Search

The Bovini tribe contains domestic and wild cattle-like species, several of which are cross-fertile. We present a completely resolved Y-chromosomal phylogeny, which is better in agreement with autosomal phylogeny, morphological data, cross-fertility and estimates of divergence times than mitochondrial data. The tree links Bos grunniens (yak) to Bison, so the commonly accepted Bos genus is not monophyletic. Therefore, we advocate the term Poephagus as designation of yak. This work illustrates the resolving power of Y-chromosomal variation for cladistic studies of closely related species. � The Willi Hennig Society 2008.

Numbers of the Asian elephants (Elephas maximus) population are declining due to poaching, human-elephant conflicts, capture of wild calves for tourism and export and habitat destruction, which also may cause inbreeding in fragmented populations. In order to contribute to a reversal of this trend, we have developed an identification and parentage test by evaluation and selection of markers from 43 microsatellite loci that have been previously described for Asian or African elephants. Testing these markers on a panel of 169 Asian elephants comprising the 23 mother-offspring, 13 father-offspring and 13 parents-offspring pairs yielded 26 polymorphic markers. However, only 14 of these were found to be suitable for an analysis of molecular diversity, 12 of which will be implemented for an identification and parentage test to control the capture of wild calves in Thailand and neighboring countries.

Summary The verification of the breed origin of animal products is relevant for food safety andauthenticity. We assessed the suitability of AFLP molecular markers in the assignment ofcattle individuals to their breed of origin. Three hundred and ninety-six animals belongingto 16 cattle breeds genotyped with 141 AFLP markers were used as reference data set.Assignment was performed with likelihood ( AFLPOP ) and Bayesian ( STRUCTURE ) methods. TheBayesian approach was superior to the likelihood algorithm with respect to (i) the correctassignment of simulated individuals to their breed of origin (93% vs. 81% respectively), (ii)the correct assignment of 44 sampled Romagnola animals (91% vs. 45% respectively) and(iii) the correct classification of animals belonging to a breed that was not included withinthe reference dataset. Thus, AFLP profiling in combination with the Bayesian approachseems a useful tool for breed assignment.Keywords AFLP, Bayesian, cattle, likelihood, traceability. Introduction

Yak domestication represents an important episode in the early human occupation of the high-altitude Qinghai-Tibet Plateau (QTP). The precise timing of domestication is debated and little is known about the underlying genetic changes that occurred during the process. Here we investigate genome variation of wild and domestic yaks. We detect signals of selection in 209 genes of domestic yaks, several of which relate to behaviour and tameness. We date yak domestication to 7,300 years before present (yr BP), most likely by nomadic people, and an estimated sixfold increase in yak population size by 3,600 yr BP. These dates coincide with two early human population expansions on the QTP during the early-Neolithic age and the late-Holocene, respectively. Our findings add to an understanding of yak domestication and its importance in the early human occupation of the QTP., Based on whole genome resequencing of more than 80 wild and domestic yaks from high-altitude Qinghai-Tibet Plateau, this study recovers demographic and genetic processes underlying the domestication of this animal. Qiu et al. also identify genes and associated pathways as candidates for selection during the domestication process.

We identified 37 single-nucleotide polymorphisms (SNPs) in sheep and screened 16 individuals from 8 different sheep breeds selected throughout Europe. Population genetic measures based on the genotyping of about 30 sheep from the same 8 breeds are reported. To date, there are no sheep SNPs documented in the National Center for Biotechnology Information dbSNP database. Therefore, the markers presented here contribute significantly to those currently available.

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.

The genetic diversity of the casein locus in cattle was studied on the basis of haplotype analysis. Consideration of recently described genetic variants of the casein genes which to date have not been the subject of diversity studies, allowed the identification of new haplotypes. Genotyping of 30 cattle breeds from four continents revealed a geographically associated distribution of haplotypes, mainly defined by frequencies of alleles at CSN1S1 and CSN3. The genetic diversity within taurine breeds in Europe was found to decrease significantly from the south to the north and from the east to the west. Such geographic patterns of cattle genetic variation at the casein locus may be a result of the domestication process of modern cattle as well as geographically differentiated natural or artificial selection. The comparison of African Bos taurus and Bos indicus breeds allowed the identification of several Bos indicus specific haplotypes (CSN1S1*C-CSN2*A2-CSN3*AI/CSN3*H) that are not found in pure taurine breeds. The occurrence of such haplotypes in southern European breeds also suggests that an introgression of indicine genes into taurine breeds could have contributed to the distribution of the genetic variation observed.

