Highly comprehensive karyotype analysis by a combination of spectral karyotyping (SKY), microdissection, and reverse painting (SKY-MD)

A technique disclosing most information about chromosome modifications is the technique of choice for the analysis of chromosome alterations. The newly developed method for microdissection of fluorescence-labeled chromosomes (FISH-MD) can improve upon this expectation in combination with 24-color spectral karyotyping (SKY). The highly efficient way to detect chromosome modifications by SKY and the detailed specification of aberrant chromosomes by FISH-MD prompted us to use both techniques in a combined approach called SKY-MD. First, an overview of chromosomal aberrations is obtained by spectral karyotyping and subsequently the derivative chromosomes recognized are characterized in a highly specific manner by microdissection and reverse painting. A small quantity of isolated material dissected directly from a 24-color metaphase is sufficient to obtain very detailed information about the chromosome regions and the breakpoints involved in the derivative chromosomes. Therefore, the combination of spectral karyotyping and microdissection in one procedure, and reverse painting can characterize chromosomal aberrations with a degree of specificity hitherto unknown from individual karyotyping experiments. In this article we compare the efficiency of both the SKY technique and that of classical microdissection with the efficiency obtained by SKY-MD.     

Reconstruction of the ancestral ferungulate karyotype by electronic chromosome painting (E-painting)

By comparing high-coverage and high-quality whole genome sequence assemblies it is now possible to reconstruct putative ancestral progenitor karyotypes, here called protokaryotypes. For this study we used the recently described electronic chromosome painting technique (E-painting) to reconstruct the karyotype of the 85 million-year-old (MYA) ferungulate ancestor. This model is primarily based on dog (Canis familiaris) and cattle (Bos taurus) genome data and is highly consistent with comparative gene mapping and chromosome painting data. The protokaryotype bears 23 autosomal chromosome pairs and the sex chromosomes and preserves most of the chromosomal associations described previously for the boreo-eutherian protokaryotype. The model indicates that five interchromosomal rearrangements occurred during the transition from the boreo-eutherian to the ferungulate ancestor. From there on 66 further interchromosomal rearrangements took place in the lineage leading to cattle and 61 further interchromosomal rearrangements in the lineage to dog. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: further insight into the ancestral karyotype and genome evolution of eutherian mammals

To better understand the evolution of genome organization of eutherian mammals, comparative maps based on chromosome painting have been constructed between human and representative species of three eutherian orders: Xenarthra, Pholidota, and Eulipotyphla, as well as between representative species of the Carnivora and Pholidota. These maps demonstrate the conservation of such syntenic segment associations as HSA3/21, 4/8, 7/16, 12/22, 14/15 and 16/19 in Eulipotyphla, Pholidota and Xenarthra and thus further consolidate the notion that they form part of the ancestral karyotype of the eutherian mammals. Our study has revealed many potential ancestral syntenic associations of human chromosomal segments that serve to link the families as well as orders within the major superordinial eutherian clades defined by molecular markers. The HSA2/8 and 7/10 associations could be the cytogenetic signatures that unite the Xenarthrans, while the HSA1/19p could be a putative signature that links the Afrotheria and Xenarthra. But caution is required in the interpretation of apparently shared syntenic associations as detailed analyses also show examples of apparent convergent evolution that differ in breakpoints and extent of the involved segments.     

Chromosomal rearrangements underlying karyotype differences between Chinese pangolin (Manis pentadactyla) and Malayan pangolin (Manis javanica) revealed by chromosome painting

