Vincenzo Russo

Ancient vs. recent processes as factors shaping the genetic variation of the European wild boar: are the effects of the last glaciation still detectable?

The European wild boar is an important game species, subjected to local extinctions and translocations in the past, and currently enormously and worryingly expanding in some areas where management is urgently required. Understanding the relative roles of ancient and recent events in shaping the genetic structure of this species is therefore not only an interesting scientific issue, but it represents also the basis for addressing future management strategies. In addition, several pig breeds descend from the European wild boar, but the geographical location of the domestication area(s) and the possible introgression of pig genomes into wild populations are still open questions. Here, we analysed the genetic variation in different wild boar populations in Europe. Ten polymorphic microsatellites were typed in 252 wild boars and the mtDNA control region was sequenced in a subset of 145 individuals. Some samples from different pig breeds were also analysed. Our results, which were obtained considering also 612 published mtDNA sequences, suggest that (i) most populations are similarly differentiated, but the major discontinuity is found along the Alps; (ii) except for the Italian populations, European wild boars show the signature of a postglacial demographic expansion; (iii) Italian populations seem to preserve a high proportion of preglaciation diversity; (iv) the demographic decline which occurred in some areas in the last few centuries did not produce a noticeable reduction of genetic variation; (v) signs of human‐mediated gene flow among populations are weak, although in some regions the effects of translocations are detectable and a low degree of pig introgression can be identified; (vi) the hypothesis of an independent domestication centre in Italy is not supported by our data, which in turn confirm that Central European wild boar might have represented an important source for domestic breeds. We can therefore conclude that recent human activities had a limited effect on the wild boar genetic structure. It follows that areas with high variation and differentiation represent natural reservoirs of genetic diversity to be protected avoiding translocations. In this context controlling some populations by hunting is not expected to affect significantly genetic variation in this species.

A first comparative map of copy number variations in the sheep genome

We carried out a cross species cattle-sheep array comparative genome hybridization experiment to identify copy number variations (CNVs) in the sheep genome analysing ewes of Italian dairy or dual-purpose breeds (Bagnolese, Comisana, Laticauda, Massese, Sarda, and Valle del Belice) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. We identified 135 CNV regions (CNVRs; 24 reported in more than one animal) covering ~10.5 Mb of the virtual sheep genome referred to the bovine genome (0.398%) with a mean and a median equal to 77.6 and 55.9 kb, respectively. A comparative analysis between the identified sheep CNVRs and those reported in cattle and goat genomes indicated that overlaps between sheep and both other species CNVRs are highly significant (P<0.0001), suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Many sheep CNVRs include genes with important biological functions. Further studies are needed to evaluate their functional relevance.

The porcine fat mass and obesity associated (FTO) gene is associated with fat deposition in Italian Duroc pigs

In humans, common variants in the fat mass and obesity associated (FTO) gene are associated with body mass index and obesity. Here we sequenced exon 4, parts of introns 3 and 4 and two portions of the 3-untranslated region of the porcine FTO gene in a panel of nine pigs of different breeds and identified three SNPs. Allele frequencies of the g.276T>G (AM931150) mutation were studied in seven pig breeds. This mutation was used to linkage-map FTO to SSC6. Association analyses between the g.276T>G polymorphism and several traits [pH of semimembranosus muscle and estimated breeding values (EBV) for average daily gain, back fat thickness, lean cuts, ham weight and feed:gain ratio] were carried out in 257 sib-tested Italian Large White pigs. Only feed:gain ratio showed P<0.05. A selective genotyping approach was applied, analysing two extreme and divergent groups of Italian Large White pigs selected on the basis of back fat thickness EBV (50 with most positive and 50 with most negative values). Fishers exact test (two-tailed) was not significant when comparing the allele frequencies of these two groups. The same approach was used in the Italian Duroc breed for which two extreme and divergent groups of animals were selected according to visible intermuscular fat EBV. Differences of allele frequencies between these two groups were highly significant (P<0.00001, P<0.001 and P<0.0001, considering all animals or only two- or three-generation unrelated animals respectively), indicating association between the analysed FTO marker and intermuscular fat deposition.

