Association and in silico assignment of sequences from turkey BACs

Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.     

Association and in Silico Assignment of Sequences from Turkey BACs

Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.     

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.     

Sponge cytogenetics - mitotic chromosomes of ten species of freshwater sponge

Porifera (sponges) are the most basal phylum of extant metazoans. To gain insight into sponge genome construction, cytogenetic analysis was performed for ten freshwater sponge species of six genera, using conventional Giemsa staining, chromosome banding, and fluorescence in-situ hybridization. The karyotypes were very similar among the ten species, exhibiting a diploid chromosome number of 2n=46 or 48, and usually consisted of microchromosomes with one or two pairs of large chromosomes. The 18S-28S rRNA genes were localized to a single pair of microchromosomes in two Ephydatia species. Hybridization signals of the telomere (TTAGGG)n sequences were observed at the ends of metaphase chromosomes. The genome sizes of Ephydatia fluviatilis and Ephydatia muelleri were estimated by flow cytometric analysis as about 0.7 pg per diploid complement. These freshwater sponge species appear to represent a fairly homogeneous group with respect to karyotypes.     

Chromosome rearrangements between chicken and guinea fowl defined by comparative chromosome painting and FISH mapping of DNA clones

Chromosome homology between chicken (Gallus gallus) and guinea fowl (Numida meleagris) was investigated by comparative chromosome painting with chicken whole chromosome paints for chromosomes 1-9 and Z and by comparative mapping of 38 macrochromosome-specific (chromosomes 1-8 and Z) and 30 microchromosome-specific chicken cosmid DNA clones. The comparative chromosome analysis revealed that the homology of macrochromosomes is highly conserved between the two species except for two inter-chromosomal rearrangements. Guinea fowl chromosome 4 represented the centric fusion of chicken chromosome 9 with the q arm of chicken chromosome 4. Guinea fowl chromosome 5 resulted from the fusion of chicken chromosomes 6 and 7. A pericentric inversion was found in guinea fowl chromosome 7, which corresponded to chicken chromosome 8. All the chicken microchromosome-specific DNA clones were also localized to microchromosomes of guinea fowl except for several clones localized to the short arm of chromosome 4. These results suggest that the cytogenetic genome organization is highly conserved between chicken and guinea fowl.     

Simultaneous painting of three genomes in hexaploid wheat by BAC-FISH.

Fluorescence in situ hybridization (FISH) is widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts, making them amenable for FISH mapping. In our BAC-FISH experiments, we selected 56 restriction fragment length polymorphism (RFLP)-locus-specific BAC clones from the libraries of Triticum monococcum and Aegilops tauschii, which are the A- and D-genome donors of wheat (Triticum aestivum, 2n = 6x = 42), respectively. The BAC clone 676D4 from the T. monococcum library contains a dispersed repeat that preferentially hybridizes to A-genome chromosomes, and two BAC clones, 9I10 and 9M13, from the Ae. tauschii library contain a dispersed repeat that preferentially hybridizes to the D-genome chromosomes. These repeats are useful in simultaneously discriminating the three different genomes in hexaploid wheat, and in identifying intergenomic translocations in wheat or between wheat and alien chromosomes. Sequencing results show that both of these repeats are transposable elements, indicating the importance of transposable elements, especially retrotransposons, in the genome evolution of wheat.     

Characterization of the turkey MHC chromosome through genetic and physical mapping

Previous studies in the chicken have identified a single microchromosome (GGA16) containing the ribosomal DNA (rDNA) and two genetically unlinked MHC regions, MHC-B and MHC-Y. Chicken DNA sequence from these loci was used to develop PCR primers for amplification of homologous fragments from the turkey (Meleagris gallopavo). PCR products were sequenced and overgo probes were designed to screen the CHORI 260 turkey BAC library. BAC clones corresponding to the turkey rDNA, MHC-B and MHC-Y were identified. BAC end and subclone sequencing confirmed identity and homology of the turkey BAC clones to the respective chicken loci. Based on subclone sequences, single-nucleotide polymorphisms (SNPs) segregating within the UMN/NTBF mapping population were identified and genotyped. Analysis of SNP genotypes found the B and Y to be genetically unlinked in the turkey. Silver staining of metaphase chromosomes identified a single pair of microchromosomes with nucleolar organizer regions (NORs). Physical locations of the rDNA and MHC loci were determined by fluorescence in situ hybridization (FISH) of the BAC clones to metaphase chromosomes. FISH clearly positioned the rDNA distal to the Y locus on the q-arm of the MHC chromosome and the MHC-B on the p-arm. An internal telomere array on the MHC chromosome separates the B and Y loci.     