The Chinese pangolin (Manis pentadactyla), a representative species of the order Pholidota, has been enlisted in the mammalian whole-genome sequencing project mainly because of its phylogenetic importance. Previous studies showed that the diploid number of M. pentadactyla could vary from 2n = 36 to 42. To further characterize the genome organization of M. pentadactyla and to elucidate chromosomal mechanism underlying the karyotype diversity of Pholidota, we flow-sorted the chromosomes of 2n = 40 M. pentadactyla, and generated a set of chromosome-specific probes by DOP-PCR amplification of flow-sorted chromosomes. A comparative chromosome map between M. pentadactyla and the Malayan pangolin (Manis javanica, 2n = 38), as well as between human and M. pentadactyla, was established by chromosome painting for the first time. Our results demonstrate that seven Robertsonian rearrangements, together with considerable variations in the quantity of heterochromatin and in the number of nucleolar organizer regions (NORs) differentiate the karyotypes of 2n = 38 M. javanica and 2n = 40 M. pentadactyla. Moreover, we confirm that the M. javanica Y chromosome bears one NOR. Comparison of human homologous segment associations found in the genomes of M. javanica and M. pentadactyla revealed seven shared associations (HSA 1q/11, 2p/5, 2q/10q, 4p+q/20, 5/13, 6/19p and 8q/10p) that could constitute the potential Pholidota-specific signature rearrangements.     

The phylogeny of howler monkeys (Alouatta, Platyrrhini): Reconstruction by multicolor cross-species chromosome painting

We performed multidirectional chromosome painting in a comparative cytogenetic study of the three howler monkey species Alouatta fusca, A. caraya and A. seniculus macconnelli (Atelinae, Platyrrhini) in order to reconstruct phylogenetic relationships within this genus. Comparative genome maps between these species were established by multicolor fluorescence in-situ hybridization (FISH) employing human, Saguinus oedipus and Lagothrix lagothricha chromosome-specific probes. The three species included in this study and previously analyzed howler monkey species were subjected to a phylogenetic analysis on the basis of a data matrix comprised of 98 discrete molecular cytogenetic characters. The results revealed that howler monkeys represent the genus with the most extensive karyotype diversity within Platyrrhini so far analyzed with high levels of intraspecific chromosomal variability. Two different multiple sex chromosome systems were identified. The phylogenetic analysis indicated that Alouatta is a monophyletic clade which can be derived from a proposed ancestral Atelinae karyotype of 2n=62 chromosomes by a chromosome fusion, a fission, a Y-autosomal translocation and a pericentric inversion. Following these suggestions, the genus Alouatta can be divided into two distinct species groups: the first includes A. caraya and A. belzebul, the second A. s. macconnelli, A. sara, A. s. arctoidea and A. fusca.     

Chromosomal studies in Callicebus donacophilus pallescens, with classic and molecular cytogenetic approaches: Multicolour FISH using human and Saguinus oedipus painting probes

This paper presents the karyotype of Callicebus donacophilus pallescens for the first time. The analysis included G-, C-, NOR-banding techniques and FISH with chromosome painting probes from Saguinus oedipus and Homo sapiens. The results were compared with the karyotypes of Callicebus moloch donacophilus and C. moloch previously published. These three karyotypes display the same diploid number (2n=50) but diverge about the number of biarmed and acrocentric chromosomes. The acrocentrics 14 and 15 from C. m. donacophilus and C. moloch have undergone an in-tandem fusion originating a large acrocentric (pair 10) in C. d. pallescens. The major submetacentric pair (pair 1) from C. d. donacophilus and C. moloch have undergone fission originating two acrocentric pairs in C. d. pallescens (pairs 15 and 22). Herein was evidence that, in spite of the high interspecific variation among Callicebus, most of the chromosomes remained conserved.     

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  ×  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. †Both authors contributed equally to this paper.     

Whole-comparative genomic hybridization (W-CGH): 1. The quick overview of repetitive DNA sequences on a genome

We present a technique (W-CGH) based on Comparative Genomic Hybridization (CGH), but using whole DNA probes, which permits the identification of chromosomal polymorphisms related to highly repetitive DNA sequences that exist between the two genomes compared. The procedure employs two differently colored whole DNA probes from two different individuals that are mixed and hybridized to metaphase chromosomes. The method provides a simple way to map whole genome differences for highly repetitive DNA sequences between two individuals, since it does not require chromosome-specific probes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pericentromeric euchromatin is conserved in minute human supernumerary chromosomes: a study using cross-species colour segmenting (RxFISH)

