned fishes, comprising 27 extant species of sturgeons and paddlefishes inhabiting waters of the Northern Hemisphere. The order has a basal position within Actinopteri (ray-finned fish minus polypterids) and is characterized by many specific morphological and genomic features, including high diploid chromosome numbers, various levels of ploidy between species, unclear sex determination, and propensity to interspecific hybridization. Recent advances in molecular genetics, genomics, and comparative cytogenetics produced novel data on different aspects of acipenseriform biology, including improved phylogenetic reconstructions and better understanding of genome structure. Here, we discuss the cytogenetic and genomic traits of acipenseriforms and their connection with polyploidization and tolerance to interspecific hybridization.

Segmental paleotetraploidy revealed in sterlet (Acipenser ruthenus) genome by chromosome painting.

BACKGROUND: Acipenseriformes take a basal position among Actinopteri and demonstrate a striking ploidy variation among species. The sterlet (Acipenser ruthenus, Linnaeus, 1758; ARUT) is a diploid 120-chromosomal sturgeon distributed in Eurasian rivers from Danube to Enisey. Despite a high commercial value and a rapid population decline in the wild, many genomic characteristics of sterlet (as well as many other sturgeon species) have not been studied. RESULTS: Cell lines from different tissues of 12 sterlet specimens from Siberian populations were established following an optimized protocol. Conventional cytogenetic studies supplemented with molecular cytogenetic investigations on obtained fibroblast cell lines allowed a detailed description of sterlet karyotype and a precise localization of 18S/28S and 5S ribosomal clusters. Localization of sturgeon specific HindIII repetitive elements revealed an increased concentration in the pericentromeric region of the acrocentric ARUT14, while the total sterlet repetitive DNA fraction (C0t30) produced bright signals on subtelomeric segments of small chromosomal elements. Chromosome and region specific probes ARUT1p, 5, 6, 7, 8 as well as 14 anonymous small sized chromosomes (probes A-N) generated by microdissection were applied in chromosome painting experiments. According to hybridization patterns all painting probes were classified into two major groups: the first group (ARUT5, 6, 8 as well as microchromosome specific probes C, E, F, G, H, and I) painted only a single region each on sterlet metaphases, while probes of the second group (ARUT1p, 7 as well as microchromosome derived probes A, B, D, J, K, M, and N) marked two genomic segments each on different chromosomes. Similar results were obtained on male and female metaphases. CONCLUSIONS: The sterlet genome represents a complex mosaic structure and consists of diploid and tetraploid chromosome segments. This may be regarded as a transition stage from paleotetraploid (functional diploid) to diploid genome condition. Molecular cytogenetic and genomic studies of other 120- and 240-chromosomal sturgeons are needed to reconstruct genome evolution of this vertebrate group.

Reconstruction of karyotype evolution in core Glires. I. The genome homology revealed by comparative chromosome painting.

Glires represent a eutherian clade consisting of rodents and lagomorphs (hares, rabbits, and pikas). Chromosome evolution of Glires is known to have variable rates in different groups: from slowly evolving lagomorphs and squirrels to extremely rapidly evolving muroids. Previous interordinal homology maps between slowly evolving Glires were based on comparison with humans. Here, we used sets of chromosome-specific probes from Tamias sibiricus (Sciuridae), Castor fiber (Castoridae) and humans to study karyotypes of six ground squirrels (genera Marmota and Spermophilus) and one tree squirrel (genus Sciurus), mountain hare (genus Lepus), and rabbit (genus Oryctolagus). These data supplemented with GTG banding comparisons allowed us to build comparative chromosome maps. Our data showed the absence of previously found squirrel associations HSA 1/8 and 2/17 in the Eurasian ground squirrels--sousliks and woodchucks, and disruptions of squirrel HSA 10/13 and HSA 8/4/8/12/22 syntenies in the four Spermophilus species studied here. We found that the karyotypes of Sciuridae and Leporidae are highly conserved and close to the Rodentia ancestral karyotype, while Castoridae chromosomes underwent many more changes. We suggest that Lagomorpha and Sciuridae (in contrast to all other rodent families) should be considered as core Glires lineages, characterized by cytogenetically conserved karyotypes which contain chromosomal elements inherent to karyotype of common Glires ancestor. Our data allowed us to further refine the putative ancestral karyotypes of Rodentia. We also describe here the putative ancestral karyotypes of Glires and lagomorphs.

Tracking genome organization in rodents by Zoo-FISH.

