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Masabanda, Julio S.; Burt, Dave W.; O'Brien, Patricia C.M.; Vignal, Alain; Villon, Valerie; Walsh, Philippa S.; Cox, Helen; Tempest, Helen G.; Smith, Jacqueline; Habermann, Felix A.; Schmidt, Michael; Matsuda, Yoichi; Ferguson-Smith, Malcolm A.; Crooijmans, Richard P. M. A.; Groenen, Martien A. M.; Griffin, Darren K. (March 2004): Molecular cytogenetic definition of the chicken genome: the first complete avian karyotype. In: Genetics, Vol. 166, No. 3: pp. 1367-1373
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Abstract

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species. THE ability to karyotype an individual or species is fundamental for any genome-mapping effort as both genetic and physical maps are made with reference to chromosome position. A karyotype provides a wealth of information about the genetic makeup of an animal or cell line, e.g., about disease status, infertility, or tumorigenesis, and is, in effect, a low-resolution map of the whole genome. For most species, chromosomes can be distinguished relatively easily by either classical (e.g., G-banding) means or molecular cytogenetics. Birds (class Aves) are a notable exception to this because, typically, the diploid number is ∼80 and because birds have many cytologically indistinguishable microchromosomes. The majority of avian genomic studies focus on the chicken (Gallus domesticus) and the chicken genome-mapping project continues apace. The genetic map now contains ∼2000 loci within 50 linkage groups, and it covers ∼4000 cM (Emara and Kim 2003). Over 235 of these loci have homology with known human or mammalian genes. The number of chicken protein sequences deposited in the SwissProt and the TrEMBL databases is between 1000 and 2000 and >600,000 chicken expressed sequence tags are deposited in the dbEST database. Large numbers of chicken full-length cDNAs are already being sequenced and it has been predicted that the chicken has 35,000 genes in total. A significant barrier to the progress of the chicken genome project, however, has been the fact that the chromosomes have not hitherto been fully classified and thus a large number of genes remain without a chromosomal assignment. The chicken genome-mapping project is also developing a number of resources essential for the study of a range of scientific disciplines. DNA microarrays are being generated to study metabolic functions and immune responses (Minet al. 2003; Neimanet al. 2003) and to analyze global gene expression in target tissues of chickens (Cogburnet al. 2003). There are also projects to target gene function by disrupting and gaining functions with the use of RNAi methods (Hudsonet al. 2002; Pekariket al. 2003). The increase in these genomic resources, easy access to the large chick embryo, and the application of sophisticated means such as RNA interference and morpholinos provide unique tools for testing gene function in all vertebrates. A resource that has been unavailable thus far, however, is a set of unique chromosome identifier probes. Single nucleotide polymorphisms within chicken genes are being exploited for the generation of candidate genes for quantitative traits (Emara and Kim 2003). Chicken accounts for 20% of meat consumption and most egg consumption worldwide. There is consequently extensive research into >200 chicken quantitative trait loci encoding for disease susceptibility, immunology, leanness, egg production, etc. (Liuet al. 2001; Marianiet al. 2001; Tatsuda and Fujinaka 2001). Many highly inbred and recombinant inbred chicken lines have large, well-defined pedigrees; thus, chicken is a primary model for the study of quantitative inheritance in humans and other vertebrates (Jeurissenet al. 2000; Leduret al. 2000; Le Bihan-Duvalet al. 2001). Mapping of quantitative traits, however, requires a chromosomal assignment and this has not yet been possible for traits that map to the smaller microchromosomes. Chicken DT40 cell lines are avian-leukosis-virus-induced B cell lines that exhibit a high ratio of targeted to random integration of transfected DNA constructs at homologous loci (Dharet al. 2001). They are suitable as a model for recombination analysis in vertebrates and are being successfully used in gene disruption experiments (Winding and Berchtold 2001). A feature of DT40 cell lines, however, is that they have a high degree of chromosomal rearrangements that, to date, could not be karyotyped. Finally, there is widespread interest in comparative genomics of birds for both genome evolution studies and comparative mapping in commercial species (Burtet al. 1999; Shettyet al. 1999). In recent years several comparative mapping studies have focused on individual chicken macro- and microchromosomes (Crooijmanset al. 2001; Suchytaet al. 2001; Buitenhuiset al. 2002; Jennenet al. 2002), expanding and refining the previously described synteny information between chicken, human, and mouse. The use of cross-species chromosome painting is well established as a quick means of generating comparative genomic data between species and thus chromosome-specific probes from at least one avian species would further this work. Given this information, it is clear that the concerted effort to complete and publish the whole chicken genome sequence is a priority (Schmidet al. 2000; Burt and Pourquie 2003). This is imminent and will provide an important anchor species between fish and mammals. The ability to distinguish all chicken chromosomes (2n = 78) is a crucial step in this project as, without it, many genes cannot have proper assignments. Moreover, such a resource has a range of other applications. In this article therefore we describe the isolation of unique chromosome identifier probes for each chicken chromosome either by mapping of individual clones or by chromosome painting.