Genetic mapping in a natural population of collared flycatchers (Ficedula albicollis): conserved synteny but gene order rearrangements on the avian Z chromosome

Data from completely sequenced genomes are likely to open the way for novel studies of the genetics of nonmodel organisms, in particular when it comes to the identification and analysis of genes responsible for traits that are under selection in natural populations. Here we use the draft sequence of the chicken genome as a starting point for linkage mapping in a wild bird species, the collared flycatcher - one of the most well-studied avian species in ecological and evolutionary research. A pedigree of 365 flycatchers was established and genotyped for single nucleotide polymorphisms in 23 genes selected from (and spread over most of) the chicken Z chromosome. All genes were also found to be located on the Z chromosome in the collared flycatcher, confirming conserved synteny at the level of gene content across distantly related avian lineages. This high degree of conservation mimics the situation seen for the mammalian X chromosome and may thus be a general feature in sex chromosome evolution, irrespective of whether there is male or female heterogamety. Alternatively, such unprecedented chromosomal conservation may be characteristic of most chromosomes in avian genome evolution. However, several internal rearrangements were observed, meaning that the transfer of map information from chicken to nonmodel bird species cannot always assume conserved gene orders. Interestingly, the rate of recombination on the Z chromosome of collared flycatchers was only approximately 50% that of chicken, challenging the widely held view that birds generally have high recombination rates.     

Comparative FISH mapping on Z chromosomes of chicken and Japanese quail

Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group.     

Deep ancestry of mammalian X chromosome revealed by comparison with the basal tetrapod Xenopus tropicalis

ABSTRACT: BACKGROUND: The X and Y sex chromosomes are conspicuous features of placental mammal genomes. Mammalian sex chromosomes arose from an ordinary pair of autosomes after the proto-Y acquired a male-determining gene and degenerated due to suppression of X-Y recombination. Analysis of earlier steps in X chromosome evolution has been hampered by the long interval between the origins of teleost and amniote lineages as well as scarcity of X chromosome orthologs in incomplete avian genome assemblies. RESULTS: This study clarifies the genesis and remodelling of the X chromosome by using a combination of sequence analysis, meiotic map information, and cytogenetic localization to compare amniote genome organization with that of the amphibian Xenopus tropicalis. Nearly all orthologs of human X genes localize to X. tropicalis chromosomes 2 and 8, consistent with an ancestral X-conserved region and a single X-added region precursor. This finding contradicts a previous hypothesis of three evolutionary strata in this region. Homologies between human, opossum, chicken and frog chromosomes suggest a single X-added region predecessor in therian mammals, corresponding to opossum chromosomes 4 and 7. A more ancient X-added ancestral region, currently extant as a major part of chicken chromosome 1, is likely to have been present in the progenitor of synapsids and sauropsids. Analysis of X chromosome gene content emphasizes conservation of single protein coding genes and the role of tandem arrays in formation of novel genes. CONCLUSIONS: Chromosomal regions orthologous to Therian X chromosomes have been located in the genome of the frog X. tropicalis. These ancestral components experienced a series of fusion and breakage events to give rise to avian autosomes and mammalian sex chromosomes. The early branching tetrapod X. tropicalis' simple diploid genome and robust synteny to amniotes greatly enhances studies of vertebrate chromosome evolution.     

The karyotype of Alligator mississippiensis,and chromosomal mapping of the ZFY/X homologue,Zfc

Comparative mapping studies of X-linked genes in mammals have provided insights into the evolution of the X chromosome. Many reptiles including the American alligator, Alligator mississippiensis, do not appear to possess heteromorphic sex chromosomes, and sex is determined by the incubation temperature of the egg during embryonic development. Mapping of homologues of mammalian X-linked genes in reptiles could lead to a greater understanding of the evolution of vertebrate sex chromosomes. One of the genes used in the mammalian mapping studies was ZFX, an X-linked copy of the human ZFY gene which was originally isolated as a candidate for the mammalian testis-determining factor (TDF). ZFX is X-linked in eutherians, but maps to two autosomal locations in marsupials and monotremes, close to other genes associated with the eutherian X. The alligator homologue of the ZFY/ZFX genes, Zfc, has been isolated and described previously. A detailed karyotype of A. mississippiensis is presented, together with chromosomal in situ hybridisation data localising the Zfc gene to chromosome 3. Further chromosomal mapping studies using eutherian X-linked genes may reveal conserved chromosomal regions in the alligator that have become part of the eutherian X chromosome during evolution.     

