Carmen Burgos

Incidence in diverse pig populations of an IGF2 mutation with potential influence on meat quality and quantity: An assay based on real time PCR (RT-PCR)

IGF2, insulin-like growth factor 2, is implicated in myogenesis and lean meat content. A mutation in a single base (A for G substitution) of the gene for IGF2 (position 3072 in intron 3) has been recently described as the cause of a major QTL effect on muscle growth in pigs [Van Laere, A. S, Nguyen, M., Braunschweig, M., Nezer, C., Collete, C., & Moreau, L. et al. (2003). Nature, 425, 832-836]. We describe here a rapid assay based on real time PCR (RT-PCR) to detect this mutation. We have evaluated the incidence of the mutation in commercial pig crosses, in three populations of purebred Iberian or Iberian×Duroc crosses, and in cured meat products and wild boars. The incidence of the mutation varies among these groups. Penetrance of the A mutation is about 80% in the commercial population. Purebred Iberian pigs were all homozygous G/G whereas crosses of Iberian pigs were heterozygous (90%) or homozygous A/A (10%). The implications of this gene for the selection of Iberian pigs are discussed.

Allelic incidence in several pig breeds of a missense variant of pig melanocortin-4 receptor (MC4R) gene associated with carcass and productive traits; its relation to IGF2 genotype

MC4R, melanocortin-4 receptor, is involved in feed intake regulation. A mutation in a single base of MC4R, a G/A substitution in position 1426, has been linked to enhanced backfat thickness, average daily gain and daily feed intake. We present in this work a method to diagnose this mutation using real time PCR (RT-PCR) which allows rapid, cheap and reliable analysis of hundreds of samples in just 2h after DNA extraction. We have used this RT-PCR based assay to study the incidence of the mutation in several pig breeds or crosses (Iberian, Duroc, Pietrain, Large White, Large White×Pietrain) and wild boars. IGF2, insuline like growth factor 2, a gene with well demonstrated effects on carcass composition, of all these animals has also been analyzed and we show, using linkage disequilibrium analysis that both genes are independent. The implications of our results for pig selection toward fatty or lean carcasses are discussed.

The effects of two alleles of IGF2 on fat content in pig carcasses and pork.

The different fat infiltration capabilities of two alleles of IGF2 (G3072A) have been investigated in pigs of a Landrace-Large White×Duroc cross. Paternally inherited G allele carrier pigs show an increased content of adipose rich meat cuts such as the lard or the belly and 4mm larger backfat thickness values. Paternally inherited A carrier pigs on the other hand contain larger muscle tissue rich cuts such as the loin, the ham and the tenderloin and have 0.19 units lower feed conversion index. No substantial differences have been found neither in intramuscular fat content in several muscles nor in meat quality in both pig groups. Hams of paternally inherited G carrier pigs are richer in both subcutaneous adipose tissue (23.1 vs 19.1mm backfat thickness) and intermuscular fat content. The suitability and the economics of using any of the two of both genotypes for cured ham production are discussed.

c.A2456C-substitution in Pck1 changes the enzyme kinetic and functional properties modifying fat distribution in pigs.

Cytosolic phosphoenolpyruvate carboxykinase, PCK1, is one of the main regulatory enzymes of gluconeogenesis and glyceroneogenesis. The substitution of a single amino acid (Met139Leu) in PCK1 as a consequence of a single nucleotide polymorphism (SNP), c.A2456C, is associated in the pig to a negative phenotype characterized by reduced intramuscular fat content, enhanced backfat thickness and lower meat quality. The p.139L enzyme shows reduced kcat values in the glyceroneogenic direction and enhanced ones in the anaplerotic direction. Accordingly, the expression of the p.139L isoform results in about 30% lower glucose and 9% lower lipid production in cell cultures. Moreover, the ability of this isoform to be acetylated is also compromised, what would increase its susceptibility to be degraded in vivo by the ubiquitin-proteasome system. The high frequency of the c.2456C allele in modern pig breeds implies that the benefits of including c.A2456C SNP in selection programs could be considerable.

Allelic frequencies of NR6A1 and VRTN, two genes that affect vertebrae number in diverse pig breeds: A study of the effects of the VRTN insertion on phenotypic traits of a Duroc × Landrace–Large White cross

A SNP (748 C>T) in the NR6A1 gene and an insertion (g.20311_20312ins291) in the VRTN gene have been shown to affect vertebrae number in the pig. The allelic frequencies of both genes were investigated in six western breeds and the effects of the VRTN insertion on some phenotypic traits in a Duroc×Landrace/Large White cross. The NR6A1 c. 748T allele, associated with higher number of vertebrae, appeared to be fixed in most studied breeds except in Iberians. The VRTN insertion (Ins allele) shows ample variability in all studied breeds although the allelic frequency of Ins seems to be larger in breeds with a greater history of genetic selection. Ins is associated with an increase in weight at slaughter, in loin and rib primal cut proportions, and with modified meat quality properties such as cooking loss, intramuscular fat content or yield after curing. We discuss the usefulness of both gene markers for pig selection.

A real time PCR (RT-PCR) alternative assay to detect the T/C mutation in position 1843 of the ryanodine receptor gene

The discovery of the causal mutation of malignant hyperthermia in pigs, a T for C substitution in base 1843 of the ryanodine receptor gene, opened the door to selection procedures based on the analysis of ryanodine receptor genotype based on PCR amplification of the region containing base 1843, subsequent digestion with specific restriction enzymes of the amplified DNA fragment, and electrophoretic analysis of the resulting bands. In this paper, we describe an assay that allows analysis of the three possible genotypes of the ryanodine receptor gene using real time PCR to amplify and detect them in a single step. Results obtained with the RT-PCR assay described in this work match 100% with those obtained using traditional PCR methods. RT-PCR methods are cheaper and faster than traditional ones allowing one to genotype up to 384 samples in a single run.

O-GlcNAcylation mediates the control of cytosolic phosphoenolpyruvate carboxykinase activity via Pgc1α

PGC1α is a coactivator of many transcription factors and cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis. PGC1α interacts with the transcription factor PPARγ to stimulate PCK1 expression and thus de novo glucose synthesis. These proteins are not only important for central energy metabolism but also for supplying intermediates for other metabolic pathways, including lipidogenesis and protein synthesis and might therefore be important factors in the ethiopathogenesis of metabolic disorders like diabetes but also in other pathologies like cancer. Since polymorphisms in these proteins have been related to some phenotypic traits in animals like pigs and PGC1α G482S polymorphism increases fat deposition in humans, we have investigated the molecular basis of such effects focusing on a commonly studied polymorphism in pig Pgc1α, which changes a cysteine at position 430 (WT) of the protein to a serine (C430S). Biochemical analyses show that Pgc1α WT stimulates higher expression of human PCK1 in HEK293T and HepG2 cells. Paradoxically, Pgc1α WT is less stable than Pgc1α p.C430S in HEK293T cells. However, the study of different post-translational modifications shows a higher O-GlcNAcylation level of Pgc1α p.C430S. This higher O-GlcNAcylation level significantly decreases the interaction between Pgc1α and PPARγ demonstrating the importance of post-translational glycosylation of PGC1α in the regulation of PCK1 activity. This, furthermore, could explain at least in part the observed epistatic effects between PGC1α and PCK1 in pigs.