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, ktonyx, 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. Copyright (C) 2002 S. KargerAG, Basel.

Karyotypic relationships of horses and zebras: results of cross-species chromosome painting

Complete sets of chromosome-specific painting probes, derived from flow-sorted chromosomes of human (HSA), Equus caballus (ECA) and Equus burchelli (EBU) were used to delineate conserved chromosomal segments between human and Equits burchelli, and among four equid species, E. przewalskii (EPR), E. caballus, E. burchelli and E. zebra hartmannae (EZH) by cross-species chromosome painting. Genome-wide comparative maps between these species have been established. Twenty-two human autosomal probes revealed 48 conserved segments in E. burchelli. The adjacent segment combinations HSA3/21, 7/16p, 16q/19q, 14/15, 12/22 and 4/8, presumed ancestral syntenies for all eutherian mammals, were also found conserved in E. burchelli. The comparative maps of equids allow for the unequivocal characterization of chromosomal rearrangements that differentiate the karyotypes of these equid species. The karyotypes of E. przewalskii and E. caballus differ by one Robertsonian translocation (ECA5 = EPR23 + EPR24); numerous Robertsonian translocations and tandem fusions and several inversions account for the karyotypic differences between the horses and zebras. Our results shed new light on the karyotypic evolution of Equidae. Copyright (C) 2003 S. Karger AG, Basel.

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 (DB1, 2n = 84), and its African sister-species the white rhinoceros, Ceratotherim 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 I 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 hetero- chromatic 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 (EBU 18, 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. Copyright (C) 2003 S. Karger AG, Base

Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype

The Afrotheria, a supraordinal grouping of mammals whose radiation is rooted in Africa, is strongly supported by DNA sequence data but not by their disparate anatomical features. We have used flow-sorted human, aardvark, and African elephant chromosome painting probes and applied reciprocal painting schemes to representatives of two of the Afrotherian orders, the Tubulidentata (aardvark) and Proboscidea (elephants), in an attempt to shed additional light on the evolutionary affinities of this enigmatic group of mammals. Although we have not yet found any unique cytogenetic signatures that support the monophyly of the Afrotheria, embedded within the aardvark genome we find the strongest evidence yet of a mammalian ancestral karyotype comprising 2n = 44. This karyotype includes nine chromosomes that show complete conserved synteny to those of man, six that show conservation as single chromosome arms or blocks in the human karyotype but that occur on two different chromosomes in the ancestor, and seven neighbor-joining combinations (i.e., the synteny is maintained in the majority of species of the orders studied so far, but which corresponds to two chromosomes in humans). The comparative chromosome maps presented between human and these Afrotherian species provide further insight into mammalian genome organization and comparative genomic data for the Afrotheria, one of the four major evolutionary clades postulated for the Eutheria.

Evolution of genome organizations of squirrels (Sciuridae) revealed by cross-species chromosome painting

With complete sets of chromosome-specific painting probes derived from flow-sorted chromosomes of human and grey squirrel (Sciurus carolinensis), the whole genome homologies between human and representatives of tree squirrels (Sciurus carolinensis, Callosciurus erythraeus), flying squirrels (Petaurista albiventer) and chipmunks (Tamias sibiricus) have been defined by cross-species chromosome painting. The results show that, unlike the highly rearranged karyotypes of mouse and rat, the karyotypes of squirrels are highly conserved. Two methods have been used to reconstruct the genome phylogeny of squirrels with the laboratory rabbit (Oryctolagus cuniculus) as the out-group: ( 1) phylogenetic analysis by parsimony using chromosomal characters identified by comparative cytogenetic approaches; ( 2) mapping the genome rearrangements onto recently published sequence-based molecular trees. Our chromosome painting results, in combination with molecular data, show that flying squirrels are phylogenetically close to New World tree squirrels. Chromosome painting and G-banding comparisons place chipmunks ( Tamias sibiricus), with a derived karyotype, outside the clade comprising tree and flying squirrels. The superorder Glires (order Rodentia + order Lagomorpha) is firmly supported by two conserved syntenic associations between human chromosomes 1 and 10p homologues, and between 9 and 11 homologues.

Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules

We have made a complete set of painting probes for the domestic horse by degenerate oligonucleotide-primed PCR amplification of flow-sorted horse chromosomes. The horse probes, together with a full set of those available for human, were hybridized onto metaphase chromosomes of human, horse and mule. Based on the hybridization results, we have generated genome-wide comparative chromosome maps involving the domestic horse, donkey and human. These maps define the overall distribution and boundaries of evolutionarily conserved chromosomal segments in the three genomes. Our results shed further light on the karyotypic relationships among these species and, in particular, the chromosomal rearrangements that underlie hybrid sterility and the occasional fertility of mules.

Comparative map between the domestic pig and dog

Cross-species chromosome painting with probes derived from flow-sorted dog and human chromosomes was used to construct a high-resolution comparative map for the pig. In total 98 conserved autosomal segments between pig and dog were detected by probes specific for the 38 autosomes and X Chromosome of the dog. Further integration of our results with the published human-dog and cat-dog comparative maps, and with data from comparative gene mapping, increases the resolution of the current pig-human comparative map. It allows for the conserved syntenies detected in the pig, human, and cat to be aligned against the putative ancestral karyotype of eutherian mammals and for the history of karyotype evolution of the pig lineage to be reconstructed. Fifteen fusions, 17 fissions, and 23 inversions are required to convert the ancestral mammalian karyotype into the extant karyotype of the pig.

Chromosome painting in the long-tailed field mouse provides insights into the ancestral murid karyotype

We report on the hybridization of mouse chromosomal paints to Apodemus sylvaticus, the long-tailed field mouse. The mouse paints detected 38 conserved segments in the Apodemus karyotype. Together with the species reported here there are now six species of rodents mapped with Mus musculus painting probes. A parsimony analysis indicated that the syntenies of nine M. musculus chromosomes were most likely already formed in the muroid ancestor: 3, 4, 7, 9, 14, 18, 19, X and Y. The widespread occurrence of syntenic segment associations of mouse chromosomes 1/17, 2/13, 7/19, 10/17, 11/16, 12/17 and 13/15 suggests that these associations were ancestral syntenies for muroid rodents. The muroid ancestral karyotype probably had a diploid number of about 2n = 54. It would be desirable to have a richer phylogenetic array of species before any final conclusions are drawn about the Muridae ancestral karyotype. The ancestral karyotype presented here should be considered as a working hypothesis. Copyright (C) 2004 S. Karger AG, Basel.

Cross-species chromosome painting in the golden mole and elephant-shrew: support for the mammalian clades Afrotheria and Afroinsectiphillia but not Afroinsectivora

Cross-species painting (fluorescence in situ hybridization) with 23 human (Homo sapiens (HSA)) chromosome-specific painting probes (HSA 1-22 and the X) was used to delimit regions of homology on the chromosomes of the golden mole (Ghrysochloris asiaticus) and elephant-shrew (Elephantulus rupestris). A cladistic interpretation of our data provides evidence of two unique associations, HSA 1/19p and 5/21/3, that support Afrotheria. The recognition of HSA 5/3/21 expands on the 3/21 synteny originally designated as an ancestral state for all eutherians. We have identified one adjacent segment combination (HSA2/8p/4) that is supportive of Afroinsectiphillia (aardvark, golden mole, elephant-shrew). Two segmental combinations (HSA 10q/17 and HSA 3/20) unite the aardvark and elephant-shrews as sister taxa. The finding that segmental syntenies in evolutionarily distant taxa can improve phylogenetic resolution suggests that they may be useful for testing sequence-based phylogenies of the early eutherian mammals. They may even suggest clades that sequence trees are not recovering with any consistency and thus encourage the search for additional rare genomic changes among afrotheres.

