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Bioinformatics and Molecular Genetic Studies of Domestic and Wild Buffalo Species: Focus on Evolutionary Relationship of the DGAT1 Gene

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Bioinformatics and Molecular Genetic Studies of Domestic and Wild Buffalo Species: Focus on Evolutionary Relationship of the DGAT1 Gene Swedish University of Agricultural Sciences Faculty of Veterinary Medicine and Animal Science Bioinformatics and molecular genetic studies of domestic and wild buffalo species: Focus on evolutionary relationship of the DGAT1 gene Farzad Hakim Javadi Examensarbete / Swedish University of Agricultural Sciences Master Thesis, 30 hp Department of Animal Breeding and Genetics Master’s Programme 427 – Animal Science Uppsala 2013 Swedish University of Agricultural Sciences Faculty of Veterinary Medicine and Animal Science Department of Animal Breeding and Genetics Bioinformatics and molecular genetic studies of domestic and wild buffalo species: Focus on evolutionary relationship of the DGAT1 gene Farzad Hakim Javadi Supervisors: Göran Andersson, SLU, Department of Animal Breeding and Genetics Erik Bongcam-Rudloff, SLU, Department of Animal Breeding and Genetics Examiner: Gabriella Lindgren, SLU, Department of Animal Breeding and Genetics Credits: 30 hp Course title: Degree project in Animal Science Course code: EX0556 Programme: Master’s Programme - Animal Science Level: Advanced, A2E Place of publication: Uppsala Year of publication: 2013 Name of series: Examensarbete / Swedish University of Agricultural Sciences, Department of Animal Breeding and Genetics, 427 On-line publicering: http://epsilon.slu.se Key words: Buffalo, DGAT1, promoter, Swamp, River Abstract Improving milk yield and production is the main purpose of the selection in dairy industry. Genotyping tools give the opportunity to increase the frequency of favorable traits in the herd. Efficient breeding programs used in dairy industry have resulted in extensive genetic progress for many economically important traits. Progeny testing helps to choose the best young males based on their genetic merit and therefore their potential for being used in breeding. During the last few years statistical tools have used the massive genetic information available for selecting the young animals with high breeding values in the early ages which is referred to genomic selection. One such trait that has been selected for is milk yield and composition that is controlled by a Quantitative trait locus (QTL) in the centromeric region of cattle (Bos taurus) chromosome 14. This SNP is not conserved between different cattle breeds and buffalo. A previously reported amino acid substitution in bovine diacylglycerol acyl transferase 1 (DGAT1) gene, K232A (lysine to alanine substitution) has been correlated with milk quality traits (Grisart et al., 2004) (Figure 16). This polymorphism is localized in exon VIII of the DGAT1 gene. DGAT is involved in the triglyceride biosynthesis and catalyzes the conversion of complex carbohydrates to triglycerides and therefore controls the lipid content in milk. Restriction fragment length polymorphism (RFLP) identified three DGAT1 genotypes (AA, AG, GG). Genotype AA is associated with higher milk fat content. Basically, dairy industry was more interested to improve milk yield and protein content of the milk, therefore cow herd should have higher proportion of G allele. Water Buffalo (Bubalus bubalis) populations have not been subjected to that selection pressure since the aim of buffalo production is more milk fat content. The aim of this project was to examine the potential association of DGAT1 genotypes in different buffalo populations. Comparison was performed between Asian subtypes (Swamp and River), and River buffaloes that were imported to Sweden from Italy. Obtained sequences were compared to those present in the publically available cattle genome sequence. The identification of the promoter region of the DGAT1 gene was done by bioinformatics approach to search for any significant differences within and between populations. Our result confirms that same SNP is available in the Swamp Buffalo population, but because of the lack of recording we have not been able to correlate genotypes with significant differences in milk fat content. Different length of promoter was observed when comparing cattle genome with human. A highly conserved region located near the DGAT1 translation start site was found using the 29 mammals’ database, which suggest that this region is important for proper translation of DGAT1 mRNA. The result of this experiment identified one SNP positioned at 857 bp 5’ of the translation initiation codon (ATG) in a potential transcriptional regulatory region. The only difference is that our River buffalo population had different SNP on that position T instead of G in the cattle reference sequence (Figure 14 and 15). The other region up or down stream of this region might be the potential region regulating transcription, which increase the importance of the SNP position to function as an regulatory region for DGAT1 transcription or the genes that surrounds the DGAT1 gene. Further functional studies can confirm our hypothesis about transcription binding site and length of promoter in the genera bovina and bubalina. I II 1 Contents 2 Introduction ..................................................................................................................................... 1 2.1 World water buffalo population .................................................................................................... 1 2.1.1 Asia ................................................................................................................................................ 1 2.1.1.1 India ................................................................................................................................... 2 2.1.1.2 China ................................................................................................................................. 4 2.1.1.3 Indonesia ........................................................................................................................... 5 2.1.2 Africa ............................................................................................................................................. 5 2.1.2.1 Egypt ................................................................................................................................. 6 2.1.3 Europe ........................................................................................................................................... 6 2.1.3.1 Italy .................................................................................................................................... 6 2.2 Buffalo breeds ............................................................................................................................... 8 2.3 Buffalo cheese and milk industry .................................................................................................. 8 2.3.1 Fat-rich milk products ................................................................................................................... 8 3 Literature review ............................................................................................................................. 9 3.1 Milk composition .......................................................................................................................... 9 3.1.1 Milk ............................................................................................................................................... 9 3.1.1.1 The globule membrane in milk ........................................................................................ 10 3.1.1.1.1 Origin ........................................................................................................................ 10 3.1.1.1.2 Composition and structure......................................................................................... 10 3.1.1.1.3 Functional and biological properties ......................................................................... 10 3.1.1.1.4 Diacylglycerol acyltransferase .................................................................................. 10 3.2 Genetic analyses .......................................................................................................................... 11 3.2.1 Breeding and selection in dairy industry ..................................................................................... 11 3.2.2 Quantitative trait analysis ............................................................................................................ 12 3.2.3 DGAT1 ........................................................................................................................................ 12 4 Materials and Methods ................................................................................................................. 13 4.1 DGAT1 Genotyping ..................................................................................................................... 13 4.1.1 Collection of hair samples and handling ..................................................................................... 13 4.1.2 Preparation of genomic DNA from hair ...................................................................................... 13 4.1.3 Primer design ............................................................................................................................... 13 4.1.4 Polymerase Chain Reaction (PCR) ............................................................................................. 13 III 4.1.5 Sequencing .................................................................................................................................. 13
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