Comparative mapping of chicken anchor loci orthologous to genes on human chromosomes 1, 4 and 9

Comparative mapping of chicken and human genomes is described, primarily of regions corresponding to human chromosomes 1, 4 and 9. Segments of chicken orthologues of selected human genes were amplified from parental DNA of the East Lansing backcross reference mapping population, and the two parental alleles were sequenced. In about 80% of the genes tested, sequence polymorphism was identified between reference population parental DNAs. The polymorphism was used to design allele-specific primers with which to genotype the backcross panel and place genes on the chicken linkage map. Thirty-seven genes were mapped which confirmed the surprisingly high level of conserved synteny between orthologous chicken and human genes. In several cases the order of genes in conserved syntenic groups differs between the two genomes, suggesting that there may have been more frequent intrachromosomal inversions as compared with interchromosomal translocations during the separate evolution of avian and mammalian genomes.     

Conserved synteny of mammalian imprinted genes in chicken, frog, and fish genomes

Conservation of synteny of mammalian imprinted genes between chicken and human suggested that highly conserved gene clusters were selected long before these genes were recruited for genomic imprinting in mammals. Here we have applied in silico mapping of orthologous genes in pipid frog, zebrafish, spotted green and Japanese pufferfish to show considerable conservation of synteny in lower vertebrates. More than 400 million years ago in a common ancestor of teleost fish and tetrapods, 'preimprinted' chromosome regions homologous to human 6q25, 7q21, 7q32, 11p15, and 15q11-->q12 already contained most present-day mammalian imprinted genes. Interestingly, some imprinted gene orthologues which are isolated from imprinted clusters in mouse and human could be linked to preimprinted regions in lower vertebrates, indicating that separation occurred during mammalian evolution. On the contrary, newly arisen genes by segmental duplication in the mammalian lineage, i.e. SNRPN and FRAT3, were transposed or translocated to imprinted clusters and recruited for parent-specific activity. By analysis of currently available sequences of non-mammalian vertebrates, the imprinted gene clusters homologous to human chromosomes 14q32 and 19q12 are only poorly conserved in chicken, frog, and fish and, therefore, may not have evolved from ancestral preimprinted gene arrays. Evidently, evolution of imprinted gene clusters is an ongoing and dynamic process in mammals. In general, imprinted gene orthologues do not show a higher degree of synteny conservation in vertebrates than non-imprinted genes interspersed with or adjacent to an imprinted cluster.     

Comparative mapping using somatic cell hybrids

Summary Comparative mapping, or ascertaining the gene linkage relationships between different species, is rapidly developing. This is possible because new techniques in chromosome identification and somatic cell hybridization, such as the generation of hybrids preferentially segregating chromosomes of any desired species including rodents, and the development of gene transfer techniques have yielded new information about the human and rodent gene maps. In addition, the discovery and characterization of mouse subspecies has generated new mouse sexual genetic linkage data. The following picture is emerging. Several X-linked genes in man are X-linked in all mammalian species tested. The linkage relationships of several tightly linked genes, less than 1 map unit apart, are also conserved in all mammalian species tested. Ape autosomal genes are assigned to ape chromosomes homologous to their human counterparts indicating extensive conservation in the 12 million years (MYR) of evolution from apes to man. Similarly, mouse and rat, 10 MYR apart in evolution, have several large autosomal synteny groups conserved. In comparing the mouse and human gene maps we find that human genes assigned to different arms of the same human chromosome are unlinked in the mouse; mouse genes large map distances (20 to 45 cM) apart are very likely to be unlinked in the human. However, several autosomal synteny groups 10 to 20 cM apart, including thePgd, Eno-1, Pgm-1 group on human chromosome arm lp, are conserved in mice and man. This suggests that homology mapping, the superimposition of one species gene map on the homologous conserved portion of another species genome may be possible, and that ancestral autosomal synteny groups should be detectable. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976.     