Investigation of marker chromosomes is one of the most challenging areas of clinical cytogenetics, especially in the prenatal scenario. A range of techniques including microdissection/reverse painting, SKY and M-FISH are available for the investigation of larger markers (>3 Mb). All these techniques rely on hybridization of unique, homologous sequences with simultaneous suppression of repeat sequences. In contrast, RxFISH is based on hybridization of cross-species syntenic sequences; repeat sequences do not hybridize due to species divergence. We have used RxFISH to analyse a group of the smallest, i.e. minute, supernumerary marker chromosomes. Our results suggest that even the smallest marker chromosomes often contain conserved pericentric euchromatin. More detailed characterization of pericentric genetic content is needed to assess the clinical significance of minute supernumerary markers.     

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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A comparative ZOO-FISH analysis in bats elucidates the phylogenetic relationships between Megachiroptera and five microchiropteran families

Fluorescence in-situ hybridization with human whole chromosome painting probes (WCPs) was applied to compare the karyotypes of members of five bat families. Twenty-five evolutionarily conserved units (ECUs) were identified by ZOO-FISH analysis. In 10 of these 25 ECUs, thorough GTG-band comparison revealed an identical banding pattern in all families studied. Differences in the remaining ECUs were used as characters to judge the phylogenetic relationships within Chiroptera. Close relations hips were found between Rhinolophidae and Hipposideridae. Also closely related are the representatives of the yangochiropteran families Phyllostomidae (genus studied: Glossophaga, Volleth et al. 1999), Molossidae and Vespertilionidae. All microchiropteran species studied here share four common features not found in the megachiropteran species Eonycteris spelaea. Two of these are considered as derived characters with a high probability of parallel evolution. On the other hand, Eonycteris shares one common, probably derived feature with the rhinolophoid families Rhinolophidae and Hipposideridae and an additional one only with Hipposideridae. At the moment, the relationships between Yangochiroptera, Rhinolophoidea and Megachiroptera must be left in an unsolved trichotomy. Comparison of neighboring segment combinations found in Chiroptera with those found in other mammalian taxa revealed six synapomorphic features for Chiroptera. Therefore, for karyological reasons, monophyly of Chiroptera is strongly supported. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH

We have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequences, greatly decreasing PCR efficiency selectively across these blocked regions. A second labelling reaction is then done and this product is resuspended in FISH hybridization mixture without further addition of blocking DNA. The hybridization produces little if any non-specific binding on any other chromosomes. We have been able to successfully use this procedure with both human and rat chromosome probes. This technique should be applicable in producing probes for CGH, M-FISH and SKY, as well as reducing the presence of repetitive DNA in genomic libraries     

The chromosome complement ofAcomys spp. (Rodentia,Muridae) from Oursi,Burkina Faso—the ancestral karyotype of thecahirinus-dimidiatus group?

We present here data on chromosome banding analysis (R- and C-bands) ofAcomys sp. (Rodentia, Muridae) from Oursi, Burkina Faso, characterized by 2n=FN=68 and comparison of its banding patterns with those ofAcomys dimidiatus from Saudi Arabia (2n=38, FN=70), studied previously. The study revealed complete homology between acrocentric chromosomes ofAcomys sp. and chromosome arms of 16 pairs of metacentric and two pairs of acrocentric chromosomes ofA. dimidiatus. In addition to monobrachial homology, one tandem translocation accompanied by a centromeric shift was identified in the karyotype of the latter species. The data obtained show that karyotypes of all the species of theAcomys cahirinus-dimidiatus group studied previously may be derived from that ofAcomys sp. from Oursi by means of numerous non-homologous Rb translocations and 1–2 tandem translocations, and thus its karyotype may be considered as ancestral for thecahirinus-dimidiatus group.accepted for publication by M. Schmid     

A fiber-FISH contig spanning the non-recombining region of the human Y chromosome

