Vol 13, Issue 4, 742-751, April 2003
RESOURCES
The First-Generation Whole-Genome Radiation Hybrid Map in the Horse Identifies Conserved Segments in Human and Mouse Genomes
Bhanu P. Chowdhary1,10,
Terje Raudsepp1,
Srinivas R. Kata2,
Glenda Goh1,
Lee V. Millon3,
Veronica Allan3,
François Piumi4,
Gérard Guérin4,
June Swinburne5,
Matthew Binns5,
Teri L. Lear6,
Jim Mickelson7,
James Murray8,
Douglas F. Antczak9,
James E. Womack2 and
Loren C. Skow1
1Department of Veterinary Anatomy and Public
Health and 2Department of Veterinary Pathobiology, College of
Veterinary Medicine, Texas A&M University, College Station, Texas
77843, USA; 3Veterinary Genetics Laboratory, University of
California, Davis, California 95616, USA; 4INRA, Centre de
Recherches de Jouy, Département de Génétique animale,
78352 Jouy-en-Josas, France; 5Animal Health Trust, Lanwades
Park, Suffolk, CB8 7UU, UK; 6Department of Veterinary
Science, M.H. Gluck Equine Research Center, University of Kentucky,
Lexington, Kentucky 40546-0099, USA; 7Department of
Veterinary Pathobiology, University of Minnesota, 295f AS/VM, St. Paul,
Minnesota 55108, USA; 8Department of Animal Science,
University of California, Davis, California 95616, USA;9
James A. Baker Institute of Animal Health, College of
Veterinary Medicine, Cornell University,
Ithaca, New York 14853, USA
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ABSTRACT
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A first-generation radiation hybrid (RH) map of the equine
(Equus caballus) genome was assembled using 92 horse x
hamster hybrid cell lines and 730 equine markers. The map is the first
comprehensive framework map of the horse that (1) incorporates type I
as well as type II markers, (2) integrates synteny, cytogenetic, and
meiotic maps into a consensus map, and (3) provides the most detailed
genome-wide information to date on the organization and comparative
status of the equine genome. The 730 loci (258 type I and 472 type II)
included in the final map are clustered in 101 RH groups distributed
over all equine autosomes and the X chromosome. The overall marker
retention frequency in the panel is  21%, and the possibility of
adding any new marker to the map is 90%. On average, the mapped
markers are distributed every 19 cR (4 Mb) of the equine genome—a
significant improvement in resolution over previous maps. With 69 new
FISH assignments, a total of 253 cytogenetically mapped loci physically
anchor the RH map to various chromosomal segments. Synteny assignments
of 39 gene loci complemented the RH mapping of 27 genes. The results
added 12 new loci to the horse gene map. Lastly, comparison of the
assembly of 447 equine genes (256 linearly ordered RH-mapped and
additional 191 FISH-mapped) with the location of draft sequences of
their human and mouse orthologs provides the most extensive
horse–human and horse–mouse comparative map to date. We expect that
the foundation established through this map will significantly
facilitate rapid targeted expansion of the horse gene map and
consequently, mapping and positional cloning of genes governing traits
significant to the equine industry.
[Supplemental material
is available online at www.genome.org. The following individuals kindly
provided reagents, samples, or unpublished information as indicated in
the paper: R. Brandon, G. Lindgren, and I. Tammen.]
The primary aim of genome analysis in the horse is to generate
composite map information for improving equine
health, reproduction, and disease resistance. To achieve these goals,
comprehensive knowledge of the genome will be instrumental in
understanding the molecular causes underlying various equine hereditary
disorders, and will be crucial in developing diagnostic and
prevention/therapeutics approaches for these conditions. Additionally,
other traits of significance such as coat color, etc., can be better
addressed by understanding the molecular cause of variation in
expression. Recent studies have shown that the mouse cannot be ‘taken
for granted’ as a model animal for all human conditions (Rieder et al.
2000 ; Heinzerling et al. 2001), and that in some cases, the horse can
be a better model (Rieder et al. 2000).
The First International Equine Gene Mapping Workshop (October 1995,
Lexington, Kentucky, USA) signaled the beginning of an organized equine
genomics program. Significant strides have since been made in expanding
the gene map of the horse (Equus caballus; ECA, reviewed in
Chowdhary and Raudsepp 2000). Currently 1200 markers have been
mapped/assigned to various equine chromosomes using approaches such as
synteny analysis (Caetano et al. 1999a,b; Shiue et al. 1999), genetic
linkage mapping (Lindgren et al. 1998; Guérin et al. 1999, 2003;
Swinburne et al. 2000a), and fluorescent in situ hybridization (FISH;
e.g., Raudsepp et al. 1999; Godard et al. 2000; Lear et al. 2001;
Mariat et al. 2001; Milenkovic et al. 2002). Among the recent prominent
developments are the generation of preliminary radiation hybrid (RH)
maps for chromosomes ECA1 and ECA10 (Kiguwa et al. 2000) and RH and
comparative maps for some of the other equine chromosomes (Chowdhary et
al. 2002; Raudsepp et al. 2002). Several years ago, a Zoo-FISH-based
landmark comparison of the organization of horse and human genomes was
provided by Raudsepp et al. (1996). Analyses of a somatic cell hybrid
(SCH) panel (Caetano et al. 1999a,b; Shiue et al. 1999) and the use of
horse and goat BAC clones as FISH probes on horse metaphase spreads
(Godard et al. 2000; Milenkovic et al. 2002) have provided additional
information.
With this progress, the current focus of equine genomics is to develop
a high-resolution ordered physical map comprising: (1) uniformly
distributed highly polymorphic markers and (2) a large set of ESTs or
human/mouse orthologs that can provide a comprehensive comparative map.
The former fulfills the need for the development of a robust genome
scan panel that can help locate gene(s) governing traits of interest,
and the latter is critical for candidate gene searches from the highly
developed human and mouse genomes. RH cell lines are excellent for this
purpose because they readily integrate markers from all sources into a
consensus map using efficient and economic PCR-based typing (e.g.,
Geisler et al. 1999; Hukriede et al. 1999; Van Etten et al. 1999;
Watanabe et al. 1999; Band et al. 2000; Murphy et al. 2000; Breen et
al. 2001). The power of this approach has recently been exploited in
the horse to obtain preliminary physical maps for some of the
chromosomes (Kiguwa et al. 2000; Chowdhary et al. 2002; Raudsepp et al.
2002).
