Athletic performance and conformation in Hanoverian warmblood horses

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University of Veterinary Medicine Hannover

Athletic performance and conformation in Hanoverian warmblood horses - population genetic and genome-wide association analyses

Thesis

Submitted in partial fulfillment of the requirements for the degree Doctor of Veterinary Medicine

- Doctor medicinae veterinariae - (Dr. med. vet.)

by

Wiebke Schröder Halle/Saale

Hannover 2010

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Academic supervision: Univ.-Prof. Dr. Dr. habil. Ottmar Distl

Institut für Tierzucht und Vererbungsforschung Bünteweg 17p

30559 Hannover

1. Referee: Univ.-Prof. Dr. Dr. habil. Ottmar Distl 2. Referee: Univ.-Prof. Dr. Karsten Feige

Day of oral examination: 22. November 2010

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To my family

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Parts of this work have been accepted or submitted for publication in the following journals:

1. The Veterinary Journal

2. Livestock Production Science 3. Archiv Tierzucht

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Table of contents

Table of contents

1 Introduction………...……… 1

2 A review on candidate genes for physical performance in the horse……… 7

2.1 Abstract……….………. 9

3 Does the proportion of genes of foreign breeds influence breeding values for performance traits in the Hanoverian warmblood horse?... 11

3.1 Abstract……….……… 13

3.2 Introduction………..……… 14

3.3 Materials and Methods……….………. 14

3.3.1 Performance Data………. 14

3.3.2 Pedigree Data……… 16

3.3.3 Statistical Analyzes……….. 17

3.3.4 Model Development………. 17

3.3.5 Genetic Analyses……….. 18

3.4 Results………..……… 19

3.4.1 Statistical Analyses………... 19

3.4.2 Analyses of Variance and Model Development……… 20

3.4.3 Genetic Analyses……….. 20

3.5 Discussion……….……… 21

3.6 Conclusion………..………. 24

3.7 References……….……….. 24

4 Genetic evaluation of Hanoverian warmblood horses for conformation traits considering the proportion of genes of foreign breeds………. 35

4.1 Abstract………. 37

4.2 Zusammenfassung……….. 38

5 A genome wide association study for quantitative trait loci of show-jumping in Hanoverian warmblood horses………. 41

5.1 Summary……….. 43

5.2 Introduction……….. 44

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Table of contents

5.3 Materials and Methods………... 45

5.3.1 Animals and Phenotypic data……….. 45

5.3.2 Genotyping SNPs……….. 46

5.3.3 Data analysis……….. 47

5.4 Results……….. 50

5.5 Discussion……… 51

5.6 References……….. 57

6 Identification of quantitative trait loci for dressage in Hanoverian warm blood horses……… 81

6.1 Summary………. 83

6.2 Introduction………. 84

6.3 Materials and Methods………. 85

6.3.1 Animals and Phenotypic data……… 85

6.3.2 Genotyping SNPs……… 87

6.3.3 Data analysis……… 87

6.4 Results……… 91

6.5 Discussion……….. 92

6.6 References………. 98

7 A genome wide association study for quantitative trait loci of conformation in Hanoverian warmblood horses………. 121

7.1 Abstract……… 123

7.2 Introduction………. 124

7.3 Results………. 125

7.4 Discussion………... 126

7.5 Materials and Methods……….. 132

7.5.1 Animals and Phenotypic data………. 132

7.5.2 Genotyping SNPs………. 133

7.5.3 Data analysis………. 134

8 General Discussion……… 169

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Table of contents

9 Summary……….. 177

10 Zusammenfassung……… 183

11 Appendix……….. 193

12 List of publications……… 197

13 Acknowledgement………. 201

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Abbreviations

List of abbreviations

A adenin

ACE angiotensin converting enzyme ACTN3 actinin alpha 3

ADP adenosine diphosphate

ADRB2 adrenergic beta-2-receptor, surface AF3BL2 ATPase family gene 3-like 2

AI auction inspection

AMPD1 adenosine monophosphate deaminase 1 ANOVA analyses of variance

Arg argentine

AT achilles tendinopathy ATI achilles tendon injury ATP adenosine triphosphate BDKRB2 bradykinin receptor B2

BDNF brain-derived neurotrophic factor BLUP Best Linear Unbiased Prediction

bp base pairs

BV breeding value

bv_dress breeding value for dressage bv_jump breeding value for jumping bv_limbs breeding value for the limbs

bv_rhp breeding value for riding horse points

C cytosine

cAMP cyclic adenosine monophosphate cDNA complementary deoxyribonucleic acid CHRM2 cholinergic receptor muscarinic 2

CK creatine kinase

CKM creatine kinase, muscle

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Abbreviations

CLUSTRALW2 multiple alignment program through sequence weights COL1A1 collagen, type I, alpha 1

COL5A1 collagen, type V, alpha 1 COL15A1 collagen, type XV, alpha 1 ConFL conformation front legs ConHL conformation hind legs

CYP27B1 cytochrome P450, family 27, subfamily B, polypeptide 1 gene David database for annotation, visualization and integrated discovery

Del deletion

Dev general impression and development DRD4 dopamine receptor D4

ECA Equus caballus autosome

Ensembl joint project between EMBL (European Molecular Biology Laboratory) – EBI (European Bioinformatics Institute) and the Wellcome Trust Sanger Institute

EPAS1 endothelial PAS domain protein 1 EquCab2 Equus caballus assembly 2

FST follistatin

G guanine

GABPA GA binding protein transcription factor, alpha subunit 60kDa GABPB GA binding protein transcription factor, bet

GDF8 growth and differential factor 8

Gly glycine

GNB5 guanine nucleotide binding protein (G protein), beta 5 GYS1 glycogen synthase 1

h² heritability

HAN proportion of Hanoverian warmblood genes

HB hemoglobin

HBB hemoglobin beta

Head conformation of the head HDC histidine decarboxylase

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Abbreviations

HFE hemochromatosis

HIF1A hypoxia inducible factor 1 alpha

HOL proportion of Holsteiner warmblood genes

HPX hemopexin

HSS Hanoverian Studbook Society IGF1 insulin-like growth factor 1

In insertion

LCORL ligand dependent nuclear receptor corepressor-like gene LD linkage disequilibrium

LSM least square means MAF minor allele frequency

MARKAPK mitogen-activated protein kinase-activated protein kinase 2

Mb megabase

MCPH1 microcephalin 1

MYL2 myosin light chain 2 regulatory, cardiac, slow gene MYL3 myosin, light chain 3, alkali; ventricular, skeletal, slow MYLK myosin light chain kinase

MYO5A myosin VA (heavy chain 12, myoxin)

MYO7B myosin VIIB

MSTN myostatin

MMP3 matrix metalloproteinase-3 MPT mare performance test n number

NCBI National Center for Biotechnology Information Neck conformation of the neck

NRAP nebulin-related anchoring protein NRF1 nuclear respiratory factor 1

NRF2 nuclear respiratory factor 2

P error probability

PAPSS2 bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthetase2 PLAGL1 pleiomorphic adenoma gene-like 1

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Abbreviations

PPARA peroxisome proliferator-activated receptor alpha

PPARGC1A peroxisome proliferator-activated receptor gamma, coactivator 1 alpha

PPARD peroxisome proliferator-activated receptor delta PRG4 proteoglycan

PT performance test

PT_Walk walk under rider evaluated at mare performance test or auction inspection

PT_Trot trot under rider evaluated at mare performance test or auction inspection

PT_Canter canter under rider evaluated at mare performance test or auction inspection

PT_FJT total score for free jumping evaluated at mare performance test or auction inspection

