Positive selection on mammalian MHC-DQ genes revisited from a multispecies perspective.

Major histocompatibility complex class II DQA and DQB genes have been shown to be under positive selection in certain mammalian species but not in others, fuelling a debate about how their polymorphism has evolved. In this study, we have analysed whether polymorphism in the peptide-binding region (PBR) of DQA (190 sequences, 11 species) and DQB (209 sequences, 7 species) molecules is positively selected by using both approximate (Nei-Gojobori, Li-Wu-Luo and Pamilo-Bianchi-Li) and maximum-likelihood methods. The results obtained with approximate methods were rather inconsistent for DQA, probably due to the high inaccuracy with which d(S) (PBR) is estimated, whereas evidence of positive selection was observed for most of the DQB PBR sequences. A parallel analysis with CodeML allowed us to demonstrate, in a very consistent way, the occurrence of positive selection in the PBR-encoding region of both DQA and DQB genes. Moreover, we have identified several DQA (alpha47, alpha55, alpha56, alpha68, alpha69, alpha76 and alpha79) and DQB (beta9, beta26 and beta57) codons that appear to be under positive selection in different, and often unrelated, mammalian species. Non-synonymous polymorphism at these sites has been evolutionarily conserved meaning that it might have functional consequences on peptide binding.

Sequence analysis of goat major histocompatibility complex class I genes.

The polymorphism of major histocompatibility complex (MHC) class I genes has been often involved in the resistance/susceptibility to a variety of infectious and parasitic diseases. In this work, the complete sequence of the coding region of a major histocompatibility complex (MHC) class I gene in goats (Cahi-N*01701, GenBank accession no. EF569216) is reported. The length of the corresponding open reading frame was 1,077 bp encoding a mature protein of 337 amino acids. Sequencing of additional clones allowed us to identify a second locus (Cahi-NC4*50301, GenBank accession no. EF569217) that, after performing a Bayesian phylogenetic analysis, happened to cluster with a bovine non-classical MHC class I gene. Nonclassical MHC class I molecules display low levels of polymorphism and fulfill an important immunoregulatory role in the placenta to inhibit maternal rejection. This initial description of the gene content of the goat MHC class I region will contribute to the characterization, in this ruminant species, of one of the most important genetic factors in the elicitation of innate and adaptive immune responses against pathogens.

Geographical partitioning of goat diversity in Europe and the Middle East.

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.

Selection and recombination drive the evolution of MHC class II DRB diversity in ungulates.

Major histocompatibility complex (MHC) antigen-presenting genes are the most variable loci in vertebrate genomes. Host-parasite co-evolution is assumed to maintain the excessive polymorphism in the MHC loci. However, the molecular mechanisms underlying the striking diversity in the MHC remain contentious. The extent to which recombination contributes to the diversity at MHC loci in natural populations is still controversial, and there have been only few comparative studies that make quantitative estimates of recombination rates. In this study, we performed a comparative analysis for 15 different ungulates species to estimate the population recombination rate, and to quantify levels of selection. As expected for all species, we observed signatures of strong positive selection, and identified individual residues experiencing selection that were congruent with those constituting the peptide-binding region of the human DRB gene. However, in addition for each species, we also observed recombination rates that were significantly different from zero on the basis of likelihood-permutation tests, and in other non-quantitative analyses. Patterns of synonymous and non-synonymous sequence diversity were consistent with differing demographic histories between species, but recent simulation studies by other authors suggest inference of selection and recombination is likely to be robust to such deviations from standard models. If high rates of recombination are common in MHC genes of other taxa, re-evaluation of many inference-based phylogenetic analyses of MHC loci, such as estimates of the divergence time of alleles and trans-specific polymorphism, may be required.

Assessment of population structure by single nucleotide polymorphisms (SNPs) in goat breeds.

Single nucleotide polymorphisms (SNPs) may be used in biodiversity studies and commercial tasks like traceability, paternity testing and selection for suitable genotypes. Twenty-seven SNPs were characterized and genotyped on 250 individuals belonging to eight Italian goat breeds. Multilocus genotype data were used to infer population structure and assign individuals to populations. To estimate the number of groups (K) to test in population structure analysis we used likelihood values and variance of the bootstrap samples, deriving optimal K from a drop in the likelihood and a rise in the variance plots against K.

