Genetic mapping of quantitative trait loci for growth and fatness in pigs.

The European wild boar was crossed with the domesticated Large White pig to genetically dissect phenotypic differences between these populations for growth and fat deposition. The most important effects were clustered on chromosome 4, with a single region accounting for a large part of the breed difference in growth rate, fatness, and length of the small intestine. The study is an advance in genome analyses and documents the usefulness of crosses between divergent outbred populations for the detection and characterization of quantitative trait loci. The genetic mapping of a major locus for fat deposition in the pig could have implications for understanding human obesity.

Linkage and comparative mapping of the locus controlling susceptibility towards E. COLI F4ab/ac diarrhoea in pigs.

In 1995, Edfors-Lilja and coworkers mapped the locus for the E. COLI K88ab (F4ab) and K88ac (F4ac) intestinal receptor to pig chromosome 13 (SSC13). Using the same family material we have refined the map position to a region between the microsatellite markers Sw207 and Sw225. Primers from these markers were used to screen a pig BAC library and the positive clones were used for fluorescent in situ hybridization (FISH) analysis. The results of the FISH analysis helped to propose a candidate gene region in the SSC13q41-->q44 interval. Shotgun sequencing of the FISH-mapped BAC clones revealed that the candidate region contains an evolutionary breakpoint between human and pig. In order to further characterise the rearrangements between SSC13 and human chromosome 3 (HSA3), detailed gene mapping of SSC13 was carried out. Based on this mapping data we have constructed a detailed comparative map between SSC13 and HSA3. Two candidate regions on human chromosome 3 have been identified that are likely to harbour the human homologue of the gene responsible for susceptibility towards E. COLI F4ab/ac diarrhoea in pigs.

Genetic variation in Con A-induced production of interleukin 2 by porcine peripheral blood mononuclear cells.

Genetic variation in concanavalin A (Con A)-induced proliferation and interleukin 2 (IL-2) production by peripheral blood mononuclear cells (PBMC) was studied in blood collected from 96 piglets, aged 7 weeks. The piglets were the offspring of seven sires and 24 dams. Pronounced differences between litters from various dams were observed in the immune parameters measured. Also, large individual differences in the magnitudes of Con A-induced proliferation and IL-2 production were seen for PBMC collected from individual pigs within each litter. Both the time course and magnitude of IL-2 activity showed genetic variation, as results from the offspring of the seven sires differed significantly. However, only the time course, not the magnitude, of proliferation differed among the offspring groups. It was possible to establish a rank order for the sires based on the IL-2 production of PBMC by their offspring. As IL-2 has a key role in regulating the immune response, mitogen-induced IL-2 activity seems to be a good candidate as a general marker for cell-mediated immunity in pigs.

Genetic variation in parameters reflecting immune competence of swine.

Genetic variation in total and differential white blood cell (WBC) counts, phagocytic capacity of polymorphonuclear leukocytes (PMNL), virus induced interferon-alpha (IFN-alpha) production, mitogen induced proliferation and interleukin 2 (IL-2) production of mononuclear cells (MNC) in vitro was studied in blood collected from 124 Yorkshire piglets, aged 8 weeks. The piglets were the offspring from 12 sires and 31 dams. Data from an earlier experiment, including 96 piglets of seven sires and 24 dams, were added when estimating heritabilities for Con A induced proliferation and IL-2 production. The highest heritability (h2 = 0.87 +/- 0.41) was estimated for the total number of PMNL. Medium high heritabilities (h2 = 0.3-0.4) were estimated for the phagocytic capacity of PMNL, Con A induced proliferation and IL-2 production and the total number of WBC, while the heritability estimates were lower (h2 = 0.00-0.08 +/- 0.12) for the total number of lymphocytes, serum concentrations of Ig and IFN-alpha production. Pronounced differences between litters from various dams were found for total number of lymphocytes, IFN-alpha production, Con A induced proliferation and IL-2 production. The Con A induced proliferation was positively correlated (r = 0.48, P < 0.001) with the IL-2 production and both these parameters were correlated (r = 0.44 and 0.37, respectively, P < 0.001) to the virus induced IFN-alpha production. Despite these positive correlations, no parental offspring group was uniformly superior across all traits measured. However, the heritabilities estimated for the immune parameters are sufficiently high to be used as genetic markers in selection for general immune competence of swine.

Influence of the Hal locus and standardized stress on antibody response and in vitro reactivity of peripheral blood lymphocytes in pigs.

