sevier.com/locate/ygeno

Genomics 87 (2

Single linkage group per chromosome genetic linkage map for the horse,

based on two three-generation, full-sibling,

crossbred horse reference families

June E. Swinburne a,1, Mike Boursnell a,*, Gemma Hill a, Louise Pettitt a, Twink Allen b,c,

Bhanu Chowdhary d, Telhisa Hasegawa e, Masahiko Kurosawa f, Tosso Leeb g, Suguru Mashima f,

James R. Mickelson h, Terje Raudsepp d, Teruaki Tozaki f, Matthew Binns a,2

a Animal Health Trust, Newmarket, Suffolk CB8 7UU, UKb T.B.A. Equine Fertility Unit, Newmarket, Suffolk CB8 9BH, UK

c Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UKd Department of Veterinary Integrative BioScience, College of Veterinary Medicine, Texas A and M University, College Station, TX 77843, USA

e Equine Research Institute, Japan Racing Association, Utsunomiya, Tochigi, Japanf Department of Molecular Genetics, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan

g Institute of Animal Breeding and Genetics, School of Veterinary Medicine Hannover, Bunteweg 17p, 30559 Hannover, Germanyh College of Veterinary Medicine, 1988 Fitch Avenue, University of Minnesota, St. Paul, MN 55108, USA

Received 14 February 2005; accepted 3 September 2005

Available online 28 November 2005

Abstract

A genetic linkage map of the horse consisting of 742 markers, which comprises a single linkage group for each of the autosomes and the X

chromosome, is presented. The map has been generated from two three-generation full-sibling reference families, sired by the same stallion, in

which there are 61 individuals in the F2 generation. Each linkage group has been assigned to a chromosome and oriented with reference to

markers mapped by fluorescence in situ hybridization. The average interval between markers is 3.7 cM and the linkage groups collectively span

2772 cM. The 742 markers comprise 734 microsatellite and 8 gene-based markers. The utility of the microsatellite markers for comparative

mapping has been significantly enhanced by comparing their flanking sequences with the human genome sequence; this enabled conserved

segments between human and horse to be identified. The new map provides a valuable resource for genetically mapping traits of interest in the

horse.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Horse; Linkage map; Genetic map; Comparative map; Radiation hybrid map

Comprehensive genetic linkage maps consisting of 1000+

microsatellites are now available for species such as cattle [1],

pig [2], sheep [3], and chicken [4]. These typically cover 2286

(pig) to 3800 cM (chicken). Molecular genetic investigations of

the horse have lagged behind these agricultural animals. One

reason for this is that the horse is not primarily considered

0888-7543/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygeno.2005.09.001

* Corresponding author.

E-mail addresses: june.swinburne@aht.org.uk (J.E. Swinburne),

mike.boursnell@aht.org.uk (M. Boursnell).1 Fax: +44 1638 750794.2 Present address: The Royal Veterinary College, Royal College Road,

London NW1 0TU, UK.

either a significant food production animal or a pet. The horse

does, however, occupy a unique position in the history of

civilized man, having played a vital role in many spheres of

human endeavor from transportation to warfare and from

communication to colonization. During the 20th century the

status of the horse largely changed from a beast of burden to a

competitor in leisure and sporting activities and, in this role, it

has become a prized possession. Many of the most valuable

individual animals in the world are Thoroughbred racehorses

and, as such, the health and welfare of the Thoroughbred horse

is of major economic concern.

There are several common conditions affecting horses that

have an inherited component, including rhabdomyolysis

006) 1 – 29

www.el

J.E. Swinburne et al. / Genomics 87 (2006) 1–292

(‘‘tying-up’’), laryngeal hemiplegia (‘‘roaring’’), osteochondro-

sis dissecans, and recurrent airway obstruction. These diseases

provide an incentive to study the genetics of the horse

generally and the Thoroughbred in particular. The identifica-

tion of the inherited components involved in such conditions

could lead to improvements in health and welfare, through

selective breeding of individuals not carrying disease muta-

tions. Studbook records should provide a valuable reference

guide for the assembling of pedigrees for mapping purposes.

For example, the General Studbook provides an account of the

pedigrees of today_s Thoroughbred horses that stretches back

over more than 30 generations, to the very foundation of the

breed. In addition, every Thoroughbred horse registered in the

General Studbook over the past 30 years has been parentage

tested, thereby providing a verified pedigree with which to

work. These two factors should facilitate genetic studies of

complex genetic diseases in the Thoroughbred breed, when

accompanied by appropriate DNA collections from accurately

phenotyped horses.

