Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes)—An atypical species with low diploid number

The chicken is the most extensively studied species in birds and thus constitutes an ideal reference for comparative genomics in birds. Comparative cytogenetic studies indicate that the chicken has retained many chromosome characters of the ancestral avian karyotype. The homology between chicken macrochromosomes (1–9 and Z) and their counterparts in more than 40 avian species of 10 different orders has been established by chromosome painting. However, the avian homologues of chicken microchromosomes remain to be defined. Moreover, no reciprocal chromosome painting in birds has been performed due to the lack of chromosome-specific probes from other avian species. Here we have generated a set of chromosome-specific paints using flow cytometry that cover the whole genome of the stone curlew (Burhinus oedicnemus, Charadriiformes), a species with one of the lowest diploid number so far reported in birds, as well as paints from more microchromosomes of the chicken. A genome-wide comparative map between the chicken and the stone curlew has been constructed for the first time based on reciprocal chromosome painting. The results indicate that extensive chromosome fusions underlie the sharp decrease in the diploid number in the stone curlew. To a lesser extent, chromosome fissions and inversions occurred also during the evolution of the stone curlew. It is anticipated that this complete set of chromosome painting probes from the first Neoaves species will become an invaluable tool for avian comparative cytogenetics.     

Comparative architectures of mammalian and chicken genomes reveal highly variable rates of genomic rearrangements across different lineages

Molecular evolution studies are usually based on the analysis of individual genes and thus reflect only small-range variations in genomic sequences. A complementary approach is to study the evolutionary history of rearrangements in entire genomes based on the analysis of gene orders. The progress in whole genome sequencing provides an unprecedented level of detailed sequence data to infer genome rearrangements through comparative approaches. The comparative analysis of recently sequenced rodent genomes with the human genome revealed evidence for a larger number of rearrangements than previously thought and led to the reconstruction of the putative genomic architecture of the murid rodent ancestor, while the architecture of the ancestral mammalian genome and the rate of rearrangements in the human lineage remained unknown. Sequencing the chicken genome provides an opportunity to reconstruct the architecture of the ancestral mammalian genome by using chicken as an outgroup. Our analysis reveals a very low rate of rearrangements and, in particular, interchromosomal rearrangements in chicken, in the early mammalian ancestor, or in both. The suggested number of interchromosomal rearrangements between the mammalian ancestor and chicken, during an estimated 500 million years of evolution, only slightly exceeds the number of interchromosomal rearrangements that happened in the mouse lineage, over the course of about 87 million years.     

Comparison of the chicken and turkey genomes reveals a higher rate of nucleotide divergence on microchromosomes than macrochromosomes

A distinctive feature of the avian genome is the large heterogeneity in the size of chromosomes, which are usually classified into a small number of macrochromosomes and numerous microchromosomes. These chromosome classes show characteristic differences in a number of interrelated features that could potentially affect the rate of sequence evolution, such as GC content, gene density, and recombination rate. We studied the effects of these factors by analyzing patterns of nucleotide substitution in two sets of chicken-turkey sequence alignments. First, in a set of 67 orthologous introns, divergence was significantly higher in microchromosomes (chromosomes 11-38; 11.7% divergence) than in both macrochromosomes (chromosomes 1-5; 9.9% divergence; P = 0.016) and intermediate-sized chromosomes (chromosomes 6-10; 9.5% divergence; P = 0.026). At least part of this difference was due to the higher incidence of CpG sites on microchromosomes. Second, using 155 orthologous coding sequences we noted a similar pattern, in which synonymous substitution rates on microchromosomes (13.1%) were significantly higher than were rates on macrochromosomes (10.3%; P = 0.024). Broadly assuming neutrality of introns and synonymous sites, or constraints on such sequences do not differ between chromosomal classes, these observations imply that microchromosomal genes are exposed to more germ line mutations than those on other chromosomes. We also find that dN/dS ratios for genes located on microchromosomes (average, 0.094) are significantly lower than those of macrochromosomes (average, 0.185; P = 0.025), suggesting that the proteins of genes on microchromosomes are under greater evolutionary constraint.     

Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates

Although several vertebrate genomes have been sequenced, little is known about the genome evolution of early vertebrates and how large-scale genomic changes such as the two rounds of whole-genome duplications (2R WGD) affected evolutionary complexity and novelty in vertebrates. Reconstructing the ancestral vertebrate genome is highly nontrivial because of the difficulty in identifying traces originating from the 2R WGD. To resolve this problem, we developed a novel method capable of pinning down remains of the 2R WGD in the human and medaka fish genomes using invertebrate tunicate and sea urchin genes to define ohnologs, i.e., paralogs produced by the 2R WGD. We validated the reconstruction using the chicken genome, which was not considered in the reconstruction step, and observed that many ancestral proto-chromosomes were retained in the chicken genome and had one-to-one correspondence to chicken microchromosomes, thereby confirming the reconstructed ancestral genomes. Our reconstruction revealed a contrast between the slow karyotype evolution after the second WGD and the rapid, lineage-specific genome reorganizations that occurred in the ancestral lineages of major taxonomic groups such as teleost fishes, amphibians, reptiles, and marsupials.     

cDNA-based gene mapping and GC3 profiling in the soft-shelled turtle suggest a chromosomal size-dependent GC bias shared by sauropsids

Mammalian and avian genomes comprise several classes of chromosomal segments that vary dramatically in GC-content. Especially in chicken, microchromosomes exhibit a higher GC-content and a higher gene density than macrochromosomes. To understand the evolutionary history of the intra-genome GC heterogeneity in amniotes, it is necessary to examine the equivalence of this GC heterogeneity at the nucleotide level between these animals including reptiles, from which birds diverged. We isolated cDNAs for 39 protein-coding genes from the Chinese soft-shelled turtle, Pelodiscus sinensis, and performed chromosome mapping of 31 genes. The GC-content of exonic third positions (GC₃) of P. sinensis genes showed a heterogeneous distribution, and exhibited a significant positive correlation with that of chicken and human orthologs, indicating that the last common ancestor of extant amniotes had already established a GC-compartmentalized genomic structure. Furthermore, chromosome mapping in P. sinensis revealed that microchromosomes tend to contain more GC-rich genes than GC-poor genes, as in chicken. These results illustrate two modes of genome evolution in amniotes: mammals elaborated the genomic configuration in which GC-rich and GC-poor regions coexist in individual chromosomes, whereas sauropsids (reptiles and birds) refined the chromosomal size-dependent GC compartmentalization in which GC-rich genomic fractions tend to be confined to microchromosomes.     

Karyotype evolution in monitor lizards: cross-species chromosome mapping of cDNA reveals highly conserved synteny and gene order in the Toxicofera clade

The water monitor lizard (Varanus salvator macromaculatus (VSA), Platynota) has a chromosome number of 2n?=?40: its karyotype consists of 16 macrochromosomes and 24 microchromosomes. To delineate the process of karyotype evolution in V. salvator macromaculatus, we constructed a cytogenetic map with 86 functional genes and compared it with those of the butterfly lizard (Leiolepis reevesii rubritaeniata (LRE); 2n?=?36) and Japanese four-striped rat snake (Elaphe quadrivirgata (EQU); 2n?=?36), members of the Toxicofera clade. The syntenies and gene orders of macrochromosomes were highly conserved between these species except for several chromosomal rearrangements: eight pairs of VSA macrochromosomes and/or chromosome arms exhibited homology with six pairs of LRE macrochromosomes and eight pairs of EQU macrochromosomes. Furthermore, the genes mapped to microchromosomes of three species were all located on chicken microchromosomes or chromosome 4p. No reciprocal translocations were found in the species, and their karyotypic differences were caused by: low frequencies of interchromosomal rearrangements, such as tandem fusions, or centric fissions/fusions between macrochromosomes and between macro- and microchromosomes; and intrachromosomal rearrangements, such as paracentric inversions or centromere repositioning. The chromosomal rearrangements that occurred in macrochromosomes of the Varanus lineage were also identified through comparative cytogenetic mapping of V. salvator macromaculatus and V. exanthematicus. Morphologic differences in chromosomes 6–8 between the two species could have resulted from pericentric inversion or centromere repositioning.     

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.     

Avian genomes: different karyotypes but a similar distribution of the GC-richest chromosome regions at interphase

The chicken karyotype, like that of the vast majority of avian species, shows a large number of dot-shaped microchromosomes that are characterized, like most telomeric regions of the macrochromosomes, by the highest GC levels and the highest gene densities. In interphase nuclei, these gene-dense regions are centrally located, and are characterized by an open chromatin structure (a similar situation also exists in mammals). Avian species belonging to the Accipitridae family (diurnal raptors) show a karyotype with no very large chromosomes, and with only a very small number of microchromosomes. To identify the GC-rich (and gene-rich) regions of the chromosomes and nuclei from Accipitridae, we performed heterologous in-situ hybridizations using chicken GC-richest isochores as probes. Our results clearly show that the gene-rich regions are prevalently located in the few microchromosome pairs and in the telomeric regions of the middle-sized chromosomes, as well as in the interior of the interphase nuclei. This result is consistent with a common organization of the genome in the nuclei of warm-blooded vertebrates. Indeed, in spite of the different size and morphology of the chromosomes, the gene-dense regions are always located in the interior of the nuclei.     