Nucleotide sequence and polymorphism of the caprine major histocompatibility complex class II DQA1 (Cahi-DQA1) gene.

The major histocompatibility class II DQ molecules are dimeric glycoproteins involved in antigen presentation to CD4(+) T cells. In the current work, we have performed the molecular analysis of the goat Cahi-DQA1 gene. Sequencing of the Cahi-DQA1 cDNA revealed a single 768bp open reading frame. The alignment of this sequence with its bovine and ovine DQA1 counterparts revealed a remarkable degree of nucleotide identity (92-93% for the most similar bovine and ovine sequences). Moreover, we amplified a region including the 3'-end of intron 1, exon 2 and the 5'-end of intron 2. We identified seven Cahi-DQA1 alleles that likely correspond to four different allelic lineages. The alignment of these seven Cahi-DQA1 alleles revealed the existence of 23 amino acid polymorphic sites, seven of which (alpha(10), alpha(55), alpha(56), alpha(68), alpha(69), alpha(71) and alpha(76)) are highly polymorphic with at least three amino acid substitutions. Ten of the 23 polymorphic amino acid sites were included in the peptide binding region and consequently they might play a crucial role in immunological processes modulating disease pathogenesis.

Structural characterization of the caprine major histocompatibility complex class II DQB1 (Cahi-DQB1) gene.

The major histocompatibility class II genes have been extensively characterized in sheep and cattle, whereas in goats the only class II genes that have been completely sequenced are DRA and DRB. Herewith, we report the complete coding sequence of the goat DQB1 gene. This gene has a single open reading frame of 786bp, being organized in five exons and displaying 95-97% nucleotide identity with its bovine and ovine cDNA orthologous sequences. The structural features of the goat DQB1 molecule are well conserved with regard to its mammalian orthologues. Conserved glycosilation sites (beta19) and cysteine residues (beta15, beta79, beta117, beta173) forming disulfide bridges have been identified in the goat DQB1 molecule. The alignment of several Cahi-DQB1 exon 2 sequences has allowed to identify five different allelic variants Neighbor-joining phylogenetic analysis of caprine, ovine and bovine DQB sequences has allowed to ascertain that the five Cahi-DQB1 alleles we have found correspond to three different allelic lineages. We have identified fifteen polymorphic positions in the Cahi-DQB1 molecule, but only six of them are located in the peptide binding region. The high degree of conservation of these polymorphic sites located outside the peptide binding region in cattle and sheep suggests that they might play a functional role in antigen-presentation to CD4+ T cells.

Microsatellite DNA and recent statistical methods in wildlife conservation management: applications in Alpine ibex [Capra ibex(ibex)].

We evaluated the usefulness of microsatellites and recently developed statistical methods for the conservation management of fragmented and reintroduced populations, using the alpine ibex (Capra ibex) as a model species. First, we assessed the effects of past reintroduction programmes on genetic diversity and population differentiation considering different population sizes and histories. We show that genetic variability in ibex populations (HE 0.13) is among the lowest reported from microsatellites in mammal species, and that the Alpi Marittime-Mercantour population has suffered from a severe genetic bottleneck associated with its reintroduction. Second, using a computer-simulation approach, we provide examples and rough guidelines for translocation programmes concerning the number and origin of individuals for future reintroductions and for the reinforcement of populations with low genetic variability. Finally, we use the ibex microsatellite data to assess the usefulness of several published statistical tests for detecting population bottlenecks and assigning individuals to their population of origin. This study illustrates that microsatellites allow: (i) evaluation of alternative translocation scenarios by simulating different numbers and origins of migrants; (ii) identification of bottlenecked populations (especially using the Wilcoxon signed-ranks test); and (iii) population assignment with a high certainty (P < 0.001) of almost 100 of the individuals (or trophies or carcasses) from two distant populations (especially using stucture or whichrun software).

Partial sequences of feline and caprine immunoglobulin epsilon heavy chain cDNA and comparative binding studies of recombinant IgE fragment-specific antibodies across different species.