Scientific interest in the genetics of the horse has increased

within the past decade. An International Equine Gene Mapping

Workshop was established in Lexington in October 1995 and

since that time substantial advances toward an integrated

genetic map suitable for the mapping of mutations and traits of

interest have been made. Specifically, the development of a cell

hybrid panel for synteny analysis [5], genetic linkage maps

[6–9], physical mapping resources [10–15], an equine BAC

library (http://bacpac.chori.org/equine241.htm), and radiation

hybrid (RH) maps [16–19] has allowed the localization of

>1000 Type I and Type II markers. In addition, ZOO

fluorescence in situ hybridization (FISH) comparison of horse

and human chromosomes has provided information on chromo-

some segments that are evolutionarily conserved between these

two species [11,20,21]. More recently, RH mapping has been

used to produce comparative maps of horse with human

Fig. 1. Pedigree structure of the horse reference family used in this study. Two pairs o

used together with a single stallion (Thinur) to generate two families of full-sibling ho

All twin embryos retrieved were found to be dizygotic in origin. The breeds used in

Queenslander, Polish Warmblood; 5, Reynir fra Holum and Thorf, Icelandic Horse

[15,17–19,22] and mouse [19,22] genomes, thereby facilitat-

ing the exploitation of the genome sequence data available for

these species for the benefit of the horse. These various resources

were utilized during the present study to develop targeted

strategies to close gaps in the last three linkage groups, resulting

in single linkage groups for each autosome and the X

chromosome.

The efficient generation of the map was dependent upon the

use of a unique mapping reference family (Fig. 1). A detailed

description of the construction of the reference pedigree, whose

effectiveness centered upon the utilization of two pairs of

identical twin mares to produce large numbers of full siblings

in the form of preimplantation embryos, can be found in the

first version of the map [9]. The production of such a pedigree

would not have been possible using traditional breeding

methods. The distinctive structure of the pedigree has meant

that a relatively small number of individuals are required to

detect linkage, compared to the large half-sibling family groups

traditionally used to map the genomes of large uniparous

mammals. From the 742 markers now mapped, panels of

markers that are polymorphic in particular breeds can be

selected for whole genome scanning.

Historically, genetic linkage maps have been composed

largely of Type II microsatellite markers. The difficulties in

generating and scoring polymorphic markers within genes,

compared to the ease with which microsatellite markers can be

isolated, has hindered the placement of genes (Type I) onto

genetic linkage maps. In contrast, both Type I and Type II

markers can be easily placed onto RH maps, where there is no

requirement for polymorphism. The lack of genes on linkage

maps, coupled with the previous lack of identifiable relation-

ships between microsatellite markers of different species, has

made it difficult to align directly the genetic linkage maps of

different species. In this paper, sequences flanking almost half

of the equine microsatellites have been found to show high

f monozygotic twin mares (Cordelia and Regan, Ophelia and Desdemona) were

rse embryos. Females are depicted by circles and males are depicted by squares.

this pedigree are 1, Arab; 2, Concha, Thoroughbred; 3, Othello, Welsh Cob; 4,

.

J.E. Swinburne et al. / Genomics 87 (2006) 1–29 3

levels of identity to unique locations on the human genome.

When the locations of these markers were compared with

existing data describing horse–human conserved segments

[19,23], the map positions were found to be consistent in the

majority of cases. We have illustrated the linkage map with the

most current RH map [19] and comparative data to draw

together these three sets of information. Using this alignment,

useful comparisons between the horse linkage map, horse RH

map, and the human genome can be made.

This study uses a unique mapping reference pedigree to add

markers efficiently to the current equine linkage map. This

paper greatly extends previous genetic linkage maps of the

horse by adding almost 400 Type II markers, to bring the total

number of markers mapped to 742, and providing a total map

length of 2772 cM.

