S. Braglia

Molecular approaches in pig breeding to improve meat quality.

This article reviews the advances in molecular genetics that have led to the identification of genes and markers associated with meat quality in pig. The development of a considerable number of annotated livestock genome sequences represents an incredibly rich source of information that can be used to identify candidate genes responsible for complex traits and quantitative trait loci effects. In pig, the huge amount of information emerging from the study of the genome has helped in the acquisition of new knowledge concerning biological systems and it is opening new opportunities for the genetic selection of this specie. Among the new fields of genomics recently developed, functional genomics and proteomics that allow considering many genes and proteins at the same time are very useful tools for a better understanding of the function and regulation of genes, and how these participate in complex networks controlling the phenotypic characteristics of a trait. In particular, global gene expression profiling at the mRNA and protein level can provide a better understanding of gene regulation that underlies biological functions and physiology related to the delivery of a better pig meat quality. Moreover, the possibility to realize an integrated approach of genomics and proteomics with bioinformatics tools is essential to obtain a complete exploitation of the available molecular genetics information. The development of this knowledge will benefit scientists, industry and breeders considering that the efficiency and accuracy of the traditional pig selection schemes will be improved by the implementation of molecular data into breeding programs.

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.

A new approach in association study of single nucleotide polymorphism of genes for carcass and meat quality traits in commercial pigs

Abstract Six batches of four commercial hybrids of heavy pigs, reared for the production of Italian dry-cured hams, were identified for having homogeneous feeding and farm conditions. For a total of 235 pigs, slaughtered in the same slaughterhouse, carcass traits and muscle composition were measured. The pigs were genotyped for single nucleotide polymorphisms (SNPs) of Na+, K+-ATPase subunit alpha 2 (ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide; ATP1A2), cystatin B (CSTB), mitochondrial 2,4-dienoyl-CoA reductase 1 (DECR1), leptin (LEP; 3 SNPs), melanocortin receptor 4 (MC4R), melanocortin receptor 5 (MC5R), sarcolipin (SLN) and titin (TTN) genes. All genes showed biallelic polymorphisms and the alleles were differently distributed between the six batches. Pigs were subsequentely clustered in “lean” and “fat” using either carcass traits (lean percentage, backfat thickness, loin muscle thickness, ham weight and ham cover fat thickness: 100 lean and 135 fat) or meat composition data (dry matter, protein, fat and ash of Biceps femoris and Vastus lateralis and pH after 24 hours: 126 lean and 109 fat). The association of gene polymorphisms with leaness and fatness of pigs was thus investigated using a logistic regression. ATP1A2, LEP (HinfI polymorphism) and MC4R, together with sex and ham weight were, included in the model to screen lean and fat pigs classified according to carcass traits data, yielding a correct classification of 71%. For the lean and fat pigs classified according to muscle composition, sex, CSTB, DECR1, MC5R and LEP (AciI/TaqI polymorphisms) were included in the regression analysis, that yielded a 66% of pigs correctly classified. These preliminary results may indicate that some of the selected candidate genes could be associated to production traits and are worth of further investigations.

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.

A gene and protein expression study on four porcine genes related to intramuscular fat deposition.

Intramuscular fat (IMF) content has a prominent role in meat quality, affecting sensory attributes such as flavour and texture. In the present research, we studied in samples of porcine Semimembranosus muscle four genes related to lipid metabolism and whose gene expressions have been associated to IMF deposition: FASN, SCD, LIPE and LPL. We analysed both mRNA and protein expressions in two groups of Italian Large White pigs divergent for Semimembranosus IMF deposition, with the aim of comparing the levels of four genes and enzymes between the two groups and identifying possible coexpression links. The obtained results suggest a prominent role of LIPE enzyme in IMF hydrolysis, as the samples with low IMF deposition show a significantly higher amount of this lipase. Finally, a poorly known correlation was found between LIPE and FASN enzymes only in female individuals. These results provide new information for the understanding of IMF deposition.

SNPs of MYPN and TTN genes are associated to meat and carcass traits in Italian Large White and Italian Duroc pigs

Myopalladin (MYPN) and titin (TTN) play key roles in skeletal muscle structural organisation and for this reason they may be functional candidates for meat production traits in pigs. We analysed by PCR–RFLP two polymorphisms previously identified and described in the 3′UTR of MYPN and TTN genes in a group of Italian Large White (ILW) and Italian Duroc (ID) pigs with extreme divergent estimated breeding values for some production traits. Significant allele frequency differences were observed for lean cuts (LC) at the MYPN locus in ILW breed and for visible intermuscular fat (VIF) at MYPN and TTN loci in ID breed. Moreover, association analysis between the two genes and some production traits (average daily gain, LC, backfat thickness, ham weight, feed conversion ratio and VIF) was performed in a sample of 409 ILW and 381 ID pigs. On the whole, the results showed significant effects of both genes on some of the considered traits in ID breed, and for TTN gene in ILW animals. These results indicate that both genes can be considered as candidates for meat and carcass traits in ID pigs.

