Homologous fission event(s) implicated for chromosomal polymorphisms among five species in the genus Equus

The genus Equus is unusual in that five of the ten extant species have documented centric fission (Robertsonian translocation) polymorphisms within their populations, namely E. hemionus onager, E. hemionus kulan, E. kiang, E. africanus somaliensis, and E. quagga burchelli. Here we report evidence that the polymorphism involves the same homologous chromosome segments in each species, and that these chromosome segments have homology to human chromosome 4 (HSA4). Bacterial artificial chromosome clones containing equine genes SMARCA5 (ECA2q21 homologue to HSA4q31. 21) and UCHL1 (ECA3q22 homologue to HSA4p13) were mapped to a single metacentric chromosome and two unpaired acrocentrics by FISH mapping for individuals possessing odd numbers of chromosomes. These data suggest that the polymorphism is either ancient and conserved within the genus or has occurred recently and independently within each species. Since these species are separated by 1-3 million years of evolution, this polymorphism is remarkable and worthy of further investigations.     

Chromosome painting between human and lorisiform prosimians: evidence for the HSA 7/16 synteny in the primate ancestral karyotype

Multidirectional chromosome painting with probes derived from flow-sorted chromosomes of humans (Homo sapiens, HSA, 2n = 46) and galagos (Galago moholi, GMO, 2n = 38) allowed us to map evolutionarily conserved chromosomal segments among humans, galagos, and slow lorises (Nycticebus coucang, NCO, 2n = 50). In total, the 22 human autosomal painting probes detected 40 homologous chromosomal segments in the slow loris genome. The genome of the slow loris contains 16 sytenic associations of human homologues. The ancient syntenic associations of human chromosomes such as HSA 3/21, 7/16, 12/22 (twice), and 14/15, reported in most mammalian species, were also present in the slow loris genome. Six associations (HSA 1a/19a, 2a/12a, 6a/14b, 7a/12c, 9/15b, and 10a/19b) were shared by the slow loris and galago. Five associations (HSA 1b/6b, 4a/5a, 11b/15a, 12b/19b, and 15b/16b) were unique to the slow loris. In contrast, 30 homologous chromosome segments were identified in the slow loris genome when using galago chromosome painting probes. The data showed that the karyotypic differences between these two species were mainly due to Robertsonian translocations. Reverse painting, using galago painting probes onto human chromosomes, confirmed most of the chromosome homologies between humans and galagos established previously, and documented the HSA 7/16 association in galagos, which was not reported previously. The presence of the HSA 7/16 association in the slow loris and galago suggests that the 7/16 association is an ancestral synteny for primates. Based on our results and the published homology maps between humans and other primate species, we propose an ancestral karyotype (2n = 60) for lorisiform primates.     

Comparative chromosome painting of four Siberian Vespertilionidae species with Aselliscus stoliczkanus and human probes

Vespertilionidae is the largest chiropteran family that comprises species of different specialization and wide geographic distribution. Up to now, only a few vespertilionid species have been studied by molecular cytogenetic approaches. Here, we have investigated the karyotypic relationships of 4 Vespertilionidae species from Siberia by G-banding and comparative chromosome painting. Painting probes from Aselliscus stoliczkanus were used to establish interspecific homologous chromosomal segments in Myotis dasycneme (2n = 44), Murina hilgendorfi (2n = 44), Plecotus auritus (2n = 32), and Vespertilio murinus (2n = 38). Robertsonian translocations and a few inversions differentiated the karyotypes of the examined species. Painting of P. auritus karyotype with human probes revealed 3 previously undetected cryptic segments homologous to human chromosomes (Homo sapiens, HSA) 8, 15, and 19, respectively. As a consequence, the existence of 2 HSA 4 + 8 syntenies in the P. auritus karyotype has been proven. In addition, a pericentric inversion or centromere shift was revealed on the smallest metacentric P. auritus chromosome 16/17 using the HSA 16 probe explaining the different G-banding pattern in comparison to the homologous Myotis chromosome 16/17.     

