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The imprints of domestication and breed development on the genomes of livestock likely differ from those of companion animals. A deep draft sequence assembly of shotgun reads from a single Hereford female and comparative sequences sampled from six additional breeds were used to develop probes to interrogate 37,470 single-nucleotide polymorphisms (SNPs) in 497 cattle from 19 geographically and biologically diverse breeds. These data show that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population, possibly due to bottlenecks associated with domestication, selection, and breed formation. Domestication and artificial selection appear to have left detectable signatures of selection within the cattle genome, yet the current levels of diversity within breeds are at least as great as exists within humans.

Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity. The porcine Mxl genes corresponding to wild type, the 3-bp deletion mutant, and the 11-bp deletion mutant were cloned and expressed in NIH3T3 cells, and the antiviral activity for influenza virus was assayed. Virus yield was observed to be 10-100-fold greater with the 11-bp deletion allele than that for wild type and the 3-bp deletion alleles. The results suggest that the 11-bp deletion type is lacking antiviral activity able to contribute to the interference of influenza virus replication.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological actions. Type I PACAP receptors (PACAPR) are specific for PACAP, whereas type II and III PACAPRs are less restricted. To localize and analyse the variation of this gene, a 559-bp long intronic fragment of the porcine PACAPR gene was amplified by polymerase chain reaction and sequenced in samples from five different pig breeds. One single nucleotide polymorphism was identified and its allele frequency was determined in all five breeds. Linkage analysis in a Berkshire x Yorkshire reference family placed the PACAPR gene on chromosome 18, between SW787 and S0062 (SW787- 8.1 cM -PACAPR- 3.0 cM -S0062). Radiation hybrid mapping confirmed that the PACAPR gene was linked to SW1682 on chromosome 18 (28.8 cR(3000); LOD = 10.4).

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.

A study in 192 entire male pigs examined the effects of breed, diet and muscle on growth, fatness, sensory traits and fatty acid composition. There were four breeds: two modern breeds, Duroc and Large White and two traditional breeds, Berkshire and Tamworth. The diets differed in energy:protein ratio, being conventional (C) and low protein (LP) diets, respectively. Muscles investigated were the `white' longissimus dorsi (LD) and the `red' psoas major (PS). Breed influenced growth rate and fatness, the modern breeds being faster-growing with leaner carcasses. However, the concentrations of neutral lipid fatty acids and marbling fat (neutral lipid+phosopholipid fatty acids) were higher in Berkshire and Duroc, in both LD and PS. Relationships between marbling fat and P2 fat thickness showed clear breed effects, with Duroc having high marbling fat at low P2 and Tamworth low marbling fat at high P2. Breed effects on sensory scores given by the trained taste panel to griddled LD and PS steaks were relatively small. Breed affected the fatty acid composition of intramuscular neutral lipid, with high % values for the saturated fatty acids, 14:0 and 16:0 in Berkshire and Tamworth (fat carcasses) and high values for polyunsaturated fatty acids in Duroc and Large White (lean carcasses). Duroc had particularly high concentrations of the long-chain polyunsaturated fatty acids, 20:5n-3 and 22:6n-3 in phospholipid of both muscles. Diet influenced growth rate and fatness, the LP diet slowing growth and producing fatter meat, more so in the two modern breeds, and particularly in intramuscular rather than subcutaneous fat. This diet produced more tender and juicy meat, although pork flavour and flavour liking were reduced. The PS muscle had higher tenderness, juiciness, pork flavour, flavour liking and overall liking scores than LD. The concentration of phospholipid fatty acids was higher in PS than LD but neutral lipid fatty acid content and marbling fat were higher in LD.

Peroxisome proliferator-activated receptor gamma (PPARG) is a key transcription factor that controls adipocyte differentiation and fat deposition in mammals. The primary goal of this study was to investigate PPARG as a candidate gene for meat quality and carcass traits in swine. Part of the PPARG promoter, along with the most 5'-proximal exon of the gene, was amplified by PCR and subsequently screened for polymorphisms by sequencing. A Met59Val substitution was detected in the porcine PPARG gene along with four polymorphisms in the promoter region of the adipose-specific PPARG2. Three of these polymorphisms were chosen for genotyping and tested for association with meat quality, carcass and growth traits, according to the candidate gene approach. More than 1500 animals from different lines and populations were used in the study with records for meat quality and carcass traits. No convincing associations were found between the traits investigated and the PPARG genotypes. It does not appear that variation at the PPARG locus is affecting meat quality, carcass or growth traits in the pig populations studied.