Results

Genotyping

New markers (n = 601) were tested on the stallion and the

twin mares to determine which were informative in the

reference family; 554 (92.2%) of them produced comprehen-

sible data. Ninety-five markers (17.1% of those scored) were

not polymorphic in the pedigree, consistent with previous

proportions of informative markers found in this crossbred

family. Nine markers were identified as duplicates of others

and were discarded. The remaining markers (n = 450) were

genotyped across the family and all the markers for which the

data were unambiguous were scored. However, a number of

markers were discarded, because the PCR amplification was

weak, the products were difficult to score, or the inheritance

was apparently non-Mendelian. Very rarely there were cases of

sporadic mutation (<10 from ¨100,000 datapoints), consistent

with an average mutation frequency for dinucleotide micro-

satellite repeats in the horse of approximately 1 � 10�4, and

there were no markers for which the identical twins differed in

genotype. Several markers segregated a null allele in this

pedigree; these were scored whenever the interpretation was

considered unambiguous. Reliable results were added to those

described by Swinburne and colleagues [9], bringing the

number of markers genotyped to 801.

Two-point analysis

Details of all the markers genotyped during this study are

listed in Supplementary Table 1, and they are described fully at

http://www.thearkdb.org/browser?species=horse and http://

locus.jouy.inra.fr/cgi-bin/horsemap/Horsemap/main.pl.

Of the 801 markers in the combined database, 794 (99.1%)

showed linkage with a LOD >3 to another marker. Linkage

groups were formed by the grouping of markers displaying

linkage greater than LOD 3 to others in the group. All bar 3 of

the 794 (i.e., 0.4%) were unambiguously assigned to a specific

linkage group. These 3 markers showed linkage to two

different groups and could not be definitively assigned to

either (Supplementary Table 1). Seven markers that showed no

linkage to another marker at LOD >3 were classed as

‘‘unassigned’’ (Supplementary Table 1). Thirty-two linkage

groups were generated, corresponding to each of the 31

autosomes and the X chromosome.

A number of spurious linkages of LOD 3–5.5 were

generated, which contradicted the overwhelming majority of

data. This is to be expected in a dataset of this size and, in

general, did not confuse the assignment of markers to linkage

groups, as these markers had much higher lod scores to other

markers. In one instance, however, the assignment of markers

to two chromosomes was not completely evident—this

involved several markers assigned to ECA13 and several

assigned to ECA22. After careful examination of the linkage

data, markers were assigned to specific chromosomes as

illustrated (Fig. 2), and this assignment agrees with the RH

map [19] (illustrated) with the exception of AHT030 and

AHT095. However, a more recent publication of a high-density

map of ECA22 [22] has repositioned these two misplaced

markers from ECA22 to ECA13, such that linkage, FISH, and

RH data now agree.

Assignment of linkage groups to chromosomes

All the linkage groups were assigned to chromosomes by

means of 159 FISH-mapped markers (not illustrated in Fig. 2).

Some of these markers were included in the previous mapping

paper [9] and, in addition, recent data were obtained from other

publications and personal communications (see Supplementary

Table 1). There were eight examples noted in the literature in

which the FISH positions contradicted the results of the linkage

analysis; these are highlighted with the word ‘‘inconsistency’’

in Supplementary Table 1. On closer examination it was clear

that some of these were mistakes made at the time of

chromosome identification, while the remainder were probably

due to chimerism in the FISH-mapped clone. Some of these

results have been double-checked and corrected in previous

publications (listed in Supplementary Table 1) and they are

described by Penedo and colleagues [24].

The linkage groups have been oriented with reference to

these FISH-mapped markers and are drawn in the same

orientation as other maps published recently [15,19,24]. For

several chromosomes there are few FISH-mapped microsa-

tellite markers, but the publication of numerous FISH-mapped

genes has allowed the unambiguous orientation of all linkage

groups, except for the small chromosomes ECA26, 27, 29,

and 30.

Targeted generation of microsatellites to close gaps in the map

At the stage in map building when there was a total of 36

linkage groups assigned to the 32 recombining chromosomes, a

targeted approach to closing the gaps was employed. At that

time ECA8 and ECA9 were each assigned two linkage groups

and ECA10 was assigned three. The strategies used the equine

BAC library (http://bacpac.chori.org/equine241.htm) to isolate

large insert clones from selected regions from which new

microsatellite markers could be isolated.

