Four-hundred million years of conserved synteny of human Xp and Xq genes on three Tetraodon chromosomes

The freshwater pufferfish Tetraodon nigroviridis (TNI) has become highly attractive as a compact reference vertebrate genome for gene finding and validation. We have mapped genes, which are more or less evenly spaced on the human chromosomes 9 and X, on Tetraodon chromosomes using fluorescence in situ hybridization (FISH), to establish syntenic relationships between Tetraodon and other key vertebrate genomes. PufferFISH revealed that the human X is an orthologous mosaic of three Tetraodon chromosomes. More than 350 million years ago, an ancestral vertebrate autosome shared orthologous Xp and Xq genes with Tetraodon chromosomes 1 and 7. The shuffled order of Xp and Xq orthologs on their syntenic Tetraodon chromosomes can be explained by the prevalence of evolutionary inversions. The Tetraodon 2 orthologous genes are clustered in human Xp11 and represent a recent addition to the eutherian X sex chromosome. The human chromosome 9 and the avian Z sex chromosome show a much lower degree of synteny conservation in the pufferfish than the human X chromosome. We propose that a special selection process during vertebrate evolution has shaped a highly conserved array(s) of X-linked genes long before the X was used as a mammalian sex chromosome and many X chromosomal genes were recruited for reproduction and/or the development of cognitive abilities. [Sequence data reported in this paper have been deposited in GenBank and assigned the following accession no: AJ308098.]     

Highly conserved linkage homology between birds and turtles: Bird and turtle chromosomes are precise counterparts of each other

The karyotypes of birds, turtles and snakes are characterized by two distinct chromosomal components, macrochromosomes and microchromosomes. This close karyological relationship between birds and reptiles has long been a topic of speculation among cytogeneticists and evolutionary biologists; however, there is scarcely any evidence for orthology at the molecular level. To define the conserved chromosome synteny among humans, chickens and reptiles and the process of genome evolution in the amniotes, we constructed comparative cytogenetic maps of the Chinese soft-shelled turtle (Pelodiscus sinensis) and the Japanese four-striped rat snake (Elaphe quadrivirgata) using cDNA clones of reptile functional genes. Homology between the turtle and chicken chromosomes is highly conserved, with the six largest chromosomes being almost equivalent to each other. On the other hand, homology to chicken chromosomes is lower in the snake than in the turtle. Turtle chromosome 6q and snake chromosome 2p represent conserved synteny with the chicken Z chromosome. These results suggest that the avian and turtle genomes have been well conserved during the evolution of the Arcosauria. The avian and snake sex Z chromosomes were derived from different autosomes in a common ancestor, indicating that the causative genes of sex determination may be different between birds and snakes.Matsuda and Nishida-Umehara contributed equally to this work.     

Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat, dog and human

Domestic cats and dogs are important companion animals and model animals in biomedical research. The cat has a highly conserved karyotype, closely resembling the ancestral karyotype of mammals, while the dog has one of the most extensively rearranged mammalian karyotypes investigated so far. We have constructed the first detailed comparative chromosome map of the domestic dog and cat by reciprocal chromosome painting. Dog paints specific for the 38 autosomes and the X chromosomes delineated 68 conserved chromosomal segments in the cat, while reverse painting of cat probes onto red fox and dog chromosomes revealed 65 conserved segments. Most conserved segments on cat chromosomes also show a high degree of conservation in G-banding patterns compared with their canine counterparts. At least 47 chromosomal fissions (breaks), 25 fusions and one inversion are needed to convert the cat karyotype to that of the dog, confirming that extensive chromosome rearrangements differentiate the karyotypes of the cat and dog. Comparative analysis of the distribution patterns of conserved segments defined by dog paints on cat and human chromosomes has refined the human/cat comparative genome map and, most importantly, has revealed 15 cryptic inversions in seven large chromosomal regions of conserved synteny between humans and cats. This revised version was published online in July 2006 with corrections to the Cover Date.     

Highly conserved chromosomes in an Asian squirrel (Menetes berdmorei, Rodentia: Sciuridae) as demonstrated by ZOO-FISH with human probes

The chromosomes of Menetes berdmorei (Rodentia, Sciuridae, Sciurinae) were studied by ZOO-FISH using whole human chromosome probes. All homoeologies between M. berdmorei and human chromosomes were determined, except for two small chromosome segments. Twelve human chromosomes are conserved in a unique block of synteny; ten are split into two and one into three blocks. Thus, a small number of interchromosomal rearrangements, about twenty, separates human from this squirrel karyotype. Homoeologies between human and the presumed ancestral chromosomes of Sciurinae could also be deduced, as well as those with the presumed ancestral chromosomes of eutherian mammals. Sciurinae chromosomes appear to be much closer to those of non-rodent mammals than those of Muridae and Cricetidae species studied so far. Thus, they provide an interesting tool to link the rodent genome to those of other mammals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Correspondence of human chromosomes 9, 12, 15, 16, 19 and 20 with donkey chromosomes refines homology between horse and donkey karyotypes

