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Fat deposition in pigs is a complex trait for which several studies in experimental populations have identified a large number of QTL, some of which segregating in outbred commercial populations. As pigs and humans have numerous physiological and phenotypic similarities for fat deposition, in order to identify the genetic factors affecting this trait in both species, it could be possible to take advantage of the results obtained in one species and, applying a comparative approach, transfer biological information to the other one, and vice versa. Therefore, such approach, on one hand, might provide useful information to improve selection efficiency in pig breeding and, on the other hand, could contribute to understand the genetic basis of human obesity and related diseases. With these aims, we designed a project based on a selective genotyping approach in commercial pig populations for which extreme and divergent groups of pigs have been chosen according to the estimated breeding value (EBV) for fat deposition traits. A candidate gene approach has been applied in order to identify markers associated with fatness in pigs. DNA markers are chosen and identified using different approaches: i) mutations already reported in pig genes affecting fat deposition; ii) discovery of new mutations in candidate genes by resequencing; iii) database mining. For the latter approach, a bioinformatic pipeline has been designed to mine pig sequences available in DNA databases. In the start up phase, about 560 Italian Large White pigs, selected according to their fatness EBV, are genotyped for more than 700 SNPs. Some preliminary results are reported. The project portal is: http://fagenomich.biocomp.unibo.it/

The increasing number of samples used for association studies in farm animals, the number of participating laboratories in such investigations and the involvement of several people and laboratory technicians in the sampling, labelling, storage, database codification, DNA isolation and analysis contribute to increase the risk of mistakes in the different sample handling stages. DNA analysis using the routine microsatellite panel for parentage verification could be used to obtain a fingerprinting of the samples but it cannot provide a quick and inexpensive way to verify if misclassification occurred. In order to verify the correct classification of a large number of male and female pigs sampled at slaughterhouses and to identify the sex of some animals for which gender was not recorded, we applied the amelogenin based sex determination method that we recently developed. The test analyses the fragment length difference between the AMELX and AMELY genes that are located on the X and Y chromosomes, respectively. The obtained PCR products can be easily resolved on polyacrylamide gels, agarose gels or by capillary electrophoresis using an automatic sequencer. Of the analysed animals, eight samples resulted misclassified, probably due to a frameshift between the sampling and the pre-available list of slaughtered animals. Their gender was also confirmed from a different DNA extraction and using other two sex determination methods based on the analysis of the ZFX and ZFY genes. Furthermore, other two samples for which gender was not recorded at the slaughterhouse were sexed using the amelogenin and ZFX/ZFY methods.

Obesity is a complex trait that, in humans, has been the objective of a large number of studies aimed to identify the genetic factors affecting different forms of this disease or being relevant for the related health problems. Mutations in several human genes have been associated to common and severe obesity. In addition, other investigations, combining gene expression and other biological evidences, have demonstrated that different genes may be important in muscle mass development. Starting from these evidences, we used a candidate gene approach to select genes that are worth to be investigated in pigs for association studies with fat and muscle deposition traits. As a first step, we mined pig expressed sequence databases and/or sequence trace records in order to identify porcine sequences for some of these genes. For a first list of selected genes, activin A receptor type II B (ACVR2B), cannabinoid receptor 1 (CNR1), cathepsin K (CTSK), myotubularin-related protein 9 (MTMR9), TBC1 domain family, member 1 (TBC1D1) and tubby (TUB), PCR primers were designed to amplify fragments containing exons and introns. Resequencing was carried out in a panel of 10-12 pigs of different breeds. Single nucleotide polymorphisms (SNPs) were identified in the ACVR2B (4 SNPs), CTSK (1 SNP) and TBC1D1 (4 SNPs) genes. These DNA markers will be used in association studies with fat and meat deposition traits in Italian heavy pigs.

In humans, mutations in the fat mass and obesity associated (FTO) gene are associated with the obesity phenotype in several populations. In pigs, we already identified three single nucleotide polymorphisms (SNPs) in the FTO gene that we genetically assigned to porcine chromosome 6 (SSC6), in an imprinted QTL region for fat deposition. Then, using a selective genotyping approach, we showed that an SNP in this gene (g.276T>G) was associated with visible intermuscular fat (VIF, a measure that resembles intermuscular fat content) in Italian Duroc pigs. Here, we refined the mapping of this gene to SSC6 using a radiation hybrid panel and evaluate the effect of the same mutation in two different pig samples. The first was constituted by 157 sib-tested Italian Duroc animals for which estimated breeding values (EBV) for average daily gain, feed:gain ratio, back-fat thickness, lean cuts, ham weight and VIF were calculated. The second group of pigs was constituted by 117 commercial hybrids measured for a total of 31 carcass and fatness traits. In the Italian Duroc pigs the FTO marker was associated only with VIF EBV (P = 0.027). In the second group of pigs, the FTO genotypes showed a tendency to be associated with several fat deposition traits. These results confirm that FTO is an important source of variability for intermuscular fat deposition in Italian Duroc pigs while the effects on fatness traits in commercial pig populations should be further investigated.

Coat colour phenotypes in European pig breeds are mainly due to alleles at the Extension and Dominant White loci. At the latter locus, several alleles are caused by mutations in the v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) gene that are mainly originated by a combination of gene duplication and single nucleotide polymorphisms. Nero Siciliano is one of the few survived local Italian pig breed reared in the Nebrodi mountains of the island of Sicily The animals are usually completely black but a few show white portions mainly in the face or in the fore legs. In order to identify the genetic factors determining the presence of white portions, we genotyped 97 Nero Siciliano pigs (25 with white portions) for the splice site mutation of the intron 17 of the KIT gene that cause the Dominant White I allele. These animals were also analysed for the presence of the duplication of the same gene. None of the pigs carried the splice site mutation while 7 out of 25 animals with white portions were positive for the duplication test, suggesting that other alleles at this locus or other genes might affect this phenotype in Nero Siciliano pigs. Then, 8 of the 21 exons of the KIT gene were sequenced in eight Nero Siciliano pigs with different colour phenotypes. Several mutations were identified indicating that new alleles of the KIT gene are present in this breed. Further studies are under way to sequence all exons and evaluate the effect on coat colour of the identified mutations.

Cathepsins are lysosomal proteinases involved in a large number of biological processes that could be directly or indirectly linked to the physiological mechanisms that might affect different production traits in farm animals. Furthermore, in pigs, an elevated cathepsin activity in fresh ham muscles and throughout the processing period has been shown to be a factor contributing to the excessive softness defect of dry cured hams. We recently showed that several cathepsin and lysosomal proteinase inhibitor genes are associated with meat production and carcass traits in Italian heavy pig populations but none of the investigated loci was associated with cathepsin activity. Here we studied two additional genes of the cathepsin family, cathepsin L (CTSL) and cathepsin S (CTSS), with the aim to identify DNA markers associated with meat production and quality traits in pigs. Single nucleotide polymorphisms in these genes were identified mining porcine expressed sequence tags and confirmed by sequencing. PCR-RFLP tests were used to genotype pigs of seven different breeds to obtain allele frequencies information for these polymorphisms. Estimated breeding values for average daily gain (ADG), back fat thickness (BFT), lean cuts (LC), ham weight (HW) and feed:gain ratio (FGR) as well as cathepsin B activity, pH1, pH2, and glycolytic potential of semimembranosus muscle were determined for 265 sib-tested Italian Large White pigs that were genotyped for the CTSL and CTSS markers. Significant associations were observed for ADG (CTSL, P