Evolutionary Molecular Cytogenetics of Catarrhine Primates: Past, Present and Future. R. Stanyon M. Rocchi(b) F. Bigoni(a) N. Archidiacono(b)

The catarrhine primates were the first group of species studied with comparative molecular cytogenetics. Many of the fundamental techniques and principles of analysis were initially applied to comparisons in these primates, including interspecific chromosome painting, reciprocal chromosome painting and the extensive use of cloned DNA probes for evolutionary analysis. The definition and importance of chromosome syntenies and associations for a correct cladistics analysis of phylogenomic relationships were first applied to catarrhines. These early chromosome painting studies vividly illustrated a striking conservation of the genome between humans and macaques. Contemporarily, it also revealed profound differences between humans and gibbons, a group of species more closely related to humans, making it clear that chromosome evolution did not follow a molecular clock. Chromosome painting has now been applied to more that 60 primate species and the translocation history has been mapped onto the major taxonomic divisions in the tree of primate evolution. In situ hybridization of cloned DNA probes, primarily BAC-FISH, also made it possible to more precisely map breakpoints with spanning and flanking BACs. These studies established marker order and disclosed intrachromosomal rearrangements. When applied comparatively to a range of primate species, they led to the discovery of evolutionary new centromeres as an important new category of chromosome evolution. BAC-FISH studies are intimately connected to genome sequencing, and probes can usually be assigned to a precise location in the genome assembly. This connection ties molecular cytogenetics securely to genome sequencing, assuring that molecular cytogenetics will continue to have a productive future in the multidisciplinary science of phylogenomics.     

Primate chromosome evolution: with reference to marker order and neocentromeres

Establishing chromosomal homology in comparative cytogenetics remained speculative until the advent of molecular cytogenetics. Chromosome sorting by flow cytometry and degenerate oligonucleotide primed-PCR (DOP-PCR) brought a significant simplification and impetus to chromosome painting. Comparative chromosome painting has permitted reasonable hypotheses for ancestral karyotypes at many points on the phylogenetic tree of mammals. Derived associations often provided landmarks that showed the route evolution took. More recently hybridization with cloned DNA has provided information on intrachromosomal rearrangements. BAC-FISH allows marker order, in addition to syntenies and associations, to be added to the ancestral karyotypes. Comparisons of marker order across species revealed that centromere shifts (evolutionary new centromeres) are frequent and important phenomena of chromosome evolution. Further comparison between evolutionary new centromeres and clinical neocentromeres shows that an evolutionary perspective can provide compelling, underlying, explicative grounds for contemporary genomic phenomena.     

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.     

The Ancestral Carnivore Karyotype As Substantiated by Comparative Chromosome Painting of Three Pinnipeds,the Walrus,the Steller Sea Lion and the Baikal Seal (Pinnipedia,Carnivora)

Karyotype evolution in Carnivora is thoroughly studied by classical and molecular cytogenetics and supplemented by reconstructions of Ancestral Carnivora Karyotype (ACK). However chromosome painting information from two pinniped families (Odobenidae and Otariidae) is noticeably missing. We report on the construction of the comparative chromosome map for species from each of the three pinniped families: the walrus (Odobenus rosmarus, Odobenidae–monotypic family), near threatened Steller sea lion (Eumetopias jubatus, Otariidae) and the endemic Baikal seal (Pusa sibirica, Phocidae) using combination of human, domestic dog and stone marten whole-chromosome painting probes. The earliest karyological studies of Pinnipedia showed that pinnipeds were characterized by a pronounced karyological conservatism that is confirmed here with species from Phocidae, Otariidae and Odobenidae sharing same low number of conserved human autosomal segments (32). Chromosome painting in Pinnipedia and comparison with non-pinniped carnivore karyotypes provide strong support for refined structure of ACK with 2n = 38. Constructed comparative chromosome maps show that pinniped karyotype evolution was characterized by few tandem fusions, seemingly absent inversions and slow rate of genome rearrangements (less then one rearrangement per 10 million years). Integrative comparative analyses with published chromosome painting of Phoca vitulina revealed common cytogenetic signature for Phoca/Pusa branch and supports Phocidae and Otaroidea (Otariidae/Odobenidae) as sister groups. We revealed rearrangements specific for walrus karyotype and found the chromosomal signature linking together families Otariidae and Odobenidae. The Steller sea lion karyotype is the most conserved among three studied species and differs from the ACK by single fusion. The study underlined the strikingly slow karyotype evolution of the Pinnipedia in general and the Otariidae in particular.     

