S. Dall’Olio

Copy Number Variation and Missense Mutations of the Agouti Signaling Protein (ASIP) Gene in Goat Breeds with Different Coat Colors

In goats, classical genetic studies reported a large number of alleles at the Agouti locus with effects on coat color and pattern distribution. From these early studies, the dominant AWt (white/tan) allele was suggested to cause the white color of the Saanen breed. Here, we sequenced the coding region of the goat ASIP gene in 6 goat breeds (Girgentana, Maltese, Derivata di Siria, Murciano-Granadina, Camosciata delle Alpi, and Saanen), with different coat colors and patterns. Five single nucleotide polymorphisms (SNPs) were identified, 3 of which caused missense mutations in conserved positions of the cysteine-rich carboxy-terminal domain of the protein (p.Ala96Gly, p.Cys126Gly, and p.Val128Gly). Allele and genotype frequencies suggested that these mutations are not associated or not completely associated with coat color in the investigated goat breeds. Moreover, genotyping and sequencing results, deviation from Hardy-Weinberg equilibrium, as well as allele copy number evaluation from semiquantitative fluorescent multiplex PCR, indicated the presence of copy number variation (CNV) in all investigated breeds. To confirm the presence of CNV and evaluate its extension, we applied a bovine-goat cross-species array comparative genome hybridization (aCGH) experiment using a custom tiling array based on bovine chromosome 13. aCGH results obtained for 8 goat DNA samples confirmed the presence of CNV affecting a region of less that 100 kb including the ASIP and AHCY genes. In Girgentana and Saanen breeds, this CNV might cause the AWt allele, as already suggested for a similar structural mutation in sheep affecting the ASIP and AHCY genes, providing evidence for a recurrent interspecies CNV. However, other mechanisms may also be involved in determining coat color in these 2 breeds.

Influence of loading method and stocking density during transport on meat and dry-cured ham quality in pigs with different halothane genotypes

The effect of loading method and stocking density in transit on meat and dry-cured ham quality was investigated in pigs with different halothane genotypes. A total of 507 Italian heavy pigs, supplied by two farms, were loaded by ramp or lift and transported unmixed for 35-55 min to the abattoir at a stocking density of either <0.4 or >0.6m(2) per 100 kg pigs. After overnight lairage in separate pens with free access to water, the pigs were slaughtered. Halothane genotype was assessed post mortem. Four hundred and thirty-nine pigs had a homozygous dominant (NN) genotype and 68 pigs were heterozygous (Nn). Carcass skin damage, meat quality traits and ham curing parameters were evaluated. Loading method and stocking density showed a negligible effect on meat and dry-cured ham quality while the predominant factor affecting these was the halothane genotype. Nn pigs produced meat with a faster rate of pH fall and lower water holding capacity as well as ham with higher weight losses in salting and greater incidence of defects in the dry-cured product. There were insignificant interactions between halothane genotype and loading method or stocking density. Overall, irrespective of pre-slaughter treatment, the Nn pigs were less suitable for the production of high quality products such as dry-cured ham.

Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes

Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172T>A, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be determined by non-functional ASIP alleles (non-agouti alleles, A(a)) and in a few cases by the E(D) Extension allele. At least three frequent ASIP haplotypes ([D(5):g.5172T], [N:g.5172A] and [D(5):g.5172A]) were detected (organized into six different diplotypes). In conclusion, the results indicated that coat colours in the Massese sheep breed are mainly derived by combining ASIP and MC1R mutations.

Deconstructing the pig sex metabolome: Targeted metabolomics in heavy pigs revealed sexual dimorphisms in plasma biomarkers and metabolic pathways

Metabolomics has opened new possibilities to investigate metabolic differences among animals. In this study, we applied a targeted metabolomic approach to deconstruct the pig sex metabolome as defined by castrated males and entire gilts. Plasma from 545 performance-tested Italian Large White pigs (172 castrated males and 373 females) sampled at about 160 kg live weight were analyzed for 186 metabolites using the Biocrates AbsoluteIDQ p180 Kit. After filtering, 132 metabolites (20 AA, 11 biogenic amines, 1 hexose, 13 acylcarnitines, 11 sphingomyelins, 67 phosphatidylcholines, and 9 lysophosphatidylcholines) were retained for further analyses. The multivariate approach of the sparse partial least squares discriminant analysis was applied, together with a specifically designed statistical pipeline, that included a permutation test and a 10 cross-fold validation procedure that produced stability and effect size statistics for each metabolite. Using this approach, we identified 85 biomarkers (with metabolites from all analyzed chemical families) that contributed to the differences between the 2 groups of pigs ( < 0.05 at the stability statistic test). All acylcarnitines and almost all biogenic amines were higher in castrated males than in gilts. Metabolites involved in tryptophan catabolism had the largest differences (i.e., delta = 20% for serotonin) between castrated males (higher) and gilts (lower). The level of several AA (Ala, Arg, Gly, His, Lys, Ser, Thr, and Trp) was higher in gilts (delta was from approximately 1.0 to approximately 4.8%) whereas products of AA catabolism (taurine, 2-aminoadipic acid, and methionine sulfoxide) were higher in castrated males (delta was approximately 5.0-6.0%), suggesting a metabolic shift in castrated males toward energy storage and lipid production. Similar general patterns were observed for most sphingomyelins, phosphatidylcholines, and lysophosphatidylcholines. Metabolomic pathway analysis and pathway enrichment identified several differences between the 2 sexes. This metabolomic overview opened new clues on the biochemical mechanisms underlying sexual dimorphism that, on one hand, might explain differences in terms of economic traits between castrated male pigs and entire gilts and, on the other hand, could strengthen the pig as a model to define metabolic mechanisms related to fat deposition.

