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.     

Chromosomal localization of seven HSA3q13-->q23 NotI linking clones on chicken microchromosomes: orthology of GGA14 and GGA15 to a gene-rich region of HSA3

Double-color fluorescence in situ hybridization was performed on chicken chromosomes using seven unique clones from the human chromosome 3-specific NotI linking libraries. Six of them (NL1-097, NL2-092, NL2-230, NLM-007, NLM-118, and NLM-196) were located on the same chicken microchromosome and NL1-290 on another. Two chicken microchromosome GGA15-specific BAC clones, JE024F14 containing the IGVPS gene and JE020G17 containing the ALDH1A1 gene, were cytogenetically mapped to the same microchromosome that carried the six NotI linking clones, allowing identification of this chromosome as GGA15. Two GGA14-specific clones, JE027C23 and JE014E08 containing the HBA gene cluster, were co-localized on the same microchromosome as NL1-290, suggesting that this chromosome was GGA14. The results indicated that the human chromosomal region HSA3q13-->q23 is likely to be orthologous to GGA15 and GGA14. The breakpoint of evolutionary conservation of human and chicken chromosomes was detected on HSA3q13.3-->q23 between NL1-290, on the one hand, and six other NotI clones, on the other hand. Considering the available chicken-human comparative mapping data, another breakpoint appears to exist between the above NotI loci and four other genes, TFRC, EIF4A2, SKIL and DHX36 located on HSA3q24-->qter and GGA9. Based on human sequences within the NotI clones, localization of the six new chicken coding sequences orthologous to the human/rodent genes was suggested to be on GGA15 and one on GGA14. Microchromosomal location of seven NotI clones from the HSA3q21 T-band region can be considered as evidence in support of our hypothesis about the functional analogy of mammalian T-bands and avian microchromosomes.     

Comparative localization of chicken five functional genes and three microsatellites on quail mitotic chromosomes by two-color FISH hybridization

To localize chicken genes and microsatellites, we used heterologous two-color FISH and chicken chromosome specific BAC clones. All BAC clones were verified by PCR. An analysis of the results has shown that maf gene forms one linkage group with the mc1r gene (CJA11), aldh1a1 forms one linkage group with the igvps gene (CJA15), pno forms one linkage group with the acaca gene (CJA19), fzf forms one linkage group with the bmp7 gene (CJA20), and cw01 forms one linkage group with the ubap2w gene (CJAW). Microsatellite ADL0254 was localized jointly with the insr gene (CJA28), and LEI0342 and MCW0330 microsatellites were localized jointly with the hspa5 gene (CJA17). If we consider that the nomenclature of quail chromosomes is the same as in chickens, their localization will correspond to the following chromosomes: CJA11 (maf), 15 (aldh1a1), 19 (pno), 20 (fzf), and W (cw01). The microsatellite ADL0254 turned out to be located on the same microchromosome as the insr gene (CJA 28), while microsatellites LEI0342 and MCW0330 were found to be in the same linkage group with the hspa5 gene (CJA17). The same work was also carried out on the chicken genome. Different results were obtained. The localization of the BAC clones containing the cw01 and fzf genes and the MCW0330 microsatellite was confirmed completely; they are located on GGAW, 20, and 17 chromosomes, respectively. Microsatellites ADL0254 and LEI0342 were each revealed to have two sites, whereas the localization of the remaining genes (maf, aldh1a1, and pno) on the GGA11, GGA15, and GGA19 chromosomes turned out to be untrue and needs further study.     

Investigation of pseudoautosomal and bordering regions in avian Z and W chromosomes with the use of large insert genomic BAC clones

To study pseudoautosomal and bordering regions in the avian Z and W chromosomes, we used seven BAC clones from genomic libraries as DNA probes of fragments of different gametologs of the ATP5A1 gene located close to the proximal border of the pseudoautosomal region (PAR) of sex chromosomes of domestic chicken and Japanese quail. Localization of BAC clones TAM31-b100C09, TAM31-b99N01, TAM31-b27P16, and TAM31-b95L18 in the short arm of Z chromosomes of domestic chicken and Japanese quail (region Zp23-p22) and localization of the BAC clones CHORI-261-CH46G16, CHORI-261-CH33F10, and CHORI-261-CH64F22 on W chromosomes of these species and in the short arm of Z chromosomes (region Zp23-p22) were determined by fluorescence in situ hybridization with the use of W-specific probes. The difference in the localization of the BAC clones on the Z and W chromosomes is probably explained by divergence of the nucleotide sequences of different sex chromosomes located beyond the pseudoautosomal region.     

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.     

Assignment of linkage groups to turkey chromosome 1 (MGA1)

Previous genetic mapping identified three linkage groups (M1, M18 and M26) in the turkey corresponding to chicken chromosome 1 (GGA1). This is inconsistent with previously described chromosomal differences between these species. FISH analysis of BAC clones corresponding to microsatellite markers from each of the three turkey linkage groups, assigned all three linkage groups to a single chromosome (MGA1).     

