Association analyses of porcine SERPINE1 reveal sex-specific effects on muscling, growth, fat accretion and meat quality.

The serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) gene encodes plasminogen activator inhibitor type 1 (PAI), which is the major physiological inhibitor of tissue-type and urokinase-type plasminogen activators and plays a role in obesity and insulin resistance in women but not in men. We detected SNP FN396538:g.566G>A in intron 3 and a non-synonymous substitution NM_213910:c.612A>G in exon 3 (p.Ile159Val) and mapped the gene to position 8.4 cM on the linkage map of chromosome 3. Association analyses were conducted on the 12th-15th generation of the Meishan × Large White (MLW) cross (n = 565), with records for weight at the end of test, lifetime daily gain, test time daily gain, loin depth and backfat depth, as well as on a European wild boar × Meishan (W × M) F(2) population (n = 333) with 47 traits recorded for carcass composition and meat quality. Analyses performed across the entire MLW population or in the male animals did not show any trait significantly associated with the loci studied. In female animals, both SNPs were associated with loin depth at nominal P < 0.05 with adjusted P values equal to 0.051 (g.566) and 0.057 (c.612). Differences between homozygotes were up to 0.65 SD. In the entire W × M population and female animals, SERPINE1 was significantly associated at adjusted P < 0.05 in descending order with muscling, growth and fat accretion and in male animals with meat quality (R-value). In the studied populations, allele effects were in opposite directions, which implies that the SNPs are markers that are in linkage disequilibrium with a causative mutation.

Porcine NAMPT gene: search for polymorphism, mapping and association studies.

NAMPT encodes an enzyme catalysing the rate-limiting step in NAD biosynthesis. The extracellular form of the enzyme is known as adipokine visfatin. We detected SNP AM999341:g.669T>C (referred to as 669T>C) in intron 9 and SNP FN392209:g.358A>G (referred to as 358A>G) in the promoter of the gene. RH mapping linked the gene to microsatellite SW944. Linkage analysis placed the gene on the current USDA ­ USMARC linkage map at position 92 cM on SSC9. Association analyses were performed in a wild boar × Meishan F2 family (W × M), with 45 traits recorded (growth and fattening, fat deposition, muscling, meat quality, stress resistance and other traits), and in a commercial Landrace × Chinese-European (LCE) synthetic population with records for 15 traits (growth, fat deposition, muscling, intramuscular fat, meat colour and backfat fatty acid content). In the W × M, SNP 669T>C was associated with muscling, fat deposition, growth and fattening, meat quality and other traits and in the LCE with muscling, meat quality and backfat fatty acid composition. In the W × M, SNP 358A>G was associated with muscling, fat deposition, growth and other traits. After correction for multiple testing, the NAMPT haplotypes were associated in the W × M with, in descending order, muscling (q = 0.0056), growth (q = 0.0056), fat deposition (q = 0.0109), fat-to-meat ratio (q = 0.0135), cooling losses (q = 0.0568) and longissimus pHU (q = 0.0695). The SNPs are hypothesized to be in linkage disequilibrium with a causative mutation affecting energy metabolism as a whole rather than fat metabolism alone.

Mapping of QTL on chromosome X for fat deposition, muscling and growth traits in a wild boar x Meishan F2 family using a high-density gene map.

Quantitative trait loci (QTL) for fat deposition, growth and muscling traits have been previously mapped on the basis of low-density linkage maps in a wild boar x Meishan F2family to the chromosome X region flanked by SW2456 and SW1943. Improved QTL resolution was possible using data for F2 animals with a marker density of 2.7 cM distance in the SW2456 to SW1943 region, including AR, SERPINA7 and ACSL4 as candidate genes. The resolution of the QTL scan was increased substantially, as evidenced by the higher F-ratio values for all QTL. Maxima of F-ratio values for fat deposition, muscling and growth traits were 28.6, 18.2 and 16.5 respectively, and those QTL positions accounted for 7.9%, 5.0% and 4.5% of the F2 phenotypic variance (VF2) respectively. QTL for fatness and growth and for most muscling traits mapped near ACSL4, with the exception of the QTL for ham traits that mapped proximally, in the vicinity of AR. An analysis performed separately for F2 male animals showed the predominant QTL affecting fat deposition traits (up to 13.6% VF2) near AR and two QTL for muscling traits (up to 9.9% VF2) mapped close to ACSL4. In the F2 female animals, QTL affecting muscling (up to 12.1% VF2) mapped at ACSL4 and SW2456, and QTL for fat deposition (10% VF2) and growth (up to 10.5% VF2) mapped at ACSL4.

