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β-lactoglobulin (β-lg) is the major whey protein in milk of ruminants and several non ruminant species, but it is lacking in rodents, lagomorphs and human. The aim of this work was sequencing the promoter region of β-lg gene in Sicilian sheep and goat dairy breeds, in order to identify polymorphisms. In these species, the promoter region was aligned using the sequences available on database. A common set of primers was designed to amplify and sequence a fragment of approximately 2.1 kb. Within this region, 12 single nucleotide polymorphisms (SNPs) were identified in goat breeds: 7 out of them were not yet reported in literature. Moreover, in sheep breeds 15 SNPs were newly identified. Using TRANSFAC database, binding sites for transcription factors (TFs) were found within the β-lg promoter region, in sheep and goat. Multiple binding sites to MPBFs, NF-1, C/EBP, and Sp1 are present within conserved regions, both in sheep and goat. Some SNPs found in the promoter region were detected within TFs sites in both species. Further studies are required to investigate the effect of these point mutations on binding affinity of TFs, their relationship with β-lg gene expression and milk production.

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 × (Landrace × Large White), Duroc × (Landrace × Large White) as well as in an experimental F2 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 ¿ 0.05; PDGFRA revealed no association reaching the P ¿ 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

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.

Six batches of four commercial hybrids of heavy pigs, reared for the production of Italian dry-cured hams, were identifiedfor 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), cystatinB (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 polymorphismsand 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 hamcover fat thickness: 100 lean and 135 fat) or meat composition data (dry matter, protein, fat and ash of Biceps femorisand Vastus lateralis and pH after 24 hours: 126 lean and 109 fat). The association of gene polymorphisms with leanessand 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 carcasstraits 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 a66% of pigs correctly classified.These preliminary results may indicate that some of the selected candidate genes could be associated to production traitsand are worth of further investigations.

Fat deposition is a crucial aspect of pig meat quality as fat content influences both organoleptic and nutritive characteristics of fresh meat, meat products and consumer acceptance. Among genes controlling fat metabolism, the gene encoding fatty acid synthase (FASN) was proposed as a candidate controlling body fat deposition as it is a central enzyme in lipogenesis. The main function of FASN enzyme is the catalysis of the biochemical process that induces the synthesis of palmitate from acetyl-CoA and malonyl-CoA, in the presence of NADPH. This work aims to study variability in the expression level of FASN gene mapped on SSc12 where different QTLs for fat composition and marbling were discovered. In particular, we analysed the SNP T265C for FASN gene identified by Munoz et al. (2003). The association study conducted on 237 Italian Large White (ILW) sib tested pigs to determine whether this polymorphism affected meat productive traits did not allow finding any associations between the SNP and the reported traits. Regarding the Italian Duroc (ID) breed, no association study was performed because in this breed T allele was very rare. Differential expression between breeds of the target gene in semimembranosus muscle and in backfat tissue was evident comparing FASN transcription level between ID and ILW pigs. In particular, ID pigs have a higher expression level of the gene in skeletal muscle than ILW (P=0.01). In backfat tissue the Italian Large White samples showed higher gene expression level than Italian Duroc pigs with a tendency to significant difference (P=0.08). If further analyses will confirm this results on a large sample, in ID breed the transcriptional level of FASN gene could be considered as marker of fat deposition.

Applicazione della real time PCR allo studio dell’espressione genica nel tessuto muscolare scheletrico di suino durante lo sviluppo embrionale nelle razze Duroc e Pietrain. La PCR quantitativa “real time”, è stata utilizzata per analizzare i livelli d’espressione di geni muscolari di suino durante lo sviluppo embrionale. A tale scopo è stato utilizzato il LightCycler™system per quantificare il numero di copie di mRNA di geni del tessuto muscolare scheletrico e confrontarne il pattern di espressione nelle razze suine Duroc e Pietrain, molto diverse per caratteristiche produttive e per la qualità della carne e della carcassa. Il livello d’espressione di questi geni è stato monitorato con quantificazione relativa, basata sul rapporto tra il numero copie cDNA/µl di ciascun gene target rispetto al gene di riferimento β2-microglobulina (B2M).

Fat deposition is a crucial aspect of pig meat quality: a suitable marbling and the right balance between lean meat and fat in carcass are both essential features to produce high quality dry cured hams. Moreover fat content influences both organoleptic and nutritive characteristics of meat products and consumer acceptance. Differences among breeds in intramuscular fat content and backfat thickness have been reported but the molecular events underlying the different aptitude to fat deposition are poorly known. The analysis of the expression profile of lipogenic genes in muscle and fat tissues in different pig breeds can contribute to a better knowledge of fat deposition control in pig meat and can provide new insights into carcass adiposity. The objective of this study is to investigate the expression level of the key adipogenic genes ATP citrate lyase and Fatty acid synthase in Italian Duroc and Italian Large White pigs with different fat deposition capacities. The obtained results suggest a tissue-specific mechanisms regulating gene transcription of FASN and ACL in the analysed samples. In adipose tissue, differential expression between the analysed breeds was observed and ILW pigs showed the highest FASN transcriptional level. In skeletal muscle tissue the greatest mRNA abundance of both lipogenic genes in ID pigs could be consistent with the high intermuscular fat deposition aptitude of this breed.