Characterization of a new repetitive sequence that is enriched on microchromosomes of turkey

We cloned and characterized a new highly repetitive, species-specific DNA sequence from turkey (Meleagris gallopavo). This repeat family, which accounts for approximately 5% of the turkey genome, consists of a 41 bp repeated element that is present in tandem arrays longer than 23 kb. In situ hybridization to turkey metaphase chromosomes (2n=80) demonstrated that this sequence was located primarily on certain microchromosomes: approximately one-third of the 66 microchromosomes showed a positive signal. With respect to the macrochromosomes, hybridization was seen only in a pericentric position on nos. 2 and 3. The turkey microchromosome (TM) sequence shares motifs (alternating A_3–5 and T_3–5 clusters separated by 6–8 bp) that have been found previously in other avian tandemly repeated elements, e.g. a chicken microchromosome sequence, and W (female) chromosome-specific sequences of chicken and turkey. However, the TM sequence does not cross-hybridize under moderately stringent conditions with these other sequence. The spread and amplification of related repetitive sequence elements on microchromosomes and W chromosomes is discussed.by E.R. Schmidt     

A genetic and cytogenetic map for the duck (Anas platyrhynchos)

A genetic linkage map for the duck (Anas platyrhynchos) was developed within a cross between two extreme Peking duck lines by linkage analysis of 155 polymorphic microsatellite markers, including 84 novel markers reported in this study. A total of 115 microsatellite markers were placed into 19 linkage groups. The sex-averaged map spans 1353.3 cM, with an average interval distance of 15.04 cM. The male map covers 1415 cM, whereas the female map covers only 1387.6 cM. All of the flanking sequences of the 155 polymorphic loci--44 monomorphic loci and a further 41 reported microsatellite loci for duck--were blasted against the chicken genomic sequence, and corresponding orthologs were found for 49. To integrate the genetic and cytogenetic map of the duck genome, 28 BAC clones were screened from a chicken BAC library using the specific PCR primers and localized to duck chromosomes by FISH, respectively. Of 28 BAC clones, 24 were detected definitely on duck chromosomes. Thus, 11 of 19 linkage groups were localized to 10 duck chromosomes. This genetic and cytogenetic map will be helpful for the mapping QTL in duck for breeding applications and for conducting genomic comparisons between chicken and duck.     

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.     

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 chicken linkage map based on microsatellite markers genotyped on a Japanese Large Game and White Leghorn cross

A detailed linkage map is necessary for efficient detection of quantitative trait loci (QTL) in chicken resource populations. In this study, microsatellite markers isolated from a (CA)n-enriched library (designated as ABR Markers) were mapped using a population developed from a cross between Japanese Game and White Leghorn chickens. In total, 296 markers including 193 ABR, 43 MCW, 31 ADL, 22 LEI, 3 HUJ, 2 GCT, 1 UMA and 1 ROS were mapped by linkage to chicken chromosomes 1-14, 17-21, 23, 24, 26-28 and Z. In addition, five markers were assigned to the map based on the chicken draft genomic sequence, bringing the total number of markers on the map to 301. The resulting linkage map will contribute to QTL mapping in chicken.     

Development of one set of chromosome-specific microsatellite-containing BACs and their physical mapping in Gossypium hirsutum L.

Fluorescence in situ hybridization (FISH), using bacterial artificial chromosome (BAC) clone as probe, is a reliable cytological technique for chromosome identification. It has been used in many plants, especially in those containing numerous small chromosomes. We previously developed eight chromosome-specific BAC clones from tetraploid cotton, which were used as excellent cytological markers for chromosomes identification. Here, we isolated the other chromosome-specific BAC clones to make a complete set for the identification of all 26 chromosome-pairs by this technology in tetraploid cotton (Gossypium hirsutum L.). This set of BAC markers was demonstrated to be useful to assign each chromosome to a genetic linkage group unambiguously. In addition, these BAC clones also served as convenient and reliable landmarks for establishing physical linkage with unknown targeted sequences. Moreover, one BAC containing an EST, with high sequence similarity to a G. hirsutum ethylene-responsive element-binding factor was located physically on the long arm of chromosome A7 with the help of a chromosome-A7-specific BAC FISH marker. Comparative analysis of physical marker positions in the chromosomes by BAC-FISH and genetic linkage maps demonstrated that most of the 26 BAC clones were localized close to or at the ends of their respective chromosomes, and indicated that the recombination active regions of cotton chromosomes are primarily located in the distal regions. This technology also enables us to make associations between chromosomes and their genetic linkage groups and re-assign each chromosome according to the corresponding genetic linkage group. This BAC clones and BAC-FISH technology will be useful for us to evaluate grossly the degree to which a linkage map provides adequate coverage for developing a saturated genetic map, and provides a powerful resource for cotton genomic researches.     

Two-dimensional screening of the Wageningen chicken BAC library

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies. Received: 26 October 1999 / Accepted: 6 January 2000     

A comparative map of chicken chromosome 24 and human chromosome 11

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.     