PT_Ride rideability as judged by the judging commission evaluated at mare performance test or auction inspection

σa² additive genetic variance

σe² residual variance

σr² event variance

R arginine RAD23B RAD23 homolog B

REML Residual Maximum Likelihood RHP riding horse points

RNF160 ring finger protein 160

RPP Reitpferde-Points

Sad saddle position

SAS statistical analysis system SCGB1A1 secretoglobin, family 1A, member 1

SD standard deviation

SE standard error

SBI studbook inspection

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Abbreviations

SBI_Walk walk at hand evaluated at studbook inspection

SBI_Imp impetus and elasticity in trot at hand evaluated at studbook inspection

SBI_Corr correctness of gaits in walk and trot at hand evaluated at studbook inspection

SLC6A4 serotonin transporter

SNP single nucleotide polymorphism SHOX2 short stature homeobox 2 T thymin

TASSEL Trait Analysis by aSSociation, Evolution and Linkage TB proportion of Thoroughbred genes

TBX4 T-box transcription factor 4 gene

TNC tenascin C

TRAK proportion of Trakehner genes TRAPPC9 trafficking protein particle complex 9 TRHR thyrotropin-releasing hormone receptor

TRPC3 transient receptor potential cation channel, subfamily C, member 3

VCE Variance Component Estimation VEGFA vascular endothelial growth factor A VDR 1,25- dihydroxyvitamin D3 receptor

VIT Vereinigte Informationssysteme Tierhaltung w.V.

VWC2 Willebrand factor C domain containing 2

WH height at withers

X stop codon

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CHAPTER 1

Introduction

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Introduction

1 Introduction

Worldwide, horses play an important role within human cultures. Since domestication, the focus of breeding has been to improve the horses’ usefulness to man. Hanoverian warmblood horses (Hanoverians) can be traced back to the 16^th century. Back then they were primarily bred for farm work and military service, requiring competitive, calm and rideable horses. In the middle of the 20^th century the usability for equitation became more of a focal point. Today, the Hanoverian represents one of the most important breeds of sport horses in the world. In particular dressage and show jumping play an economically important role in Hanoverian breeding. A favourable conformation is an additional asset for sales, especially at young ages. Hence, the Hanoverian studbook society (HSS) aims at selecting animals with best performance and most favourable conformation values for the next generation. In order to refine conformation of future progeny, Thoroughbreds and Trakehners are commonly used, while the intended use of Holsteiner warmblood horses (Holsteiner) is to improve the show-jumping performance. Population genetic analyses of performance traits and conformation are performed regularly to ease performance orientated matings.

Currently, many genes are in discussion to influence human athletic capability, while such studies in horses are rare. However, due to the rapid development in the equine molecular genetics that has evolved around the second assembled genome sequence of the horse (EquCab2.0), the prospects of dissecting the genetic components of multigenic traits such as performance and conformation have increased dramatically. In addition, the strong conserved synteny between human and equine chromosomal structure and new bioinformatic tools should facilitate identifying genes involved in equine performance. Our approach is focused on genetic factors that play a major role for equine physical capability and conformation.

Molecular genetic studies in particular on heterogeneous populations are highly sensitive to data stratification. Hence a careful model choice is required to avoid false positive results. In particular the proportions of genes and relationship are important factors for stratification. The model regularly used for population genetic analyses of Hanoverians does not consider the proportion of foreign breeds. Hence, one aim of

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Introduction

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this study was to determine whether the inclusion of the proportion of genes could improve the model for genetic evaluation for performance and conformation of Hanoverians.

The second purpose of this study was to identify genomic regions harbouring candidate genes for physical performance and conformation of Hanoverians. In order to achieve this objective, we performed whole genome association analyses of single nucleotide polymorphism (SNP) with the aim to further define quantitative trait loci (QTL) for different traits of performance and conformation employing the Illumina Equine SNP50 BeadChip.

Overview of chapter contents

The content of the thesis is presented in single papers according to § 8 Abs. 3 of the Rules of Graduation (Promotionsordnung) of the University of Veterinary Medicine Hanover.

Chapter 2 reviews the literature for specialities of the equine physical performance and represent 28 candidate genes for equine performance.

Chapter 3 and 4 contain an update on performance and conformation related breeding values of Hanoverian warmblood horses and investigates whether the inclusion of the proportion of genes could improve in the model.

Chapter 5, 6 and 7 present whole genome association analyses for performance and conformation employing the Illumina Equine SNP50 BeadChip, in order to determine genomic regions responsible for show-jumping, dressage and conformation in Hanoverians.

Chapter 8 comprises a general discussion and conclusions referring to chapters 2-7.

Chapter 9 is a concise English summary of this thesis, while Chapter 10 is an expanded detailed German summary which takes into consideration the overall research context.

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Introduction

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CHAPTER 2

A review on candidate genes for physical performance in the horse

Wiebke Schröder, Andreas Klostermann, Ottmar Distl

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany

Article in Press, Corrected Proof (doi:10.1016/j.tvjl.2010.09.029)

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A review on candidate genes for physical performance in the horse

2 A review on candidate genes for physical performance in the horse

2.1 Abstract

Intense selection for speed, endurance or pulling power in the domestic horse (Equus caballus) has resulted in a number of adaptive changes in the phenotype required for elite athletic performance. To date, studies in human have revealed a large number of genes involved in elite athletic performance, but studies in horses are rare. The horse genome assembly and bioinformatic tools for genome analyses have been used to compare human performance genes with their equine orthologues, to retrieve pathways for these genes and to investigate their chromosomal distribution. We represent 28 candidate genes for equine performance that have polymorphisms associated with human elite athletic performance and may have impact on athletic performance in horses. A significant accumulation of candidate genes was found on horse chromosomes 4 and 12. Genes involved in pathways for focal adhesion, regulation of actin cytoskeleton, neuroactive ligand- receptor interaction, and calcium signalling were overrepresented. Genome-wide association studies for athletic performance in horses may benefit from the strong conserved synteny of the chromosomal arrangement of genes among human and horse.

Keywords: Equus caballus; Genome; Performance; Candidate genes; Genetic polymorphisms; Pathway analysis

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CHAPTER 3

Does the proportion of genes of foreign breeds influence breeding values for performance traits in the Hanoverian warmblood horse?

W. Schröder, K.F. Stock, O. Distl

Department of Animal Breeding and Genetics, University of Veterinary Medicine Hannover (Foundation), Hannover; Germany

Submitted for publication

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Genetic evaluation for performance

3 Does the proportion of genes of foreign breeds influence breeding values for performance traits in the Hanoverian warmblood horse?

3.1 Abstract

Performance data of in total 36,441 Hanoverian warmblood horses (Hanoverians) were used to determine whether genetic evaluation for performance in the Hanoverian could benefit from the inclusion of the proportion of genes of foreign breeds in the model. For our analyses we considered all Hanoverians born from 1992 to 2005, for which records from mare performance tests, auction inspections or studbook inspections were available. Genetic parameters were estimated univariately for five traits evaluated at mare performance tests and auction inspections (walk, trot and canter under saddle, free jumping, and rideability) and for three traits evaluated at studbook inspections (walk, elasticity and correctness of gaits for walk and trot in hand) in a linear animal model using Residual Maximum Likelihood. Genetic evaluation was subsequently performed using Best Linear Unbiased Prediction. To investigate the effect of correcting for the proportion of genes of stallions from foreign breeds, two different models were used for the analyses. In Model 1, the fixed effects sex (for the auction inspection data only) and age, and the random effect date-place interaction were considered. In Model 2, proportions of genes of Thoroughbred, Trakehner and Holsteiner stallions were additionally included as fixed effects.