To control the spread of bovine spongiform encephalopathy, several DNA methods have been described for the detection of the species origin of meat and bone meal. Most of these methods are based on the amplification of a mitochondrial DNA segment. We have developed a semiquantitative method based on real-time PCR for detection of ruminant DNA, targeting an 88-bp segment of the ruminant short interspersed nuclear element Bov-A2. This method is specific for ruminants and is able to detect as little as 10 fg of bovine DNA. Autoclaving decreased the amount of detectable DNA, but positive signals were observed in feeding stuff containing 10% bovine material if this had not been rendered in accordance with the regulations, i.e., heated at 134 degrees C for 3 instead of 20 min.

Feline amyloidosis is an uncommon disorder caused by deposits of amyloid in a variety of organs. Most frequently encountered types are amyloid derived of pancreatic islet amyloid polypeptide (AIAPP) in older cats and of the apolipoprotein, apo-serum amyloid A (AA) in Abyssinian/Somali (Aby) and Siamese/Oriental (Siam) cats occurring at a relatively young age. For the AA protein of the Aby, Siam and domestic shorthair cat (DSH) breed different amino acid sequences have been described. It is not yet clear, however, whether the tendency to develop AA amyloidosis in Aby and Siam is associated with specific apoSAA protein sequences and whether this is breed specific. In this study, DNA from one Siam and two DSH cats revealed on Southern blot three bands suggesting at least three genes or gene clusters. The SAA cDNAs of hepatic mRNA from three Abys, five Siams and five DSHs were amplified by RT-PCR, cloned and sequenced. Siams and Abys had limited SAA sequence variability. All five Siams, three of which were positive for amyloid, had the amyloidogenic Siam SAA and the amyloidogenic Aby SAA sequence. Two of the Abys, both with amyloid, had the amyloidogenic Aby SAA sequence. The third Aby, without amyloid, missed its amyloidogenic sequence. The SAA sequences of the DSHs found in the present preliminary survey, suggested a possible tendency for more variability, whereas the amyloidogenic Siam as well as the amyloidogenic Aby sequence were found once. Up to five different sequences were found in a single animal. All five DSHs, moreover, had a specific sequence lacking in the Siams and Abys. The present results, especially those of the Siams, favor that in addition to the occurrence of amyloid associated SAA genes other factors such as infections and inflammatory processes are involved in the development of phenotypical amyloidosis.

Livestock conservation practice is changing rapidly in light of policy developments, climate change and diversifying market demands. The last decade has seen a step change in technology and analytical approaches available to define, manage and conserve Farm Animal Genomic Resources (FAnGR). However, these rapid changes pose challenges for FAnGR conservation in terms of technological continuity, analytical capacity and integrative methodologies needed to fully exploit new, multidimensional data. The final conference of the ESF Genomic Resources program aimed to address Bruford et al. Challenges in livestock conservation these interdisciplinary problems in an attempt to contribute to the agenda for research and policy development directions during the coming decade. By 2020, according to the Convention on Biodiversity’s Aichi Target 13, signatories should ensure that “…the genetic diversity of …farmed and domesticated animals and of wild relatives …is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity.” However, the real extent of genetic erosion is very difficult to measure using current data. Therefore, this challenging target demands better coverage, understanding and utilization of genomic and environmental data, the development of optimized ways to integrate these data with social and other sciences and policy analysis to enable more flexible, evidence-based models to underpin FAnGR conservation. At the conference, we attempted to identify the most important problems for effective livestock genomic resource conservation during the next decade. Twenty priority questions were identified that could be broadly categorized into challenges related to methodology, analytical approaches, data management and conservation. It should be acknowledged here that while the focus of our meeting was predominantly around genetics, genomics and animal science, many of the practical challenges facing conservation of genomic resources are societal in origin and are predicated on the value (e.g., socio-economic and cultural) of these resources to farmers, rural communities and society as a whole. The overall conclusion is that despite the fact that the livestock sector has been relatively well-organized in the application of genetic methodologies to date, there is still a large gap between the current state-of-the-art in the use of tools to characterize genomic resources and its application to many non-commercial and local breeds, hampering the consistent utilization of genetic and genomic data as indicators of genetic erosion and diversity. The livestock genomic sector therefore needs to make a concerted effort in the coming decade to enable to the democratization of the powerful tools that are now at its disposal, and to ensure that they are applied in the context of breed conservation as well as development

We tested the use of biallelic Amplified fragment length polymorphism (AFLP) polymorphisms for the estimation of relative genetic distances of cattle individuals within or across breeds. An allele permutation procedure was developed to estimate the stochastic variation of the genetic distance that is inherent to a given dataset. In a panel of 47 Holstein-Friesian cattle analysed with 248 polymorphic markers, the average genetic distance of bulls selected for breeding was slightly lower than the distance of the cows. The observed standard deviation (SD) of the distance indicated genetic subdivision, which for the bulls was explained by variation in the additive relationship derived from herdbook data. Animals from three different breeds, the highly selected Holstein-Friesian, the Italian Brown and the historic Maremmana, were compared on the basis of 106 polymorphic markers. No breed-specific fragments were observed. The mean pair-wise genetic distance within breeds was 85% of the value across breeds, but principal coordinates analysis clustered the animals according to their breed of origin. Calculation of distances between the breeds indicated a relatively divergent position of the Maremmana, relative to the two other breeds. However, biallelic markers indicate that the process of breed formation had only a limited effect on the diversity at marker loci.

