Fine-mapping of the F4ab/F4ac receptor locus on pig chromosome 13

Abstract

Enterotoxigenic Escherichia coli (ETEC) with F4ab/F4ac fimbriae are an important cause of diarrhea in pigs and can therefore be responsible for considerable economic losses and negative impacts on animal welfare. Since some pigs are resistant to adhesion of ETEC F4ab/F4ac in the intestine, a genetic approach to implement F4ab/F4ac resistance in breeding schemes is under development. The receptor locus for ETEC F4ab/F4ac has been mapped to a region around the gene MUC13 on the pig chromosome 13, and recent research gave high evidence that MUC13 is responsible for resistance/susceptibility against F4ab/F4ac adhesion. Due to technical difficulties in amplification and sequencing in parts of MUC13, the causative mutation has not yet been found. Therefore, genotyping of marker single nucleotide polymorphisms (SNPs) is used to predict the receptor genotype for F4ab/F4ac susceptibility/resistance. For efficient and cost-effective SNP genotyping, alternative methods to sequencing and polymerase chain reaction – restriction fragment length polymorphism tests (PCR-RFLP tests) are desired. The methods Kompetitive Allele-Specific PCR (KASP), Melting Temperature (Tm)-Shift, and High Resolution Melting (HRM), which are based on allele-specific PCR or melting curve analysis, are thus tested on reliability and applicability in the present thesis. With the method KASP, we genotyped pigs from different sample groups on the marker SNPs ALGA5, CFCH1 and CFCH2. For a possible replacement of the current PCR-RFLP test for E. coli F18 resistance, a KASP assay was developed and successfully tested. The established labour intensive PCR-RFLP test can therefore be replaced for genotyping purposes. The KASP assays for the different SNPs achieved 100% concordant results with previous genotyping data. Importantly, no samples got a wrong genotype assigned. Some samples did not amplify well, due to poor DNA quality or amount, and remained without assigned genotype. Similar results were achieved for Tm-shift. The two Tm-shift assays for ALGA5 and CFCH2 both genotyped all samples concordant to previous data. With HRM, we were unable to reliably genotype samples for ALGA5 and CFCH2. It is concluded, that HRM makes higher demands on assay development and result interpretation compared to KASP and Tm-shift. In terms of applicability, KASP and Tm-shift are comparable and both simple to perform. Tm-shift has the advantage to be more flexible due to the primer design being done directly in the lab. Furthermore Tm-shift is more suitable to genotype few samples while KASP may be more suitable to genotype many samples, due to different demands on the minimal number of samples and the number of necessary control samples. KASP and Tm-shift are considered both suitable to genotype marker SNPs for ETEC F4ab/F4ac resistance, while HRM is considered less suitable due to extra costs for the analysis software and the suggested higher demands on assay development. Based on our experiences with KASP assays for CFCH1 and CFCH2, the SUISAG decided to genotype 271 boars in use for artificial insemination (AI) in a routine lab. The two markers were completely concordant in all 196 Large White boars, whereas in the breeds Pietrain, Duroc and Landrace up to 40% showed discordant genotypes. Fifteen recombinant boars were genotyped with additional markers and phenotype information from three Landrace boars revealed the SNP CFCH1 to be prioritized in this breed. Duroc and Pietrain pigs can only be reliably genotyped if CFCH1 and CFCH2 give concordant results. Phenotypic data of discordant genotypes are needed in these breeds in order to select a suitable marker.