Painting the chromosomes of <Emphasis Type="Italic">Brachypodium</Emphasis>—current status and future prospects

Chromosome painting is one of the most powerful and spectacular tools of modern molecular cytogenetics, enabling complex analyses of nuclear genome structure and evolution. For many years, this technique was restricted to the study of mammalian chromosomes, as it failed to work in plant genomes due mainly to the presence of large amounts of repetitive DNA common to all the chromosomes of the complement. The availability of ordered, chromosome-specific BAC clones of Arabidopsis thaliana containing relatively little repetitive genomic DNA enabled the first chromosome painting in dicotyledonous plants. Here, we show for the first time chromosome painting in three different cytotypes of a monocotyledonous plant—the model grass, Brachypodium distachyon. Possible directions of further detailed studies are proposed, such as the evolution of grass karyotypes, the behaviour of meiotic chromosomes, and the analysis of chromosome distribution at interphase.     

New insights into porcine-human synteny conservation

Eleven genes were mapped to the porcine genome with the aim of improving the human-porcine comparative gene map. Five of these genes were from regions of the human genome painted by porcine chromosomal probes; of these, two mapped to chromosomes not expected from the painting results. Among the six genes from human regions not painted by porcine chromosomal probes, three genes did not map where expected by the principle of parsimony. Several of the gene assignments indicate the existence of small regions of conserved synteny not detected by heterologous chromosome painting, especially in telomeric regions. We have also detected new rearrangements in gene order within the regions of correspondence between human Chromosome (HSA) 15 and porcine Chromosome (SSC) 1 as well as between HSA4 and SSC8. Received: 30 September 1998 / Accepted: 3 December 1998     

Interpretation of karyotype evolution should consider chromosome structural constraints

Comparative genetics, genomics and cytogenetics provide tools to trace the evolutionary history of extant genomes. Yet, the interpretation of rapidly increasing genomic data is not always done in agreement with constraints determined by chromosome structural features and by insights obtained from chromosome mutagenesis. The terms 'non-reciprocal chromosome translocation', 'chromosome fusion' and 'centromere shift' used to explain genomic differences among organisms are misleading and often do not correctly reflect the mechanisms of chromosome rearrangements underlying the evolutionary karyotypic variation. Here, we (re)interpret evolutionary genome alterations in a parsimonious way and demonstrate that results of comparative genomics and comparative chromosome painting can be explained on the basis of known primary and secondary chromosome rearrangements. Therefore, some widespread terms used in comparative and evolutionary genomics should be either avoided (e.g. non-reciprocal translocation) or redefined (e.g. chromosome fusion and centromere shift).     

Ancestral chromosomal blocks are triplicated in Brassiceae species with varying chromosome number and genome size

The paleopolyploid character of genomes of the economically important genus Brassica and closely related species (tribe Brassiceae) is still fairly controversial. Here, we report on the comparative painting analysis of block F of the crucifer Ancestral Karyotype (AK; n = 8), consisting of 24 conserved genomic blocks, in 10 species traditionally treated as members of the tribe Brassiceae. Three homeologous copies of block F were identified per haploid chromosome complement in Brassiceae species with 2n = 14, 18, 20, 32, and 36. In high-polyploid (n >or= 30) species Crambe maritima (2n = 60), Crambe cordifolia (2n = 120), and Vella pseudocytisus (2n = 68), six, 12, and six copies of the analyzed block have been revealed, respectively. Homeologous regions resembled the ancestral structure of block F within the AK or were altered by inversions and/or translocations. In two species of the subtribe Zillineae, two of the three homeologous regions were combined via a reciprocal translocation onto one chromosome. Altogether, these findings provide compelling evidence of an ancient hexaploidization event and corresponding whole-genome triplication shared by the tribe Brassiceae. No direct relationship between chromosome number and genome size variation (1.2-2.5 pg/2C) has been found in Brassiceae species with 2n = 14 to 36. Only two homeologous copies of block F suggest a whole-genome duplication but not the triplication event in Orychophragmus violaceus (2n = 24), and confirm a phylogenetic position of this species outside the tribe Brassiceae. Chromosome duplication detected in Orychophragmus as well as chromosome rearrangements shared by Zillineae species demonstrate the usefulness of comparative cytogenetics for elucidation of phylogenetic relationships.     