Methods currently used for the identification of the species origin of meat or tissue samples have not been validated for other bovine species than taurine cattle or water buffalo. These methods also do not discriminate between the different bovine species that are used as source of beef. Here, we describe two complementary methods for detection and differentiation of bovine species, which are based on mutations in mitochondrial DNA and centromeric satellite DNA, respectively. The analysis of satellite DNA is especially relevant for the identification of animals that are of hybrid origin.

It is known that a satisfactory clinical outcome can follow the implantation of cardiac valve allografts in spite of the loss of living cells in the tissue. If viable cells are not required for long term graft function, then effective disinfection of the tissue might become possible. In an earlier paper in this series we reported that peracetic acid (PAA) is an effective antimicrobial agent for the treatment of valve allografts; it was lethal to the cells but at a concentration of 0.21% had little effect on the mechanical properties or extracellular morphology of the valve leaflets. It was also found that PAA-treatment could be combined with storage in 85% glycerol at 4 °C, or cryopreservation with 10%Me2SO, without substantial further impairment of microscopic structure or mechanical properties. In this paper we describe the implantation of processed ovine aortic valves in the descending thoracic aorta of sheep. The experimental groups included control untreated valves and valves that had been treated with antibiotics or PAA and either cryopreserved, or stored in 85%glycerol. The recipient sheep showed good clinical appearances until the experiment was terminated at six months. The explanted grafts were examined by standard morphological and mechanical testing methods. The PAA-treated valves were clearly recognisable as valves: the leaflets had fair to medium morphology in both the unpreserved and the cryopreserved groups. All leaflets had a superficial overgrowth of cells. Microsatellite analysis for allelic differences were performed on samples of donor and recipient tissues using three markers of tissue source. Only one valve, which had been treated with PAA, revealed allelic differences between donor and recipient. It is suggested that DNA-fragments may have remained after the destruction of donor cells and six months of implantation: the overgrowing cells were almost certainly of recipient origin. We conclude that our experiments, in which PAA-treatment was combined with preservation, are sufficiently encouraging to justify further studies to refine the technique, but in our opinion they are not sufficient to justify a clinical trial at this time.

Both Bos indicus (zebu) and Bos javanicus (banteng) contribute to the Indonesian indigenous livestock, which is supposedly of a mixed species origin, not by direct breeding but by secondary cross breeding. Here, the analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed banteng introgression of 10–16% in Indonesian zebu breeds with East-Javanese Madura and Galekan cattle having higher levels of autosomal banteng introgression (20–30%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Ychromosomal haplotypes, presumably of French Limousin origin. There was no evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng.

The mammalian tribe Bovini (subfamily Bovinae, family Bovidae) contains all the most important of the world’s larger domestic species. The domestications of these cattle and cattle-like taxa were among the most significant advances of the neolithic transition. With their specialized digestive system, these species can utilize cellulose as an energy source, generating the benefits of dairy, meat and hide production. In addition, cattle provided, for several millennia, the animal draught power that underpinned agriculture in the Old World and have assumed a cultural and even religious importance, which is preserved to the present day in some societies. In this chapter we describe the wild and domestic cattle species and their systematics, and we review the phylogenetic analyses that have been performed on this closely related and poorly resolved group.

The available data on the domestication and history of cattle are summarized in this chapter, including the appearance of phenotypes that today are the subject of genetic research. Focus is given on taurine cattle spread over the old world, zebus around the equator, large taurines during classical antiquity, small cattle in medieval Europe, modernization of cattle husbandry and industrial cattle, crossbreds and local breeds.

For the first time we used amplified fragment length polymorphism on individual nematode parasites to analyse the genetic diversity between and within isolates during consecutive stages of increased benzimidazole resistance and of increased levamisole resistance of Haemonchus contortus. The genetic diversity of the H. contortus genome turned out to be unusually high, within and between the isolates. The difference between individuals of an isolate could be as high as between individuals of two different mammalian species that do not interbreed. During benzimidazole selection the genetic constitution of the population was changed, but did not lead to a decrease in the genetic diversity. The selection for levamisole resistance resulted in a limited reduction of the genetic diversity only after the first selection step. The extensive genetic diversity apparently has allowed a fast and flexible response of H. contortus to drug selection as shown by the appearance of drug resistant isolates. This selection however has little or no effect on the extent of the genetic diversity of these resistant isolates. Implications for more sustainable control methods are discussed.