Chicken genome: current status and future opportunities

The chicken genome sequence is important for several reasons. First, the chicken shared a common ancestor with mammals approximately 310 million years ago (Mya) at a phylogenetic distance not previously covered by other genome sequences. It therefore fills a gap in our knowledge and understanding of the evolution and conservation of genes, regulatory sequences, genomes, and karyotypes. The chicken is also a major source of protein in the world, with billions of birds used in meat and egg production each year. It is the first livestock species to be sequenced and so leads the way for others. The sequence and the 2.8 million genetic polymorphisms defined in a parallel project are expected to benefit agriculture and cast new light on animal domestication. Also, as the first bird to be sequenced, it is a model for the 9600 avian species thought to exist today. Many of the features of the chicken genome and its biology make it an ideal organism for studies in development and evolution, along with applications in agriculture and medicine.     

Salmonella enterica serovar Gallinarum: addressing fundamental questions in bacteriology sixty years on from the 9R vaccine

Sixty years on from Smith’s seminal work on Fowl Typhoid vaccines, there is renewed interest in experimental avian salmonellosis and in particular the use of Salmonella enterica serovar Gallinarum as a tool to understand key features of bacterial evolution and host adaptation. In this short review we outline some of the recent advances in avian salmonellosis research that have coupled both the power of whole genome analysis and new tools to understand the host response to existing experimental infection models. These approaches are underpinning a fundamental understanding of Salmonella biology relevant to both the chicken and other avian and mammalian species.     

Characterization of chromosome structures of Falconinae (Falconidae, Falconiformes, Aves) by chromosome painting and delineation of chromosome rearrangements during their differentiation

Karyotypes of most bird species are characterized by around 2n = 80 chromosomes, comprising 7–10 pairs of large- and medium-sized macrochromosomes including sex chromosomes and numerous morphologically indistinguishable microchromosomes. The Falconinae of the Falconiformes has a different karyotype from the typical avian karyotype in low chromosome numbers, little size difference between macrochromosomes and a smaller number of microchromosomes. To characterize chromosome structures of Falconinae and to delineate the chromosome rearrangements that occurred in this subfamily, we conducted comparative chromosome painting with chicken chromosomes 1–9 and Z probes and microchromosome-specific probes, and chromosome mapping of the 18S–28S rRNA genes and telomeric (TTAGGG) _n sequences for common kestrel (Falco tinnunculus) (2n = 52), peregrine falcon (Falco peregrinus) (2n = 50) and merlin (Falco columbarius) (2n = 40). F. tinnunculus had the highest number of chromosomes and was considered to retain the ancestral karyotype of Falconinae; one and six centric fusions might have occurred in macrochromosomes of F. peregrinus and F. columbarius, respectively. Tandem fusions of microchromosomes to macrochromosomes and between microchromosomes were also frequently observed, and chromosomal locations of the rRNA genes ranged from two to seven pairs of chromosomes. These karyotypic features of Falconinae were relatively different from those of Accipitridae, indicating that the drastic chromosome rearrangements occurred independently in the lineages of Accipitridae and Falconinae.     

Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (<Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, Trionychidae)

A novel family of repetitive DNA sequences that are components of constitutive heterochromatin were cloned from BglI-digested genomic DNA of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae), and characterized by filter hybridization and chromosome in-situ hybridization. The BglI-family of repetitive sequences were classified into four types by their genome organization and chromosomal distribution as follows: the repeated sequences located on (1) two pairs of microchromosomes, (2) four pairs of microchromosomes,(3) about half the number of microchromosomes and (4) the interstitial region of the short arm of chromosome 2. The presence of microchromosome-specific repetitive sequences has also been reported in the Struthioniformes and Galliformes, suggesting that turtle chromosomes retain some similarity to the chromosome structure as well as the karyotypes of avian species     

