Stefania Dall'Olio

An initial comparative map of copy number variations in the goat (Capra hircus) genome

BackgroundThe goat (Capra hircus) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome.ResultsWe identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P < 0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals.ConclusionsWe describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs and their effects on behavior, production, and disease resistance traits in goats.

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.

Combined effects of pre-slaughter treatments and lairage time on carcass and meat quality in pigs of different halothane genotype

The effect of two different lairage times on meat quality was investigated in pigs with different halothane genotype previously submitted to controlled pre-slaughter treatments. One hundred and ninety nine Italian heavy pigs were loaded by ramp or lift and transported unmixed for 1 h to the abattoir at a stocking density of either <0.4 or >0.6 m(2) per 100 kg pigs. After unloading, an equal number of animals within each previous treatment was held in lairage for 2 h or overnight (22 h) before slaughter. Carcass and meat quality and incidence of skin damage were evaluated. Longer lairage did not negatively affect carcass traits and reduced the incidence of PSE meat without increased DFD occurrence. It also seriously increased the amount of skin damage due to fighting, resulting in long fasting. Pre-slaughter treatments and halothane genotype showed a negligible effect on the response to the resting time.

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.

Analysis of Horse Myostatin Gene and Identification of Single Nucleotide Polymorphisms in Breeds of Different Morphological Types

Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus) MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T>C and GQ183900:g.156T>C, the latter located within a conserved TATA-box like motif) were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed). The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA) on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds.

A melanocortin 1 receptor ( MC1R ) gene polymorphism is useful for authentication of Massese sheep dairy products

Massese is an Italian sheep breed, with black or grey coat colour, mainly reared in the Tuscany and Emilia Romagna regions. Recently, the emerging interests in this breed have resulted in the production of Pecorino cheese obtained with only Massese milk. In order to be profitable, this marketing link between Massese breed and its products should be defended against fraudsters who could include milk of other sheep breeds or cow milk in Massese labelled productions. To identify the genetic factors affecting coat colour in sheep, we have recently analysed the melanocortin 1 receptor (MC1R) gene and identified several single nucleotide polymorphisms (SNPs). In this work, as a first step to set up a DNA based protocol for authentication of Massese dairy products, we further investigated the presence and distribution of one of these SNPs (c.-31G>A) in 143 Massese sheep and in another 13 sheep breeds (for a total of 351 animals). The Massese breed was fixed for allele c.-31A, whereas in all other breeds allele c.-31 G was the most frequent or with frequency of 0·50. At the same nucleotide position the cattle MC1R gene carries the G nucleotide. Using these data we developed a method to detect adulterating milk (from other sheep breeds or from cow) in Massese dairy products based on the analysis of the c.-31G>A SNP. We first tested the sensitivity of the protocol and then applied it to analyse DNA extracted from ricotta and Pecorino cheese obtained with only Massese milk or obtained with unrestricted sheep and cattle milk. To our knowledge, this system represents the first one that can be used for breed authentication of a sheep production and that, at the same time, can reveal frauds derived from the admixture of milk of an unreported species.

A Missense Mutation in the Rabbit Melanocortin 4 Receptor (MC4R) Gene is Associated with Finisching Weight in a Meat Rabbit Line

In this study we resequenced 1729 bp of the rabbit melanocortin 4 receptor (MC4 R) gene in 31 rabbits from different breeds/lines and identified ten polymorphisms: one was an indel and 9 were single nucleotide polymorphisms (SNPs). The indel and 5 SNPs were in the 5′-flanking region, 3 were synonymous SNPs and one was a missense mutation (c.101G>A; p.G34D), located in a conserved position of the extracellular tail of the MC4 R protein. The missense mutation was analyzed in a panel of 74 rabbits of different breeds and in 516 performance tested rabbits of a commercial paternal line under selection for growth efficiency. Association analysis indicated that rabbits with the less frequent genotype in this population (DD) had a lighter weight at 70 postnatal days than animals with genotype GD (P < 0.10) and animals with genotype GG (P < 0.05). This is the third study on candidate genes, after those on GH1 and IGF2 that reported a marker associated with finishing weight. Therefore, it seems that a candidate gene approach in rabbit based on previous information accumulated in other livestock species could be useful to identify genes explaining a fraction of variability of performance traits with potential application on rabbit breeding and selection.

Bovine β-lactoglobulin H: isolation by preparative isoelectric focusing in immobilized pH gradients and preliminary characterization

In spite of the fact that beta-lactoglobulin (beta-lg) was first discovered in bovine milk more than fifty years ago, and that it represents the main whey protein component in all the milks in which it has been found, its biological role and genetic evolution still remain rather uncertain. From comparative studies of the primary and tertiary structures of beta-lg and of other proteins of a similar size, the existence of a new superfamily of proteins with the function of transporter of hydrophobic molecules has been conjectured. The elucidation of the structure of beta-lg either from different species or from different genetic variants of the same species should give useful information on the evolution and function of this protein family. With this aim in mind we have now undertaken the isolation and characterization of a recently discovered, new genetic variant of bovine beta-lg. A two-step purification procedure involving preparative HPLC gel filtration and preparative IEF-IPG has been successfully carried out; it affords a good recovery of the new beta-lg in highly purified form.

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.