R. Davoli

Perilipin 1 and perilipin 2 protein localization and gene expression study in skeletal muscles of European cross-breed pigs with different intramuscular fat contents

This study investigated the lipid droplet coat proteins perilipin 1 (PLIN1) and perilipin 2 (PLIN2) localization in pig skeletal muscle and their relationship with intramuscular fat (IMF) content. PLIN1 and PLIN2 proteins were immunostained in semimembranosus muscle cross-sections from two groups of samples divergent for IMF and the gene expression was quantified. PLIN1 localized in the periphery of intramuscular adipocytes, whereas PLIN2 localized within myofibers with high lipid content. The high IMF group showed higher total cross-sectional area of PLIN1-stained adipocytes compared with the low IMF group (P<0.05), while the cross-sectional area and percentage of PLIN2-positive myofibers did not differ between IMF-divergent groups. This suggested that IMF content is mainly determined by extra-myocellular lipids. At mRNA level, PLIN2 expression was higher in high IMF muscles (P<0.05). The results indicate for the first time that in pig muscle PLIN1 and PLIN2 proteins are localized in correspondence with extra and intra-myocellular lipids, respectively.

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.

Association of PPARGC1A and CAPNS1 gene polymorphisms and expression with meat quality traits in pigs.

This study aimed to investigate the genes PPARGC1A (peroxisome proliferator-activated receptor gamma-coactivator 1A) and CAPNS1 (calpain small subunit 1) as candidate genes affecting meat quality traits in pigs. Four polymorphisms were identified in PPARCG1A and three in CAPNS1. The PPARGC1A polymorphism c.1288T>A was associated with pH and cooking loss in a F2 Duroc×Pietrain experimental cross (DuPi, n=313) and with pH values in Italian Large White (ILW, n=380) and Italian Landrace (ILA, n=158) populations (P<0.05). The CAPNS1 polymorphism c.429A>C was associated with pH and conductivity in DuPi and with meat color in ILA (P<0.05). PPARGC1A mRNA expression associated with drip loss (P<0.01) and the same tendency was found for CAPNS1 (P=0.06). The promoter methylation profiling suggested that methylation is not involved in CAPNS1 expression regulation. In conclusion, porcine PPARGC1A and CAPNS1 genes may affect meat quality traits, with breed-specific differences, and they could be used as markers for the improvement of meat quality in pigs.

A whole genome scan for QTL affecting milk protein percentage in Italian Holstein cattle, applying selective milk DNA pooling and multiple marker mapping in a daughter design

We report on a complete genome scan for quantitative trait loci (QTL) affecting milk protein percentage (PP) in the Italian Holstein-Friesian cattle population, applying a selective DNA pooling strategy in a daughter design. Ten Holstein-Friesian sires were chosen, and for each sire, about 200 daughters, each from the high and low tails of estimated breeding value for PP, were used to construct milk DNA pools. Sires and pools were genotyped for 181 dinucleotide microsatellites covering all cattle autosomes. Sire marker allele frequencies in the pools were obtained by shadow correction of peak height in the electropherograms. After quality control, pool data from eight sires were used for all subsequent analyses. The QTL heterozygosity estimate was lower than that of similar studies in other cattle populations. Multiple marker mapping identified 19 QTL located on 14 chromosomes (BTA1, 2, 3, 4, 5, 6, 8, 9, 12, 14, 17, 20, 23 and 27). The sires were also genotyped for seven polymorphic sites in six candidate genes (ABCG2, SPP1, casein kappa, DGAT1, GHR and PRLR) located within QTL regions of BTA6, 14 and 20 found in this study. The results confirmed or excluded the involvement of some of the analysed markers as the causative polymorphic sites of the identified QTL. The QTL identified, combined with genotype data of these candidate genes, will help to identify other quantitative trait genes and clarify the complex QTL patterns observed for a few chromosomes. Overall, the results are consistent with the Italian Holstein population having been under long-term selection for high PP.

Polymorphisms in an obesity-related gene (PCSK1) are associated with fat deposition and production traits in Italian heavy pigs.

The proprotein convertase subtilisin/kexin type 1 (PCSK1) gene encodes the prohormone convertase 1/3 enzyme that processes prohormones into functional hormones that, in turn, regulate central and peripheral energy metabolism. Mutations in the human PCSK1 gene cause severe monogenic obesity or confer risk of obesity. We herein investigated the porcine PCSK1 gene with the aim of identifying polymorphisms associated with fat deposition and production traits in Italian heavy pigs. By re-sequencing about 5.1 kb of this gene in 21 pigs of different breeds, we discovered 14 polymorphisms that were organized in nine haplotypes, clearly distributed in two clades of putative European and Asian origin. Then we re-mapped this gene on porcine chromosome 2 and analysed its expression in several tissues including gastric oxyntic mucosa of weanling pigs in which PCSK1 processes the pre-pro-ghrelin into ghrelin, which in turn is involved in the control of feed intake and energy metabolism. Association analyses between PCSK1 single-nucleotide polymorphisms (SNPs) and production, carcass and several other traits were conducted on five groups of pigs from three different experimental designs, for a total of 1221 animals. Results indicated that the analysed SNPs were associated (P < 0.01 or P < 0.05) with several traits including backfat thickness and visible intermuscular fat in Italian Duroc (ID) and growth performances in Italian Large White (ILW) and in ILW × Italian Landrace pigs. However, the effects estimated in the ILW were opposite to the effects reported in the ID pigs. Suggestive association (P < 0.10) was observed with muscle cathepsin B activity, opening, if confirmed, potential applications to reduce the excessive softness defect of the green hams that is of particular concern for the processing industry. The results obtained supported the need to further investigate the PCSK1 gene to fully exploit the value of its variability and apply this information in pig breeding programmes.

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.

Association mapping of quantitative trait loci for carcass and meat quality traits at the central part of chromosome 2 in Italian Large White pigs.

Association mapping of the central part of porcine chromosome 2 harboring QTLs for carcass and meat quality traits was performed with 17 gene-tagged SNPs located between 44.0 and 77.5 Mb on a physical map (Sscrofa10.2) in Italian Large White pigs. For the analyzed animals records of estimated breeding values for average daily gain, back fat thickness, lean cuts, ham weight, feed conversion ratio, pH1, pHu, CIE L*, CIE a*, CIE b* and drip loss were available. A significant QTL for fat deposition (adjusted P=0.0081) and pH1 (adjusted P=0.0972) to MYOD1 at position 44.4 Mb and a QTL for growth and meatiness (adjusted P=0.0238-0.0601) to UBL5 at position 68.9 Mb were mapped. These results from association mapping are much more accurate than those from linkage mapping and facilitate further search for position candidate genes and causative mutations needed for application of markers through marker assisted selection.

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.