FISH Mapping and Identification of Canine
Chromosomes
M. Breen, C. F. Langford, N. P. Carter, N. G. Holmes, H. F. Dickens,
R. Thomas, N. Suter, E. J. Ryder, M. Pope, and M. M. Binns
The karyotype of the domestic dog (Canis familiaris) is widely accepted as one of
the most difficult mammalian karyotypes to work with. The dog has a total of 78
chromosomes; all 76 autosomes are acrocentric in morphology and show only a
gradual decrease in length. Standardization of the canine karyotype has been performed in two stages. The first stage dealt only with chromosomes 1–21 which can
be readily identified by conventional G-banding techniques. The remaining 17 autosomal pairs have proven to be very difficult to reliably identify by banding alone.
To facilitate the identification of all canine chromosomes, chromosome-specific
paint probes have been produced by DOP-PCR from flow-sorted dog chromosomes. Each paint probe has been used for FISH to identify the corresponding
chromosome(s), allowing precise identification of all 78 canine chromosomes. The
identification of the undesignated 17 autosomal pairs has been agreed upon by the
standardization committee during the second stage of their role. Cosmid clones
containing microsatellite markers may now be conclusively assigned to their chromosomal origin by simultaneous dual-color FISH with the corresponding paint
probe. In this way a collection of chromosome-specific cosmid clones is being
constructed, comprising at least one marker per chromosome, which will allow
anchoring of existing and future linkage groups to the physical map.
From the Centre for Preventive Medicine, Animal
Health Trust, PO Box 5, Newmarket, Suffolk CB8 7DW,
U.K. ( Breen, Holmes, Dickens, Ryder, Pope, and Binns),
the Sanger Centre, Wellcome Trust Genome Campus,
Hinxton, Cambridge, U.K. ( Langford and Carter), and
the Department of Biochemistry, University of Leicester, Leicester, U.K. ( Thomas and Suter). We gratefully
acknowledge the support of the Guide Dogs for the
Blind Association for canine genetics research at the
Animal Health Trust. Cordelia Langford and Nigel Carter are supported by a grant from the Wellcome Trust.
Address correspondence to Matthew Breen at the address above or e-mail: [email protected]. This
paper was delivered at the International Workshop on
Canine Genetics at the College of Veterinary Medicine,
Cornell University, Ithaca, New York, July 12–13, 1997.
q 1999 The American Genetic Association 90:27–30
The construction of genome maps for
mammalian species has been under way
for a number of years. Although man and
mouse have received considerably more
attention than other mammalian species,
large international efforts are in place to
produce maps for a range of economically
important species such as cattle, sheep,
pigs, and chickens. In addition more recent collaborations have been established
to produce genome maps for companion
animals such as horse and dog. A key feature of all the above genome mapping efforts has been the use of microsatellite
loci which, with significant levels of polymorphism and an even distribution
throughout the genome, have been adopted to produce the framework on which
more detailed maps may be developed.
The international collaboration, DogMap,
was established in 1993 to initially develop a low-resolution marker map of the canine genome. Similar studies are also being carried out in the United States.
As with other mapping projects, canine
genome mapping requires the establishment of a linkage map with the same number of linkage groups as chromosomes
within the karyotype. The dog has a par-
ticularly difficult karyotype to work with,
with a total of 78 chromosomes as 38 autosomal pairs plus the sex chromosomes.
All 38 autosomal pairs are acrocentric in
morphology with a gradual decrease in
their length. The largest chromosome is X,
which has been estimated to be approximately 137 Mb in size, with the tiny Y
chromosome a mere 27 Mb ( Langford et
al. 1996). Due to the similarities in the size
and GTG-banding appearance of many of
the canine chromosomes, the establishment of a globally recognized standard
karyotype has been perhaps more difficult
than for any other commonly studied domestic/laboratory animal. At a meeting
which took place as part of the 11th European Colloquium on Cytogenetics of Domestic Animals, the Committee for the
Standardized Karyotype of the Dog (Canis
familiaris) was established. The initial aim
of this committee was to produce an acceptable standard for those chromosomes
which could readily be identified using
conventional G-banding. This work resulted in the publication of a standard karyotype restricted to the largest 21 autosomes and for the sex chromosomes (Switonski et al. 1996).
27
Due to the difficulty in recognizing the
remaining 17 pairs of autosomes, it was
decided that those chromosomes would
need to be identified by the use of chromosome-specific probes, either whole
chromosome paint probes (WCPP) or
unique sequence probes ( USP). Due to the
lack of chromosome-specific USPs at that
time, it was decided to adopt the use of
WCPP.