The antibody response to two Escherichia coli antigens (O149 and K88) and the in vitro reactivity of peripheral blood lymphocytes (PBLs) to pokeweed mitogen was studied in growing pigs after exposure to varying degrees of stress. Three experimental groups were used; transportation by lorry, transportation by lorry after a previous injection of a tranquillizing drug (amperozide), no transportation but an amperozide injection. Another group was used as a control group. This group was not transported and received no amperozide injection. The animals were the offspring of boars and sows heterozygous (Nn) for the Hal gene, and all 3 Hal genotypes (NN, Nn, nn) were identified and could thus be compared within litters. Immediately after the experimental treatment, the highest PBL reactivity was found for the amperozide-treated animals and for the non-transported animals, with no differences in reactivity between Hal genotypes. Two weeks later, the treatments caused different effects on pigs of the 3 Hal genotypes, both with regard to the PBL reactivity and the IgG response to O149. The NN pigs had a higher PBL reactivity than the nn pigs for all treatments except the 'drug + transport' class where the reverse rank order was found. The NN pigs also had a higher IgG response to O149 than the nn pigs in the 'drug + no transport' class. The amperozide treatment was followed by a higher PBL reactivity in non-transported NN pigs and in transported nn pigs. The amperozide-treated non-transported NN pigs also had a higher IgG response to O149. The highest PBL reactivity and IgG response to O149 were found mainly in pigs with the lowest cortisol levels. Pronounced differences between litters were found for both the antibody response and the PBL reactivity.

Effects of transport stress on concentrations of cortisol, corticosteroid-binding globulin and glucocorticoid receptors in pigs with different halothane genotypes.

Effects of stress on concentrations of cortisol and corticosteroid-binding globulin (CBG) in blood plasma and on glucocorticoid receptor concentrations in muscle cytosol were studied in pigs representing three Halothane (Hal) genotypes (NN, Nn, nn). At 12 wk of age, animals were divided into four groups: pigs subjected to transport (5 h in a truck), pigs treated with amperozide prior to transport, pigs not transported but treated with amperozide and pigs neither transported nor given amperozide. Animals were slaughtered the week they reached 100 kg live weight (3 mo later). The Hal gene showed no major influence on the variables studied except for cortisol concentrations (P = .06) measured directly after transport at 12 wk of age (NN = 66.8 nM, Nn = 61.4 nM, nn = 69.4 nM). However, the response in each Hal genotype differed, depending on whether or not the pigs had been exposed to transport. Two weeks after transport, NN pigs developed higher cortisol concentrations than untransported animals, whereas the response was reversed in nn animals; Nn pigs showed no difference in this regard. At slaughter, the effect of transport (12 wk of age) on cortisol and CBG was still apparent. In NN pigs cortisol and CBG concentrations were elevated (P less than or equal to .05, P = .08, respectively), whereas concentrations tended to be lower in nn pigs (P = .17, P = .07, respectively) when compared with untransported pigs. Transported pigs had lower receptor concentrations at slaughter (P less than or equal to .01) than untransported pigs. However, pigs given amperozide in connection with transport had a receptor concentration comparable to that in untransported pigs. Our study shows conclusively that transport stress had long-term effects on cortisol, CBG and glucocorticoid receptor concentrations. In addition, amperozide had long-term effects on cortisol and receptor concentrations.

Effect of halothane genotype on muscle metabolism at slaughter and its relationship with meat quality: A within-litter comparison.

The effects of halothane genotype on muscle metabolism at slaughter and its relationship with meat quality were studied within 16 litters. Heterozygous boars and sows were mated and the offspring were halothane tested and bloodtyped to reveal the halothane (Hal) genotype of the 120 animals used (NN, Nn or nn). Following slaughter at 100kg live weight, muscle samples from M. longissimus dorsi (LD) and M. quadriceps (Qu) were taken immediately after exsanguination and analysed for glycogen, glucose-6-phosphate, lactate, creatine phosphate (CP), and adenosine triphosphate (ATP), as well as for enzyme activities representing both the oxidative and glycolytic pathways. The enzyme activities were similar for all genotypes. All muscle metabolites differed significantly between samples from NN and nn animals, with higher lactate and glucose-6-phosphate and lower glycogen, CP and ATP in the nn muscles. The heterozygote animals were intermediate or close to either of the homozygotes. Meat quality characteristics (drip loss, surface and internal reflectance and dielectric loss factor) were studied only in the LD muscle. Meat quality of the muscle from the heterozygote (Nn) animals was inferior to that from NN animals (no difference for internal reflectance) but better than that from nn animals. When reflectance and drip loss were combined into an index, very few values from the nn-animals were better than the total mean. Indexes from the dominant homozygotes were generally better than the mean and those of heterozygotes were approximately normally distributed around the mean.