Quantitative trait loci affecting milk yield and protein percentage in a three-country Brown Swiss population.

Quantitative trait loci (QTL) mapping projects have been implemented mainly in the Holstein dairy cattle breed for several traits. The aim of this study is to map QTL for milk yield (MY) and milk protein percent (PP) in the Brown Swiss cattle populations of Austria, Germany, and Italy, considered in this study as a single population. A selective DNA pooling approach using milk samples was applied to map QTL in 10 paternal half-sib daughter families with offspring spanning from 1,000 to 3,600 individuals per family. Three families were sampled in Germany, 3 in Italy, 1 in Austria and 3 jointly in Austria and Italy. The pools comprised the 200 highest and 200 lowest performing daughters, ranked by dam-corrected estimated breeding value for each sire-trait combination. For each tail, 2 independent pools, each of 100 randomly chosen daughters, were constructed. Sire marker allele frequencies were obtained by densitometry and shadow correction analyses of 172 genome-wide allocated autosomal markers. Particular emphasis was placed on Bos taurus chromosomes 3, 6, 14, and 20. Marker association for MY and PP with a 10% false discovery rate resulted in nominal P-values of 0.071 and 0.073 for MY and PP, respectively. Sire marker association tested at a 20% false discovery rate (within significant markers) yielded nominal P-values of 0.031 and 0.036 for MY and PP, respectively. There were a total of 36 significant markers for MY, 33 for PP, and 24 for both traits; 75 markers were not significant for any of the traits. Of the 43 QTL regions found in the present study, 10 affected PP only, 8 affected MY only, and 25 affected MY and PP. Remarkably, all 8 QTL regions that affected only MY in the Brown Swiss, also affected MY in research reported in 3 Web-based QTL maps used for comparison with the findings of this study (http://www.vetsci.usyd.edu.au/reprogen/QTL_Map/; http://www.animalgenome.org/QTLdb/cattle.html; http://bovineqtl.tamu.edu/). Similarly, all 10 QTL regions in the Brown Swiss that affected PP only, affected only PP in the databases. Thus, many QTL appear to be common to Brown Swiss and other breeds in the databases (mainly Holstein), and an appreciable fraction of QTL appears to affect MY or PP primarily or exclusively, with little or no effect on the other trait. Although QTL information available today in the Brown Swiss population can be utilized only in a within family marker-assisted selection approach, knowledge of QTL segregating in the whole population should boost gene identification and ultimately the implementation and efficiency of an individual genomic program.

Missense and nonsense mutations in melanocortin 1 receptor (MC1R) gene of different goat breeds: association with red and black coat colour phenotypes but with unexpected evidences

BackgroundAgouti and Extension loci control the relative amount of eumelanin and pheomelanin production in melanocytes that, in turn, affects pigmentation of skin and hair. The Extension locus encodes the melanocortin 1 receptor (MC1R) whose permanent activation, caused by functional mutations, results in black coat colour, whereas other inactivating mutations cause red coat colour in different mammals.ResultsThe whole coding region of the MC1R gene was sequenced in goats of six different breeds showing different coat colours (Girgentana, white cream with usually small red spots in the face; Maltese, white with black cheeks and ears; Derivata di Siria, solid red; Murciano-Granadina, solid black or solid brown; Camosciata delle Alpi, brown with black stripes; Saanen, white; F1 goats and the parental animals). Five single nucleotide polymorphisms (SNPs) were identified: one nonsense mutation (p.Q225X), three missense mutations (p.A81V, p.F250V, and p.C267W), and one silent mutation. The stop codon at position 225 should cause the production of a shorter MC1R protein whose functionality may be altered. These SNPs were investigated in a larger sample of animals belonging to the six breeds. The Girgentana breed was almost fixed for the p.225X allele. However, there was not complete association between the presence of red spots in the face and the presence of this allele in homozygous condition. The same allele was identified in the Derivata di Siria breed. However, its frequency was only 33%, despite the fact that these animals are completely red. The p.267W allele was present in all Murciano-Granadina black goats, whereas it was never identified in the brown ones. Moreover, the same substitution was present in almost all Maltese goats providing evidence of association between this mutation and black coat colour.ConclusionAccording to the results obtained in the investigated goat breeds, MC1R mutations may determine eumelanic and pheomelanic phenotypes. However, they are probably not the only factors. In particular, the surprising not complete association of the nonsense mutation (p.Q225X) with red coat colour raises a few hypotheses on the determination of pheomelanic phenotypes in goats that should be further investigated.

