Comparative chromosome painting in carnivora and pholidota

The order of Carnivora has been very well characterized with over 50 species analyzed by chromosome painting and with painting probe sets made for 9 Carnivora species. Representatives of almost all families have been studied with few exceptions (Otariidae, Odobenidae, Nandiniidae, Prionodontidae). The patterns of chromosome evolution in Carnivora are discussed here. Overall, many Carnivora species retained karyotypes that only slightly differ from the ancestral carnivore karyotype. However, there are at least 3 families in which the ancestral carnivore karyotype has been severely rearranged - Canidae, Ursidae and Mephitidae. Here we report chromosome painting of yet another Carnivora species with a highly rearranged karyotype, Genetta pardina. Recurrent rearrangements make it difficult to define the ancestral chromosomal arrangement in several instances. Only 2 species of pangolins (Pholidota), a sister order of Carnivora, have been studied by chromosome painting. Future use of whole-genome sequencing data is discussed in the context of solving the questions that are beyond resolution of conventional banding techniques and chromosome painting.     

The pattern of phylogenomic evolution of the Canidae

Canidae species fall into two categories with respect to their chromosome composition: those with high numbered largely acrocentric karyotypes and others with a low numbered principally metacentric karyotype. Those species with low numbered metacentric karyotypes are derived from multiple independent fusions of chromosome segments found as acrocentric chromosomes in the high numbered species. Extensive chromosome homology is apparent among acrocentric chromosome arms within Canidae species; however, little chromosome arm homology exists between Canidae species and those from other Carnivore families. Here we use Zoo-FISH (fluorescent in situ hybridization, also called chromosomal painting) probes from flow-sorted chromosomes of the Japanese raccoon dog (Nyctereutes procyonoides) to examine two phylogenetically divergent canids, the arctic fox (Alopex lagopus) and the crab-eating fox (Cerdocyon thous). The results affirm intra-canid chromosome homologies, also implicated by G-banding. In addition, painting probes from domestic cat (Felis catus), representative of the ancestral carnivore karyotype (ACK), and giant panda (Ailuropoda melanoleuca) were used to define primitive homologous segments apparent between canids and other carnivore families. Canid chromosomes seem unique among carnivores in that many canid chromosome arms are mosaics of two to four homology segments of the ACK chromosome arms. The mosaic pattern apparently preceded the divergence of modern canid species since conserved homology segments among different canid species are common, even though those segments are rearranged relative to the ancestral carnivore genome arrangement. The results indicate an ancestral episode of extensive centric fission leading to an ancestral canid genome organization that was subsequently reorganized by multiple chromosome fusion events in some but not all Canidae lineages.     

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.     

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.     

Comparative genomics: tracking chromosome evolution in the family ursidae using reciprocal chromosome painting

The Ursidae family includes eight species, the karyotype of which diverges somewhat, in both chromosome number and morphology, from that of other families in the order Carnivora. The combination of consensus molecular phylogeny and high-resolution trypsin G-banded karyotype analysis has suggested that ancestral chromosomal fissions and at least two fusion events are associated with the development of the different ursid species. Here, we revisit this hypothesis by hybridizing reciprocal chromosome painting probes derived from the giant panda (Ailuropoda melanoleuca), domestic cat (Felis catus), and man (Homo sapiens) to representative bear species karyotypes. Comparative analysis of the different chromosome segment homologies allowed reconstruction of the genomic composition of a putative ancestral bear karyotype based upon the recognition of 39 chromosome segments defined by painting as the smallest conserved evolutionary unit segments (pSCEUS) among these species. The different pSCEUS combinations occurring among modern bear species support and extend the postulated sequence of chromosomal rearrangements and provide a framework to propose patterns of genome reorganization among carnivores and other mammal radiations.     

Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.     

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.     

Chromosomal polymorphism in mammals: an evolutionary perspective

Although chromosome rearrangements (CRs) are central to studies of genome evolution, our understanding of the evolutionary consequences of the early stages of karyotypic differentiation (i.e. polymorphism), especially the non‐meiotic impacts, is surprisingly limited. We review the available data on chromosomal polymorphisms in mammals so as to identify taxa that hold promise for developing a more comprehensive understanding of chromosomal change. In doing so, we address several key questions: (i) to what extent are mammalian karyotypes polymorphic, and what types of rearrangements are principally involved? (ii) Are some mammalian lineages more prone to chromosomal polymorphism than others? More specifically, do (karyotypically) polymorphic mammalian species belong to lineages that are also characterized by past, extensive karyotype repatterning? (iii) How long can chromosomal polymorphisms persist in mammals? We discuss the evolutionary implications of these questions and propose several research avenues that may shed light on the role of chromosome change in the diversification of mammalian populations and species.     