Mitochondrial DNA evolution in the genus Equus

Employing mitochondrial DNA (mtDNA) restriction-endonuclease maps as the basis of comparison, we have investigated the evolutionary affinities of the seven species generally recognized as the genus Equus. Individual species' cleavage maps contained an average of 60 cleavage sites for 16 enzymes, of which 29 were invariant for all species. Based on an average divergence rate of 2%/Myr, the variation between species supports a divergence of extant lineages from a common ancestor approximately 3.9 Myr before the present. Comparisons of cleavage maps between Equus przewalskii (Mongolian wild horse) and E. caballus (domestic horse) yielded estimates of nucleotide sequence divergence ranging from 0.27% to 0.41%. This range was due to intraspecific variation, which was noted only for E. caballus. For pairwise comparisons within this family, estimates of sequence divergence ranged from 0% (E. hemionus onager vs. E. h. kulan) to 7.8% (E. przewalskii vs. E. h. onager). Trees constructed according to the parsimony principle, on the basis of 31 phylogenetically informative restriction sites, indicate that the three extant zebra species represent a monophyletic group with E. grevyi and E. burchelli antiquorum diverging most recently. The phylogenetic relationships of E. africanus and E. hemionus remain enigmatic on the basis of the mtDNA analysis, although a recent divergence is unsupported.      

High-resolution gene maps of horse chromosomes 14 and 21: additional insights into evolution and rearrangements of HSA5 homologs in mammals

High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse x hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improved resolution is vital for rapid chromosomal localization of traits of interest on these chromosomes and for facilitating candidate gene searches. The comparative gene mapping data on ECA14 and ECA21 finely align the chromosomes to sequence/gene maps of a range of evolutionarily distantly related species. It also demonstrates that compared to ECA14, the ECA21 segment corresponding to HSA5 is a more conserved region because of preserved gene order in a larger number of and more diverse species. Further, comparison of ECA14 and the distal three-quarters region of ECA21 with corresponding chromosomal segments in 50 species belonging to 11 mammalian orders provides a broad overview of the evolution of these segments in individual orders from the putative ancestral chromosomal configuration. Of particular interest is the identification and precise demarcation of equid/Perissodactyl-specific features that for the first time clearly distinguish the origins of ECA14 and ECA21 from similar-looking status in the Cetartiodactyls.     

Construction of a medium-density horse gene map

A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH) (using an equine RH(5000) panel) and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified. An enhanced resolution of conserved and rearranged chromosomal segments presented in this study provides clarification of chromosome evolution history.     

Chromosome homologies between man and mountain zebra (Equus zebra hartmannae) and description of a new ancestral synteny involving sequences homologous to human chromosomes 4 and 8

Using human chromosome painting probes, we looked for homologies between human and mountain zebra (Equus zebra hartmannae, Equidae, Perissodactyla) karyotypes. Except for two very short segments, all euchromatic regions were found to have a human homologous chromosome segment. Conserved syntenies previously described in various mammalian orders were detected. Each synteny corresponded to a chromosomal region homologous to two parts of human chromosomes: HSA3 and HSA21, HSA7 and HSA16, HSA12 and HSA22, and HSA16 and HSA19. Chromosomal segments homologous to a part of HSA11 and HSA19p are found syntenic in zebra, horse and donkey, suggesting that this group of synteny has been inherited from an Equidae or Perissodactyla common ancestor. A synteny of segments homologous to parts of HSA4 and HSA8 was observed in zebra and horse. It also exists in the rabbit (Lagomorpha) and several Carnivora species. A second group of taxa which does not have this region of synteny is composed of primates, Chiroptera and Insectivora, and possibly also Cetacea and Scandantia. Thus, the presence or absence of this region of synteny may separate two groups of eutherian mammals.     

FISH analysis comparing genome organization in the domestic horse (Equus caballus) to that of the Mongolian wild horse (E. przewalskii)

Przewalski's wild horse (E. przewalskii, EPR) has a diploid chromosome number of 2n = 66 while the domestic horse (E. caballus, ECA) has a diploid chromosome number of 2n = 64. Discussions about their phylogenetic relationship and taxonomic classification have hinged on comparisons of their skeletal morphology, protein and mitochondrial DNA similarities, their ability to produce fertile hybrid offspring, and on comparison of their chromosome morphology and banding patterns. Previous studies of GTG-banded karyotypes suggested that the chromosomes of both equids were homologous and the difference in chromosome number was due to a Robertsonian event involving two pairs of acrocentric chromosomes in EPR and one pair of metacentric chromosomes in ECA (ECA5). To determine which EPR chromosomes were homologous to ECA5 and to confirm the predicted chromosome homologies based on GTG banding, we constructed a comparative gene map between ECA and EPR by FISH mapping 46 domestic horse-derived BAC clones containing genes previously mapped to ECA chromosomes. The results indicated that all ECA and EPR chromosomes were homologous as predicted by GTG banding, but provide new information in that the EPR acrocentric chromosomes EPR23 and EPR24 were shown to be homologues of the ECA metacentric chromosome ECA5.     