Major structural genomic alterations can be associated with hybrid speciation in Aegilops markgrafii (Triticeae)

During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hybridization, resulting in allopolyploidy; whereas homoploid hybrid speciation was found only in rye. Homoeologous chromosomes within the Triticeae preserved cross‐species macrocolinearity, except for a few species with rearranged genomes. Aegilops markgrafii, a diploid wild relative of wheat (2n = 2x = 14), has a highly asymmetrical karyotype that is indicative of chromosome rearrangements. Molecular cytogenetics and next‐generation sequencing were used to explore the genome organization. Fluorescence in situ hybridization with a set of wheat cDNAs allowed the macrostructure and cross‐genome homoeology of the Ae. markgrafii chromosomes to be established. Two chromosomes maintained colinearity, whereas the remaining were highly rearranged as a result of inversions and inter‐ and intrachromosomal translocations. We used sets of barley and wheat orthologous gene sequences to compare discrete parts of the Ae. markgrafii genome involved in the rearrangements. Analysis of sequence identity profiles and phylogenic relationships grouped chromosome blocks into two distinct clusters. Chromosome painting revealed the distribution of transposable elements and differentiated chromosome blocks into two groups consistent with the sequence analyses. These data suggest that introgressive hybridization accompanied by gross chromosome rearrangements might have had an impact on karyotype evolution and homoploid speciation in Ae. markgrafii.     

Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats.

Reciprocal chromosome painting between mouse and rat using complete chromosome probe sets of both species permitted us to assign the chromosomal homology between these rodents. The comparative gene mapping data and chromosome painting have a better than 90% correspondence. The reciprocal painting results graphically show that mouse and rat have strikingly different karyotypes. At least 14 translocations have occurred in the 10-20 million years of evolution that separates these two species. The evolutionary rate of chromosome translocations between these two rodents appears to be up to 10 times greater than that found between humans and cats, or between humans and chimpanzees, where over the last 5-6 million years just one translocation has occurred. Outgroup comparison shows that the mouse genome has incorporated at least three times the amount of interchromosomal rearrangements compared to the rat genome. The utility of chromosome painting was also illustrated by the assignment of two new chromosome homologies between rat and mouse unsuspected by gene mapping: between mouse 11 and rat 20 and between mouse 17 and rat 6. We conclude that reciprocal chromosome painting is a powerful method, which can be used with confidence to chart the genome and predict the chromosome location of genes. Reciprocal painting combined with gene mapping data will allow the construction of large-scale comparative chromosome maps between placental mammals and perhaps other animals.     

Chromosome rearrangements between chicken and guinea fowl defined by comparative chromosome painting and FISH mapping of DNA clones

Chromosome homology between chicken (Gallus gallus) and guinea fowl (Numida meleagris) was investigated by comparative chromosome painting with chicken whole chromosome paints for chromosomes 1-9 and Z and by comparative mapping of 38 macrochromosome-specific (chromosomes 1-8 and Z) and 30 microchromosome-specific chicken cosmid DNA clones. The comparative chromosome analysis revealed that the homology of macrochromosomes is highly conserved between the two species except for two inter-chromosomal rearrangements. Guinea fowl chromosome 4 represented the centric fusion of chicken chromosome 9 with the q arm of chicken chromosome 4. Guinea fowl chromosome 5 resulted from the fusion of chicken chromosomes 6 and 7. A pericentric inversion was found in guinea fowl chromosome 7, which corresponded to chicken chromosome 8. All the chicken microchromosome-specific DNA clones were also localized to microchromosomes of guinea fowl except for several clones localized to the short arm of chromosome 4. These results suggest that the cytogenetic genome organization is highly conserved between chicken and guinea fowl.     

Fourteen chromosomal localizations and an update of the cytogenetic map of the rabbit

In order to improve the informativeness of the cytogenetic map of the rabbit genome, fourteen markers were regionally mapped to individual chromosomes. The localizations comprise eleven gene loci (PRLR, GHR, HK1, ACE, TF, 18S+28S rDNA, CYP2C4, PMP2, TCRB, ALOX15 and MT1) and three microsatellite loci (Sat13, Sol33 and D1Utr6). Five of the genes contain known microsatellite sequences. To achieve these localizations, homologous and heterologous small insert clones, and clones from a rabbit Bacterial Artificial Chromosome (BAC) library were used as probes for fluorescence in situ hybridization experiments. Results indicate that especially BAC clones are a valuable tool for cytogenetic mapping. Some of the genes were selected for mapping on the basis of human- rabbit comparative painting data, to achieve localizations on gene-poor rabbit chromosomes. Our data are, in general, in agreement with the human-rabbit comparative painting data. By mapping microsatellite sequences that have also been used in linkage studies, links are provided between the genetic and physical maps of the rabbit genome. Linkage groups I, VI and XI could be assigned to chromosomes 1, 5 and 3 respectively. Moreover, in this paper we give an overview of the current status of the rabbit cytogenetic map. This map now comprises 62 physically mapped genes, which are scattered over all autosomes, except chromosome 2, and the X chromosome.     

