Baldassare Portolano

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.

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.

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.

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.

Estimation of the genetic and phenotypic variance of several growth traits of the Sicilian Girgentana goat

Abstract The possibility of increasing the age at slaughter of kids, from 25 to 45–60 days, without affecting meat quality, led to this study of the source of variation of several growth traits in the Girgentana goat. Data used in this study were collected between 1998 and 2000 from a total of 276 kids (118 males and 158 females), the progeny of 23 sires and 109 dams. For each individual the identification number, pedigree, sex, body weight at birth and every 15 days up to the age of 60 days and the individual’s maternal litter size were recorded. A sire model was used in order to estimate genetic parameters and correlations between all traits considered, including, in total, 94 animals from 23 sires. This data-set was made up of nine generations. As fixed effects, the mixed sire model included year of birth, age of dam at birth of the offspring and individual’s maternal litter size. Sire within year of birth and the residuals were used as random effects. The genetic correlations between body weight at birth (BW0)/body weight at 30 days (BW30) and body weight at 60 days (BW60) showed the possibility of indirect selection for BW60, by selecting for BW0 or BW30. These genetic correlations with BW60 were 0.86 and 0.90, respectively for BW0 and BW30. Furthermore, body weight heritability ranged from 0.20 to 0.49 at different ages. Average daily gain (ADG) was also considered in four successive 15 day periods starting at birth and up to 60 days (ADG1–4). The ADG heritability estimates were very low and ranged from 0.06±0.026 (ADG2 and 3) to 0.16±0.040 (ADG4). The genetic correlations between ADG1 with 2–4 were negative and tended to zero with increasing age. The phenotypic correlations, on the contrary, increased (from 0.33 to 0.82) with the increase of age. The genetic correlations between BW and ADG traits ranged from −0.32 to 0.93; the phenotypic correlations were lower than the genetic correlations, but showed an analogous trend.

Application of the Wood model to lactation curves of Comisana sheep

Abstract Ninety-two ewes of the Comisana breed were used in a study to verify the forecasting capacity of the mathematical model proposed by Wood for fitting sheep data, and to evaluate the effect of the environmental factors affecting the parameters of the lactation curve. The Wood model underestimated the total lactation milk production with an error margin of 12.2 ± 7.8 kg of milk corresponding to 10.9 ± 6.6%. There was a strong influence of order of lambing × lambing season interaction on the lactation parameters. The curves for the winter lambing ewes demonstrated a higher peak production than autumn lambing ewes (1728.4 vs. 1082.6 g, P ≤ 0.05 for the second lambing ewes and 1772.8 vs. 1383.7 g, P ≤ 0.01 for ≥ 3 lambing ewes); the season persistence difference was small, 4.88 vs. 5.60 for second lambing and 5.32 vs. 5.33 for ≥ 3 lambings. Simple correlations within lambing season showed negative correlation between the peak production and the persistence of lactation ( r a = −0.32 and r w = −0.15). The time at peak and the persistence were positively correlated ( r a = 0.88 and r w = 0.97). Parity was positively correlated with peak production ( r a = 0.41and r w = 0.50) and negatively correlated with the time at the peak ( r a = −0.22 and r w = −0.39) and the persistence ( r a = −0.25 and r w = −0.41). The linear regression of peak production on total production referring to the period from the 31st day post-partum to lactation end was significant. The type of lambing did not significantly influence the shape of the lactation curve although sheep lambing to twins produced more milk than those having single lambs.

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.

Demographic characterization and genetic variability of the Girgentana goat breed by the analysis of genealogical data

Abstract The aim of this paper is to present an overview of the actual Sicilian Girgentana population structure by the analysis of genealogical data. Statistics show that in 1983 the population consisted of 30,000 Girgentana goats; ten years later almost 98% of the entire Girgentana population disappeared. The remaining population consists of 461 individuals (134 males and 327 females), with 368 living animals. The effective population size is 380 individuals. The inbreeding rate per generation was equal to 0.13%. The average estimated inbreeding level within the living male population was equal to 0.8% (0-15%); and the average inbreeding level within the living female population was equal to 0.7% (0-31%). The average relationship between males and females estimated on 27,772 possible matings was equal to 0.5% (0-8.7%). The estimated inbreeding level was not high due to the lack of pedigree information. This is resulting in a ratio between the number of founder equivalents (ƒe = 22,94) and the number of absolute founders (ƒa = 93) equal to 25%.

The hairless (hr) gene is involved in the congenital hypotrichosis of Valle del Belice sheep

Congenital hypotrichosis in mammalian species consists of partial or complete absence of hair at birth. The hairless gene is often responsible for this disorder in men, mice and rats. Recent experimental data on Valle del Belice sheep reared in Sicily for milk production, support the genetic control of the ovine hypotrichosis as a Mendelian recessive trait. The ovine hairless gene was chosen as the candidate gene involved in this disorder. Blood samples were collected from Valle del Belice sheep with the normal and hypotrichotic phenotypes. Almost the entire hairless gene was successfully amplified using the long PCR technique. Unrelated sheep with differing phenotypes were randomly chosen for sequencing the amplified products. Different mutations related to the hypotrichotic phenotype were found in exon 3. In fact, sequencing revealed an A/T transversion at position 739, a G/A transition at position 823, and a C/T transition at position 1312. From these nucleotide exchanges, three substitutions of the processed mature protein were deduced at the amino acid positions 247 (Thr/Ser), 275 (Ala/Thr), and 438 (Gln/Stop). A PCR-SSCP based test was developed in order to detect the last mutation, which is responsible for the hypotrichotic phenotype.

Genome wide linkage disequilibrium and genetic structure in Sicilian dairy sheep breeds

BackgroundThe recent availability of sheep genome-wide SNP panels allows providing background information concerning genome structure in domestic animals. The aim of this work was to investigate the patterns of linkage disequilibrium (LD), the genetic diversity and population structure in Valle del Belice, Comisana, and Pinzirita dairy sheep breeds using the Illumina Ovine SNP50K Genotyping array.ResultsAverage r2 between adjacent SNPs across all chromosomes was 0.155 ± 0.204 for Valle del Belice, 0.156 ± 0.208 for Comisana, and 0.128 ± 0.188 for Pinzirita breeds, and some variations in LD value across chromosomes were observed, in particular for Valle del Belice and Comisana breeds. Average values of r2 estimated for all pairwise combinations of SNPs pooled over all autosomes were 0.058 ± 0.023 for Valle del Belice, 0.056 ± 0.021 for Comisana, and 0.037 ± 0.017 for Pinzirita breeds. The LD declined as a function of distance and average r2 was lower than the values observed in other sheep breeds. Consistency of results among the several used approaches (Principal component analysis, Bayesian clustering, FST, Neighbor networks) showed that while Valle del Belice and Pinzirita breeds formed a unique cluster, Comisana breed showed the presence of substructure. In Valle del Belice breed, the high level of genetic differentiation within breed, the heterogeneous cluster in Admixture analysis, but at the same time the highest inbreeding coefficient, suggested that the breed had a wide genetic base with inbred individuals belonging to the same flock. The Sicilian breeds were characterized by low genetic differentiation and high level of admixture. Pinzirita breed displayed the highest genetic diversity (He, Ne) whereas the lowest value was found in Valle del Belice breed.ConclusionsThis study has reported for the first time estimates of LD and genetic diversity from a genome-wide perspective in Sicilian dairy sheep breeds. Our results indicate that breeds formed non-overlapping clusters and are clearly separated populations and that Comisana sheep breed does not constitute a homogenous population. The information generated from this study has important implications for the design and applications of association studies as well as for development of conservation and/or selection breeding programs.