A dual fluorescent multiprobe assay for prion protein genotyping in sheep

BACKGROUND: Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep. METHODS: A dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above. RESULTS: The assay is succesfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep. CONCLUSIONS: We report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations

Molecular cloning and characterization of the porcine prostaglandin transporter (SLCO2A1): evaluation of its role in F4 mediated neonatal diarrhoea

<p>Abstract</p> <p>Background</p> <p>Because prostaglandins are involved in many (patho)physiological processes, <it>SLCO2A1 </it>was already characterized in several species in an attempt to unravel specific processes/deficiencies. Here, we describe the molecular cloning and characterization of the porcine ortholog in order to evaluate its possible involvement in F4 enterotoxigenic <it>E. coli </it>mediated neonatal diarrhoea, based on a positional candidate gene approach study.</p> <p>Results</p> <p>Porcine <it>SLCO2A1 </it>is organized in 14 exons, containing an open reading frame of 1935 bp, encoding a 12-transmembrane organic anion cell surface transporter of 644 aa. The -388 to -5 upstream region comprises a (CpG)₄₈island containing a number of conserved promoter elements, including a TATA box. A potential alternative promoter region was found in the conserved -973 to -700 upstream region. No consensus polyadenylation signal was discovered in the 3' UTR. Repeat sequences were found in 15% of all the non coding sequences.</p> <p>As expected for a multifunctional protein, a wide tissue distribution was observed. mRNA expression was found in the adrenal gland, bladder, caecum, colon (centripetal coil/centrifugal coil), diaphragm, duodenum, gallbladder, heart, ileum, jejunum, kidney, liver, longissimus dorsi muscle, lung, lymph node, mesenterium, rectum, spleen, stomach, tongue and ureter, but not in the aorta, oesophagus and pancreas.</p> <p>The promoter region and the exons (including the splice sites) of <it>SLCO2A1 </it>were resequenced in 5 F4ab/ac receptor positive and 5 F4ab/ac receptor negative pigs. Two silent and 2 missense (both S → L at position 360 and 633) mutations were found, but none was associated with the F4ab/ac receptor phenotype. In addition, no phenotype associated differential mRNA expression or alternative/abberant splicing/polyadenylation was found in the jejunum.</p> <p>Conclusion</p> <p>The molecular cloning and characterization of porcine <it>SLCO2A1 </it>not only contributes to the already existing knowledge about the transporter in general, but enables studies on porcine prostaglandin related processes/deficiencies as patient and/or model. Here we examined its possible involvement as receptor in F4 enterotoxigenic <it>E. coli </it>mediated neonatal diarrhoea. Because no phenotype associated differences could be found in the gene sequence nor in its jejunal transcription profile of F4ab/ac receptor positive/negative pigs, SLCO2A1 can most likely be excluded as receptor for F4 bacteria.</p

Association analysis of PPARGC1A mutations with meat quality parameters in a commercial hybrid pig population

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) is a promising candidate gene for selection on meat and carcass quality traits in the pig industry. In the pig, several SNPs have been reported in both coding and regulatory regions of this gene, some of which were associated with fat characteristics, but none of these associations have been confirmed and many SNPs have not yet been evaluated. Therefore, 18 PPARGC1A SNPs were genotyped in 65 slaughter pigs of a commercial hybrid population and used in an association analysis with multiple muscle and carcass traits. Several SNPs located in the 3'UTR and exon 8 and 9 of PPARGC1A were significantly (P < 0.05-0.001) associated with various carcass composition, tenderness and muscle fibre traits. They could potentially serve as DNA selection markers, if their impact was to be confirmed by a functional analysis.status: publishe

Evaluation Of The Genetic Variability Of 23 Bovine Microsatellite Markers In Four Belgian Cattle Breeds

The polymorphism of 23 microsatellites in the four main cattle breeds in Belgium (Holstein Friesian, Belgian Blue, Belgian Red Pied and East Flemish) was analysed. Heterozygosity, polymorphism information content, the effective number of alleles, exclusion probability and the probability of genotypic identity for two random individuals were calculated for all microsatellites and all breeds. The Belgian Blue breed is generally a little less polymorphic in comparison with the other three breeds, Estimates of the genetic distances between these breeds confirmed the widely accepted proposition that the Belgian Blue is the most genetically distinct of these breeds. The three other breeds are likely to become one population, given current breeding strategies. Exclusion probabilities in parentage control cases are >0.9999 in all four breeds when all 23 microsatellites are used and >0.98 with only the two most polymorphic multiplexes

Population genetic structure of and inbreeding in an insular cattle breed, the Jersey, and its implications for genetic resource management

The Jersey is a ubiquitous and successful breed of cattle that originates from the UK Channel Island of Jersey. While the breed has been exported extensively, no imports have taken place to the island since 1789, leading to a concern regarding possible losses of genetic diversity and increased inbreeding. We have conducted the first large-scale genetic analysis of the Jersey cattle using only samples from the island. A total of 223 cattle from all parishes except one were genotyped for 12 microsatellite loci. The average number of alleles per locus and expected heterozygosity were found to be comparatively high (nA=4, He=0.64) with respect to that observed in a number of continental breeds. Only breeds that have been upgraded and are therefore the result of admixture are clearly more variable than the Jersey. We also found a significant but limited amount of genetic differentiation between parishes (Fst=0.013), or even between farms (Fst=0.035) despite an apparent lack of movement. This is confirmed by the application of two recent statistical methods. A Bayesian partition analysis shows that the most probable value of K, the number of possible hidden partitions, is 1 (P0.98). K=2 has a much lower probability (P0.02) while other values are essentially zero. Similarly, we were able to show that there was no support for departure from panmixia other than due to population structure, and thus that there is sufficient background gene flow across the island to overcome local drift. Overall, it appears that the current level of genetic diversity and its distribution within the island means it is unnecessary to import unrelated genetic material to the island for management purposes