Differences in gene density on chicken macrochromosomes and microchromosomes

The chicken karyotype comprises six pairs of large macrochromosomes and 33 pairs of smaller microchromosomes^1. Cytogenetic evidence suggests that microchromosomes may be more gene-dense than macrochromosomes. In this paper, we compare the gene densities on macrochromosomes and microchromosomes based on sequence sampling of cloned genomic DNA, and from the distribution of genes mapped by genetic linkage and physical mapping. From these different approaches we estimate that microchromosomes are twice as gene-dense as macrochromosomes and show that sequence sampling is an effective means of gene discovery in the chicken. Using this method we have also detected a conserved linkage between the genes for serotonin 1D receptor ( HTR1D ) and the platelet-activating factor receptor protein gene ( PTAFR ) on chicken chromosome 5 and human chromosome 1p34 ·3. Taken together with its advantages as an experimental animal, and public access to genetic and physical mapping resources, the chicken is a useful model genome for studies on the structure, function and evolution of the vertebrate genome.     

Origin and evolution of avian microchromosomes

The origin of avian microchromosomes has long been the subject of much speculation and debate. Microchromosomes are a universal characteristic of all avian species and many reptilian karyotypes. The typical avian karyotype contains about 40 pairs of chromosomes and usually 30 pairs of small to tiny microchromosomes. This characteristic karyotype probably evolved 100-250 million years ago. Once the microchromosomes were thought to be a non-essential component of the avian genome. Recent work has shown that even though these chromosomes represent only 25% of the genome; they encode 50% of the genes. Contrary to popular belief, microchromosomes are present in a wide range of vertebrate classes, spanning 400-450 million years of evolutionary history. In this paper, comparative gene mapping between the genomes of chicken, human, mouse and zebrafish, has been used to investigate the origin and evolution of avian microchromosomes during this period. This analysis reveals evidence for four ancient syntenies conserved in fish, birds and mammals for over 400 million years. More than half, if not all, microchromosomes may represent ancestral syntenies and at least ten avian microchromosomes are the product of chromosome fission. Birds have one of the smallest genomes of any terrestrial vertebrate. This is likely to be the product of an evolutionary process that minimizes the DNA content (mostly through the number of repeats) and maximizes the recombination rate of microchromosomes. Through this process the properties (GC content, DNA and repeat content, gene density and recombination rate) of microchromosomes and macrochromosomes have diverged to create distinct chromosome types. An ancestral genome for birds likely had a small genome, low in repeats and a karyotype with microchromosomes. A "Fission-Fusion Model" of microchromosome evolution based on chromosome rearrangement and minimization of repeat content is discussed.     

Localization of the NotI clones from human chromosome 3 on quail microchromosomes

For the purpose of comparative mapping of quail (Coturnix c. japonica) and human (Homo sapiens) genomes, DNA fragments from human chromosome 3 (HSA3p14-21 and HSA3q13-23) were localized on quail mitotic chromosomes. Using the method of double-color fluorescence DNA-DNA in situ hybridization, these fragments were mapped to two different microchromosomes. Earlier, similar studies were performed using chicken mitotic chromosomes. There it was demonstrated that the clones of interest were distributed among three microchromosomes (GGA12, GGA14, and GGA15). Thus, interspecific difference in the location of human chromosome 3 DNA fragments in the genomes of closely related avian species was discovered. A new confirmation of the hypothesis on the preferable localization of the gene-rich human chromosome regions on avian microchromosomes was obtained. At the same time, a suggestion on the localization of some orthologous genes in the genome of the organism under study was made: ARF4, SCN5A, PHF7, ABHD6, ZDHHC3, MAPKAPK3, ADSYNA (homolog of chicken chromosome 12), DRD2, PP2C-ETA, RAB7, CCKAR, and PKD1 (homolog of chicken chromosome 15). However, localization of the corresponding quail genes needs to be confirmed, as far as the sequences used were only the orthologs of the corresponding chicken genes.     

Mapping the beta NGF gene in situ to a microchromosome in chicken.

The chicken nerve growth factor (beta NGF) gene has been mapped by fluorescent in situ hybridization to a pair of microchromosomes, confirming previous reports of the existence of a single gene locus. A 39-kb genomic fragment cloned in a cosmid vector and including the 5' end of the beta NGF locus was biotinylated for nonradioactive detection of the gene. This report adds to the increasing evidence proving microchromosomal localization of highly conserved and biologically fundamental genes. The implications of such genes belonging to very small linkage groups for the transmission of alleles from generation to generation together with the relevance of nonisotopic in situ hybridization for avian gene mapping are considered.     