Chromosome paints are complex mixtures of DNA probes which, when used in
fluorescence in situ hybridization ( FISH)
assays, allow the direct visualization of
specific chromosomes. Traditionally chromosome paint probes were made by labeling of cloned DNA libraries produced
from flow-sorted chromosomes (Cremer et
al. 1988; Lichter et al. 1988; Pinkel et al.
1988). More recently, chromosome paints
have been prepared by collecting a much
smaller number of flow sorted chromosomes and then applying degenerate oligonucleotide primed PCR ( DOP-PCR), as
described by Telenius et al. (1992), to allow the production of paint-probes. The
specificity of such probes may then be
verified by reverse painting (Carter et al.
1992). This approach has been applied to
assign flow karyotype peaks to their corresponding chromosome in a number of
species including mouse (Rabbitts et al.
1995) and pig ( Langford et al. 1993). Dual
laser, bivariate flow sorting, in which ATrich DNA is stained with Hoechst 33258
and GC-rich DNA is stained with chromomycin A3, allows for the highest resolution
flow karyotypes in which the chromosomes are sorted by both DNA content
and base pair ratio. The first successful
production of a high-resolution flow karyotype from the domestic dog was reported by Langford et al. (1996), from which
paint probes were produced for all canine
chromosomes. The availability of such a
collection of paint probes enables the
identification of all canine chromosomes
and the correlation to their corresponding
G-banding patterns.
Another highly desirable resource for
genome mapping is the development of a
panel of chromosome-specific USP markers. Following the development of chromosome paint probes cosmid clones containing microsatellite markers may now be
conclusively assigned to their chromosomal origin by simultaneous dual-color
FISH with the corresponding paint probe.
In this way a collection of chromosomespecific cosmid clones is being constructed, comprising at least one marker per
chromosome, which will allow anchoring
28 The Journal of Heredity 1999:90(1)
of existing and future linkage groups to the
physical map.
Materials and Methods
Preparation of Canine Chromosome
Paint Probes
Chromosome preparations were made by
mitogenic stimulation of canine peripheral
lymphocytes and prepared for flow analysis and sorting on a dual-laser Elite ESP
flow sorter (Coulter Electronics) [for a detailed description see Langford et al.
(1996)]. Five hundred chromosomes from
each distinguishable peak of the flow karyotype were collected and amplified by
two rounds of DOP-PCR to produce the resulting biotinylated or fluorochrome-labeled paint probes. One microliter (approximately 75 ng) of each of the labeled
DOP-PCR products was mixed with 10 mg
of sonicated dog DNA, 5 mg of sonicated
salmon sperm DNA, and mixed with 15 ml
of hybridization buffer [50% formamide,
23 SSC, 0.1% Tween-20, 10% (w/v) dextran
sulfate].
Preparation of Canine Metaphase
Spreads
Canine metaphase preparations were prepared by phytohemagglutinin (PHA) stimulation of peripheral lymphocytes in RPMI
(1640) supplemented with 20% fetal bovine serum and 100 mM L-glutamine according to routine methods. Slide preparations were produced by conventional
colcemid, hypotonic, and fixation treatments. Slides were aged for 5 days prior
to being used for FISH.
Isolation of Microsatellite Containing
Cosmid Clones
One thousand five hundred clones from a
canine genomic cosmid library (constructed in the vector pWE15, Stratagene; average insert size 35–41 kb) were screened
for the presence of the microsatellite repeat (CA)n. The hybridization conditions
were 16 h at 658C in 0.5 M disodium phosphate, 1 mM EDTA, 7% SDS, 0.1% BSA pH
7.2 with post-hybridization washes of 13
SSC:0.1% SDS at 658C. This level of stringency ensured that the motif selected was
not less than (CA)12 in length and was
therefore almost certain to result in the
isolation of a polymorphic marker ( Breen
et al. 1997b). Three hundred positive
clones were initially selected for further
analysis and DNA preparations made using Quiagen columns.
Preparation of Cosmid FISH Probes
For each cosmid DNA preparation, 1 mg
was indirectly labeled with bio-14-dATP
and/or dig-11-dUTP or directly labeled
with rhodamine-4-dUTP and/or fluorescein11-dUTP in nick translation reactions optimized to result in fragment sizes suitable
for efficient hybridization (ranging from
100 to 500 bp). In each case, the labeled
DNA was purified through a G-50 sephadex
column equilibrated in 13 TNE (0.2 M
NaCl; 10 mM Tris-HCl; 1 mM EDTA). Fifty
nanograms of labeled cosmid DNA was coprecipitated with 5 mg of sonicated canine
DNA, 10 mg of sonicated salmon sperm
DNA, and resuspended in 15 ml of hybridization buffer (as above). For confirmation
of chromosome assignment, 50 ng of corresponding paint probe was also added to
the above mixture.
