Genome scan for quantitative trait loci (QTL) for osteochondrosis in Hanoverian Warmblood horses using an optimised microsatellite marker set

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Aus dem Institut für Tierzucht und Vererbungsforschung der Tierärztlichen Hochschule Hannover

Genome scan for Quantitative Trait Loci (QTL) for osteochondrosis in Hanoverian Warmblood horses using

an optimised microsatellite marker set

I

NAUGURAL

-D

ISSERTATION

zur Erlangung des Grades einer D

OKTORIN DER

V

ETERINÄRMEDIZIN

(Dr. med. vet.)

durch die Tierärztliche Hochschule Hannover

Vorgelegt von

Kathrin Löhring

aus Stadtlohn

Hannover 2003

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Scientific supervision: Univ.-Prof. Dr. Dr. habil. O. Distl

Examiner: Univ.-Prof. Dr. Dr. habil. O. Distl

Co-examiner: Prof. Dr. Dr. habil. Dr. h.c. F. Ellendorf

Date of oral examination: 18.11.2003

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To my family

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Index of contents I

Index of contents

Introduction 1

Osteochondrosis in horses 3

Definition of the term osteochondrosis 3

Pathogenesis 3

Age of onset 4

Clinical signs and diagnosis 5

Prevalence of OC 7

Biomechanical trauma and exercise 10

Nutrition and mineral imbalance 11

Influence of weight, body size and sex 12

Endocrinologic, enzymatic and metabolic components in cartilage maturation 13

Genetic factors 16

Molecular genetic methods for identification of Quantitative Trait Loci (QTL) in horses 20

The equine genome 20

Physical gene maps 21

Genetic or linkage map 22

Present status of gene maps in horses 23

Mapping of QTL 24

Characterisation of a microsatellite marker set for genome-wide screens of the equine

genome 27

Introduction 27

Material and methods 27

Markers 27

Genotyping of microsatellites 28

Statistical parameters for the derived marker set 29

Results 30

Discussion 33

Summary 36

Genome scan for Quantitative Trait Loci (QTL) of osteochondrosis in Hanoverian Warmblood horses using an optimised microsatellite marker set 37

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Index of contents II

Introduction 37

Material and methods 38

Family material 38

Genome scan panel 39

Genotyping 39

Statistical analysis 40

Results 41

Linkage analysis 41

Discussion 46

Summary 49

Summary 50

Erweiterte Zusammenfassung 52

References 59 Appendix

Acknowledgements

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List of abbreviations III

List of abbreviations

AcP acid phosphatase

ADG average daily gain

ALP alkaline phosphatase

AMP adenosine monophosphate

APS ammoniumperoxidsulfat ATM animal threshold model

BAC bacterial artificial chromosome

BC backcrossing BMD bone mineral density

bp base pairs

CART1 cartilage homeoprotein 1

cM centiMorgan

CP crude protein

cR centiRay

CS chondroitin sulfate

dATP deoxy adenine triphosphate

DCP dyschondroplasia

dCTP deoxy cytosine triphosphate

DE digestible energy

DF dorsal fragments

dGTP deoxy guanine triphosphate

DMSO dimethylsulfoxid DNA deoxyribonuclein acid

dNTP deoxy nucleoside triphosphate DOD developmental orthopaedic disease dTTP deoxy thymine triphosphate

ECA chromosome of equus caballus

ECM extracellular matrix

EDTA ehtylenediamine-tetraaceticacid EST expressed sequence tag

FISH flourescent in situ hybridization

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List of abbreviations IV

GAG glycosaminoglycan

GS Gibbs sampling

HE expected heterozygosity

HO observed heterozygosity

HP hydroxylysylpyridinoline HSA chromosome of homo sapiens

h² heritability

IBD identical-by-descent IGF insulin-like growth factor

Ihh Indian hedgehog

IHRFP international horse reference family panel INRA Institut National de la Recherche Agronomique kb kilobase

KS keratan sulfate

KWPN Koninklijke Vereniging Warmbloed Paardenstamboek Nederland

LAM linear animal model

LG linkage group

LP lysylpyridinoline

LSM linear sire model

Mb megabases

ML maximum likelihood

MMP matrix metalloproteinase

mRNA messenger ribonucleic acid MSA multiplication stimulating activity NRC National Research Council

OC osteochondrosis

OCD osteochondrosis dissecans

PCR polymerase chain reaction PG proteoglycan PIC polymorphism information content PIT1 pituitary specific transcription factor 1 POF palmar/plantar osteochondral fragments

PTH parathyroid hormone

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List of abbreviations V

PTHR parathyroid hormone receptors

PTH-rP parathyroid hormone-related peptide QTL quantitative trait loci

RH radiation hybrid

REML restricted maximum likelihood

RFLP restriction fragment length polymorphismus

SCH somatic cell hybrid

sd standard deviation

SSCP single strand conformation polymorphism STM sire threshold model

STRP short tandem repeat polymorphisms

Ta annealing temperature

TBE TRIS - Boric acid - EDTA

TD tibial dyschondroplasia

TEMED tetramethylendiamine

TGF transforming growth factor

T3 triiodothyronine

T4 thyroxine

UPE united palmar/plantar eminence

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Introduction 1

Introduction

Osteochondrosis appears to be the most common primary cause of degenerative joint diseases in domestic animals and is one of the most important skeletal problems affecting young horses. Articulations that can be affected are the fetlock, hock, carpal, stifle, elbow, hip and vertebral joints. In affected joints, osteochondrotic lesions can be identified as subchondral fractures, cyst-like lesions, wearlines, chondromalacia, cartilage flaps, synovitis, and osteochondrosis dissecans (free joint bodies). These lesions can further develop into chronic degenerative joint diseases such as osteoarthrosis or arthropathy in the cervical spine, which can lead to vertebral stenosis and wobbler syndrome.

Available epidemiological data suggest that osteochondrosis is present in the horse population across a range of different breeds at unacceptably high levels, i.e. between 10 and 25%. Since the first report of osteochondrosis 56 years ago, the international importance of the condition has been well recognised, as its incidence appears to be steadily increasing, and OC is therefore now associated with enormous economic losses on a global scale.

OC is defined as a disturbance in the process of endochondral ossification. It occurs in growing cartilage of either the growth plates or the articular/epiphyseal complex. The specific causes are still unknown, but this disorder appears to be multifactorial in origin. Influencing factors include growth rate, body size, nutrition, mineral imbalance, endocrinological dysfunction, and biomechanical trauma. Furthermore, a hereditary disposition to osteochondrosis has been demonstrated in the Trotter, Coldblood, and Warmblood horse breeds, but no responsible genes have as yet been identified.

Osteochondrosis may be assumed to be a quantitatively inherited trait, as it is likely that its development is influenced by different genes and environmental factors.

In the course of this study, which was carried out in collaboration with the Association of Hanoverian Warmblood Breeders e.V., a number of different possible influences were investigated: nutrition, metabolic and endocrinological factors, housing conditions, management, population, and molecular genetics. The radiological examinations and interpretations of the radiographs were carried out by N. Krekeler, M. Reininghaus, P. Arnan and V. Welker of the Department of Veterinary Medicine, Clinic for Horses, Surgery and Radiology, Freie Universität Berlin (under the direction of Professor B. Hertsch). The influences of nutrition and metabolic and endocrinological factors were examined by A.