Integration of the cytogenetic and physical maps of chicken chromosome 17

The chicken genome, like those of most avian species, contains numerous microchromosomes that cannot be distinguished by size alone. Unique properties attributed to the microchromosomes include high GC content and gene density, and an enhanced recombination rate. Previously, microchromosome GGA 17 was shown to align with the consensus genetic linkage group E41W17, and bacterial artificial chromosome (BAC) clones containing E41W17 markers were isolated and assigned on the physical BAC map as well as the recently assembled draft chicken genome sequence. For this study, these same BACS were utilized as probes for fluorescence in-situ hybridization (FISH) to develop the GGA 17 cytogenetic map. Here we detail the chromosome order of ten BAC DNAs, thereby deriving a cytogenetic map of GGA 17 that is simultaneously integrated with both the linkage map and genome sequence. The location of the FISH probes together with the morphological appearance of the chromosome suggested that GGA 17 is an acrocentric chromosome whose cytogenetic map orientation is reversed from that currently indicated by the linkage map and draft genome sequence. The reversed orientation and the centromere location of GGA 17 were confirmed experimentally by dual-colour FISH hybridization using terminal BACs and the centromere-specific CNM oligonucleotide as probes. An advantage of this cyto-genomic approach is the improved alignment of the sequence and linkage maps with cytogenetic features such as the centromere, telomeres, p and q arms, and staining patterns indicating GC versus AT content.     

Comparative genome analyses of Arabidopsis spp.: inferring chromosomal rearrangement events in the evolutionary history of A. thaliana

Comparative genome analysis is a powerful tool that can facilitate the reconstruction of the evolutionary history of the genomes of modern-day species. The model plant Arabidopsis thaliana with its n = 5 genome is thought to be derived from an ancestral n = 8 genome. Pairwise comparative genome analyses of A. thaliana with polyploid and diploid Brassicaceae species have suggested that rapid genome evolution, manifested by chromosomal rearrangements and duplications, characterizes the polyploid, but not the diploid, lineages of this family. In this study, we constructed a low-density genetic linkage map of Arabidopsis lyrata ssp. lyrata (A. l. lyrata; n = 8, diploid), the closest known relative of A. thaliana (MRCA approximately 5 Mya), using A. thaliana-specific markers that resolve into the expected eight linkage groups. We then performed comparative Bayesian analyses using raw mapping data from this study and from a Capsella study to infer the number and nature of rearrangements that distinguish the n = 8 genomes of A. l. lyrata and Capsella from the n = 5 genome of A. thaliana. We conclude that there is strong statistical support in favor of the parsimony scenarios of 10 major chromosomal rearrangements separating these n = 8 genomes from A. thaliana. These chromosomal rearrangement events contribute to a rate of chromosomal evolution higher than previously reported in this lineage. We infer that at least seven of these events, common to both sets of data, are responsible for the change in karyotype and underlie genome reduction in A. thaliana.     

Origin of amphibian and avian chromosomes by fission, fusion, and retention of ancestral chromosomes

Amphibian genomes differ greatly in DNA content and chromosome size, morphology, and number. Investigations of this diversity are needed to identify mechanisms that have shaped the evolution of vertebrate genomes. We used comparative mapping to investigate the organization of genes in the Mexican axolotl (Ambystoma mexicanum), a species that presents relatively few chromosomes (n = 14) and a gigantic genome (>20 pg/N). We show extensive conservation of synteny between Ambystoma, chicken, and human, and a positive correlation between the length of conserved segments and genome size. Ambystoma segments are estimated to be four to 51 times longer than homologous human and chicken segments. Strikingly, genes demarking the structures of 28 chicken chromosomes are ordered among linkage groups defining the Ambystoma genome, and we show that these same chromosomal segments are also conserved in a distantly related anuran amphibian (Xenopus tropicalis). Using linkage relationships from the amphibian maps, we predict that three chicken chromosomes originated by fusion, nine to 14 originated by fission, and 12-17 evolved directly from ancestral tetrapod chromosomes. We further show that some ancestral segments were fused prior to the divergence of salamanders and anurans, while others fused independently and randomly as chromosome numbers were reduced in lineages leading to Ambystoma and Xenopus. The maintenance of gene order relationships between chromosomal segments that have greatly expanded and contracted in salamander and chicken genomes, respectively, suggests selection to maintain synteny relationships and/or extremely low rates of chromosomal rearrangement. Overall, the results demonstrate the value of data from diverse, amphibian genomes in studies of vertebrate genome evolution.     

The avian Toll-Like receptor pathway—Subtle differences amidst general conformity

The Toll-Like receptor (TLR) pathway plays a core role in innate immunity and is maintained with remarkable consistency across all vertebrate species. Amidst this background of overall conservation, subtle differences in the components that make up this pathway may have important implications for species-specific defense against key pathogens. Here we employ a homology-based comparative method to characterize the TLR pathway in the recently sequenced chicken and zebra finch genomes, which represent two distantly related bird species. The key features of the TLR pathway are conserved in birds and mammals, although some clear differences exist. The TLR receptors show a pattern of gene duplication and gene loss in both avian species when compared to mammals. In particular, we observe avian specific duplication of both TLR1 and TLR2 as well and a recent duplication of the TLR7 gene in the zebra finch lineage. Both positive selection and gene conversion shape the evolution of the avian specific TLR2 genes. In addition, there are notable differences in the zebra finch repertoire of antimicrobial peptides (AMPs) when compared to those of the chicken. Bioinformatic analysis reveals no evidence of cathelicidins in the zebra finch genome but does identify a cluster of 12 novel defensins which map to the avian beta-defensin locus on chromosome 3. These findings contribute to the characterization of the differing immune response systems that have evolved in individual vertebrate species in response to their microbiological environment.     

Evolution of gene order in the genomes of two related yeast species.

Changes in gene order between the genomes of two related yeast species, Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum were studied. From the dataset of a previous low coverage sequencing of the S. bayanus var. uvarum genome, 35 different synteny breakpoints between neighboring genes and two cases of local gene inversion were characterized in detail. The number and the type of the chromosomal rearrangements that have led to these differences were identified. We show that evolution of gene order in the genomes of these two yeast species is driven mainly by gene duplication onto different chromosomes followed by differential loss of the repeated copies. In addition, local gene inversions also would result from a mechanism of gene duplication, but in an inverted orientation, followed by loss of the original copy. The identification of traces of anciently duplicated genes, called relics, show that the loss of duplicates is more frequently caused by the accumulation of numerous mutations in one of the two copies than by DNA deletion. Surprisingly, gross chromosomal rearrangements such as translocations have only a minor effect on gene order reshuffling as they account for <10% of the synteny breakpoints.     

Global patterns of apparent copy number variation in birds revealed by cross-species comparative genomic hybridization

There is a growing interest in copy number variation (CNV) and the recognition of its importance in phenotype, disease, adaptation and speciation. CNV data is usually ascertained by array-CGH within-species, but similar inter-species comparisons have also been made in primates, mice and domestic mammals. Here, we conducted a broad appraisal of putative cross-species CNVs in birds, 16 species in all, using the standard array-CGH approach. Using a chicken oligonucleotide microarray, we detected 790 apparent CNVs within 135 unique regions and developed a bioinformatic tool ‘CNV Analyser’ for analysing and visualising cross-species data sets. We successfully addressed four hypotheses as follows: (a) Cross-species CNVs (compared to chicken) are, as suggested from preliminary evidence, smaller and fewer in number than in mammals; this ‘dogma’ was rejected in the light of the new evidence. (b) CNVs in birds are likely to have a functional effect through an association with genes; a large proportion of detected regions (70 %) were indeed associated with genes (suggesting functional significance), however, not necessarily more so than in mammals. (c) There are more CNVs in birds with more rearranged karyotypes; this hypothesis was rejected. Indeed, Falco species contained fewer than most with relatively standard (chicken-like) karyotypes. (d) There are more CNVs per megabase on micro-chromosomes than macrochromosomes; this hypothesis was accepted. Indeed, in species with rearranged karyotypes characterised by chromosomal fusions, the fused former microchromosomes still ‘behaved’ as though they were their microchromosomal ancestors. Gene ontology analysis of CNVRs revealed enrichment in immune response and antigen presentation genes and five CNVRs were perfectly correlated with the unique loss of sexual dichromatism in one Galliformes species.     

Compositional mapping of chicken chromosomes and identification of the gene-richest regions

Compositional chromosomal mapping, namely the assessment of the GC level of chromosomal bands, led to the identification, in the human chromosomes, of the GC-richest H3⁺ bands and of the GC-poorest L1⁺ bands, which were so called on the basis of the isochore family predominantly present in the bands. The isochore organization of the avian genome is very similar to those of most mammals, the only difference being the presence of an additional, GC-richest, H4 isochore family. In contrast, the avian karyotypes are very different from those of mammals, being characterized, in most species, by few macrochromosomes and by a large number of microchromosomes. The compositional mapping of chicken mitotic and meiotic chromosomes by in-situ hybridization of isochore families showed that the chicken GC-richest isochores are localized not only on a large number of microchromosomes but also on almost all telomeric bands of macrochromosomes. On the other hand, the GC-poorest isochores are generally localized on the internal regions of macrochromosomes and are almost absent in microchromosomes. Thus, the distinct localization of the GC-richest and the GC-poorest bands observed on human chromosomes appears to be a general feature of chromosomes from warm-blooded vertebrates.