Combined analyses of data from quantitative trait loci mapping studies. Chromosome 4 effects on porcine growth and fatness.

For many species several similar QTL mapping populations have been produced and analyzed independently. Joint analysis of such data could be used to increase power to detect QTL and evaluate population differences. In this study, data were collated on almost 3000 pigs from seven different F(2) crosses between Western commercial breeds and either the European wild boar or the Chinese Meishan breed. Genotypes were available for 31 markers on chromosome 4 (on average 8.3 markers per population). Data from three traits common to all populations (birth weight, mean backfat depth at slaughter or end of test, and growth rate from birth to slaughter or end of test) were analyzed for individual populations and jointly. A QTL influencing birth weight was detected in one individual population and in the combined data, with no significant interaction of the QTL effect with population. A QTL affecting backfat that had a significantly greater effect in wild boar than in Meishan crosses was detected. Some evidence for a QTL affecting growth rate was detected in all populations, with no significant differences between populations. This study is the largest F(2) QTL analysis achieved in a livestock species and demonstrates the potential of joint analysis.

Polymorphic AP-1 binding site in bovine CSN1S1 shows quantitative differences in protein binding associated with milk protein expression.

Polymorphisms in 5'-flanking regions of milk protein encoding genes can influence the binding activity of the affected response elements and thus have an impact on the expression of the gene products. However, precise quantitative data concerning the binding properties of such variable response elements have so far not been described. In this study we present the results of a quantitative fluorescent electromobility shift assay comparing the allelic variants of a polymorphic activator protein-1 binding site in the promoter region of the bovine alphas1-casein encoding gene (CSN1S1), which is affected by an A-->G exchange at -175 bp (CSN1S1(-175bp)). A supershift assay using a commercial c-jun antibody was carried out to verify the specificity of protein binding. The gel shift analysis revealed specific and significantly reduced protein binding of oligonucleotides containing the G variant of the CSN1S1(-175bp) binding site. Further investigations comprised genotyping of the variable CSN1S1(-175bp) activator protein-1 element by an NmuCl restriction fragment length polymorphism in 62 cows of the breed Simmental and 80 cows of the breed German Holstein. Single milk proteins from at least 4 milk samples per cow were quantified by alkaline urea polyacrylamide gel electrophoresis. Homozygotes for CSN1S1(-175bp)*G were not observed, and the allele frequencies were 0.19 in Simmental and 0.05 in German Holstein. Carriers of CSN1S1(-175bp)*G showed higher content (%) as well as quantity (g/d) of alphas1-casein than CSN1S1(-175bp)*A homozygotes, independent of breed. We assume that the positive association of the CSN1S1(-175bp)*G variant with CSN1S1 expression is likely to be caused by a reduced affinity of the affected response element to a c-jun-containing CSN1S1 dimer with repressor properties.

Application of polymorphic DNA sequences to differentiate the origin of decomposed bovine meat.

Two bovine meat samples were analysed although the samples were already in a state of advanced decomposition. The isolated DNA was extremely degraded and not suitable for conventional DNA fingerprinting (profiling), thus the polymerase chain reaction (PCR) was applied to screen for a Y-chromosome specific fragment and the loci of the two milk proteins kappa-casein and beta-lactoglobulin. Both samples contained Y-chromosome specific DNA and the kappa-casein genotype AA but they differed at the beta-lactoglobulin locus (BB versus AB). Thus a different origin (carcass) could be verified for the two samples and a subject was exonerated from a case of meat larceny.

Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana.

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite interactions depend to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5x10(4) sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1-2 standard deviations. The study lays the foundation for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite interaction in protozoan infectious disease.

Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR.

A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.

Production and use of bovine DNA libraries: DNA-sequencing.