Genetic structure of candidate genes for litter size in Italian Large White pigs

The aim of this work was to verify whether polymorphisms in candidate genes for litter size segregate in Italian Large White (ITLW) pigs. We genotyped 120 sows that belonged to six different farms for 10 single nucleotide polymorphisms (SNPs) of 10 different genes. Polymorphisms in the chosen genes had already been associated with litter-size traits in other pig populations and were candidates for function and/or chromosomal location. The results indicated that the CLGN, pDAZL, and RFN4 SNPs were not segregating in the genotyped samples. The remaining seven markers were polymorphic with minor allele frequencies ranging from 0.10 (AFP) to 0.48 (RBP4). Because of the observed genetic variabilities in the investigated loci, the polymorphisms in the AFP, BMPR1B, CXCL10, ESR2, GNRHR, MAN2B2, and RBP4 genes can be considered suitable markers for association studies with litter-size traits in ITLW pigs.

The BovMAS Consortium: investigation of bovine chromosome 14 for quantitative trait loci affecting milk production and quality traits in the Italian Holstein Friesian breed

Riassunto Studio del cromosoma 14 di bovino per l’identificazione di QTL per la produzione e la qualità del latte nella razza Frisona Italiana. Nel presente studio, utilizzando le metodologie del daughter design e del selective DNA pooling, abbiamo verificato la presenza di un importante QTL per la produzione di latte nella regione prossimale del cromosoma 14 nella razza Frisona Italiana. Inoltre, abbiamo effettuato la ricerca di altri QTL per la percentuale di proteine, la percentuale di grasso e la produzione di latte su questo cromosoma. I risultati mettono in evidenza che il QTL nella regione prossimale è dovuto solo in parte alla mutazione A232K del gene DGAT1 e che probabilmente un altro o altri QTL segregano nella regione centrale-distale del cromosoma. Ulteriori studi sono necessari per confermare e chiarire questi risultati.

Effect of Different Rates of Postmortem pH Decline on the Technological Quality of Calabrian Capocollo

Ninety carcasses from cross-bred pigs [Duroc × (Landrace × Large White)] were examined to study the effect of postmortem acidification rate on weight loss during the processing of protected designation of origin Calabrian Capocollo. The pH was measured in the loin at the 6th rib at 1, 3, and 6 h postmortem. Thirty-four carcasses were selected on the basis of pH decline at 3 h: slow (pH3 > 6.20, n = 11), intermediate (5.91 > pH3 ≤ 6.20, n = 11), and fast (pH3 ≤ 5.90, n = 12). Before and after curing and ripening, the Capocollo weights were recorded and the respective losses (%) were calculated. At 1, 3, and 7 days postmortem, measurements of pH, color, and water holding capacity (WHC) were recorded from adjacent loin samples. Fast acidification was associated with a lighter color and lower WHC of fresh meat but did not affect weight loss after curing and ripening, which were very similar between the three groups.

Investigation of the premelanosome protein ( PMEL or SILV ) gene and identification of polymorphism excluding it as the determinant of the dilute locus in domestic rabbits ( Oryctolagus cuniculus )

Abstract. After the rediscovery of the Mendel’s laws, the domesticated European rabbit (Orycolagus cuniculus) has been the objective of pioneering studies on coat colour genetics. However, despite the early role of this species in defining genetic mechanisms determining this phenotypic trait, only recently a few loci have been characterized at the molecular level analysing also in rabbits genes already shown to affect coat colour in mice. We herein investigated the rabbit premelanosome protein (PMEL) gene, also known as melanocyte protein Pmel 17 (PMEL17) or silver (SILV), as mutations in the homologous gene in mice and other species produce phenotypic effects similar to what is observed in the dilute coat colour in rabbit. The rabbit dilute locus is determined by a recessive coat colour mutation that dilutes the black to blue (grey) interacting with the basic colours influenced by the agouti and extension loci. To investigate this candidate gene, we isolated and sequenced cDNAs as well as portions of intronic and exonic regions of the PMEL gene in several rabbits with different coat colours and identified single nucleotide polymorphisms, including several missense mutations. One polymorphism, positioned in intron 7, was genotyped in a family in which there was segregation of the dilute coat colour. The results excluded PMEL as the causative gene for the dilute locus in rabbits, shortening the list of candidate genes that should be analysed to identify the mutation determining this phenotypic trait.