Investigation of candidate genes for glycolytic potential of porcine skeletal muscle: Association with meat quality and production traits in Italian Large White pigs

The objective of this study was to investigate the association of DNA markers in candidate genes for glycolytic potential on meat quality parameters (pH(1), pH(u), glycogen and lactate content and glycolytic potential of semimembranosus muscle) and estimated breeding values (EBVs) for average daily gain, lean cuts, back fat thickness, ham weight, and feed:gain ratio in 272 Italian Large White pigs. Three mutations in the PRKAG3 gene (T30N, G52S and I199V) were investigated as well as single nucleotide polymorphisms in two other skeletal muscle genes (PGAM2 and PKM2) involved in the glycolytic pathway. Association analysis with the PRKAG3 markers showed significant results (P<0.05) only for pH(1) (I199V, with significant additive effect) and lactate content (T30N), confirming, at least in part, the effects of this gene on meat quality traits. Significant association (P<0.05) was also observed for PGAM2 and ham weight EBV with significant additive and dominance effects. PKM2 was associated with average daily gain, lean cuts (P<0.001), back fat thickness and feed:gain ratio (P<0.05), with significant additive and/or dominance effects on these traits. PKM2 encodes for a key enzyme of the muscle glycolytic pathway and maps on porcine chromosome 7 where other studies have reported important QTL for the same traits. These data might suggest an important function of this gene in the mechanisms that produce the observed effects. The results will be important to evaluate the inclusion of some of these DNA polymorphisms in marker assisted selection programs.

Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth, carcass, and production traits in Italian Large White pigs

To identify DNA markers associated with performance, carcass, and meat production traits including muscle postmortem cathepsin activity, several porcine genes encoding for lysosomal proteinases (cathepsin B, CTSB; cathepsin D, CTSD; cathepsin F, CTSF; cathepsin H, CTSH; cathepsin L, CTSL; and cathepsin Z, CTSZ) and for a cathepsin inhibitor (cystatin B) were investigated. Single nucleotide polymorphisms were identified in CTSD, CTSH, CTSL, and CTSZ genes with a combination of in silico expressed sequence tag database mining and single-strand conformation polymorphism analysis. Sequencing and PCR-RFLP protocols were used to validate the identified polymorphisms. Allele frequencies at these loci were investigated in Italian Large White, Landrace, Duroc, Piétrain, Belgian Landrace, Hampshire, and Meishan breeds. Genotyping CTSD and CTSH markers made it possible to genetically map these genes to SSC 2 and 7, respectively. Markers in CTSD, CTSH, CTSL, and CTSZ genes, together with mutations we previously reported in cystatin B, CTSB, and CTSF genes, were genotyped in an Italian Large White sib-tested population (272 or 482 animals). For these animals, meat quality traits (cathepsin B activity, pH measured at 2 h postmortem, pH measured at 24 h postmortem, glycogen, lactate, and glycolytic potential of semimembranosus muscle) and EBV for ADG, lean cuts (LC), backfat thickness (BFT), ham weight (HW), and feed:gain ratio (FGR) were determined. Analyzed markers did not show any association with muscle cathepsin B activity. Thus, it could be possible that different genes, other than these investigated candidates, affect this trait, which is correlated with the excessive softness defect of dry-cured hams. The results of association analysis confirmed the effects we already reported in another study for CTSF on ADG (P = 0.008), LC (P = 0.001), and BFT (P = 0.02). Moreover, CTSD was associated with ADG, LC (P < 0.0001), BFT, HW, and FGR (P < 0.001); CTSH was associated with FGR (P = 0.026); and CTSZ was associated with ADG (P = 0.006), LC (P = 0.01), HW (P = 0.024), and FGR (P = 0.029). The biochemical and physiological functions of the lysosomal proteinases, together with the results obtained in our investigation, suggest that the cathepsin gene family might play important roles affecting economic traits in pigs.