Heritabilities of analyzed performance traits in both models ranged between 0.11 and 0.34, with standard errors of 0.01. Pearson correlation coefficients determined between corresponding breeding values from Model 1 and 2 were highly positive (>0.98), indicating little effect of the model on the results of genetic evaluation. Our results indicate that using a model which includes the proportion of genes of Thoroughbred, Trakehner and Holsteiner as fixed effects will not relevantly improve genetic evaluation for performance in the Hanoverian.

Keywords: Hanoverian, genetic evaluation, performance, proportion of genes.

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3.2 Introduction

The Hanoverian warmblood horse is primarily bred to be a rideable and talented sport horse with good abilities for the disciplines dressage, jumping, eventing and driving (Koenen et al., 2004). Therefore, an early performance evaluation is not just beneficial for a preselection of talented youngster sport horses (Ducro et al., 2007;

Wallin et al., 2003), but also plays an important role for the selection of breeding horses. Performance traits, evaluated at mare performance tests (MPT), auction inspections (AI) and studbook inspections (SBI) represent a suitable selection base.

Estimation of breeding values for performance traits recorded at MPT and SBI have been described for the Hanoverian warmblood as well as for other German warmblood breeds (Christmann, 1996; Lührs-Behnke et al., 2006). In the models used for genetic evaluation, environmental effects which principally influence performance, i.e. age at evaluation, sex, place and date of evaluation were considered separately and combined.

For dairy cattle, Vanderick et al. (2009) could show that breeding values that account for breed proportions provide a theoretically better tool to evaluate crossbred dairy cattle populations. Similar results were obtained by Stewart et al. (2009) for the sport horse population of Great Britain. They considered a model including breed classes as most appropriate for the estimation of breeding values for dressage performance. For the Hanoverian warmblood horse (Hanoverian), the influence of the proportion of foreign breeds on performance has not been investigated in depth.

Thoroughbred, Trakehner and Holstein warmblood (Holsteiner) represent the most common stallions employed for crossbreeding in the Hanoverian population (Hamann and Distl, 2008). Hence, the aim of our study was to determine whether genetic evaluation for performance could benefit from inclusion of the proportion of genes of these three breeds in the Hanoverian.

3.3 Materials and Methods 3.3.1 Performance Data

Results of MPT, AI and SBI of the Hanoverian Studbook Society (HSS) were made available for this study. Information on 36,441 Hanoverian warmblood horses

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born between 1992 and 2005 were considered. All performance data and pedigree information were made available by the HSS through the national unified animal ownership database (Vereinigte Informationssysteme Tierhaltung w.V., VIT) in Verden at the Aller, Germany.

Mare performance test. MPT data included information on 16,814 performance tested mares. Of these mares 14,500 accomplished their MPT in a one day event in the field (MPTF), whilst only 2,314 mares completed their MPT in stationary performance tests (MPTS). Tests at station included 26 days of standardized training and a final test. If a mare participated in more than one MPT, only the last test result was considered for subsequent analyses. Included MPTs took place in 1995 to 2008.

The number of performance tested mares per year ranged between 855 and 1,286 in MPTF and between 126 and 210 in MPTS. The mares were judged for quality of gaits (walk, trot, and canter under rider), jumping talent (style and ability of free jumping), rideability, and character using a 0.5 scale from 0 (not shown) to 10 (excellently shown). Style and ability of free jumping were scored individually and subsequently averaged to a total score for free jumping. Rideability was separately scored by a judging commission and by a test rider. The character was only judged at MPTS.

Most of the mares completed their MPT until the completion of their fourth year of age (mean age of 3.58 ± 0.91 years in MPTF and 3.54 ± 0.78 years in MPTS).

Performance tests in the field were held at 63 places with on average 26.94 ± 13.19 (1 to 82) judged Hanoverian warmblood mares per date and place. MPTS took place in only 5 places with on average 18.52 ± 6.47 (1 to 30) mares per date and place.

Auction inspection. Horses offered for sale at riding horse auctions of the HSS are preselected by a judging commission. Auction candidates are chosen based on their preliminary performance evaluation. Between 1999 and 2008, 8,081 Hanoverians (5,567 males, 2,514 females) were judged at auction inspections (AI) in a procedure similar to MPTF. Accordingly, presented horses were scored for quality of gaits (walk, trot and canter under rider), jumping talent (style and ability of free jumping, total

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score for free jumping), and rideability. Mean age of evaluated horses at AI was 4.21

± 0.82 years. AI were held at 111 places, with on average 16.77 ± 18.68 (range 1 to 96) inspected horses per date and place.

Studbook inspection. All mares intended to be used for breeding under the HSS must be registered in the Hanoverian studbook. At studbook inspection (SBI) a judging commission gives individual scores for several conformation traits as well as for walk, correctness of gaits in walk and trot at hand, and impetus and elasticity in trot at hand. Scores on a scale from 0 (not shown) to 10 (excellently shown) were assigned for each trait. For more details see Stock and Distl (2006). For this study we considered the SBI results with respect to the three gait-related traits of 29,053 mares, presented between 1995 and 2008 at a mean age of 3.79 ± 1.66 years. There were 182 places of SBI with on average 11.73 ± 12.44 (range 1 to 83) inspected mares per date and place.

3.3.2 Pedigree Data

For the genetic analyses, four ancestral generations of all horses with performance data (SBI, MPT or AI results) were considered. The relationship matrix comprised 80,746 individuals, including 7,486 base animals. The 29,053 mares judged at SBI descended from 1,079 sires and 1,706 maternal grandsires. The sires were on average represented by 26.91 ± 63.5 (range 1 to 998) horses and the maternal grandsires were on average represented by 17.02 ± 41.59 (range 1 to 704) horses. The 24,895 horses that performed at MPT or AI descended from 935 sires and 1,485 maternal grandsires. The sires were on average represented by 26.61 ± 62.59 (range 1 to 941) horses and the maternal grandsires were on average represented by 16.76 ± 40.61 (range 1 to 686) horses. The performance tested Hanoverian population had an average proportion of 0.58 (median = 0.59) Hanoverian genes, 0.23 (median = 0.20) Thoroughbred genes, 0.07 (median = 0.05) Trakehner genes, and 0.05 (median = 0) Holsteiner genes. The proportions of genes provided by Thoroughbred, Trakehner and Holsteiner were calculated for the tested

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Hanoverian population. For this calculation, all available pedigree information was used. Details are described elsewhere (Hamann and Distl, 2008).

3.3.3 Statistical Analyzes

Statistical analyses included three traits evaluated at SBI, i.e. walk at hand (SBI_Walk), correctness of gaits in walk and trot at hand (SBI_Corr) and impetus and elasticity in trot at hand (SBI_Imp), and five performance test (PT) traits evaluated at MPT and AI, i.e. walk under rider (PT_Walk), trot under rider (PT_Trot), canter under rider (PT_Canter), total score of free jumping (PT_FJT) and rideability scored by judging commission (PT_Ride). Because for some horses individual scores for style and ability of free jumping were not recorded, only the total score for free jumping was considered for all individuals. Horses evaluated at AI were, unlike the ones at MPTs, only scored for rideability by a judging commission, but not by a test rider. For that reason we only included rideability scores from the judging commission for our analyses.