Fluorescence in situ Hybridization
Probe/competitor DNA mixtures were denatured at 708C for 10 min and preannealed at 378C for 30 min. Chromosome
preparations were denatured in 70% formamide:23 SSC at 658C for 2 min, quenched
in ice cold 70% ethanol, and dehydrated
through an ethanol series (70%, 90%,
100%). Preannealed probe was added to
the denatured chromosome preparations
and allowed to hybridize for 17 h at 378C.
Slides were washed in three exchanges of
50% formamide:23 SSC at 428C, followed
by three exchanges of 23 SSC at 428C. The
hybridization sites for indirectly labeled
probes were detected by immunocytochemical detection as described by Breen
et al. (1997a). The chromosomes were
counterstained with 49-69 diaminidino-2phenylindole ( DAPI) and image detection
was performed with a SmartCapture FISH
workstation ( Digital Scientific) comprising
a fluorescence microscope (Axiophot,
Zeiss) equipped with FITC and DAPI filters
(Chroma Technologies) and a highly sensitive CCD camera (Photometrics, Tucson,
AZ).
Results
Assignment of Flow-Sorted Canine
Paint Probes
The bivariate flow karyotype of the dog was
resolved into 33 peaks, from which chromosome paints were produced by DOP-PCR.
Reverse painting analysis revealed that all
chromosomes in the karyotype were represented in the flow karyotype peaks. Twentyfive peaks contained material from single
chromosomes, with the remaining eight
peaks (H, J, L, S, V, W, Z, and cc) containing
material from two chromosomes. Detailed
analyses of the image-enhanced DAPI banding of the reverse painted chromosomes
from peaks A–R and X and Y were reported
by Langford et al. (1996), while chromosome
identification from the remaining peaks, S–
ff, has been made by the Committee for the
Standardized Karyotype of the Dog (Canis
familiaris) (Breen et al., in preparation).
Cosmid Localization
To date 300 cosmid clones, each containing a microsatellite repeat, have been isolated for use in FISH studies. Of these, 85
have thus far been localized to their chromosomal origin. Of the 40 chromosomes
comprising the canine karyotype, 26 (1–
10, 14, 18–20, 24–28, 30–32, 34, 36, X, and
Y) have to date been associated with a
cosmid and 14 remain to be associated.
Those cosmids which have thus far been
localized are at the various stage of characterization required to determine their
usefulness for genetic mapping. An example of the use of simultaneous FISH using
both cosmid- and chromosome-specific
paint probe is shown in Figure 1.
Discussion
The development of a set of chromosome
paints for the canine karyotype has, as expected, proven to be an extremely valuable resource in the precise identification
of chromosomes in what is considered by
many to be one of the most difficult common karyotypes to study. All 38 pairs of
autosomes and the sex chromosomes of
the dog were represented in the flow karyotype, with 25 peaks representing single
chromosomes and a further eight peaks
each representing two chromosomes. The
existence of the latter eight paints, with
five of these targeting some of the smallest
chromosomes, has meant that additional
resources such as cosmid markers will be
required for precise identification of the
chromosomes involved. Our primary aim
in this study has been to identify at least
one polymorphic microsatellite marker associated with each of the 40 canine chromosomes.
Due to the necessity to obtain reliable
FISH data, cosmids have been the clones
of choice to conduct this study. Evidence
from other species indicates that CA/GT
microsatellites are evenly dispersed
throughout the genome, and since approximately one in three of the canine cosmids
(insert size 35–41 kb) screened contained
at least one (CA)121 repeat, it is anticipated that there will be no shortage of suit-
Figure 1 Simultaneous fluorescence in situ hybridization ( FISH) for the conclusive assignment of canine cosmids
to their chromosome of origin: (a) canine metaphase spread with no visible signal; (b) signal resulting from the
hybridization of a cosmid clone, AHTk338 (arrows); (c) signal resulting from the hybridization of a paint probe to
the same chromosome (chromosome 1) as in ( b); (d) DAPI banded metaphase spread showing sites of hybridization of cosmid AHTk338 (arrows) to the centromeric end of canine chromosome 1.
able markers. Estimations from the flow
karyotype indicate that the total size of a
haploid set of chromosomes (from a male
dog) is in the region of 2800 Mb, with the
largest seven autosomes representing
over 25% of the estimated genome size.