Borchers, M. Granel and S. Winkelsett of the Department of Nutrition, School of Veterinary

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Introduction 2

Medicine Hannover (under the scientific supervision of Professor M. Coenen). Management and population genetics were investigated by M. Schober and A. Wilke of the Department of Animal Breeding and Genetics, University of Göttingen (scientific supervision: Professor E.

Bruns). The molecular-genetic aspects of the development of OC were examined by the author at the Institute of Animal Breeding and Genetics, School of Veterinary Medicine Hannover (under the scientific supervision of Professor O. Distl).

The objective of this work was to establish a genome-wide microsatellite marker set suitable for whole-genome scans in order to identify quantitative trait loci (QTL) that significantly influence the development of osteochondrosis in Hanoverian Warmblood horses.

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Osteochondrosis in horses 3

Osteochondrosis in horses

Definition of the term osteochondrosis

Osteochondrosis (OC) is defined as a disturbance in the process of endochondral ossification of growing cartilage of the growth plates and/or the articular/epiphyseal complex (VAN DE LEST et al. 1999). The disease belongs to the complex of developmental orthopaedic diseases (DOD), which includes all skeletal problems associated with growth and development in foals. Osteochondrosis occurs in a variety of animal species, and is a common condition in young horses. Clinical signs in affected animals vary (see clinical signs and diagnosis) depending on the affected articulation, the location of the lesions in the joints and the type of alterations. According to the severity of the clinical signs (e.g. joint swelling, pain, recurrent lameness) osteochondrosis can be associated with reduced performance, which may ultimately lead to loss of the animal.

No specific aetiopathogenesis of osteochondrosis is as yet known. The condition appears to be multifactorial in origin, including the factors heredity, growth rate, body size, nutrition, mineral imbalance, endochrinological dysfunction and biomechanical trauma (JEFFCOTT 1996). This chapter discusses the different aspects of osteochondrosis and the influences and factors that may play a role in its aetiopathogenesis.

Pathogenesis

It is generally accepted that osteochondrosis (OC) develops due to a defect in the process of endochondral ossification in the growing horse (ROONEY 1975; STROMBERG 1979;

MOHAMMED 1990; JEFFCOTT 1996). Endochondral ossification is an ordered process, including cartilage proliferation, maturation and calcification followed by osseous replacement, and is responsible for longitudinal bone growth and enlargement of the epiphysis. In osteochondrosis, cartilage cells appear to proliferate normally, but maturation and differentiation are abnormal (TROTTER and McILWRAITH 1981). The initial lesion occurs in the proliferative or hypertrophic zone in growth cartilage adjacent to the joint surface (articular/epiphyseal cartilage). The loss of normal differentiation of cartilage cells means that transitional calcification of the matrix does not occur and that capillary sprouts fail to penetrate the distal region of the hypertrophic zone. The altered process of endochondral ossification leads to retention and thickening of cartilage, resulting in the development of a

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cartilage core, which is the primary lesion. As the primary lesion occurs in growing cartilage, JEFFCOTT (1996) prefers the term dyschondroplasia (DCP) to describe these early lesions.

The primary lesions can progress to further damage within the joint so that signs of OC can occur, such as subchondral fractures, subchondral cyst-like lesions, chondromalacia, wear lines, cartilage flaps, osteochondritis and synovitis. Osteochondrosis dissecans (OCD) refers to the separation of (osteo)chondral fragments. Articulations most commonly affected are the fetlock, hock and stifle joints, but other articulations (e.g. vertebral joints) can be affected, as well. Finally, these lesions can further develop into chronic degenerative joint diseases such as osteoarthrosis or arthropathy in the cervical spine, which can lead to vertebral stenosis and wobbler syndrome.

Age of onset

Articular lesions due to OC can develop very early in life. As growing cartilage is a tissue undergoing complex processes of proliferation and maturation especially during the first months of life, it is very important to distinguish between the time of the first detection of an osteochondrotic lesion and the time from which it is definitively permanent. Abnormal radiographic presentations and growth irregularities are commonly detected in the horse at the age of one month, particularly in hock joints (DIK et al. 1999) but most of these abnormalities disappear during the first months of life. The point of no return can be defined as the time at which no resolution of abnormal findings can be detected and when shifts from normal to abnormal findings become rare.

Several authors have reported on the age of onset of osteochondrotic lesions in different articulations of young horses. STROMBERG (1979) mentioned to a three-day-old Standardbred colt with a dissecting lesion. YOVICH et al. (1986) described a six-month-old male Quarter Horse foal with OCD lesions in the fetlock joints. SMALLWOOD and KELLY (1991) found radiological signs of OCD in the fetlock joints of a ten-week-old Quarter Horse foal which was examined radiologically at regular intervals since its birth. KROLL et al.

(2001) demonstrated lesions of OC in the fetlock joints in horses younger than one month of age, and found free joint bodies in the fetlock joints one to two months later. DIK et al. (1999) found that hock OC could be diagnosed at the age of one month, whereas the majority of osteochondrotic lesions in the stifles developed later (above the age of three months). In a longitudinal study CARLSTEN et al. (1993) examined 77 Standardbred foals six times from

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birth to the age of 16 months. The horses were classified as having permanent hock or fetlock OC if the findings were present at the age of 12 months. Eight foals showed permanent hock OC at the age of 12 months. In all eight foals the defects had already been visible before the age of three months, and four of those foals showed radiographic changes indicating early OC before one month of age. Eleven foals showed radiographic changes of hock OC that were first detected between one and three months of age and that reverted to normal at the examination at seven or eight months. In no case were additional significant radiographic findings detected between the ages of 8 and 16 months. Furthermore, eleven horses showed significant radiographic signs of fetlock OC at 12 months of age. In all these horses, early indications of these changes could be found before the age of five months. Seven horses showed early signs of abnormal ossification in the fetlock joints, which reverted to normal by the age of eight months. No additional significant radiographic findings were detected in fetlock or in hock joints after the age of seven to eight months. Although the horses in the study of CARLSTEN et al. (1993) were classified as having permanent hock or fetlock OC at the age of 12 months, all permanent lesions had already been visible earlier. These findings correspond to the results of DIK et al. (1999), who showed that the majority of abnormal radiographic appearances found in the hock joints at the age of one month disappeared by the time when the foals were five months old. Normal appearances rarely became abnormal during this time. As no shifts from normal to abnormal or vice versa were detected from the age of five months, the authors concluded that hock OC is permanent at the age of five months. In the stifle joints, no shifts from normal to abnormal appearances were detected above the age of eight months, so that lesions of stifle OC were considered to be permanent at that age. Growth irregularities in hock and fetlock joints were also noted by KROLL et al.

(2001), who found that fetlock and hock OC became manifest by the age of four months.

