The present Pyrenean population of bearded vulture (Gypaetus barbatus): its genetic characteristics.

The Pyrenean population of the endangered bearded vulture (Gypaetus barbatus) is the largest natural population in Europe. In this study, its current genetic variability was assessed using 110 animals of the recent population in order to know what the present situation. Sex identification by DNA methodology in the 110 bearded vultures, mitochondrial DNA (mtDNA) and eight microsatellite markers in 87 bearded vultures have been analysed. Our results for sex identification present a number of 49 males and 61 females; no significant differences for number of males and females in this population have been observed. mtDNA studies indicate that nucleotide and haplotype diversities and number of variable sites were low. Tajima's D test and Fu and Li's D* and F* tests suggest that mutations are selectively neutral and the population is expanding. A mean number of alleles per locus and a mean observed heterozygosity have been obtained by microsatellite analysis. FIS is not high, and inbreeding depression could be discarded in the near future. The results suggest that the Pyrenean population of bearded vultures have to be controlled in order to avoid the loss of genetic variability. This data should be taken into account when considering conservation plans for the species.

Molecular approach of the fragile chromosomal region Xq31-34 in cattle (Bos taurus) by microdissection and DOP-PCR / Aproximação molecular da região cromossômica frágil Xq31-34 em bovinos (Bos taurus) utilizando microdissecação cromossômica e DOP-PCR

Fragile sites (FS) are chromosomal regions where the normal compactation of chromatine is not observed. FRAXA (Fra Xq27.3, X sexual chromosome) is one of the most studied FS in humans. FRAXA is an expansion of the trinucleotide CGG located in the gene FMR-1. In cattle, sites of chromosomal fragility were reported in BTAX, associated with different pathologies and fertility impairment. Chromosomal microdissection has became a valuable tool for isolating chromatine fragments. In this work, it was combined the chromosomal microdissection technique with DOP-PCR in order to carry out a molecular analysis of the fragile chromosomal region BTAXq31-34. In that region, polymorphic DNA-RAPD sequences (GC rich) are present and sequences of the gene FMR-1 are missing. The results showed the usefulness of the microdissection-DOP-PCR technique for molecular characterization of fragile chromosomal sites in cattle.

Os sítios frágeis (FS) são regiões de cromossomo onde a compactação normal da cromatina não é realizada. O FRAXA (Fra Xq27.3, cromossomo sexual X) é um dos FS mais estudados em seres humanos. O FRAXA apresenta expansão do trinucleotídeo CGG localizado no gene FMR-1. Em bovinos, existem estudos informando sobre fragilidade cromossômica em BTAX associada com diversas patologias e alterações na fertilidade. A microdissecação cromossômica é uma valiosa técnica para isolar fragmentos de cromatina. Neste trabalho, combinou-se a técnica de microdissecação de cromossomo com DOP-PCR para executar a análise molecular da região do sitio frágil cromossômico BTAXq31-34. Naquela região estão presentes seqüências do polimorfo DNA-RAPD (rico em GC), em que as seqüências do gene FMR-1 estão ausentes. Os resultados mostram a utilidade da técnica de microdissecação-DOP-PCR para a caracterização molecular de sítios frágeis cromossômicos em bovinos.

Cytogenetic screening of livestock populations in Europe: an overview.

Clinical animal cytogenetics development began in the 1960's, almost at the same time as human cytogenetics. However, the development of the two disciplines has been very different during the last four decades. Clinical animal cytogenetics reached its 'Golden Age' at the end of the 1980's. The majority of the laboratories, as well as the main screening programs in farm animal species, presented in this review, were implemented during that period, under the guidance of some historical leaders, the first of whom was Ingemar Gustavsson. Over the past 40 years, hundreds of scientific publications reporting original chromosomal abnormalities generally associated with clinical disorders (mainly fertility impairment) have been published. Since the 1980's, the number of scientists involved in clinical animal cytogenetics has drastically decreased for different reasons and the activities in that field are now concentrated in only a few laboratories (10 to 15, mainly in Europe), some of which have become highly specialized. Currently between 8,000 and 10,000 chromosomal analyses are carried out each year worldwide, mainly in cattle, pigs, and horses. About half of these analyses are performed in one French laboratory. Accurate estimates of the prevalence of chromosomal abnormalities in some populations are now available. For instance, one phenotypically normal pig in 200 controlled in France carries a structural chromosomal rearrangement. The frequency of the widespread 1;29 Robertsonian translocation in cattle has greatly decreased in most countries, but remains rather high in certain breeds (up to 20-25% in large beef cattle populations, even higher in some local breeds). The continuation, and in some instances the development of the chromosomal screening programs in farm animal populations allowed the implementation of new and original scientific projects, aimed at exploring some basic questions in the fields of chromosome and/or cell biology, thanks to easier access to interesting biological materials (germ cells, gametes, embryos ...).

Bilateral Leydig cell tumor in a six-year-old intersex goat affected by Polled Intersex Syndrome.

