Genetic characterisation and breed assignment in Austrian sheep breeds using microsatellite marker information.

Samples from 717 sheep of 11 Austrian sheep breeds were genotyped for 25 microsatellite loci. Twenty-one loci showing no deviation from Hardy-Weinberg equilibrium were used to calculate pairwise genetic distances (Nei's minimum distance and Reynolds' distance). All breeds could be clearly distinguished through these genetic distances. The shortest genetic distance was found between Alpines Steinschaf (AS) and Waldschaf (WS). Within the so-called 'Steinschaf' group [AS, Montafoner Steinschaf (MS), Krainer Steinschaf (KS) and Tiroler Steinschaf (TS)] the MS adopted an extreme status with the largest distance to the other breeds in the group. This finding resulted in the decision to consider the MS no longer as subpopulation of Alpines Steinschaf but as an independent breed. A correct breed assignment using a Bayesian approach was possible for only 66% of all individuals belonging to Alpines Steinschaf, but for at least 90% of individuals for all other breeds investigated.

Development of the Lipizzan horse breed.

The development and a brief history of the Lipizzan horse breed are reviewed. The contribution of several breeds, some of them already extinct, to the development of the Lipizzan horse, gives it a special status representing an important gene pool. This well-documented breed is a part of the common European natural and cultural heritage. Breeding practices establishing stallion and mare family lines as well as availability of pedigrees are described. Molecular analysis of mitochondrial DNA (mtDNA) and microsatellite data allowed us to analyse the structure of the Lipizzan population, to estimate genetic variability within the population and to test the reliability of the pedigree data. DNA sequence analysis of the mtDNA control region confirmed relative high variability of the gene pool, containing majority of mtDNA haplotypes found in horse populations worldwide. Microsatellite analysis showed that the level of heterozygosity in the Lipizzan population is comparable with the heterozygosity in other populations. The fact that majority of the Lipizzan population is bred on eight state studs in the Central and Eastern Europe contributes to the structuring of the population which results in three clusters: classical cluster, represented by studs Lipica, Piber and Monterotondo, transition cluster, represented by studs Szilvasvarad, Djakovo and Topolcianky and eastern cluster represented by studs Beclean and Fagaras. The molecular markers also allowed verification of pedigree data, and the rough estimation of pedigree errors was about 10%.

Microsatellite diversity, population subdivision and gene flow in the Lipizzan horse.

Blood samples of 561 Lipizzan horses from subpopulations (studs) of seven European countries representing a large fraction of the breed's population were used to examine the genetic diversity, population subdivision and gene flow in the breed. DNA analysis based on 18 microsatellite loci revealed that genetic diversity (observed heterozygosity = 0.663, gene diversity = 0.675 and the mean number of alleles = 7.056) in the Lipizzan horse is similar to other horse breeds as well as to other domestic animal species. The genetic differentiation between Lipizzan horses from different studs, although moderate, was apparent (pairwise F(ST) coefficients ranged from 0.021 to 0.080). Complementary findings explaining the genetic relationship among studs were revealed by genetic distance and principal component analysis. One genetic cluster consisted of the subpopulations of Austria, Italy and Slovenia, which represent the classical pool of Lipizzan horse breeding. A second cluster was formed by the Croatian, Hungarian and Slovakian subpopulations. The Romanian subpopulation formed a separate unit. The largest genetic differentiation was found between the Romanian and Italian subpopulation. Genetic results are consistent with the known breeding history of the Lipizzan horse. Correct stud assignment was obtained for 80.9% and 92.1% of Lipizzan horses depending on the inclusion or exclusion of migrant horses, respectively. The results of the present study will be useful for the development of breeding strategies, which consider classical horse breeding as well as recent achievements of population and conservation genetics.

Isolation of Y chromosome-specific microsatellites in the horse and cross-species amplification in the genus Equus.

Y chromosome polymorphisms such as microsatellites or single nucleotide polymorphisms represent a paternal counterpart to mitochondrial DNA (mtDNA) for evolutionary and phylogeographic studies. The use of Y chromosome haplotyping in natural populations of species other than humans is still hindered by the lack of sequence information necessary for polymorphism screening. Here we used representational difference analysis (RDA) followed by a screen of a bacterial artificial chromosome (BAC) library for repetitive sequences to obtain polymorphic Y-chromosomal markers. The procedure was performed for the domestic horse (Equus caballus) and we report the first six Y-chromosomal microsatellite markers for this species. Three markers were also useful for haplotyping taxa of the zebra/ass lineage. Y-chromosomal microsatellite markers show a single haplotype in the domestic horse, whereas notable variation has been observed in the other members of the genus Equus.

Fixed nucleotide differences on the Y chromosome indicate clear divergence between Equus przewalskii and Equus caballus.

The phylogenetic relationship between Equus przewalskii and E. caballus is often a matter of debate. Although these taxa have different chromosome numbers, they do not form monophyletic clades in a phylogenetic tree based on mtDNA sequences. Here we report sequence variation from five newly identified Y chromosome regions of the horse. Two fixed nucleotide differences on the Y chromosome clearly display Przewalski's horse and domestic horse as sister taxa. At both positions the Przewalski's horse haplotype shows the ancestral state, in common with the members of the zebra/ass lineage. We discuss the factors that may have led to the differences in mtDNA and Y-chromosomal observations.

