P. Zambonelli

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.

Detecting population structure and recent demographic history in endangered livestock breeds: the case of the Italian autochthonous donkeys

Since its domestication, about 5000 years ago, the donkey (Equus asinus) has been extensively used as a work or draft animal in agricultural activities and for the transportation of people and goods. In the last century, technology improvement and growing mechanization strongly affected agriculture and the management and use of this livestock species in the industrialized countries. Nowadays, the use of donkeys for work or transport has almost disappeared, together with the need for mules or hinny breeding. During the last five decades, Italian autochthonous donkey populations suffered from a severe reduction in population size, which led to the extinction of several breeds. At present, eight breeds remain, all classified by FAO as critically endangered or endangered: Asinara, Pantesco, Grigio Siciliano, Romagnolo, Amiatino, Sardo Grigio, Martina Franca, and Ragusano. To evaluate the extant genetic variability of Italian donkeys, we typed 16 microsatellite loci in 258 individuals from these breeds. The results highlighted moderate levels of inbreeding ( F (IS) = 0.127) and a significant partition of genetic variation into breeds, as suggested by fixation index ( F (ST) = 0.109) and analysis of molecular variance (10.86% of total variation assigned to the between-breeds level) analyses. This was confirmed by a Bayesian clustering procedure that also highlighted a further partitioning at lower hierarchical levels corresponding to the farms of origin. This evidence suggests that an effective management strategy for Italian donkey populations should focus on breeds as conservation units. However, this requires a synergic management strategy at the farm level to maintain diversity and avoid inbreeding.

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.

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.

Genetic analysis of seven Italian horse breeds based on mitochondrial DNA D-loop variation

To understand the origin and genetic diversity of Italian horses, mitochondrial DNA D-loop sequences were generated for 163 horses from seven breeds. Sequence analysis of a 480-bp segment revealed a total of 84 haplotypes with 57 polymorphic sites, indicating multiple maternal origins and high genetic diversity. Comparison of the haplotypes with the equine mtDNA haplotype/haplogroup nomenclature showed a haplogroup distribution in the Italian breeds more similar to that found in the Middle East breeds than in the European breeds, probably due to the economic and cultural relationship with the Middle East in the past centuries.

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.

Bovine chromosome 20: milk production QTL and candidate gene analysis in the Italian Holstein-Friesian breed

Abstract Bovine chromosome 20 (BTA20) was studied to identify QTL for milk yield and protein percentage in the Italian Holstein-Friesian breed using a selective milk DNA pooling strategy in a daughter design with sire haplotype analysis. Several QTL were identified. The effect of the known GHR F279Y and PRLR S18N mutations were in for the most part confirmed. However, it was also shown that these markers cannot explain all significant effects observed on BTA20 for the investigated traits.

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.