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

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.

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%.

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.

Molecular basis for the dominant white phenotype in the domestic pig.

The change of phenotypic traits in domestic animals and crops as a response to selective breeding mimics the much slower evolutionary change in natural populations. Here, we describe that the dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene encoding the mast/stem cell growth factor receptor (MGF), one gene duplication associated with a partially dominant phenotype and a splice mutation in one of the copies leading to the fully dominant allele. The splice mutation is a G to A substitution in the first nucleotide of intron 17 and leads to skipping of exon 17. The duplication is most likely a regulatory mutation affecting KIT expression, whereas the splice mutation is expected to cause a receptor with impaired or absent tyrosine kinase activity. Immunocytochemistry showed that this variant form is expressed in 17- to 19-day-old pig embryos. Hundreds of millions of white pigs around the world are assumed to be heterozygous or homozygous for the two mutations. [The EMBL accession numbers for porcine KIT1*0101, KIT1*0202, KIT2*0202, and KIT2*0101 are AJ223228-AJ223231, respectively.]

Variable number of pig MHC class I genes in different serologically defined haplotypes identified by a 3'-untranslated region probe.

To estimate the number of porcine class I major histocompatibility genes, a short class I cDNA probe from the 3'-untranslated region was developed to be used in restriction fragment length polymorphism analysis. Six clones isolated from a pig spleen cDNA library were sequenced from their 3'-untranslated region. Three different transcripts were identified, one probably derived from the class I PD7 locus and two showing highest homology to the PD1 and the PD14 genes, respectively. Class I typing was performed both by restriction fragment length polymorphism and serology. Segregation of class I haplotypes was followed in one three-generation family (European Wild Boar x Large White: Swedish Yorkshire) and in six two-generation families (Duroc, Yorkshire and Chester White), for a total of 266 pigs. Twenty different class I haplotypes were identified either with restriction fragment length polymorphism and/or serological typing. Furthermore, previously unpublished serological haplotypes H62, H67 and H68 were identified. Two to seven polymorphic and three monomorphic fragments were detected in different restriction fragment length polymorphism haplotypes indicating that the number of class I genes in the investigated haplotypes varies.

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.

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.

The porcine intestinal receptor for Escherichia coli K88ab, K88ac: regional localization on chromosome 13 and influence of IgG response to the K88 antigen.

The loci encoding the porcine intestinal receptors for Escherichia coli K88ab and K88ac (K88abR and K88acR) were firmly assigned to chromosome 13 by linkage analysis using a three-generation pedigree. The linear order of these loci and seven other markers on chromosome 13 was determined by multipoint analyses. The K88abR and K88acR loci were tightly linked (theta = 0.01, zeta = 41.06) with the K88abR locus localized 7.4 cM (sex average) proximal to the transferrin locus. The results, together with previous reports from two other groups, provide an unequivocal assignment of the K88 receptor loci to chromosome 13, and reject a previous assignment to chromosome 4. Pigs possessing the receptor had a slightly higher specific IgG response to the K88 antigen after an intramuscular immunization with an E. coli vaccine.

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.

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.

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.

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.

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.

Assignment of 20 microsatellite markers to the porcine linkage map.

Twenty-one porcine microsatellite markers were developed by screening DNA libraries and by a computer search of databases. The microsatellites were typed in a large three-generation family established by a cross between the European wild pig and a Swedish Yorkshire breed. Linkage analysis benefited from the fact that due to the divergence between the parental populations, the degree of microsatellite polymorphism was significantly higher in the F1 animals than in either of the parental populations. Parallel typing of a set of 35 restriction fragment length polymorphism, protein, and blood group markers rendered it possible to assign as many as 20 of the microsatellites to the porcine linkage map. Fourteen microsatellites were localized to a chromosome segment, whereas six constituted parts of unassigned linkage groups. Analysis of four microsatellites within genes allowed the assignment of the endoplasmic reticulum Ca2+ transport ATPase locus to chromosome 14, the assignment of the interferon-gamma and the diacylglycerol kinase loci to a new linkage group (XI), and the localization of the tumor necrosis factor beta locus close to the major histocompatibility complex (SLA) on chromosome 7 to be confirmed. Fluorescence in situ hybridization mapping of two microsatellite-containing cosmids assigned two linkage groups to chromosomes 9 and 12, respectively. In total, 27 new markers were added to the porcine linkage map, thereby almost doubling the number of markers on the map. Linkage groups are now present on 10 of 18 of the pig autosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.