Biodiversity of Arabian horses in Syria Dissertation zur Erlangung des akademischen Grades Doctor rerum agriculturarum (Dr. rer. agr.) eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt Universität zu Berlin von M.Sc. Saria Almarzook Präsidentin der Humboldt-Universität zu Berlin Prof. Dr. Sabine Kunst Dekan der Humboldt-Universität zu Berlin Prof. Dr. Bernhard Grimm Gutachterin/Gutachter Prof. Dr. Gudrun Brockmann Prof. Dr. Dirk Hinrichs Prof. Dr. Armin Schmitt Tag der mündlichen Prüfung: 17. September 2018 Dedication This research is dedicated to my homeland …Syria Contents Zusammenfassung ................................................................................................................... I Summary ............................................................................................................................... VI List of publications and presentations .................................................................................. XII List of abbreviations ............................................................................................................ XIII List of figures ....................................................................................................................... XIV List of tables ......................................................................................................................... XV 1. General introduction and literature review ..................................................................... 1 1.1. Domestication and classification of horses ........................................................ 1 1.2. Arabian horses .................................................................................................. 5 1.2.1. History and origin .............................................................................................. 5 1.2.2. Population structure and stud-books ................................................................ 6 1.2.3. Arabian horses in Syria ...................................................................................... 6 1.2.3.1. Location and climatic conditions ....................................................................... 6 1.2.3.2. Registries and strains ......................................................................................... 8 1.2.3.3. Breeding system ................................................................................................ 9 1.3. Phenotypic characterization of Arabian horses ............................................... 12 1.3.1. Morphological traits ........................................................................................ 12 1.3.2. Endurance performance .................................................................................. 14 1.3.3. Coat color ........................................................................................................ 16 1.4. Molecular genetic characterization of Arabian horses ..................................... 17 1.4.1. Genetic diversity ............................................................................................. 17 1.4.2. Measures of genetic diversity and population structure ................................. 18 1.4.2.1. Genetic variation within population ................................................................ 19 1.4.2.2. Genetic variation between populations ........................................................... 20 1.4.3. Molecular markers for genetic diversity studies in horses ............................... 23 1.4.3.1. Mitochondrial markers .................................................................................... 24 1.4.3.2. Y chromosome markers ................................................................................... 27 1.4.3.3. Whole genome chromosomal markers ........................................................... 28 1.4.4. Candidate genes for endurance performance ................................................. 33 1.4.4.1. Mitochondrial candidate genes ....................................................................... 34 1.4.4.1.1. MT-ND4 ........................................................................................................... 37 1.4.4.1.2. MT-CYB ........................................................................................................... 37 1.4.4.1.3. MT-COX3 ......................................................................................................... 38 1.4.4.1.4. MT-ATP6 ......................................................................................................... 38 1.4.4.2. Autosomal candidate genes ............................................................................ 39 1.4.4.2.1. ACTN3 ............................................................................................................. 41 1.4.4.2.2. ADRB2 ............................................................................................................. 42 1.4.4.2.3. MSTN .............................................................................................................. 43 1.4.4.2.4. PPARGC1A ....................................................................................................... 45 1.4.4.2.5. CALCA .............................................................................................................. 46 1.4.4.2.6. CYP2R1 ............................................................................................................ 47 1.4.5. Genome-wide association studies (GWAS) ...................................................... 48 2. Objectives of the study .................................................................................................. 52 3. Materials and methods ................................................................................................. 54 3.1. Materials ......................................................................................................... 54 3.1.1. Chemicals ........................................................................................................ 54 3.1.2. Instruments ..................................................................................................... 55 3.1.3. Arabian horse data .......................................................................................... 56 3.1.3.1. Animals ........................................................................................................... 56 3.1.3.2. Morphological traits ........................................................................................ 59 3.1.3.3. Endurance records .......................................................................................... 62 3.1.3.4. Ethics ............................................................................................................... 63 3.2. Molecular methods ......................................................................................... 63 3.2.1. DNA extraction ................................................................................................ 63 3.2.2. DNA fragment amplification ............................................................................ 64 3.2.3. DNA quantification .......................................................................................... 64 3.2.4. Sequencing of mitochondrial PCR amplicons ................................................... 65 3.2.5. Microsatellites genotyping .............................................................................. 66 3.2.6. Genotyping with the equine SNP array ............................................................ 69 3.2.7. Sequencing of candidate genes ....................................................................... 69 3.3. Analyses of diversity and Statistics .................................................................. 74 3.3.1. Genetic diversity indices and phylogeny .......................................................... 74 3.3.2. SNP array data analyses................................................................................... 75 3.3.3. Genome-wide association study ...................................................................... 76 4. Results ........................................................................................................................... 77 4.1. Diversity of mitochondrial DNA in three Arabian horse strains ........................ 77 4.1.1. Genetic variation and haplotypes of the mitochondrial D-loop ....................... 77 4.1.2. Maternal phylogenetic relationship ................................................................. 79 4.2. Genetic diversity based on 12 microsatellite loci ............................................. 81 4.2.1. Within the three strains .................................................................................. 81 4.2.2. Between the three strains ............................................................................... 83 4.3. Genetic diversity using the equine SNP70K BeadChip ..................................... 85 4.3.1. Diversity indices .............................................................................................. 85 4.3.2. Population structure ....................................................................................... 86 4.4. Genome-wide association study of morphological traits in Arabian horses ..... 88 4.5. Test of candidate genes polymorphisms in endurance Arabian horses ........... 89 4.5.1. Mitochondrial genes ......................................................................................