3.3.4 Model Development

The following effects were tested for their influence on distribution of performance trait scores from MPT/AI and SBI: Age at MPT/AI or SBI evaluation as covariate or fixed effect (3-, 4- or ≥ 5 years old); evaluation year (individual years from 1995- 2008), evaluation month (individual months), evaluation season (February through April, May through July, August through October, November through January), and evaluation place (182 places of SBI, 111 places of AI, 63 places of MPTF and 5 places of MPTS) as fixed effects; combined date-place effect (2,476 levels for SBI, 482 levels for AI, 537 levels for MPTF, 126 levels for MPTS) as random effect;

proportion of genes (Hanoverian or Thoroughbred, Trakehner, and Holsteiner) as covariate or fixed effect (low, moderate and high proportion of genes of the respective breed). The sex effect (male, female) was tested for the AI traits.

Simple and multiple analyses of variance (ANOVA) were performed using the procedures GLM and MIXED of the Statistical Analysis System (SAS), Version 9.2 (SAS Institute Inc., Cary, NC, USA, 2010). Model choice was based on the model fit

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test statistics and the significance tests. In the final model (Model 1), sex (only for AI) and age group at SBI or MPT/AI evaluation were considered as fixed effects, and the combined date-place effect was considered as random effect. To investigate the impact of accounting for the proportion of genes on results of the genetic analyses, an alternative model (Model 2) was used which additionally included the proportions of genes of Thoroughbred, Trakehner and Holsteiner as fixed effects. Class were formed from the proportions of genes with the aim of having similar numbers of horses in each of three effect levels. Given the uneven representation of breeds, boundaries were set independently for Thoroughbred (≤0.13, >0.13 and <0.30,

≥0.30), Trakehner (≤0.2, >0.2 and <0.8, ≥0.8), and Holsteiner (0.0, >0.0 and <0.3,

≥0.3).

Distributions of performance scores and residuals were analyzed for including tests for normality using Kolmogorov-Smirnov statistics of the UNIVARIATE procedure of SAS. For all analyses, the significance limit was set to 0.05.

3.3.5 Genetic Analyses

Genetic parameters were estimated univariately in a linear animal model with (Residual Maximum Likelihood (REML) using VCE-5, version 5.1.2 (Variance Component Estimation; Kovač et al., 2003). Estimates for environmental and genetic effects were obtained under the same models using Best Linear Unbiased Prediction (BLUP) with the software PEST (Groeneveld et al., 1990).

yijnopq= μ + AGEAI/MPT/SBIi + SEXj + dateAI/MPT/SBI x placeAI/MPT/SBIno [1]

+ ap + eijnopq

yijklmnopq= μ + AGEAI/MPT/SBIi + SEXj + TBk + TRAKl + HOLm [2]

+ dateAI/MPT/SBI x placeAI/MPT/SBIno + ap + eijklmnopq

with yi…q = MPT/AI or SBI score, μ = model constant, AGEAI/MPT/SBIi = fixed effect of age group at performance evaluation (i = 1-3), SEXj = fixed effect of sex (j = 1-2), TBk = proportion of Thoroughbred genes (k = 1-3), TRAKl = proportion of

Trakehner genes (l = 1-3), HOLm = proportion of Holsteiner genes (m = 1-3),

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dateAI/MPT/SBI x placeAI/MPT/SBIno = random effect of interaction between date of performance evaluation and place of MPT/AI or SBI, ap = random additive genetic effect of the individual horse ( r = 1-36,441) and ei…q = residual.

To study the genetic correlations between the analyzed traits and to test the influence of the model on the results of genetic evaluation, Pearson correlation coefficients between breeding values were calculated using the procedure CORR of SAS.

3.4 Results

3.4.1 Statistical Analyses

Between 1995 and 2008, 16,814 Hanoverian mares participated in MPT, gaining 83,923 scores for the considered performance traits (PT_Walk, PT_Trot, PT_Canter, PT_FJT and PT_Ride). Between 1999 and 2008, 8,081 Hanoverians were evaluated at AI, gaining 38,976 scores for the same five traits. Concerning SBI 29,053 Hanoverian mares were presented between 1995 and 2008, gaining 87,037 scores for the tree considered performance traits (SBI_Walk, SBI_Imp, and SBI_Corr).

Development of mean scores of for the gait related performance traits (MPT_Walk, MPT_Trot and MPT_Canter evaluated at MPT and AI; SBI_Walk, SBI_Imp and SBI_Corr evaluated at SBI) are shown in Figures 1 and 2. Mean scores for MPT_Ride and MPT_FJT ranged between 4.9 (PT_FJT at AI in 2008) and 7.6 (PT_Ride at MPT in 2008). Mean scores from MPTs were considerably higher than those from AI. Mean scores for gaits and rideability differed by 0.49-0.76, jumping scores differed by up to 1.92 between AI and MPT. Considering gaits, lowest mean was determined for PT_Trot (6.2) at AI in 2002 and 2006, and highest mean was determined for PT_Canter at MPT in 2008 (7.5). Scores ranged between 3 and 10 at SBI, and between 1.5 and 10 at MPT/AI. Neither MPT or AI scores nor SBI scores were distributed normally (P<0.01). Skewness coefficients were in the range of

|s|=0.02-0.68.

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3.4.2 Analyses of Variance and Model Development

In all subsets of data (MPTF, MPTS, AI, and SBI) the fixed effects proportions of genes of Thoroughbred, Trakehner and Holsteiner, age group at evaluation, additionally sex for AI, and the random effect (combined date-place) were significant for all traits, as previously found by Stock and Distl (2007).

Table 1 shows least square means (LSM) with their standard errors (SE) of performance scores evaluated at MPT/AI and SBI for the proportion of genes of Thoroughbred, Trakehner, and Holsteiner. Figure 3 shows cumulative percentages of Hanoverian, Thoroughbred, Trakehner and Holsteiner genes. The relation between performance trait scores of MPT/AI and SBI, and the proportion of genes of the considered horse breeds is illustrated in Supplementary Figures 1 and 2. Proportions of genes of the considered breeds differed markedly between the upper and lower 10%-quantiles for SBI_Walk, PT_Walk and PT_FJT with respect to the Holsteiner, and for PT_Trot with respect to Holsteiner and Trakehner.

3.4.3 Genetic Analyses

Results of univariate genetic analyses of performance traits evaluated at MPT/AI and SBI without (Model 1) or with (Model 2) correction for the proportion of genes of Thoroughbred, Trakehner and Holsteiner are shown in Table 2. Heritabilities estimated for the SBI traits ranged between h²= 0.11 (SBI_Corr in both models) and h²= 0.34 (SBI_Imp in both models). Similar heritabilities were estimated for the MPT/AI traits, ranging between 0.22 (MPT_Walk in Model 2) and 0.34 (MPT_Trot in Model 1).

The comparison of heritability estimates from the two models showed only slight differences of 0.0007 (SBI_Imp) to 0.0058 (MPT_Walk). Additive genetic variances (σa²) were slightly smaller in Model 1 than in Model 2 (0.0007-0.0075). The event variances (σr²) were identical in both models for the performance traits evaluated at SBI and for walk at MPT/AI, and only slight differences (range between 0.0001- 0.0017) were seen for MPT_Trot, MPT_Canter, MPT_FJT and MPT_Ride. Residual variances (σe²) for all traits but MPT_Walk were slightly higher (by 0.0002-0.0048) in Model 1 than in Model 2 (Table 2).

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Pearson correlation coefficients calculated between the breeding values for all traits estimated using Model 1 and Model 2 are given in Table 3. Except for PT_FJT correlations between all performance traits were significantly positive, ranging between 0.35 and 0.79. However, PT_FJT was negatively correlated with all other traits with -0.16 to -0.14.

