Valentina Riggio

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.

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.

Genetic parameters for early lamb survival and growth in Scottish Blackface sheep.

The objectives of this study were 1) to estimate the heritability of lamb survival and growth in the Scottish Blackface breed; 2) to examine the relationship between lamb survival and live BW; and 3) to investigate the possibility of using lamb survival in a breeding program for this breed. The data used for the analyses contained information about survival and live BW at different ages on 4,459 animals. The records were collected from 1988 to 2003 in a Scottish Blackface flock. Live BW was recorded every 4 wk from birth to 24 wk. Survival was defined either by perinatal or postnatal mortality (up to weaning at 12 wk), or as cumulative survival to 1, 4, 8, and 12 wk. The pedigree file comprised 1,416 dams and 178 sires. A sire model was used to estimate genetic parameters for binary survival traits. Heritabilities of BW traits, and phenotypic and genetic correlations between BW and between survival and BW were estimated by fitting an animal model. Further, correlations of survival with live BW were estimated by using a Markov chain Monte Carlo threshold model, implemented by Gibbs sampling. The heritability estimates for cumulative lamb survival declined from birth onward (from 0.33 to 0.08), and postnatal survival had a heritability of 0.01. The direct and maternal heritabilities for BW traits ranged from 0.08 to 0.26 and from 0.06 to 0.21, respectively, whereas the maternal environmental component was between 0.04 and 0.16. The genetic correlations between BW traits at different ages were high. The genetic and phenotypic correlations between survival and BW were always positive (ranging from 0.04 to 0.54), so there was no antagonism between these traits. Therefore, it is possible to simultaneously improve both survival and live BW in a breeding program for this breed.

Genome-wide association and regional heritability mapping to identify loci underlying variation in nematode resistance and body weight in Scottish Blackface lambs

The genetic architecture underlying nematode resistance and body weight in Blackface lambs was evaluated comparing genome-wide association (GWA) and regional heritability mapping (RHM) approaches. The traits analysed were faecal egg count (FEC) and immunoglobulin A activity against third-stage larvae from Teladorsagia circumcincta, as indicators of nematode resistance, and body weight in a population of 752 Scottish Blackface lambs, genotyped with the 50k single-nucleotide polymorphism (SNP) chip. FEC for both Nematodirus and Strongyles nematodes (excluding Nematodirus), as well as body weight were collected at approximately 16, 20 and 24 weeks of age. In addition, a weighted average animal effect was estimated for both FEC and body weight traits. After quality control, 44 388 SNPs were available for the GWA analysis and 42 841 for the RHM, which utilises only mapped SNPs. The same fixed effects were used in both analyses: sex, year, management group, litter size and age of dam, with day of birth as covariate. Some genomic regions of interest for both nematode resistance and body weight traits were identified, using both GWA and RHM approaches. For both methods, strong evidence for association was found on chromosome 14 for Nematodirus average animal effect, chromosome 6 for Strongyles FEC at 16 weeks and chromosome 6 for body weight at 16 weeks. Across the entire data set, RHM identified more regions reaching the suggestive level than GWA, suggesting that RHM is capable of capturing some of the variation not detected by GWA analyses.

Genome-wide analysis of Italian sheep diversity reveals a strong geographic pattern and cryptic relationships between breeds.

Italy counts several sheep breeds, arisen over centuries as a consequence of ancient and recent genetic and demographic events. To finely reconstruct genetic structure and relationships between Italian sheep, 496 subjects from 19 breeds were typed at 50K single nucleotide polymorphism loci. A subset of foreign breeds from the Sheep HapMap dataset was also included in the analyses. Genetic distances (as visualized either in a network or in a multidimensional scaling analysis of identical by state distances) closely reflected geographic proximity between breeds, with a clear north-south gradient, likely because of high levels of past gene flow and admixture all along the peninsula. Sardinian breeds diverged more from other breeds, a probable consequence of the combined effect of ancient sporadic introgression of feral mouflon and long-lasting genetic isolation from continental sheep populations. The study allowed the detection of previously undocumented episodes of recent introgression (Delle Langhe into the endangered Altamurana breed) as well as signatures of known, or claimed, historical introgression (Merino into Sopravissana and Gentile di Puglia; Bergamasca into Fabrianese, Appenninica and, to a lesser extent, Leccese). Arguments that would question, from a genomic point of view, the current breed classification of Bergamasca and Biellese into two separate breeds are presented. Finally, a role for traditional transhumance practices in shaping the genetic makeup of Alpine sheep breeds is proposed. The study represents the first exhaustive analysis of Italian sheep diversity in an European context, and it bridges the gap in the previous HapMap panel between Western Mediterranean and Swiss breeds.

A joint analysis to identify loci underlying variation in nematode resistance in three European sheep populations.