Histomorphometric study of bone microstructure of primates and domestic animal with the goal of species identification with reference to the effects of domestication

Functional bone microstructure of long limb bones is a function of species-specific biomechanical properties such as locomotion and weight. Histomorphometry and statistics were used to identify various primate species (Hylobates moloch, Pongo satyrus borneensis, Pan tr. troglodytes, Gorilla g. gorilla, Homo sapiens), equid species (Equus caballus, Equus asinus, Equus mulus, Equus hemionus kulan, Equus ferus przewalskii) and also extinct horses e.g. iron age, medieval and neolithic forms on the microstructural level. Furthermore, bones from domesticated cattle, their Neolithic forms, pigs, sheep and goats (Bos taurus, Sus scrofa, Ovis aries, Capra hircus) were examined. Thin sections from proximal metacarpi or radii per species were taken in case of the domesticated animals and from distal humeri in case of the primates. Areas, perimeters, minimal and maximal axis of Haversian canals and secondary osteons were measured on digital images. Canonical discriminant analysis permits a differentiation of the species by these parameters of bone microstructure. Thus it is possible to distinguish between the different primate species, sheep and goats, horses, extinct horses, donkeys, mules and kulans on the microstructural level, however not between cattle and pig, E. f. przewalskii and Equus caballus, medieval and iron age horses. Neolithic cattle and horses do overlap, yet they are different from the modern forms.     

Variation in skin surface lipid composition among the Equidae

Skin surface lipids from Equus caballus, E. przewalskii, E. asinus, E. grevyi, E. hemionus onager and a mule (E. asinus/E. caballus) were analyzed in detail. In all species the surface lipid mixtures consisted of giant-ring lactones, cholesterol, cholesteryl esters and minor amounts of wax diesters. In E. caballus, the lactone hydroxyacids were entirely branched chained, while in E. asinus and E. grevyi they were almost exclusively straight chained. In E. przewalskii, the onager and the mule there were both straight and branched chain hydroxyacid lactones. These results are in harmony with published interpretations of the evolutionary relationships among Equus species.     

Chromosome Evolution in Bats as Revealed by FISH: The Ongoing Search for the Ancestral Chiropteran Karyotype

Chiroptera, the second largest order of mammals, comprises more than 1,000 species in 18 highly morphologically diverse families. Chromosome painting with human probes has been applied to 10 bat species from 8 families. Except for the combination 10/12pq/22q, all syntenic segmental associations proposed for the mammalian ancestor have been found in Chiroptera. Bat-specific painting probes, established from 4 species of 3 families, have been used in whole chromosome painting experiments in 29 species from 8 families. The results show that the prevailing mode of chromosomal evolution in bats is Robertsonian translocation with a large number of convergent events. Given our present knowledge of chiropteran karyotypes, only a few elements of the ancestral chiropteran karyotype can be reconstructed with confidence.     

Cross-species chromosome painting in the Perissodactyla: delimitation of homologous regions in Burchell's zebra (Equus Burchellii) and the white (Ceratotherium Simum) and black rhinoceros (Diceros Bicornis)

