Mammalian Genome 11, 803–805 (2000).
DOI: 10.1007/s003350010146
Incorporating Mouse Genome
© Springer-Verlag New York Inc. 2000
A horse whole-genome–radiation hybrid panel: Chromosome 1 and 10
preliminary maps
Susanna L. Kiguwa,1 Patrick Hextall,1 Angela L. Smith,1 Ricky Critcher,1 June Swinburne,2 Lee Millon,3
Matthew M. Binns,2 Peter N. Goodfellow,1,4 Linda C. McCarthy,1,5 Christine J. Farr,1 E. Ann Oakenfull1
1
Department of Genetics, University of Cambridge, Downing Site, Cambridge CB2 3EH, UK
Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK
Veterinary Genetics Laboratory, University of California–Davis, Davis, California 95616-8744, USA
4
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Harlow, Essex CM19 5AW, UK
5
Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
2
3
Received: 24 January 2000 / Accepted: 14 April 2000
In recent years there has been increasing interest in mapping the
horse genome, particularly to identify disease and performanceenhancing genes. Although a number of horse mapping tools have
been developed and have proved very useful (genetic maps, somatic cell hybrid panels, and a BAC library), the benefits of
whole-genome–radiation hybrid (WG-RH) mapping have not been
available. Its advantages over genetic linkage mapping are: (i) the
resolving power is not lost in regions of the genome with a low
recombination rate, because it does not rely on meiotic recombination events; (ii) it is especially useful in animals such as the
horse with relatively long generation times and single births; and
(iii) genetic and physical maps can be integrated, as both polymorphic and non-polymorphic markers can be placed on the same
map. The WG-RH panel constructed in this study is the first such
panel to be reported for the horse, and its preliminary characterization demonstrates its usefulness for horse genome mapping.
The panel was constructed by the fusion of horse embryonic
endothelial primary lung cells (male) to the established hamster
fibroblast cell line A23 (Westerveld et al. 1971) by using the
method described in McCarthy et al. (1997). A series of fusions,
involving irradiation (3000 rads) of donor cells prior to fusion with
equal numbers of recipient cells, generated ∼160 hybrids in total.
From these 160 hybrids, 94 were selected at random and screened
by using 20 widely distributed markers (representing 17 chromosomes). Any hybrids that did not produce an amplification product
with these markers were screened by FISH to determine whether
they contained horse DNA from other chromosomes or regions of
chromosomes (not represented by the 20 markers); hybrids found
to be negative by the FISH screen were replaced at random from
the remaining unused hybrids, and the same PCR and FISH screening procedure was repeated until 94 hybrids were assembled.
These 94 hybrids were used for preliminary characterization as a
mapping panel. It is expected that the majority of the hybrids
(>95%) characterized here will be represented in the TM99 panel
of 94 hybrids that is being grown on a large scale by Research
Genetics Inc. (Huntsville, Ala. 35801).
The amount of horse DNA retained by the hybrids in the panel
was evaluated by examining the retention of the 20 widely distributed horse markers. On average each marker was retained in
27.8% of the hybrids (ranging from 10.6% to 71.3%; Table 1),
implying that the panel as a whole retains the equivalent of approximately 26 horse genomes. These retention frequencies compare well with those found for the human and mouse RH panels,
which have been used successfully for creating whole-genome
maps (Gyapay et al. 1996; McCarthy et al. 1997).
Correspondence to: E.A. Oakenfull; E-mail: [email protected]
The mapping ability of the panel was assessed by producing
RH maps for the two horse chromosomes with the most markers
available, and comparing these with the latest genetic maps (Swinburne et al. 2000a). In total, 39 markers on Chromosome (Chr) 1
and 15 markers on Chr 10 were analyzed (Table 1). The average
retention of markers was 15.4% (ranging from 5.3% to 29.8%) on
Chr 1, and was higher, 25.4% (ranging from 16.0% to 44.7%) on
Chr 10. An increase in the retention frequency on smaller chromosomes was also noted in human and mouse RH panels (Gyapay
et al. 1996; McCarthy et al. 1997). For each chromosome, linkage
groups with at least 4-LOD units support were identified; four such
groups were found on Chr 1 and two were detected on Chr 10
(Figs. 1 and 2). Within each of these linkage groups, framework
markers were ordered with 3-LOD units support, and most of the
non-framework markers were ordered with 2-LOD units support.
