Molecular Strategies in the Analysis of the Porcine Genome

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The main goal of genome research in livestock is to map and characterize trait loci controlling various phenotypic characters like disease resistance, reproduction and other production traits of economic importance (e.g. meat quality). Although identification and mapping of functional genes and quantitative trait locus (QTL) regions associated with traits of interest increased significantly in pigs, the number of genes mapped is still small compared with human and mouse. Comparative analysis and bioinformatic tools combined with genomic and post-genomic techniques enable to achieve greater progress in the analysis of the porcine genome. The present thesis focuses on structural and functional analysis of genes of the porcine phosphoglycerate kinase (PGK) family which have previously been identified as candidate genes for inherited male fertility along with other inherited disorders in human and mouse. The second part of the thesis describes the isolation of microsatellite markers either from specific chromosomal region of interest, or from large-insert library clones harboring targeted trait genes, and the application of these informative markers in phylogenetic studies.The porcine functional PGK genes, i.e. the ubiquitously expressed X-linked PGK1 gene and the autosomally determined testis-specific PGK2 gene, were identified and mapped on SSCXq12-q13 and SSC7q14-q15, respectively. The 1665 bp of full-length PGK2 cDNA were compiled using the modified rapid amplification 5 -RACE and 3 -RACE information. The results of genomic and cDNA sequences of the porcine PGK2 gene demonstrated that it is a single-exon intronless gene with a complete open reading frame of 1251 bp encoding a PGK protein of 417 amino acids. Real time PCR results showed that PGK2 mRNA was solely expressed in the testis. There was a lower amount of PGK2 expression in the testis from a herniated boar and a very small amount of PGK2 expression in the testis of an 8-week-old piglet compared to an adult boar. Two SNPs of PGK2 gene (SNP-A: T427C; SNP-B: C914A) resulting in amino acid substitutions (SNP-A: Ser102-Pro102; SNP-B: Thr264-Lys264) were detected and genotyped among six pig breeds. The SNP-A mutation of proline could lead to the loss of a casein kinase II (CK2) phosphorylation site in the PGK2 peptide. Association analyses between the PGK2 gene and some traits of sperm quantity and quality were performed. The results showed that SNP-B has a significant effect on semen volume in the Pietrian breed (p = 0.08).Fifteen (GT)n porcine microsatellites (S0701 S0715) were isolated from nine PAC clones by an improved efficient and rapid flanking sequence-based approach. Eight of the newly developed microsatellites were gene-associated, and the other seven microsatellites within the poorly covered chromosome region SSC16q were isolated to refine this region. The microsatellite markers were genotyped for a panel of 245 unrelated animals representing 15 European and Chinese indigenous pig breeds. A set of eleven microsatellite loci with a total of 136 alleles was used to analyze the phylogenetic relationships between the pig populations. Breed differentiation was significant as shown by the high average fixation index (FST = 0.271). Neighbor-joining (NJ) tree and unweighted pair group method with arithmetic mean (UPGMA) dendrogram were constructed. The results revealed that European and Chinese indigenous pig breeds had diverged into two distinct groups, apart from the German Göttingen Minipig and the European wild boar, which clustered into the branches of Chinese pig populations.