Whole chromosome paints for human (HSA) chromosomes 9, 12, 15 and 20 and arm-specific paints for HSA16p, 19p and 19q were applied on donkey metaphase spreads. All probes, except HSA19p, gave distinct hybridization signals on donkey chromosomes/chromosomal segments. The results show direct segmental homology between human and donkey genomes, and enable refinement of correspondence between donkey and horse karyotypes. Of specific interest is the identification of hitherto unknown correspondence between four equine acrocentric chromosomes (ECA22, 23, 25 and 28) and the donkey chromosomes. Overall, the findings mark the beginning of an ordered study of comparative organization of genomes/karyotypes of the equids, that can shed light on karyotype evolution and ancestral chromosomal condition in the Perissodactyls. This revised version was published online in July 2006 with corrections to the Cover Date.     

Non-homologous sex chromosomes of birds and snakes share repetitive sequences

Snake sex chromosomes provided Susumo Ohno with the material on which he based his theory of how sex chromosomes differentiate from autosomal pairs. Like birds, snakes have a ZZ male/ZW female sex chromosome system, in which the snake Z is a macrochromosome much the same size as the bird Z. However, the gene content shows clearly that the snake and bird Z chromosomes are completely non-homologous. The molecular aspect of W chromosome degeneration in snakes remains largely unexplored. We used comparative genomic hybridization to identify the female-specific region of the W chromosome in representative species of Australian snakes. Using this approach, we show that an increasingly complex suite of repeats accompanies the evolution of W chromosome heteromorphy. In particular, we found that while the python Liasis fuscus exhibits no sex-specific repeats and indeed, no cytologically recognizable sex-specific region, the colubrid Stegonotus cucullatus shows a large domain on the short arm of the W chromosome that consists of female-specific repeats, and the large W of Notechis scutatus is composed almost entirely of repetitive sequences, including Bkm and 18S rDNA-related elements. FISH mapping of both simple and complex probes shows patterns of repeat amplification concordant with the size of the female-specific region in each species examined. Mapping of intronic sequences of genes that are sex-linked in both birds (DMRT1) and snakes (CTNNB1) reveals massive amplification in discrete domains on the W chromosome of the elapid N. scutatus. Using chicken W chromosome paint, we demonstrate that repetitive sequences are shared between the sex chromosomes of birds and derived snakes. This could be explained by ancestral but as yet undetected shared synteny of bird and snake sex chromosomes or may indicate functional homology of the repeats and suggests that degeneration is a convergent property of sex chromosome evolution. We also establish that synteny of snake Z-linked genes has been conserved for at least 166 million years and that the snake Z consists of two conserved blocks derived from the same ancestral vertebrate chromosome.     

Parallel Radiation Hybrid Mapping: A Powerful Tool for High-Resolution Genomic Comparison

Comparative gene mapping in mammals typically involves identification of segments of conserved synteny in diverse genomes. The development of maps that permit comparison of gene order within conserved synteny has not advanced beyond the mouse map that takes advantage of linkage analysis in interspecific backcrosses. Radiation hybrid (RH) mapping provides a powerful tool for determining order of genes in genomes for which gene-based linkage mapping is impractical. Comparative RH mapping of 24 orthologous genes in this study revealed internal structural rearrangements between human chromosome 17 (HSA17) and bovine chromosome 19 (BTA19), two chromosomes known previously to be conserved completely and exclusively at level of synteny. Only six of the 24 genes had been previously ordered on the human G3 RH map. The use of the G3 panel to map the other 18, however, produced parallel RH maps for comparison of gene order at a resolution of <5 Mb on the bovine linkage map and from 1 to 3 Mb in the human physical map.     