Expanding considerably on our earlier work, herein we report the use of
a variety of markers—some already mapped using different approaches,
and others newly generated—to develop the first physically ordered RH
maps for all equine chromosomes, except the Y. The aim of this
"first-generation RH map" is to integrate data from linkage,
synteny, and FISH maps into a single consensus map for each of the
chromosomes. The map thus generated will provide the most extensive
coverage currently available for the equine genome with a variety of
markers. Incorporation of equine orthologs for human genes in the RH
map considerably enhances the comparative status of the horse genome in
relation to human/mouse genomes. This will not only form the basis for
initiating searches for genes of economic value to the equine
industry, but will also be valuable in understanding the
comparative organization and evolution of this perissodactyl genome in
relation to other mammals or vertebrates.
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RESULTS
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The RH Map
A total of 901 markers were typed on the 5000rad
International Equine Whole Genome RH panel. Of these, 40 markers
(5%) either showed no amplification or were considered
‘unreliable’ because of inconsistent results, and therefore were
discarded from further analysis. An additional 131 markers (14.5%)
were considered unlinked at the threshold set for analysis (lod  7;
RHMAP), leaving 730 markers (81%) clustered in 101 RH groups. Of
these, 258 represented specific genes/ESTs (type I) and 472 were
microsatellites (type II). The final or comprehensive map is comprised
of 259 framework markers, around which the remaining 471 markers were
positioned.
Estimated Size, Genome Coverage, Marker Distribution, and Retention Frequency
The estimated size of the RH map in this study, summed up over all
the chromosomes (except the Y), is 14,587 centiRay (cR). The total map
distance for individual chromosomes ranged from 102 (ECA26) to 958 cR
(ECA1). Assuming that, like other mammals, the physical size of the
equine genome is 3000 Mb, the map currently provides, on average,
one marker every 4 Mb (19cR) of the equine genome. Further, 40 of
the 901 markers could not be typed in the panel, indicating the
coverage of the equine genome to be only 95%. However, considering
that a total of 171 markers dropped out of the final analysis, this
estimate reduces to 81%. The latter is certainly an
underestimation, because the statistic excludes even those ‘unlinked’
markers (the group of 131 mentioned above) that are just short of the
accepted threshold for inclusion in the RH groups. It is expected that
several (if not all) of them will be incorporated in the map as its
density improves. Hence, a realistic estimate of the genome coverage in
the panel is probably 90%.
Of the markers incorporated in the RH map, the highest number of
markers is located on ECA1 (63 markers; ECA1 is the largest
chromosome), whereas only six markers were localized to ECA28 (the
lowest number/chromosome). The latter is among the smallest equine
chromosomes. However, if viewed in terms of density of markers per
unit length of the chromosome, ECA11 appears to be the most densely
mapped chromosome (13 markers/unit length), followed by ECA4 and
ECA20 (10 markers/unit length). The corresponding value for ECA1 is
8.9, and for ECA28 is 3–4. Despite the differences, the entire
horse genome is reasonably well covered with markers, especially for a
first-generation RH map.
On average, 3–4 RH groups were found on each of the equine
chromosomes, with the number ranging from 10 on ECA1 to only one each
on, for example, ECA26–ECA29 (Fig. 1, enclosed poster). The relatively
large number of RH groups on ECA1 could be attributed to more
irradiation-induced breakages expected in large-sized chromosomes than
in the smaller ones. The overall retention frequency of the markers in
the panel is 21%, with a range of 11% for ECA1 to 37% for
ECA11. Retention of markers was slightly higher than average in the
pericentromeric regions of a number of meta- and submetacentric
chromosomes (e.g., ECA1–10) and a few acrocentric chromosomes (ECA23).
A similar marginally high retention frequency was observed towards
the telomeric regions of some chromosomes (e.g., ECA3p, ECA6p,
ECA13p, ECA16q, and ECA20q). On ECA11, the overall retention of
markers was much higher on the short arm than the long arm, most likely
due to the presence of the TK1 gene on ECA11p,
which is preferentially retained in all cell hybrids.
FISH Mapping and Alignment of RH Maps
A total of 69 RH-mapped loci (33 genes and 36 microsatellites) were
FISH-mapped. These represent new cytogenetic data on the equine genome.
The majority of the loci mapped to ECA1 (19), ECA10 (12), ECA14 (12),
and ECA17/21 (5 each), where the number of RH-typed markers is also
relatively high. A complete list of all the markers FISH-mapped in this
study is presented in Table 1. These
localizations, together with published FISH data on 191 markers (see
Chowdhary et al. 2002; Milenkovic et al. 2002; Raudsepp et al. 2002),
provided 253 ‘anchors,’ 118 type I and 135 type II, that aligned 88
of the 101 RH groups to individual chromosomes, thus facilitating their
physical placement and orientation. A distribution of the number of
anchor markers by chromosome is presented in Table
2. The remaining 13 RH groups were placed
using available genetic linkage information (Lindgren et al. 1998;
Swinburne et al. 2000a; Guérin et al. 2003) and the
RH2pt data for end markers with markers in the adjacent RH groups (Fig.
1, enclosed poster; see linkage groups with white bars).
Synteny Mapping
A total of 39 new equine genes/ESTs were mapped using the UC-Davis
somatic hybrid cell panel (see Caetano et al. 1999a; Shiue et al.
1999). The loci were distributed on 20 equine chromosomes. Twenty-seven
of these loci are also on the RH map presented here. Except for
HDAC1, the SCH mapping data for all loci are in agreement with
the RH localizations. FISH data on 12 of the 39 loci (Lear et al. 2001;
Mariat et al. 2001; Milenkovic et al. 2002) strongly support the
synteny results.
Comparative Mapping
The RH map presented here incorporates a total of 258 type I
markers. When compared to the estimated length of the RH map (14,587
cR), the markers are, on average, spaced every 50 cR (or 10 Mb) of
the equine genome. To improve the density of this comparative
framework, all previously FISH-mapped equine genes (see Chowdhary et
al. 2002; Milenkovic et al. 2002; Raudsepp et al. 2002) were ‘placed’
in the physically ordered scaffold of the RH type I markers (Fig. 1,
enclosed poster; see loci arranged next to colored vertical bars). The
253 ‘anchors’ aligning the RH and cytogenetic maps acted as guides to
deduce the most likely location of these FISH-mapped genes in
the assembly. For example, on ECA31, the FISH location of ‘anchor’
AHT34 helped to deduce the likely location of ESR as
distal to PCMT1. Similarly, the location of IGF2R was
inferred as proximal to VIP through the ‘anchor’
AHT033. Accordingly, a plausible order of three RH-mapped
(PLG, VIP, and PCMT1) and two previously
FISH-mapped genes (IGF2R and ESR) was derived. This
improved the comparative power of the ECA31 map from three to five
loci. Following this, a physically ordered collection of a total of 447
equine genes was obtained for comparison with the human and mouse gene
maps.
