Genetic diversity within and between European pig breeds using microsatellite markers.

An important prerequisite for a conservation programme is a comprehensive description of genetic diversity. The aim of this study was to use anonymous genetic markers to assess the between- and the within-population components of genetic diversity for European pig breeds at the scale of the whole continent using microsatellites. Fifty-eight European pig breeds and lines were analysed including local breeds, national varieties of international breeds and commercial lines. A sample of the Chinese Meishan breed was also included. Eleven additional breeds from a previous project were added for some analyses. Approximately 50 individuals per breed were genotyped for a maximum of 50 microsatellite loci. Substantial within-breed variability was observed, with the average expected heterozygosity and observed number of alleles per locus being 0.56 [range 0.43-0.68] and 4.5 respectively. Genotypic frequencies departed from Hardy-Weinberg expectations (P < 0.01) in 15 European populations, with an excess of homozygotes in 12 of them. The European breeds were on average genetically very distinct, with a Wright F(ST) index value of 0.21. The Neighbour-Joining tree drawn from the Reynolds distances among the breeds showed that the national varieties of major breeds and the commercial lines were mostly clustered around their breeds of reference (Duroc, Hampshire, Landrace, Large White and Piétrain). In contrast, local breeds, with the exception of the Iberian breeds, exhibited a star-like topology. The results are discussed in the light of various forces, which may have driven the recent evolution of European pig breeds. This study has consequences for the interpretation of biodiversity results and will be of importance for future conservation programmes.

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers.

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.

Genetic linkage mapping of quantitative trait loci for behavioral and neuroendocrine stress response traits in pigs.

A QTL analysis of behavioral and neuroendocrine responses to a "novel environment" stress was conducted in a three-generation experimental cross between Meishan and Large White pig breeds. A total of 186 F2 males and 182 F2 females were studied for their behavioral and neuroendocrine reactivity to a novel environment test at 6 wk of age. Locomotion, vocalization, and defecation rate, as well as exploration time, were measured for 10 min. Blood samples were taken immediately before and after the test to measure plasma levels of ACTH, cortisol, and glucose. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using two interval mapping methods: a line-cross regression method, where founder lines were assumed to be fixed for different QTL alleles, and a half-/full-sib maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Both methods revealed a highly significant gene effect for poststress cortisol level (P < 0.001) and a significant effect for basal cortisol level (P < 0.05) at the end of the q arm of chromosome 7, explaining, respectively, 20% and 7% of the phenotypic variance. Meishan alleles are associated with higher cortisol levels and are partially dominant (for poststress levels) over Large White alleles. Other significant gene effects on biological measures were detected on chromosomes 1 and 17 (ACTH response to stress), 3, 5, and 8 (glucose levels). The SSC 17 QTL explains 12% of the phenotypic variance of poststress ACTH levels, with a suggestive evidence of imprinting effects. Meishan alleles are associated with lower poststress ACTH levels. Gene effects of low amplitude only were found for behavioral reactivity traits. Considering the effects of stress neuroendocrine systems on energy fluxes and protein deposition, and the importance of stress reactivity for meat quality and animal welfare, these results open new perspectives for pig selection.

Molecular genetic approaches to investigate individual variations in behavioral and neuroendocrine stress responses.

A large response range can be observed in both behavioral and neuroendocrine responses to environmental challenges. This variation can arise from central mechanisms such as those involved in the shaping of general response tendencies (temperaments) or involves only one or the other output system (behavioral vs. endocrine response). The participation of genetic factors in this variability is demonstrated by family and twin studies in humans, the comparison of inbred strains and selection experiments in animals. Those inbred strains diverging for specific traits of stress reactivity are invaluable tools for the study of the molecular bases of this genetic variability. Until recently, it was only possible to study biological differences between contrasting strains, such as neurotransmitter pathways in the brain or hormone receptor properties, in order to suggest structural differences in candidate genes. The increase of the power of molecular biology tools allows the systematic screening of significant genes for the search of molecular variants. More recently, it was possible to search for genes without any preliminary functional hypothesis (mRNA differential expression, nucleic acid arrays, QTL search). The approach known as quantitative trait loci (QTL) analysis is based on the association between polymorphic anonymous markers and the phenotypical value of the trait under study in a segregating population (such as F2 or backcross). It allows the location of chromosomal regions involved in trait variability and ultimately the identification of the mutated gene(s). Therefore, in a first step, those studies skip the 'black box' of intermediate mechanisms, but the knowledge of the gene(s) responsible for trait variability will point out to the pathway responsible for the phenotypical differences. Since variations in stress-related responses may be related to numerous pathological conditions such as behavioral and mood disorders, drug abuse, cardiovascular diseases or obesity, and production traits in farm animals, these studies can be expected to bring significant knowledge for new therapeutic approaches in humans and improved efficiency of selection in farm animals.