Comparative compositional mapping of chicken and quail chromosomes

The distribution of various isochore families on mitotic chromosomes of domestic chicken and Japanese quail was studied by the method of fluorescence in situ DNA--DNA hybridization (FISH). DNA of various isochore families was shown to be distributed irregularly and similarly on chromosomes of domestic chicken and Japanese quail. The GC-rich isochore families (H2, H3, and H4) hybridized mainly to microchromosomes and a majority of macrochromosome telomeric regions. In chicken, an intense fluorescence was also in a structural heterochromatin region of the Z chromosome long arm. In some regions of the quail macrochromosome arms, hybridization was also with isochore families H3 and H4. On macrochromosomes of both species, the pattern of hybridization with isochores of the H2 and H3 families resembled R-banding. The light isochores (L1 and L2 families) are mostly detected within macrochromosome internal regions corresponding to G bands, whereas microchromosomes lack light isochores. Although mammalian and avian karyotypes differ significantly in organization, the isochore distribution in genomes of these two lineages of the warm-blooded animals is similar in principle. On macrochromosomes of the two avian species studied, a pattern of isochore distribution resembled that of mammalian chromosomes. The main specific feature of the avian genome, a great number of microchromosomes (about 30% of the genome), determines a compositional specialization of the latter. This suggests the existence of not only structural but also functional compartmentalization of the avian genome.     

Inference of the Protokaryotypes of Amniotes and Tetrapods and the Evolutionary Processes of Microchromosomes from Comparative Gene Mapping

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.     

Comparative Compositional Mapping of Chicken and Quail Chromosomes

The distribution of various isochore families on mitotic chromosomes of domestic chicken and Japanese quail was studied by the method of fluorescence in situ DNA–DNA hybridization (FISH). DNA of various isochore families was shown to be distributed irregularly and similarly on chromosomes of domestic chicken and Japanese quail. The GC-rich isochore families (H2, H3, and H4) hybridized mainly to microchromosomes and a majority of macrochromosome telomeric regions. In chicken, an intense fluorescence was also in a structural heterochromatin region of the Z chromosome long arm. In some regions of the quail macrochromosome arms, hybridization was also with isochore families H3 and H4. On macrochromosomes of both species, the pattern of hybridization with isochores of the H2 and H3 families resembled R-banding. The light isochores (L1 and L2 families) are mostly detected within macrochromosome internal regions corresponding to G bands, whereas microchromosomes lack light isochores. Although mammalian and avian karyotypes differ significantly in organization, the isochore distribution in genomes of these two lineages of the warm-blooded animals is similar in principle. On macrochromosomes of the two avian species studied, a pattern of isochore distribution resembled that of mammalian chromosomes. The main specific feature of the avian genome, a great number of microchromosomes (about 30% of the genome), determines a compositional specialization of the latter. This suggests the existence of not only structural but also functional compartmentalization of the avian genome.     

Chromosomal localization of the <Emphasis Type="Italic">NotI</Emphasis> clones from human chromosome 3 on quail microchromosomes

For the purpose of comparative mapping of quail (Coturnix c. japonica) and human (Homo sapiens) genomes, DNA fragments from human chromosome 3 (HSA3p14-21 and HSA3q13-23) were localized on quail mitotic chromosomes. Using the method of double-color fluorescence DNA-DNA in situ hybridization, these fragments were mapped to two different microchromosomes. Earlier, similar studies were performed using chicken mitotic chromosomes. There it was demonstrated that the clones of interest were distributed among three microchromosomes (GGA12, GGA14, and GGA15). Thus, interspecific difference in the location of human chromosome 3 DNA fragments in the genomes of closely related avian species was discovered. A new confirmation of the hypothesis on the preferable localization of the gene-rich human chromosome regions on avian microchromosomes was obtained. At the same time, a suggestion on the localization of some orthologous genes in the genome of the organism under study was made: ARF4, SCN5A, PHF7, ABHD6, ZDHHC3, MAPKAPK3, ADSYNA (homolog of chicken chromosome 12), DRD2, PP2C-ETA, RAB7, CCKAR, and PKD1 (homolog of chicken chromosome 15). However, localization of the corresponding quail genes needs to be confirmed, as far as the sequences used were only the orthologs of the corresponding chicken genes.     

Microchromosomal assignment of the chicken ovotransferrin and adenylate kinase genes.