Genome assembly,sex-biased gene expression and dosage compensation in the damselfly Ischnura elegans

The evolution of sex chromosomes, and patterns of sex-biased gene expression and dosage compensation, are poorly known among early winged insects such as odonates. We assembled and annotated the genome of Ischnura elegans (blue-tailed damselfly), which, like other odonates, has a male-hemigametic sex-determining system (X0 males, XX females). By identifying X-linked genes in I. elegans and their orthologs in other insect genomes, we found homologies between the X chromosome in odonates and chromosomes of other orders, including the X chromosome in Coleoptera. Next, we showed balanced expression of X-linked genes between sexes in adult I. elegans, i.e. evidence of dosage compensation. Finally, among the genes in the sex-determining pathway only fruitless was found to be X-linked, while only doublesex showed sex-biased expression. This study reveals partly conserved sex chromosome synteny and independent evolution of dosage compensation among insect orders separated by several hundred million years of evolutionary history.     

Bird and mammal sex-chromosome orthologs map to the same autosomal region in a salamander (ambystoma)

We tested hypotheses concerning the origin of bird and mammal sex chromosomes by mapping the location of amniote sex-chromosome loci in a salamander amphibian (Ambystoma). We found that ambystomatid orthologs of human X and chicken Z sex chromosomes map to neighboring regions of a common Ambystoma linkage group 2 (ALG2). We show statistically that the proportion of human X and chicken Z orthologs observed on ALG2 is significantly different from the proportion that would be expected by chance. We further show that conserved syntenies between ALG2 and amniote chromosomes are identified as overlapping conserved syntenies when all available chicken (N = 3120) and human (N = 14,922) RefSeq orthologs are reciprocally compared. In particular, the data suggest that chromosomal regions from chicken chromosomes (GGA) Z and 4 and from human chromosomes (HSA) 9, 4, X, 5, and 8 were linked ancestrally. A more distant outgroup comparison with the pufferfish Tetraodon nigroviridis reveals ALG2/GGAZ/HSAX syntenies among three pairs of ancestral chromosome duplicates. Overall, our results suggest that sex chromosomal regions of birds and mammals were recruited from a common ancestral chromosome, and thus our findings conflict with the currently accepted hypothesis of separate autosomal origins. We note that our results were obtained using the most immediate outgroup to the amniote clade (mammals, birds, and other reptiles) while the currently accepted hypothesis is primarily based upon conserved syntenies between in-group taxa (birds and mammals). Our study illustrates the importance of an amphibian outgroup perspective in identifying ancestral amniote gene orders and in reconstructing patterns of vertebrate sex-chromosome evolution.     

Different origins of bird and reptile sex chromosomes inferred from comparative mapping of chicken Z-linked genes

Recent progress of chicken genome projects has revealed that bird ZW and mammalian XY sex chromosomes were derived from different autosomal pairs of the common ancestor; however, the evolutionary relationship between bird and reptilian sex chromosomes is still unclear. The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes have been identified. In order to investigate this further, we performed molecular cytogenetic analyses of this species, and thereby identified ZZ/ZW-type micro-sex chromosomes. In addition, we cloned reptile homologues of chicken Z-linked genes from three reptilian species, the Chinese soft-shelled turtle and the Japanese four-striped rat snake (Elaphe quadrivirgata), which have heteromorphic sex chromosomes, and the Siam crocodile (Crocodylus siamensis), which exhibits temperature-dependent sex determination and lacks sex chromosomes. We then mapped them to chromosomes of each species using FISH. The linkage of the genes has been highly conserved in all species: the chicken Z chromosome corresponded to the turtle chromosome 6q, snake chromosome 2p and crocodile chromosome 3. The order of the genes was identical among the three species. The absence of homology between the bird Z chromosome and the snake and turtle Z sex chromosomes suggests that the origin of the sex chromosomes and the causative genes of sex determination are different between birds and reptiles.     

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.     

Conservation of avian Z chromosomes as revealed by comparative mapping of the Z-linked aldolase B gene

A chicken Z-linked BAC probe containing the aldolase B gene was used for fluorescence in-situ hybridization (FISH) mapping in four different avian species. The biotinylated BAC clone showed distinct unique hybridization sites on the structurally different Z chromosomes. This result, together with previous data, lends credence to the notion that, despite undergoing structural rearrangements, the gene content of the avian Z chromosome remained conserved during evolution. Our study also demonstrates the feasibility of using large genomic clones for comparative mapping of Z-linked genes in birds.     