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.     

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.     

Integration of chicken cytogenetic and genetic maps: 18 new polymorphic markers isolated from BAC and PAC clones

As an approach to integrate the chicken genetic and cytogenetic maps, bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones were localized by fluorescence in situ hybridization (FISH) on chromosomes and by genetic mapping on the East Lansing and Compton reference families. Some of the clones used in this study were previously selected for the presence of potentially polymorphic (CA)n repeats and a microsatellite marker was developed when possible for genetic mapping. For other clones, a single strand conformational polymorphism (SSCP) was developed and used for this purpose. Between the two approaches, 18 markers linking the cytogenetic and genetic maps, seven on macrochromosomes and 11 on microchromosomes, were generated. Our results enabled the assignment and orientation of a linkage group to chromosome 3, together with the assignment of linkage groups to eight different microchromosomes, a fraction of the genome lacking mapping data and for which the degree of coverage by the genetic map was not well estimated previously.     

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.     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

DNA repeat arrays in chicken and human genomes and the adaptive evolution of avian genome size

Background

Birds have smaller average genome sizes than other tetrapod classes, and it has been proposed that a relatively low frequency of repeating DNA is one factor in reduction of avian genome sizes.

Results

DNA repeat arrays in the sequenced portion of the chicken (Gallus gallus) autosomes were quantified and compared with those in human autosomes. In the chicken 10.3% of the genome was occupied by DNA repeats, in contrast to 44.9% in human. In the chicken, the percentage of a chromosome occupied by repeats was positively correlated with chromosome length, but even the largest chicken chromosomes had repeat densities much lower than those in human, indicating that avoidance of repeats in the chicken is not confined to minichromosomes. When 294 simple sequence repeat types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human.

Conclusions

The fact that the chicken simple sequence repeat arrays were consistently smaller than arrays of the same type in human is evidence that the reduction in repeat array length in the chicken has involved numerous independent evolutionary events. This implies that reduction of DNA repeats in birds is the result of adaptive evolution. Reduction of DNA repeats on minichromosomes may be an adaptation to permit chiasma formation and alignment of small chromosomes. However, the fact that repeat array lengths are consistently reduced on the largest chicken chromosomes supports the hypothesis that other selective factors are at work, presumably related to the reduction of cell size and consequent advantages for the energetic demands of flight.     

Karyotype study of 5 species of Costa Rican snakes of the colubrid family

The karyotypes of five species of colubrid snakes from Costa Rica are as follows: Imantodes cenchoa and Drymobius margaritiferus have a diploid number of 36, with 16 macro- and 20 microchromosomes. The fourth pair is heteromorphic in females of I. cenchoa, with a metacentric Z and a submetacentric W chromosomes. Karyotypes of Erythrolampius bizonus and Leimadophis epinephalus have 28 chromosomes, without a clearcut separation between macro- and microchromosomes. In the case of E. bizomus, the fourth pair contains the sex chromosomes Z and W, both are submetacentric, but the W is smaller. Xenedon rabdocephalus has a diploid number of 34 chromosomes (22 macro- and 12 microchromosomes); pair 3 is heteromorphic in females, with a submetacentric Z and a smaller metacentric W. The karyotype of X. rabdocephalus may be derived from a primitive karyotype by means of reduction in the number of microchromosomes and centric fissions of two pairs of metacentric autosomes.     

Reassignment of chicken W chromosome sequences to the Z chromosome by fluorescence in situ hybridization (FISH)

There is much interest in the gene content of the small heterochromatic W chromosome of the chicken, on the supposition that it may contain sex-determining genes. A considerable region in the chicken genome has been assigned to the W chromosome on the basis of its repetitive sequences. Using fluorescent in situ hybridization (FISH) we localized five Bacterial Artificial Chromosomes (BACs) onto female chicken metaphase spreads. We physically mapped these BACs to the Z chromosome. The chicken genome database, however, assigned all five BACs to the W chromosome. Our results demonstrate that the 17 genes on these BACs are Z-specific, and points to the inadequacy of assigning regions of the genome based exclusively on repetitive sequences.     