Clinical signs and diagnosis

The clinical signs of osteochondrosis (OC) are difficult to characterise in the horse because of the wide range of lesions and sites involved (JEFFCOTT 1996). Clinical or radiographic signs can be found in the fetlock, hock, carpal, stifle, elbow, hip and vertebral joints. The articulations most commonly affected are the fetlock, hock and stifle joints. The predilection sites of the particular joints are indicated below. Not all lesions of OC produce clinical signs.

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In case of superimposed biomechanical stress or trauma, joint damage may lead to pain and consequently to lameness or reduced performance.

The most common sign of equine OC irrespective of the age of the animal is a non-painful distension of the affected joint. Clinical signs can be classified into two categories, those presented by foals younger than six months of age and those in older animals. Often the first sign noted in foals is a tendency to spend more time lying down, frequently accompanied by joint swelling, stiffness and difficulties in keeping up with other animals in the paddock. An upright conformation of the limbs may also be apparent. Marked lameness is not usually a feature of equine OC, which frequently occurs in correlation with lesions in the shoulder and can be present in some cases of subchondral bone cysts in the medial femoral condyle. Pain and lesions of OC do not always occur simultaneously, and horses often exhibit very severe pathological changes without showing much pain or distress.

The main signs in yearlings or older animals are stiffness of joints, positive responses to flexion tests and in some cases varying degrees of lameness. These signs usually accompany the onset of training and thus indicate a biomechanical influence and an activation of subclinical or “silent” lesions.

The diagnosis of OC is usually based on the results of radiographic examinations.

Radiographic signs of OC are detected by examination of the outline of the bone contour, the density of the subchondral bone and the existence and size of visible fragments at the predilection sites (DIK et al. 1999). Radiographic findings indicative of OC are signs of irregular bone trabeculation with variable radiopacity (e.g. radioluceny of the subchondral bone) and changes of the regular bone contour such as exostoses, cartilage flaps, irregular bone margins and flattened or concave outlining. Radiographic appearances in the sense of osteochondrosis dissecans (OCD) are bony fragments (joint mice) with or without a defect in the underlying bone, visible as isolated radiodense areas in the joint space (CARLSTEN et al.

1993; KROLL et al. 2001).

The predilection sites in hock joints are the intermediate ridge of the distal tibia, the lateral/medial trochlear ridge of the talus and the lateral/medial malleolus of the tibia (McILWRAITH 1993). In the stifle joints, the predilection sites are the lateral/medial trochlear ridge of the femur, the trochlear groove of the femur and the patella. In the scapulohumeral joint the humeral head and the glenoid may be involved. The intervertebral articular processes are most commonly affected in the vertebral joints (VAN WEEREN and BARNEVELD 1999). Lesions of OC in the fetlock joints are often characterised as POF

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(palmar/plantar fragments and/or bone defects at the site of attachment of the short sesamoidean ligaments to the proximal phalanx), UPE (ununited palmar/plantar eminence of the proximal phalanx, including intra- and extraarticular fragments), or DF (dorsal fragments at the dorsoproximal rim of the proximal phalanx and/or fragments or defects) (CARLSTEN et al. 1993). According to their findings CARLSTEN et al. (1993) regard fetlock POF and UPE as one entity. The affiliation of fetlock POF and UPE to the OC complex has been questioned by several authors (e.g. DALIN et al. 1993; SANDGREN et al. 1993b; NIXON and POOL 1995). After histological examination of osteochondral fragments from the proximoplantar/proximopalmar region of the proximal phalanx of 30 horses NIXON and POOL (1995) suggested that these fragments may be a result of fracture rather than a manifestation of OC. DALIN et al. (1993) assumed that fetlock POF are the result of outwardly rotated hindlimb axes and subsequent point loading in the medial fetlock area, and concluded that the origin of these lesions was traumatic. SANDGREN et al. (1993b) stated that the differences in body weights, body measurements and clinical signs between horses affected by tarsocrural OC and horses affected with fetlock POF or UPE in their study are indications of the different pathogeneses of these lesions. Another question is whether fragments in the dorsal recessus of the fetlock joints belong to the OC complex. YOVICH et al. (1986) classified osteocartilaginous bodies associated with the dorsal saggittal ridge in young horses as a form of OC. SCHUBE et al. (1991) found free joint bodies of different origin seven fetlock joints, two of which were considered to be chip fractures of the phalangeal dorsal margins; the remaining five fragments obviously developed in the squamous ruptures of the hyperplastic chondroblastema. GRØNDAHL (1992) reported that opinions differ as to whether bony fragments located at the dorsoproximal end of the proximal phalanx are traumatic in origin or a manifestation of OC.

Prevalence of OC

Radiographic examinations have been carried out in different horse populations to estimate prevalences of OC (Table 1). The problem with radiographic surveys of young horses is that radiographic lesions will not be visible if there is only cartilaginous damage present with no subchondral bone involvement and no ossification of cartilage flaps (as in mild or early cases) (JEFFCOTT 1993). Thus, the actual prevalence of OC might be somewhat higher than that indicated by the incidence of animals showing clinical signs.

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Table 1. Prevalences of OC/OCD in different horse populations

Horse population Age Sites of OC/OCD % affected

horses Authors fetlock

(forelimb) 7.7%

fetlock

(hindlimb) 22.2%

German Warmblood Horses

(n = 200)

3 years

hock 11.5%

HARFST 1986

Danish Trotters (n = 325)

18 months to

2 years hock 12.0% SCHOUGAARD

et al. 1990 hock 10.4%

Swedish Standardbred Trotters

(foals, n = 77)

12 months

fetlock 14.3%

CARLSTEN et al. 1993

hock 14.3%

Norwegian Trotters (n = 753)

6 to 21 months

fetlock 11.8%

GRØNDAHL &

DOLVIK 1993

fetlock 31.8%

Holstein Warmblood Horses (n = 220, resp.151)

5 to 9 months

hock 8.7%

HEINZ 1993

hock 10.6%

( n = 1917)

3 to 8 years

fetlock 9.3%

MERZ 1993

hock 10.5%

Swedish Standardbred Trotters ¹ (n = 674)

11 to 24 months

fetlock 21.5%

hock 16.8%

Swedish Standardbred Trotters ² (n = 119)

11 to 24 months

fetlock 27.7%

PHILIPSSON et al. 1993

Dutch Warmblood Horses

(mares, n = 590)

3 years hock 13.7% KWPN 1994

hock 13.9%

Holstein Warmblood Horses

(n = 402)

3 years

fetlock 27.6%

MÜLLER 1994

fetlock 48.4%

Holstein Warmblood Horses (n = 190, resp.151)

1 year

hock 11.3%

THOMSEN 1995

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Table 1. continued

Horse population Age Sites of OC/OCD % affected

horses Authors fetlock 76.8%

Holstein Warmblood Horses

(n = 151)

2 years

hock 12.7%

KIRCHNER 1996

hock 12.3%

( n = 2269)

3 to 7 years

fetlock 30.1%

LEONHARDT 1996

(n = 3566)

3 to 8 years hock

fetlock 11.0% WINTER et al. 1996

(mares, n = 472)

- hock 5.3%

German Warmblood Horses (foals, n = 151 - 220)

6 months to

2 years hock 0.7 - 78.8%

WILLMS et al. 1999

hock 11.7%

fetlock

(forelimb) 6.0%

(n = 669)

2 years

fetlock

(hindlimb) 11.5%

KAHLER 2001

Italian Maremmano

horses (n = 350)

2 to 3 years

hock stifle fetlock

16.6% PIERAMATI et al. 2003

1 progeny of randomly selected stallions

2 progeny of selected stallions (stallions affected with OC/OCD)

HEINZ (1993) x-rayed 220 Holstein foals from five to nine months of age, which were then examined as yearlings and at the age of two years by THOMSON (1995) and KIRCHNER (1996). The prevalences of OC/OCD in the studies of HEINZ (1993) and THOMSEN (1995) were calculated on the basis of the number of foals (n = 151) that remained for the study of KIRCHNER (1996). In the study of HEINZ (1993), 31.8% of the foals showed radiographic signs of OC/OCD in the fetlock joints, and 8.7% showed signs of hock OC/OCD.