Using fluorescence in-situ hybridization on interphase chromatin fibers (fiber-FISH), we have constructed an overlapping fiber-FISH contig spanning the non-recombining region of the human Y chromosome (NRY). We first established a standard FISH-signal pattern for a distinct panel of DNA clones on prometaphase Y chromosomes in six healthy fertile men. Clones in the panel were selected from all R-bands as well as deletion intervals 1 through 7 plus PAR1 and PAR2 of the human Y chromosome. We next used signals of these marker clones to build a fiber-FISH contig for the multicopy gene families, CDY, DAZ, RBMY, TSPY and XKRY, along the NRY. Our fiber-FISH contig of human NRY may help to close the four gaps that still exist in the current physical map of the human Y chromosome. Furthermore, it provides a more complete picture with respect to the positions and arrangements of the multicopy gene families along the human NRY.     

Conservation of aphidicolin-induced fragile sites in Papionini (Primates) species and humans

Fragile sites are considered structural features of mammalian chromosomes and a commonly repeated hypothesis is that they are evolutionarily conserved. We tested this hypothesis by establishing the subchromosomal homology of regions harbouring fragile sites in the chromosomes of humans, Macaca fascicularis (MFA) and Mandrillus sphinx (MSP). We delineated the interspecific homology of chromosome bands expressing aphidicolin-induced fragile sites of homologues to human chromosomes 1, 3, 5, 7, 18 and X by the comparative FISH of human BAC and YAC clones. Notably, two YAC clones known to span human chromosome regions containing fragile sites were shown to also span fragile sites in macaques and mandrills. The present comparative BAC/YAC mapping data represent, up to now, the most precise evidence of fragile site conservation during primate evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Localization of Repetitive DNAs to Zebrafish (Danio rerio) Chromosomes by Fluorescence in situ Hybridization (FISH)

The genome of the zebrafish, Danio rerio, contains two major classes of tandem repetitive elements (AT-rich and GC-rich). The AT-rich repeats can be further subdivided into two subgroups which differ by about 10% of 185 bp in the repeating unit. The chromosomal location of these sequences and the moderately repetitive 5S rDNA sequences was determined in two diploid zebrafish cell lines using in-situ hybridization with fluorochrome-labeled probes. The AT-rich sequences were found at the centromeres of all chromosome pairs and the GC-rich sequences were found in paracentromeric location on over half of the chromosomal pairs. Different patterns of hybridization were found for the two subgroups of the AT-rich family. One type hybridized primarily to centromeres of one half to two thirds of the chromosomal pairs and the other type to centromeres of about three fourths of the chromosomal pairs. The pattern of hybridization with the GC-rich sequences varied somewhat between the cell lines consistent with interindividual variation in the location of paracentromeric heterochromatin. The 5S rRNA genes are found on the long arm of chromosome 3. Most of this chromosome arm is late replicating, but apparently does not contain either the AT-rich or GC-rich repetitive sequences.     

Characterization of the atypical karyotype of the black-winged kite Elanus caeruleus (Falconiformes: Accipitridae) by means of classical and molecular cytogenetic techniques

The karyotype of the black-winged kite (Elanus caeruleus), a small diurnal raptor living in Africa, Asia and southern Europe, was studied with classical (G-, C-, R-banding, and Ag-NOR staining) and molecular cytogenetic methods, including primed in-situ labelling (PRINS) and fluorescence in-situ hybridization (FISH) with telomeric (TTAGGG) and centromeric DNA repeats. The study revealed that the genome size, measured by flow cytometry (3.1pg), is in the normal avian range. However, the black-winged kite karyotype is particularly unusual among birds in having a moderate diploid number of 68 chromosomes, and containing only one pair of dot-shaped microchromosomes. Moreover, the macrochromosomes are medium-sized, with the Z and W gonosomes being clearly the largest in the set. C-banding shows that constitutive heterochromatin is located at the centromeric regions of all chromosomes, and that two pairs of small acrocentrics and the pair of microchromosomes are almost entirely heterochromatic and G-band negative. The distribution pattern of a centromeric repeated DNA sequence, as demonstrated by PRINS, follows that of C-heterochromatin. The localization of telomeric sequences by FISH and PRINS reveals many strong telomeric signals but no extratelomeric signal was observed. The atypical organization of the karyotype of the black-winged kite is considered in the context of the modes of karyotypic evolution in birds.     