Behavior and pituitary-adrenal function in large white and Meishan pigs.

Six-wk-old piglets of both sexes from European Large White (LW, n = 36) and Chinese Meishan (MS, n = 24) breeds were individually exposed to a novel environment, a stressful stimulation. Behavioral and pituitary-adrenal reactivity were investigated. When compared with LW, MS pigs displayed low locomotion (18.5 +/- 2.2 vs. 41.0 +/- 3.8 squares crossed/10 min; P < 0.0001), and defecation scores (0.58 +/- 0.15 vs. 4.86 +/- 0.37 fecal boli; P < 0.0001). Basal concentrations of cortisol were higher in MS (96.1 +/- 1.1 vs. 44.9 +/- 1.1 ng/ml; P < 0.0001), although no differences between breeds were found in basal concentrations of adrenocorticotropic hormone (ACTH). In response to novel environment exposure, the ACTH increase was greater in LW than in MS, but the cortisol response was not different on a log scale. To further investigate the pituitary-adrenal differences between the two breeds, the 24-hr profile of ACTH and cortisol plasma concentrations, a corticotropin-releasing factor (CRF) and a coupled dexamethasone-ACTH test were studied. Five castrated male 9-wk-old piglets from each breed were fitted surgically with a jugular vein catheter. A classic marked circadian rhythm of cortisol and a weak nycthemeral variation of ACTH were found. Cortisol concentrations were approximately twice higher in MS exclusively during the early light phase (from 0800-1200 hr) of the cycle, but no significant interbreed difference was found in the circadian rhythm of ACTH. Administration of CRF (1 microgram/kg iv) induced the same significant increase in plasma ACTH and cortisol concentrations in both breeds. Administration of ACTH (10 micrograms/kg i.v.) increased significantly cortisol concentrations and revealed no difference in plasma cortisol response to ACTH. These data suggest that the hypercortisolism of MS pigs is of adrenal origin, and related to extrapituitary factors that control the adrenal sensitivity during the light phase of the diurnal cycle.

Genetic study of behavioral and pituitary-adrenocortical reactivity in response to an environmental challenge in pigs.

The adaptive response to environmental challenges involves both behavioral and neuroendocrine adjustments, and genetic factors have been shown to partly determine the intra- and interspecific variability observed in stress responses. To gain access to the biological and genetic basis of this variability, differences in neuroendocrine and behavioral responses to a 10-min novel environment exposure were studied in Meishan (MS) and Large White (LW) pig breeds, as well as in their F1 (MS x LW), F1R (LW x MS), and F2 (F1 x F1) crossings. Different behavioral scores were recorded and blood was taken by venipuncture, before and after the test, to measure levels of stress hormones (adrenocorticotropic hormone: ACTH and cortisol). MS pigs exhibited low vocalization, locomotion, and defecation scores when compared to LW. F1s showed intermediate locomotion scores. The vocalization scores of F1s were not significantly different from the respective scores of their parental MS and LW breeds. The defecation scores in F1s showed that there was some degree of dominance in the MS direction. Basal and poststress cortisol levels were higher in MS, F1s, and F2 than in LW, suggesting the dominance of this trait. Basal ACTH levels did not differ between the genetic types, whereas LW displayed higher poststress ACTH levels than MS. Phenotypic correlations were analyzed in the F2 segregating cross to study a possible link between behavioral and neuroendocrine traits. All behavioral variables were intercorrelated with 3 levels of association. The correlations between vocalization and locomotion scores and poststress ACTH levels suggest that these measures reflect the level of reactivity to the environmental challenge, and that they may share a common genetic control.