There were only minor differences of 0.0015 to 0.0928 between the Pearson correlation coefficients determined between breeding values for the analyzed traits in Model 1 and Model 2. The largest differences between the two models were seen for the correlations involving breeding values for PT_FJT. Comparisons of breeding values estimated in Model 1 and Model 2 for the same trait revealed for all traits Pearson correlation coefficients close to unity (r > 0.98; results not shown).

3.5 Discussion

The aim of this study was to determine whether including the proportions of genes of foreign breeds in the model could improve genetic evaluation for performance in the Hanoverian as indicated by the results of Stewart et al. (2009), with respect to genetic evaluation for dressage of the sport horse population in Great Britain.

Possible optimization of genetic evaluation for performance through inclusion of some breed effect in the model has been previously shown by Vanderick et al. (2009) for a cross-breed dairy cattle population in New Zealand. For the German warmblood horse, especially the Hanoverian, the impact of different proportions of genes of other horse breeds on prediction of breeding values has not been investigated yet.

The breeding aim of the Hanoverian is defined as follows: a rideable, noble, big framed and correct warmblood horse, which, on the basis of its natural abilities, its temperament and character is suitable as a performance horse as well as a pleasure horse. On this basis the HSS strives for the breeding of talented sport horses for the disciplines dressage, jumping, eventing and driving. Genetic evaluation for performance in the main disciplines of riding sport, dressage and jumping is routinely performed using records from competitions and performance tests (Von Velsen- Zerweck, 1998).

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Publication of breeding values allows performance-oriented matings with regard to dressage, jumping or both. Development of MPT scores over time, as shown for the time period 1995 to 2008 in this study, illustrate the breeding progress particularly in the dressage related traits. In this connection, possible sale benefits may have caused that breeders tend to put more weight on dressage than on jumping talent of their foals. At young age, high prices are primarily achieved for foals with good movements.

Results from SBI, MPT and AI have been used for this study. SBI is obligatory for all Hanoverian broad mares, as well as MPT for dams of stallions born after 1990.

For all other mares, MPT is voluntary. Training for and participation in MPT is costly and time consuming, so breeder may only present their best mares. The much lower increase of SBI scores than of MPT scores may therefore also reflect preselection effects.

Unlike MPT, AI gives a more representative cross-section through the Hanoverian horse population, because riding horse auctions are organized by the HSS as a sale platform for their breeders. Horses of any gender and at different training level are presented. AI averages accordingly increased on a lower level than the MPT averages, but also indicated the continuing breeding process. The highly positive additive genetic correlations between analogous performance traits evaluated on the occasion of MPT and AI (Stock and Distl, 2006) justified the combined use of MPT and AI data in this study.

Moderate heritabilities, mostly ranging between 0.15 and 0.58 have been reported for those performance traits evaluated at MPT/AI and SBI in large numbers of horses (e.g. Lührs-Behnke et al., 2006; Stock and Distl, 2006; Stock and Distl, 2007).

Results of this study agree with previous estimates, regardless of which of the two models have been used. In the Hanoverian, breeding use of stallions and mares from other breeds is possible, given the agreement with the breeding directives.

Thoroughbreds and Trakehners are commonly used to make future progeny nobler, the intended use of Holsteiners is to improve jumping performance. Our data support that higher proportions of genes of Thoroughbred and Trakehner had a positively impact performance, particularly rideability. This is in line with the current

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recommendation of the HSS to increase the use of Thoroughbred stallions for breeding. The Holsteiner is especially bred for show jumping (Koenen et al., 2004), so that increased proportions of genes of this breed should be beneficial for free jumping performance in the present analysis.

Accordingly, extension of the model used for routine genetic evaluation by effects correcting for the influence of other breeds, resulted in slightly lower estimates of genetic variances. However, decrease of heritabilities was very small, and the impact on genetic evaluation was negligible. Correlations between corresponding breeding values of >0.98 indicate, that identification of genetically superior individuals will not be relevantly improved by using the extended model. Different results were recently obtained with regard to competition data for dressage performance (Stewart et al., 2009). However, the analyzed horse population was much smaller and more heterogeneous than the Hanoverian population analyzed here. Use of preselected data (scores better than 60% and of internationally competing horses) for a very wide range of horse types with limited pedigree information that performed under different and unknown conditions makes it plausible that stratifications by breed proportions can influence the results of genetic analyses. In the Hanoverian data analyzed here possible breed-related differences may have already been sufficiently accounted for by the relationship matrix. Therefore, genetic evaluation in the linear animal model without explicit consideration of the proportion of genes from Thoroughbred, Trakehner and Holsteiner resulted almost in identical results as the extended model.

These results indicate that analyses of performance data collected under standardized conditions for a large number of horses from the same breed will not relevantly benefit from model extension by breed class effects. Given the positive correlations between young horse performances in performance tests with later success in competitions (Wallin et al., 2003), breeding values estimated on the basis of MPT/AI and SBI information in the standard model should allow reliable identification of individuals for favourable breeding use. Performance-orientated mating will then ensure further breeding progress of the Hanoverian in the main disciplines of riding sport.

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Consideration of the proportions of genes of foreign breeds may be required when using different data sources (e.g., competition data from horses from different breeds) or focussing on identification of genome regions influencing performance.

Stratification of data by the proportion of genes of the most important foreign breeds may then facilitate using molecular genetic tools to speed up the selection response with respect to performance.

3.6 Conclusion

Genetic parameters estimated for performance traits in the Hanoverian without and with the consideration of the proportion of genes of foreign breeds produced almost identical results. Thus the results of this study show that for the Hanoverian population, genetic evaluation for performance in the linear animal model will probably not benefit from including the proportion of genes from Thoroughbred, Trakehner and Holsteiner.

3.7 References

Ducro, B.J., Koenen, E.P.C., van Tartwijk, J.M.F.M., Bovenhuis, H., 2007. Genetic relations of movement and free-jumping traits with dressage and show-jumping performance in competition of Dutch Warmblood horses. Livest. Sci.107, 227- 234.

Groeneveld, E, Kovač, M, Wang, T., 1990. PEST, a general purpose BLUP package for multivariate prediction and estimation. In: World Congress on Genetics Applied to Livestock Production, 488-491. Edinburgh, UK Garsi, Madrid.

Hamann, H., Distl, O., 2008. Genetic variability in Hanoverian warmblood horses using pedigree analysis. J. Anim Sci. 86, 1503-1513.

Koenen, E. P. C., Aldridge, L. I., Philipsson, J., 2004. An overview of breeding objectives for warmblood sport horses. Livest. Prod. Sci. 88, 77-84.

Kovač, M, Groeneveld, E., Garcia-Cortez, A., 2003. VCE-5 User’s Guide and Reference Manual Version 5.1.2. Institute for Animal Science and Animal Husbandry, Federal Agricultural Research Centre (Bundesforschungsanstalt für Landwirtschaft, FAL), Mariensee / Neustadt, Germany.

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Lührs-Behnke, H., Röhe, R., Kalm, E., 2006. Genetische Parameter für Zuchtstutenprüfungsmerkmale der verschiedenen deutschen Warmblutzuchtverbände. Züchtungskunde 78, 271-280.

Stewart, I.D., Woolliams, J.A., Brotherstone, S., 2009. Genetic evaluation of horses for performance in dressage competitions in Great Britain. Livest. Sci., doi:10.1016/j.livsci.2009.10.011.

Stock, K.F., Distl, O., 2007. Genetic correlations between performance traits and radiographic findings in the limbs of German Warmblood riding horses. J. Anim Sci. 85, 31-41.

Stock, K.F., Distl, O., 2006. Genetic correlations between conformation traits and radiographic findings in the limbs of German Warmblood riding horses. Gen. Sel.

Evol. 38, 657-671.