The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2-10 tandems CA/GT repeats that are interrupted by sequences of 1-10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes.

Expressed sequence tags from the parasitic nematode Haemonchus contortus were generated in order to identify anchor loci for comparative mapping between nematode genomes and candidate targets for future control measures. In total, 370 SL1 trans-spliced cDNAs from different developmental stages representing 195 different genes were partially sequenced. From these expressed sequence tags 50% were similar to genes with a known or predicted function and 19% were similar to nematode sequences with no ascribed function. From the first, free-living L1 and L3 stages relatively many cDNAs matched to housekeeping genes, and 11% (L1) or 23% (L3) of the encoded proteins were predicted to contain signal peptides. In contrast, no function could be ascribed to most of the cDNAs from the early L5 and adult parasitic stages, but for 30% (L5) or 55% (adult) of the encoded proteins a signal sequence was predicted. This limited analysis suggests that during the transition from the free-living to parasitic stages gene expression shifts towards the synthesis of less conserved extracellular proteins. These proteins offer the best perspectives for vaccine development and the development of anthelmintic drugs. In contrast, cDNAs from the first larval stages may be most suitable for comparative mapping with the free-living nematode Caenorhabditis elegans.

Otsen, M., Plas, M. E., Groeneveld, J., Roos, M. H., Lenstra, J. A., and Hoekstra, R. 2000. Genetic markers for the parasitic nematode Haemonchus contortus based on intron sequences. Experimental Parasitology95, 226–229

We describe satellite DNA variation that detects hybridization of Bos indicus (zebu or indicine cattle) and Bos taurus (taurine cattle) in African cattle populations. On Southern blots hybridized to a satellite III probe, relative intensities of HinfI fragments correlated with the taurine-zebu composition in hybrid animals as deduced from AFLP genotyping of the same animals and previous data on microsatellite allele frequencies. Similar results were obtained by PCR-RFLP analysis of a zebu-specific mutation in the repeat unit of satellite 1·711b. Analysis of individuals from 20 African cattle breeds indicate that the centromeric satellites of the sanga breeds are of the taurine type and that several East-African zebu breeds are hybrids between taurine and zebu. These satellite RFLP, or SFLP, markers provide a fast method to screen the genetic makeup of African cattle.

Hctc1, a member of the Tc1-family of transposable elements was isolated from the parasitic nematode Haemonchus contortus. Hctc1 is 1590 bp long, is flanked by 55 bp inverted repeats and carries a single open reading frame of a 340 amino acid transposase-like protein. Hctc1 is similar to Tc1 of Caenorhabditis elegans and elements Tcb1 and Tcb2 of Caenorhabditis briggsae in the inverted terminal repeats, the open reading frame, as well as the target insertion sequence. Furthermore, the copy number of Hctc1 is comparable with the Tc1 copy number in low copy strains of C. elegans. The sequence of Hctc1 is highly variable in H. contortus due to deletions, insertions and point mutations, with at least five distinct length variants of Hctc1. Most of the Hctc1 variation was within rather than between H. contortus populations. The high level of sequence variation is probably due to variation generally found for members of the Tc1-family, as well as a high background level of genetic variation of H. contortus.

A convenient DNA-based identification system is described for testing the species origin of meat samples. Probes are generated by PCR with primers binding to species-specific satellite DNA and hybridized to DNA purified from meat. This method is more robust and versatile than methods based on oligonucleotide hybridization. With the exception of a slight cross-reaction of mutton and beef, each probe only recognized the species from which it was derived. Purifying the DNA with a DNA-binding resin improved the sensitivity. Admixtures of 0.1–0.5% can be detected in raw meat and 0.5–5% in autoclaved meat samples. The method can be adapted to detect any eukaryotic species for which species-specific DNA sequences are available. This method has proven its value in the routine inspection of meat samples by revealing more cases of deliberate or accidental species substitution and admixture than conventional techniques.

A PCR assay for the detection of small ruminant lentiviral gag DNA (provirus) in the white blood cells of sheep and goats was developed and compared with a serological test (AGIDT). A sample of the DNA prepared from the white blood cells in 3 ml of blood from 208 sheep and goats from 18 different flocks was subjected to PCR assay. One of 85 animals from flocks accredited under the Dutch national MVV/CAEV control programme was positive by PCR while none was positive by AGIDT. In infected flocks, the AGIDT appeared slightly more sensitive, but preliminary results show that the sensitivity of the PCR assay may be further improved by increasing the number of monocytes tested. The PCR assay, however, was clearly more sensitive in detecting animals in the early stages of infection. With the use of a set of mixed primers and probes, the assay was able to detect the variety of CAEV and MVV strains occurring in the field.