Based on the comparative location of the 447 equine loci, a total of 44
conserved syntenies (two or more pairs of homologous genes
located on the same chromosome regardless of order; Nadeau and Sankoff
1998; denoted by color bars next to the RH map in Fig. 1, enclosed
poster) were identified between the human and the horse genomes.
Additionally, eight smaller homology segments (one pair of
homologous genes in two species; Nadeau and Sankoff 1998) comprising a
single locus were found on some chromosomes, for example, ECA1
(COMT) and ECA2 (ACADL). Five of these segments
originate from results compiled from Milenkovic et al. (2002), and two
are novel. The latter are based on the mapping of FABP3
(ECA2q-HSA1) and ARPC3 (ECA9q-HSA12). Compared to this, the
horse and mouse genomes demonstrated a total of 71 conserved syntenies
(comprising 2 loci; see mouse chromosomal location of equine
orthologs in Fig. 1, enclosed poster) and 41 homology segments (each
comprising a single comparatively mapped locus) between the two
species. Overall, the horse–human conserved syntenies were larger than
the horse–mouse conserved syntenies. Nonetheless, some entire
chromosomes or chromosome arms (e.g., ECA17-HSA13-MMU11 and
ECA22-HSA20, part of MMU2) were completely conserved among the three
species.
In order to produce a refined comparative map, the precise sequence
locations of human and mouse orthologs for all the 447 physically
arranged equine genes were obtained from the available draft sequence
data (http://genome.ucsc.edu; version June 2002 for human and February
2002 for mouse). If sequence locations of a group of human or mouse
orthologs indicated conservation of gene order in relation to the
derived order of equine genes, the data were clustered in boxes (see
Fig. 1, enclosed poster). These clusters, referred to as conserved
linkages (maximally contiguous chromosomal region with identical gene
content and order; Nadeau and Sankoff 1998), showed a group of genes
with similar physical order in horse–human or horse–mouse. In the
majority of the cases, clustering highlighted smaller evolutionarily
conserved segments within the larger conserved syntenies.
Within the 44 horse–human conserved syntenies, 84 distinct clusters or
conserved linkages of human loci were established where the gene order
was conserved between the two species. These conserved linkages
included 87% (391/447) of the compared loci. Conversely, the 71
horse–mouse conserved syntenies split into 80 conserved linkages that
shared highly conserved gene order with the horse. However, it is
noteworthy that, on average, these conserved linkages were smaller than
those observed between horse and human, and included only 66%
(297/452) of the compared loci. It was observed that for some of the
equine chromosomes (e.g., ECA18 and ECA21), the comparative order of
the human and mouse orthologs was exceptionally conserved with
available equine order. Contrary to this, considerable rearrangements
were detected in a few other chromosomes (e.g., ECA7 and ECA16).
Lastly, comparison of gene order of the 447 loci across horse, human,
and mouse showed 85 clusters of genes that had highly conserved gene
order in all three species (Fig. 1, enclosed poster; see yellow
horizontal shades across horse–human–mouse gene order). These
clusters of conserved linkages were demarcated when the gene order was
disrupted between syntenic loci in human or mouse. For example, on
ECAXq, the two distal clusters are separated because of gene order
disruption in the mouse at PAK3 and IGSF1. Of the 85
clusters, 59 had 3–10 ordered loci each, and 23 had two loci each
that were located close to each other among horse, human, and mouse.
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DISCUSSION
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This study provides the first whole-genome radiation hybrid map of
the equine genome. The map comprises a total of 730 markers that are
distributed over 31 equine autosomes and the X chromosome. This
represents a greater than twofold expansion over the previously
presented linkage (Guérin et al. 1999, 2003; Swinburne et al.
2000a) and comparative (Milenkovic et al. 2002) maps. Although the
locus order in the RH map is in general agreement with that reported in
published maps (Guérin et al. 1999, 2003; Swinburne et al.
2000a), some differences, mainly attributed to improved resolution, are
evident. Examples of these are shown in later sections. Most
significantly, for the first time, a true integration of the equine
genetic linkage, syntenic, and cytogenetic maps has been achieved, thus
producing a map that is, to date, the most comprehensive for this
species. Moreover, with greater than 258 physically ordered
gene-specific/EST markers, the map is hitherto the most current
linearly ordered comparative map of the horse genome.
In terms of number of mapped type I and type II loci, the RH map
generated in this study is comparable to contemporary first-generation
maps in several species (see Table 3; pig,
Hawken et al. 1999; cat, Murphy et al. 2000; dog, Priat et al. 1998;
zebrafish, Hukriede et al. 1999). Some of the maps, for example, those
for ECA1, 10, 11, 14, 16, and X are exceptionally well covered with
markers compared to the current status for these chromosomes in the
horse. However, other chromosomes (e.g., ECA12 and ECA23 to ECA30) need
new gene-specific markers, and there are still others that require type
II polymorphic markers (e.g., ECA28). On the whole, the map is an
important foundation upon which a detailed map of the equine genome can
be built.
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Table 3. Comparative Statistics on Major Parameters of Whole-Genome RH Maps
(First Generation and Subsequent Maps) in Horse and Other
Species
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The number of RH groups reported in this study (101) is higher than
those reported for first-generation RH maps in other domesticated
species such as the dog (57, Priat et al. 1998) and cattle (61, Band et
al. 2000), but lower than those reported in pigs (128, Hawken et al.
1999) and zebrafish (190, Geisler et al. 1999). The average retention
frequency of markers in the horse RH panel is similar to the estimates
reported for dog, cattle, human, and zebrafish. However, higher
estimates have been reported for pig, cat, human, rat, and mouse (see
Table 3 for details).
Resolution
The resolution of the panel, often referred to as the shortest
physical distance that can be resolved using the panel (McCarthy et al.
1997), is projected as a function of average retention frequency and
the total length of the map in cR. In this study, 1 cR corresponds to
200 kb, implying that the average fragment size of retained horse
chromosomes in the panel is around 20 Mb. This divided by the number of
hybrid lines (92) x retention frequency (22/100 or .22) suggests a
resolution of 1 Mb for our panel. To our understanding, this
estimate is tentative, because actual cR distances are not available
for the entire length of all horse chromosomes. Presuming that at least
50 cR separates current RH groups, the 50–60 gaps between different RH
groups alone will contribute an additional 3000cR. This is bound to
deflate the current kb/cR estimates and show a lower
estimate (better resolution) than is now predicted. Thus, true
resolution of the panel will only be known when the density of markers
is improved and an enhanced framework map signifying accurate
intermarker distances is constructed.