In commercial livestock populations, QTL detection methods often use existing half-sib family structures and ignore additional relationships within and between families. We reanalyzed the data from a large QTL confirmation experiment with 10 pig lines and 10 chromosome regions using identity-by-descent (IBD) scores and variance component analyses. The IBD scores were obtained using a Monte Carlo Markov Chain method, as implemented in the LOKI software, and were used to model a putative QTL in a mixed animal model. The analyses revealed 61 QTL at a nominal 5% level (out of 650 tests). Twenty-seven QTL mapped to areas where QTL have been reported, and eight of these exceeded the threshold to claim confirmed linkage (P < 0.01). Forty-two of the putative QTL were detected previously using half-sib analyses, whereas 46 QTL previously identified by half-sib analyses could not be confirmed using the variance component approach. Some of the differences could be traced back to the underlying assumptions between the two methods. Using a deterministic approach to estimate IBD scores on a subset of the data gave very similar results to LOKI. We have demonstrated the feasibility of applying variance component QTL analysis to a large amount of data, equivalent to a genome scan. In many situations, the deterministic IBD approach offers a fast alternative to LOKI.

Population-wide associations between loci due to linkage disequilibrium can be used to map quantitative trait loci (QTL) with high resolution. However, spurious associations between markers and QTL can also arise as a consequence of population stratification. Statistical methods that cannot differentiate between loci associations due to linkage disequilibria from those caused in other ways can render false-positive results. The transmission-disequilibrium test (TDT) is a robust test for detecting QTL. The TDT exploits within-family associations that are not affected by population stratification. However, some TDTs are formulated in a rigid form, with reduced potential applications. In this study we generalize TDT using mixed linear models to allow greater statistical flexibility. Allelic effects are estimated with two independent parameters: one exploiting the robust within-family information and the other the potentially biased between-family information. A significant difference between these two parameters can be used as evidence for spurious association. This methodology was then used to test the effects of the fourth melanocortin receptor (MC4R) on production traits in the pig. The new analyses supported the previously reported results; i.e., the studied polymorphism is either causal or in very strong linkage disequilibrium with the causal mutation, and provided no evidence for spurious association.

We have constructed a radiation hybrid (RH) map of the porcine genome using an RH panel generated by an irradiation dose of 5000-rad (Sus scrofa radiation hybrid map, SSRH map). Normal porcine aortic endothelial cells were irradiated and fused with a thymidine kinase-deficient mouse cell line, L-M (TK-). A total of 110 cell lines were selected and used for further analysis. Among 1091 microsatellite (MS) markers selected for mapping, 842 markers (77%) could be typed on the panel. The framework map comprised 342 MS markers and an additional 247 MS markers were then added to generate the whole-genome map. The average retention frequency for the data set was 30.6%. The total map length was 5596.2 centiRay (cR). Using an estimated physical length of 2718 Mbp, the average ratio between cR and physical distance over the porcine genome was estimated to be 0.49 Mb/cR.

Porcine skeletal muscle fibres were molecularly classified, using in situ hybridisation and immunocytochemistry, into four types, according to the isoform of myosin heavy chain (MyHC) that was present in each fibre (MyHC slow/I, MyHC 2a, MyHC 2x and MyHC 2b). The relationship between MyHC fibre types and meat quality traits between two phenotypically divergent muscles [longissimus dorsi (LD) and psoas], and between the same muscles of different breeds (traditional Berkshire and Tamworth, and modern Duroc-based and Large White-based) were examined. We found that the greater abundance of fast oxidative-glycolytic MyHC 2a and 2x fibres in the psoas was associated with superior meat quality traits, and that the greater presence of fast glycolytic MyHC 2b fibres in the LD could account for less favourable quality traits, both in terms of pH, drip loss, grain, colour, yield force and work done. Although significant correlations were found between specific fibre types and quality traits, within either the psoas or LD muscle of some breeds, no consistent correlation was found across both muscles and all breeds. This finding was in line with the view that a given fibre type could have considerable differences in phenotype between breeds, and between muscles. The observed inverse compositional and functional-meat quality relationship between MyHC 2b and 2x fibres, and MyHC 2b and 2a fibres could form a basis of fibre type manipulation to improve meat quality.

Progress in animal nutrition, reproduction, quantitative genetics, and the development of molecular genetics, proteomics, and functional genomics open new perspectives for the meat sector. The most promising developments include a wider utilisation of molecular markers, the possibilities of semen sexing and the targeted use of nutrition to modify the composition of meat. The increased use of biotechnology will have a considerable impact on the economics of production of meat and further processed products. New technologies will increase the possibilities for product differentiation and improve homogeneity of live animals. The consumer and society in general will influence the direction of these developments. This review will focus on the long-term impact of new technologies for the meat production chain.

Thirteen pairs of primers were designed for the entire porcine MSTN gene to enable PCR amplification for the detection of single nucleotide polymorphisms (SNPs) by a PCR-SSCP approach. Altogether 96.5% (1089/1128) of the encoding regions and 971 bp of the non-coding regions were screened. A total of three polymorphisms were identified with PCR-SSCP. They were located in the promoter, intron one and exon three regions of the gene. These polymorphisms were then confirmed to be point mutations (T --> A transversion, G --> A transition and C --> T transition respectively) by sequencing. Allele frequencies were determined for all three SNPs in several different pig breed populations. The polymorphisms were found to be rare in Western breeds, but much more common in Chinese breeds. Whether they have any relationship with the marked difference in lean meat mass between Western and Chinese breeds requires further study.

Several quantitative trait loci (QTL) affecting muscle glycogen content and related traits were mapped to pig chromosome 15 using a three-generation intercross between Berkshire x Yorkshire pigs. On the basis of the QTL location the PRKAG3 (protein kinase, AMP-activated, gamma(3)-subunit) gene was considered to be a good candidate for the observed effects. Differences in the PRKAG3 gene sequences of the founder animals of the intercross were analyzed. The RN(-) mutation previously reported was not present in the cross but three missense substitutions and a polymorphic short interspersed element (SINE) were identified. To confirm the hypothesis that at least one of these mutations was associated with differences in meat quality, >1800 animals from several unrelated commercial lines were genotyped for the candidate substitutions and an association study was performed. The results demonstrate the presence of new economically important alleles of the PRKAG3 gene affecting the glycogen content in the muscle and the resulting meat quality. Haplotype analysis was shown to resolve the effects of PRKAG3 more clearly than analysis of individual polymorphisms. Because of their prevalence in the more common commercial breeds, the potential implications for the pig industry and consumers are considerably greater than the original discovery of the RN(-) mutation. Furthermore, these results illustrate that additional alleles of genes involved in major mutations may play a significant role in quantitative trait variation.

Black spotting on a red or white background in pigs is determined by the E(P) allele at the MC1R/Extension locus. A previous comparison of partial MC1R sequences revealed that E(P) shares a missense mutation (D121N) with the E(D2) allele for dominant black color. Sequence analysis of the entire coding region now reveals a second mutation in the form of a 2-bp insertion at codon 23 (nt67insCC). This mutation expands a tract of six C nucleotides to eight and introduces a premature stop codon at position 56. This frameshift mutation is expected to cause a recessive red color, which was in fact observed in some breeds with the E(P) allele present (Tamworth and Hereford). RT-PCR analyses were conducted using skin samples taken from both spotted and background areas of spotted pigs. The background red area had transcript only from the mutant nt67insCC MC1R allele, whereas the black spot also contained a transcript without the 2-bp insertion. This indicates that black spots are due to somatic reversion events that restore the frame and MC1R function. The phenotypic expression of the E(P) allele is highly variable and the associated coat color ranges from red, red with black spots, white with black spots, to almost completely solid black. In several breeds of pigs the phenotypic manifestation of this allele has been modified by selection for or against black spots.

Our knowledge of the genetic factors affecting obesity is increasing, but information about the individual gene effects remains limited in humans as well as in animal models. The melanocortin-4 receptor gene (MC4R) has been implicated in the regulation of feeding behavior and body weight in humans and mice. We have studied MC4R as a candidate gene for the control of economically important growth and performance traits in the pig. A missense mutation was identified in a region highly conserved among melanocortin receptor (MCR) genes. To determine whether there was an association of this MC4R polymorphism with phenotypic variation, we tested the mutation in a large number of individual animals from several different pig lines. Analyses of growth and performance test records showed significant associations of MC4R genotypes with backfat and growth rate in a number of lines as well as feed intake overall. It is probable that the variant amino acid residue of the MC4R mutation (or a closely linked mutation) causes a significant change of the MC4R function. These results support the functional significance of a pig MC4R missense mutation and suggest that comparative genomics based on model species may be equally important for application to farm animals as they are for human medicine.

A white belt is a common coat color phenotype in pigs and is determined by a dominant allele (Be). Here we present the result of a genome scan performed using a Hampshire (Belt)/Pietrain (non-Belt) backcross segregating for the white belt trait. We demonstrate that Belt maps to the centromeric region of pig Chromosome (Chr) 8 harboring the Dominant white (I/KIT) locus. Complete cosegregation between Belt and a single nucleotide polymorphism in the KIT gene was observed. Another potential candidate gene, the endothelin receptor type A gene (EDNRA), was excluded as it was assigned to a different region (SSC8q21) by FISH analysis. We argue that Belt is a regulatory KIT mutation on the basis of comparative data on mouse KIT mutants and our previous sequence analysis of the KIT coding sequence from a Hampshire pig. Quantitative PCR analysis revealed that Belt is not associated with a KIT duplication, as is the case for the Patch and Dominant white alleles. Thus, Belt is a fourth allele at the Dominant white locus, and we suggest that it is denoted I(Be).

We compare two strategies for ELISA detection of restriction site polymorphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1(hal)). In both procedures, target DNA is amplified by PCR with one primer that is 5' biotinylated and a second primer that is 5' fluoresceinylated. PCR products are captured in duplicate wells on a streptavidin-coated, 96-well plate. The duplicates may be treated in two ways. In a single restriction enzyme assay, one duplicate is exposed to a restriction enzyme that cuts one allele specifically, and the second duplicate is exposed to no restriction enzyme. In a dual restriction enzyme assay, the second replicate is exposed to a second restriction enzyme that cuts the alternate allele specifically. Thereafter, the two procedures are similar; anti-fluorescein antibodies conjugated to peroxidase are allowed to bind to the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in the two wells is used to classify subjects by genotype. When the dual restriction enzyme assay is run, three genotype groups are easily distinguishable. When the single restriction enzyme assay is run, heterozygotes generate values that may overlap with those of the homozygotes that are not cut by the restriction enzyme. Dual restriction enzyme assays are more accurate than single restriction enzyme assays; however, single restriction enzyme assays are sufficient for identifying pigs that carry RYR1(hal).

Identification of individual major genes affecting quantitative traits in livestock species has been limited to date. By using a candidate gene approach and a divergent breed cross involving the Chinese Meishan pig, we have shown that a specific allele of the estrogen receptor (ER) locus is associated with increased litter size. Female pigs from synthetic lines with a 50% Meishan background that were homozygous for this beneficial allele produced 2.3 more pigs in first parities and 1.5 more pigs averaged over all parities than females from the same synthetic lines and homozygous for the undesirable allele. This beneficial ER allele was also found in pigs with Large White breed ancestory. Analysis of females with Large White breed background showed an advantage for females homozygous for the beneficial allele as compared to females homozygous for the other allele of more than 1 total pig born. Analyses of growth performance test records detected no significant unfavorable associations of the beneficial allele with growth and developmental traits. Mapping of the ER gene demonstrated that the closest known genes or markers were 3 centimorgans from ER. To our knowledge, one of these, superoxide dismutase gene (SOD2), was mapped for the first time in the pig. Analysis of ER and these linked markers indicated that ER is the best predictor of litter size differences. Introgression of the beneficial allele into commercial pig breeding lines, in which the allele was not present, and marker-assisted selection for the beneficial allele in lines with Meishan and Large White background have begun.