Investigation of SNPs in the ATP1A2, CA3and DECR1 genes mapped to porcine chromosome 4: analysis in groups of pigs divergent for meat production and quality traits

Abstract Three genes (ATPase, Na+/K+ transporting, α 2(+) polypeptide, ATP1A2; carbonic anhydrase III, CA3; 2,4-dienoyl CoA reductase 1, mitochondrial, DECR1), isolated from a porcine skeletal muscle cDNA library and mapped on porcine chromosome 4 (SSC4), were investigated. A new single nucleotide polymorphism (SNP) was identified in the 3’-untranslated region of the CA3 gene and used to genetically map this locus on SSC4 together with the ATP1A2 and DECR1 loci for which SNPs were already reported. Allele frequencies of the three loci were reported for 11 pig breeds (Italian Large White, Italian Landrace, Italian Duroc, Belgian Landrace, Hampshire, Piùtrain, Meishan, Cinta Senese, Casertana, Calabrese and Nero Siciliano). Radiation hybrid mapping of these genes confirmed the linkage mapping results as well as mapping information reported by other authors. Then, the SNPs identified in the ATP1A2, CA3 and DECR1 genes were genotyped in Italian Large White and Italian Duroc animal groups with extreme and divergent estimated breeding value for several production traits. For CA3 significant differences in allele frequencies (P< 0.05) were observed between the extreme groups of pigs for the lean cuts (Italian Large White) and visible intermuscular fat (Italian Duroc) traits. For DECR1, a significant difference in allele frequencies was observed only for the visible intermuscular fat trait. ATP1A2, which maps close to the FAT1 locus, did not show any significant difference. A very high linkage disequilibrium (D’= 0.967; P< 0.0001) was identified between CA3 and DECR1 in the Italian Duroc population. Further investigations are needed to evaluate the effect of CA3 and DECR1 on the considered traits.

SNPs detection in DHPS-WDR83 overlapping genes mapping on porcine chromosome 2 in a QTL region for meat pH

BackgroundThe pH is an important parameter influencing technological quality of pig meat, a trait affected by environmental and genetic factors. Several quantitative trait loci associated to meat pH are described on PigQTL database but only two genes influencing this parameter have been so far detected: Ryanodine receptor 1 and Protein kinase, AMP-activated, gamma 3 non-catalytic subunit. To search for genes influencing meat pH we analyzed genomic regions with quantitative effect on this trait in order to detect SNPs to use for an association study.ResultsThe expressed sequences mapping on porcine chromosomes 1, 2, 3 in regions associated to pork pH were searched in silico to find SNPs. 356 out of 617 detected SNPs were used to genotype Italian Large White pigs and to perform an association analysis with meat pH values recorded in semimembranosus muscle at about 1 hour (pH1) and 24 hours (pHu) post mortem.The results of the analysis showed that 5 markers mapping on chromosomes 1 or 3 were associated with pH1 and 10 markers mapping on chromosomes 1 or 2 were associated with pHu. After False Discovery Rate correction only one SNP mapping on chromosome 2 was confirmed to be associated to pHu. This polymorphism was located in the 3’UTR of two partly overlapping genes, Deoxyhypusine synthase (DHPS) and WD repeat domain 83 (WDR83). The overlapping of the 3’UTRs allows the co-regulation of mRNAs stability by a cis-natural antisense transcript method of regulation. DHPS catalyzes the first step in hypusine formation, a unique amino acid formed by the posttranslational modification of the protein eukaryotic translation initiation factor 5A in a specific lysine residue. WDR83 has an important role in the modulation of a cascade of genes involved in cellular hypoxia defense by intensifying the glycolytic pathway and, theoretically, the meat pH value.ConclusionsThe involvement of the SNP detected in the DHPS/WDR83 genes on meat pH phenotypic variability and their functional role are suggestive of molecular and biological processes related to glycolysis increase during post-mortem phase. This finding, after validation, can be applied to identify new biomarkers to be used to improve pig meat quality.

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.

Skeletal muscle expression analysis of fat metabolism genes in pig

Abstract Fat content and backfat thickness are important polygenic parameters influencing meat quality and carcass traits in pigs. Up to now, there is a lack of knowledge on the expression level of the genes encoding for enzymes involved in fatty acid metabolism in porcine skeletal muscle. In the present study we analysed, by quantitative real time PCR, the expression of three genes, acetyl-CoA carboxylase (ACACA), ATP citrate lyase (ACL) and fatty acid synthase (FASN) in skeletal muscle tissue samples of Italian Large White and Italian Duroc pigs with divergent breeding values for backfat thickness or visible intramuscular fat. Significant differences of the expression level for ACACA gene (P=0.04) and for ACL gene (P=0.02) were observed between the two breeds, comparing the samples selected for backfat thickness trait. The expression analysis of FASN gene in the samples with different genotype at the SNP c.265C>T showed that the TT genotype presented the lowest values in both breeds. The differences observed between breeds should be further considered to investigate the putative involvement of these genes on fat deposition traits.