Analysis of melanocortin 1 receptor (MC1R) gene polymorphisms in some cattle breeds: their usefulness and application for breed traceability and authentication of Parmigiano Reggiano cheese

Abstract In cattle, the MC1R gene has been the subject of several studies with the aim to elucidate the biology of coat colour. Then, polymorphisms of this gene have been proposed as tools for breed identification and animal products authentication. As a first step to identify breed specific DNA markers that can be used for the traceability of mono-breed dairy cattle products we investigated, using PCR-RFLP and PCR-APLP protocols, the presence and distribution of some alleles at the MC1R locus in 18 cattle breeds for a total of 1360 animals. For each of seven breeds (Italian Holstein, Italian Brown, Italian Simmental, Rendena, Jersey, Reggiana and Modenese) a large number of animals (>70) was genotyped so the obtained results can be considered with more confidence. Allele Ed was identified only in black pied cattle (Italian Holstein and Black Pied Valdostana). Allele E (this nomenclature includes all alleles except Ed, E1 and e) was observed in Italian Brown, Rendena, Jersey, Modenese, Italian Simmental, Grigio Alpina, Piedmontese, Chianina, Romagnola, Marchigiana, Swedish Red and White and Danish Red. Allele E1 was identified in Italian Brown, Rendena, Grigio Alpina, Piedmontese, Swedish Red and White and Danish Red. The recessive allele e, known to cause red coat colour, was fixed in Reggiana and almost fixed in Italian Simmental. This allele was observed also in Italian Holstein, Italian Brown, Rendena, Jersey and Modenese albeit with low frequency. Moreover, this allele was detected in Valdostana, Pezzata Rossa d’Oropa, Piedmontese, Romagnola, Swedish Red and White, Danish Red, Charoleis and Salers. In the case of the Reggiana breed, which is fixed for allele e, the MC1R locus is highly informative with respect to breeds that carry other alleles or in which allele e is at very low frequency. In theory, using the MC1R locus it is possible to identify the presence of milk from some other breeds in Parmigiano Reggiano cheese labelled as exclusively from the Reggiana breed. This possibility was practically tested by setting up protocols to extract and analyse polymorphisms of the MC1R locus in several dairy products, including Parmigiano Reggiano cheese cured for 30 months. The lower detection limit was estimated to be 5% of non expected DNA. This test can represent a first deterrent against fraud and an important tool for the valorisation and authentication of Parmigiano Reggiano cheese obtained from only Reggiana milk.

The relationship between carcass bruising in bulls and behaviour observed during pre-slaughter phases

The bruises often observable on bull carcasses after skinning are due to rupture of the vascular supply and accumulation of blood in tissues. They are produced by impact against the handling facilities and/or other bulls, or by violent contact with prods or sticks used improperly by handlers. Bruises reflect poor handling practices during the pre-slaughter operations and result in economic damage due to loss of edible parts, labour cost for trimming and general carcass value depreciation. The aim of this work was to evaluate the possible relationship between some behaviour observed during handling before slaughter and the presence of bruises on bull carcasses.

Identification of polymorphisms in the rabbit (Oryctolagus cuniculus) myostatin (MSTN) gene and association analysis with finishing weight in a commercial rabbit population

Background: Myostatin (MSTN or GDF8) is a member of the transforming growth factor-b superfamily that actively represses skeletal muscle growth. Myostatin null mice generated by gene targeting showed a 2to 3-fold increase in muscle mass compared to wild type mice, primarily because of an increased number of muscle fibres, followed by muscle cell hypertrophy and suppression of body fat accumulation. Several mutations disrupting myostatin function or expression cause doublemuscle phenotypes, muscle hypertrophy, birthing difficulties, increased growth rate and increased feed conversion efficiency in cattle and sheep. Following the initiative of the Broad Institute shotgun sequencing of the rabbit genome (Ensembl Gene ID ENSOCUG00000012663 in the OryCun2 rabbit genome assembly), the rabbit MSTN gene sequence was recently assembled. No study has been carried out so far to investigate its variability or the effects of MSTN polymorphisms on rabbit production traits.