Cytogenetic Alignment of RH Maps
While generating RH maps, accurate alignment of RH groups to
specific chromosomal regions is crucial. This alignment becomes even
more significant if there is limited support from the meiotic maps. The
currently available meiotic maps in the horse (Newmarket: Swinburne et
al. 2000a; International: Guérin et al. 1999, 2003)
are comprised of about 325 markers, of which only 85 have been
cytogenetically mapped (on average 2–3 markers/chromosome). The RH map
presented here marks a threefold increase in the number of markers,
with a total of 253 of the typed markers aligned by FISH, and 69
markers reported for the first time. This significantly facilitated the
placement and orientation of different RH groups, and provided
validation for the suggested order. Although the cytogenetic alignment
of the RH map is fairly uniform along the length of the majority of the
chromosomes, some chromosomes (e.g., ECA25–ECA30) and chromosomal
regions (e.g., middle part of ECA6, ECA7, ECA10, and ECA17) require
additional FISH markers.
FISH localizations of some of the loci reported earlier do not agree
with the proposed RH order. For example, assignment of microsatellite
AHT27 to ECA12q (Swinburne et al. 2000b) does not agree with
the RH markers assigned to this arm. Instead, the locus shows close
linkage with microsatellites SG10 and RKJ12, of which
the former is FISH-mapped to the short arm. It is therefore likely that
due to misidentification of the arm (common for small metacentric
chromosomes), AHT27 was allocated to the long arm. By moving
the locus to a corresponding position on the short arm, the FISH and RH
maps align perfectly. Whether a similar situation also exists for
MT2A on ECA13p should be verified, because the human ortholog
for this equine gene is on HSA16, which shares homology with the long
arm of the chromosome and not the short arm (Raudsepp et al. 1996).
Localization of F11 is discussed in greater detail in the
Comparative Map section below.
Integration of Different Maps: A Step Towards a Consensus Map
The RH map presented in this study represents the first organized
effort aimed at genome-wide integration of the syntenic, cytogenetic,
and meiotic maps of the horse into a single consensus map. Until now,
the synteny mapping approach in the horse (Caetano et al. 1999a; Shiue
et al. 1999) has been instrumental in assigning markers to specific
equine chromosomes, whereas meiotic linkage mapping (Newmarket:
Swinburne et al. 2000a; International: Guérin et al. 1999,
2003) has played an important role in identifying and
ordering linked markers. However, because very few linked markers are
cytogenetically aligned, it is difficult to ascertain the precise
physical span of individual linkage groups on the chromosomes. Earlier
attempts to improve this status were restricted to ECA1 and ECA10 on a
preliminary level (Kiguwa et al. 2000) and ECA11 (Chowdhary et al.
2002) and ECAX (Raudsepp et al. 2002) on a more extended level. Thus,
for the first time, 340 meiotic, 395 syntenic, and >400 cytogenetic
markers are integrated, resulting in consensus maps for each of the
equine chromosomes. These maps will be extremely useful in devising
strategies to close in on genes governing traits of interest in the
horse.
Improved Resolution Over Previous Linkage, Cytogenetic, and RH Maps
The RH map presented herein helps resolve the order of a number of
markers clustered at one location in the reported cytogenetic (Godard
et al. 2000; Mariat et al. 2001; Milenkovic et al. 2002; this study)
and linkage maps (Lindgren et al. 1998; Swinburne et al. 2000a;
Guérin et al. 1999, 2003). For example, the RH map
linearly orders markers that are FISH-mapped to overlapping bands
(e.g., COR46-LEX58-TJP1-1CA43 on ECA1p) or even to the same
band of the chromosome (e.g., 1CA44-1CA30 and
CA487-VHL134 on ECA1p and COR006-HMS7 on ECA1q).
Likewise, compared to previously published linkage (Swinburne et al.
2000a) and RH (Kiguwa et al. 2000) maps, the current RH map of, for
example, ECA1 resolves and rearranges the order of a number of markers
clustered at the proximal (e.g., 1CA44-ASB41-1CA30) and distal
ends (e.g., 1CA16, HMS7, COR006, and
1CA40) of the chromosome (see Fig. 1, enclosed poster).
Similarly, the RH map readily resolves the order of meiotic map markers
UCD304-HMS05-HTG15 clustered on ECA5 (Swinburne et al. 2000a),
and assigns them to the proximal part of ECA5q—which could not be done
previously.
The first-generation RH map shows marked improvement over the recently
published 3000rad preliminary RH maps of ECA1 and ECA10 (Kiguwa et al.
2000). On both chromosomes, the total number of mapped markers is
almost doubled, new polymorphic markers are included, and several new
FISH localizations have been added. With regard to the very recently
published maps of ECA11 (Chowdhary et al. 2002) and ECAX (Raudsepp et
al. 2002), the new maps show reasonable improvement. Major
contributions include the addition of eight genes/ESTs to ECA11 and
four new genes and five microsatellite markers to ECAX. Except for
minor flips involving closely linked loci, the overall order between
the new and the previous RH maps is preserved on both chromosomes.
The Comparative Map
Together with the 258 linearly ordered type I loci in the RH map,
the compilation of all the mapped equine genes provided a total of 447
gene-based markers that facilitated the generation of the most
comprehensive comparative map between horse and human to date, and the
first detailed comparative map between horse and mouse. This represents
185 more genes compared to the most recent horse–human comparative
map (Milenkovic et al. 2002). Although the assembly of the equine genes
does not provide precise linear order, it does offer a reliable working
framework for comparison. Within this framework, no drastic locus order
changes are expected, especially within the conserved syntenic blocks,
because the overall order is strongly supported with 256 linearly
arranged RH-mapped loci and the 191 moderately well positioned FISH
markers.
Of considerable significance is the mapping of the first equine gene
(F11) to ECA27. This helped infer likely correspondence of the
latter with part of HSA4. Earlier comparative studies (Raudsepp et al.
1996; Caetano et al. 1999a; Godard et al. 2000; Lear et al. 2001;
Mariat et al. 2001; Milenkovic et al. 2002) could not detect
equivalence of this chromosome with any of the human or mouse
chromosomes. It is noteworthy that using a goat BAC clone, Milenkovic
et al. (2002) FISH-mapped F11 to ECA3. However, we found the
locus to be tightly linked to the RH group assigned to ECA27. Further,
most of the loci in the RH group are also in the genetic linkage group
assigned to this chromosome (Swinburne et al. 2000a). Thus it seems
that the goat BAC used by Milenkovic et al. (2002) resulted in an
erroneous assignment of the locus to ECA3. Sequencing of the PCR
product obtained during RH typing further verified the locus to be
indeed F11.