A fast procedure for species identification in heated meat is described. Deoxyribonucleic acid (DNA) was isolated from meat samples by an alkaline extraction method and hybridized to a conjugate of a specific oligonucleotide and alkaline phosphatase. The oligonucleotide probes are based on satellite DNA, tandem-repeated sequences, which are highly specific for species. Probes are developed for the identification of meat from cattle, sheep/goat, horse, deer, pig, chicken and turkey. Differentiation between closely related species like chicken and turkey is possible. Admixtures of 1-5% of meat of one species in another could be detected. The complete assay of up to 50 samples takes 4 h. Heated meat samples could be analysed.

The history of the abundant repeat elements in the bovine genome has been studied by comparative hybridization and PCR. The Bov-A and Bov-B SINE elements both emerged just after the divergence of the Camelidae and the true ruminants. A 31-bp subrepeat motif in satellites of the Bovidae species cattle, sheep, and goat is also present in Cervidae (deer) and apparently predates the Bovidae. However, the other components of the bovine satellites were amplified after the divergence of the cattle and the Caprinae (sheep and goat). A 23-bp motif, which as subrepeat of two major satellites occupies 5% of the cattle genome, emerged only after the split of the water buffalo and other cattle species. During the evolution of the Bovidae the satellite repeat units were shaped by recombination events involving subrepeats, other satellite components, and SINE elements. Differences in restriction sites of homologous satellites indicate a continuing rapid horizontal spread of new sequence variants.

SummaryIn 2002, it was discovered that several Cika cattle in the mountain areasof Slovenia had escaped the official policy of cross-breeding. Here, wereport a genetic characterization to assess their status as autochthonousbreed. We compared genotypes for 14 microsatellite markers in 150 Cikacattle individuals with data from 16 Central European cattle breeds. Weshow that Cika cattle are genetically as diverse as other Eastern Alpinebreeds, are more diverse than Austrian Simmental but less than the Bal-kan Busha cattle. STRUCTURE analysis showed Pinzgauer admixture inseveral individuals but also indicated a unique genetic identity for Cika.This analysis also allowed a selection of the most genetically pure Cikaindividuals as assessed by the panel of microsatellites. These original Cikacattle form an Eastern Alpine breed cluster together with Pinzgauer andPustertaler cattle. Cika cattle should be considered as an authentic andvaluable genetic resource, which offers clear opportunities for sustainableagriculture and landscape conservation in marginal and mountain areas.IntroductionSlovenia is with other Balkan countries locatedbetween the Southwest Asian domestication centresand Northwest Europe, where the most productivecattle breeds have been developed. During the agricul-tural colonization of Europe repetitive founder effectsdecreased livestock genetic diversity with increasingdistance from the domestication centre (Cymbronet al. 2005; Ajmone-Marsan et al. 2010). Approxi-mately two hundred years ago, homogeneous andgeneticallyisolatedbreedsemergedwithmoresystem-atic selection and the standardization of morphologyandperformance(Felius1995;Medugoracet al.2009).Slovenian cattle were described for the first time in1872 (Schollmayr 1873) as widespread but unnamed,yellow, red, red-brown or brown-black cattle fromthe Alpine part of the Austro-Hungarian provinceCarniola (present north-west Slovenia). These cattlewere small due to very poor feeding and rearingconditions. In 1878, the brown-red Bohinj cattle inthe north-west Slovenian Bohinj valley (Figure 1)had an elegant body frame, light bones, and a rela-tively high milk production of 1.200 to 1.500 l/yearconsidered a body weight of only 225–280 kg andpoor rearing conditions (Hitz 1878). These cattle werekept pure, but were from 1869 also cross-bred withCarinthian M€olltaler sires. The M €olltaler was devel-oped from light-red-pied cattle cross-bred to Pustertal-er and Tyrolean Pinzgauer (Felius 1995) and becamein 1925 absorbed by the popular Pinzgauer. It had arelatively light body frame (300-400 kg if kept in themountains), a dark-red coat colour and a high milkproduction (Felius 1995; Sambraus 1999). In 1894,Tolmin and Bovec cattle from the Soca valley weredescribed as a lighter type of M€olltaler cattle with ahigh milk production (Povse 1894). There wasintensive trading in cattle between different Austro-Hungarian provinces. Bohinj cows were appreciatedas milk cows in the Carinthia and Salzburg provincesin Austria (Zan Lotri c 2012), and Bovec cows weresold in the M€oll valley.