Associations between major histocompatibility complex genes and production traits in White Leghorns.

The frequency of the MHC haplotype B15 had been found in a previous study to be more than two times higher in a White Leghorn line selected for high egg production compared with the unselected control strain. To further evaluate these findings, matings were performed between chickens with the same heterozygous B genotypes, being combinations of the most frequent haplotypes, i.e., B15, B19, and B21. In total, more than 1,300 observations from two generations were analyzed. In each generation, approximately one half of the chickens were derived from the line selected for total egg mass, the other half from the control strain. The MHC genotypes were determined serologically. Additive and dominance effects of B haplotypes on production traits were analyzed using an individual animal model. The estimation of genotypic values, together with the analysis of gene substitution effects, showed that the B15 haplotype was associated with early sexual maturity and low egg production during the late production period, i.e., between 43 and 63 wk of age, whereas B19 was associated with later onset of sexual maturity. The association of B15 with early sexual maturity would thus explain the high frequency of the B15 haplotype previously observed in a line selected for high early egg production. No dominance effect of the B system was observed for any of the traits, suggesting that the present results were due predominantly to additive gene effects.

Genetic and stress-mediated influence on Aujeszky's disease virus induced interferon-alpha production in porcine leukocytes.

The ability to produce interferon-alpha (IFN-alpha) in vitro was measured in blood from 200 F2-crosses between European wild boar and Swedish Yorkshire pigs, originating from a reference pedigree for gene mapping. A total of 200 pigs of 44 litters, descendent from 4 boars and 22 sows, were stressed by transportation together with non-littermates for 5 h. Blood samples were collected from each individual twice, i.e. immediately before transportation and the day after transportation. IFN-alpha production was induced in whole blood cultures by a monolayer of fixed, Aujeszky's disease virus infected, porcine kidney cells. In general, the amount of IFN-alpha produced was significantly lower (p = 0.02) the day after transportation, although the ability to produce IFN-alpha showed a large individual variation (p < 0.001). However, both the levels of IFN-alpha produced and the decrease after transportation varied between the four parental offspring groups. Also, indications of single genes with significant effects on the ability to produce IFN-alpha were found. These results confirm a genetic influence on the ability to produce IFN-alpha. In addition, stress, such as transportation and mixing, may decrease the level of IFN-alpha produced.

Polymorphism at the porcine Dominant white/KIT locus influence coat colour and peripheral blood cell measures.

We have examined the phenotype of different KIT genotypes with regard to coat colour and several blood parameters (erythrocyte numbers and measures, total and differential leucocyte numbers, haematocrit and haemoglobin levels and serum components). The effect of two different iron supplement regimes (one or two iron injections) on the blood parameters was also examined. For a total of 184 cross-bred piglets (different combinations of Hampshire, Landrace and Yorkshire) blood parameters were measured four times during their first month of life, and the KIT genotypes of these and 70 additional cross-bred piglets were determined. Eight different KIT genotypes were identified, which confirms the large allelic diversity at the KIT locus in commercial pig populations. The results showed that pigs with different KIT genotypes differ both in coat colour and in haematological parameters. In general, homozygous Dominant white (I/I) piglets had larger erythrocytes with lower haemoglobin concentration, indicating a mild macrocytic anaemia. The effect of two compared with one iron injection was also most pronounced for the I/I piglets.

Confirmation of QTL on porcine chromosomes 1 and 8 influencing leukocyte numbers, haematological parameters and leukocyte function.

A genome wide search in European Wild Boar x Swedish Yorkshire (W x Y) inter-cross pigs has earlier identified quantitative trait loci (QTL) for leucocyte number and function on porcine chromosomes 1 and 8 (SSC 1 and 8). To verify the involvement of these chromosomal regions in the regulation of haematocrit (Hem) and haemoglobin (Hb) levels, leucocyte numbers and in vitro leukocyte functions (mitogen induced proliferation and IL-2 production, virus induced interferon-alpha production and neutrophil phagocytosis), animals of different genetic backgrounds were analysed. The animals comprised a back-cross sire family (n=47) of W x Y pigs and six crossbred [Y x Landrace (L)] sire families (n=191). They were genotyped for 16 genetic markers and an interval analysis was performed. On SSC1, a QTL close to S0082 on the q-arm that influenced numbers of white blood cells in L x Y pigs and numbers of band neutrophils and CD8(+) cells in W x Y pigs was identified (P

Binding and detection of glycosaminoglycans immobilized on membranes treated with cationic detergents.