Chromosomal phylogeny of 7 species of Sciurinae

The sequence of chromosomal rearrangements that leads to the karyotypes of living species of Sciurinae is hardly compatible with a dichotomic evolution. The most probable hypothesis is that of a populational chromosomal evolution: the different lineages would have been isolated successively from an ancestral population in which several chromosomal rearrangements would have spread to more or less important fractions of the population. The proposed order in the succession of these isolations (Marmota monax, Sciurus vulgaris, Callosciurus flavimanus, Heliosciurus gambianus, Atlantoxerus getulus, Eutamias sibiricus then Menetes berdmorei) fits the paleontological data.     

Molecular cytogenetic studies in strepsirrhine primates, dermoptera and scandentia

Since the first chromosome painting study between human and strepsirrhine primates was performed in 1996, nearly 30 species in Strepsirrhini, Dermoptera and Scandentia have been analyzed by cross-species chromosome painting. Here, the contribution of chromosome painting data to our understanding of primate genome organization, chromosome evolution and the karyotype phylogenetic relationships within strepsirrhine primates, Dermoptera and Scandentia is reviewed. Twenty-six to 43 homologous chromosome segments have been revealed in different species with human chromosome-specific paint probes. Various landmark rearrangements characteristic for each different lineage have been identified, as cytogenetic signatures that potentially unite certain lineages within strepsirrhine primates, Dermoptera and Scandentia.     

Chromosomal phylogeny and karyotype evolution in x=7 crucifer species (Brassicaceae)

Karyotype evolution in species with identical chromosome number but belonging to distinct phylogenetic clades is a long-standing question of plant biology, intractable by conventional cytogenetic techniques. Here, we apply comparative chromosome painting (CCP) to reconstruct karyotype evolution in eight species with x=7 (2n=14, 28) chromosomes from six Brassicaceae tribes. CCP data allowed us to reconstruct an ancestral Proto-Calepineae Karyotype (PCK; n=7) shared by all x=7 species analyzed. The PCK has been preserved in the tribes Calepineae, Conringieae, and Noccaeeae, whereas karyotypes of Eutremeae, Isatideae, and Sisymbrieae are characterized by an additional translocation. The inferred chromosomal phylogeny provided compelling evidence for a monophyletic origin of the x=7 tribes. Moreover, chromosomal data along with previously published gene phylogenies strongly suggest the PCK to represent an ancestral karyotype of the tribe Brassiceae prior to its tribe-specific whole-genome triplication. As the PCK shares five chromosomes and conserved associations of genomic blocks with the putative Ancestral Crucifer Karyotype (n=8) of crucifer Lineage I, we propose that both karyotypes descended from a common ancestor. A tentative origin of the PCK via chromosome number reduction from n=8 to n=7 is outlined. Comparative chromosome maps of two important model species, Noccaea caerulescens and Thellungiella halophila, and complete karyotypes of two purported autotetraploid Calepineae species (2n=4x=28) were reconstructed by CCP.     

Chromosome repatterning in three representative parrots (Psittaciformes) inferred from comparative chromosome painting

Parrots (order: Psittaciformes) are the most common captive birds and have attracted human fascination since ancient times because of their remarkable intelligence and ability to imitate human speech. However, their genome organization, evolution and genomic relation with other birds are poorly understood. Chromosome painting with DNA probes derived from the flow-sorted macrochromosomes (1-10) of chicken (Gallus gallus, GGA) has been used to identify and distinguish the homoeologous chromosomal segments in three species of parrots, i.e., Agapornis roseicollis (peach-faced lovebird); Nymphicus hollandicus (cockatiel) and Melopsittacus undulatus (budgerigar). The ten GGA macrochromosome paints unequivocally recognize 14 to 16 hybridizing regions delineating the conserved chromosomal segments for the respective chicken macrochromosomes in these representative parrot species. The cross-species chromosome painting results show that, unlike in many other avian karyotypes with high homology to chicken chromosomes, dramatic rearrangements of the macrochromosomes have occurred in parrot lineages. Among the larger GGA macrochromosomes (1-5), chromosomes 1 and 4 are conserved on two chromosomes in all three species. However, the hybridization pattern for GGA 4 in A. roseicollis and M. undulatus is in sharp contrast to the most common pattern known from hybridization of chicken macrochromosome 4 in other avian karyotypes. With the exception of A. roseicollis, chicken chromosomes 2, 3 and 5 hybridized either completely or partially to a single chromosome. In contrast, the smaller GGA macrochromosomes 6, 7 and 8 displayed a complex hybridization pattern: two or three of these macrochromosomes were found to be contiguously arranged on a single chromosome in all three parrot species. Overall, the study shows that translocations and fusions in conjunction with intragenomic rearrangements have played a major role in the karyotype evolution of parrots. Our inter-species chromosome painting results unequivocally illustrate the dynamic reshuffling of ancestral chromosomes among the karyotypes of Psittaciformes.     