Some non-framework markers had lower statistical support for
their order (shown in italics, Figs. 1 and 2). Similarly, the relative
order of some linkage groups was suggested by linkage analysis
but had low statistical support [Chr 1 (groups B and C) and Chr 10
(groups A and B)]. The relative order of the other RH linkage
groups was determined by comparison with the genetic map and
FISH localizations.
The genetic map can give a misleading impression of marker
density because genetic distances may be small owing to regions
having a low meiotic recombination rate. In such regions the markers may actually be physically far apart and, therefore, at a lower
density than predicted by the genetic map. Since RH panels require
a high density of markers, this might account for the low statistical
support for the ordering of some markers and linkage groups. A
low density of available markers could also explain why two markers (HLM5, ICA22) could not be placed on the RH map. HLM5 has
already been shown to be 24 cM from its nearest neighboring
marker on the genetic map. Three other markers could not be
placed on the RH map (UM004, HTG12, and HMS15), but were
able to be placed on the genetic map in a region equivalent to RH
linkage group D; this discrepancy between the two maps should be
resolved as both maps are characterized further.
This study has demonstrated the ability of the horse WG-RH
panel to produce an accurate genome map as there is good agreement of the genetic and physical maps with the RH maps for Chrs
1 and 10 (Figs. 1 and 2). Only a few differences between the maps
were observed, and experiences with human mapping suggest that
such differences are common and are resolved as more markers are
incorporated into the maps (Walter et al. 1994). The WG-RH panel
was able to order some markers that co-segregate on the genetic
linkage map, i.e., Chr 1 markers LEX39 and ICA18, and ICA41 and
ICA32, and Chr 10 markers LEX8 and COR015, and NVHEQ18
and AHT15.
804
S.L. Kiguwa et al.:
Horse whole-genome–radiation hybrid panel
Table 1. Marker retention frequency across the genome.
Chr
Marker Name/
GenBank Accession No.
Retention
Frequency (%)
Chr
Marker Name/
GenBank Accession No.
Retention
Frequency (%)
Chr
Marker Name/
GenBank Accession No.
Retention
Frequency (%)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ICA30/AF043209
LEX30/AF075632
HP27a
ASB41/AF004771
LEX39/AF075641
ICA18/AF043201
ICA12/AF043199
AHT26/AJ271511
VHL134/Y08448
UM002/AF195124
ASB12/X93526
ICA27/AF043207
ICA01/AF043198
SGCV02/U90585
ICA21/AF043203
ICA36/AF043211
SGCV25/U90603
AHT21b
AHT40/AJ271525
ICA41/AF043214
ASB8/X93522
ICA32/AF043210
LEX58/AF075665
ICA20/AF043202
19.1
8.5
7.4
9.6
6.4
11.7
5.3
6.4
29.8
28.7
21.3
22.3
25.5
17.0
18.1
14.9
17.0
17.0
13.8
9.6
10.6
12.8
14.9
14.9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
4
6
8
9
10
10
10
10
ICA28/AF043208
ICA43/AF043215
ICA25/AF043205
RC09c
UM026/AF195573
ICA16/AF043200
COR006/AF083449
HMS7/X74636
ICA44/AF043216
HLM5/U36497
HMS15/U35401
HTG12/AF169296
ICA22/AF043204
ICA40/AF043213
*UM004/AF195126
ASB18/X93532
AHT6/Y07734
UM015/AF195133
COR012/AF083455
LEX19/AF075621
*LEX62/AF075662
LEX9/AF075612
SGCV17/U90595
HMS2/X74631
20.2
21.3
22.3
13.8
16.0
13.8
16.0
14.9
14.9
14.9
5.3
17.0
11.7
20.2
16.0
12.8
36.2
25.5
22.3
39.4
44.7
22.3
22.3
22.3
10
10
10
10
10
10
10
10
10
10
10
11
11
12
15
15
16
18
20
21
21
22
30
X
NVHEQ7/AF011402
LEX17/AF075619
COR015/AF083458
LEX8/AF075611
SGCV30/U90605
NVHEQ18/AF011404
ASB6/X93520
UCDEQ482/U67416
COR020/AF083463
AHT15b
HMS23/U89810
SGCV13/U90592
UCDEQ39/U25168
SGCV10/U90591
AHT2/Y07730
COR014/AF083457
ASB42/AF004772
SGCV07/U90589
HTG5/AF169166
SGCV14/U90593
SGCV16/U90594
SGCV03/U90586
LEX25/AF075627
LEX24/AF075626
25.5
31.9
29.8
29.8
28.7
21.3
21.3
22.3
22.3
16.0
20.2
48.9
71.3
23.4
17.0
17.0
18.1
21.3
27.7
30.8
28.7
19.1
24.5
10.6
* These markers were included in the twenty genome wide markers along with the other markers not on Chrs 1 and 10.