Vanderick, S., Harris, B.L., Pryce, J.E., Gengler, N., 2009. Estimation of test-day model (co)variance components across breeds using New Zealand dairy cattle data. J. Dairy Sci. 92, 1240-1252.

Von Velsen-Zerweck, A., 1998. Integrierte Zuchtwertschätzung für Zuchtpferde. Diss.

agr. Georg August Universität Göttingen.

Wallin, L., Strandberg, E., Philipsson, J., 2003. Genetic correlations between field test results of Swedish Warmblood Riding Horses as 4-year-olds and lifetime performance results in dressage and show jumping. Livest. Prod. Sci. 82, 61-71.

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Table 1 Least square means (LSM) with their standard errors (SE) of performance scores from performance tests (PT) and studbook inspections (SBI) for the proportions of genes of Thoroughbred, Trakehner and Holsteiner, estimated in 36,441 Hanoverians from birth years 1992 to 2005.

PT traits SBI traits

Breed

Breed class PT_Walk PT_Trot PT_Canter PT_FJT PT_Ride SBI_Walk SBI_Imp SBI_Corr

1 6.58 ± 0.017 6.49 ± 0.017 6.78 ± 0.016 6.80 ± 0.038 6.98 ± 0.016 6.67 ± 0.014 6.72 ± 0.014 6.63 ± 0.011 2 6.72 ± 0.014 6.60 ± 0.015 6.88 ± 0.014 6.61 ± 0.035 7.07 ± 0.014 6.77 ± 0.012 6.80 ± 0.012 6.62 ± 0.010 TB

3 6.77 ± 0.017 6.55 ± 0.017 6.89 ± 0.016 6.35 ± 0.038 7.05 ± 0.016 6.80 ± 0.015 6.74 ± 0.014 6.64 ± 0.012 1 6.66 ± 0.015 6.44 ± 0.016 6.81 ± 0.015 6.71 ± 0.037 6.98 ± 0.015 6.70 ± 0.013 6.69 ± 0.012 6.61 ± 0.010 2 6.68 ± 0.015 6.57 ± 0.015 6.87 ± 0.014 6.59 ± 0.036 7.05 ± 0.014 6.75 ± 0.015 6.78 ± 0.012 6.65 ± 0.010 TRAK

3 6.73 ± 0.018 6.63 ± 0.017 6.87 ± 0.017 6.47 ± 0.038 7.08 ± 0.016 6.78 ± 0.015 6.80 ± 0.014 6.63 ± 0.012 1 6.77 ± 0.012 6.64 ± 0.013 6.84 ± 0.013 6.15 ± 0.034 7.06 ± 0.012 6.81 ± 0.01 6.79 ± 0.009 6.61 ± 0.008 2 6.79 ± 0.024 6.60 ± 0.023 6.88 ± 0.022 6.51 ± 0.045 7.09 ± 0.021 6.81 ± 0.02 6.79 ± 0.020 6.62 ± 0.017 HOL

3 6.52 ± 0.016 6.40 ± 0.016 6.83 ± 0.016 7.11 ± 0.037 6.97 ± 0.015 6.61 ± 0.01 6.69 ± 0.014 6.66 ± 0.011

PT_Walk = walk under rider evaluated at mare performance test (MPT) or auction inspection (AI); PT_Trot = trot under rider evaluated at MPT/AI; PT_Canter = canter under rider evaluated at MPT/AI; PT_FJT = total score for free jumping evaluated at MPT/AI; PT_Ride = rideability as judged by the judging commission evaluated at MPT/AI; SBI_Walk = walk at hand evaluated at studbook inspection (SBI); SBI_Imp = impetus and elasticity in trot at hand evaluated at SBI; SBI_Corr = correctness of gaits in walk and trot at hand evaluated at SBI; TB = Thoroughbred (1= ≤0.13; 2 = >0.13 and <0.30; 3 = ≥0.30 of Thoroughbred genes); TRAK = Trakehner (1= ≤0.2; 2 = >0.2 and <0.8; 3 = ≥0.8 of Trakehner genes); HOL = Holsteiner warmblood (1 = 0.0; 2 = >0.0 and <0.3; 3 =

≥0.3 of Holsteiner genes).

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Table 2 Comparison of residual variances (σe²), event variances (σr²) and additive genetic variances (σa²) and of heritabilities (h²) which were univariately estimated for performance traits from performance tests (PT) and studbook inspections (SBI) of 36,415 Hanoverian Warmblood horses from birth years 1992-2005 without (Model 1) or with (Model 2) correction for the proportion of genes of Thoroughbred, Trakehner and Holsteiner in the model.

Model 1 Model 2

Trait

σe² σr² σa² h² σe² σr² σa² h²

PT_Walk 0.6126 ± 0.0087

0.0416 ± 0.0028

0.2207 ± 0.0113

0.2523 ± 0.0114

0.6161 ± 0.0087

0.0416 ± 0.0029

0.2152 ± 0.0113

0.2465 ± 0.0115 PT_Trot 0.4113 ±

0.0073 0.1008 ±

0.0051 0.2627 ±

0.0099 0.3391 ±

0.0107 0.4116 ±

0.0073 0.1012 ±

0.0051 0.2620 ±

0.0099 0.3382 ± 0.0107 PT_Cante

r

0.3889 ± 0.0077

0.1006 ± 0.0056

0.2141 ± 0.0105

0.3043 ± 0.0114

0.3907 ± 0.0076

0.1008 ± 0.0056

0.2112 ± 0.0102

0.3006 ± 0.0113 PT_Ride . 0.3934 ±

0.0070

0.0973 ± 0.0052

0.1461 ± 0.0089

0.2295 ± 0.0107

0.3944 ± 0.0070

0.0974 ± 0.0052

0.1446 ± 0.0088

0.2272 ± 0.0107 PT_FJT 0.7561 ±

0.0084 1.1011 ±

0.0232 0.5455 ±

0.0118 0.2271 ±

0.0080 0.7609 ±

0.0084 1.1028 ±

0.0232 0.5380 ±

0.0118 0.2240 ± 0.0080 SBI_Imp 0.4106 ±

0.0073

0.0428 ± 0.0024

0.2354 ± 0.0099

0.3418 ± 0.0107

0.4108 ± 0.0073

0.0428 ± 0.0024

0.2348 ± 0.0099

0.3411 ± 0.0107 SBI_Corr 0.4279 ±

0.0056 0.0228 ±

0.0019 0.0578 ±

0.0058 0.1137 ±

0.0079 0.4291 ±

0.0056 0.0228 ±

0.0019 0.0560 ±

0.0057 0.1104 ± 0.0079 SBI _Walk 0.5180 ±

0.0079

0.0565 ± 0.0027

0.1890 ± 0.0101

0.2475 ± 0.0107

0.5195 ± 0.0081

0.0565 ± 0.0027

0.1869 ± 0.0102

0.2450 ± 0.0109 PT_Walk = walk under rider evaluated at mare performance test (MPT) or auction inspection (AI); PT_Trot = trot under rider evaluated at MPT/AI; PT_Canter = canter under rider evaluated at MPT/AI; PT_FJT = total score for free jumping evaluated at MPT/AI; PT_Ride = rideability as judged by the judging commission evaluated at MPT/AI; SBI_Walk = walk at hand evaluated at studbook inspection (SBI); SBI_Imp = impetus and elasticity in trot at hand evaluated at SBI; SBI_Corr = correctness of gaits in walk and trot at hand evaluated at SBI.