Identification by R-banding and FISH of chromosome arms involved in Robertsonian translocations in several deer species

We constructed and analyzed the RBG-banded karyotype of five deer species: Chital (Axis axis), White-lipped deer (Cervus albirostris), Rusa deer (Cervus timorensis russa), Sambar deer (Cervus unicolor) and Eld's deer (Cervus eldi siamensis). Among these five species, only Eld's deer had been previously karyotyped using R-banding. In order to identify all the chromosome correspondences with cattle and precisely which chromosome arms are involved in Robertsonian translocations, we compared the karyotypes of these five species with those of the closely related and well-characterized species, cattle (Bos taurus) and Vietnamese Sika deer (Cervus nippon pseudaxis). Among these six deer species (the five above plus the Vietnamese Sika deer), we found thirteen different Robertsonian translocations involving nineteen different chromosome arms. Thirteen chromosome arms were identified by comparison of R-banding patterns only and the remaining six were either confirmed or identified by FISH-mapping of bovine or caprine probes previously localized in cattle. Finally, we observed that five of the thirteen Robertsonian translocations are shared by at least two species and that some chromosome arms are more frequently involved in Robertsonian translocations than others. This revised version was published online in July 2006 with corrections to the Cover Date.     

High chromosome conservation detected by comparative chromosome painting in chicken, pigeon and passerine birds

Chicken chromosome paints for macrochromosomes 1-10, Z, and the nine largest microchromosomes (Griffin et al. 1999) were used to analyze chromosome homologies between chicken (Gallus gallus domesticus: Galliformes), domestic pigeon (Columba livia: Columbiformes), chaffinch (Fringilla coelebs Passeriformes), and redwing (Turdus iliacus: Passeriformes). High conservation of syntenies was revealed. In general, both macro- and microchromosomes in these birds showed very low levels of interchromosomal rearrangements. Only two cases of rearrangements were found. Chicken chromosome 1 corresponds to chromosome 1 in pigeon, but to chromosomes 3 and 4 in chaffinch and chromosomes 2 and 5 in redwing. Chicken chromosome 4 was shown to be homologous to two pairs of chromosomes in the karyotypes of pigeon and both passerine species. Comparative analysis of chromosome painting data and the results of FISH with (TTAGGG)n probe did not reveal any correlation between the distribution of interstitial telomere sites (ITSs) and chromosome rearrangements in pigeon, chaffinch and redwing. In chaffinch, ITSs were found to co-localize with a tandem repeat GS (Liangouzov et al. 2002), monomers of which contain an internal TTAGGG motif.     

Chromosomal localization of the telomeric (TTAGGG)<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> sequence in four species of Armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra

The distribution of the vertebrate telomeric sequence (TTAGGG)_n in four species of armadillos (Dasypodidae, Xenarthra), i.e. Chaetophractus villosus (2n = 60), Chaetophractus vellerosus (2n = 62), Dasypus hybridus (2n = 64) and Zaedyus pichiy (2n = 62) was examined by FISH with a peptide nucleic acid (PNA) probe. Besides the expected telomeric hybridization, interstitial (centromeric) locations of the (TTAGGG)n sequence were observed in one chromosome pair of Chaetophractus vellerosus and Zaedyus pichiy, suggesting chromosome fusion of ancestral chromosomes occurring during the evolution of Dasypodidae. In addition, all the species analysed showed one to four apparently telocentric chromosomes, exhibiting only two telomeric signals. However, the immunodetection study of kinetochore proteins on synaptonemal complex spreads from C. villosus showed that the apparently telocentric chromosomes have a tiny short arm that can be resolved only in the more elongated pachytene bivalents. This finding suggests that none of the species of armadillos possess true telocentric chromosomes. Our present results support a reduction in the diploid number by fusion of acrocentrics with loss of chromosome material as a tendency in Dasypodidae.