We have estimated the incidence of microand mini-satellites in the dog genome. A genomic phage library from canine liver, with an average insert size of 16 kb, was screened to detect potentially polymorphic microand mini-satellite sequences, which may be useful for the development of markers of inherited diseases, for fingerprinting, or for population genetics. Synthetic oligonucleotide probes were used to search for microsatellite sequences, and minisatellites were investigated with eight heterologous VNTR probes. (CA)n.(GT)n sequences were by far the most frequent, with a calculated average distance between consecutive loci of 42 kb. The average distance between loci of tri- or tetra-nucleotide repeats was about 330 kb. Mean inter-locus distances were 320 kb for (GGC)n, 205 kb for (GTG)n, 563 kb for (AGG)n, 320 kb for (TCG)n, 233 kb for (TTA)n, 384 kb for (CCTA)n, 368 kb for (CTGT)n, 122 kb for (TTCC)n, 565 kb for (TCTA)n, and 229 kb for (TAGG)n. Cross-hybridization with eight human minisatellite probes was found at average distances of 1400 kb; only one did not hybridize at all. We conclude that the di-, tri and tetra-nucleotide short tandem repeats, as well as some minisatellite sequences, are potentially useful as genetic markers, for mapping of the canine genome, and also for paternity testing and the analysis of population characteristics.

We sequenced and analyzed 97.6 kb of new DNA sequence containing the human TCRAC (C alpha) and TCRDC (C delta) genes as well as the TCRDV3 (V delta 3) and 61 different TCRAJ (J alpha) gene segments and compared its organization and structure to the previously described mouse T-cell receptor TCRAC/TCRDC (C alpha/C delta) region. A comprehensive nomenclature, consistent with the IUIS nomenclature committee recommendations, for both human and mouse TCRAJ gene segments is presented. In the human sequence, we identified 20 new TCRAJ gene segments and obtained the germline sequence for 23 additional TCRAJ gene segments known from cDNA clones. Using the sequence data obtained from the human TCRAC/TCRDC region, we have extended a polymerase chain reaction-based assay to test for the expression of the individual TCRAJ gene segments. At least five TCRAJ pseudogene segments were identified by sequence criteria. Like the murine TCRAC/TCRDC sequence, this sequence contains a high level of coding sequence, with over 6.6% of the total sequence being transcribed. Comparison of the human sequence with the previously reported mouse DNA sequence reveals homologous counterparts for the variable and joining (J) gene segments and both constant genes. Eleven new J pseudogene segments have been identified in the mouse TCRAC/TCRDC sequence through the use of human and mouse sequence comparisons. In terms of structure and organization, this region of the human and mouse genome appears to be remarkably conserved.

The major satellite of the horse genome consists of about 1 million copies of a 221-bp tandem repeat unit. By fluorescence in situ hybridization it has been localized in the centromeres of 58 of the 64 horse chromosomes. The donkey genome contains a similar but not identical satellite. Strikingly, the equine repeat did not hybridize to DNA of the Grevy zebra, despite the divergence of the horse and zebra only 3 to 5 million years ago and the ability of these species to crossbreed. The evolution of satellite DNA in the Equidae is more rapid than that in other mammalian families, which may be explained by their rapid karyotypic evolution.

The images for Figures 1 and 3 were incorrectly switched. The image that appears as Figure 1 should be Figure 3, and the image that appears as Figure 3 should be Figure 1. The figure legends appear in the correct order.

Summary Several systems are available for the expression of foreign gene sequences inEscherichia coli. We describe the use of prokaryotic expression products of viral gene fragments in order to identify the regions that specify the binding sites of antibodies. This approach is particulary successful if the antigenicity does not depend on the native protein, but only on the amino acid sequence, i.e., if the epitope is sequential. Combining prokaryotic expression with the use of synthetic peptides often permits a fast and accurate mapping of an epitope. The occurrence of immunodominant sequential epitopes on the surface of viruses seems to be a widespread phenomenon.

Information on the genetic structure and variability of autochthonous livestock breeds is essential for effective conservation programs. Here we present a molecular characterization on the basis of 30 microsatellite markers of 5 Spanish endangered cattle breeds Betizu (BET), Mallorquina (MAL), Menorquina, Monchina (MON), and Serrana de Teruel (ST) and of 2 fighting bull populations, Casta Navarra (CN) and Casta Vistahermosa. The feral and critically endangered BET is divided into 2 subpopulations, one of which has exceptionally low diversity values. A low number of alleles was also observed in the island population MAL. Although the small population size and genetic drift have caused a considerable divergence between the breeds, phylogenetic analysis is in accordance with historical and geographical data. The 2 northern Spanish feral breeds BET and MON cluster together. The local fighting breed CN is relatively close to the more inbred Casta Vistahermosa, which is the progenitor of most other fighting bulls in Spain. Comparison with nonendangered breeds suggests admixture of Alpine and/or Pyrenean mountain cattle in the ST, which may contribute to the high level of linkage disequilibrium in this population.

For the control of the transmission of bovine spongiform encephalopathy in cattle via feedstuff, a real-time polymerase chain reaction assay was developed with ruminant-specific Bov-B SINE primers, SYBR Green fluorescence detection, and melting curve analysis. In formulated cattle and chicken feed samples spiked with pure bovine and sheep meat and bone meal heated at 133 degrees C for 20 min, a contamination level of 0.1% was detected.