UDP-glucose pyrophosphorylase 2 (UGP2) is a direct precursor of glycogen in liver and muscle tissue catalyzing the first step of glycogen biosinthesys by transferring a glucose moiety from glucose-1-phosphate to UTP and forming UDP-glucose. For its important function, this gene could have an effect on the control of glycogen content in skeletal muscle. In humans two main isoforms are produced by alternative splicing of the gene in the 5’UTR and other splice variants are obtained by an alternative utilization of small exons, as illustrated in GeneCards database. In pigs only the two main splice variants are reported in UniGene. No data about the expression level are described neither in humans nor in pigs for specific variants in any tissue. The aim of the present work was to investigate the presence of UGP2 splice variants in the pig skeletal muscle transcriptome and to study the expression level of the detected isoforms. Primer pairs were designed on porcine sequence NM_213980 homologous to human splice variant 1 and BW982424 homologous to human splice variant 2 to amplify the 5’UTR regions specific for each isoform. Moreover, the sequence of a new splice variant, found in the porcine EST BF075430 by aligning the ESTs available in GenBank for this gene and not present in human ESTs, was considered designing an additional primer pair. Finally, a fourth primer pair was obtained within the coding region of the gene, present in all splice variants. The four amplicons were sequenced using genomic DNA and/or cDNA from porcine skeletal muscle in order to confirm the amplification of the correct variant. The expression level of the different UGP2 transcripts was investigated by quantitative Real-Time PCR in sixteen porcine Semimembranosus skeletal muscle samples of Italian Large White pigs using the same primer pairs designed for the sequence analysis. The samples were chosen as having high (8 samples) or low (8 samples) glycolitic potential values. The detected level of expression of each UGP2 products was normalised using the geometric means of the expression level of two stably expressed reference genes (Beta 2 microglobulin, B2M and RNA polymerase II largest subunit, POLR2A). The analysis showed the expression of all four transcripts of UGP2 in porcine skeletal muscle. The results obtained in the two groups of samples divergent for the glycolitic potential showed no expression differences. We analysed the expression level of each variant pooling the results of the 16 samples and the most expressed variants was the one corresponding to human splice variant 1. Its level of expression was significant different compared to the new detected variant (P

The pH of pig meat is an important parameter involved in the assessment of the quality of fresh and seasoned meat products. This trait is is under genetic control and is also strongly influenced by environmental factors, mainly the pre-slaughter events. Nevertheless the genetic effects are very important; only two major genes affecting this parameter, RYR1 and PRKAG3, have been identified so far. Several researches have reported QTL regions in at least 10 porcine chromosomes influencing meat pH and some of them have been confirmed by more Authors. With the aim to contribute to the fine mapping of these regions and to locate the genes responsible of the effect of the QTLs we chose three of the most relevant QTL regions so far detected, in order to find SNPs in silico in transcribed sequences. The selected QTL regions were: SSC1 (60-80 cM), SSC2 (55-66 cM), SSC3 (42-60 cM). We then localised more than 2000 porcine Unigene clusters of genes/ESTs in the selected regions comparing the homologous part of pig and human chromosomes using the NCBI map viewer tool. We filtered the identified clusters selecting only those with a minimum of eight ESTs or mRNAs then discarding the clusters without at least one mRNA. On the whole we considered 634 clusters and the sequences corresponding to each one were aligned using BLAST in order to find polymorphisms. We marked as putative SNP a mutation detected in at least three sequences to avoid considering sequencing errors and also to exclude SNPs with a very low frequency of the rarest allele. A first use of this strategy carried out on 264 clusters located in the three analysed chromosomes, allowed us to find 159 putative SNPs with an percentage of SNP detected of 60.2. For some of the SNPs detected in silico primer pairs were designed in order to confirm the presence of the polymorphisms. The proposed approach will be applied to detect several hundreds SNPs in large regions spanning 10-20 cM each were QTLs for meat pH were reported. Moreover, the detection of SNPs within transcribed sequences will be useful for further researches aimed to fine maps the considered QTL in order to narrow the regions where the genes responsible to the QTL effect are located.