The zebrafish gene map defines ancestral vertebrate chromosomes

Genetic screens in zebrafish (Danio rerio) have identified mutations that define the roles of hundreds of essential vertebrate genes. Genetic maps can link mutant phenotype with gene sequence by providing candidate genes for mutations and polymorphic genetic markers useful in positional cloning projects. Here we report a zebrafish genetic map comprising 4073 polymorphic markers, with more than twice the number of coding sequences localized in previously reported zebrafish genetic maps. We use this map in comparative studies to identify numerous regions of synteny conserved among the genomes of zebrafish, Tetraodon, and human. In addition, we use our map to analyze gene duplication in the zebrafish and Tetraodon genomes. Current evidence suggests that a whole-genome duplication occurred in the teleost lineage after it split from the tetrapod lineage, and that only a subset of the duplicates have been retained in modern teleost genomes. It has been proposed that differential retention of duplicate genes may have facilitated the isolation of nascent species formed during the vast radiation of teleosts. We find that different duplicated genes have been retained in zebrafish and Tetraodon, although similar numbers of duplicates remain in both genomes. Finally, we use comparative mapping data to address the proposal that the common ancestor of vertebrates had a genome consisting of 12 chromosomes. In a three-way comparison between the genomes of zebrafish, Tetraodon, and human, our analysis delineates the gene content for 11 of these 12 proposed ancestral chromosomes.     

Zebrafish comparative genomics and the origins of vertebrate chromosomes

To help understand mechanisms of vertebrate genome evolution, we have compared zebrafish and tetrapod gene maps. It has been suggested that translocations are fixed more frequently than inversions in mammals. Gene maps showed that blocks of conserved syntenies between zebrafish and humans were large, but gene orders were frequently inverted and transposed. This shows that intrachromosomal rearrangements have been fixed more frequently than translocations. Duplicated chromosome segments suggest that a genome duplication occurred in ray-fin phylogeny, and comparative studies suggest that this event happened deep in the ancestry of teleost fish. Consideration of duplicate chromosome segments shows that at least 20% of duplicated gene pairs may be retained from this event. Despite genome duplication, zebrafish and humans have about the same number of chromosomes, and zebrafish chromosomes are mosaically orthologous to several human chromosomes. Is this because of an excess of chromosome fissions in the human lineage or an excess of chromosome fusions in the zebrafish lineage? Comparative analysis suggests that an excess of chromosome fissions in the tetrapod lineage may account for chromosome numbers and provides histories for several human chromosomes.     

Defining the ancestral eutherian karyotype: A cladistic interpretation of chromosome painting and genome sequence assembly data

A cladistic analysis of genome assemblies (syntenic associations) for eutherian mammals against two distant outgroup species—opossum and chicken—permitted a refinement of the 46-chromosome karyotype formerly inferred in the ancestral eutherian. We show that two intact chromosome pairs (corresponding to human chromosomes 13 and 18) and three conserved chromosome segments (10q, 19p and 8q in the human karyotype) are probably symplesiomorphic for Eutheria because they are also present as unaltered orthologues in one or both outgroups. Seven additional syntenies (4q/8p/4pq, 3p/21, 14/15, 10p/12pq/22qt, 19q/16q, 16p/7a and 12qt/22q), each involving human chromosomal segments that in various combinations correspond to complete chromosomes in the ancestral eutherian karyotype, are also present in one or both outgroup taxa and thus are probable symplesiomorphies for Eutheria. Interestingly, several of the symplesiomorphic characters identified in chicken and/or opossum are present in more distant outgroups such as pufferfish and zebrafish (for example 3p/21, 14/15, 19q/16q and 16p/7a), suggesting their retention since vertebrate common ancestry ∼450 million years ago. However, eight intact pairs (corresponding to human chromosomes 1, 5, 6, 9, 11, 17, 20 and the X) and three chromosome segments (7b, 2p-q13 and 2q13-qter) are derived characters potentially consistent with eutherian monophyly. Our analyses clarify the distinction between shared-ancestral and shared-derived homology in the eutherian ancestral karyotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The syntenic relationship of the zebrafish and human genomes

The zebrafish is an important vertebrate model for the mutational analysis of genes effecting developmental processes. Understanding the relationship between zebrafish genes and mutations with those of humans will require understanding the syntenic correspondence between the zebrafish and human genomes. High throughput gene and EST mapping projects in zebrafish are now facilitating this goal. Map positions for 523 zebrafish genes and ESTs with predicted human orthologs reveal extensive contiguous blocks of synteny between the zebrafish and human genomes. Eighty percent of genes and ESTs analyzed belong to conserved synteny groups (two or more genes linked in both zebrafish and human) and 56% of all genes analyzed fall in 118 homology segments (uninterrupted segments containing two or more contiguous genes or ESTs with conserved map order between the zebrafish and human genomes). This work now provides a syntenic relationship to the human genome for the majority of the zebrafish genome.     

