Chromosome Research 7: 401406, 1999. # 1999 Kluwer Academic Publishers.

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The DAPI banded karyotype of the domestic dog (Canis familiaris) generated using chromosome-specic paint probes
Matthew Breen1 , Joern Bullerdiek2 & Cordelia F. Langford3 Animal Health Trust, Lanwades Park, Kentford, Suffolk, CB8 7UU, U.K.; Tel. (44) 1638-750659; Fax (44) 1638-750794. E-mail: mtbreen@hgmp.mrc.ac.uk; 2 Centre for Human Genetics and Genetic Counselling, University of Bremen, Bremen, Germany; 3 The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, U.K.
1

Received 3 June 1999; received in revised form and accepted for publication by H. Macgregor 25 June 1999

Key words: chromosome, chromosome painting, dog, karyotype

Abstract The domestic dog (Canis familiaris) is widely used as a model in the study of human disease. However, many of the 78 chromosomes comprising the canine karyotype are extremely difcult to identify reliably by classical cytogenetics. This has been a major hindrance to molecular cytogenetic studies of this species. The Animal Health Trust and the Sanger Centre have developed a set of canine whole chromosomespecic uorescence in situ hybridisation (FISH) probes (chromosome paints). We have used these chromosome paints to identify unequivocally each chromosome in a metaphase spread. An increasing number of laboratories are making use of cooled CCD cameras and sophisticated software for FISH mapping. Consequently, there is a major trend towards the use of DAPI banding for concurrent chromosome identication during FISH analyses in a range of species. Here we present, for the rst time, a complete DAPI banded karyotype of the dog in which each chromosome has been accurately placed, together with a 460-band DAPI ideogram. These data will facilitate the accurate assignment of FISHmapped loci to all chromosomes comprising the karyotype and form the basis for an agreed standard of the dog karyotype.

Introduction It is widely accepted that a comprehensive genome map of the domestic dog would be uniquely suited to complement those of humans and mice. Genome mapping of the dog has thus far resulted in low resolution linkage maps, concentrating primarily on linkage analysis of Type II markers (Lingaas et al. 1997, Mellersh et al. 1998 and Neff et al. 1999). In addition, grouping of such markers into syntenic groups with Type I loci has been facilitated by the

use of radiation hybrid mapping (Priat et al. 1998). Relatively few Type I or Type II loci have been assigned conclusively to the physical map of the dog genome (see for example Fischer et al. 1995; Dutra et al. 1996; Guevara-Fujita et al. 1996; Thomas et al. 1997; Werner et al. 1997). Many linkage groups also remain to be assigned. An accurate karyotype of the dog is clearly a fundamental prerequisite for the chromosomal assignment of existing and future linkage and syntenic groups. A number of attempts have been made over the

402 past 25 years to produce reliable, complete karyotypes of the domestic dog using a variety of conventional banding techniques. Examples include: Selden et al. (1975, GTG-banding); Manolache et al. (1976, GTG- and Q-banding); Howard-Peebles and Pryor (1980, RGB-banding); Mayr et al. (1983, counterstain-enhanced banding); Wayne et al. (1987, GTG-banding); Stone et al. (1991, GTGand Q-banding); Graphodatsky et al. (1995, GTGbanding) and more recently, by image analysis methodology Christian et al. (1998, D/C R-banding). In all cases the authors drew attention to the difculty in identifying reliably many of the smaller autosomal pairs in the karyotype due to their similarity in size and banding patterns. This has led to a number of discrepancies with regard to the nomenclature of the dog karyotype. The Committee for the Standardisation of the Canine Karyotype agreed upon the identity of the rst 21 pairs of autosomes in 1995 (Switonski et al. 1996). A further advance towards standardisation of the entire dog karyotype was made by Reimann et al. (1996) who were able to correctly orientate the chromosomes by the use of a centromeric repeat probe. These authors described an extended nomenclature of the canine karyotype and proposed a revised GTG-banded ideogram at the 460-band level. However, the application of GTG banding to FISH studies requires multiple time-consuming steps and is often unreliable. With an increasing number of laboratories making use of cooled CCD cameras and sophisticated software for FISH analyses involving a range of species, there is now a denite trend toward the use of 49,6-diamidino-2phenylindole (DAPI) banding. The major advantage of DAPI banding is that not only do the bands closely resemble those produced by GTG banding (which has historically been the method of choice), but it is also uorochrome-based, allowing simultaneous visualisation of the bands with FISH signals. In the present study we have unambiguously identied every dog chromosome using a set of chromosome specic paint probes (Langford et al. 1996) and present, for the rst time, an accurate DAPI-banded karyotype and ideogram. For the purposes of continuity, the layout of the karyotype and nomenclature of the DAPI-banded ideogram have retained maximum identity with the existing GTG-banded nomenclature of Reimann et al. (1996), following Selden et al. (1975). Materials and Methods Cell culture