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THOMSON (1995) found radiographic changes due to OC/OCD in the fetlock joints in 48.4% of the horses examined. OC/OCD of the hock joint was diagnosed in 11.3% of the yearlings. In a radiographic survey, KIRCHNER (1996) reported findings in the fetlock joints of 76.8% of the two-year-old horses that could be classified as OC/OCD, and signs of OC/OCD in the hock joints in 12.7% of the horses. These three studies indicate that the prevalence of OC/OCD in the hock joint may remain almost the same, whereas the prevalence of OC/OCD in the fetlock joints might increase with age. One reason for this high prevalence in older horses could be that THOMSON (1995) and KIRCHNER (1996) predominantly examined affected animals of the 220 foals from the study of HEINZ (1993). Nevertheless, it is questionable if this can explain an increase in the prevalence of more than 50%.

Biomechanical trauma and exercise

The importance of biomechanical trauma for the development of OC/OCD was discussed very early (BAKER 1963; ADAMS 1966; DIETZ et al. 1976). However, it was not possible to establish a mechanical experimental model to reproduce lesions of OC (TROTTER and McILWRAITH 1981). SCHUBE et al. (1991) recognised that focal necroses, ruptures and clefts of the hyperplastic chondroblastema on the phalangeal dorsal margin appeared as a result of traumatic injuries caused by recidivating hyperextension of the fetlock joint. It has also been suggested that the type and duration of movement/activity have an influence on the incidence of OC. The effects of exercise on bone development and quality have been studied by several authors (e.g. JEFFCOTT et al. 1988; McCARTHY and JEFFCOTT 1991; VAN DE LEST 2003). It has been shown that exercise has an influence on the appearance and the distribution of lesions of OC (VAN WEEREN and BARNEVELD 1999). KNAAP and GERDING (1999) reported that the way young foals are kept had an influence on the course of the disease. Foals which grew up in stables had a significantly higher incidence of OC than foals at pastures. Furthermore, it has been shown that exercise influences cartilage metabolism. BRAMA et al. (2002) found out that there were significantly lower increases in calcium content and in the lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) crosslink levels in the box-stabled foals than in pastured foals. VAN DEN HOOGEN et al.

(1999b) showed alterations in the proteoglycan metabolism with different exercise regimens.

There is also a proven effect of exercise on bone mineral density (BMD) (FIRTH et al. 1999;

BARNEVELD and VAN WEEREN 1999). VAN DE LEST (2003) found that an overall

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increase in bone mass was apparently related to continuous, evenly distributed exercise (pasture group). They suggested that short, heavy bouts of exercise superimposed on a basic resting regimen might have a long-term deleterious effect. CORNELISSEN et al. (1999) concluded that box rest during the first months of life results in a retardation of normal development, which is compensated for when the restriction of exercise is lifted. VAN DEN HOOGEN et al. (1999b) stated that pasture exercise is best for the development of healthy cartilage.

Nutrition and mineral imbalance

Nutrition is also considered to play an important role in the development of osteochondrosis (OC). Over-nutrition seems to be an especially predisposing factor in the production of this disease. GLADE and BELLING (1986) were able to induce lesions of physeal dysplasia and OC by feeding young Thoroughbred horses approximately 130% of the National Research Council (NRC) (1989) recommendations for both protein and digestible energy (DE).

SAVAGE et al. (1993a) came to similar results on the basis of thirty mixed-breed foals aged between 2.5 and 6.5 months and fed different diets. The diets were composed of rice-based pellets, maize oil and oaten chaff. Six of the twelve foals fed with 129% of NRC levels for DE showed clinical and radiographic signs of dyschondroplasia (DCP). None of the six foals fed with ~126% of NRC levels for crude protein (CP) showed any sign of DCP. The high energy diet did not manifest its effects through increases in average daily gain (ADG), as no significant increases occurred in foals fed the high energy diet compared to those fed the basal control diet.

Imbalances of mineral homeostasis may also be involved in the pathogenesis of osteochondrosis. The most important minerals which are assumed to have an influence are calcium, phosphorus, copper and zinc. KROOK and MAYLIN (1988) proposed that diets with an excessive content of calcium were responsible for hypercalcitoninism, which was assumed to cause disturbances of chondrocytic maturation and cartilage replacement by bone, leading to DCP. SAVAGE et al. (1993b) showed in their study that foals fed high phosphorus and high calcium/DE diets were affected more frequently and severely with DCP. In a blind randomised study, 18 Quarter Horse foals, divided into two groups, were fed with two different diets with different copper contents (HURTIG et al. 1993). The foals in the low- copper-diet group had lower liver copper values (detected by liver biopsies), and developed

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epiphysitis, limb deformities and osteochondritis. Biochemical studies indicated significantly reduced collagen cross-linking in articular cartilage, growth plate cartilage and bone in severely affected foals.

A relationship has also been shown between nutrition and endocrinology. GLADE (1986) postulated that any nutritionally induced effects on cartilage growth are mediated by the endocrine system. High energy diets lead to altered thyroid hormone levels and to altered insulin metabolism. So the effects of nutrition and endocrinology cannot only be seen as individual factors in the development of OC but must also be considered as interacting components in a complex system.

Influence of weight, body size and sex

The influences of weight, body size and sex have been described in several studies.

SANDGREN et al. (1993a) examined 793 Standardbred trotters at between 11 and 24 months of age and found no significant sex differences in the incidence of OC and POF. However, males were significantly more often affected when both lesions were considered simultaneously. Furthermore, the incidence of OC findings was related to body size. Affected horses had a greater circumference of the carpus and were taller at the withers. The incidence of hock OC was also related to the date of birth, with higher prevalences in foals born in the later part of the foaling season. In the study of SANDGREN et al. (1993b) 77 Standardbred foals were examined six times from birth to the age of 16 months. Foals affected with hock OC (n = 8) had a higher body weight at birth and continued to be heavier, with significantly higher average daily weight gains up to an age of 12 months when compared with unaffected foals. The affected foals also had a larger frame, including a greater height at the croup and at the withers, and had a markedly larger circumference of the cannon bone and the carpus.