A Comparative Genomic Analysis of Two Distant Diptera, the Fruit Fly, Drosophila melanogaster, and the Malaria Mosquito, Anopheles gambiae

Genome evolution entails changes in the DNA sequence of genes and intergenic regions, changes in gene numbers, and also changes in gene order along the chromosomes. Genes are reshuffled by chromosomal rearrangements such as deletions/insertions, inversions, translocations, and transpositions. Here we report a comparative study of genome organization in the main African malaria vector, Anopheles gambiae, relative to the recently determined sequence of the Drosophila melanogaster genome. The ancestral lines of these two dipteran insects are thought to have separated approximately 250 Myr, a long period that makes this genome comparison especially interesting. Sequence comparisons have identified 113 pairs of putative orthologs of the two species. Chromosomal mapping of orthologous genes reveals that each polytene chromosome arm has a homolog in the other species. Between 41% and 73% of the known orthologous genes remain linked in the respective homologous chromosomal arms, with the remainder translocated to various nonhomologous arms. Within homologous arms, gene order is extensively reshuffled, but a limited degree of conserved local synteny (microsynteny) can be recognized.     

The genome phylogeny of domestic cat, red panda and five mustelid species revealed by comparative chromosome painting and G-banding

Genome-wide homology maps among stone marten (Martes foina, 2n = 38), domestic cat (Felis catus, 2n = 38), American mink (Mustela vison, 2n = 30), yellow-throated marten (Martes flavigula, 2n = 40), Old World badger (Meles meles, 2n = 44), ferret badger (Melogale moschata, 2n = 38) and red panda (Ailurus fulgens, 2n = 36) have been established by cross-species chromosome painting with a complete set of stone marten probes. In total, 18 stone marten autosomal probes reveal 20, 19, 21, 18 and 21 pairs of homologous chromosomal segments in the respective genomes of American mink, yellow-throated marten, Old World badger, ferret badger and red panda. Reciprocal painting between stone marten and cat delineated 21 pairs of homologous segments shared in both stone marten and cat genomes. The chromosomal painting results indicate that most chromosomes of these species are highly conserved and show one-to-one correspondence with stone marten and cat chromosomes or chromosomal arms, and that only a few interchromosomal rearrangements (Robertsonian fusions and fissions) have occurred during species radiation. By comparing the distribution patterns of conserved chromosomal segments in both these species and the putative ancestral carnivore karyotype, we have reconstructed the pathway of karyotype evolution of these species from the putative 2n = 42 ancestral carnivore karyotype. Our results support a close phylogenetic relationship between the red panda and mustelids. The homology data presented in these maps will allow us to transfer the cat gene mapping data to other unmapped carnivore species.     

Zoo-FISH delineates conserved chromosomal segments in horse and man

Human chromosome specific libraries (CSLs) were individually applied to equine metaphase chromosomes using the fluorescencein situ hybridization (FISH) technique. All CSLs, except Y, showed painting signals on one or several horse chromosomes. In total 43 conserved chromosoma segments were painted. Homoeology could not, however, be detected for some segments of the equine genome. This is most likely related to the very weak signals displayed by some libraries, rather than to the absence of similarity with the human genome. In spite of divergence from the human genome, dated 70–80 million years ago, a fairly high degree of synteny conservation was observed. In seven cases, whole chromosome synteny was detected between the two species. The comparative painting results agreed completely with the limited gene mapping data available in horses, and also enabled us provisionally to assign one linkage group (U2) and one syntenic group (NP, MPI, IDH2) to specific equine chromosomes. Chromosomal assignments of three other syntenic groups are also proposed. The findings of this study will be of significant use in the expansion of the hitherto poorly developed equine gene map.accepted for publication by M. Schmid     

Census of vertebrate Wnt genes: isolation and developmental expression of Xenopus Wnt2, Wnt3, Wnt9a, Wnt9b, Wnt10a, and Wnt16.

The Wnt family of growth factors regulate many different aspects of embryonic development. Assembly of the complete mouse and human genome sequences, plus expressed sequence tag surveys have established the existence of 19 Wnt genes in mammalian genomes. However, despite the importance of model vertebrates for studies in developmental biology, the complete complement of Wnt genes has not been established for nonmammalian genomes. Using genome sequences for chicken (Gallus gallus), frog (Xenopus tropicalis), and fish (Danio rerio and Tetraodon nigroviridis), we have analyzed gene synteny to identify the orthologues of all 19 human Wnt genes in these species. We find that, in addition to the 19 Wnts observed in humans, chicken contained an additional Wnt gene, Wnt11b, which is orthologous to frog and zebrafish Wnt11 (silberblick). Frog and fish genomes contained orthologues of the 19 mammalian Wnt genes, plus Wnt11b and several duplicated Wnt genes. Specifically, the Xenopus tropicalis genome contained 24 Wnt genes, including additional copies of Wnt7-related genes (Wnt7c) and 3 recent Wnt duplications (Wnt3, Wnt9b, and Wnt11). The Danio rerio genome contained 27 Wnt genes with additional copies of Wnt2, Wnt2b, Wnt4b, Wnt6, Wnt7a, and Wnt8a. The presence of the additional Wnt11 sequence (Wnt11b) in the genomes of all ancestral vertebrates suggests that this gene has been lost during mammalian evolution. Through these studies, we identified the frog orthologues of the previously uncharacterized Wnt2, Wnt3, Wnt9a, Wnt9b, Wnt10a, and Wnt16 genes and their expression has been characterized during early Xenopus development.     