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Elongated metaphase chromosomes from clinically normal dogs were prepared by mitogenic stimulation of peripheral lymphocytes and denatured as described previously (Breen et al. 1999). Fluorescence in situ hybridisation (FISH) The bivariate ow karyotype of the dog described by Langford et al. (1996) was resolved into 32 distinct peaks. An additional peak, V9, was later resolved exploiting the presence of polymorphic heterochromatin within chromosome 28 of a single animal. Chromosome paint probes were produced from each of these 33 peaks by incorporation of either biotin16-dUTP or digoxygenin-11-dUTP using DOP-PCR as described by Carter (1994). Fifty nanograms of a biotin- or a digoxygenin-labelled paint probe were precipitated with 10 g of sonicated genomic dog DNA and resuspended in 15 l of hybridisation buffer (50% deionized formamide, 2 3 SSC and 10% (wav) dextran sulphate). The probe plus competitor DNA mix was denatured at 708C for 10 min and allowed to preanneal at 378C for 30 min prior to being added to the denatured chromosomes. Posthybridisation stringency washes and immunocytochemical detection of hybridisation sites were as described by Breen et al. (1999). Chromosomes were counterstained with 80 ngaml DAPI, mounted with antifade (Vectashield, Vector Labs.) and sealed with a coverslip. Images were captured using a uorescence microscope (Axiophot, Carl Zeiss) equipped with a triple band pass lter, an FITC/Texas Red/DAPI lter set (Chroma Technologies) and a cooled CCD camera (KAF1400, Photometrics, Tuscon, AZ, USA). Images were captured using SmartCapture (Vysis Inc.) software and the digital image of each metaphase spread was processed using a high-pass spatial lter to reveal enhanced DAPI bands. Production of an accurate DAPI-banded karyotype and ideogram Following image capture from 30 suitably well spread metaphase preparations, the same metaphases were re-used for repeat hybridisation for all paints (following successive redenaturation steps for 1560 sec-

The DAPI banded karyotype of the domestic dog onds at 658C). Using this process, all chromosome pairs were conclusively identied in each metaphase spread. Accurate karyotypes were produced from 30 mid-metaphase spreads and used to derive a 460band DAPI ideogram. The ideogram was differentially shaded with up to ve grey levels to provide a more realistic representation of the banded chromosomes themselves, as described by Francke (1994). For continuity, wherever possible the precise nomenclature of each schematic chromosome, including landmark bands, was retained as that proposed by Reimann et al. (1996).

403 paint probes, under identical conditions, to their own banded metaphase preparations in order to deduce a consensus of the banded appearance of each painted chromosome. The results of that consensus study (Breen et al. 1998) were in agreement with those of our own, which are presented in Table 1. Figure 1 shows a complete DAPI-banded karyotype in which each chromosome has been veried as a result of hybridisation with the corresponding chromosome paint probe. Although eight paint probes hybridised to two pairs of chromosomes, those chromosome pairs are sufciently different in their banding appearance to allow condent identity. Figure 2 shows our DAPI-banded ideogram at the 460band stage. This ideogram has now been incorporated into the karyotyper software, which forms part of the Vysis QuipsTM Image Analysis Software.