FISH mapping of 57 BAC clones reveals strong conservation of synteny between Galliformes and Anseriformes

Karyotypes of chicken (Gallus gallus domesticus; 2n = 78) and mallard duck (Anas platyrhynchos; 2n = 80) share the typical organization of avian karyotypes including a few macrochromosome pairs, numerous indistinguishable microchromosomes, and Z and W sex chromosomes. Previous banding studies revealed great similarities between chickens and ducks, but it was not possible to use comparative banding for the microchromosomes. In order to establish precise chromosome correspondences between these two species, particularly for microchromosomes, we hybridized 57 BAC clones previously assigned to the chicken genome to duck metaphase spreads. Although most of the clones showed similar localizations, we found a few intrachromosomal rearrangements of the macrochromosomes and an additional microchromosome pair in ducks. BAC clones specific for chicken microchromosomes were localized to separate duck microchromosomes and clones mapping to the same chicken microchromosome hybridized to the same duck microchromosome, demonstrating a high conservation of synteny. These results demonstrate that the evolution of karyotypes in avian species is the result of fusion and/or fission processes and not translocations.     

Evolutionary strata on the chicken Z chromosome: implications for sex chromosome evolution

The human X chromosome exhibits four "evolutionary strata," interpreted to represent distinct steps in the process whereby recombination became arrested between the proto X and proto Y. To test if this is a general feature of sex chromosome evolution, we studied the Z-W sex chromosomes of birds, which have female rather than male heterogamety and evolved from a different autosome pair than the mammalian X and Y. Here we analyze all five known gametologous Z-W gene pairs to investigate the "strata" hypothesis in birds. Comparisons of the rates of synonymous substitution and intronic divergence between Z and W gametologs reveal the presence of at least two evolutionary strata spread over the p and q arms of the chicken Z chromosome. A phylogenetic analysis of intronic sequence data from different avian lineages indicates that Z-W recombination ceased in the oldest stratum (on Zq; CHD1Z, HINTZ, and SPINZ) 102-170 million years ago (MYA), before the split of the Neoaves and Eoaves. However, recombination continued in the second stratum (on Zp; UBAP2Z and ATP5A1Z) until after the divergence of extant avian orders, with Z and W diverging 58-85 MYA. Our data suggest that progressive and stepwise cessation of recombination is a general feature behind sex chromosome evolution.     

The Staurotypus Turtles and Aves Share the Same Origin of Sex Chromosomes but Evolved Different Types of Heterogametic Sex Determination

Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines) and the giant musk turtle (Staurotypus salvinii) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis) revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.     

Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence

The divergence of lineages leading to extant squamate reptiles (lizards, snakes, and amphisbaenians) and birds occurred about 275 million years ago. Birds, unlike squamates, have karyotypes that are typified by the presence of a number of very small chromosomes. Hence, a number of chromosome rearrangements might be expected between bird and squamate genomes. We used chromosome-specific DNA from flow-sorted chicken (Gallus gallus) Z sex chromosomes as a probe in cross-species hybridization to metaphase spreads of 28 species from 17 families representing most main squamate lineages and single species of crocodiles and turtles. In all but one case, the Z chromosome was conserved intact despite very ancient divergence of sauropsid lineages. Furthermore, the probe painted an autosomal region in seven species from our sample with characterized sex chromosomes, and this provides evidence against an ancestral avian-like system of sex determination in Squamata. The avian Z chromosome synteny is, therefore, conserved albeit it is not a sex chromosome in these squamate species.     

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: implications for the evolutionary derivation of human chromosome 19.

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri x X. clemenciae F1 hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, chi 2 = 35.37, P less than 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.     

Comparative cytogenetic analysis of sex chromosomes in several Canidae species using zoo-FISH

Sex chromosome differentiation began early during mammalian evolution. The karyotype of almost all placental mammals living today includes a pair of heterosomes: XX in females and XY in males. The genomes of different species may contain homologous synteny blocks indicating that they share a common ancestry. One of the tools used for their identification is the Zoo-FISH technique. The aim of the study was to determine whether sex chromosomes of some members of the Canidae family (the domestic dog, the red fox, the arctic fox, an interspecific hybrid: arctic fox x red fox and the Chinese raccoon dog) are evolutionarily conservative. Comparative cytogenetic analysis by Zoo-FISH using painting probes specific to domestic dog heterosomes was performed. The results show the presence of homologous synteny covering the entire structures of the X and the Y chromosomes. This suggests that sex chromosomes are conserved in the Canidae family. The data obtained through Zoo-FISH karyotype analysis append information obtained using other comparative genomics methods, giving a more complete depiction of genome evolution.