The number of rodent species examined by modern comparative genomic approaches, particularly chromosome painting, is limited. The use of human whole-chromosome painting probes to detect regions of homology in the karyotypes of the rodent index species, the mouse and rat, has been hindered by the highly rearranged nature of their genomes. In contrast, recent studies have demonstrated that non-murid rodents display more conserved genomes, underscoring their suitability for comparative genomic and higher-order systematic studies. Here we provide the first comparative chromosome maps between human and representative rodents of three major rodent lineages Castoridae, Pedetidae and Dipodidae. A comprehensive analysis of these data and those published for Sciuridae show (1) that Castoridae, Pedetidae and Dipodidae form a monophyletic group, and (2) that the European beaver Castor fiber (Castoridae) and the birch mouse Sicista betulina (Dipodidae) are sister species to the exclusion of the springhare Pedetes capensis (Pedetidae), thus resolving an enduring trifurcation in rodent higher-level systematics. Our results together with published data on the Sciuridae allow the formulation of a putative rodent ancestral karyotype (2n = 50) that is thought to comprise the following 26 human chromosomal segments and/or segmental associations: HSA1pq, 1q/10p, 2pq, 2q, 3a, 3b/19p, 3c/21, 4b, 5, 6, 7a, 7b/16p, 8p/4a/8p, 8q, 9/11, 10q, 12a/22a, 12b/22b, 13, 14/15, 16q/19q, 17, 18, 20, X and Y. These findings provide insights into the likely composition of the ancestral rodent karyotype and an improved understanding of placental genome evolution.

Cross-species chromosome painting among camel, cattle, pig and human: further insights into the putative Cetartiodactyla ancestral karyotype.

The great karyotypic differences between camel, cattle and pig, three important domestic animals, have been a challenge for comparative cytogenetic studies based on conventional cytogenetic approaches. To construct a genome-wide comparative chromosome map among these artiodactyls, we made a set of chromosome painting probes from the dromedary camel (Camelus dromedarius) by flow sorting and degenerate oligonucleotide primed-PCR. The painting probes were first used to characterize the karyotypes of the dromedary camel (C. dromedarius), the Bactrian camel (C. bactrianus), the guanaco (Lama guanicoe), the alpaca (L. pacos) and dromedary x guanaco hybrid karyotypes (all with 2n = 74). These FISH experiments enabled the establishment of a high-resolution GTG-banded karyotype, together with chromosome nomenclature and idiogram for C. dromedarius, and revealed that these camelid species have almost identical karyotypes, with only slight variations in the amount and distribution patterns of heterochromatin. Further cross-species chromosome painting between camel, cattle, pig and human with painting probes from the camel and human led to the establishment of genome-wide comparative maps. Between human and camel, pig and camel, and cattle and camel 47, 53 and 53 autosomal conserved segments were detected, respectively. Integrated analysis with previously published comparative maps of human/pig/cattle enabled us to propose a Cetartiodactyla ancestral karyotype and to discuss the early karyotype evolution of Cetartiodactyla. Furthermore, these maps will facilitate the positional cloning of genes by aiding the cross-species transfer of mapping information.

Karyotype evolution and phylogenetic relationships of hamsters (Cricetidae, Muroidea, Rodentia) inferred from chromosomal painting and banding comparison.

The evolutionary success of rodents of the superfamily Muroidea makes this taxon the most interesting for evolution studies, including study at the chromosomal level. Chromosome-specific painting probes from the Chinese hamster and the Syrian (golden) hamster were used to delimit homologous chromosomal segments among 15 hamster species from eight genera: Allocricetulus, Calomyscus, Cricetulus, Cricetus, Mesocricetus, Peromyscus, Phodopus and Tscherskia (Cricetidae, Muroidea, Rodentia). Based on results of chromosome painting and G-banding, comparative maps between 20 rodent species have been established. The integrated maps demonstrate a high level of karyotype conservation among species in the Cricetus group (Cricetus, Cricetulus, Allocricetulus) with Tscherskia as its sister group. Species within the genera Mesocricetus and Phodopus also show a high degree of chromosomal conservation. Our results substantiate many of the conclusions suggested by other data and strengthen the topology of the Muroidea phylogenetic tree through the inclusion of genome-wide chromosome rearrangements. The derivation of the muroids karyotypes from the putative ancestral state involved centric fusions, fissions, addition of heterochromatic arms and a great number of inversions. Our results provide further insights into the karyotype relationships of all species investigated.

Reciprocal chromosome painting between three laboratory rodent species.

The laboratory mouse (Mus musculus, 2n = 40), the Chinese hamster (Cricetulus griseus, 2n = 22), and the golden (Syrian) hamster (Mesocricetus auratus, 2n = 44) are common laboratory animals, extensively used in biomedical research. In contrast with the mouse genome, which was sequenced and well characterized, the hamster species has been set aside. We constructed a chromosome paint set for the golden hamster, which for the first time allowed us to perform multidirectional chromosome painting between the golden hamster and the mouse and between the two species of hamster. From these data we constructed a detailed comparative chromosome map of the laboratory mouse and the two hamster species. The golden hamster painting probes revealed 25 autosomal segments in the Chinese hamster and 43 in the mouse. Using the Chinese hamster probes, 23 conserved segments were found in the golden hamster karyotype. The mouse probes revealed 42 conserved autosomal segments in the golden hamster karyotype. The two largest chromosomes of the Chinese hamster (1 and 2) are homologous to seven and five chromosomes of the golden hamster, respectively. The golden hamster karyotype can be transformed into the Chinese hamster karyotype by 15 fusions and 3 fissions. Previous reconstructions of the ancestral murid karyotype proposed diploid numbers from 2n = 52 to 2n = 54. By integrating the new multidirectional chromosome painting data presented here with previous comparative genomics data, we can propose that syntenies to mouse Chrs 6 and 16 were both present and to hypothesize a diploid number of 2n = 48 for the ancestral Murinae/Cricetinae karyotype.