Conserved chromosomal segments in the black rhinoceros, Diceros Bicornis (DBI, 2n = 84), and its African sister-species the white rhinoceros, Ceratotherium Simum (CSI, 2n = 82), were detected using Burchell's zebra (Equus Burchellii, EBU, 2n = 44) chromosome-specific painting probes supplemented by a subset of those developed for the horse (Equus Caballus, ECA, 2n = 64). In total 41 and 42 conserved autosomal segments were identified in C. Simum and D. Bicornis respectively. Only 21 rearrangements (20 fissions and 1 fusion) are necessary to convert the Burchell's zebra karyotype into that of the white rhinoceros. One fission distinguishes the D. Bicornis and C. Simum karyotypes which, excluding heterochromatic differences, are identical in all respects at this level of resolution. Most Burchell's zebra chromosomes correspond to two rhinoceros chromosomes although in four instances (EBU18, 19, 20 and 21) whole chromosome synteny has been retained among these species. In contrast, one rhinoceros chromosome (DBI1, CSI1) comprises two separate Burchell's zebra chromosomes (EBU11 and EBU17). In spite of the high diploid numbers of the two rhinoceros species their karyotypes are surprisingly conserved offering a glimpse of the putative ancestral perissodactyl condition and a broader understanding of genome organization in mammals.     

Gene assignment in the spider monkey (Ateles paniscus chamek--APC): APE-MYH7 to 2q; AR-GLA-F8C to the X chromosome.

Comparative gene assignment between the spider monkey species Ateles paniscus chamek (APC) and man (HSA) showed conserved syntenic associations despite extensive karyotypic rearrangement between species. Two HSA 14q genes were allocated to APC 2q, being syntenic to other HSA 14q and HSA 15q markers previously assigned to APC 2q, and to HSA 12q genes previously assigned to APC 2p. These findings were consistent with A. geoffroyi chromosome painting with human whole-chromosome probes, indicating that the genus Ateles is karyotypically very rearranged. On the other hand, three human X-linked markers were assigned to the Ateles X chromosome, indicating that this chromosome is evolutionary stable.     

Zoo-FISH with microdissected arm specific paints for HSA2, 5, 6, 16, and 19 refines known homology with pig and horse chromosomes

Microdissected arm specific paints (ASPs) for human (HSA) chromosomes (Chrs) 2, 5, 6, 16, and 19 were used as probes on pig (SSC) and horse (ECA) metaphase chromosomes. Regions homologous to individual human arms were delineated in the two species studied. Of the ten ASPs used, HSA6 and 16 ASPs showed complete synteny conservation of individual arms as single blocks/arms both in pig and horse. A similar trend was, in general, also observed for HSA19 ASPs. However, contrary to these observations, synteny conservation of individual arms of HSA2 and HSA5 was not observed in pig and horse. The arm specific painting data, coupled with the available gene mapping data, showed that, although HSA2 corresponded to two arms/chromosomes each in pig and horse, the breakpoint of this synteny in humans was not located at the centromere, but at HSA2q13 band. Similarly, arm specific paints for HSA5 showed that of the two blocks/chromosomes painted in pig and horse, one corresponded to HSA5q13-pter, the other to HSA5q13-qter. The findings suggest that 5q13 band may also be an evolutionary break point, similar to the one detected on HSA2q13. The microdissected human arm specific painting probes used in the present work provide more accurate and refined comparative information on pig and horse chromosomes than that available through the use of human whole chromosome specific paints. Received: 1 June 1997 / Accepted: 5 September 1997     

Chromosomal assignment of six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) in four species of the genus Equus

We mapped six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) on the chromosomes of Equus caballus, Equus asinus, Equus grevyi, and Equus burchelli by fluorescence in situ hybridization. Our results add six type I markers to the cytogenetic map of these species and provide new information on the comparative genomics of the genus Equus.     

Common and species-specific esterases of Equidae--IV. Horse of przewalski, onager and Zebra hartmannae.

1. Among several species of Equidae only E. przewalskii possesses a serum esterase identical with that of E. caballus. 2. The esterases of Hemionidae differ slightly from that of domestic horse by electrophoretic migration and by antigenic structure. 3. Zebras (grevyi, burchelli) appear devoid of this component but Z. hartmannae possesses an esterase of high enzymatic activity, differing notably from that of horse by electrophoretic and antigenic properties.     