Compared to the foals affected with hock OC, the foals affected with fetlock POF and/or UPE weighed slightly less at the age of 12 months, but this difference was not significant. There was no difference in the average daily weight gain or in the different body measurements between the foals affected with fetlock POF/UPE and the unaffected foals. VAN WEEREN et al. (1999) examined the influence of sex, birth weight, final achieved height and monthly weight gains on the occurrence of OC in the femoropatellar and the tarsocrural joints in a group of 43 foals and found no influence of sex on the prevalences of OC in hock and stifle joints at the ages of five months and eleven months. There were also no relationships found

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between birth weight, final achieved height, monthly weight gain rates and hock OC. Foals positive for femoropatellar OC had significantly higher weight gains during the first eleven months of life, were heavier at eleven months of age and taller at the croup and at the withers than non-affected foals.

As the number of foals and the age at examination differed and the results were somewhat conflicting in the studies mentioned above, it is difficult to draw consistent conclusions. There may be some influence of sex on the development of OC, but the influences of the remaining factors remain uncertain.

Endocrinologic, enzymatic and metabolic components in cartilage maturation

The endocrinological control of skeletal development and growth is extremely complex. The hormones that are most likely to participate in endochondral ossification are insulin, thyroxine, growth hormone, parathyroid hormone and calcitonin (JEFFCOTT 1997). GLADE (1986) described different metabolic components that are involved in cartilage maturation during endochondral ossification. They mentioned growth hormone, somatomedin-C, insulin- like growth factor I (IGF-I), multiplication stimulating activity (MSA II), insulin, thyroxine (T4), triiodothyronine (T3), glucocorticoids, estrogen, calcitonin, parathyroid hormone (PTH), cyclic adenosine monophosphate (cAMP) and prostaglandins. A survey on the factors involved in endochondral ossification has been given by JEFFCOTT and HENSON (1998).

It has been shown that transforming growth factor ß (TGF-ß) plays an important role in growth cartilage metabolism, particularly in the control of chondrocyte differentiation and hypertrophy (HENSON et al. 1997a). TGF-ß is also known to inhibit proteoglycan production, to regulate collagen synthesis and to decrease alkaline phosphatase (ALP) activity (THORP et al. 1993). THORP et al. (1993) found that transitional chondrocytes usually contain high concentrations of TGF-ß and the product of the C-myc proto-oncogene. They showed that transitional chondrocytes in animals affected with avian tibial dyschondroplasia (TD) are deficient in TGF-ß and C-myc. HENSON et al. (1997a) identified reductions in TGF-ß1 mRNA and protein in focal lesions of dyschondroplasia (DCP). SEMEVOLOS et al.

(2001) found an intralesional increase of the expression of TGF-ß1 mRNA and suggested a reparative response to the OC lesion.

Insulin has a direct effect on equine chondrocytes in culture, which leads to an increase in their survival time (HENSON et al. 1997b), as well as an indirect effect via the metabolism of

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other systemic and local factors and hormones, including IGFs (HALL et al. 1989), TGF-ß (BADILLO et al. 1994), T3 and T4 (JEFFCOTT and HENSON 1998). HENSON et al.

(1997b) suggested that relative hyperinsulinaemia may be a contributing factor to equine dyschondroplasia.

T3 and T4 are involved in the final stages of chondrocyte differentiation and in the metaphyseal invasion of blood vessels into the growth cartilage. Thus, a lack of capillary penetration can be related to low T3 and T4 levels in young horses.

IGFs play an important role in cartilage metabolism and growth, including the introduction of increasing cellular proliferation and the synthesis of cartilage aggrecan and collagen (SEMEVOLOS et al. 2001). HENSON et al. (1997b) demonstrated in their study that both IGF-I and IGF-II act as mitogens in isolated foetal and neonatal equine chondrocytes.

SLOET VAN OLDRUITENBORGH-OOSTERBAAN et al. (1999) found that foals affected with OC had a significantly lower IGF-I plasma concentration during the first months of life.

In the research of SEMEVOLOS et al. (2001) cartilage obtained from osteochondrotic lesions had a significantly higher expression of IGF-I. As most of the horses were between eight and twelve months old, the OC lesions were frequently mature and contained secondary changes associated with repair tissue, so that the increased IGF-I expression was interpreted as reparative response.

Recent studies by SLOET VAN OLDRUITENBORGH-OOSTERBAAN et al. (1999) report that foals suffering from OCD had increased serum levels of PTH and 1,25-dihydroxy-vitamin D.

Recently, the paracrine factors, parathyroid hormone-related peptide (PTH-rP) and Indian hedgehog (Ihh), have been implicated in controlling cartilage differentiation and hypertrophy in the growth plate. SCHIPANI et al. (1995) identified a single heterozygous nucleotide exchange in exon M2 of the gene encoding the PTH-PTHrP receptor in a human patient with Jansen-type metaphyseal dyschondroplasia. SEMEVOLOS et al. (2002) examined the expression of PTHrP and Ihh in equine OC and found a significantly increased PTHrP protein and mRNA expression compared to control cartilages.

In growth cartilage, cells (chondrocytes) are embedded within a complex extracellular matrix (ECM), which is produced and regulated by the chondrocytes themselves. The cartilage matrix consists primarily of water, collagens, proteoglycans and glycoproteins. Collagen types I, II, VI and X can be found in the cartilage matrix. HENSON et al. (1996) detected collagen type II mRNA in all chondrocytes of growth cartilage, whereas the localisation of

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both collagen types VI and X varied during cartilage development. Type-II collagen expression remained unchanged in dyschondroplasia (THORP et al. 1993). The increased type-X collagen mRNA concentration showed an elevated transcription of the type-X gene in DCP, indicating that the low concentrations of type-X collagen in dyschondroplastic cartilage are a result of a defect in its secretion or incorporation of the collagen into the ECM, but not in its production.

The cartilage that has to be calcified is predominantly composed of proteoglycans (able to bind calcium) and collagen type II (VAN DEN HOOGEN et al. 1999a). Proteoglycans (PG) are polydisperse molecules composed of a core protein to which glycosaminoglycans (GAG), mainly chondroitin sulphate (CS) and keratan sulphate (KS), are linked covalently.

LILLICH et al. (1997) showed that articular cartilage from horses with naturally acquired distal tibial OC had significantly lower quantities of uronic acid, total GAG, and CS. VAN DEN HOOGEN et al. (1999a) showed that osteochondrotic cartilage of 11-month-old foals had a markedly decreased sulphate incorporation and assumed a decrease in proteoglycan (PG) production. Since the synthesis of PGs is one of the major tasks of chondrocytes, PG production can be considered as a measure of the activity/vitality of the chondrocytes. The authors concluded that the chondrocytes of the 11-month-old osteochondrotic cartilage had lost most of their vitality. Furthermore, in the osteochondrotic cartilage of the 11-month-old foals, there was a remarkable increase in the release of newly synthesised PGs. The altered turnover time of the newly synthesised PGs in the absence of a change in total PG release indicated a change in the composition of the PG population. It was suggested that the newly synthesised PGs were not of optimal quality and had a reduced ability to aggregate, which made them more easily removable. LAVERTY et al. (2000) found that there was a significant decrease in the epitope 846 and the keratan sulphate epitope on the cartilage proteoglycan aggrecan in young horses with OC. In contrast, the concentration of the C propeptide of cartilage type-II procollagen increased. LAVERTY et al. (2002) showed a significant increase in type-II collagen cleavage by collagenases, but found no evidence for an increased proteoglycan degradation.