The overall number of major horse–human conserved syntenies
reported in the present study is in close agreement with earlier
Zoo-FISH findings (Raudsepp et al. 1996). However, the figure is less
than half of the 113 conserved segments reported in the two species by
Milenkovic et al. (2002). This large discordance is primarily
related to the way the segments were counted in the two studies.
Schibler et al. (1998), Band et al. (2000), Pinton et al. (2000), and
Milenkovic et al. (2002) divided large conserved syntenies into smaller
segments on the basis of preserved or disrupted chromosome band
order of the mapped loci in the human. This resulted in a
significantly higher count of conserved segments in all four species
(goat, 107; cattle, 105; pig, 84; horse, 113). Contrary to this,
Watanabe et al. (1999) disrupted conserved syntenies only when linearly
arranged rat orthologs were from different human/mouse chromosomes, an
approach followed in the present study. This explains why, for example,
with only 12 mapped markers each on ECA11 and ECAX, Milenkovic et al.
(2002) show 6–7 conserved segments for individual human counterparts
(HSA17 and HSAX, respectively), compared to only one shown in the
present study despite twice the amount of data. However, if the 84
horse–human conserved linkages we observed are compared with the
number of conserved segments reported in different livestock species
(Schibler et al. 1998; Band et al. 2000; Pinton et al. 2000; Milenkovic
et al. 2002), our estimates are at the lower end of the spectrum.
The most striking genome conservation between horse and human was seen
on ECA4, 5, 17, 18, 21, 25, and 31 and their human counterparts (see
Fig. 1, enclosed poster), where the derived equine gene order closely
corresponded to the order reported in the draft sequence of the human
genome. A similar situation was also observed on ECA3p, ECA6p, and
ECA14. However, of greatest significance was the striking conservation
of gene order among ECA3p, ECA6p, and ECA22 genes and their human and
mouse homologs: HSA16q/part MMU8, HSA2q/part MMU1, and HSA20/MMU2,
respectively. Incidentally, HSA20 represents one of the most conserved
mammalian autosomes (Chowdhary et al. 1998; Haig 1999). Similarly,
genomic segments corresponding to HSA16q and HSA2q are considered
evolutionarily highly conserved (Chowdhary et al. 1998;
http://www.informatics.jax.org/menus/homology_menu.shtml, Murphy et al.
2000). Detection of conserved gene order for these segments in the
horse is novel and reiterates their ancestral status. Contrary to this,
gene order within some of the large conserved syntenies in the horse
(e.g., entire ECA16 corresponds to HSA3p or 3q) was found to be
considerably rearranged in relation to the observed order of the same
genes in human and mouse. This is evident from the greater number of
conserved linkages for this chromosome compared to those seen on other
chromosomes (see Fig. 1, enclosed poster).
The identification of 85 clusters of loci demonstrating gene order
conservation (conserved linkages) across horse, human, and mouse
(yellow-shaded regions, Fig. 1, enclosed poster) was extremely
significant. Basically, these clusters are core blocks within the
conserved syntenies, where the gene order is reasonably well preserved
across evolutionarily diverged species. Hence it is tempting to
speculate that these segments represent potentially the most conserved
genomic regions of the ancestor common to horse, human, and mouse.
Discovery of these conserved linkages provides a quick comparative
overview of smaller genomic blocks shared among the three species and
signifies an important advance in accurate alignment of the three
genomes.
Overview
The RH and comparative map presented here is the most
elaborate and dense map in the horse produced to date. Among farm
animals, the map ranks fifth after cattle, dog, chicken and pig.
Translating the quote of Flaherty and Herron (1998) for the mouse RH
map, the horse map is the "new kid on the block". This new
comprehensive and integrated map will enable equine geneticists to
perform a range of studies at a resolution not available earlier.
Because the map is linearly ordered and reasonably well aligned along
the length of individual equine chromosomes, its overall ability to
facilitate accurate mapping of markers/traits of interest is greatly
enhanced. Moreover, with the largest assembly of type I markers in the
horse, the alignment of the horse genome with human and mouse genomes
is markedly refined. This will facilitate the use of the highly
developed human and mouse gene maps in searching for candidate gene(s)
implicated in various inherited conditions important to the equine
industry. In evolutionary terms, the findings are significant in
providing upgraded comparative information on the genome organization
of an additional mammalian order—the Perissodactyla, for which such
detailed information was not available earlier.
Finally, with the availability of an informative RH panel to the
equine gene-mapping community worldwide, and the installation of an
instant two-point linkage output Web interface (installation in
progress), investigators in any country will be able to readily map any
locus of interest to the equine genome. This will certainly speed up
the rapid determination of likely locations of candidate genes, and
facilitate positional cloning.
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METHODS
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|---|
Markers for RH Mapping
A total of 901 markers chosen from a variety of sources were used
in this study. Primer pairs for the majority of the markers were
available either from databases (HorseBase:
http://locus.jouy.inra.fr/cgi-bin/lgbc/mapping/common/intro2.pl?BASE=horse,
http://roslin.thearkdb.org/; NCBI: http://www.ncbi.nlm.nih.gov) or
from published papers and personal resources (Dr. R.
Brandon, University of Queensland, Australia). ESTs were generated from
an equine testis cDNA library (kindly provided by Dr. N.
Ing, Texas A&M University, Texas). Sequencing of 3360
clones yielded 2090 high-quality sequences. Both ends of each sequence
were compared against dbEST and databases of GenBank using BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/) to produce 1732 equine sequences
with significant hits (90% or more sequence similarity) to human genes
or ESTs. Redundant sequences, especially those representing
mitochondrial DNA or genome-wide gene families such as ribosomal
RNA, were discarded. On the basis of human orthologs and horse-human
Zoo-FISH data (Raudsepp et al. 1996), the sequences were classified
into groups that would most likely map to a specific horse chromosome.
Primer pairs were designed in the 3'UTR, using Primer3 software
(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) to
produce a PCR amplification product of 150–300 bp in size. This
yielded 76 primer pairs, of which 28 were typed on the RH panel.
Lastly, primer pairs were designed for two putative equine orthologs
using alignments of human, mouse, and other mammalian sequences to
identify conserved regions. A chromosome-wise list of all the markers
included in this study, along with details on origin/source, symbols,
primer sequences, PCR conditions, and references is presented in Table
1.