The emerging data clearly demonstrate
that, as expected, a random selection of
cosmid clones are more likely to be derived from the larger chromosomes than
from the smaller ones. Currently almost
one-third of all cosmids mapped have
been assigned to the first seven chromosomes, the remainder being fairly evenly
distributed on chromosomes 8–38. With
this figure in mind we anticipate being
able to produce at least one cosmid per
chromosome from our current panel of
300 clones. If additional clones are needed, either due to residual “naked” chromosomes or lack of polymorphism in assigned markers, we are in a position to
rapidly select further (CA)n clones from
our library screen. By determining the
chromosomal localization of such clones
prior to the lengthy process of microsat-
ellite characterization, it has been possible to ensure that progress toward the
completion of this study and the production of a set of chromosome-specific microsatellite markers is as swift as possible.
Once a panel of chromosome-specific microsatellite-containing cosmids is available, it is our intention to make them available for use to the canine genome mapping community, although the mechanism
by which this will proceed has yet to be
determined.
Our initial aim of at least one cosmid
per chromosome will permit correlation of
the emerging linkage map with the physical map. As the number of cosmids per
chromosome increases, orientation of the
linkage groups will become possible and
we will be in a position to verify that the
linkage groups span the entire length of
each chromosome. A combination of chromosome painting and fine mapping with
type I markers will provide detailed information with regard to synteny with other
species. Data from syntenic mapping will
give valuable information for the study of
Breen et al • FISH Mapping of Canine Chromosomes 29
canine evolution. In addition, when the
chromosomal location of a marker linked
to a disease is known, the map position in
the canine karyotype may be used to investigate syntenic regions from more comprehensively mapped species such as
man, thereby greatly accelerating the
search for candidate genes.
integration of anchored markers in horse genome mapping. Mamm Genome 8:267–273.
References
Langford CF, Fischer PE, Binns MM, Holmes NG, and
Carter NP, 1996. Chromosome-specific paints from a
high resolution flow karyotype of the dog. Chromosome Res 4:115–123.
Breen M, Langford CF, Carter NP, Fischer PE, Marti E,
Gerstenberg C, Allen WR, Lear TL, and Binns MM,
1997a. Detection of equine X chromosome abnormalities using a horse X whole chromosome paint probe
(WCPP). Vet J 154:235–238.
Breen M, Lindgren G, Binns MM, Norman J, Irvin Z, Bell
K, Sandberg K, and Ellegren H, 1997b. Genetical and
physical assignments of equine microsatellites—first
30 The Journal of Heredity 1999:90(1)
Langford CF, Telenius H, Miller NG, Thomsen PD, and
Tucker EM, 1993. Preparation of chromosome-specific
paints and complete assignment of chromosomes in
the pig flow karyotype. Anim Genet 24:261–267.
DC, 1988. Delineation of individual human chromosomes in metaphase and interphase cells by in situ
suppression hybridization using recombinant DNA libraries. Hum Genet 80:224–234.
Pinkel D, Landegent J, Collins C, et al., 1988. Fluorescence in situ hybridization with human chromosomespecific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 85:
9138–9142.
Rabbitts P, Impey H, Heppell-Parton A, Langford C,
Tease C, Lowe N, Bailey D, Ferguson-Smith M, and Carter N, 1995. Chromosome specific paints from a high
resolution flow karyotype of the mouse. Nat Genet 9:
369–37.
Switonski M, Reimann N, Bosma AA, Long S, Bartnitzke
S, Pienkowska A, Moreno-Milan MM, and Fischer P,
1996. Report on the progress of standardization of the
G-banded canine (Canis familiaris) karyotype. Chromosome Res 4:306–309.
Telenius H, Pelmear AH, Turncliffe A, et al., 1992. Cytogenetic analysis by chromosome painting using DOPPCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4:257–263.
Lichter P, Cremer T, Borden J, Manueldis L, and Ward
Corresponding Editor: Gustavo Aguirre
Carter NP, Ferguson-Smith MA, Perryman MT, Telenius
H, Pelmear AH, Leversha MA, Glancy MT, Wood SL,
Cook K, Dyson HM, Ferguson-Smith ME, and Willatt LR,
1992. Reverse chromosome painting: a method for the
rapid analysis of aberrant chromosomes in clinical cytogenetics. J Med Genet 29:299–307.
Cremer T, Lichter P, Borden J, Ward DC, and Manueldis
L, 1988. Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome specific library probes. Hum
Genet 80:235–246.