J.E. Swinburne et al. / Genomics 87 (2006) 1–294

BAC clones were isolated using overgo probes derived from

the following sequences: (1) microsatellite markers that were

placed in the regions on other maps, but were uninformative in

our family; (2) microsatellite markers at the ends of the linkage

groups on both sides of the gaps that were informative on only

one-half of the family, with a view to developing new markers

Fig. 2. Sex-averaged genetic linkage maps of the horse autosomes and female-specif

The positions of markers along the chromosome are shown in centimorgans (cM) to

ordered with a threshold of LOD >1 are shown to the right in vertical text. The adjac

indicated by a short horizontal line. The RH map [19] is shown to the far right. Color

the horse linkage map and the human genome sequence. The identity of the human

the human chromosome is shown in megabases. The maps were oriented by referen

from these BACs that were informative on both sides of the

family; (3) genes that were in gap regions based on the RH

map; and (4) equine BAC end sequences that were identified

by comparison with the human genome sequence as being

positioned in the gap regions. In addition, new equine

microsatellite sequences submitted to the sequence databases

ic map of the X chromosome are shown as gray bars in the center of each map.

the left. Framework markers are shown in bold italics. Markers that cannot be

ent vertical line describes their probable location with their most likely position

ed bars to the far left indicate proposed segments of conserved synteny between

chromosome is shown adjacent to the colored bars. The position of match with

ce to FISH-mapped markers (refer to Supplementary Table 1) and genes [19].

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–29 5

were screened by comparing them with the human genome

sequence to identify those markers likely to be situated on

ECA8, 9, and 10. Each of these strategies contributed new

microsatellite markers that led to the closure of all four gap

regions.

Construction of the map

Sex-averaged maps were constructed for each of the 31

autosomes and a female-specific map was made for the X

chromosome. Forty-nine markers were discarded during the

process of map construction due to them not being placed

unequivocally in one region of a linkage group. The maps

constructed from the remaining 742 markers are depicted in

Fig. 2. The linkage groups vary in length from 24 cM for

ECA26 to 194 cM for ECA1. The whole map measures

2772 cM.

The framework map, the order of which is significant at

LOD 3 (i.e., it is 1000 times more likely than any other

order), consists of 169 markers (22.8% of those assigned to

linkage groups) and is illustrated in bold italics in Fig. 2. The

comprehensive map, which is built around the framework

map and has an order significant at LOD 1, (i.e., it is 10

times more likely than any other order), consists of 507

markers (68.3% of those assigned to linkage groups). The

remaining markers, numbering 235 (31.7% of those assigned

to linkage groups), have been assigned their most probable

location.

There are 174 (23.4%) markers that share a map position

with one or more other markers. These markers were examined

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–296

in detail and eight pairs were found to be identical (UMNe448 =

HP027, UM002 = TKY297, LEX015 = LEX038, UMNe259 =

AHT076, AHT063 = AHT081, TKY328 = AHT102, AHT018 =

AHT100, TKY288 = LEX005). These are in addition to markers

that were already known to be identical and for which only one

of each pair was used in developing the present map (COR041 =

COR065, LEX024 = UM001, LEX062 = LEX066, COR055 =

COR060, COR83 = COR102, UM038 = SGCV031).

Comparative mapping

Of the 742 microsatellites included in this map, the

sequences of 610 (82.2%) had been submitted to GenBank

and were therefore available for comparative mapping using

the method described (see Materials and methods). The

BLAST comparison of their flanking sequences with the

human genome yielded 312 (51.1%) markers with significant

and unique matches and these are listed in Supplementary

Table 2.

When the conserved segments generated with these data

were compared with the most recent comparative maps (see

Materials and methods) it was found that 288 (92.3%) of the

markers described here fell into the same human segments as

those described in the previous publications. The remaining 24

markers (7.7%) were all single markers that did not agree with

adjacent markers. It may be that these are small regions that

have not previously been identified, or they could be spurious

results suggesting an incorrect match. Because these 24

markers did not have additional data to support their human

match, they have been included only as an Appendix to

Supplementary Table 2 and their matches have been omitted

from Fig. 2.

We did not observe several small segments identified by

Milenkovic and colleagues [15] and Chowdhary and collea-

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–29 7

gues [19], (i.e., HSA22 on ECA1, HSA2 and HSA8 on ECA2,

HSA3 on ECA3, HSA22 on ECA5, HSA19 on ECA7, HSA12

on ECA9, HSA9 on ECA19, HSA22 on ECA28), although all

but two of these segments were defined by only one marker.

These may be small conserved segments that were not detected

by the markers in this study. Similarly we did not observe four

segments of homology identified by ZOO-FISH [21], namely

HSA8 on ECA2, HSA19 on ECA7 and ECA21, and HSA22 on

ECA28.