Immobilization of molecules on surfaces is used for preparative, quantitative, and qualitative studies. Glycosaminoglycans (GAGs) are strongly hydrophilic and negatively charged molecules that do not bind well to either polystyrene surfaces or hydrophobic blotting membranes. Hydrophobic membranes were derivatized with cationic detergents to become hydrophilic and positively charged. The ability of the polyvinylidene fluoride and nitrocellulose membranes to retain GAGs increased up to 12.8 microg per spot in the dot blot assay when the membrane was treated with a cationic detergent. Immobilized GAGs were stained with alcian blue, and the staining intensity was quantitated by scanning and densitometry. The derivatized membranes were used for solid-phase extraction of GAGs in blood plasma, urine, or cerebrospinal fluid. The detection sensitivity was equal for different types of GAGs but there was a slight negative interference from fibrinogen in blood plasma. The immobilized GAGs could also be released from the membrane using a nonionic detergent at high ionic strength. Recovery of different proteoglycan populations, separated by electrophoresis and detected by reversible staining with toluidine blue, was 70-100%.

Structure and organization of pig MHC class II DRB genes: evidence for genetic exchange between loci.

The pig major histocompatibility complex DRB genes were studied by polymerase chain reaction (PCR) amplification of exon 2 from eight domestic pigs and two European wild boars. Sequence comparisons together with a phylogenetic analysis showed the existence of at least three DRB genes of which only one appears to be expressed. The two putative DRB pseudogenes contained deletions in exon 2, making it possible to confirm the presence of three non-allelic DRB genes by analyzing the length polymorphism of the amplified PCR products. The expressed gene shows allelic polymorphism at the same positions as in the human DRB1 gene. In addition, this pig gene shows extensive allelic polymorphism at positions 84-88, whereas, e.g., human DRB genes do not. Surprisingly, the two putative DRB pseudogenes also display a considerable amount of allelic polymorphism, albeit of a different character as compared with the expressed DRB gene. Short stretches of sequences are shared between individual alleles at different loci. These sequence similarities cannot be due to natural selection, since two of the three DRB genes involved are polymorphic pseudogenes constituting allelic series that have diverged after the inactivation event. Instead, the results indicate that the sequences have been exchanged between the DRB genes by intergenic recombination.

Mapping quantitative trait loci for stress induced alterations in porcine leukocyte numbers and functions.

To identify quantitative trait loci (QTLs) with effects on 'stress' induced alterations of porcine immune functions, a number of immune capacity traits were analysed in the F2 generation of a Wild Boar--Yorkshire intercross. All traits were measured prior, and one day after, exposure to experimental 'stress' (mixing and transport). The 'stress' protocol induced a decrease in numbers of circulating neutrophils and in spontaneous proliferation in vitro, whereas phagocytic capacity, mitogen induced proliferation and spontaneous IL-2 activity increased. The IFN-alpha production tended to decrease, although the individual variation was pronounced. More than 200 genetic markers have been scored in the entire pedigree and were used to trace the inheritance of individual chromosome segments. Wild Boar alleles were on average associated with higher mitogen induced IL-2 activity and a slightly lower decrease in IFN-alpha production after mixing and transport. Four QTLs with significant effects were identified; one influencing 'stress' induced alteration in numbers of neutrophils (chromosome 8), one influencing spontaneous proliferation after 'stress' (chromosome 2), one influencing mitogen induced IL-2 activity after 'stress' (chromosome 6) and one influencing 'stress' induced alterations in mitogen induced IL-2 activity (chromosome 12). In addition, several suggestive QTLs were indicated.

The relationship between bovine major histocompatibility complex class II polymorphism and disease studied by use of bull breeding values.

The predictive value of class II DQ and DYA polymorphisms of the bovine major histocompatibility (MHC) complex (BoLA) for the incidence of disease in dairy cattle was estimated in a sample of 196 progeny-tested AI bulls of the Swedish Red and White breed. The BoLA DQ and DYA types of the bulls were determined by analysing restriction fragment length polymorphisms (RFLPs). Breeding values of bulls for clinical mastitis, all diseases including clinical mastitis and diseases other than clinical mastitis were used as measures of disease resistance or susceptibility. The relationship between MHC polymorphism and bull breeding values for disease resistance was evaluated statistically by linear regression analysis. A significant association between the haplotype DQ1A and susceptibility to clinical mastitis was revealed. No other DQ haplotype nor the DYA locus has a significant effect on any of the disease traits studied.

Genetic variation at a pig serum protein locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group.