Phylogenomic study of the subfamily Caprinae by cross-species chromosome painting with Chinese muntjac paints

Chromosomal homologies have been established between the Chinese muntjac (Muntiacus reevesi, MRE, 2n = 46) and five ovine species: wild goat (Capra aegagrus, CAE, 2n = 60), argall (Ovis ammon, OAM, 2n = 56), snow sheep (Ovis nivicola, ONI, 2n = 52), red goral (Naemorhedus cranbrooki, NCR, 2n = 56) and Sumatra serow (Capricornis sumatraensis, CSU, 2n = 48) by chromosome painting with a set of chromosome-specific probes of the Chinese muntjac. In total, twenty-two Chinese muntjac autosomal painting probes detected thirty-five homologous segments in the genome of each species. The chromosome X probe hybridized to the whole X chromosomes of all ovine species while the chromosome Y probe gave no signal. Our results demonstrate that almost all homologous segments defined by comparative painting show a high degree of conservation in G-banding patterns and that each speciation event is accompanied by specific chromosomal rearrangements. The combined analysis of our results and previous cytogenetic and molecular systematic results enables us to map the chromosomal rearrangements onto a phylogenetic tree, thus providing new insights into the karyotypic evolution of these species.

Karyotypic conservatism in the suborder Feliformia (Order Carnivora)

Multidirectional comparative chromosome painting was used to investigate the karyotypic relationships among representative species from three Feliformia families of the order Carnivora ( Viverridae, Hyaenidae and Felidae). Complete sets of painting probes derived from flow-sorted chromosomes of the domestic dog, American mink, and human were hybridized onto metaphases of the spotted hyena (Crocuta crocuta, 2n = 40) and masked palm civet (Paguma larvata, 2n = 44). Extensive chromosomal conservation is evident in these two species when compared with the cat karyotype, and only a few events of chromosome fusion, fission and inversion differentiate the karyotypes of these Feliformia species. The comparative chromosome painting data have enabled the integration of the hyena and palm civet chromosomes into the previously established comparative map among the domestic cat, domestic dog, American mink and human and improved our understanding on the karyotype phylogeny of Feliformia species. Copyright (C) 2005 S. Karger AG, Basel.

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, a

The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia : Anura): a new gene order in the vertebrate mtDNA

The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNA(Thr), tRNA(Pro), tRNA(Leu) ((CUN))) a tandem duplication of tRNA(Mer) gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNA(Thr)/tRNA(Pro)/tRNA(Leu)/tRNA(Phe). The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAM(Met) and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNA(Met) were first observed in the vertebrate mitochondrial genomes. (c) 2004 Elsevier B.V. All rights reserved.

Construction, characterization and chromosomal mapping of bacterial artificial chromosome (BAC) library of Yunnan snub-nosed monkey (Rhinopithecus bieti)

We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136 320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100 kb) is 2.74%, and only 2.67% non-recombinant clones were observed. Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and assigned the accession number CG891489-CG891703.

Production of triploid African catfish, Clarias gariepinus (Burchell), using chromosome manipulation techniques

An experiment was conducted to induce triploidy in African catfish, Clarias gariepinus, using heat shock and cold shock techniques. Cold shock at a temperature of 0± 1°C and 5±1°C for a duration of 15, 30, 45 and 60 min and heat shock at a temperature of 40±0.5°C and 41 ±OS C for a duration of 1, 2 and 3 min was given to induce triploidy 5 min after fertilization. Maximum percentage of triploids (91.4%) were obtained in the heat shock at a temperature of 40±0SC for a duration of 1 min whereas cold shock at 0± 1 C for a duration of 60 min yielded 90% of triploids. Chromosome analysis revealed that diploids have 54 chromosomes and triploids have 81 chromosomes. The erythrocyte measurements of the minor axis and major axis were 1.17 times larger in treated fish than in controls. The growth studies showed that the growth rate was not significantly affected in triploids.