Analysis of a polymorphism within a TATA box of horse myostatin gene promoter in different breeds

Abstract Myostatin or growth and differentiation factor 8 (MSTN or GDF8, respectively) is a member of the transforming growth factor-β superfamily that acts as a negative regulator of skeletal muscle development and growth. The myostatin encoding gene (MSTN) and the 5’ regulative region in different species has been investigated. The MSTN gene consists of three exons and two intronic regions. In cattle, the gene has well-characterized mutations determining phenotypes with increased muscle mass. In a previous study, by alignment of nucleotide sequences of PCR products of horse MSTN gene, we identified some SNPs of which two T=C transitions in the promoter of the gene. The aim of this work is to study in different horse breeds the frequency of the polymorphism located 516 nt upstream of the ATG start codon. This SNP is within a TATA box sequence (NATAAAA, where N= T or C) and the comparative sequence analysis of mammals myostatin gene promoters reveals that this TATA-box motive is conserved in position and sequence among some species including cattle, sheep and goat. The point mutation is not recognised by restriction enzymes, then the genotyping was done by restriction site insertion-PCR. The primer pair was designed on the basis of the obtained nucleotide sequences. The reverse primer was modified and designed with a single nucleotide mismatched with respect to target sequence to introduces an SspI artificial restriction site when the T nucleotide occurred. The PCR products of 203 bp were digested and the resulting fragments were resolved on 3.5% agarose gel. Two hundred and six samples of DNA belonging to 18 horse breeds (Bardigiano, 20; Breton, 5; Criollo, 10; Esperia Pony, 4; Haflinger, 9; Italian Heavy Draught Horse, 24; Italian Saddle, 21; Italian Trotter, 16; Lipizzan, 11; Maremmano, 13; Murgese, 12; Norico, 10; Purebred Spanish Horse, 10; Salernitano, 12; San Fratellano, 3; Tolfetano, 7; Thoroughbred, 11 and Ventasso Horse, 8) were genotyped. The T allele occurs in all the breeds. The C allele, which eliminates TATA box motif, was detected in 11 breeds and its value frequency ranged from 0.006 (Thoroughbred) to 0.400 (Norico). The homozygous C/C genotype was detected in Haflinger, Bardigiano, Norico and Tolfetano breeds. The presence of the polymorphism within a conserved region of promoter of horse myostatin gene could affect expression gene. This marker could be used for association studies with performance traits in horses.

Study of Fatty Acid Synthase and Adiponectin SNPs in the Italian Duroc breed

Abstract Fatty acid synthase (FASN) is a multifunctional enzyme that plays a central role in fatty acid biosynthesis catalysing the conversion of acetyl-CoA and malonyl-CoA into long-chain saturated fatty acids and has an important role in energy homeostasis. Pig FASN gene has been assigned to chromosome 12p1.5 and a T>C polymorphism in the fourth exon was found. Adiponectin (ADN) is a fat-derived hormone involved in insulin sensitivity, in lipid and glucose metabolism. In literature is reported that the gene was mapped on chromosome 13 at 53.6 cM, in a region containing QTL for intramuscolar fat (IMF). In this gene several SNPs were identified and one of these polymorphisms (a G>A missense mutation within the 60th codon) determining the Val-Ile substitution in the protein, has been previously reported. The aim of this work is to analyse the variability of polymorphisms of fatty acid synthase described by Munoz et al., 2003 (Anim. Genet. 34:234) and adiponectin genes, candidates for meat and carcass quality, in Italian Duroc pigs. Up to now researches on these functional genes in this pig breed have not been performed. In order to estimate allele frequencies, the FASN T>C polymorphism and the ADN G>A mutation were studied by PCR-RFLP in 134 and 257 Italian Duroc, respectively. At the FASN locus the allele frequency was 0.041 for T allele; at the ADN locus the frequency of the alleles A was 0.136. We investigated the effect of FASN and ADN polymorphisms on Estimated Breeding Values (EBVs) for single traits (average daily gain, feed to gain ratio, weight of lean cuts, ham weight, backfat thickness and visible intermuscular fat) and for selection indexes (Total Index and Terminal Index) estimated by ANAS. The analyses were performed using the GLM procedure of SAS System and a model with the genotype at FASN or ADN locus as fixed factor. At the FASN locus only few pigs carried the T allele; therefore, we tried to perform the analysis comparing the pigs with both genotypes T/T plus C/T versus those genotyped C/C at this locus. The results didn’t show any association of the polymorphism with the examined traits. The same model was used for the ADN locus and the G/A genotyped pigs versus the G/G ones showed a significant higher estimated mean value for Total Index (P= 0.0114). For both the analysed genes the obtained results should be verified on a larger sample even if the present findings could suggest a possible role of ADN on the traits included in Total Index in the Duroc breed.