FISH mapping of the IGF2 gene in horse and donkey—detection of homoeology with HSA11

Three genomic subclones derived from a phage clone containing the equine IGF2 gene were used to FISH map the gene on horse (ECA) and donkey (EAS) metaphase chromosomes. The gene mapped on ECA 12q13 band and is the first locus mapped to this horse chromosome. In donkey the gene mapped very terminal on the long arm of one small submetacentric chromosome that shows almost identical DAPI-banding pattern with ECA12. This is the first locus mapped in donkey genome. Cross species chromosome painting of equine metaphase chromosomes with human Chromosome (Chr) 11-specific probe showed homoeology of this human chromosome with ECA12 and ECA7. The novel ECA12 comparative painting results are thus in accordance with the localization of the equine IGF2 gene. Comparison of the hitherto known physical locations of IGF2 in different species, viz. human, cattle, sheep, horse, and donkey, shows that this gene tends to maintain a terminal location on the chromosome arm. Received: 12 January 1997 / Accepted: 17 March 1997     

Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids

Human and sheep chromosome-specific probes were used to construct comparative painting maps between the pig (Suiformes), cattle and sheep (Bovidae), and humans. Various yet unknown translocations were observed that would assist in a more complete reconstruction of homology maps of these species. The number of homologous segments that can be identified with sheep probes in the pig karyotype exceeds that described previously by chromosome painting between two non-primate mammals belonging to the same order. Sheep probes painted 62 segments on pig autosomes and delineated not only translocations, but also 9 inversions. All inversions were paracentric and indicate that these rearrangements may be characteristic for chromosomal changes in suiforms. Hybridizations of all sheep painting probes to cattle chromosomes confirmed the chromosome conservation in bovids. In addition, we observed a small translocation that was previously postulated from linkage mapping data, but was not yet described by physical mapping. The chromosome painting data are complemented with a map of available comparative gene mapping data between pig and sheep genomes. A detailed table listing the comparative gene mapping data between pig and cattle genomes is provided. The reanalysis of the pig karyotype with a new generation of human paint probes provides an update of the human/pig comparative genome map and demonstrates two new chromosome homologies. Seven conserved segments not yet identified by chromosome painting are also reported. Received: 2 October 2000 / Accepted: 15 January 2001     

Mapping homology between human and black and white colobine monkey chromosomes by fluorescent in situ hybridization

We used in situ hybridization of chromosome specific DNA probes (“chromosome painting”) of all human chromosomes to establish homologies between the human and the white and black colobus (Colobus guereza 2n = 44). The 24 human paints gave 31 signals on the autosomes (haploid male chromosome set). Robertsonian translocations between chromosomes homologus to human 14 and 15, 21 and 22, form colobine chromosomes 6 and 16, respectively. Reciprocal translocations were found between human chromosomes 1 and 10, 1 and 17, as well as 3 and 19. The alternating hybridization signals between human 3 and 19 on Colobus chromosome 12 show that in this case a reciprocal translocation was followed by a pericentric inversion. The hybridization data show that in spite of the same diploid number and similar Fundamental Numbers, the black and white colobine monkey differs from Presbytis cristata, an Asian colobine, by 6 reciprocal translocations. Comparisons with the hybridization patterns in other primates show that some Asian colobines have a more derived karyotype with respect to African colobines, macaques, great apes, and humans. Chromosome painting also clearly shows that similarities in diploid number and chromosome morphology both between colobines and gibbons are due to convergence. Am. J. Primatol. 42:289–298, 1997. © 1997 Wiley-Liss, Inc.     