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Table 3 Pearson correlation coefficients determined between breeding values which were univariately estimated for performance traits from studbook inspections (SBI) and performance tests (PT) of 36,415 Hanoverian Warmblood horses from birth years 1992-2005 without (Model 1) or with (Model 2) correction for the proportion of genes of Thoroughbred, Trakehner and Holsteiner in the model; correlations based on breeding values of all 80,746 horses in the relationship matrix.

Model Trait

SBI_Corr SBI_Walk PT_Walk PT _Trot PT_Canter PT_Ride PT_FJT

Model 1

SBI_Elast 0.5648 0.5749 0.5313 0.7769 0.6836 0.6874 -0.2978 SBI_Corr 0.4176 0.3950 0.5130 0.4738 0.5160 -0.2487 SBI_Walk 0.7432 0.5563 0.5490 0.5976 -0.3742 PT_Walk 0.6338 0.6170 0.6930 -0.4499

PT _Trot 0.7669 0.7892 -0.4146

PT_Canter 0.7884 -0.2219

PT_Ride -0.3143

Model 2

SBI_Elast 0.5566 0.5603 0.5150 0.7670 0.6807 0.6835 -0.2557 SBI_Corr 0.3764 0.3492 0.4841 0.4804 0.5062 -0.1559 SBI_Walk 0.7496 0.5494 0.5314 0.5924 -0.3855 PT_Walk 0.6223 0.6028 0.6870 -0.4484

PT _Trot 0.7540 0.7860 -0.3877

PT_Canter 0.7878 -0.1694

PT_Ride -0.2810

PT_Walk = walk under rider evaluated at mare performance test (MPT) or auction inspection (AI) ; PT_Trot = trot under rider evaluated at MPT/AI; PT_Canter = canter under rider evaluated at MPT/AI; PT_FJT = total score for free jumping evaluated at MPT/AI; PT_Ride = rideability as judged by the judging commission evaluated at MPT/AI; SBI_Walk = walk at hand evaluated at studbook inspection (SBI); SBI_Imp = impetus and elasticity in trot at hand evaluated at SBI; SBI_Corr = correctness of gaits in walk and trot at hand evaluated at SBI.

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Figure 1 Development of performance-related scores from studbook inspections (SBI) between 1995 and 2008 in 29,031 Hanoverians mares from birth years 1992- 2005.

SBI_Walk = walk at hand evaluated at studbook inspection (SBI); SBI_Imp = impetus and elasticity in trot at hand evaluated at SBI; SBI_Corr = correctness of gaits in walk and trot at hand evaluated at SBI.

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Figure 2 Development of scores from mare performance tests (MPT) and auction inspections (AI) between 1995 and 2008 in 24,882 Hanoverians from birth years 1992-2005.

MPT_Walk = walk under rider under evaluated at mare performance test (MPT);

MPT_Trot = trot under rider evaluated at MPT; MPT_Canter = canter under rider valuated at MPT/AI; AI_Walk = walk under rider evaluated at auction inspection (AI);

AI_Trot = trot under rider evaluated at AI; AI_Canter = canter under rider evaluated at AI.

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Figure 3

Cumulative percentage of the proportion of Hanoverian warmblood (Han), Thoroughbred (TB), Trakehner (Trak) and Holsteiner warmblood (Hol) genes in 36,415 Hanoverian Warmblood horses from birth years 1992-2005.

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0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

SBI_Corr (lower 10%) SBI_Corr (20-80%) SBI_Corr (upper 10%) SBI_Imp (lower 10%) SBI_Imp (20-80%) SBI_Imp (upper 10%) SBI_Walk (lower 10%) SBI_Walk (20-80%) SBI_Walk (upper 10%)

Han TB Trak Hol Others

Supplementary Figure 1 Proportions of genes of Hanoverian warmblood (Han), Thoroughbred (TB), Trakehner (Trak), Holsteiner warmblood (Hol), and other breeds (Others) by quantiles of scores for correctness of gaits in walk and trot at hand (SBI_Corr), impetus and elasticity in trot at hand (SBI_Imp) and walk at hand (SBI_Walk) evaluated at studbook inspections between 1995 and 2008 in 29,031 Hanoverians mares from birth years 1992-2005.

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0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

PT_Ride (lower 10%) PT_Ride (20-80%) PT_Ride (upper 10%) PT_FJT (lower 10%) PT_FJT (20-80%) PT_FJT (upper 10%) PT_Canter (lower 10%) PT_Canter (20-80%) PT_Canter (upper 10%) PT_Trot (lower 10%) PT_Trot (20-80%) PT_Trot (upper 10%) PT_Walk (lower 10%) PT_Walk (20-80%) PT_Walk (upper 10%)

Han TB Trak Hol Others

Supplementary Figure 2 Proportions of genes of Hanoverian warmblood (Han), Thoroughbred (TB), Trakehner (Trak), Holsteiner warmblood (Hol), and other breeds (Others) by quantiles of scores for walk under rider (PT_Walk), trot under rider (PT_Trot), canter under rider (PT_Canter), total score for free jumping (PT_FJT), and rideability as judged by the judging commission (PT_Ride) evaluated at mare performance tests and auction inspections between 1995 and 2008 in 24,882 Hanoverians mares from birth years 1992-2005.

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CHAPTER 4

Genetic evaluation of Hanoverian warmblood horses for conformation traits considering the proportion of genes of foreign breeds

WIEBKE SCHRÖDER, KATHRIN FRIEDERIKE STOCK and OTTMAR DISTL

Department of Animal Breeding and Genetics, University of Veterinary Medicine Hannover (Foundation), Hannover; Germany

Archiv Tierzucht 53 (2010) 4, 377-387, ISSN 0003-9438

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Genetic evaluation for conformation

4 Genetic evaluation of Hanoverian warmblood horses for conformation traits considering the proportion of genes of foreign breeds

4.1 Abstract

Conformation data of in total 29,053 Hanoverian warmblood mares were used to determine whether genetic evaluation for conformation in the Hanoverian could benefit from the inclusion of the proportion of genes of foreign breeds in the model.

For our analyses, we considered all Hanoverian mares born from 1992 to 2005 with available studbook inspection data. Genetic parameters were estimated univariately for eight routinely scored conformation traits (head, neck, saddle position, frontlegs, hindlegs, type, frame, and general impression and development), and height at withers from studbook inspections, in a linear animal model using Residual Maximum Likelihood (REML). Genetic evaluation was subsequently performed using Best Linear Unbiased Prediction. To investigate the effect of correcting for the proportion of genes of foreign breeds, two different models were used for the analyses. In Model 1, the fixed effect age at studbook inspection, and the random effect date-place interaction were considered. In Model 2, proportions of genes of Thoroughbred, Trakehner and Holsteiner were additionally included as fixed effects. Heritabilities of analyzed conformation traits and withers height ranged in both models between 0.10 and 0.57, with standard errors of ≤0.01. Pearson correlation coefficients determined between breeding values of corresponding traits using Model 1 and 2 were highly positive (>0.99), indicating little effect of the model on the results of genetic evaluation. According to our results using a model which includes the proportion of genes of Thoroughbred, Trakehner and Holsteiner as fixed effects will not relevantly improve genetic evaluation for conformation in the Hanoverian.