Comparison of horse Chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping

The melanocortin 1 receptor (MC1R), mast/stem cell growth factor receptor (KIT), and platelet-derived growth factor receptor α (PDGFRA) are loci that all belong to equine linkage group 2 (LG2). Of these, KIT was fluorescent in situ hybridization (FISH) mapped to ECA3q21 with equine cDNA and heterologous porcine BAC probes, while MC1R was localized to ECA3p12 and PDGFRA to ECA3q21 with heterologous porcine BAC probes. A three-step comparison between ECA3 and donkey chromosomes was carried out. First, microdissected ECA3 painting probe was used on donkey chromosomes, which showed disruption of the equine synteny. Next, human (HSA) Chromosomes (Chrs) 16q and 4 specific paints, known to be homologous to ECA3p and 3q, respectively, were applied to detect homologous chromosomal segment(s) in donkey. Finally, four genes (MC1R, ALB, PDGFRA, KIT) and two equine microsatellite markers (SGCV18 and SGCV33) located on ECA3 were FISH mapped to donkey chromosomes. The findings refined the cross species painting homology results and added six new markers to the nascent donkey gene map. The hypothesis that Tobiano coat color in horses may be associated with a chromosomal inversion involving genes within LG2 was tested by G-banding-based cytogenetic analysis and ordering of four loci—KIT, PDGFRA, albumin (ALB), and MC1R—in Tobiano and non-tobiano (homozygous as well as heterozygous) horses. However, no difference either in banding patterns or location/relative order of the genes was observed in the three classes. The study highlights successful FISH mapping of BAC probes across evolutionarily diverged species, viz., pig and horse/donkey, and represents the first use of large-sized individual clones across distantly related farm animals. Received: 2 September 1998 / Accepted: 20 October 1998     

A cytogenetic study of the Caspian pony

The group of Caspian ponies studied contained some animals with 65 chromosomes and others with 64 chromosomes. The morphology and G-banding pattern of the chromosomes resembled those of Equus caballus and E. przewalskii. The karyogram of animals with 65 chromosomes was identical to that of the cross between E. caballus and E. przewalskii. It is suggested that the Caspian pony is the product of natural hybridization between E. caballus and E. prezwalskii. Low reproductive effeciency of the Caspian pony is suggested as the cause of decline in the population of these animals.     

Chromosomal distribution of the telomere sequence (TTAGGG)(n) in the Equidae

Telomeres are a class of repetitive DNA sequences that are located at chromosome termini and that act to stabilize the chromosome ends. The rapid karyotypic evolution of the genus Equus has given rise to ten taxa, all with different diploid chromosome numbers. Using fluorescence in situ hybridization (FISH) we localized the mammalian telomere sequence, (TTAGGG)(n), to the chromosomes of nine equid taxa. TTAGGG signal was located at chromosome termini in all species, however additional signal was seen at interstitial sites on some chromosomes in the Burchell's zebra, Equus quagga burchelli, the Hartmann's zebra, Equus zebra hartmannae, and at large heterochromatin-associated regions on the chromosomes of the donkey, Equus asinus. The interstitial signal in the zebras may be a relic of an ancient telomere-telomere fusion and mark the point at which two ancestral chromosomes may have fused. For the donkey, the heterochromatin-associated signal may represent degenerate telomere-like satellite sequences and identify a second type of satellite DNA for this taxon.     

Comparative molecular cytogenetic studies in the order Carnivora: mapping chromosomal rearrangements onto the phylogenetic tree

We have made a set of chromosome-specific painting probes for the American mink by degenerate oligonucleotide primed-PCR (DOP-PCR) amplification of flow-sorted chromosomes. The painting probes were used to delimit homologous chromosomal segments among human, red fox, dog, cat and eight species of the family Mustelidae, including the European mink, steppe and forest polecats, least weasel, mountain weasel, Japanese sable, striped polecat, and badger. Based on the results of chromosome painting and G-banding, comparative maps between these species have been established. The integrated map demonstrates a high level of karyotype conservation among mustelid species. Comparative analysis of the conserved chromosomal segments among mustelids and outgroup species revealed 18 putative ancestral autosomal segments that probably represent the ancestral chromosomes, or chromosome arms, in the karyotype of the most recent ancestor of the family Mustelidae. The proposed 2n = 38 ancestral Mustelidae karyotype appears to have been retained in some modern mustelids, e.g., Martes, Lutra, Ictonyx, and Vormela. The derivation of the mustelid karyotypes from the putative ancestral state resulted from centric fusions, fissions, the addition of heterochromatic arms, and occasional pericentric inversions. Our results confirm many of the evolutionary conclusions suggested by other data and strengthen the topology of the carnivore phylogenetic tree through the inclusion of genome-wide chromosome rearrangements.