Discussion

This genetic linkage map has been constructed using a

unique reference pedigree of two related full-sibling families,

the structure and composition of which have been refined in a

number of ways: (1) the founding horses represent a number of

disparate breeds in an attempt to maximize the polymorphism

present in the pedigree (Fig. 1); in particular the Icelandic

Horse, which has been geographically separated from other

European breeds for 900 years, should demonstrate significant

genetic divergence; (2) the use of full siblings required that

many fewer animals needed to be genotyped (n = 71) to detect

linkage, compared to previously published maps for the horse

that involved 271 and 458 half-sibling individuals [6–8]; (3)

the three-generation structure allowed phase to be determined;

(4) the genotyping of the dams in the pedigree allowed the

production of an X-chromosome map.

The resulting map improves upon the previous iteration [9]

by adding an additional 383 markers to bring the total number

mapped to 742. The markers comprise 717 that are autosomal

in origin and 25 that are X-linked (Fig. 2). Only 7 markers

(0.9%) did not display linkage with another marker at LOD >3.

These may lie within the existing linkage groups in regions

where the map is based on information from one side of the

reference family only, such that the recombination distance to

the next adjacent marker sharing information is too great to

produce a significant lod score. This situation was seen during

the cloning of markers in the last few gaps between linkage

groups on ECA8, 9, and 10, where two linkage groups

overlapped but did not join due to the lack of shared

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–298

information at the ends of the groups. In fact, 6 of these 7

markers are informative on one side of the family only and, in

addition, have an average number of informative meioses of

only 33.4 (range 18–61), compared to an average of 71.8

(range 16–144) among the whole dataset.

The generation of a single linkage group for each

chromosome is a great improvement on previously published

linkage maps and suggests that map coverage is extensive. The

average distance between markers on the map is 3.7 cM and the

total length of the linkage groups is 2772 cM. This is a

significantly shorter map length than the recent compilation

map described by Penedo and colleagues [24] of 3826 cM.

Possible reasons for the difference are discussed by Penedo and

colleagues [24] and principally include genotyping errors in the

data. Our map length of 2772 cM is, however, very close to the

total map length of 2720 cM estimated from chiasma counts in

horses [25].

As the number of typed markers increases, marker order can

be determined less accurately. This is due to fewer recombina-

tions occurring in the reference family between adjacent

markers. In some instances there will be no recombinations

between close markers, and this will result in these markers

being assigned the same map position. Indeed the stage will be

reached at which the number of recombinations present in this

pedigree is a limiting factor and the reward for adding

additional markers does not justify the effort. It may be that

this stage is approaching, though there remains value in rapidly

and accurately mapping markers to defined regions on the map,

to be used for fine mapping in other populations.

Marker placement was compared with the most recent

published RH map of the horse genome [19], and two

contradictions were observed. The first is COR006, which is

assigned to ECA20 here, but ECA1 on the RH map. The

linkage data placing COR006 on ECA20 is not unquestionable

(COR006�LEX071 Q = 0.12, LOD = 3.25; COR006 –UM031

Q = 0.12, LOD = 3.25), and the BLAST match of this marker to

HSA14 does suggest that it should be assigned to the p arm of

ECA1, which is where it is located on the RH map. The second

is TKY275, which is assigned to ECA8 here, but ECA26 on the

RH map. The linkage assignment of TKY275 to ECA8 is

strong, however, suggesting mislabeling is to blame. Compar-

ison with a recent compilation of linkage data from several

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–29 9

sources [24] shows only four discrepancies. These include our

assignment of TKY281 to ECA1 rather than ECA28, of UM031

to ECA20 rather than ECA26, of SGCV003 to ECA22 rather

than ECA13, and of TKY320 to ECA23 rather than ECA28.

Further examination has shown our linkage data to be solid for

the first two of these markers, though weaker for the second

two. It may well be that SGCV003 and TKY320 are incorrectly

assigned on this map. The discrepancies with TKY281 and

UM031 may lie with reagent or data mislabeling, but these

minor discrepancies are likely to be resolved with further

refinements of the maps.

In Fig. 2 the most recently published RH map [19] has been

cross-referenced with the linkage map. On the whole the

marker orders agree, and although there are localized regions

where marker order disagrees, this may merely be due to

anomalies in the maps or markers not being placed in an order

exceeding LOD 1. There are other occasions, however, in

which one RH group is flipped with respect to the linkage map;

obvious examples are the RH groups delineated by UMNe054

and COL1A2 on ECA4 and by AHT063 and COR039 on

ECA16. Occasionally, the linkage map suggests that the order

of RH groups along a chromosome should be changed, e.g.,

ECA15 and 16.