Keywords: horse; Hanoverian; genetic parameters; blood proportion; breeding values; conformation; type

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4.2 Zusammenfassung

Genetische Evaluierung von Exterieur Merkmalen des Hannoveraners, unter Berücksichtigung von Fremd-Genanteilen

Untersucht wurde, ob die Berücksichtigung von Fremdgenanteilen ein Modell für genetische Analysen von Exterieurmerkmalen verbessern kann. Zu diesem Zweck standen Stutbuchaufnahmedaten von insgesamt 29.053 Hannoveraner Stuten der Jahrgänge 1992 bis 2005 des Hannoveraner Verbandes zur Verfügung. Genetische Parameter wurden für acht, routinemäßig bei Stutbuchaufnahmen beurteilte, Exterieurmerkmale (Kopf, Hals, Sattellage, Vorderhand, Hinterhand, Typ, Rahmen und Gesamteindruck und Entwicklung) sowie die Widerristhöhe univariat in einem linearen Tiermodel mittels Residual Maximum Likelihood geschätzt. Die Zuchtwertschätzung wurde anschließend mittels Best Linear Unbiased Prediction und der geschätzten Varianzen durchgeführt. Um die Auswirkungen einer Berücksichtigung von Fremdgenanteilen auf genetische Analysen prüfen zu können, wurden zwei unterschiedliche Modelle verwendet. In Modell 1 wurde das Alter bei Aufnahme ins Stutbuch als fixer Effekt und die Kombination aus Datum und Ort als zufälliger Effekt berücksichtigt. Ein zweites Modell wurde zusätzlich um die Genanteilsklassen von Englischen Vollblut, Trakehner und Holsteiner als fixen Effekten erweitert. Heritabilitäten lagen in beiden Modellen zwischen 0,10 und 0,57 bei Standardfehlern ≤0.01 für die analysierten Exterieurmerkmale sowie der Widerristhöhe. Die Zuchtwerte, geschätzt mit Modell 1 und Modell 2, waren hoch positiv miteinander korreliert (Korrelationskoeffizienten nach Pearson >0.99).

Zusammenfassend zeigen unsere Ergebnisse, dass eine Berücksichtigung von Fremdgenanteilen in einem Zuchtwertschätzmodell für Exterieurmerkmale des Hannoveraners keine wesentlichen Vorteile bringt.

Schlüsselwörter: Pferd; Hannoveraner; genetische Parameter; Blutanteile;

Zuchtwerte; Exterieur; Typ

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GWAS for show-jumping

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41 CHAPTER 5

A genome wide association study for quantitative trait loci of show-jumping in Hanoverian warmblood horses

W. Schröder, A. Klostermann, K. F. Stock and O. Distl

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany

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5 A genome wide association study for quantitative trait loci of show- jumping in Hanoverian warmblood horses

5.1 Summary

Show-jumping is an economical important breeding goal in Hanoverian warmblood horses. The aim of this study was a genome-wide association (GWA) study for quantitative trait loci (QTL) of show-jumping in Hanoverian warmblood horses employing the Illumine equine SNP50 Beadchip. For our analyses we genotyped 115 stallions of the National State stud of Lower Saxony. The show- jumping talent of a horse includes style and ability of free jumping. To control spurious associations based on population stratification, two different mixed linear animal model approaches were employed besides linear models with adaptive permutations for correcting multiple testing. Population stratification was explained best in the mixed linear animal model considering Hanoverian, Thoroughbred, Trakehner and Holsteiner genes and the marker identity-by-state relationship matrix.

We identified six QTL for show-jumping on horse chromosomes (ECA) 1, 8, 9 and 26 (-log10 P-value > 5) and further putative QTL with -log10 P-values of 3-5 on ECA1, 2, 3, 11, 17 and 21. Within six QTL regions, we identified human performance related genes including PAPSS2 on ECA1, MYL2 on ECA8, TRHR on ECA9 and NRF2 on ECA26 and within the putative QTL regions NRAP on ECA1, and TBX4 on ECA11.

The results of our GWA suggest that genes involved in muscle structure, development and metabolism are crucial for elite show jumping performance. Further studies are necessary to validate these QTL in larger data sets and other horse populations.

Keywords: horse, show-jumping, quantitative trait loci, GWA, single nucleotide polymorphisms

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5.2 Introduction

Performance in show jumping is an economical important trait in warmblood horse breeding. An athletic horse, suitable for dressage and show-jumping is the main aim of the Hanoverian Studbook Society (HSS). Primarily bred to be a horse suitable for the military usage, since the foundation of the HSS as early as 1735, the Hanoverian warmblood (Hanoverian) was intensely selected for an athletic and competitive phenotype that is required for an intensely used riding horse. Therefore, the Hanoverian represents one of the most important breeds of sport horses in the world today. Heritabilities for show-jumping in Hanoverian horses were estimated at 0.39 to 0.61 (Stock & Distl 2007). In 1993, a breeding program for show-jumpers "Programm Hannoveraner Springpferdezucht" was initiated with the aim to give more impact to breed elite Hanoverian show-jumping horses.

Due to the long generation interval, genetic improvement in horses needs longer time-spans to be realised, so the application of genetic markers in selection schemes to improve physical performance appears highly desirable. However, even population genetic analyses are performed routinely nowadays, studies on QTL and candidate genes contributing to equine performance are still in the beginnings. Gu et al. (2009) localized genomic regions in the Thoroughbred genome potentially containing genes that influence exercise-related phenotypes by using a hitchhiking mapping approach based on microsatellites. Recently, Hill et al. (2010) could show a single nucleotide polymorphism (SNP) in the myostatin gene (MSTN) which is strongly associated with best results on short and long race distances among Thoroughbreds.

In humans, numerous studies have been performed to identify QTL and candidate genes affecting physical performance in different sports. Therefore, related to physical performance the human is the best studied species so far (Bray et al. 2009).

For human and a few other species e.g. cattle and dogs, genotyping arrays containing SNP markers were successfully used for mapping QTL for quantitative traits (Karlsson et al. 2007; Myles et al. 2008; Kolbehdari et al. 2009). With the completion of the equine genome assembly, SNP assays covering the whole equine genome have been developed to scan genetic variations in horses at a very high resolution.

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The objectives of our study were to perform a genome-wide association (GWA) analysis for show-jumping in Hanoverian horses using the equine SNP50 BeadChip (Illumina, San Diego, CA, USA) and to screen the potential QTL for possible candidate genes known from human studies.

5.3 Materials and Methods 5.3.1 Animals and phenotypic data

Blood samples were collected from 115 Hanoverian warmblood stallions of the National State stud of Lower Saxony. These stallions were born between 1972 and 2000 and represent a random sample from all Hanoverian stallions born in last 20-30 years. Pedigree data were made available by the HSS through the national unified animal ownership database (Vereinigte Informationssysteme Tierhaltung w.V., VIT).

Pedigree records of these stallions allowed us to assign the 115 stallions into 16 families which included a total of 798 stallions. We employed the latest breeding values (BVs) for show-jumping (Mai 2009) provided by HSS. BVs for show-jumping were estimated based on results recorded at mare performance tests (MPTs) since 1987 and inspections before auctions (PAIs) since 1999 including 35,512 animals.

Show-jumping is a composed trait resulting from scores for style and ability of free- jumping. At MPTs mares are scored by a judging commission for show-jumping using a scale from 0 (not shown) to 10 (excellently shown) with 0.5 intervals. Style and ability of free jumping are separately scored and then averaged to a total score for show-jumping. Horses pre-selected for sale at riding horse auctions of the HSS are scored for show-jumping by a judging commission. Between 1999 and 2008, 8,081 Hanoverians (5,567 males, 2,514 females) were judged at PAIs. Auction candidates are scored for the same traits and at the same scale like the mares at MPTs. The same commission judges the mares at MPTs and the auction candidates to ensure comparable results. If a mare took part in a PAI as well as in a MPT, then the result of the MPT is included in the BV estimation. For mares which repeated the MPT, the last result is included. BVs are estimated yearly through VIT for show-jumping employing a bivariate BLUP (best linear unbiased prediction) animal model (Christmann 1996).

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