A 6-year-old, sterile, Blanca Celtibérica breed adult doe was referred to our faculty. The doe had external female genitalia, a short anogenital distance, and normally shaped udders. Masculinization signs in the head shape and male behavior were also noted at the time of referral. Genetic analysis demonstrated normal 2n = 60 XX karyotype and an absence of the sex-determining region Y (SRY). The animal was homozygous for a DNA deletion responsible for the Polled Intersex Syndrome (PIS). A uterus and 2 uterine horns were present at the postmortem examination. Gartner's ducts and degenerated Wolffian derivatives persisted. There were 2 intra-abdominal testicle-like structures, one of which consisted of epididymal and deferent ducts. An advanced Leydig cell tumor, resulting in almost total destruction of the intratesticular structures, was also observed. Leydig cell tumors usually produce testosterone. Thus, these histologic findings are compatible with the evident virilization.

Microsatellite markers for the analysis of genetic variability and relatedness in red-legged partridge (Alectoris rufa) farms in Spain.

The usefulness of several microsatellites in the management of Alectoris rufa restocking farms is evaluated in the present report. We analysed seven microsatellite loci in 147 randomly chosen individuals from four Spanish farms. Global F(IS) values were not significant. Slight genetic differentiation was only found between Farms 1 and 4. The global first and second parent exclusionary powers were 0.742 and 0.930, respectively. Microsatellite analysis can estimate the genetic relatedness between pairs of individuals by means of the Identity index. Since genealogies are unknown in most farms, pairwise Identity index values proved to be helpful in the management of matings, and the improvement of reproductive success and fitness of the descendants. Mean Identity index values showed that individuals within farms were not genetically more related than expected under random mating. Variance of the Identity index values suggest the absence of closed familial groups in these farms.

Scrapie resistance alleles are not associated with lower prolificity in Rasa Aragonesa sheep.

Scrapie is a prion disease characterised by the accumulation of the pathological associated form of cellular prion protein (PrP(SC)) in the central nervous system. Susceptibility to scrapie is associated with polymorphism in the ovine prion protein (PrP) gene. The European Union has implemented scrapie control programs, relying on selective breeding for scrapie resistance; the use of ARR-carrier and the exclusion of VRQ-carrier were recommended. In this study, 4323 individuals from Rasa Aragonesa Sheep breed were genotyped for the PrP gene and the individual estimated breeding values (EBV) for prolificity were calculated. Most represented PrP alleles do not work against prolificity. Only a significant association between VRQ/VRQ genotype and a lower EBV was observed (p = 0.027, eta2 = 0.002). Therefore, avoiding reproduction of VRQ/VRQ individuals would not cause negative effect regarding prolificity.

Phylogenetic relationships among Spanish goats breeds.

We partially sequenced the mitochondrial hypervariable region 1 (HVR1) in 60 goats belonging to six Spanish breeds. The analysis of these and previously published sequences reveals a weak phylogeographical structure in the Iberian Peninsula breeds. Individuals from a single breed did not group into a single cluster. Furthermore, individuals from different breeds often shared single phylogenetic tree branches after UPGMA analysis. This could reflect the non-existence of breed isolation because of traditional seasonal pastoralism and annual long-distance migrations. Three goats belonging to the C maternal lineage were found, demonstrating a wider than previously thought distribution for this lineage.

Chromosome homology between chicken (Gallus gallus domesticus) and the red-legged partridge (Alectoris rufa); evidence of the occurrence of a neocentromere during evolution.

Chromosome-specific paints from macrochromosomes 1-9 and Z of the chicken were hybridised to metaphases of the red-legged partridge and revealed no inter-chromosomal rearrangements. The results from chromosome painting are similar to previous studies on the Japanese quail but different from findings in guinea fowl and several species of pheasant. The difference in centromere position in chicken and partridge chromosome 4, previously assumed to be the result of an inversion, was confirmed. However, FISH mapping of BAC clones from chicken chromosome 4 revealed that the order of loci was the same in both species, indicating the occurrence of a neocentromere during divergence.

Analysis of microsatellites and paternity testing in Rasa Aragonesa sheep.

Four microsatellite loci (MAF50, MAF18, OarFCB20 and MCM527) were studied in Rasa Aragonesa sheep in order to evaluate their use in paternity testing. Several population characteristics were estimated [allele frequencies. effective allele number (Ne), polymorphism informative content (PIC) and probability of excluding wrong paternities (Pe)]. In 32 randomly chosen individuals, four alleles were detected for MAF50, with 2.55 effective alleles, 0.58 PIC and 0.35 Pe. For MAF18, five alleles were identified, with 2.99 effective alleles, 0.51 PIC and 0.32 Pe. For oarFCB20, 10 alleles were observed, with 6.06 effective alleles, 0.82 PIC and 0.68 Pe. Finally, for MCM527, six alleles were found, with 3.75 effective alleles, 0.69 PIC and 0.50 Pe. When these loci were used together with serum transferrin locus, Pe rose to 97.20 per cent. Field trials confirmed the real usefulness of these techniques.

Population genetics and paternity in Rasa Aragonesa sheep using DNA fingerprinting.