Comparative analysis of the cattle and human genomes: detection of ZOO-FISH and gene mapping-based chromosomal homologies

Comparative chromosome painting with individual human chromosome-specific libraries (CSLs) on cattle metaphase chromosomes delineated 46 homologous chromosomal segments between the two species. Continuous arrangement of these segments on individual cattle chromosomes demonstrates a nearly complete coverage of the bovine karyotype and shows physical boundaries of bovine chromosomal segments homologous to individual human chromosomes. Alignment of the available comparative gene mapping data with the homologous segments strongly supports the detected gross homologies between the karyotypes of the two species. In addition to cattle, four human CSLs were hybridized to sheep metaphase chromosomes also, to further verify the known karyotype homology within the Bovidae. Besides its application to karyotype evolution research, the comparative knowledge provides for rapid expansion of the much needed Type I locus-based bovine gene map. Received: 9 September 1995 / Accepted: 4 December 1995     

Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA

An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG/CGG_n trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human fragile-X probes on cattle chromosomes was however, medium-prominent sub-centromeric signal on two homologues. BrdU administration in 12 h before harvesting identified these homologues to be chromosome number 5. In addition retrospective slides of cattle and sheep chromosomes used for fragile site studies showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X.     

A high-resolution comparative radiation hybrid map of ovine chromosomal regions that are homologous to human chromosome 6 (HSA6)

In this study, we constructed high-resolution radiation hybrid (RH) and comparative maps of ovine chromosomes or chromosomal segments that are homologous to human chromosome 6 (HSA6). A total of 251 markers were successfully genotyped across the recently developed USUoRH5000 whole-genome panel; 208 of these markers were assigned to five RH linkage groups distributed on three ovine chromosomes (OAR8, 9 and 20). The RH maps have good correspondence with previous chromosome painting data, although a small centromeric region on OAR9 that is homologous to HSA6 had not been previously detected using human chromosome paints on ovine chromosomal spreads. High percentages of the ovine markers were identified as orthologues in the bovine (86.3%), dog (85.8%), horse (69.3%) and human (88.7%) genomes. These maps contribute to investigations in mammalian chromosome evolution and the search for economic trait loci in sheep.     

Comparative Chromosome Painting in Mammals: Human and the Indian Muntjac (Muntiacus muntjak vaginalis)

We have used human chromosome-specific painting probes forin situhybridization on Indian muntjac (Muntiacus muntjak vaginalis,2n= 6, 7) metaphase chromosomes to identify the homologous chromosome regions of the entire human chromosome set. Chromosome rearrangements that have been involved in the karyotype evolution of these two species belonging to different mammalian orders were reconstructed based on hybridization patterns. Although, compared to human chromosomes, the karyotype of the Indian muntjac seems to be highly rearranged, we could identify a limited number of highly conserved homologous chromosome regions for each of the human chromosome-specific probes. We identified 48 homologous autosomal chromosome segments, which is in the range of the numbers found in other artiodactyls and carnivores recently analyzed by chromosome painting. The results demonstrate that the reshuffling of the muntjac karyotype is mostly due to fusions of huge blocks of entire chromosomes. This is in accordance with previous chromosome painting analyses between various Muntjac species and contrasts the findings for some other mammals (e.g., gibbons, mice) that show exceptional chromosome reshuffling due to multiple reciprocal translocation events.     

Physical mapping confirms that sheep chromosome 10 has extensive conserved synteny with cattle chromosome 12 and human chromosome 13

The following loci, on human chromosome 13, have been newly assigned to sheep chromosome 10 using chromosomally characterized sheep-hamster cell hybrids: gap junction protein, beta 2, 26 kDa (connexin 26) (GJB2); gap junction protein, alpha 3, 46 kDa (connexin 46) (GJA3), and esterase D/formylglutathione hydrolase (ESD). This assignment of ESD is consistent with comparative mapping evidence, but not with an earlier report of it on sheep chromosome 3p26-p24. Cell hybrid analysis confirmed the location of another human chromosome 13 locus, retinoblastoma 1 (including osteosar-coma) (RBI), and the anonymous ovine genomic sequence RP11 on sheep chromosome 10. Isotopic in situ hybridization was used to regionally localize RP11 on to sheep 10q15-q22. The location of microsatellites AGLA226, OarDB3, OarHH41, OarVH58, and TGLA441, previously assigned to sheep chromosome 10 by linkage analysis, was confirmed by polymerase chain reaction using the cell hybrid panel. These mapping data provide further evidence that sheep chromosome 10 is the equivalent of cattle chromosome 12, and that these chromosomes show extensive conserved synteny with human chromosome 13.     