Results In our bivariate ow karyotype of the dog the 33 peaks were designated AZ followed by aaff, with the additional peak labelled V9 (Table 1). The ow karyotype peaks corresponding to chromosomes 1 to 21, plus the X and the Y, were identied by Langford et al. (1996). Having completed our own analyses of all 33 paints, probes for the remaining 17 undesignated chromosomes were distributed to members of the Committee for the Standardisation of the Canine Karyotype. Each laboratory was required to apply the

Discussion Accurate identication of chromosomes within the karyotype of any species is a fundamental prerequisite to physical genome mapping. Dog genome mapping is still in its relative infancy, with few markers thus far physically assigned by FISH. With recent

Table 1. Assignment of each dog chromosome to its corresponding bivariate ow karyotype peak, reported by Langford et al. (1996). Assignment of peaks AM, OR and X and Y are taken from Langford et al. (1996). Assignment of peaks N, SW and Zff are from this report. Numbering of the chromosomes follows that suggested by Reimann et al. (1996), following Selden et al. (1975). Peak Number of homologues 2 2 2 2 2 2 2 4 2 4 2 4 2 2 2 2 2 Chromosome assignment 1 3 4 2 5 7 6 8, 11 12 10, 17 9 13, 15 16 22 14 20 18 Peak Number of homologues 2 4 2 2 4 2 4 1 1 4 2 2 4 2 2 2 Chromosome assignment 19 21, 23 25 27 24, 28 28 29, 32 X Y 31, 34 26 30 33, 36 3 38 35

A B C D E F G H I J K L M N O P Q

R S T U V V9 W X Y Z aa bb cc dd ee ff

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Figure 1. DAPI-banded karyotype of the dog. All chromosomes have been accurately placed in the karyotype following successive hybridisations with a panel of chromosome-specic paint probes.

advancements in technology, more molecular cytogenetic laboratories have access to sophisticated image acquisition and analysis workstations. Genome mapping projects for many species, including the human, now routinely use DAPI banding for whole chromosome and chromosome band identication. This current study offers a 460-band differentially shaded ideogram as a means of reliably identifying all dog chromosomes. This ideogram

was derived from DAPI-banded karyotypes in which, for the rst time, each chromosome has been accurately identied. This ideogram provides an accurate framework upon which a physical genome map of the dog may be constructed and also forms a basis for an internationally recognised standardised karyotype. With the data in this paper we are now in the process of producing a complete set of chromosome-specic microsatellite markers contained in

The DAPI banded karyotype of the domestic dog

405

Figure 2. DAPI-banded ideogram of the domestic dog (Canis familiaris) at the 460-band stage. The ideogram has been differentially shaded with up to ve grey levels to provide a more realistic interpretation of the actual chromosomes. The size of each chromosome in Mb is taken from Langford et al. (1996).

406 cosmid clones. These clones will serve as chromosome-specic single-locus FISH probes, and the markers that they contain will allow the anchoring of linkage groups to specic dog chromosomes. This will ensure that all chromosomes are included in the evolving dog genome map(s) (Breen/Holmes et al., manuscript in preparation). Although, for the purposes of continuity, we have arranged our karyotype as described previously, it is interesting to note that this order of the chromosomes bears little relation to the actual physical size of each chromosome (megabase pairs), as determined by comparative bivariate ow karyotype analysis (Langford et al. 1996). Perhaps discussion could be invited to consider the possibility of renumbering the chromosomes according to their physical size. This would most certainly provide an easier basis on which to develop automated classiers for the DAPI-banded karyotype of the dog. Acknowledgements Dog genome studies at the Animal Health Trust (MB) are generously supported by the Guide Dogs for the Blind Association. Cordelia Langford is supported by funds from the Wellcome Trust. We are grateful to Nicola Reimann and Sabine Bartnitske for useful discussions. We thank Anna Bristow for technical assistance.

M. Breen et al.
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