DNA fingerprinting was used to study the population genetics and paternity of the Rasa Aragonesa sheep (Ovis aries). Using a combination of Hae III and the M13 derived probe pV47, the mean number of bands per individual (5.422 +/- 0.309), the background band sharing coefficient (0.347), the mean population frequency of alleles (0.192) and the mean heterozygosity for bands (0.893) were calculated for 45 individuals from eight different farms in Aragon, northern Spain. Therefore, the estimated probability of missing a wrong paternity was 0.126 for unrelated males, and this probability was 0.438 for full-sibs males. In addition, in a field trial of this technique, paternity was assigned for three ewe-lamb pairs with a probability of 97 per cent in two cases and 77 per cent in one case.

Cloning of the bovine activin receptor type II gene (ACVR2) and mapping to chromosome 2 (BTA2).

The cDNA for the bovine activin receptor type II (ACVR2) gene has been cloned and sequenced. It encodes a protein of 513 amino acids which is highly homologous (approximately 98% identity) to the human, mouse, and rat proteins. Using PCR analysis of bovine x hamster somatic cell lines, the ACVR2 gene was mapped to syntenic group U17, which has been localised to bovine chromosome 2. Comparative mapping has shown that the genes within U17 are also in a syntenic group on the long arm of human and sheep chromosome 2, as well as on mouse chromosome 1. This group of genes represents an evolutionarily conserved mammalian chromosomal segment. Genotyping a highly polymorphic microsatellite locus, (AT)4(GT)9(AT)11, found within an intron of this gene confirmed the localisation by linking the ACVR2-associated microsatellite to the region of chromosome 2 flanked by CSSM42 and TGLA226. This gene locus, which has the characteristics of a type I and type II mapping locus, represents the first localisation of this gene in any species to date.

Sister-chromatid exchanges in cattle: breed, sex and BrdU dose effects.

The spontaneous incidence of sister-chromatid exchanges (SCE) was investigated in a group of cattle, composed of 21 animals of both sexes and from two different breeds (Fleckvieh and Pirenaica). Peripheral lymphocytes of these animals were cultured in three different bromodeoxyuridine (BrdU) concentrations: 5, 15 and 30 micrograms/ml. The work was carried out following a randomized block design. Among the analyzed sources of variability, group, breed and BrdU dose factors had significant effects on the SCE frequency. No differences between sexes were found. Comparisons of the BdrU doses showed that the 5 micrograms/ml dose differed from both the 15 and 30 micrograms/ml doses, whereas the 15 and 30 micrograms/ml doses did not differ from each other. The results indicate that the breed of cattle as well as the BrdU dose chosen for the analysis must be considered when the SCE test is used for the biomonitoring of environmental mutagens.

Sister-chromatid exchanges induced by chloramphenicol on bovine lymphocytes.

The induction of sister-chromatid exchanges (SCE) was studied in bovine lymphocyte cultures treated with chloramphenicol (CAP), an antibiotic agent in wide use in human and animal therapy. A total of six individuals, matched for sex, race, age and environmental conditions, were used for the analysis. Chloramphenicol was tested at four different concentrations (5, 10, 20 and 40 micrograms/ml) and acted for the last 24 h of the culture. Each experiment included two animals, each of which was exposed to all chloramphenicol doses, for a total of three repetitions. The results of the corresponding analysis of variance showed that this chemical had a small but statistically significant effect on the SCE frequency. In addition, the lymphocyte cultures responded strangely to this chemical: the highest SCE induction was produced by the lowest dose. However, the study of high frequency cells did not show the presence of this kind of cell which could explain this chloramphenicol response. In addition, chloramphenicol induced a high delay in the cell cycle.

Effect of chloramphenicol on sister chromatid exchange in bovine fibroblasts.

The genotoxic potential of different chloramphenicol concentrations (5, 20, 40 and 60 micrograms ml-1) was investigated in bovine fibroblast primary lines by sister chromatid exchange assay. Chloramphenicol acted for long enough to ensure similar effects to persistent storage in the kidney. In this experiment 10 micrograms ml-1 of 5-bromodeoxyuridine was added for 60 hours for all doses of chloramphenicol and to the control. When the tissue culture cells were exposed to increasing doses, increased numbers of sister chromatid exchanges developed. Differences were significantly different to the control.

Confirmation of assignment of the bovine K-casein locus by PCR.

The provision of a bovine gene map will allow the ready identification of genetic disease in cattle and will lead to the identification of the genetic loci responsible for quantitative traits of economic importance. An extension of the polymerase chain reaction to the identification of linkage in bovine-Chinese hamster cell hybrids has improved the speed and facility of the assignment of genes to linkage groups and thus makes it easier to achieve a bovine linkage map.

Assignment of two markers carried by human chromosome 1 to different cattle synteny groups: FH to U1 and PEPC to U17 (chromosome 8).

Two loci located on human chromosome 1 were mapped in cattle by means of interspecific (hamster x cattle) somatic cell hybridization. FH is assigned to the U1 synteny group together with PGD, ENO1, AT3, and REN, while PEPC is found to belong to the U17 group (chromosome 8), like FN1, CRYG, and VIL1.