The impact of chromosome sorting and painting on the comparative analysis of primate genomes

Chromosome sorting by flow cytometry is the main source of chromosome-specific DNA for the production of painting probes. These probes have been used for cross-species in situ hybridization in the construction of comparative maps, in the study of karyotype evolution and phylogenetics, in delineating territories in interphase nuclei, and in the analysis of chromosome breakpoints. We review here the contributions that this technology has made to the analysis of primate genomes.     

Chromosome evolution in perissodactyla

Comparative painting has provided a wealth of useful information and helped to reconstruct the pathways of karyotype evolution within major eutherian phylogenetic clades. New data have come from gene localizations, BAC mapping and high throughout sequencing projects that enrich and provide new details of genome evolution. Extensive research on perissodactyl genomes has revealed not only increased rates of chromosomal rearrangements, but also an exceptionally high number of centromere repositioning events in equids. Here were combined new physical mapping, comparative painting and genome sequencing data to refine the putative ancestral karyotype maps and to revise the previously proposed scenario of perissodactyl karyotype evolution.     

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     

Karyotypic evolution of the family Sciuridae: inferences from the genome organizations of ground squirrels

Cross-species chromosome painting has made a great contribution to our understanding of the evolution of karyotypes and genome organizations of mammals. Several recent papers of comparative painting between tree and flying squirrels have shed some light on the evolution of the family Sciuridae and the order Rodentia. In the present study we have extended the comparative painting to the Himalayan marmot (Marmotahimalayana) and the African ground squirrel (Xerus cf. erythropus), i.e. representative species from another important squirrel group--the ground squirrels--, and have established genome-wide comparative chromosome maps between human, eastern gray squirrel, and these two ground squirrels. The results show that 1) the squirrels so far studied all have conserved karyotypes that resemble the ancestral karyotype of the order Rodentia; 2) the African ground squirrels could have retained the ancestral karyotype of the family Sciuridae. Furthermore, we have mapped the evolutionary rearrangements onto a molecular-based consensus phylogenetic tree of the family Sciuridae.     

Genomic restructuring in the Tasmanian devil facial tumour: chromosome painting and gene mapping provide clues to evolution of a transmissible tumour

Devil facial tumour disease (DFTD) is a fatal, transmissible malignancy that threatens the world's largest marsupial carnivore, the Tasmanian devil, with extinction. First recognised in 1996, DFTD has had a catastrophic effect on wild devil numbers, and intense research efforts to understand and contain the disease have since demonstrated that the tumour is a clonal cell line transmitted by allograft. We used chromosome painting and gene mapping to deconstruct the DFTD karyotype and determine the chromosome and gene rearrangements involved in carcinogenesis. Chromosome painting on three different DFTD tumour strains determined the origins of marker chromosomes and provided a general overview of the rearrangement in DFTD karyotypes. Mapping of 105 BAC clones by fluorescence in situ hybridisation provided a finer level of resolution of genome rearrangements in DFTD strains. Our findings demonstrate that only limited regions of the genome, mainly chromosomes 1 and X, are rearranged in DFTD. Regions rearranged in DFTD are also highly rearranged between different marsupials. Differences between strains are limited, reflecting the unusually stable nature of DFTD. Finally, our detailed maps of both the devil and tumour karyotypes provide a physical framework for future genomic investigations into DFTD.     

Chromosome painting in the manatee supports Afrotheria and Paenungulata

Background  

Sirenia (manatees, dugongs and Stellar's sea cow) have no evolutionary relationship with other marine mammals, despite similarities in adaptations and body shape. Recent phylogenomic results place Sirenia in Afrotheria and with elephants and rock hyraxes in Paenungulata. Sirenia and Hyracoidea are the two afrotherian orders as yet unstudied by comparative molecular cytogenetics. Here we report on the chromosome painting of the Florida manatee.