Abstract

In this paper, we present a radiation hybrid framework map of BTA13 composed of nine microsatellite loci, six genes and one EST. The map has been developed using a recently constructed 12'000 rad bovine-hamster whole-genome radiation hybrid panel. Moreover, we present a comprehensive map of BTA13 comprising 72 loci, of which 45 are microsatellites, 20 are genes and seven are ESTs. The map has an estimated length of 2694.7 cR_12'000. The proposed order is in general agreement with published maps of BTA13. Our results only partially support previously published information of five blocks of conserved gene order between cattle and man. We found no evidence for the existence of an HSA20 homologous segment of coding DNA on BTA13 located centromeric of a confirmed HSA10 homologous region. The present map increases the marker density and the marker resolution on BTA13 and enables further insight into the evolutionary development of the chromosome as compared to man.

Genetic relationships between Swiss sheep breeds were estimated on the basis of microsatellite analysis. In addition to the Swiss breeds wild-type Mou¯on was included in this investigation. Polymerase chain reaction ampli®cations of 31 ovine, bovine and caprine microsatellites were performed in a total of 307 animals representing eight populations. The average heterozygosity within each population was high in the domestic breeds (0.60±0.71) and lower in Mou¯on 0.45. The average coef®cient of gene differentiation G ST over all loci was 0.17, i.e. a small part of the variability at the 31 microsatellite loci analysed must be ascribed to between-breed variability. Genetic distances between breeds were obtained, which were used to construct a phylogenetic tree. Microsatellites developed from closely related species of cattle and goat are useful for estimating genetic relationships among sheep breeds. ZusammenfassungGenetische Verwandtschaft bei Schweizer Schafrassen aufgrund der Analyse von Mikrosatelliten Die genetische Diversita Èt zwischen den Schweizer Schafrassen wurde anhand der Analyse von 31 ovinen, bovinen und caprinen Mikrosatelliten gescha Ètzt. DNA von 307 zufa Èllig ausgewa Èhlten Tieren wurde untersucht. Diese Tiere geho È ren den sieben Rassen Braunko È p®ges Fleischschaf, Schwarzbraunes Bergschaf, Schwarznasen-Schaf, Weisses Alpenschaf, Engadiner Schaf, Spiegelschaf und Walliser Landschaf an. Zusa Ètzlich wurde der Wildtyp des Hausschafes, der Muf¯on in die Untersuchung ein Veinbezogen bezogen. Der mittlere Heterozygotiegrad innerhalb der Rassen war fu È r die Hausschafe hoch (0.60±0.71), und beim Muf¯on lag dieser Wert etwas tiefer (0.45). Nur ein kleiner Teil der Gesamtvariation der genetischen Diversita Èt (G ST 0.17) konnte fu È r die Unterschiede zwischen den Rassen verantwortlich gemacht werden. Die gescha Ètzten genetischen Distanzen zwischen den Rassen wurden fu È r die Erstellung von phylogenetischen Ba Èumen verwendet. Die Untersuchung hat gezeigt, dass die ovinen, bovinen und caprinen Mikrosatelliten sich gleich gut fu È r die Analyse der genetischen Diversita Èt bei Schafen eignen.

BackgroundPorcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD) controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2).ResultsBisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele.ConclusionsOur results indicate that the IGF2 imprinting status is transcript-specific. Biallelic IGF2 expression in adult porcine liver and relaxation of IGF2 imprinting in porcine muscle were a common feature. These results were consistent with the IGF2 promoter P1 usage in adult liver and IGF2 promoter P2, P3 and P4 usages in muscle. The results showed further that bialellic IGF2 expression in liver and relaxation of imprinting in muscle and kidney were not associated with DNA methylation variation at and around at least one CTCF binding site in H19 DMD. The imprinting status in adult liver, muscle and kidney tissues were also not reflected in the methylation patterns of IGF2 DMRs 1 and 2.

The Italian wolf is in the process of regaining the Alpine region which comes into conflict with the extensive sheep keeping practiced in Switzerland during the summer. As in Switzerland, the wolf is a protected species, the government reimburses losses caused by wolves. Therefore we wanted to know whether the Italian wolf could be distinguished from the domestic dog by microsatellite analysis if DNA samples of the predators could be secured. The evaluation of combined genotypes for the microsatellites CanBern6, CPH4, CPH7, CPH9, CPH12, CPH22 and ZuBeCa1 made it possible to identify an individual as either a domestic dog or an Italian wolf. The assignment of an individual to either one of the two populations is based on the logarithm of the likelihood ratio of an individual being an Italian wolf rather than a domestic dog, given a specific combined genotype. The distribution of the Italian wolf combined genotypes (n = 42) is clearly distinct from the distribution of the domestic dog combined genotypes (n = 90). The likelihood ratio for the "worst" Italian wolf combined genotype was 2.3 E+5 and for the "worst" domestic dog combined genotype was 3.8 E-5.

Six previously unassigned genes have been assigned to the BTA13 by means of somatic cell hybrid analysis. Polymerase chain reaction primer design for the amplification of AHCY, PLTP, PPGB and PCK1 was based on nucleic acids sequence information of homologous genes on HSA20. Primers for PDYN and ASIP were designed from porcine and bovine sequence information, respectively. Homology was established by sequence analysis. These assignments support the previous finding of a conserved syntenic relationship between HSA20 and BTA13 and contribute to the understanding of the chromosomal evolution of BTA13. This is significant since the prion protein gene, thought to play a key rule in the development and course of bovine spongiforme encephalopathy is also located on BTA13.