The use of BLAST comparisons to identify comparative

positions between Type II markers and the human genome

sequence has added greatly to the comparative map available

for the horse and complements the comparative map illustrated

by Chowdhary and colleagues [19], in which Type I markers

were used to provide points of homology between the two

species. The position has now been reached at which accurate

predictions can often be made as to the location of candidate

genes in the horse genome. Markers that are likely to be in the

correct region can be chosen for linkage mapping from the

2000+ Type II markers now available for the horse genome. In

addition, the comparative map will have great value in

suggesting candidate genes from genetic mapping projects

for which a critical interval has been narrowed to a manageable

size. The comparative map will therefore play a valuable role in

both candidate gene and positional cloning approaches to

disease gene identification, until a genome sequence is

available for the horse.

The conserved segments identified in the present study have

been drawn as contiguous segments (Fig. 2) wherever the data

suggest that this may be the case. It is premature to begin

estimating how many evolutionary break-points have occurred

between the horse and the human, as the maps are not yet

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–2910

detailed enough for this. However, 59 segments have been

drawn using this approach, with the reservation that 9 segments

are represented by only one marker.

Ideally those groups now actively involved in mapping in

the horse need to strive to integrate the data that are currently

available from linkage, RH, cytogenetic, and comparative

sources. The recent publications of an expansive RH map,

which integrates linkage, RH, and cytogenetic data [19], and an

integration of several linkage maps [24] have begun this

process. The amalgamation of mapping efforts across the

community is necessary to bring together the varied strands of

information to one centralized location for easy viewing and

interrogation. A possible vehicle for this may be the recently

developed viewing tool for Web-based access to published

maps (http://www.vgl.ucdavis.edu/equine/caballus/), which

provides a means for cross-referencing mapping data.

The maps generated for the horse genome are now reaching

a stage at which they can be utilized for the mapping of genes

involved in inherited diseases and traits. Panels of markers are

being developed for use in whole genome scanning projects

and, in addition, the emerging comparative map between horse

and human in particular provides access to suggestive

candidate genes in positional cloning experiments.

Materials and methods

Production of reference family DNA

The generation of the reference family material and the isolation of DNA

from it are described by Swinburne and colleagues [9]. The pedigree comprises

71 individuals consisting of 10 adult horses and 61 F2 offspring. The structure

of the family is shown in Fig. 1, and the name, breed, and sex of each

individual are listed.

Genotyping of microsatellites

Microsatellite markers were developed for the horse genome in a number of

laboratories worldwide and the primer pairs or primer sequences were sent to

the Animal Health Trust in Newmarket, England, for genotyping. A list of the

markers used in this study can be found in Supplementary Table 1 and they are

described in detail at http://www.thearkdb.org/browser?species=horse and

http://locus.jouy.inra.fr/cgi-bin/horsemap/Horsemap/main.pl.

Initially, markers were tested on the reference family stallion and mares

to confirm that they were informative in the pedigree. They were then

pooled into groups of either two or three pairs of primers prior to PCR or

were combined post-PCR, to maximize efficiency and reduce costs.

Fluorescence labeling of the PCR amplicon was achieved, by labeling

one primer either with 6-FAM, TET, HEX, TAMRA, JOE, or NED (for use

with the ABI3700 automated sequencer) or with 6-FAM, VIC, NED, or

PET (for use with the ABI3100 automated sequencer). Recently, the three-

primer methodology described by Schuelke [26] has been employed as an

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–29 11

extremely cost-effective and efficient means of fluorescently labeling PCR

fragments.

PCRs were performed in 0.2-ml thin-walled PCR plates (Alpha

Laboratories Ltd., Hampshire, UK), using 12-Al reaction volumes and the

MgCl2 concentrations suggested in the original marker report. All PCRs

were performed using 1.5 units AmpliTaq Gold (Applied Biosystems, Foster

City, CA, USA), 1� GeneAmp PCR buffer II (Applied Biosystems), and

200 AM each dNTP. For the majority of markers 10 pmol of each primer

was added to the reaction, except when the methodology of Schuelke [26]

was employed, in which case 5 pmol of reverse, 2 pmol of tailed forward,

and 10 pmol of the labeled third primer were added. An MJ Tetrad PCR

cycler (Bio-Rad Laboratories, Hercules, CA, USA) and a general PCR

program with variable annealing temperature (Ta) were used for all PCR

amplifications: 95-C for 10 min, followed by 30 cycles of 95-C for 30 s,

Ta for 30 s, and 72-C for 1 min and then 72-C for 10 min. However, when

the methodology of Schuelke [26] was employed the PCR program that

they specified was used (94-C for 4 min; followed by 30 cycles of 94-Cfor 1 min, 55-C for 1 min, and 72-C for 1 min; followed by 8 cycles of

94-C for 1 min, 50-C for 1 min, and 72-C for 1 min and then 72-C for

10 min).