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.     

Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes

Examining the relationships among DNA sequence, meiotic recombination, and chromosome structure at a genome-wide scale has been difficult because only a few markers connect genetic linkage maps with physical maps. Here, we have positioned 1195 genetically mapped expressed sequence tag (EST) markers onto the 10 pachytene chromosomes of maize by using a newly developed resource, the RN-cM map. The RN-cM map charts the distribution of crossing over in the form of recombination nodules (RNs) along synaptonemal complexes (SCs, pachytene chromosomes) and allows genetic cM distances to be converted into physical micrometer distances on chromosomes. When this conversion is made, most of the EST markers used in the study are located distally on the chromosomes in euchromatin. ESTs are significantly clustered on chromosomes, even when only euchromatic chromosomal segments are considered. Gene density and recombination rate (as measured by EST and RN frequencies, respectively) are strongly correlated. However, crossover frequencies for telomeric intervals are much higher than was expected from their EST frequencies. For pachytene chromosomes, EST density is about fourfold higher in euchromatin compared with heterochromatin, while DNA density is 1.4 times higher in heterochromatin than in euchromatin. Based on DNA density values and the fraction of pachytene chromosome length that is euchromatic, we estimate that approximately 1500 Mbp of the maize genome is in euchromatin. This overview of the organization of the maize genome will be useful in examining genome and chromosome evolution in plants.     

Reconstructing the genomic architecture of ancestral mammals: lessons from human, mouse, and rat genomes

Recent analysis of genome rearrangements in human and mouse genomes revealed evidence for more rearrangements than thought previously and shed light on previously unknown features of mammalian evolution, like breakpoint reuse and numerous microrearrangements. However, two-way analysis cannot reveal the genomic architecture of ancestral mammals or assign rearrangement events to different lineages. Thus, the "original synteny" problem introduced by Nadeau and Sankoff previously, remains unsolved, as at least three mammalian genomes are required to derive the ancestral mammalian karyotype. We show that availability of the rat genome allows one to reconstruct a putative genomic architecture of the ancestral murid rodent genome. This reconstruction suggests that this ancestral genome retained many previously postulated chromosome associations in the placental ancestor and reveals others that were beyond the resolution of cytogenetic, radiation hybrid mapping, and chromosome painting techniques. Three-way analysis of rearrangements leads to a reliable reconstruction of the genomic architecture of specific regions in the murid ancestor, including the X chromosome, and for the first time allows one to assign major rearrangement events to one of human, mouse, and rat lineages. Our analysis implies that the rate of rearrangements is much higher in murid rodents than in the human lineage and confirms the existence of rearrangement hot-spots in all three lineages.     

A comparative map of the zebrafish genome

Zebrafish mutations define the functions of hundreds of essential genes in the vertebrate genome. To accelerate the molecular analysis of zebrafish mutations and to facilitate comparisons among the genomes of zebrafish and other vertebrates, we used a homozygous diploid meiotic mapping panel to localize polymorphisms in 691 previously unmapped genes and expressed sequence tags (ESTs). Together with earlier efforts, this work raises the total number of markers scored in the mapping panel to 2119, including 1503 genes and ESTs and 616 previously characterized simple-sequence length polymorphisms. Sequence analysis of zebrafish genes mapped in this study and in prior work identified putative human orthologs for 804 zebrafish genes and ESTs. Map comparisons revealed 139 new conserved syntenies, in which two or more genes are on the same chromosome in zebrafish and human. Although some conserved syntenies are quite large, there were changes in gene order within conserved groups, apparently reflecting the relatively frequent occurrence of inversions and other intrachromosomal rearrangements since the divergence of teleost and tetrapod ancestors. Comparative mapping also shows that there is not a one-to-one correspondence between zebrafish and human chromosomes. Mapping of duplicate gene pairs identified segments of 20 linkage groups that may have arisen during a genome duplication that occurred early in the evolution of teleosts after the divergence of teleost and mammalian ancestors. This comparative map will accelerate the molecular analysis of zebrafish mutations and enhance the understanding of the evolution of the vertebrate genome.