Chromosome mapping of Miller-Diecker,Smith-Magenis and RARA loci in non-human primates: implications in the evolution of human chromosome 17

Molecular cytogenetics allows to verify chromosomal homologies previously hypothesised on the base of banding pattern comparison in different species. So far only the chromosome painting technique has been extensively used in studies of chromosomal evolution. This technique allows to detect only interchromosomal rearrangements. Human and Great Apes chromosomes basically differ by intrachromosomal rearrangements, in particular inversions; with chromosome painting it has just been possible to confirm the origin by fusion of human chromosome 2 and a reciprocal translocation in Gorilla, involving the homologous of chromosome 5 and 17. In order to verify intrachromosomal rearrangements in human chromosomal evolution, chromosome mapping of human loci in non-human primates is a useful approach. We mapped Miller-Diecker, Smith-Magenis and RARA loci localised on human chromosome 17, in Gorilla gorilla, Pongo pygmaeus, Macaca fascicularis and Cercopithecus aethiops. On the base of the obtained results it was possible to verify chromosomal rearrangements previously identified by banding, to achieve new informations about the controversial evolution of human chromosome 17, and to detect the occurrence of a paracentric inversion in the homologous in Cercopithecus aethiops.     

Single‐copy gene‐based chromosome painting in cucumber and its application for chromosome rearrangement analysis in Cucumis

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single‐copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome‐specific gene probes. This single‐copy gene‐based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis‐aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross‐species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.     

Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome‐specific painting

Reliable identification of individual chromosomes in eukaryotic species is the foundation for comparative chromosome synteny and evolutionary studies. Unfortunately, chromosome identification has been a major challenge for plants with small chromosomes, such as the Citrus species. We developed oligonucleotide‐based chromosome painting probes for all nine chromosomes in Citrus maxima (Pummelo). We were able to identify all C. maxima chromosomes in the same metaphase cells using multiple rounds of sequential fluorescence in situ hybridization with the painting probes. We conducted comparative chromosome painting analysis in six different Citrus and related species. We found that each painting probe hybridized to only a single chromosome in all other five species, suggesting that the six species have maintained a complete chromosomal synteny after more than 9 million years of divergence. No interchromosomal rearrangement was identified in any species. These results support the hypothesis that karyotypes of woody species are more stable than herbaceous plants because woody plants need a longer period to fix chromosome structural variants in natural populations.     

Comparative painting of mammalian chromosomes

Comparative chromosome painting has shown that synteny has been conserved for large segments of the genome in various placental mammals. Advances such as spectral karyotyping and multicolour ‘bar coding’ lend speed and precision to comparative molecular cytogenetics. Reciprocal chromosome painting and hybridisations with probes such as yeast artificial chromosomes, cosmids, and fibre fluorescence in situ hybridisation allow subchromosomal assignments of chromosome regions and can identify breakpoints of rearranged chromosomes. Advances in molecular cytogenetics can now be used to test the hypothesis that chromosome rearrangement breakpoints in human pathology and in evolution are correlated.     

Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double‐stranded labelled oligos, which produced much stronger signals than single‐stranded labelled oligos, by amplification using fluorophore‐conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross‐species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo‐painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.     