Physical anchorage and orientation of equine linkage groups by FISH mapping BAC clones containing microsatellite markers

A horse bacterial artificial chromosome (BAC) library was screened for 19 microsatellite markers from unassigned or non-oriented linkage groups. Clones containing 11 (AHT20, EB2E8, HMS45, LEX005, LEX014, LEX023, LEX044, TKY111, UCDEQ425, UCDEQ464 and VIASH21) of these were found, which were from eight different linkage groups. The BAC clones were used as probes in dual colour FISH to identify their precise chromosomal origin. The microsatellite markers are located on nine different horse chromosomes, four of which (ECA6, ECA25, ECA27 and ECA28) had no previously in situ assigned markers.     

Candidate gene analysis of GH1 for effects on growth and carcass composition of cattle

We present an approach to evaluate the support for candidate genes as quantitative trait loci (QTLs) within the context of genome-wide map-based cloning strategies. To establish candidacy, a bacterial artificial chromosome (BAC) clone containing a putative candidate gene is physically assigned to an anchored linkage map to localise the gone relative to an identified QTL effect. Microsatellite loci derived from BAC clones containing an established candidate gone are integrated into the linkage map facilitating the evaluation by interval analysis of the statistical support for QTL identity. Permutation analysis is employed to determine experiment-wise statistical support. The approach is illustrated for the growth hormone 1 ( GH1 ) gene and growth and carcass phenotypes in cattle. Polymerase chain reaction (PCR) primers which amplify a 441 bp fragment of GH1 were used to systematically screen a bovine BAC library comprising 60 000 clones and with a 95% probability of containing a single copy sequence. The presence of GH1 in BAC-110R2C3 was confirmed by sequence analysis of the PCR product from this clone and by the physical assignment of BAC110R2C3 to bovine chromosome 19 (BTA19) band 22 by fluorescence in situ hybridisation (FISH). Microsatellite KHGH1 was isolated from BAC110R2C3 and scored in 529 reciprocal backcross and F₂ fullsib progeny from 41 resource families derived from Angus ( Bos taurus ) and Brahman ( Bos indicus ). The microsatellite KHGH1 was incorporated into a framework genetic map of BTA19 comprising 12 microsatellite loci, the erythrocyte antigen T and a GH1-TaqI restriction fragment length polymorphism (RFLP). Interval analysis localised effects of taurus vs. indicus alleles on subcutaneous fat and the percentage of ether extractable fat from the longissimus dorsi muscle to the region of BTA19 harbouring GH1 .     

Isolation, characterization and FISH assignments of horse BAC clones containing type I and II markers

In order to increase the number of markers on the horse cytogenetic map and expand the integration with the linkage map, an equine BAC library was screened for genes and for microsatellites. Eighty-nine intra-exon primers were designed from consensus gene sequences in documented species. After PCR screening, 38 clones containing identified genes were isolated and FISH mapped. These data allowed us to refine the available Zoo-FISH results, to define ten new conserved cytogenetic segments and expand two others, thus leading to the identification of a total of 26 conserved segments between horse and human. Interestingly, a new homeology segment was detected between ECA6p and HSA2q. Screening BAC clones for dinucleotide repeats led to the isolation of 33 microsatellites. Ten of the clones each contained at least a polymorphic microsatellite and one specific gene. The success of the approach in the production of integrative anchor loci and their potential use in localization and analysis of traits of interest by the candidate gene and positional cloning approach, are discussed.     

Integration of chicken genomic resources to enable whole-genome sequencing

Different genomic resources in chicken were integrated through the Wageningen chicken BAC library. First, a BAC anchor map was created by screening this library with two sets of markers: microsatellite markers from the consensus linkage map and markers created from BAC end sequencing in chromosome walking experiments. Second, HINdIII digestion fingerprints were created for all BACs of the Wageningen chicken BAC library. Third, cytogenetic positions of BACs were assigned by FISH. These integrated resources will facilitate further chromosome-walking experiments and whole-genome sequencing.     

Comparative map between chicken Chromosome 15 and human chromosomal region 12q24 and 22q11-q12

The physical and comparative map of GGA15 was improved by the construction of 9 BAC contigs around loci previously mapped on GGA15 by linkage analysis. In total, 240 BAC clones were isolated, covering 30–35% of GGA15, and 120 STS were developed (104 STS derived from BAC end sequences and 18 STS derived within genes). Seventeen chicken orthologues of human genes located on human Chr 22q11-q12 were directly mapped within BAC contigs of GGA15. Furthermore, the partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases and revealed matches to 26 genes, ESTs, and genomic clones located on HSA22q11-q12 and HSA12q24. These results provide a better alignment of GGA15 with the corresponding regions in human and mouse, and improve our knowledge of the evolution and dynamics of the vertebrate genome. GenBank Accession Numbers: The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers BZ592394-BZ592544.     

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.