RH Panel Typing and Analysis
A 5000rad whole-genome RH panel comprised of 92 hybrid cell lines
was typed by PCR as described (Chowdhary et al. 2002). PCR conditions
for each of the primer pairs were optimized so that only horse-specific
amplification products were obtained. All markers were typed in
duplicate along with horse, hamster, and a ‘no-DNA’ control. PCR
products were resolved on 2.5% agarose gels (containing 0.25µg/mL
ethidium bromide) and scored manually.
Initially markers were assigned to groups on the basis of confirmed
mapping data (linkage, synteny, cytogenetic, and Zoo-FISH). Using
RHMAPPER software (Slonim et al. 1997), markers with unknown locations
were assigned to individual chromosomes at lod 11.0. Following
this, RHMAP 3.0 software (Boehnke 1992; Lunetta et al. 1995) was used
for all analyses. RH groups within individual chromosomes were obtained
at lod 7.0 using the RH2PT program. Frameworks were obtained at
lod 3 (1000:1), and a comprehensive map was built by placing the
remaining markers in relation to the framework map. RH groups with no
framework markers were ordered at a threshold of lower than 1000:1.
Orientation of multiple RH groups on a single chromosome was
accomplished using cytogenetic and linkage data and 2pt lod score
values of the end marker with markers in adjacent groups. RH groups
with no cytogenetic alignment were placed solely on the basis of
linkage data (Lindgren et al. 1998; Swinburne et al. 2000a;
Guérin et al. 1999, 2003).
BAC Library Screening and Fluorescent In Situ Hybridization (FISH)
The INRA equine BAC library was screened by PCR for 78 markers (37
genes and 41 microsatellites) as described in detail elsewhere (see
Milenkovic et al. 2002). Briefly, PCR primers for individual markers
were used to identify positive clones. These clones were grown
overnight, and DNA was isolated from each of them. Approximately 1 µg
DNA from each of the BACs was biotin-labeled, dissolved in 20 µL
hybridization mix (50% formamide, 2 x SSC, 10% dextran sulfate),
and hybridized to horse metaphase spreads. The signals were detected
with FITC-conjugated antibodies, and chromosomes were counterstained
with DAPI. Hybridization results were examined and analyzed using a
Zeiss Axioplan2 fluorescent microscope and Cytovision/Genus application
software version 2.7 (Applied Imaging).
Synteny Mapping
The UC-Davis somatic cell hybrid synteny panel is described
elsewhere (Caetano et al. 1999a,b; Shiue et al. 1999). A modified panel
representing a more unique set of 70 clones (vs. the 108 hybrid clones
present in the original panel) was used. A total of 39 equine
genes/ESTs were synteny-mapped. Primer pairs for 33 ESTs were from a
horse fetal cDNA library (60-day whole fetus; Dr. R. Brandon,
University of Queensland, Australia). Primers for six genes
were generated in the following way: CATS (Lyons et al. 1997) or
universal primers (Venta et al. 1996) were used to amplify
horse-specific PCR product. The PCR product was sequenced, and
horse-specific primers were then designed. Details on gene names,
primer pairs, PCR conditions, etc. are provided in Table 1. Following
typing of the panel by PCR, the products were run on a 2% agarose gel,
and the results were scored as + or –. Correlation coefficients were
calculated between all markers as described (Caetano et al. 1999a,b). A
correlation value of 0.70 was accepted as evidence for synteny between
two markers (Chevalet and Corpet 1986).
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WEB SITE REFERENCES
|
|---|
http://genome.ucsc.edu; human genome browser.
http://www.informatics.jax.org/menus/homology_menu.shtml; mammalian
homology and comparative maps.
http://locus.jouy.inra.fr/cgi-bin/lgbc/mapping/common/intro2.pl?
BASE=horse;
horsemap database.
http://roslin.thearkdb.org/; ARKdb farm animal genome database.
http://www.ncbi.nlm.nih.gov; National Center for Biotechnology
Information (NCBI) databases.
http://www.ncbi.nlm.nih.gov/BLAST/; BLAST search programs.
http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi; NCBI
database for primer design.
|
Acknowledgements
|
|---|
This project was funded by grants from the Texas Higher Education
Board (ARP 010366-0162-2001), NRICGP/USDA Grant 2000-03510 (LCS), Texas
Equine Research Foundation (BPC, LCS), Link Endowment (BPC, LCS), The
Morris Animal Foundation, and the Dorothy Russell Havemeyer Foundation.
Additional support was available from the USDA-NRSP-8 Coordinators
Fund. We are extremely grateful to Drs. Richard Brandon, Gabriella
Lindgren, and Imke Tammen for providing equine primer pairs for various
markers. Dee Honeycutt is gratefully acknowledged for her excellent
management of the horse RH panel.
The publication costs of this article were defrayed in part by payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
|
Footnotes
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|---|
10 Corresponding author. 
E-MAIL bchowdhary{at}cvm.tamu.edu; FAX (979) 845-9972.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.917503.
|
REFERENCES
|
|---|
Band, M.R., Larson, J.H., Rebeiz, M., Green, C.A., Heyen, D.W., Donovan, J., Windish, R., Steining, C., Mahyuddin, P., Womack, J.E., et al. 2000. An ordered comparative map of the cattle and human genomes. Genome Res. 10: 1359-1368.[Abstract/Free Full Text]
Boehnke, M. 1992. Multipoint analysis for radiation hybrid mapping. Ann. Med. 24: 383-386.[Medline]
Breen, M., Lindgren, G., Binns, M.M., Norman, J., Irvin, Z., Bell, K., Sandberg, K., and Ellegren, H. 1997. Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping. Mamm. Genome 8: 267-273.[CrossRef][Medline]
Breen, M., Jouquand, S., Renier, C., Mellersh, C.S., Hitte, C., Holmes, N.G., Cheron, A., Suter, N., Vignaux, F., Bristow, A.E., et al. 2001. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res. 11: 1784-1795.[Abstract/Free Full Text]
Caetano, A.R., Shiue, Y.L., Lyons, L.A., O'Brien, S.J., Laughlin, T.F., Bowling, A.T., and Murray, J.D. 1999a. A comparative gene map of the horse (Equus caballus). Genome Res. 9: 1239-1249.[Abstract/Free Full Text]
Caetano, A.R., Lyons, L.A., Laughlin, T.F., O'Brien, S.J., Murray, J.D., and Bowling, A.T. 1999b. Equine synteny mapping of comparative anchor tagged sequences (CATS) from human Chromosome 5. Mamm. Genome 10: 1082-1084.[CrossRef][Medline]
Chevalet, C. and Corpet, F. 1986. Statistical decision rules concerning synteny or independence between markers. Cytogenet. Cell Genet. 43: 132-139.[Medline]
Chowdhary, B.P. and Raudsepp, T. 2000. Cytogenetics and physical gene maps. In: In The genetics of the horse (eds. A.T. Bowling & A. Ruvinsky), pp. 171–243. CABI Publishing, New York, NY.