PCRs were stored at �20-C before being analyzed on an ABI3700

(Applied Biosystems) or ABI3100 (Applied Biosystems) according to the

manufacturer_s instructions. Filter sets were chosen according to the dyes used

in the reaction. Dilution of the sample prior to loading on the ABI3700 was

determined empirically when required. Occasionally, the injection time was

altered when the ABI3100 was used to optimize the signal strength.

Genotyping data were collected and analyzed initially with GeneScan

(Applied Biosystems) and then with either Genotyper (Version 3.6 NT;

Applied Biosystems) or, more recently, GeneMapper (Version 3.0; Applied

Biosystems).

Biallelic marker typing

An SNP was identified in the equine IL4 gene and this was mapped onto

the reference family by sequencing PCR-amplified products. The PCR program

involved 94-C for 2 min; then 30 cycles of 94-C for 1 min, 55-C for 1 min, and

72-C for 4 min; and then 72-C for 10 min, which generated a product of about

2.5 kb. The G/A polymorphism was identified at a position about 300 bp into

the sequence generated using the reverse primer for sequencing

(TCTCTGGGGCG/AGTGTGTCA).

A randomly generated SNP named S22 was characterized by sequencing a

PCR product. The PCR program involved 95-C for 10 min; then 30 cycles of

95-C for 30 s, 55-C for 1 min, and 72-C for 1 min; and then 72-C for 5 min,

which generated a product of about 300 bp. The SNP produces a restriction site

for NlaIII (CATG) that can be assayed by restriction digest (CATCTTTAA-

TAACA/TTGTGGACCAATTGT).

Selection of BAC clones, subcloning, and the characterization of

microsatellites

Overgo probes [27] were designed from sequences flanking microsatellite

repeats and from equine BAC end sequences placed in gap regions by

comparative mapping. The two probes were combined in a stock solution at 10

pmol/Al and heated at 90-C for 2 min and then placed at room temperature for

10 min before being stored on ice. The labeling reaction was performed in a

10-Al volume with 1 Al oligonucleotide mix, 0.5 Al BSA (2 mg/ml), 4 Al ofoligo ligation buffer [27], 5 ACi [a-32P]dATP (110 TBq/mmol) (Amersham), 5

ACi [a-32P]dCTP (110 TBq/mmol) (Amersham), 2 units of Klenow fragment

DNA polymerase (Roche), and deionized water. The reaction was allowed to

proceed for 30 min at 37-C. After the addition of 2 Al cold dATP/dCTP mix

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–2912

(100 AM) the reaction was incubated for a further 30 min at 37-C. The probe

was purified by passing it through a G50 Sephadex column (Amersham) and

eluting it in 400 Al 1� SSC.

Equine BAC library filters (BACPAC Resources, Children_s Hospital,

Oakland, CA, USA; http://bacpac.chori.org/equine241.htm) were prehybri-

dized for at least 1 h at 60-C in 40 ml prewarmed Church buffer [28]. Overgo

probes were denatured for 10 min at 95-C and cooled on ice for 1 min before

being added to the prehybridization solution. Hybridization continued overnight

at 60-C with constant rotation. Filters were rinsed with 50 ml 2� SSC, 0.1% (w/

v) SDS; washed twice in 50 ml 2� SSC, 0.1% (w/v) SDS for 15 min at 60-C;

wrapped in Saran wrap; and exposed to X-ray film for 24 h at room temperature.

After processing, positive clone IDs were ascertained from the filters using the

grid position on the filter. Multiple clones were selected for each probe.

BAC DNAwas prepared using a PSIC Clone, Big BAC DNA kit (Princeton

Separations, Adelphia, NJ, USA) following the manufacturer_s instructions.

