SNP mapping of QTL affecting growth and fatness on chicken GGA1

An F2 chicken population was established from a crossbreeding between a Xinghua line and a White Recessive Rock line. A total of 502 F2 chickens in 17 full-sib families from six hatches was obtained, and phenotypic data of 488 individuals were available for analysis. A total of 46 SNP on GGA1 was initially selected based on the average physical distance using the dbSNP database of NCBI. After the polymorphism levels in all F0 individuals (26 individuals) and part of the F1 individuals (22 individuals) were verified, 30 informative SNP were potentially available to genotype all F2 individuals. The linkage map was constructed using Cri-Map. Interval mapping QTL analyses were carried out. QTL for body weight (BW) of 35 d and 42 d, 49 d and 70 d were identified on GGA1 at 351–353 cM and 360 cM, respectively. QTL for abdominal fat weight was on GGA1 at 205 cM, and for abdominal fat rate at 221 cM. Two novel QTL for fat thickness under skin and fat width were detected at 265 cM and 72 cM, respectively.     

Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH

Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).     

High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6

Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.     

Chromosomal mapping of chicken mega-telomere arrays to GGA9, 16, 28 and W using a cytogenomic approach

Four mega-telomere loci were mapped to chicken chromosomes 9, 16, 28, and the W sex chromosome by dual-color fluorescence in situ hybridization using a telomeric sequence probe and BAC clones previously assigned to chicken chromosomes. The in-common features of the mega-telomere chromosomes are that microchromosomes are involved rather than macrochromosomes; in three cases (9, 16, 28) acrocentrics are involved with the mega-telomeres mapping to the p arms. Three of the four chromosomes (9, 16, W) encode tandem repeats which in two cases (9 and 16) involve the ribosomal DNA arrays (the 5S and 18S-5.8S-28S gene repeats, respectively). All involved chromosomes have a typical-sized telomere on the opposite terminus. Intra- and interindividual variation for mega-telomere distribution are discussed in terms of karyotype abnormalities and the potential for mitotic instability of some telomeres. The diversity and distribution of telomere array quantity in the chicken genome should be useful in contributing to research related to telomere length regulation - how and by what mechanism genomes and individual chromosomes establish and maintain distinct sets of telomere array sizes, as well as for future studies related to stability of the chicken genome affecting development, growth, cellular lifespan and disease. An additional impact of this study includes the listing of BAC clones (26 autosomal and six W BACs tested) that were cytogenetically verified; this set of BACs provide a useful tool for future cytogenetic analyses of the 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.     

Comparative FISH mapping on Z chromosomes of chicken and Japanese quail

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

Refined mapping of the three DNA repair genes, ERCC1, ERCC2, and XRCC1, on human chromosome 19

Three DNA repair genes, ERCC1, ERCC2, and XRCC1, have been regionally mapped on human chromosome 19. ERCC2 and XRCC1 have been assigned to bands q13.2----q13.3 by in situ hybridization using fluorescently-labeled cosmid probes. ERCC1 and ERCC2 have been found to be separated by less than 250 kb by large fragment restriction enzyme site mapping.     

Cytogenetic assignment of 29 functional genes to chicken microchromosomes by FISH

We assigned 29 functional genes to chicken microchromosomes by fluorescence in situ hybridization (FISH). Two linkage groups in the genetic linkage map of the East Lansing breed were identified in this study by localizing the genes AGRN and H2FA to microchromosomes. The frequency of the genes mapped on 30 pairs of microchromosomes, which account for roughly 30% of the whole chicken genome, was about 40% of the 73 genes randomly mapped in our laboratory. This result confirms the important role of microchromosomes for avian genome function and supports the likelihood of a high gene density on avian microchromosomes.     

Multiple phosphorylation events regulate the subcellular localization of GGA1

GGAs comprise a family of Arf-dependent coat proteins or adaptors that regulate vesicle traffic from the trans -Golgi network (TGN). GGAs bind activated Arf, cargo, and additional components necessary for vesicle budding through interactions with their four functional domains: VHS, GAT, hinge, and GAE. We identified three sites of phosphorylation in GGA1 by tandem mass spectrometry: S268 and T270 in the GAT domain and S480 in the hinge. Expression of HA-GGA1 in mammalian cells and comparison to endogenous GGA1 confirmed their localization to late Golgi compartments. In contrast, mutations that mimic the phosphoprotein (HA-GGA1[S268D] or HA-GGA1[T270D]) at either of the sites in the GAT domain caused a decrease in the colocalization with markers of the Golgi and TGN and an increase in puncta in cytoplasm. Quantitative comparisons of the extent of colocalization of GGA1 proteins with the known components of GGA1 vesicles revealed that the composition of those markers tested in HA-GGA1[S268D] and HA-GGA1[T270D] vesicles were indistinguishable from those of HA-GGA1 vesicles. We conclude that phosphorylation of the GAT domain can stabilize the coat proteins bound and thus regulate the rate of coat protein dissociation.     

Comparative FISH mapping of Gab1 and Gab2 genes in human, mouse and rat.

Gab1 and Gab2 are members of the Gab family which act as adapters for transmitting various signals in response to stimuli through cytokine and growth factor receptors, and T- and B-cell antigen receptors. We determined chromosome locations of the two genes in human, mouse and rat by fluorescence in situ hybridization. The Gab1 gene was localized to chromosome 4q31.1 in human, 8C3 in mouse and 19q11.1--> q11.2 in rat, and the Gab2 gene was located on chromosome 11q13.4-->q13.5 in human, 7E2 in mouse and 1q33.2-->q33.3 in rat. All human, mouse and rat Gab1 and Gab2 genes were localized to chromosome regions where conserved homology has been identified among the three species.     

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

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