Genomic characterisation and polymorphism analysis of candidate genes for milk production traits and association studies in three cattle breeds [Elektronische Ressource] / Franz Reinhold Seefried

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 13
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Tierzucht
Genomic Characterisation and Polymorphism Analysis of Candidate Genes for Milk
Production Traits and Association Studies in Three Cattle Breeds
Franz Reinhold Seefried
Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzende: Univ.-Prof. A. Schnieke, Ph.D. (Univ. of Edinburgh/UK)
Prüfer der Dissertation:
1. Univ.-Prof. Dr. Dr. H.-R. Fries
2. Univ.-Prof. Dr. G. A. Thaller (Christian-Albrechts-Universität zu Kiel)
Die Dissertation wurde am 10.12.2007 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum für Ernährung, Landnutzung und Umwelt am
11.06.2008 angenommeniiTable of contents
List of figures ......................................................................................................................vii
List of tables ..........................................................................................................................x
Abbreviations......................................................................................................................xiv
1. Introduction and goals...................................................................................................1
1. Literature review............................................................................................................2
1.1. Analysis of quantitative traits...................................................................................................2
2.2. QTL mapping approaches on cattle chromosome 6 (BTA6)................................................3
2.2. Previous association studies with candidate genes on cattle chromosome 6 (BTA6) ........4
2. Animals, Materials and Methods...................................................................................8
3.1. Animal panels for SNP screening and association studies....................................................8
3.2. Materials and Methods..............................................................................................................9
3.1.1. In-silico analyses...............................................................................................................................9
3.1.1.1. Selection of candidate genes........................................................................................................9
3.2.1.2. Gene identification in the bovine genomic sequence .................................................................9
3.2.1.3. Gene annotation..........................................................................................................................10
3.2.1.4. Genomic characterisation of candidate genes...........................................................................11
3.2.2. Primer design...................................................................................................................................12
3.2.1. Preparation of genomic DNA .........................................................................................................12
3.2.4. Polymerase chain reaction (PCR)...................................................................................................13
3.2.4.1. Standard PCR .............................................................................................................................13
3.2.4.1. PCR optimisation steps..............................................................................................................13
3.2.5. DNA sequencing .............................................................................................................................14
3.2.5.1. PCR cleaning..............................................................................................................................14
3.2.5.2. Sequencing reaction ...................................................................................................................14
3.2.5.3. Sequencing on an ABI377 automated sequencer......................................................................14
3.2.5.4. Detection of DNA variants ........................................................................................................15
3.2.6. Selection of SNPs for association studies ......................................................................................15
3.2.4. Genotyping with iPLEX Assay for Matrix-assisted Laser-Desorption / Ionisation Time-of-
Flight Mass spectrometry (MALDI-TOF) .........................................................................................................15
3.2.4.1. Primer design for iPLEX Assay ................................................................................................17
3.2.4.2. PCR for iPLEX Assay................................................................................................................18
3.2.4.3. Treatment with shrimp alakaline phosphatase (SAP)...............................................................18
3.2.4.4. iPLEX reaction...........................................................................................................................18
3.2.4.5. Desalting the iPLEX reaction ....................................................................................................19
3.2.4.6. Chip dispensing and MALDI-TOF Analysis ............................................................................19
iiiTable of contents
3.2.8. Data processing ...............................................................................................................................20
3.2.4.1. Phenotypic data ..........................................................................................................................20
3.2.8.2. Genotypic data............................................................................................................................21
3.2.9. Statistical analyses ..........................................................................................................................22
3.2.4.1. Testing for Hardy-Weinberg Equilibrium.................................................................................22
3.2.9.2. Analysing Linkage Disequilibrium between candidate genes..................................................22
3.2.9.3. Haplotype construction ..............................................................................................................23
3.2.9.4. Linear regression models ...........................................................................................................23
3.2.9.4.1. Linear regression models in single marker analysis (SMA)..............................................23
3.2.9.4.2. Linear regression models in haplotype analysis (HA) .......................................................25
3.2.9.4.3. Linear regression models in multi marker analysis (MMA)..............................................26
3.2.9.4.4. Haplotype analysis with final marker set ...........................................................................27
3. Results .........................................................................................................................28
4.1. Selection of candidate genes....................................................................................................28
4.2. In-silico gene structure derivation .........................................................................................28
4.2.1. Positioning of candidate genes in bovine draft sequences.............................................................28
4.2.2. Annotation of candidate genes........................................................................................................31
4.3. Characterisation of candidate genes......................................................................................33
4.2.1. ATP-binding cassette sub-family G member 2 gene (ABCG2) ....................................................33
4.3.2. Osteopontin gene (OPN).................................................................................................................36
4.3.3. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha gene (PPARGC1A) .....39
4.3.4. Phosphoglucomutase-2 gene (PGM2)............................................................................................43
4.3.5. Alpha-s1-casein gene (CSN1S1).....................................................................................................46
4.3.6. Facilitated glucose transporter member 9 gene (SLC2A9) ............................................................50
4.3.7. Summary of candidate gene characterisation.................................................................................53
4.4. Polymorphism analysis............................................................................................................54
4.4.1. Identified polymorphisms ...............................................................................................................54
4.4.2. Polymorphisms selected for association studies ............................................................................56
4.5. Genotyping result and test for Hardy-Weinberg Equilibrium ..........................................58
4.6. Linkage Disequi