Gastrointestinal nematode infections are one of the main health/economic issues in sheep industries, worldwide. Indicator traits for resistance such as faecal egg count (FEC) are commonly used in genomic studies; however, published results are inconsistent among breeds. Meta (or joint)-analysis is a tool for aggregating information from multiple independent studies. The aim of this study was to identify loci underlying variation in FEC, as an indicator of nematode resistance, in a joint analysis using data from three populations (Scottish Blackface, Sarda × Lacaune and Martinik Black-Belly × Romane), genotyped with the ovine 50k SNP chip. The trait analysed was the average animal effect for Strongyles and Nematodirus FEC data. Analyses were performed with regional heritability mapping (RHM), fitting polygenic effects with either the whole genomic relationship matrix or matrices excluding the chromosome being interrogated. Across-population genomic covariances were set to zero. After quality control, 4123 animals and 38 991 SNPs were available for the analysis. RHM identified genome-wide significant regions on OAR4, 12, 14, 19 and 20, with the latter being the most significant. The OAR20 region is close to the major histocompatibility complex, which has often been proposed as a functional candidate for nematode resistance. This region was significant only in the Sarda × Lacaune population. Several other regions, on OAR1, 3, 4, 5, 7, 12, 19, 20 and 24, were significant at the suggestive level.

Genome-wide association study of tick resistance in South African Nguni cattle.

Ticks and tick-borne diseases are among the main causes of economic loss in the South African cattle industry through high morbidity and mortality rates. Concerns of the general public regarding chemical residues may tarnish their perceptions of food safety and environmental health when the husbandry of cattle includes frequent use of acaricides to manage ticks. The primary objective of this study was to identify single nucleotide polymorphism (SNP) markers associated with host resistance to ticks in South African Nguni cattle. Tick count data were collected monthly from 586 Nguni cattle reared in four herds under natural grazing conditions over a period of two years. The counts were recorded for six species of ticks attached in eight anatomical locations on the animals and were summed by species and anatomical location. This gave rise to 63 measured phenotypes or traits, with results for 12 of these traits being reported here. Tick count (x) data were transformed using log10(x+1) and the resulting values were examined for normality. DNA was extracted from hair and blood samples and was genotyped using the Illumina BovineSNP50 assay. After quality control (call rate >90%, minor allele frequency >0.02), 40,436 SNPs were retained for analysis. Genetic parameters were estimated and association analysis for tick resistance was carried out using two approaches: a genome-wide association (GWA) analysis using the GenABEL package and a regional heritability mapping (RHM) analysis. The Bonferroni genome-wide (P<0.05) corrected significance threshold was 1.24×10(-6), with 2.47×10(-5) as the suggestive significance threshold (P<0.10) (i.e., one false positive per genome scan) in the GWA analysis. Likelihood ratio test (LRT) thresholds for genome-wide and suggestive significance were 13.5 and 9.15 for the RHM analysis. Six ixodid tick species were identified, with Amblyomma hebraeum (the vector for Heartwater disease) being the dominant species. Heritability estimates (h(2)) from the fitted animal and sire models ranged from 0.02±0.00 to 0.17±0.04 for the transformed tick count data. Several genomic regions harbouring quantitative trait loci (QTL) were identified for different tick count traits by both the GWA and RHM approaches. Three genome-wide significant regions on chromosomes 7, 10 and 19 were identified for total tick count on the head, total body A. hebraeum tick count and total A. hebraeum on the perineum region, respectively. Additional regions significant at the suggestive level were identified on chromosomes 1, 3, 6, 7, 8, 10, 11, 12, 14, 15, 17, 19 and 26 for several of the traits. The GWA approach identified more genomic regions than did the RHM approach. The chromosomal regions identified here as harbouring QTL underlying variation in tick burden form the basis for further analyses to identify specific candidate genes and polymorphisms related to cattle tick resistance and provide the potential for marker-assisted selection in Nguni cattle.

Study of polymorphisms in the promoter region of ovine β-lactoglobulin gene and phylogenetic analysis among the Valle del Belice breed and other sheep breeds considered as ancestors.

The aim of this work was to sequence the promoter region of β-lactoglobulin (BLG) gene in four sheep breeds, in order to identify polymorphisms, infer and analyze haplotypes, and phylogenetic relationship among the Valle del Belice breed and the other three breeds considered as ancestors. Sequencing analysis and alignment of the obtained sequences showed the presence of 36 single nucleotide polymorphisms (SNPs) and one deletion. A total of 22 haplotypes found in “best” reconstruction were inferred considering the 37 polymorphic sites identified. Haplotypes were used for the reconstruction of a phylogenetic tree using the Neighbor-Joining algorithm. The number of polymorphisms identified showed high variability within breeds. Analysis of genetic diversity indexes showed that the Sarda breed presented the lowest nucleotide diversity, whereas the Comisana breed presented the highest one. Comparing the nucleotide diversity among breeds, the highest value was obtained between Valle del Belice and Pinzirita breeds, whereas the lowest one was between Valle del Belice and Sarda breeds. Considering that polymorphisms in the promoter region of BLG gene could have a functional role associated with milk composition, the lowest value of nucleotide diversity between Valle del Belice and Sarda breeds may be related to a higher similarity of milk composition of these two breeds compared to the others. Further analyses will be conducted in order to evaluate the possible correlation between the genetic diversity indexes and the BLG content in milk of our breeds.

Breeding for resistance to gastrointestinal nematodes - the potential in low-input/output small ruminant production systems.

Highlights • Resistance to anthelmintics in small ruminants is documented worldwide.• Genetic resistance in GIN have been studied in experimental and commercial flocks.• Limited in low-input low-output farming systems.• Nematode eradication has evolved to manipulation of host-parasite equilibrium.• Selection for resistant hosts can be considered a sustainable control strategy.