Chowdhary, B.P., Raudsepp, T., Fronicke, L., and Scherthan, H. 1998. Emerging patterns of comparative genome organization in some mammalian species as revealed by Zoo-FISH. Genome Res. 8: 577-589.[Abstract/Free Full Text]
Chowdhary, B.P., Raudsepp, T., Honeycutt, D., Owens, E.K., Piumi, F., Guérin, G., Matise, T.C., Kata, S.R., Womack, J.E., and Skow, L.C. 2002. Construction of a 5000(rad) whole-genome radiation hybrid panel in the horse and generation of a comprehensive and comparative map for ECA11. Mamm. Genome 13: 89-94.[CrossRef][Medline]
Flaherty, L. and Herron, B. 1998. The new kid on the block—A whole genome mouse radiation hybrid panel. Mamm. Genome 9: 417-418.[CrossRef][Medline]
Geisler, R., Rauch, G.J., Baier, H., van Bebber, F., Brobeta, L., Dekens, M.P., Finger, K., Fricke, C., Gates, M.A., Geiger, H., et al. 1999. A radiation hybrid map of the zebrafish genome. Nat. Genet. 23: 86-89.[CrossRef][Medline]
Godard, S., Vaiman, A., Schibler, L., Mariat, D., Vaiman, D., Cribiu, E.P., and Guérin, G. 2000. Cytogenetic localization of 44 new coding sequences in the horse. Mamm. Genome. 11: 1093-1097.[CrossRef][Medline]
Guérin, G., Bailey, E., Bernoco, D., Anderson, I., Antczak, D.F., Bell, K., Binns, M.M., Bowling, A.T., Brandon, R., Cholewinski, G., et al. 1999. Report of the International Equine Gene Mapping Workshop: Male linkage map. Anim. Genet. 30: 341-354.[CrossRef][Medline]
Guérin, G., Bailey, E., Bernoco, D., Anderson, I., Antczak, D.F., Bell, K., Biros, I., Bjornstad, G., Bowling, A.T., Brandon, R., et al. 2003. The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map. Anim. Genet. (in press).
Gyapay, G., Schmitt, K., Fizames, C., Jones, H., Vega-Czarny, N., Spillett, D., Muselet, D., Prud'homme, J.F., Dib, C., Auffray, C., et al. 1996. A radiation hybrid map of the human genome. Hum. Mol. Genet. 5: 339-346.[Abstract/Free Full Text]
Haig, D. 1999. A brief history of human autosomes. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 354: 1447-1470.[CrossRef][Medline]
Hawken, R.J., Murtaugh, J., Flickinger, G.H., Yerle, M., Robic, A., Milan, D., Gellin, J., Beattie, C.W., Schook, L.B., and Alexander, L.J. 1999. A first-generation porcine whole-genome radiation hybrid map. Mamm. Genome 10: 824-830.[CrossRef][Medline]
Heinzerling, L.M., Feige, K., Rieder, S., Akens, M.K., Dummer, R., Stranzinger, G., and Moelling, K. 2001. Tumor regression induced by intratumoral injection of DNA coding for human interleukin 12 into melanoma metastases in gray horses. J. Mol. Med. 78: 692-702.[CrossRef][Medline]
Hopman, T.J., Han, E.B., Story, M.R., Schug, M.D., Aquadro, C.F., Bowling, A.T., Murray, J.D., Caetano, A.R., and Antczak, D.F. 1999. Equine dinucleotide repeat loci COR001-COR020. Anim. Genet. 30: 225-226.[CrossRef][Medline]
Hukriede, N.A., Joly, L., Tsang, M., Miles, J., Tellis, P., Epstein, J.A., Barbazuk, W.B., Li, F.N., Paw, B., Postlethwait, J.H., et al. 1999. Radiation hybrid mapping of the zebrafish genome. Proc. Natl. Acad. Sci. 96: 9745-9750.[Abstract/Free Full Text]
Irvin, Z., Giffard, J., Brandon, R., Breen, M., and Bell, K. 1998. Equine dinucleotide repeat polymorphisms at loci ASB 21, 23, 25 and 37–43. Anim. Genet. 29: 67.
ISCNH 1997. International System for Cytogenetic Nomenclature of the Domestic Horse. Bowling, A.T., Breen, M., Chowdhary, B.P., Hirota, K., Lear, T., Millon, L.V., Ponce de Leon, F.A., Raudsepp, T., and Stranzinger, G. (Committee). Chromosome Res. 5: 433-443.[CrossRef][Medline]
Kiguwa, S.L., Hextall, P., Smith, A.L., Critcher, R., Swinburne, J., Millon, L., Binns, M.M., Goodfellow, P.N., McCarthy, L.C., Farr, C.J., et al. 2000. A horse whole-genome-radiation hybrid panel: Chromosome 1 and 10 preliminary maps. Mamm. Genome 11: 803-805.[CrossRef][Medline]
Lear, T.L., Brandon, R., and Bell, K. 1999. Physical mapping of ten equine dinucleotide repeat microsatellites. Anim. Genet. 30: 235.