Cytogenetic features of rRNA genes across land plants: analysis of the Plant rDNA database

The online resource http://www.plantrdnadatabase.com/ stores information on the number, chromosomal locations and structure of the 5S and 18S‐5.8S‐26S (35S) ribosomal DNAs (rDNA) in plants. This resource was exploited to study relationships between rDNA locus number, distribution, the occurrence of linked (L‐type) and separated (S‐type) 5S and 35S rDNA units, chromosome number, genome size and ploidy level. The analyses presented summarise current knowledge on rDNA locus numbers and distribution in plants. We analysed 2949 karyotypes, from 1791 species and 86 plant families, and performed ancestral character state reconstructions. The ancestral karyotype (2n = 16) has two terminal 35S sites and two interstitial 5S sites, while the median (2n = 24) presents four terminal 35S sites and three interstitial 5S sites. Whilst 86.57% of karyotypes show S‐type organisation (ancestral condition), the L‐type arrangement has arisen independently several times during plant evolution. A non‐terminal position of 35S rDNA was found in about 25% of single‐locus karyotypes, suggesting that terminal locations are not essential for functionality and expression. Single‐locus karyotypes are very common, even in polyploids. In this regard, polyploidy is followed by subsequent locus loss. This results in a decrease in locus number per monoploid genome, forming part of the diploidisation process returning polyploids to a diploid‐like state over time.     

Origins of primate chromosomes - as delineated by Zoo-FISH and alignments of human and mouse draft genome sequences

This review examines recent advances in comparative eutherian cytogenetics, including Zoo-FISH data from 30 non-primate species. These data provide insights into the nature of karyotype evolution and enable the confident reconstruction of ancestral primate and boreo-eutherian karyotypes with diploid chromosome numbers of 48 and 46 chromosomes, respectively. Nine human autosomes (1, 5, 6, 9, 11, 13, 17, 18, and 20) represent the syntenies of ancestral boreo-eutherian chromosomes and have been conserved for about 95 million years. The average rate of chromosomal exchanges in eutherian evolution is estimated to about 1.9 rearrangements per 10 million years (involving 3.4 chromosome breaks). The integrated analysis of Zoo-FISH data and alignments of human and mouse draft genome sequences allow the identification of breakpoints involved in primate evolution. Thus, the boundaries of ancestral eutherian conserved segments can be delineated precisely. The mapping of rearrangements onto the phylogenetic tree visualizes landmark chromosome rearrangements, which might have been involved in cladogenesis in eutherian evolution.     

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 Equus 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.     

Molecular cytogenetic and genomic insights into chromosomal evolution

This review summarizes aspects of the extensive literature on the patterns and processes underpinning chromosomal evolution in vertebrates and especially placental mammals. It highlights the growing synergy between molecular cytogenetics and comparative genomics, particularly with respect to fully or partially sequenced genomes, and provides novel insights into changes in chromosome number and structure across deep division of the vertebrate tree of life. The examination of basal numbers in the deeper branches of the vertebrate tree suggest a haploid (n) chromosome number of 10-13 in an ancestral vertebrate, with modest increases in tetrapods and amniotes most probably by chromosomal fissioning. Information drawn largely from cross-species chromosome painting in the data-dense Placentalia permits the confident reconstruction of an ancestral karyotype comprising n=23 chromosomes that is similarly retained in Boreoeutheria. Using in silico genome-wide scans that include the newly released frog genome we show that of the nine ancient syntenies detected in conserved karyotypes of extant placentals (thought likely to reflect the structure of ancestral chromosomes), the human syntenic segmental associations 3p/21, 4pq/8p, 7a/16p, 14/15, 12qt/22q and 12pq/22qt predate the divergence of tetrapods. These findings underscore the enhanced quality of ancestral reconstructions based on the integrative molecular cytogenetic and comparative genomic approaches that collectively highlight a pattern of conserved syntenic associations that extends back ～360 million years ago.     

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.     

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.     

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.