Parts of the feline and caprine IgE epsilon heavy chain cDNA (third and fourth constant domains, IgEf3/4) were cloned, sequenced, and expressed to raise antibodies (Abs). The DNA and derived protein sequences of the feline recombinant IgEf (rIgEf) shared high homology with the analogous canine parts (81% at the nucleotide and 71% at the protein levels) and the caprine with the ovine ones (95%/84%), respectively. The polyclonal Abs raised in chickens against the feline and caprine rIgEf3/4 were subjected to a comparative binding study utilizing an ELISA including rIgEf and specific Abs to these rIgEf from dog and horse (rIgEf2 and rIgEf3/4) and sheep (rIgEf3/4). All but the ovine-specific rIgEf3/4 Ab were polyclonal, which had been raised in chickens, and bound to most applied rIgEf; the ovine-specific monoclonal mouse Ab recognized only in addition to ovine rIgEf3/4 the closely related caprine rIgEf3/4. Significant, positive correlations were detected between binding reactions of the polyclonal Abs in ELISA and percentage protein sequence homology (p<0.01). Thus, the newly described feline and caprine IgE nucleotide sequences and corresponding Abs represent useful tools for further species-specific and comparative allergy and disease-associated research.

Genetic diversity in Swiss goat breeds based on microsatellite analysis.

Genetic diversity in eight Swiss goat breeds was estimated using PCR amplification of 20 bovine microsatellites on 20-40 unrelated animals per breed. In addition, the Creole breed from the Caribbean and samples of Ibex and Bezoar goat were included. A total of 352 animals were tested. The bovine microsatellites chosen amplified well in goat. The average heterozygosity within population was higher in domestic goat (0.51-0.58) than in Ibex (0.17) and Bezoar goat (0.19). Twenty-seven per cent of the genetic diversity in the total population could be attributed to differences between the populations. However, with the exclusion of Ibex from the total population, this proportion dropped to 17%. Principal component analysis showed that all Swiss goat breeds are closely related, whereas the Creole breed, Ibex and Bezoar goat are clearly distinct from all eight Swiss breeds.

Caprine T-cell receptor variable beta-chain (TCRV beta) repertoire analysis and potential applications in cowdriosis immune response studies.

Anchor polymerase chain reaction has been applied to the study of caprine TCR V beta-chain repertoire at the mRNA level in peripheral blood of a Saanen goat. Single stranded, g-tailed cDNA synthesized from total RNA was PCR-amplified using a sheep V beta constant region primer (3') and a poly(dC) anchor primer (5') at the variable region end of the TCR V beta-chain. The obtained amplicon was subsequently cloned into Bluescript plasmid vector. A total of 72 recombinant clones whereof 61 contained an insert of appropriate size were harvested. Up to now, the full length sequences of a total of 55 clones have been obtained. Nine clones were rearranged but not functional due to stop codons. Forty-five sequences were functionally rearranged and further analyzed. They were classified into 15 different V beta families on the basis of V-region sequence homology with their human counterpart. V beta families corresponding to the 9 published bovine families were represented in our library. This complexity enables us to develop V beta family-specific primers in order to study the TCR V beta repertoire of other goat breeds of interest, i.e., the Creole goat. There the TCR V beta repertoire analysis and kinetic study of the cowdriosis model will provide insight into the type of the immune response and the status of protection during the immunization process and challenge.

Lack of association of bovine MHC class I alleles with carcass and reproductive traits.

The present study was carried out to examine whether a relationship between bovine major histocompatibility complex (BoLA) class I alleles and carcass traits or reproductive performance exists in Braunvieh and Fleckvieh AI (artificial insemination) bulls. The influence of BoLA class I (BoLA-A) alleles on deregressed breeding values for net growth rate, carcass index and thigh volume was assessed in Braunvieh crosses and Fleckvieh bulls with a gene substitution model. The reproductive traits: non-return rate and interval between first and last insemination of daughters (female fertility), as well as non-return rate of inseminated cows (male fertility), were only investigated in Fleckvieh animals. No influence of the BoLA-A region on the traits evaluated could be demonstrated. An improper, i.e. less restrictive analysis would have led to spurious results.