The turnover of ECM macromolecules is controlled by chondrocyte-derived enzymes: the matrix metalloproteinases (MMPs) (collagenases, stromelysins, gelatinases) and the cathepsins, which degrade both the collagenous and non-collagenous components of ECM (JEFFCOTT and HENSON 1998). GLÄSER et al. (2003) found that cathepsin B was characteristically located in chondrocytes at the articular surface and in the hypertrophic zone

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in all growing horses. On the other hand, cathepsin L was predominantly present in proliferating chondrocytes in foetal and neonatal cartilage. Based on the age-related fluctuation levels and the significantly different patterns in the distribution of both enzymes, the authors proposed that cathepsin B could be more important in post-natal, mechanically- induced turnover of cartilage ECM, whereas cathepsin L might be involved in the early stages of endochondral ossification.

Other enzymes that may play a role in the transition of cartilage into bone are alkaline phosphatase (ALP), lysyl oxidase and acid phosphatase (AcP) (VAN DE LEST et al. 1999).

ALP is essential for the formation of hydroxyapatite molecules by liberating the necessary phosphate molecules (ANDERSON 1995). Lysyl oxidase, a copper-dependent enzyme, is involved in the enzymatic cross-linking of type-I collagen matrix. AcP plays a role in the solubilisation of hydroxyapatite molecules. VAN DE LEST et al. (1999) showed that in subchondral bone affected with OC, lysyl oxidase and total and bone ALP activity were significantly increased. In avian tibial dyschondroplasia, transitional chondrocytes show an increased concentration of ALP (THORP et al. 1993).

All endocrinologic, enzymatic and metabolic factors that can be clearly identified as playing a role in the proliferation and maturation of growing cartilage can be suspected of having an influence on the development of OC. As mentioned above, mutations in receptor-coding genes or genes coding for hormones, enzymes or other factors might be involved in the pathogenesis of OC. Factors involved only in secondary reparative responses of the affected tissues should not be considered. As endochondral ossification is a very complex process, it may be difficult to identify single factors playing a role in pathogenesis.

Genetic factors

SCHOUGAARD et al. (1990) performed a study based on 325 yearlings representing the majority of the progeny of nine Trotter stallions. Heritability of hock OC was estimated by means of the χ² value for binominal data (ROBERTSON and LERNER 1949) (Table 2). Of the 325 examined yearlings 39 (12%) showed significant lesions. The incidence within progeny groups varied from 3.4% to 30% and the heritability was estimated at 0.26.

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Table 2. Heritability estimations for OC/OCD in different limb joints Population

and number of investigated

horses

Radiographic

finding Heritability Method of analysis Author Danish Trotters

(n = 325) OC (hock) 0.26 ± 0.14 STM (χ²- heterogeneity test ¹)

SCHOUGAARD et al. 1990 OCD (fetlock) 0.52

Norwegian Trotters

(n = 644) OC (hock) 0.21

STM (expectation maximum-REML ²)

GRØNDAHL &

DOLVIK 1993

0.09 → 0.19 0.09 → 0.24

LSM (χ²- heterogeneity test ¹

→ transformation ³) 0.08 → 0.17

Swedish Standardbred

Trotters (n = 793)

OC (hock) OCD (fetlock)

0.09 → 0.27

LSM (Henderson III ⁴

→ transformation ³)

PHILIPSSON et al. 1993

0.14 ± 0.17 LAM (REML, transformation ⁷) 0.01 ± 0.06

→ 0.02 ± 0.14

LSM (REML

→ transformation ⁷) Dutch Warmblood

Horses (mares, n = 590)

OC (hock)

0.02 ± 0.06 STM (REML ⁸)

KWPN 1994

0.07 ± 0.03 LAM (REML) German

Riding Horses (n = 2407

or 3566)

OCD (hock) OCD (fetlock)

0.06 ± 0.04 LSM (Henderson III ⁴)

WINTER et al. 1996

0.45 ± 0.12 LSM (GS) 0.64 STM (REML-type

algorithm ⁹) German

Riding Horses (mares, n = 401

or 456)

OCD (hock)

0.34 ± 0.06 ATM (GS) 0.58 ± 0.15 LSM (GS) German

Riding Horses (foals, n = 144)

OCD (hock)

0.19 ± 0.02 ATM (GS)

WILLMS et al. 1996

0.13 - 0.14

±0.22 - 0.24

LAM (REML ⁵, transformation ³) Italian

Maremmano horses (n = 350)

OCD (hock, stifle,

fetlock) 0.08 - 0.09

± 0.23 - 0.24

ATM (Average Informat. REML ⁶)

PIERAMATI et al. 2003

ATM: animal threshold model LSM: linear sire model

GS: Gibbs sampling REML: restricted maximum likelihood LAM: linear animal model STM: sire threshold model

1 ROBERTSON and LERNER 1949 ⁴ HARVEY 1985 ⁷ GIANOLA 1982

² GIANOLA and FOULLEY 1983 ⁵ BOLDMAN et al. 1993 ⁸ MISZTAL et al. 1989

³DEMPSTER and LERNER 1950 ⁶ WANG 1994 ⁹ MISZTAL 1989

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In the study of PHILIPSSON et al. (1993) 793 horses sired by 24 Swedish Standardbred Trotter stallions were used for examinations of radiographic findings of OC in the tarsocrural joints and of palmar/plantar osteochondral fragments (POF) in metacarpo- and metatarsophalangeal joints. Hock joint OC was diagnosed in 71 (10.5%) offspring of the randomly selected stallions; fetlock joint POF was found in 145 (21.5%) offspring. In the group of offspring of selected stallions which were affected with OC and/or POF themselves, OC was diagnosed in 20 (16.8%) offspring and POF in 33 (27.7%) offspring. The heritability of OC using Henderson’s method III was 0.09 on the observed linear scale, corresponding to 0.24 on the underlying scale. For POF the estimates were 0.09 and 0.19, respectively.

Estimating the heritability by means of the χ² value for binominal data (ROBERTSON and LERNER 1949), the heritability for OC was estimated at 0.09 on the threshold scale and 0.27 on the quantitative scale; corresponding estimates for POF were 0.08 and 0.17, respectively.

GRØNDAHL and DOLVIK (1993) radiographed 753 Standardbred Trotters for OC. OC in the tibiotarsal joint was found in 108 (14.3%) horses, and POF was diagnosed in 89 (11.8%) horses. The heritability analysis was restricted to 644 horses so as to include five or more progeny from 39 sires. Heritability of OC in the tibiotarsal joint was 0.52 (nonlinear model) and 0.32 (linear); heritability of POF was lower with estimates of 0.21 and 0.13, respectively.

WINTER et al. (1996) and WILLMS et al. (1999) estimated the heritability of OC for both hock and fetlock joints. WINTER et al. (1996) assumed that low heritabilities of h² between 0.06 and 0.07 indicate environmental components in the development of the disease, unlike WILLMS et al. (1999), who estimated values of h² between 0.19 and 0.64 and concluded there was a predominantly genetic influence on the development of osteochondrosis.

