This diploma thesis follows the bachelor thesis and it?s task is to processing analysis of the occurrence of PSE meat in a population of the Czech white breed as a pigs stress syndrome impact. General problem of congenital diseases is processed in literature review. Further, this work deals with pigs stress syndrome and stress, which causes the meat defects as an external factor. Molecular - genetic methods, which are used in the practical part, are described as well. The research focused on genotyping of loci selected panel of samples, to determine the frequency of alleles and genotypes of selected locus and the statistical relationship between genotype and expression of PSE meat. Results were statistically processed and evaluated at the end.

Malignant hyperthermia (MH) is a dominant skeletal muscle disorder caused by mutations in the ryanodine receptor skeletal muscle calcium release channel (RyR1). Allele-specific differences in RyR1 expression levels might provide insight into the observed incomplete penetrance and variations in MH phenotypes between individuals. Firstly, an H4833Y allele-specific PCR (AS-PCR) assay was designed that allowed for the relative quantification of the two RYR1 mRNA alleles in heterozygous samples. In four MHS skeletal muscle samples and two lymphoblastoid cell lines (LCLs), the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. These differences were not caused by variations in RYR1 mRNA stabilities. Secondly, high-throughput amplicon sequencing was employed for the quantification of both the T4826I and H4833Y causative MH mutations in heterozygous MHS samples. With the exception of one, all detected H4833Y and T4826I mutation frequencies were about 50%. This included a control, which was constructed and proven to have a 3:1 ratio of the wild type (H4833) versus the mutant (Y4833) RYR1 allele. This suggested that that the high-throughput amplicon sequencing approach as used here, was not suitable for accurate quantification of the two RyR1 alleles in heterozygous H4833Y MHS samples. To detect possible variations in RyR1 alleles at the protein level, the RyR1 was to be isolated from microsomes prepared from a H4833Y MHS frozen skeletal muscle tissue. Microsomes isolated from MHS skeletal muscle tissues lacked the immunoreactive band that was believed to be the full length RyR1. Poor muscle quality, due to long term storage was believed to be the main cause of RyR1 depletion. Faster and less expensive screening methodologies are required for the identification of genetic variants in MH research. Thus, in an additional project inexpensive and high-throughput high-resolution melting (HRM) assays were developed to allow screening of the RYR1 gene, for mutations associated with MH and/or central core disease (CCD).