An important part in the use of genomic DNA libraries is the sequencing of identified clones for detailed information. In this study, methods for DNA sequence analysis were elaborated and employed for the k-casein gene, a bovine milk protein. The results encourage further research.

Effect of misidentification on the estimation of breeding value and heritability in cattle.

For daughter groups of 15 test bulls, controls of paternity were performed by using blood group factors and biochemical polymorphisms. Data of incorrectly assigned daughters influenced the estimation of breeding values, heritabilities and correlations for milk performance traits. Formulae are given that show the effects of variable misidentification rates on estimation of breeding values, selection intensities, heritabilities, and genetic gains. For example, for milk fat yield, the genetic gains drop at a misidentification rate of 15% between 8.7% (for h2 = .5) and 16.9% (for h2 = .2) below values attained without misidentifications. Consequently, decreasing misidentification rates in progeny of test bulls can be used to diminish the progeny size per test bull for constant genetic gain, to achieve more precise ranking of all or distinct test bulls according to their "true" breeding values and(or) to increase the number of test bulls by using the same amount of test inseminations and the same precision of ranking. Actions to reduce misidentification rates in cattle populations are discussed.

Identification of QTL affecting important traits on porcine chromosome 12.

To screen the whole porcine chromosome 12 for QTL affecting economically important traits, ten genetic markers were genotyped in two F2 populations generated from the cross of genetically diverse breeds: European Wild pig and commercial pig breed Pietrain (W x P), and Chinese Meishan and Pietrain (M x P). Fifty-one traits were recorded. A least squares method was used for chromosome-wide screening for QTL. An association analysis between genotypes at the GH locus and traits was also carried out. The least squares analysis did not reveal the presence of genome-wide significant QTL affecting the traits, while the association study showed significant (P < 0.01) associations between GH genotypes and fatness traits in M x P, but not in W x P. F2 pigs carrying the genotype C1A2/C4A2 at the GH locus displayed the thinnest backfat (21.76 mm), while the ones carrying the genotype C2A2/C2A2 had the thickest (31.41 mm).

[Analysis of gene effects on performance characteristics]. / Analyse von Genwirkungen auf Leistungsmerkmale.

In farm animals, associations between individually identified genotypes and the values of performance traits were investigated since more than 30 years. The topic of research was largely determined by the Veterinary Institute of the University of Göttingen. For the experimental analysis of gene loci, for which allelic variants are connected with alterations of trait values, new techniques of DNA diagnostic were of crucial significance. Thereby, two approaches of analysis of quantitative trait loci (QTL) can be distinguished. By considering informative groups of animals, the first approach uses marker loci in order to trace the inheritance of their alleles and thus simultaneously the transfer of specific chromosome sections to individuals of the offspring generation. By this manner, the associations between the marked chromosome regions and trait values are calculated. Results are shown for examples from experiments with milk performance in cattle and with fatting and carcass traits in pigs. In the second approach, genetic effects of trait values are assigned to distinct genes or gene clusters. For this purpose, variants of the gene structure are identified and then analysed for their effects on the formation of specific trait values. As an example of such a functional analysis of single gene positions, the milk protein coding genes in cattle are given. From the data we see that DNA techniques allow a direct access to genotypic information and so far-reaching potential for tracing back effects on trait values to single nucleotide differences. However, such a functional analysis need specific test systems which are able to consider the complex net work between single gene effects and the multifactorially caused values of performance traits. This will be possible by proceedings, which identify the gene effects in vivo and the balancing forces of haplotype combinations in populations. Genetic parameters of such investigations are needed for farm animal populations before variants of genotypes are applied for breeding.

[Gene technology in animal husbandry, new research approaches exemplified by the milk protein gene of cattle]. / Gentechnologie in der Tierzucht, neue Forschungsansätze am Beispiel der Milchprotein-Gene beim Rind.

For investigation of bovine milk protein genes several methods of recombinant DNA techniques are presented. Possible applications of genome research in animal breeding are given including the characterization of structure and function for single genes, gene mapping as well as screening for gene variants in populations. Hence it follows that scientific and practical developments can be expected and will be an influence on future animal production.