Campylobacter jejuni isolated from poultry and humans in Styria, Austria: epidemiology and ciprofloxacin resistance.

Sixty-six broiler flocks were sampled to determine the presence of Campylobacter spp. at slaughter in 1998. Thirty flocks (45%) tested positive and C. jejuni was identified in all isolates. Combined pulsed-field gel electrophoresis/amplified fragment length polymorphism (PFGE/AFLP) subtyping of 177 isolates from 24 positive flocks revealed 62 subtypes; 16 flocks harboured more than one subtype. When subtyping 101 clinical C. jejuni isolates collected in the same time period and area, 60 PFGE/AFLP types were identified. Comparison of subtypes from poultry and human isolates revealed three shared PFGE/AFLP types, which were present in 11 human isolates. Fifty per cent of all poultry isolates and 39% of all human isolates were resistant to ciprofloxacin. The present study confirms the increase in ciprofloxacin resistance in both human and poultry C. jejuni isolates in Austria, as observed in several countries worldwide. A small number of human isolates shared PFGE/AFLP types with poultry isolates, however, further studies should also focus on the identification of other sources of C. jejuni infection in humans.

Inbreeding, microsatellite heterozygosity, and morphological traits in Lipizzan horses.

While the negative effects of inbreeding and reduced heterozygosity on fecundity and survival are well established, only a few investigations have been carried out concerning their influence on morphological traits. This topic is of particular interest for a small and closed population such as the Lipizzan horse. Thus, 27 morphological traits were measured in 360 Lipizzan mares and were regressed on the individual inbreeding coefficients, as well as on the individual heterozygosity and mean squared distances (mean d(2)) between microsatellite alleles within an individual. Both individual heterozygosity and mean d(2) were based on 17 microsatellite loci dispersed over 14 chromosomes. The results obtained by multivariate analysis reveal significant effects of stud (P <.0001), age at measurement (P <.0001), and mean d(2) (P =.0143). In univariate analyses, significant associations were obtained between length of pastern-hindlimbs and inbreeding coefficient (P <.01), length of cannons-hindlimb and mean d(2) (P <.01), and length of neck and mean d(2) (P <.001). After adjustment of single-test P values for multiple tests (Hochberg's step-up Bonferroni method), only the association of the length of neck and mean d(2) remained significant (P =.0213). Thus, no overall large effects of inbreeding, microsatellite heterozygosity, and mean d(2) on morphological traits were observed in the Lipizzan horse.

PrP genotyping of Austrian sheep breeds.

Scrapie, an ovine and caprine transmissible spongiforme encephalopathy, is widely spread among sheep populations in many European countries. As it is known that susceptibility to scrapie is determined genetically, breeding programmes aiming at providing scrapie-resistant flocks have been established. Selection is based on the prion protein (PrP) genotype, which is used to classify animals into risk groups of susceptibility (R1-R5) according to the amino acids encoded by codons at positions 136, 154 and 171, respectively. At position 136 (136V-->136A) alanine and at position 154 (154H-->154R) as well as 171 (171Q-->171R) arginine are the favoured amino acids. Whereas PrP genotyping data are available for many of the European sheep breeds, comparable data for local Austrian sheep breeds are missing. The most known among these are Tyrolean mountain sheep, forest sheep. Tyrolean stone sheep and Carynthian sheep. The genotypes of 112 sheep from these four local breeds were determined. In terms of PrP genetics, Austrian breeds belong to the group of non-valine-breeds, with the exception of the Carynthian sheep, that exhibited a frequency of 136V of 4.2%. The most frequent allele was ARQ with 64.6-71.2% (depending on the breed), followed by ARR (14.8-25.8%). In contrast to the above-mentioned findings, scrapie has never been diagnosed in any of the Austrian sheep breeds. Native Austrian sheep breeds exhibit a very robust constitution, a pronounced adaptation to harsh climates and good reproduction parameters as well as a marked mother instinct. Therefore, these breeds are often used in crossbreeding programmes. Beside the above-mentioned characteristics, our results indicate that the investigated breeds may be effectively used in crossing-out breeding programmes for eliminating valine at position 136 of PrP.

Microsatellite analysis of sperm-use patterns in the bushcricket Requena verticalis.

The proportion of offspring sired by the second male to mate with a doubly mated female, P2, is a ubiquitously measured statistic in the study of insect sperm competition. Nevertheless, the underlying mechanisms of sperm transfer, storage, and use that determine P2 are poorly understood. Typically the second male to mate gains moderate to high paternity. More rarely, the first male to mate gains the majority of fertilizations. Here we examine the transfer, storage, and use of sperm in the bushcricket Requena verticalis, a species with male parental investment and almost complete first male paternity. Sperm drain from an externally attached spermatophore into the female's reproductive tract, where they are transported to the sperm store or spermatheca. We find that only sperm from the first male to mate are transported to the spermatheca. We provide some data that address a number of different mechanisms that might account for the lack of storage of second-male sperm. DNA microsatellite markers are developed to assign paternity. By manipulating the numbers of sperm transferred by first and second males, we show that the size of the ejaculate transferred by the first male has a major impact on paternity; increasing ejaculate size of the first male assures his paternity. Paternity assurance in R. verticalis holds significant implications for the evolution of paternal investment via the male's nuptial gift.

Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning.

Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported.