Genome-wide study on intramuscular fat in Italian Large White pig breed using the PorcineSNP60 BeadChip.

Genome-wide association study results are presented for intramuscular fat in Italian Large White pig breed. A total of 886 individuals were genotyped with PorcineSNP60 BeadChip. After quality control performed with plink software and in R environment, 49 208 markers remained for the association analysis. The genome-wide association studies was conducted using linear mixed model implemented in GenABEL. We detected seven new SNPs of genes till now not found associated to intramuscular fat (IMF). Three markers map in a wide intergenic region rich of QTL linked to fat traits, one map 388 kb upstream the gene SDK1, one map inside PPP3CA gene, one inside SCPEP1 gene and the last is not mapped in the porcine genome yet. Associations here presented indicate a moderate effect of these genes on IMF. In particular, PPP3CA, that is involved in the oxidative metabolism of skeletal muscle, could be considerated as an interesting candidate gene for IMF content in pigs. However, further studies are needed to clarify the role of these genes on the physiological processes involved in IMF regulation. These results may be useful to control this trait that is important in terms of nutritional, technological and organoleptic characteristics of fresh meat and processed products.

Relationship between perilipin genes polymorphisms and growth, carcass and meat quality traits in pigs.

The perilipins (PLIN) belong to a family of structural proteins that play a role regulating intracellular lipid storage and mobilization. Here, PLIN1 and PLIN2 have been evaluated as candidate genes for growth, carcass and meat quality traits in pigs. A sample of 607 Duroc pigs were genotyped for two single-nucleotide polymorphisms, one in intron 2 of the PLIN1 gene (JN860199:g.173G>A) and the other at the 3' untranslated region of the PLIN2 gene (GU461317:g.98G>A). Using a Bayesian approach, we have been able to find evidence of additive, dominant and epistatic associations of the PLIN1 and PLIN2 polymorphisms with early growth rate and carcass length. However, the major effects were produced by the dominant A allele at the PLIN2 polymorphism, which also affected the carcass lean weight. Thus, pigs carrying an additional copy of the A allele at the g.98G>A PLIN2 polymorphism had a probability of at least 98% of producing carcasses with heavier lean weight (+0.41 kg) and ham weight (+0.10 kg). The results obtained indicate that the PLIN2 polymorphism could be a useful marker for lean growth. In particular, it may help to reduce the undesired negative correlated response in lean weight to selection for increased intramuscular fat content, a common scenario in some Duroc lines involved in the production of high quality pork products.

Polymorphisms of beta-lactoglobulin promoter region in three Sicilian goat breeds.

Several beta-lactoglobulin (BLG) polymorphisms have been described within the proximal promoter region and coding region of the caprine gene, although no genetic variants affecting the protein amino acid composition and/or expression level have been characterized so far. Binding sites for several transcription factors (TFs) are present in the BLG promoter region. The aims of this work were to sequence the full-length promoter region of three Sicilian goat breeds in order to identify polymorphisms, analyze the identified haplotypes, search for differences between breeds for the presence of polymorphisms in this gene region, search for putative TFs binding sites, and check if polymorphisms lay within the identified TFs binding sites. The promoter region of BLG gene in Sicilian goat breeds showed high level of polymorphism due to the presence of 36 single nucleotide polymorphisms (SNPs). Association between polymorphic sites was computed within the whole sample analyzed and 18 haplotypes were inferred. Binding sites for three milk protein binding factors (MPBFs) and four nuclear factor-I (NF-I) were found within BLG promoter region based on the ovine sequence. The identification of some SNPs within TFs binding sites allowed hypothesizing the loss of TFs. Further studies are in progress to evaluate the effect of these mutations on binding affinity of TFs, the functional interaction of the TFs with the goat BLG promoter, and the relationship of the polymorphisms with BLG gene expression and milk production and composition.

Analysis of MC4R polymorphism in Italian Large White and Italian Duroc pigs: association with carcass traits.

The melanocortin-4 receptor (MC4R) gene codes for a G protein transmembrane receptor playing an important role in energy homeostasis control. In pig a single nucleotide polymorphism c.1426G>A has been identified and associated to average daily gain, feed intake and fatness traits but a lack of agreement on the effects of the gene on carcass traits in different breeds comes out from many studies. In the present study the c.1426G>A polymorphism is analysed in two Italian pig breeds, Large White and Duroc to study the association of the MC4R gene with some carcass traits. The results show that the c.1426G>A polymorphism affects daily gain, feed conversion ratio and ham weight in both breeds, lean cuts in the Italian Duroc and backfat thickness in the Italian Large White. The presence of MC4R mRNA transcript in different porcine tissues was analysed.

Expression profiling of functional genes in prenatal skeletal muscle tissue in Duroc and Pietrain pigs.

In livestock, skeletal muscle is a tissue of major economic importance for meat production and muscle mass is largely determined during the prenatal period by the number and the size of muscle fibres. The understanding of gene expression changes during prenatal pig muscle development is still limited. In this study, genes identified as differentially expressed in a previous microarray research and chosen for the function of the coded protein as putative candidate involved in myogenesis were considered to analyse their expression profile during foetal growth of Duroc and Pietrain pigs. The eleven genes were considered by real-time PCR for a time-course evaluation of the transcription level at six stages of prenatal longissimus dorsi development. The results suggest that the most relevant variations in mRNA levels of the analysed genes seem to follow temporal waves of gene expression. Significant changes of transcription were observed at 21-35 and 63-91 days, the two main phases of skeletal muscle development. During the early phases of Pietrain embryos' development, 10 of the 11 genes showed an induction. In Duroc embryos, a second phase of gene up-regulation can be identified in the phase 63-77 days. These results provide new data on developmental changes of expression profile of 11 genes involved in different functional pathways related to prenatal myogenic processes in Duroc and Pietrain pigs.

Associations of functional candidate genes derived from gene-expression profiles of prenatal porcine muscle tissue with meat quality and muscle deposition.

Ten genes (ANK1, bR10D1, CA3, EPOR, HMGA2, MYPN, NME1, PDGFRA, ERC1, TTN), whose candidacy for meat-quality and carcass traits arises from their differential expression in prenatal muscle development, were examined for association in 1700 performance-tested fattening pigs of commercial purebred and crossbred herds of Duroc, Pietrain, Pietrain x (Landrace x Large White), Duroc x (Landrace x Large White) as well as in an experimental F(2) population based on a reciprocal cross of Duroc and Pietrain. Comparative sequencing revealed polymorphic sites segregating across commercial breeds. Genetic mapping results corresponded to pre-existing assignments to porcine chromosomes or current human-porcine comparative maps. Nine of these genes showed association with meat-quality and carcass traits at a nominal P-value of < or = 0.05; PDGFRA revealed no association reaching the P < or = 0.05 threshold. In particular, HMGA2, CA3, EPOR, NME1 and TTN were associated with meat colour, pH and conductivity of loin 24 h postmortem; CA3 and MYPN exhibited association with ham weight and lean content (FOM) respectively at P-values of < 0.003 that correspond to false discovery rates of < 0.05. However, none of the genes showed significant associations for a particular trait across all populations. The study revealed statistical-genetic evidence for association of the functional candidate genes with traits related to meat quality and muscle deposition. The polymorphisms detected are not likely causal, but markers were identified that are in linkage disequilibrium with causal genetic variation within particular populations.

Comparison of genetic models for analysing the effects of a PvuII polymorphism in the oestrogen receptor 1 (ESR1) gene on prolificacy in an Iberian x Meishan pig population.

The effect of a previously reported PvuII polymorphism in oestrogen receptor 1 (ESR1) was analysed in an F(2) population of Iberian x Meishan pigs. We tested three hypotheses: (1) that a causal mutation was fixed in the parental populations, (2) that a causal mutation existed that was in complete linkage disequilibrium with the alleles of the PvuII polymorphism and (3) that a causal mutation existed in linkage disequilibrium within each parental population. The third model was the most plausible based on the available data. ESR1 alleles displayed different patterns of linkage disequilibrium with the causal mutation in each of the parental populations and the PvuII polymorphism was clearly not the causal mutation. As a consequence, the use of the ESR1 mutation for selection must be evaluated for a particular pig population before it is applied.

Mapping, identification of polymorphisms and analysis of allele frequencies in the porcine skeletal muscle myopalladin and titin genes.

Genes coding for sarcomeric proteins may play a key role in muscle mass accretion and meat production. Screening a skeletal muscle cDNA library we isolated two partial sequences coding for the sarcomeric myopalladin and titin genes. In the present work we identified three SNPs in the 3' untranslated region, two at the myopalladin locus and one at the titin locus. Myopalladin was mapped on porcine chromosome (SSC) 14 using a somatic cell hybrid panel, a radiation hybrid panel and by linkage mapping. The linkage mapping of titin confirmed the position on SSC15. Then we analysed the allelic distribution of the alleles at both loci in six different porcine breeds. The analysis of the allele frequencies for these two loci in extremely divergent groups of pigs selected according to lean cuts (LC) and average daily gain (ADG) approached the significance level for myopalladin and LC trait. Further studies are needed to test the presence of a putative effect of myopalladin on lean meat content.

Isolation of porcine expressed sequence tags for the construction of a first genomic transcript map of the skeletal muscle in pig.

To identify genes with effects on meat quality and production traits we developed an adult porcine skeletal muscle cDNA library. After pre-screening this library with seven genes highly expressed in skeletal muscle, 385 non-hybridizing clones were sequenced from both ends to yield 510 expressed sequence tags (ESTs). Together with those ESTs previously generated from this library, we have produced 701 porcine skeletal muscle ESTs. These ESTs were grouped into 306 different cDNA species and compared with the human skeletal muscle transcriptional profiles obtained from different databases. Furthermore we mapped 107 of these cDNAs using a somatic cell hybrid panel with genes mapping over all the autosomes (except on chromosome 11) and on chromosome X. The mapping of these cDNAs contributed to the construction of a first genomic transcript map of the skeletal muscle tissue in pig.

Detection of rifampin resistance in Mycobacterium tuberculosis by double gradient-denaturing gradient gel electrophoresis.

We applied double gradient-denaturing gradient gel electrophoresis (DG-DGGE) for the rapid detection of rifampin (RMP) resistance from rpoB PCR products of Mycobacterium tuberculosis isolates and clinical samples. The results of this method were fully concordant with those of DNA sequencing and susceptibility testing analyses. DG-DGGE is a valid alternative to the other methods of detecting mutations for predicting RMP resistance.

Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system.

Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.