Ovotransferrin, an egg-white protein implicated in the transfer of trace elements from the hen oviduct to the developing avian embryo, and cytosolic adenylate kinase, an essential enzyme involved in the interconversion of adenine nucleotides in energetically active tissues, have been mapped to two separate chicken microchromosomes by fluorescent in situ hybridization. Considering present and previous data, the possibility of loss of intron material resulting in the compactation of genes in chicken microchromosomes is briefly discussed.     

Sequence scanning chicken cosmids: a methodology for genome screening

The chicken genome is relatively poorly studied at the molecular level. The karyotype 2n=78 is divided into three main chromosomal sub-groups: the macrochromosomes (six pairs), the intermediate microchromosomes (four pairs) and the microchromosomes (29 pairs). Whilst the microchromosome group comprise only 25% of the DNA, increasing evidence is proving that this is disproportionate to their gene content. This paper demonstrates the utility of cosmid sequence scanning as a potential method for analysing the chicken genome, providing an economical method for the production of a molecular map. The GC content, gene density and repeat distribution are analysed relative to chromosomal origin. Results indicate that gene density is higher on the microchromosomes. During the scanning process an example of conserved linkage between chicken and human (12q34.2) has been demonstrated.     

The avian genome uncovered

Our knowledge of avian genomics has increased rapidly over the past few years, culminating in the recent publication of a draft sequence of the chicken genome, a milestone event in avian genetics and evolutionary biology. Comparative analysis reveals a compact avian genome structure containing a similar number of genes as found in mammals but with shorter intergenic DNA sequences and fewer repeats. Recombination is at a higher rate than in mammals, particularly for microchromosomes. These also differ from macrochromosomes in their GC and gene content, and their substitution rate. The avian genome has remained unusually stable during evolution and contrasts sharply with the frequent chromosomal rearrangements seen in the rodent lineage. Detailed analyses of polymorphism levels in chickens, including a genome-wide screening in three chicken breeds yielding a set of 2.8 million SNP markers, reveal unexpectedly high levels of genetic diversity. As a notable exception, the female-specific W chromosome is very low in diversity, a probable consequence of the effect of selection on non-recombining chromosomes. The chicken genome promises to be a useful resource for ecological and evolutionary studies of other bird species.     

Dispersed chromosomal localization of the proto-oncogenes transduced into the genome of Mill Hill 2 or E26 leukemia virus.

Both Mill Hill 2 and E26 retroviruses have transduced two cellular genes--c-myc and c-mil/mht (Mill Hill 2) and c-myb and c-ets (E26). We localized the genes transduced by these viruses to different chromosomes: c-myc and c-myb to relatively large chromosomes and c-mil/mht and c-ets to microchromosomes. Thus, like avian erythroblastosis virus, each of these retroviruses has transduced two cellular genes unlinked in the chicken genome.     

A comparative cytogenetic study of chromosome homology between chicken and Japanese quail

In order to construct a chicken (Gallus gallus) cytogenetic map, we isolated 134 genomic DNA clones as new cytogenetic markers from a chicken cosmid DNA library, and mapped these clones to chicken chromosomes by fluorescence in situ hybridization. Forty-five and 89 out of 134 clones were localized to macrochromosomes and microchromosomes, respectively. The 45 clones, which localized to chicken macrochromosomes (Chromosomes 1-8 and the Z chromosome) were used for comparative mapping of Japanese quail (Coturnix japonica). The chromosome locations of the DNA clones and their gene orders in Japanese quail were quite similar to those of chicken, while Japanese quail differed from chicken in chromosomes 1, 2, 4 and 8. We specified the breakpoints of pericentric inversions in chromosomes 1 and 2 by adding mapping data of 13 functional genes using chicken cDNA clones. The presence of a pericentric inversion was also confirmed in chromosome 8. We speculate that more than two rearrangements are contained in the centromeric region of chromosome 4. All 30 clones that mapped to chicken microchromosomes also localized to Japanese quail microchromosomes, suggesting that chromosome homology is highly conserved between chicken and Japanese quail and that few chromosome rearrangements occurred in the evolution of the two species.     

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.     

In vivo - in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue

Background

By comparing the quail genome with that of chicken, chromosome rearrangements that have occurred in these two galliform species over 35 million years of evolution can be detected. From a more practical point of view, the definition of conserved syntenies helps to predict the position of genes in quail, based on information taken from the chicken sequence, thus enhancing the utility of this species in biological studies through a better knowledge of its genome structure. A microsatellite and an Amplified Fragment Length Polymorphism (AFLP) genetic map were previously published for quail, as well as comparative cytogenetic data with chicken for macrochromosomes. Quail genomics will benefit from the extension and the integration of these maps.