Two horses (a 7-year-old Groninger warmblood gelding and a six-month-old Trakehner mare) with pathologically confirmed rhabdomyolysis were diagnosed as suffering from multiple acyl-CoA dehydrogenase deficiency (MADD). This disorder has not been recognised in animals before. Clinical signs of both horses were a stiff, insecure gait, myoglobinuria, and finally recumbency. Urine, plasma, and muscle tissues were investigated. Analysis of plasma showed hyperglycemia, lactic acidemia, increased activity of muscle enzymes (ASAT, LDH, CK), and impaired kidney function (increased urea and creatinine). The most remarkable findings of organic acids in urine of both horses were increased lactic acid, ethylmalonic acid (EMA), 2-methylsuccinic acid, butyrylglycine (iso)valerylglycine, and hexanoylglycine. EMA was also increased in plasma of both animals. Furthermore, the profile of acylcarnitines in plasma from both animals showed a substantial elevation of C4-, C5-, C6-, C8-, and C5-DC-carnitine. Concentrations of acylcarnitines in urine of both animals revealed increased excretions of C2-, C3-, C4-, C5-, C6-, C5-OH-, C8-, C10:1-, C10-, and C5-DC-carnitine. In addition, concentrations of free carnitine were also increased. Quantitative biochemical measurement of enzyme activities in muscle tissue showed deficiencies of short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and isovaleryl-CoA dehydrogenase (IVD) also indicating MADD. Histology revealed extensive rhabdomyolysis with microvesicular lipidosis predominantly in type 1 muscle fibers and mitochondrial damage. However, the ETF and ETF-QO activities were within normal limits indicating the metabolic disorder to be acquired rather than inherited. To our knowledge, these are the first cases of biochemical MADD reported in equine medicine.

Thirty microsatellite markers were analysed in 1426 goats from 45 traditional or rare breeds in 15 European and Middle Eastern countries. In all populations inbreeding was indicated by heterozygosity deficiency (mean FIS = 0.10). Genetic differentiation between breeds was moderate with a mean FST value of 0.07, but for most (c. 71%) northern and central European breeds, individuals could be assigned to their breeds with a success rate of more than 80%. Bayesian-based clustering analysis of allele frequencies and multivariate analysis revealed at least four discrete clusters: eastern Mediterranean (Middle East), central Mediterranean, western Mediterranean and central/northern Europe. About 41% of the genetic variability among the breeds could be explained by their geographical origin. A decrease in genetic diversity from the south-east to the north-west was accompanied by an increase in the level of differentiation at the breed level. These observations support the hypothesis that domestic livestock migrated from the Middle East towards western and northern Europe and indicate that breed formation was more systematic in north-central Europe than in the Middle East. We propose that breed differentiation and molecular diversity are independent criteria for conservation.

The tribe Bovini comprises cattle and cattle-like species. Reconstructions of their phylogeny have so far been incomplete and have yielded conflicting conclusions about the relationship of American bison and wisent (European bison). We have compared the sequences of three mitochondrial and two Y-chromosomal DNA segments. Mitochondrial DNA indicates that four distinct maternal lineages diverged after an early split-off of the buffalo species, leading to (1) taurine cattle and zebu, (2) wisent, (3) American bison and yak, and (4) banteng, gaur, and gayal, respectively. At a higher level, lineages (1) and (2) and lineages (3) and (4) are probably associated. In contrast, Y-chromosomal sequences indicate a close association of American and European bison, which is in agreement with their morphological similarity, complete fertility of hybrid offspring, and amplified fragment length polymorphism (AFLP) fingerprints of nuclear DNA. One explanation for the anomalous divergence of the mitochondrial DNA from the two bison species is lineage sorting, which implies that two distinct mitochondrial lineages coexisted in the bison-yak branch until the recent divergence of American bison and wisent. Alternatively, the wisent may have emerged by species hybridization initiated by introgression of bison bulls in another ancestral species. This "transpatric" mode of species formation would be consistent with the recent appearance of the wisent in the fossil record without clearly identifiable ancestors.

Haemonchus contortus is a sheep parasitic nematode that causes severe economic losses. Previous studies have indicated a high degree of genetic heterogeneity, which is hardly affected by selection for drug resistance. As a tool for the analysis of the population dynamics of H. contortus and its response to drug resistance, we designed a strategy to study the inbreeding of a benzimidazole-sensitive and a benzimidazole-resistant strain. After 15 generations, a theoretical inbreeding coefficient of 0.87 was achieved. The different stages of inbreeding were analysed using restriction fragment polymorphism, microsatellite variability and amplified fragment length polymorphism. Model-based clustering of the amplified fragment length polymorphism genotypes showed that the allele frequencies of the benzimidazole-resistant strain were stable during the last eight generations. In the sensitive strain a gradual shift of allele frequencies was observed, which led to a temporary increase of the genetic diversity around the eight generations.