BAC DNA was digested with AluI and size fractionated by electrophoresis to

select fragments of 600–1000 bp, and the isolated fragments were ligated into

pUC18 that had been digested with SmaI. Following transformation into

Escherichia coli TG1 [29], recombinant clones were gridded in duplicate and

grown overnight at 37-C. One set of clones was then transferred to Hybond

N+filters (Amersham Biosciences, Buckinghamshire, UK), denatured, neutral-

ized, and fixed by UV irradiation. The filters were prehybridized and hybridized

with radioactively labeled (dA–dC)n I (dG–dT)n polynucleotide probe (Pfizer,

New York, NY, USA) and washed as described above for overgo probing of the

BAC library. Plasmid DNA was isolated from positive clones using the REAL

Prep 96 Plasmid Kit (Qiagen, Valencia, CA, USA) and the insert DNA was

sequenced in both directions using universal M13 forward and reverse primers.

Microsatellites were identified in the sequence traces and oligonucleotide

primers designed to amplify the microsatellites using PRIMER (Version 0.5;

Whitehead Institute for Biomedical Research).

Data handling and two-point linkage analysis

Pedigree information and genotypes were entered into CYRILLIC 2.1.3

(Family Genetix Ltd., Wallingford, Oxford, UK) and exported to CRI-MAP

(Phil Green, Version 2.4 (3/26/1990)) for linkage analysis. Initially, two-point

analysis, with a cut-off threshold of LOD 3, was performed to identify

linkage groups. At this point, any cases of non-Mendelian inheritance were

highlighted. Markers isolated from large, FISH-mapped clones such as

cosmids or BACs were used to identify to which chromosome each linkage

group belonged. These locations were all obtained from published sources

and details can be found in Supplementary Table 1.

Construction of the map

MULTIMAP [30] was used to generate a linkage map for each chromosome

by identifying the most likely marker order and estimating the distance between

each marker. Initially framework maps of at least four markers were generated

that identified markers the order of which was significant at LOD 3. For some

chromosomes (ECA12, 26, 27, and X) the stringency was relaxed to LOD 2 to

generate a framework, and for others, particularly the smaller ones, fewer than

four markers were accepted as the framework (ECA7, 12, 25, 26, 29, and 31).

Comprehensive maps were then generated, based on the frameworks in which

the order of the markers was significant at LOD >1. This process resulted in

chromosome maps with marker orders 10-fold more likely than any other. The

most likely positions of markers that could not be inserted into the map in an

order significant at LOD 1 were identified by MULTIMAP, and these have been

depicted separately in Fig. 2 (see figure legend for details). Sex-averaged map

distances were generated for all autosomes and female-specific map distances

for the X chromosome.

Fig. 2 (continued).

J.E. Swinburne et al. / Genomics 87 (2006) 1–29 13

Comparative mapping

GenBank was searched for all entries with ‘‘microsatellite’’ and ‘‘horse’’ and

1708 sequences were identified. These were compared with the human genome

sequence by using BLAST in a batch format. The top five matches for each were

listed and examined in turn and the results were split into two groups—those for

which a significant match to a unique position on the human genome was found

and those for which such a match was not found. The criteria used were not rigid;

high BLAST matches were not included if there were also high matches to other

genomic locations, and moderately high BLAST matches were considered

significant if there were no other moderately high matches, i.e., it was the

difference between the top scoring location and the second scoring location that

was important, rather than the absolute score. No matches less than e�2 were

considered. Markers that were linkage mapped and had significant unique

matches were picked out and ordered as they appeared along the horse

chromosome. Conserved segments in which adjacent markers on the horse

map corresponded with contiguous stretches of the human genome were

identified. These data are given in Supplementary Table 2 and illustrated in

Fig. 2.

The conserved segments generated with these data were compared with the

most recent comparative maps [15,19,21].

Acknowledgments

We thank the Horserace Betting Levy Board and the

Childwick Trust for generous financial support. We also

acknowledge the Dorothy Russell Havemeyer Foundation for

their continuing support of collaboration in this field by

financing International Workshops. We are grateful to the

laboratories that contributed to this effort by providing primers

for microsatellite markers, including INRA Centre de

Recherches de Jouy, France; the College of Veterinary

Medicine, University of Minnesota; the Equine Research

Institute, Japan Racing Association, Japan; Uppsala University,

Sweden; and the Norwegian College of Veterinary Medicine,

Oslo, Norway.

Appendix A. Supplementary data

Supplementary data associated with this article can be found

in the online version at doi:10.1016/j.ygeno.2005.09.001.

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Fig. 2 (continued).

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Fig. 2 (continued).

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Fig. 2 (continued).

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Fig. 2 (continued).

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