Results

The integrated linkage map presented here is based on segregation analysis of both anonymous markers and functional gene loci in 1,050 quail from three independent F2 populations. Ninety-two loci are resolved into 14 autosomal linkage groups and a Z chromosome-specific linkage group, aligned with the quail AFLP map. The size of linkage groups ranges from 7.8 cM to 274.8 cM. The total map distance covers 904.3 cM with an average spacing of 9.7 cM between loci. The coverage is not complete, as macrochromosome CJA08, the gonosome CJAW and 23 microchromosomes have no marker assigned yet. Significant sequence identities of quail markers with chicken enabled the alignment of the quail linkage groups on the chicken genome sequence assembly. This, together with interspecific Fluorescence In Situ Hybridization (FISH), revealed very high similarities in marker order between the two species for the eight macrochromosomes and the 14 microchromosomes studied.

Conclusion

Integrating the two microsatellite and the AFLP quail genetic maps greatly enhances the quality of the resulting information and will thus facilitate the identification of Quantitative Trait Loci (QTL). The alignment with the chicken chromosomes confirms the high conservation of gene order that was expected between the two species for macrochromosomes. By extending the comparative study to the microchromosomes, we suggest that a wealth of information can be mined in chicken, to be used for genome analyses in quail.     

A preliminary linkage map of the chicken genome.

We have used backcross progeny from a cross between two inbred lines of chickens to construct a linkage map of the chicken. The map currently consists of 100 loci, identified using either anonymous cloned fragments of genomic DNA or sequences corresponding to cloned genes. Parent birds were derived from two lines of White Leghorn chickens, which differ in susceptibility to a number of diseases. Restriction fragment length variants were identified by comparison of the DNA of these two parent birds using a panel of seven restriction enzyme digests and the segregation pattern observed in progeny of these two birds. Restriction fragment length variants were detected for approximately 41% of the clones tested, whether these were known genes or random genomic fragments. This high level of variability was also reflected in the presence of variation within the parental lines for some clones. The overall size of the linkage groups and the progressively higher incidence of linkage as further clones were added suggests that the map covers the majority of the genome, although it is unlikely that there are marker loci on all the microchromosomes. The present map will be of use in locating genes affecting disease resistance, but also illustrates the relative ease with which such maps for the chicken can be constructed.     

Microsatellites from the linkage groups E26C13 and E50C23 are located on the Gallus gallus domesticus microchromosomes 20 and 21

Using the method of dual color fluorescence in situ hybridization and a set of chromosome-specific BAC clones, localization of microsatellites LEI0345 and LEI0336 on chicken (Gallus gallus domesticus) mitotic chromosomes was performed. Microsatellite LEI0345 (TAM 32, BAC clones r49A10 and r55M23) from the linkage group E26C13 was mapped to microchromosome 20, while microsatellite LEI0336 (TAM 32, BAC clones r19E22 and r13C08) from the linkage E50C23 was assigned to microchromosome 21. Using the PCR technique, an attempt to assign the suitable markers to chromosome-specific BAC clones was made. The PCR data confirmed the microsatellite localization performed with the help of FISH technique and showed the presence of the LEI0345 microsatellite sequence on many other chicken microchromosomes, except for microchromosomes 19 and 22. Linkage groups E26C13 and E50C23 were assigned to microchromosomes 20 and 21, respectively.     

Localization of the muscular dystrophy AM locus using a chicken linkage map constructed with the Kobe University resource family

A chicken linkage map, constructed with the Kobe University (KU) resource family, was used to locate the genetic locus for muscular dystrophy of abnormal muscle type (AM). The KU resource family is a backcross pedigree with 55 offspring produced from the mating of a White Leghorn F-line (WL-F) male and a hybrid female produced from a cross between the WL-F male and a female of the Fayoumi OPN line who was homozygous for the AM gene. In total, 872 loci were genotyped on the pedigree; 749 (86%) were informative and mapped to 38 linkage groups. These informative loci included 649 AFLPs, 93 MS, three functional genes, the AM locus, sex phenotype, and two red blood cell loci. The remaining 123 markers were unlinked. Nineteen of the 38 KU linkage groups were assigned to macrochromosomes 1-8 and 11 microchromosomes including chromosome W, while 19 linkage groups were unassigned. The total map was 3569 cM in length, with an average marker interval of 4.8 cM. The AM locus was mapped 130 cM from the distal end of chromosome 2q.