Hybridization between wild and domestic bovine species occurs worldwide either spontaneously or by organized crossing. We have analysed hybridization of banteng (Bos javanicus) and zebu (Bos indicus) in south-east Asian cattle using mitochondrial DNA (PCR-RFLP and sequencing), AFLP, satellite fragment length polymorphisms (SFLP or PCR-RFLP of satellite DNA) and microsatellite genotyping. The Indonesian Madura zebu breed is reputed to be of hybrid zebu-banteng origin, but this has never been documented and Bali cattle are considered to be a domesticated form of banteng. The banteng mitochondrial type was found in all animals sampled on the isle of Bali, Indonesia, but only in 35% of the animals from a Malaysian Bali-cattle population. The Madura animals also carried mitochondrial DNA of either zebu and banteng origin. In both populations, zebu introgression was confirmed by AFLP and SFLP. Microsatellite analysis of the Malaysian Bali population revealed for 12 out of 15 loci screened, Bali-cattle-specific alleles, several of which were also found in wild banteng animals. The tools we have described are suitable for the detection of species in introgression studies, which are essential for the genetic description of local breeds and the preservation of their economic and cultural value.

The genetic integrity of crossfertile bovine- or cattle-like species may be endangered by species hybridization. Previously, amplified fragment length polymorphism, satellite fragment length polymorphism and microsatellite assays have been used to analyze the species composition of nuclear DNA in taurine cattle, zebu, banteng and bison populations, while mitochondrial DNA reveals the origin of the maternal lineages. Here, we describe species-specific markers of the paternally transmitted Y-chromosome for the direct detection of male-mediated introgression. Convenient PCR-restriction fragment length polymorphism and competitive PCR assays are shown to differentiate the Y-chromosomes of taurine cattle, American bison and European bison, and to detect the banteng origin of Indonesian Madura and Bali cattle bulls.

Myophosphorylase deficiency in cattle is a muscle disease induced by a C-->T point mutation in codon 489 of the myophosphorylase gene, which until now has only been diagnosed in the Charolais breed. The disease seems to be inherited in an autosomal monogenic recessive manner. A calf of double muscled phenotype was suspected of suffering from myophosphorylase deficiency based on typical symptoms, i.e. brown-coloured, transparent urine, occurring after exercise; exercise intolerance; symptoms of pain; and an elevated level of plasma creatine kinase. The presence of the previously described mutation was excluded using a newly developed, improved polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure to identify easily heterozygous carriers and homozygous affected animals.

The Bovini species comprise both domestic and wild cattle species. Published phylogenies of this tribe based on mitochondrial DNA contain anomalies, while nuclear sequences show only low variation. We have used amplified fragment length polymorphism (AFLP) fingerprinting in order to detect variation in loci distributed over the nuclear genome. Computer-assisted scoring of electrophoretic fingerprinting patterns yielded 361 markers, which provided sufficient redundancy to suppress stochastic effects of intraspecies polymorphisms and length homoplasies (comigration of non-homologous fragments). Tree reconstructions reveal three clusters: African buffalo with water buffalo, ox with zebu, and bison with wisent. Similarity values suggest a clustering of gaur and banteng, but bifurcating clustering algorithms did not assign consistent positions to these species and yak. We propose that because of shared polymorphisms and reticulations, tree topologies are only partially adequate to represent the phylogeny of the Bovini. Principal-coordinate analysis positions zebu between a gaur/banteng cluster and taurine cattle. This correlates with the region of origin of these species and suggests that genomic distances between the cattle species have been influenced by genetic exchange between neighbouring ancestral populations.

The cattle genome contains several distinct centromeric satellites with interrelated evolutionary histories. We compared these satellites in Bovini species that diverged 0.2 to about 5 Myr ago. Quantification of hybridization signals by phosphor imaging revealed a large variation in the relative amounts of the major satellites. In the genome of water buffalo this has led to the complete deletion of satellite III. Comparative sequencing and PCR-RFLP analysis of satellites IV, 1.711a, and 1.711b from the related Bos and Bison species revealed heterogeneities in 0.5 to 2% of the positions, again with variations in the relative amounts of sequence variants. Restriction patterns generated by double digestions suggested a recombination of sequence variants. Our results are compatible with a model of the life history of satellites during which homogeneity of interacting repeat units is both cause and consequence of the rapid turnover of satellite DNA. Initially, a positive feedback loop leads to a rapid saltatory amplification of homogeneous repeat units. In the second phase, mutations inhibit the interaction of repeat units and coexisting sequence variants amplify independently. Homogenization by the spreading of one of the variants is prevented by recombination and the satellite is eventually outcompeted by another, more homogeneous tandem repeat sequence.