The phenotypic and genetic correlations between the findings in different articulations have been examined in some studies. SANDGREN et al. (1993a) showed that horses with UPE had a significantly higher incidence of POF. However, no correlation was found between hock OC and fetlock POF/UPE. This corresponds to the findings of GRØNDAHL and DOLVIK (1993), who found no significant correlation between hock OC and fetlock POF/UPE.

STOCK et al. (2003) estimated the correlations between hock and fetlock OCD. The additive genetic correlation between these traits was moderately negative both with a linear animal model and a linear sire model. For male horses, the estimates were about rg = -0.4, whereas the additive genetic correlation was close to zero in female horses. Thus, hock and fetlock OCD have to be considered as two genetically different traits.

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The results of the different heritability estimations substantiate the presence of a genetic influence on the development of equine OC. A candidate gene approach could be used to examine the genetic influence more precisely. It has not yet been possible to identify obvious candidate genes for equine OC in physiological studies. Candidate genes may be proposed on the basis of known pathogenesis of equine OC or on the basis of a close functional relationship to a gene known to be involved in a similar disease in other species. However, little is known about candidate genes of osteochondrosis in other species, as well.

In a recent study, ANDERSSON-EKLUND et al. (2000) identified one genome-wide and a chromosome-wide QTL for OC in pigs. Possible candidate genes might be pituitary specific transcription factor 1 (PIT1), which codes for a transcriptional factor of growth hormone, genes coding for parathyroid hormone receptors (PTHR), insulin-like growth factor I (IGF-I), and cartilage homeoprotein1 (CART1). These genes were selected on the one hand for their indicated role in the development of OC or cartilage growth and on the other hand due to their locations in the vicinity of an identified QTL or in the homologous region of the human genome.

Another possibility is a whole genome scan using a set of highly polymorphic markers in order to identify the chromosomal locations of quantitative trait loci (QTL). These chromosomal regions can then be refined to locate genes responsible for OC.

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Molecular genetic methods for identification of QTL in horses 20

Molecular genetic methods for identification of Quantitative Trait Loci (QTL) in horses

The equine genome

The equine genome is estimated to contain approximately 3000 megabases (Mb) of DNA distributed on 31 pairs of autosomes and the X and Y chromosomes (MURRAY and BOWLING 2000). At present, 439 genes and 735 microsatellite markers are registered at the INRA Horsemap homepage.

The aim of gene mapping is to identify the location of genes and markers on chromosomes and to obtain information about the distance between them. According to the different methods used to establish gene maps we can distinguish between genetic linkage maps and physical maps. Dense gene maps are prerequisites for mapping of phenotypic traits using DNA marker data.

Two types of markers are classified in the horse, specific genes or expressed sequence tag (EST) gene sequences (type I), and microsatellites (type II) (CHOWDHARY et al. 2003). The majority of the markers are microsatellite markers. Microsatellites are short arrays of simple repeated sequences consisting of tandemly repeated di-, tri- or hexanucleotides. The most common microsatellites in the horse are poly (CA) repeats. Microsatellite markers are often highly polymorphic, which is a feature of their high mutation rate to new alleles (changes in the number of repeats in the array). These short tandem repeat polymorphisms (STRPs) are interspersed throughout the whole genome. They are usually situated in non-coding regions of the DNA and accordingly are not expressed phenotypically. Due to their shortness, microsatellites can easily be assayed using PCR amplification followed by electrophoresis, in order to demonstrate the marker genotype. As microsatellites are codominant, heterozygotes can be distinguished from homozygotes.

Microsatellite markers are important tools in parentage analysis and genetic linkage studies (e.g. analyses of quantitative trait loci).

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Physical gene maps

Physical maps are based on direct assignment of genes or markers to chromosomes or to chromosome regions, bands, or base pairs. There are different methods that can be used to construct physical maps.

Synteny mapping

A synteny map (syntenous = located on the same chromosome) represents loci that reside on the same chromosome. The basic method for constructing synteny maps is the establishment of somatic cell hybrid (SCH) panels by fusing cell lines of two species. Analysis of a pair of genes in such an SCH panel makes it possible to show concordance or discordance of their retention, which indicates their synteny or asynteny.

Radiation hybrid mapping

Radiation hybrid mapping is basically an SCH technique with the difference that, before fusion of the cell lines, the whole or partial genome of the species of interest is exposed to high doses of X-ray irradiation leading to fragmentation of the chromosomes (COX et al.

1990). RH mapping shows synteny between loci as well as the physical distance between them. The closer two markers are situated on a chromosome the smaller is the probability that they will be separated by X-ray treatment, and vice versa. The distance between two loci can be calculated from the retention frequency and is denoted in centiRay (cR). The resolution of RH maps depends on the doses of X-ray irradiation. Due to the higher breakage frequency a 5000-rad panel has a higher resolution than a 3000-rad panel.

In situ hybridisation

The technique of in situ hybridisation allows direct visualisation of the position of genes or markers on the chromosomes. One of the two major components of in situ hybridisation is the target, which is an interphase, metaphase or prometaphase chromosome. The other component comprises the probes, which are DNA segments of various lengths. The length of the DNA segments ranges between the minimum of 1000 bp and approximately 300 kilobases (kb), which can be cloned for example in a bacterial artificial chromosome (BAC) vector.

Depending on the type of in situ hybridisation, the probes can be labelled radioactively, enzymatically or with the help of fluorochrome conjugates. The technique of labelling the

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probe with fluorescent colours is also called flourescent in situ hybridisation (FISH). FISH is the most common technique for anchoring markers or genes on chromosomes or chromosomal segments.

Chromosome painting

When flow-sorted or microdissected whole chromosomes or parts of them are used as a composite probe in FISH experiments, the technique is called chromosome painting. A special type of chromosome painting is comparative chromosome painting or Zoo-FISH, as the painting is carried out across species. Zoo-FISH helps to construct comparative maps between different mammalian species (WIENBERG and STANYON 1997; CHOWDHARY 1998). Conservation of synteny or linkage between genes or markers can be taken as an indication of cross-species genome homology for the segments lying between them.

Genetic or linkage map

Genetic maps are established using linkage analysis in reference populations. They reflect the relative order and distance of markers (type I and type II) along a chromosome in centiMorgan (cM) units. The genetic distance is calculated using the recombination frequency between two or more markers. A recombination frequency of 1% corresponds to 1 cM. The transformation of the recombination frequency to map units is performed by using mapping functions. As crossing-over during meiosis does not occur completely at random in the genome, a direct comparison between physical and genetic maps is not immediately possible.

Crossovers are often suppressed in particular chromosomal regions (near the centromere and telomeres). Furthermore, the presence of a crossover can inhibit further crossovers in adjacent regions (interference), so there may be variations in the ratio base pairs/centiMorgan among populations and among individuals. The recombination frequency varies between sexes.

Chiasmata during meiosis occur more often in female cells than in male cells. A decrease in the rate of recombination increases the number of base pairs per cM.