Two-dimensional agarose gel (pH 5.4)-polyacrylamide gel (pH 9.0) electrophoresis of pig serum samples revealed a new serum protein (postalbumin-2, PO-2) polymorphism. Family data supported the hypothesis that the three PO-2 phenotypes observed were controlled by two codominant, autosomal alleles (Po-2F and Po-2s) at a single locus. The frequency of Po-2F in Swedish Landrace and in Swedish Yorkshire breeds was estimated at 0.74 and 0.65, respectively. Evidence was presented for close genetic linkage between Po-2 and the red cell phosphohexose isomerase locus (Phi). A recombination frequency of 3.2% was obtained from double backcross material. Data obtained in a Danish Landrace material showing linkage between the Po-2 locus and the H blood group locus, the Pgd locus and Hal (locus for halothane sensitivity) are also given. A total of seven recombinants were observed. They show that Po-2 is a new locus in a previously established linkage group. The likely sequence of the loci is: Phi, Hal, S, H, Po-2, Pgd.

Major histocompatibility genes in egg-laying hens.

A common base population of White Leghorn was "synthesized" for a joint project on the genetics of egg-laying, undertaken by animal breeding geneticists in 4 Scandinavian countries. After 6 to 7 generations of line selection for various egg-laying parameters, MHC typing was undertaken of both the selection lines in Denmark, Norway, and Sweden and the respective control lines representing the common base population. Ten MHC haplotypes were defined which jointly accounted for about 95% of the MHC gene pool of the base population. The 2 haplotypes which were predominant in the base population, B15 and B19, responded very differently to the selection pressures applied.

Conserved synteny between pig chromosome 8 and human chromosome 4 but rearranged and distorted linkage maps.

The porcine genes encoding interleukin 2, alcohol dehydrogenase (class I) gamma polypeptide, and osteopontin were mapped to chromosome 8 by linkage analysis. Together with previous assignments to this chromosome (the albumin, platelet-derived growth factor receptor A, and fibrinogen genes), an extensive syntenic homology with human chromosome 4 was discovered. Loci from about three-quarters of the q arm of human chromosome 4 are on pig chromosome 8. However, the linear order of the markers is not identical in the two species, and there are several examples of interspecific differences in the recombination fractions between adjacent markers. The conserved synteny between man and the pig gives strong support to a previous suggestion that a synteny group present in the ancestor of mammalian species has been retained on human chromosome 4q. Since loci from this synteny group are found on two cattle chromosomes, the bovine rearrangement must have occurred after the split of Suidae and Bovidae within Artiodactyla.

The gene for dominant white color in the pig is closely linked to ALB and PDGRFRA on chromosome 8.

White is a widespread coat color among domestic pig breeds and is controlled by an autosomal dominant gene I. The segregation of this gene was analyzed in a reference pedigree for gene mapping developed by crossing the European wild pig and a Large White domestic breed. The gene for dominant white color was shown to be closely linked to the genes for albumin (ALB) and platelet-derived growth factor receptor alpha (PDGFRA) on chromosome 8. An unexpected phenotype with patches of colored and white coat was observed among the F1 and F2 animals. The segregation data indicated that the phenotype was controlled by a third allele, denoted patch (Ip), most likely transmitted by one of the Large White founder animals. It is shown that the ALB, PDGFRA, I linkage group shares homologies with parts of mouse chromosome 5, human chromosome 4, and horse linkage group II, all of which contain dominant genes for white or white spotting. Candidate genes for the dominant white and patch mutations in the pig are proposed on the basis on these linkage homologies and the recent molecular definition of the dominant white spotting (W) and patch (Ph) mutations in the mouse.

Mapping quantitative trait loci for immune capacity in the pig.

Immune capacity traits show considerable genetic variation in outbred populations. To identify quantitative trait loci (QTLs) for immune capacity in the pig, various measures of immune function (total and differential leukocyte counts, neutrophil phagocytosis, mitogen-induced proliferation, IL-2 production, and virus induced IFN-alpha production in whole blood cultures, and Ab responses to two Escherichia coli antigens) were determined in 200 F2 animals from a wild pig-Swedish Yorkshire intercross. The pedigree has been typed for 236 genetic markers covering all autosomes, the X chromosome and the X/Y pseudoautosomal region. Through interval mapping using a least-squares method, four QTLs with significant effects were identified; one for total leukocyte counts, one for mitogen-induced proliferation, one for prevaccination levels of Abs to E. coli Ag K88, and one for Ab response to the O149 Ag. In addition, several putative QTLs were indicated. The results from the present study conclusively show that it is possible to identify QTLs for immune capacity traits in outbred pig populations by genome analysis.