a
Unpublished marker (L. Skow pers. comm.).
b
Swinburne et al. 1997.
c
Unpublished marker (S. Mashima pers. comm.).
Fig. 1. Comparison of the RH map, genetic map, and G-banded idiogram
for horse Chr 1. The RH map is shown on the right, the genetic map in the
centre, and a G-banded idiogram on the left. The RH linkage groups and
their likelihood support are shown on the far right. Within these groups the
framework markers, whose order is supported by 3-LOD units, are shown
in bold and underlined; markers whose order is supported by 2-LOD units
are shown in plain text, and the order of the markers that are shown in
italics is suggested by linkage analysis, but is not statistically well supported. The genetic map markers joined by a solid line are linked together
with 3-LOD units support, although the support for their order is sometimes lower, and the markers joined by a broken line are linked by 2-LOD
units. The distances in cR3000 and cM are shown to the right and left of the
RH and genetic maps respectively. Markers common to both RH and the
genetic maps are joined by a solid line, and those whose order has only
been suggested on the RH map are joined by a dashed line. A solid line also
joins those markers on the genetic map that have been physically mapped
by FISH to their location on the idiogram (Sakagami et al. 1995; Breen et
al. 1997; Godard et al. 1997, 1998; Lear et al. 1999; L. Skow pers comm.;
Swinburne et al. 2000a, 2000b). PCR conditions were optimized for each
primer set, the annealing temperature ranged from 50 to 60°C, and the
Mg2+ concentration ranged from 1 to 2 mM. The temperature/time profile
for PCR was an initial 94°C for 3 min, followed by 36 cycles of 94°C for
30 s and the optimum annealing temperature for 30 s; and a final 3 min at
72°C. A touchdown procedure was used to decrease nonspecific hybridization with hamster DNA and involved a reduction in the annealing temperature by 1°C for each of the first seven cycles until the optimum annealing temperature was reached. The products were resolved on 4% agarose gels, and all markers were screened in duplicate. The RH map was
constructed using RADMAP (Matise et al. 1994). A two-point analysis
identified markers linked by at least 4-LOD unit support, and multipoint
analysis was used to order the markers within each linkage group.
<
The preliminary characterization and mapping of this horse
WG-RH panel compares well with initial characterization of human and mouse WG-RH panels (Walter et al. 1994; McCarthy et
al. 1997). The availability of more markers will provide a frame-
S.L. Kiguwa et al.:
Horse whole-genome–radiation hybrid panel
Fig. 2. Comparison of the RH map, genetic map, and G-banded idiogram
for horse Chr 10. The details for this figure are the same as those described
in Fig. 1.
work RH map of the whole genome that will allow the ordering of
co-segregating markers on the genetic map, and will also enable
non-polymorphic markers, such as expressed sequence tags (ESTs)
and bacterial artificial chromosome (BAC) clones, to be integrated
with markers on genetic maps, thus accelerating positional cloning
of target genes.
Acknowledgments. This work was funded by the Medical Research Council of Great Britain, grant number G9503936. We thank Maria Davis and
Sally Debenham for technical assistance.
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