Markers located on the same chromosome are considered to be syntenic markers. The most common method of identifying synteny groups is by using an SCH clone panel created by the fusion of primary cells from the species under study (donor cell) with cells from a transformed cell line. Chromosomes from the donor cell line are lost randomly over time, for example in the UC Davis horse x mouse SCH panel through between 40 and 45 passages

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(SHIUE et al. 1999). Synteny can be determined by following the loss or retention of associated markers across multiple, clonally derived SCH cell lines.

Linkage mapping can also aid in the construction of comparative gene maps. Therefore, it is necessary to type genes (type I markers) as opposed to type-II markers occurring in non- coding DNA, because the former are under evolutionary constraints to be conserved in order to preserve gene function.

Present status of gene maps in horses

Gene mapping in horses has become increasingly important because gene maps will aid in the study of, and in the establishment of tests for, inherited defects and quantitative traits of the horse (SWINBURNE et al. 2000). This development started eleven years ago with the publication of the first equine microsatellite markers by ELLEGREN et al. (1992).

Subsequently, MARKLUND et al. (1994) described initial linkage observations, and BREEN et al. (1997) and GODARD et al. (1997) reported physical position assignments for a limited number of markers.

The first genetic linkage map was published by LINDGREN et al. (1998). The marker set included 121 microsatellite markers, eight protein polymorphisms, five RFLPs, three blood group polymorphisms, two PCR-RFLPs, and one single-strand conformation polymorphism (SSCP). The average distance between linked markers was 12.6 cM and the total map distance obtained within linkage groups was 679 cM. The linkage map created by GUERIN et al. (1999) was tested on the international horse reference family panel (IHRFP). It contained seven blood group loci, ten biochemical loci and 144 microsatellite DNA loci which were assigned to 26 autosomes. Of the 161 markers, 124 could be arranged in 29 linkage groups.

The average interval between loci was 14.2 cM, and the total map distance was 936.5 cM.

The most complete linkage map as yet is that published by SWINBURNE et al. (2000). Thus far, 353 equine microsatellites and six biallelic markers have been assigned to all 31 autosomes and the X chromosome. A total of 334 markers (93%) have been significantly linked to at least one other marker, so that 42 linkage groups have been formed. In addition, the physical location of 85 markers is now known, which allows the anchoring of 37 linkage groups to the physical map. The average distance between the markers is 10.5 cM, and the total genetic distance covered by all linkage groups is 1780 cM. Assuming that each linkage group (n = 42) and each unlinked marker (n = 25) could cover a flanking area of 5 cM on each

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side, this map covers 2450 cM. Recently a second half-sibling linkage map was published by GUERIN et al. (2003). This low-density, male-based linkage map includes 310 markers on all 31 autosomes arranged in 34 linkage groups. The maps spans 2262 cM with an average interval between the loci of 10.1 cM.

MILENKOVIC et al. (2002) used fluorescent in situ hybridisation (FISH) to map 136 genes in the horse. RAUDSEPP et al. (1999), GODARD et al. (2000), LEAR et al. (2001), MARIAT et al. (2001) and LINDGREN et al. (2001) mapped further markers to equine chromosomes using FISH. Zoo-FISH was carried out by RAUDSEPP et al. (1996), CHOWDHARY et al. (1998) and CAETANO et al. (1999b), who identifed conserved chromosomal segments between the horse and human genome.

With the development of a horse-mouse somatic cell hybrid panel it was possible to assign large numbers of markers to horse chromosomes by synteny analysis (CAETANO et al.

1999a, b; SHIUE et al. 1999).

KIGUWA et al. (2000) generated preliminary radiation hybrid (RH) maps for chromosomes ECA1 and ECA10, and these were followed by RH and comparative maps for some of the other equine chromosomes (CHOWDHARY et al. 2002, RAUDSEPP et al. 2002). A first- generation whole-genome radiation hybrid map in the horse was recently published by CHOWDHARY et al. (2003). This map was established using 92 horse x hamster hybrid cell lines and 730 equine markers. The 730 loci (258 type I and 472 type II) were clustered in 101 RH groups distributed over all equine autosomes and the X chromosome, on average every 19 cR. This map is the first comprehensive framework map of the horse that includes type I and type II markers, integrates synteny, cytogenetic, and meiotic maps into a consensus map, and provides the most detailed genome-wide information of the equine genome to date.

Mapping of QTL

Many traits of economic interest in animals are of a quantitative genetic nature, which means that the influences of many genes combine to contribute to a particular phenotype. Unlike qualitative (i.e., Mendelian) traits, which are generally controlled by a single gene, quantitative traits show continuous variation, which is present in all populations of eukaryotes and is caused by environmental or genetic factors, or by both genes and the environment (RAPP 2000). According to GELDERMANN et al. (1985) a quantitative trait locus (QTL) is a single gene locus, or a marked DNA region that contains the gene, with a measurable effect

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on the genetic variance of a trait. Such a QTL or so-called major gene should determine more than 10% of the phenotypic variance of the targeted trait.

Two basic approaches or a combination of them are used in molecular biology to detect quantitative trait loci (QTL): the candidate gene approach (ROTHSCHILD et al. 1994), linkage to molecular markers (ANDERSSON et al. 1994), or a combined approach using microsatellites of the candidate genes and the genomic regions surrounding them.

With the availability of dense, highly informative marker maps, it is now possible to map QTL accounting for part of the variance of quantitative traits (GEORGES 1997). The identification of QTL with genetic markers is based on the following principle. During meiosis, each haploid gamete receives n chromosomes resulting from a random sampling of large segments within each parental chromosome pair. Two DNA loci which are located close to each other (linked) on the same chromosome are likely to be transmitted together to the gametes and then to progeny. Considering two linked polymorphic loci called M (marker) and Q (QTL), and a doubly heterozygous parent MQ/mq, the alleles M and Q are on the same chromosome strand, and m and q are on the homologous chromosome strand. When the loci are closely linked (frequency of recombination ~ 0), Q and q will mostly be associated to M and m, respectively, in the progeny of this parent. If the locus Q affects the trait, the effect of substitution of allele q by allele Q can be detected by comparing the progeny which received m or M. Through its genetic linkage, marker M can thus indicate the QTL.

Initially, most of the analytical methods were developed for populations derived from inbred lines; these methods include backcrossing (BC) populations and F2 populations. The advantage of inbred line crosses is that they offer ideal properties for mapping. When two parental strains which are homozygous for alternate alleles of two loci are crossed, all F1 individuals have identical genotypes (including the same linkage phase) and are heterozygous at all loci that differ between the crossed lines, so that these F1 individuals are all fully informative. Marker linkage association is more efficiently evaluated in populations (e.g. BC populations, F2 populations) that are in linkage disequilibrium (DU and WOODWARD 1997), which means that the relative frequencies of marker alleles in affected individuals differ from those in the general population.

It is impractical in most species of domestic animals to produce inbred lines because of the long duration of gestation, the small number of offspring per pregnancy, the long generation interval and the negative side effects of inbreeding. Inbreeding depression results in reduced fitness and increases the frequency of abnormalities due to the presence of recessive,