Lear, T.L., Brandon, R., Piumi, F., Terry, R.R., Guérin, G., Thomas, S., and Bailey, E. 2001. Mapping of 31 horse genes in BACs by FISH. Chromosome Res. 9: 261-262.[CrossRef][Medline]
Lindgren, G., Sandberg, K., Persson, H., Marklund, S., Breen, M., Sandgren, B., Carlsten, J., and Ellegren, H. 1998. A primary male autosomal linkage map of the horse genome. Genome Res. 8: 951-966.[Abstract/Free Full Text]
Lunetta, K.L., Boehnke, M., Lange, K., and Cox, D.R. 1995. Experimental design and error detection for polyploid radiation hybrid mapping. Genome Res. 5: 151-163.[Abstract/Free Full Text]
Lyons, L.A., Laughlin, T.F., Copeland, N.G., Jenkins, N.A., Womack, J.E., and O'Brien, S.J. 1997. Comparative anchor tagged sequences (CATS) for integrative mapping of mammalian genomes. Nat Genet. 15: 47-56.[CrossRef][Medline]
Mariat, D., Oustry-Vaiman, A., Cribiu, E.P., Raudsepp, T., Chowdhary, B.P., and Guérin, G. 2001. Isolation, characterization, and FISH assignments of horse BAC clones containing type I and II markers. Cytogenet. Cell Genet. 92: 144-148.[CrossRef][Medline]
McCarthy, L.C., Terrett, J., Davis, M.E., Knights, C.J., Smith, A.L., Critcher, R., Schmitt, K., Hudson, J., Spurr, N.K., and Goodfellow, P.N. 1997. A first-generation whole genome-radiation hybrid map spanning the mouse genome. Genome Res. 7: 1153-1161.[Abstract/Free Full Text]
Milenkovic, D., Oustry-Vaiman, A., Lear, L.T., Billault, A., Mariat, D., Piumi, F., Schibler, L., Cribiu, E., and Guérin, G. 2002. Cytogenetic localization of 136 genes in the horse: Comparative mapping with the human genome. Mamm. Genome 13: 524-534.[CrossRef][Medline]
Murphy, W.J., Sun, S., Chen, Z., Yuhki, N., Hirschmann, D., Menotti-Raymond, M., and O'Brien, S.J. 2000. A radiation hybrid map of the cat genome: Implications for comparative mapping. Genome Res. 10: 691-702.[Abstract/Free Full Text]
Nadeau, J.H. and Sankoff, D. 1998. Counting on comparative maps. Trends Genet. 14: 495-501.[CrossRef][Medline]
Pinton, P., Schibler, L., Cribiu, E., Gellin, J., and Yerle, M. 2000. Localization of 113 anchor loci in pigs: Improvement of the comparative map for humans, pigs, and goats. Mamm. Genome. 11: 306-315.[CrossRef][Medline]
Priat, C., Hitte, C., Vignaux, F., Renier, C., Jiang, Z., Jouquand, S., Cheron, A., Andre, C., and Galibert, F. 1998. A whole-genome radiation hybrid map of the dog genome. Genomics 54: 361-378.[CrossRef][Medline]
Raudsepp, T., Frönicke, L., Scherthan, H., Gustavsson, I., and Chowdhary, B.P. 1996. Zoo-FISH delineates conserved chromosomal segments in horse and man. Chromosome Res. 4: 218-225.[CrossRef][Medline]
Raudsepp, T., Kijas, J., Godard, S., Guérin, G., Andersson, L., and Chowdhary, B.P. 1999. Comparison of horse chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping. Mamm. Genome 10: 277-282.[CrossRef][Medline]
Raudsepp, T., Kata, S.R., Piumi, F., Swinburne, J., Womack, J.E., Skow, L.C., and Chowdhary, B.P. 2002. Conservation of gene order between horse and human X chromosomes as evidenced through radiation hybrid mapping. Genomics 79: 451-457.[CrossRef][Medline]
Rieder, S., Strickler, C., Jörg, H., Dummer, R., and Stranzinger, G. 2000. A comparative genetic approach for the investigation of aging gray horse melanoma. J. Anim. Breed. Genet. 117: 73-82.[CrossRef]
Ruth, L.S., Hopman, T.J., Schug, M.D., Aquadro, C.F., Bowling, A.T., Murray, J.D., Caetano, A.R., and Antczak, D.F. 1999. Equine dinucleotide repeat loci COR041-COR060. Anim. Genet. 30: 320-321.[CrossRef][Medline]
Schibler, L., Vaiman, D., Oustry, A., Giraud-Delville, C., and Cribiu, E.P. 1998. Comparative gene mapping: A fine-scale survey of chromosome rearrangements between ruminants and humans. Genome Res. 8: 901-915.[Abstract/Free Full Text]
Shiue, Y.L., Bickel, L.A., Caetano, A.R., Millon, L.V., Clark, R.S., Eggleston, M.L., Michelmore, R., Bailey, E., Guérin, G., Godard, S., et al. 1999. A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers. Anim. Genet. 30: 1-9.[CrossRef][Medline]
Slonim, D., Kruglyak, L., Stein, L., and Lander, E. 1997. Building human genome maps with radiation hybrids. J. Comput. Biol. 4: 487-504.[Medline]
Stewart, E.A., McKusick, K.B., Aggarwal, A., Bajorek, E., Brady, S., Chu, A., Fang, N., Hadley, D., Harris, M., Hussain, S., et al. 1997. An STS-based radiation hybrid map of the human genome. Genome Res. 7: 422-433.[Abstract/Free Full Text]
Swinburne, J.E., Marti, E., Breen, M., and Binns, M.M. 1997. Characterization of twelve new horse microsatellite loci: AHT12-AHT23. Anim. Genet. 28: 453.
Swinburne, J.E., Gerstenberg, C., Breen, M., Aldridge, V., Lockhart, L, Marti, E., Antczak, D., Eggleston-Stott, M., Bailey, E., Mickelson, J., et al. 2000a. First comprehensive low-density horse linkage map based on two 3-generation, full-sibling, cross-bred horse reference families. Genomics 66: 123-134.[CrossRef][Medline]
Swinburne, J.E., Lockhart, L., Aldridge, V., Marti, E., Breen, M., and Binns, M.M. 2000b. Characterization of 25 new physically mapped horse microsatellite loci: AHT24++-48. Anim. Genet. 31: 237-238.[Medline]
Swinburne, J.E., Turner, A., Alexander, L.J., Mickleson, J.R., and Binns, M.M. 2003. 61 new horse microsatellite loci: AHT49-109. Anim. Genet. (in press).
Van Etten, W.J., Steen, R.G., Nguyen, H., Castle, A.B., Slonim, D.K., Ge, B., Nusbaum, C., Schuler, G.D., Lander, E.S., and Hudson, T.J. 1999. Radiation hybrid map of the mouse genome. Nat. Genet. 22: 384-387.[CrossRef][Medline]
Venta, P.J., Brouillette, J.A., Yuzbasiyan-Gurkan, V., and Brewer, G.J. 1996. Gene-specific universal mammalian sequence-tagged sites: Application to the canine genome. Biochem. Genet. 34: 321-341.[CrossRef][Medline]
Watanabe, T.K., Bihoreau, M.T., McCarthy, L.C., Kiguwa, S.L., Hishigaki, H., Tsuji, A., Browne, J., Yamasaki, Y., Mizoguchi-Miyakita, A., Oga, K., et al. 1999. A radiation hybrid map of the rat genome containing 5,255 markers. Nat. Genet. 22: 27-36.[CrossRef][Medline]
Williams, J.L., Eggen, A., Ferretti, L., Farr, C.J., Gautier, M., Amati, G., Ball, G., Caramorr, T., Critcher, R., Costa, S., et al. 2002. A bovine whole-genome radiation hybrid panel and outline map. Mamm. Genome 13: 469-474.[CrossRef][Medline]
Received October 16, 2002;
accepted in revised format December 30, 2002.
13:742-751 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00
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ABSTRACT
RESULTS