Lipogenesis is a complex process mainly influenced by genes coding for enzymes involved in de novo fatty acids synthesis. Changes in mRNA abundance of these genes have been associated to obesity related traits but the genetic regulation of fat deposition is not completely known. The variability at DNA and mRNA level of FASN (Fatty Acid Sinthase), ACL (ATP Citrate Lyase) and ACACA (Acetyl CoA Carboxylase) genes, involved in key steps of fatty acids metabolism, was investigated in porcine skeletal muscle and fat tissues. We genotyped Italian Duroc (IDU) and Italian Large White (ILW) pigs both for the SNP T265C of FASN gene (Munoz et al 2003) and the SNP T2956C of ACL gene that we newly identified by in silico analisys and sequencing. We localized ACL gene on SSC12 (RH panel, LOD: 11.49 with SW943 marker) where FASN and ACACA genes map and where QTLs for fat related traits were detected. The mRNA level of the three genes was determined in skeletal muscle and fat tissues samples of ILW and IDU pigs. Differential expression between breeds (FASN P=0.057; ACL P=0.021; ACACA P=0.041) only in skeletal muscle tissue was observed and IDU pigs showed the higher level. Studies are in progress to investigate on relationship between DNA variation and gene expression. The transcription of FASN, ACL and ACACA genes was found to be regulated in both tissues and breeds suggesting that these genes can represent promising functional and positional candidates for fat deposition in meat and carcass.

In pigs, several studies have been focused on structural and regulatory muscle genes and their possible relationship with meat production trait as they may play key role in muscle mass accretion. In the present work, we investigated four skeletal muscle genes, calpain 1 (CAPN1), calpastatin (CAST), myopalladin (MYPN) and titin (TTN). These genes were mapped on porcine chromosomes 2p14-p17, 2q21-q24, 14q25-q29 and 15q23-q26, respectively, and located in regions where quantitative trait loci for carcass traits were found. The calpain protein (coded by CAPN1 gene and expressed in skeletal muscle and other tissues) is involved in the early post mortem protein degradation promoting the initial disruption and destabilization of the myofibrillar structures. Calpastatin is a specific inhibitor of ubiquitous calpains and its post mortem activity influences meat tenderization process. Myopalladin is a sarcomeric protein which has central roles in the organization and assembly of the Z-line and seems to affect regulatory mechanisms of muscle gene expression. TTN encodes a large abundant protein of striated muscle that is involved in muscle assembly and ultrastructure and it is also implicated in muscle elasticity. To evaluate the putative effects of these four genes on carcass quality traits, we analysed single nucleotide polymorphisms (SNP) identified in the 3’ untranslated regions (MYPN and TTN) and in introns (CAST and CAPN1) in 272 sib tested Italian Large White (ILW) pigs and 114 sib tested Italian Duroc (ID). General linear model procedure of the SAS package was used to search for association between the SNPs and the productive traits average daily gain (ADG), backfat thickness (BFT), lean cuts (LC), ham weight (HW), feed conversion ratio (FCR) for both breeds and visible intramuscular fat (VIF) only for the ID breed. Statistically significant results for the association analyses were found for CAST in the ILW breed, with ADG, LC and FCR (P < 0.05) and in the ID breed with ADG, LC, BFT, HW (P < 0.05). Moreover, in the ID breed, the MYPN gene was associated with all considered traits (P < 0.01). The results confirm that CAST and MYPN could be considered positional and biological candidate genes affecting carcass traits, as previously reported in literature.

The use of molecular markers in functional genes as tool to characterize the genetic diversity in livestock populations can provide new opportunities for the identification of putative alleles of economic value. The aim of this study was to investigate on the genetic diversity among eleven pig breeds by typing SNPs in 23 genes coding for protein. DNA samples of five Italian native pig breeds (Calabrese, Casertana, Cinta Senese, Mora Romagnola and Nero Siciliano) and six cosmopolite breeds (Italian Large White, Italian Landrace, Italian Duroc, Belgian Landrace, Hampshire and Piétrain) were used. A sample of Meishan breed was also included. Allele frequencies, heterozygosity, Fst, Fis, Fit, and Reynold’s genetic distances were calculated and Neighbour-Joining tree with bootstrap resampling was obtained. The average expected heterozygosity over all loci ranged from 0.187 in Mora Romagnola to 0.342 in Italian Large White. Mora Romagnola and Meishan breeds showed the lowest within-population diversity. The results obtained for SNPs were compared with those produced previously in Pig Biodiversity project that analysed DNA samples of the same breeds of the present study, except for Mora Romagnola, using microsatellites markers. The SNP marker-based regression of heterozygosity vs distance from each population to origin produced a similar trend to that obtained with microsatellites. This analysis indicates that the founder expected heterozygosity is 0.382 for SNPs and 0.773 for microsatellites.