Identification of a homeologous chromosome pair by in situ DNA hybridization to ribosomal RNA loci in meiotic chromosomes of cotton (Gossypium hirsutum).

In situ DNA hybridization with 18S-28S and 5S ribosomal DNA probes was used to map 18S-28S nucleolar organizers and tandem 5S repeats to meiotic chromosomes of cotton (Gossypium hirsutum L.). Mapping was performed by correlating hybridization sites to particular positions in translocation quadrivalents. Arm assignment required translocation quadrivalents with at least one interstitial chiasma and sufficient distance between the hybridization site and the centromere. We had previously localized a major 18S-28S site to the short arm of chromosome 9; here we mapped two additional major 18S-28S sites to the short arm of chromosome 16 and the left arm of chromosome 23. We also identified and mapped a minor 18S-28S site to the short arm of chromosome 7. Two 5S sites of unequal size were identified, the larger one near the centromere of chromosome 9 and the smaller one near the centromere of chromosome 23. Synteny of 5S and 18S-28S sites indicated homeology of chromosomes 9 and 23, while positions of the other two 18S-28S sites supplement genetic evidence that chromosomes 7 and 16 are homeologous.     

A comparative karyological study of the blue-breasted quail (Coturnix chinensis, Phasianidae) and California quail (Callipepla californica, Odontophoridae)

We conducted comparative chromosome painting and chromosome mapping with chicken DNA probes against the blue-breasted quail (Coturnix chinensis, CCH) and California quail (Callipepla californica, CCA), which are classified into the Old World quail and the New World quail, respectively. Each chicken probe of chromosomes 1-9 and Z painted a pair of chromosomes in the blue-breasted quail. In California quail, chicken chromosome 2 probe painted chromosomes 3 and 6, and chicken chromosome 4 probe painted chromosomes 4 and a pair of microchromosomes. Comparison of the cytogenetic maps of the two quail species with those of chicken and Japanese quail revealed that there are several intrachromosomal rearrangements, pericentric and/or paracentric inversions, in chromosomes 1, 2 and 4 between chicken and the Old World quail. In addition, a pericentric inversion was found in chromosome 8 between chicken and the three quail species. Ordering of the Z-linked DNA clones revealed the presence of multiple rearrangements in the Z chromosomes of the three quail species. Comparing these results with the molecular phylogeny of Galliformes species, it was also cytogenetically supported that the New World quail is classified into a different clade from the lineage containing chicken and the Old World quail.     

Cytogenetic analysis of California condor (Gymnogyps californianus) chromosomes: comparison with chicken (Gallus gallus) macrochromosomes

The California condor is the largest flying bird in North America and belongs to a group of New World vultures. Recovering from a near fatal population decline, and currently with only 197 extant individuals, the species remains listed as endangered. Very little genetic information exists for this species, although sexing methods employing chromosome analysis or W-chromosome specific amplification is routinely applied for the management of this monomorphic species. Keeping in mind that genetic conditions like chondrodystrophy have been identified, preliminary steps were undertaken in this study to understand the genome organization of the condor. This included an extensive cytogenetic analysis that provided (i) a chromosome number of 80 (with a likelihood of an extra pair of microchromosomes), and (ii) information on the centromeres, telomeres and nucleolus organizer regions. Further, a comparison between condor and chicken macrochromosomes was obtained by using individual chicken chromosome specific paints 1-9 and Z and W on condor metaphase spreads. Except for chromosomes 4 and Z, each of the chicken (GGA) macrochromosomes painted a single condor (GCA) macrochromosome. GGA4 paint detected complete homology with two condor chromosomes, viz., GCA4 and GCA9 providing additional proof that the latter are ancestral chromosomes in the birds. The chicken Z chromosome showed correspondence with both Z and W in the condor. The homology suggests that the condor sex chromosomes have not completely differentiated during evolution, which is unlike the majority of the non-ratites studied up till now. Overall, the study provides detailed cytogenetic and basic comparative information on condor chromosomes. These findings significantly advance the effort to study the chondrodystrophy that is responsible for over ten percent mortality in the condor.     

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.