Chromosome reshuffling in birds of prey: the karyotype of the world's largest eagle (Harpy eagle, Harpia harpyja) compared to that of the chicken (Gallus gallus)

Like various other diurnal birds of prey, the world's largest eagle, the Harpy (Harpia harpyja), presents an atypical bird karyotype with 2n=58 chromosomes. There is little knowledge about the dramatic changes in the genomic reorganization of these species compared to other birds. Since recently, the chicken provides a “default map” for various birds including the first genomic DNA sequence of a bird species. Obviously, the gross division of the chicken genome into relatively gene-poor macrochromosomes and predominantly gene-rich microchromosomes has been conserved for more than 150 million years in most bird species. Here, we present classical features of the Harpy eagle karyotype but also chromosomal homologies between H. harpyja and the chicken by chromosome painting and comparison to the chicken genome map. We used two different sets of painting probes: (1) chicken chromosomes were divided into three size categories: (a) macrochromosomes 1–5 and Z, (b) medium-sized chromosomes 6–10, and (c) 19 microchromosomes; (2) combinatorially labeled chicken chromosome paints 1–6 and Z. Both probe sets were visualized on H. harpyja chromosomes by multicolor fluorescence in situ hybridization (FISH). Our data show how the organization into micro- and macrochromosomes has been lost in the Harpy eagle, seemingly without any preference or constraints.     

Defining the ancestral karyotype of all primates by multidirectional chromosome painting between tree shrews, lemurs and humans

We used multidirectional chromosome painting with probes derived by bivariate fluorescence-activated flow sorting of chromosomes from human, black lemur (Eulemur macaco macaco) and tree shrew (Tupaia belangeri, order Scandentia) to better define the karyological relationship of tree shrews and primates. An assumed close relationship between tree shrews and primates also assists in the reconstruction of the ancestral primate karyotype taking the tree shrew as an ”outgroup” species. The results indicate that T. belangeri has a highly derived karyotype. Tandem fusions or fissions of chromosomal segments seem to be the predominant mechanism in the evolution of this tree shrew karyotype. The 22 human autosomal painting probes delineated 40 different segments, which is in the range found in most mammals analyzed by chromosome painting up to now. There were no reciprocal translocations that would distinguish the karyotype of the tree shrew from an assumed primitive primate karyotype. This karyotype would have included the chromosomal forms 1a, 1b, 2a, 2b, 3/21, 4–11, 12a/22a, 12b/22b, 13, 14/15, 16a, 16b, 17, 18, 19a, 19b, 20 and X and Y and had a diploid chromosome number of 2n=50. Of these forms, chromosomes 1a, 1b, 4, 8, 12a/22a, and 12b/22bmay be common derived characters that would link the tree shrew with primates. To define the exact phylogenetic relationships of the tree shrews and the genomic rearrangements that gave rise to the primates and eventually to humans further chromosome painting in Rodentia, Lagomorpha, Dermoptera and Chiroptera is needed, but many of the landmarks of genomic evolution are now known. Received: 11 February 1999; in revised form: 17 June 1999 / Accepted: 20 July 1999     

Regional Assignment of Conserved Reference Loci Anchors Unassigned Linkage and Syntenic Groups to Ovine Chromosomes

Seven loci that have been previously mapped to human and mouse chromosomes have now been regionally assigned to six sheep chromosomes. Nerve growth factor β (NGFB), antigen CD3 ζ polypeptide (CD3Z), inhibin β A (INHBA), estrogen receptor (ESR), rhodopsin (RHO), insulin-like growth factor 2 (IGF2), and myelin basic protein (MBP) were mapped by in situ hybridization to sheep chromosomes 1p24-p21, 1p14-p11, 4q26-q31, 8q25-q27, 19q23-qter, 21q21-qter, and 23q11-q12.3, respectively. ESR, RHO, IGF2, and MBP are the first markers to be assigned to their respective sheep chromosomes. These new data allow the previously unassigned sheep linkage groups H, J, K, and S to be provisionally assigned to chromosomes 21, 19, 4, and 8, respectively. The unassigned sheep syntenic groups U8 and U13 are provisionally assigned to sheep chromosomes 8 and 21, respectively. The new assignments support the emerging picture that there is extensive conservation of human chromosomal segments in the sheep and cattle genomes. The position of another evolutionary breakpoint on human chromosome 1q is suggested.     

Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting

This study presents a whole-genome comparison of human and a representative of the Afrotherian clade, the African elephant, generated by reciprocal Zoo-FISH. An analysis of Afrotheria genomes is of special interest, because recent DNA sequence comparisons identify them as the oldest placental mammalian clade. Complete sets of whole-chromosome specific painting probes for the African elephant and human were constructed by degenerate oligonucleotide-primed PCR amplification of flow-sorted chromosomes. Comparative genome maps are presented based on their hybridization patterns. These maps show that the elephant has a moderately rearranged chromosome complement when compared to humans. The human paint probes identified 53 evolutionary conserved segments on the 27 autosomal elephant chromosomes and the X chromosome. Reciprocal experiments with elephant probes delineated 68 conserved segments in the human genome. The comparison with a recent aardvark and elephant Zoo-FISH study delineates new chromosomal traits which link the two Afrotherian species phylogenetically. In the absence of any morphological evidence the chromosome painting data offer the first non-DNA sequence support for an Afrotherian clade. The comparative human and elephant genome maps provide new insights into the karyotype organization of the proto-afrotherian, the ancestor of extant placental mammals, which most probably consisted of 2n=46 chromosomes.     

Chromosome Painting Technique Contributes to Constructions of Evolutionary Trees of Lemurs

We used human-specific probes for chromosomes 3, 11, 14, 15, 17, 19, 21 to paint chromosomes of four species of Indridae (Avahi laniger, Indri indri, Propithecus verreauxi, and P. diadema). All human chromosome probes hybridized specifically to chromosome segments or to whole arms of indrid chromosomes. In general, the results obtained by chromosome painting confirm those obtained by R-banding except for some reciprocal translocations undetected by R-banding. Our findings confirm that in the evolution of the Indridae, Avahi laniger first emerges, then Indri and Propithecus share a common trunk. After populational evolution, this sister clade diverged.     

A novel source of highly specific chromosome painting probes for human karyotype analysis derived from primate homologues

We have established a series of highly specific painting probes for human acrocentric chromosomes. These chromosomes are involved in the formation of the nucleolar organizer region (NOR) and show DNA sequence homologies within their pericentric heterochromatin. To date, these chromosomes have shown considerable cross hybridization in chromosome painting experiments. Our probe set has been established from primate homologues that are not involved in the NOR in that particular species or from species in which highly repetitive sequences have undergone rapid sequence divergence. The new painting probes should be of particular value for automated microscopy, for which highly specific signals are required as they are recorded at low magnification, e.g. when scoring chromosome 21 domains in interphase nuclei. Received: 22 May 1997 / Accepted: 16 June 1997     

A brief history of human autosomes.

Comparative gene mapping and chromosome painting permit the tentative reconstruction of ancestral karyotypes. The modern human karyotype is proposed to differ from that of the most recent common ancestor of catarrhine primates by two major rearrangements. The first was the fission of an ancestral chromosome to produce the homologues of human chromosomes 14 and 15. This fission occurred before the divergence of gibbons from humans and other apes. The second was the fusion of two ancestral chromosomes to form human chromosome 2. This fusion occurred after the divergence of humans and chimpanzees. Moving further back in time, homologues of human chromosomes 3 and 21 were formed by the fission of an ancestral linkage group that combined loci of both human chromosomes, whereas homologues of human chromosomes 12 and 22 were formed by a reciprocal translocation between two ancestral chromosomes. Both events occurred at some time after our most recent common ancestor with lemurs. Less direct evidence suggests that the short and long arms of human chromosomes 8, 16 and 19 were unlinked in this ancestor. Finally, the most recent common ancestor of primates and artiodactyls is proposed to have possessed a chromosome that combined loci from human chromosomes 4 and 8p, a chromosome that combined loci from human chromosomes 16q and 19q, and a chromosome that combined loci from human chromosomes 2p and 20.