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Polymorphism in Myostatin Gene and Athletic Performance in Nordic Horse Breeds

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Polymorphism in Myostatin Gene and Athletic Performance in Nordic Horse Breeds Swedish University of Agricultural Sciences Faculty of Veterinary Medicine and Animal Science Polymorphism in myostatin gene and athletic performance in Nordic horse breeds Agnese Viluma Examensarbete / Swedish University of Agricultural Sciences, Master’s Thesis, 30 HEC Department of Animal Breeding and Genetics, Erasmus Mundus Programme 378 – European Master in Animal Uppsala 2012 Breeding and Genetics Swedish University of Agricultural Sciences Faculty of Veterinary Medicine and Animal Science Department of Animal Breeding and Genetics Polymorphism in myostatin gene and athletic performance in Nordic horse breeds Agnese Viluma Supervisor: Gabriella Lindgren, SLU, Department of Animal Breeding and Genetics Lisa Andersson, SLU, Department of Animal Breeding and Genetics Dag Inge Våge, UMB, Husdyr- og akvakulturvitenskap Examiner: Leif Andersson, SLU, Department of Animal Breeding and Genetics Credits: 30 HEC Course title: Degree project in Animal Science Course code: EX0556 Programme: Erasmus Mundus Programme – European Master in Animal Breeding and Genetics Level: Advanced, A2E Place of publication: Uppsala Year of publication: 2012 Cover picture: Lasse Fransson Name of series: Examensarbete / Swedish University of Agricultural Sciences, Department of Animal Breeding and Genetics, 378 On-line publication: http://epsilon.slu.se Key words: myostatin, horse, athletic performance, Coldblooded Trotter, Icelandic Horse, Nordic breeds European Master in Animal Breeding and Genetics POLYMORPHISM IN MYOSTATIN GENE AND ATHLETIC PERFORMANCE IN NORDIC HORSE BREEDS Agnese Viluma 860627-P107 THESIS ANIMAL BREEDING AND GENETICS (EX0556) Jun 2012 Department of Animal Breeding and Genetics SUPERVISORS Supervisor 1 Gabriella Lindgren, SLU Supervisor 2 Lisa Andersson, SLU Supervisor 3 Dag Inge Våge, UMB CONTENTS Abbreviations ......................................................................................................................................... 3 Abstract .................................................................................................................................................. 4 1. Introduction ........................................................................................................................................ 5 2. Materials and Methods ....................................................................................................................... 8 2.1. Study animals .............................................................................................................................. 8 2.3. Molecular analysis ...................................................................................................................... 9 2.4. Data analysis ............................................................................................................................. 11 2.5. Phenotypic associations ............................................................................................................ 11 2.5.1. Frequency differences ........................................................................................................ 12 2.5.2. Haplotype association ........................................................................................................ 12 3. Results .............................................................................................................................................. 12 3.1. SNP marker segregation in different horse breeds .................................................................... 12 3.2. Detection of Ins227 ................................................................................................................... 16 2.3. Phenotypic associations ............................................................................................................ 17 2.3.1. Comparison of allele and haplotype frequencies between groups ..................................... 17 2.3.2. Associations with phenotypic data ..................................................................................... 19 4. Discussion ........................................................................................................................................ 20 References ............................................................................................................................................ 23 Acknowledgements .............................................................................................................................. 26 ABBREVIATIONS BLUP Best linear unbiased prediction bp Base pairs BRD Best race distance DNA Deoxyribonucleic acid ECA Equine chromosome HWE Hardy-Weinberg equilibrium LD Linkage disequilibrium MAF Minor allele frequency miRNA Micro Ribonucleic acid MSTN Myostatin PCR Polymerase chain reaction UTR Untranslated region WRD Win-race distance 3 ABSTRACT Athletic performance including working ability, riding, racing and endurance has played a leading role in horse breeding possibly since their domestication. Even though traits of athletic performance are considered as complex and dependant on environment, management and training, it has been shown that heritability of some performance traits can be considerably high. Skeletal muscle system seems to be a key factor to exceptional athletic performance and numerous studies in other species (dogs, humans, sheep, cattle etc.) have shown that polymorphisms in MSTN gene can be responsible for phenotypic changes in muscle tissues. Several studies focusing on horse MSTN gene region have been made and some significant associations discovered in Thoroughbred populations. The main objective of this study is to estimate the allele frequencies of the g.66493737 SNP, which is the strongest associated SNP with the optimim race distance, and some additional SNPs (two in a promoter region and one downstream the gene) in MSTN gene of Nordic horse breeds and correlate them with performance trait data to create a deeper understanding of the gene’s influence on performance ability in horses. For analysis 25 horse breeds and four Donkey and Przewalski’s horse individuals were selected. Four SNP marker positions (g.65868604G>T, g.66493737C>T, g.66495696G>A, g.66495826G>A) and one insertion (Ins227) were chosen based on previously published research results. For genotyping SNPs real time PCR TaqMan approach and touchdown PCR for detection of Ins227 was used. Phenotypic data of performance traits were correlated with inferred haplotypes of Icelandic Horse and Coldblooded Trotters using R software. Results revealed segregation in all SNP marker positions, having variable allele frequencies between breeds and lacking g.66495696G homozygous individuals. No Ins227 allele was detected, except those in Thoroughbred horses and one heterozygous individual of Swedish Warmblood. Haplotype association resulted in some significant haplotype effects on proportions (p-value<0.05) and tolt - running walk - ability (p-value<0.05) in Icelandic horse. More significant effect was detected for Coldblooded Trotters where both haplotypes having mutation in proximal promoter region had a significant effect on total BLUP value and BLUP values for Position, Earnings and Time. Haplotype having g.66495696G allele shoved the most significant association having p-values from 0.029 for Start status BLUP value to 0.001 for Position and Earnings. Finally, we can conclude that tested polymorphisms in MSTN gene are widespread in Nordic Horse breeds and there are indications that some of them might have a functional effect on horse performance traits. In addition, we have obtained specific directions of further research possibilities. Keywords: myostatin, horse, athletic performance, Coldblooded Trotter, Icelandic Horse, Nordic breeds 4 1. INTRODUCTION In modern horse (Equus ferus caballus) breeding athletic performance has been considered as the most important criteria for selection and horses with superior athletic performance are of the highest value. They have been selected for working, riding, racing and endurance performance since their domestication and possess a range of functional and structural adaptations for exceptional athletic performance (Schröder et al., 2011). Traits of athletic performance are of complex nature and are highly dependent on such factors as environment, management and training (Bower et al., 2012). However, since the end of 20th century it has been accepted that there are underlying genetic factors and the heritability of various measures of racing performance have been considered as high (Gaffney & Cunningham; 1988). In addition, studies in humans, rats and mice have revealed a large number of genes that are involved in elite athletic performance (Schröder et al. 2011). Thoroughbreds is one of the most investigated horse populations and the racing performance of Thoroughbreds is believed to be polygenic - being influenced by genes that contribute to the wide range of anatomical, metabolic and physiological adaptations (Bower et al., 2012). Myostatin has been proved to be one of the genes with an important role in horse athletic performance and polymorphism of this gene (g.66493737) has been associated with the best race distance in Thoroughbred horses (Hill et. al., 2010a). Myostatin (MSTN) or transforming growth factor-8 (TGF-8) is a member of TGF-B superfamily and was discovered by McPherron et al. (1997) during the search of new TGF-B superfamily members. Genomic location of MSTN gene is on equine chromosome 18 (ECA18) 66,490208
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