A comparison of genetic and genomic breeding values in Saanen and Alpine goats.

Nowadays, several countries are developing or adopting genomic selection in the dairy goat sector. The most used method to estimate breeding values is Single-Step Genomic Best Linear Unbiased Prediction (ssGBLUP) which offers several advantages in terms of computational process and accuracy of the estimated breeding values (EBVs). Saanen and Alpine are the predominant dairy goat breeds in Italy, and both have similar breeding programs where EBVs for productive traits are currently calculated using BLUP. This work describes the implementation of genomic selection for these two breeds in Italy, aligning with the selection practices already carried out in the international landscape. The available dataset included 3 611 genotyped animals, 11 470 lactation records, five traits (milk, protein and fat yields, and fat and protein percentages), and three-generation pedigrees. EBVs were estimated using BLUP, GBLUP, and ssGBLUP both with single and multiple trait approaches. The methods were compared in terms of correlation between EBVs and genetic trends. Results were also validated with the linear regression method excluding part of the phenotypic data. In both breeds, EBVs and GEBVs were strongly correlated and the trend of each trait was similar comparing the three methods. The average increase in accuracy across traits and methods amounted to +13 and +10% from BLUP to ssGBLUP for Alpine and Saanen breeds, respectively. Results indicated higher prediction accuracy and correlation for GBLUP and ssGBLUP compared to BLUP, implying that the use of genotypes increases the accuracy of EBVs, particularly in the absence of phenotypic data. Therefore, ssGBLUP is likely to be the most effective method to enhance genetic gain in Italian Saanen and Alpine goats.

Investigating the population structure and genetic differentiation of livestock guard dog breeds.

Livestock guarding dogs are a valuable adjunct to the pastoral community. Having been traditionally selected for their working ability, they fulfil their function with minimal interaction or command from their human owners. In this study, the population structure and the genetic differentiation of three Italian livestock guardian breeds (Sila's Dog, Maremma and Abruzzese Sheepdog and Mannara's Dog) and three functionally and physically similar breeds (Cane Corso, Central Asian Shepherd Dog and Caucasian Shepherd Dog), totalling 179 dogs unrelated at the second generation, were investigated with 18 autosomal microsatellite markers. Values for the number of alleles per locus, observed and expected heterozygosity, Hardy-Weinberg Equilibrium, F stats, Nei's and Reynold's genetic distances, clustering and sub-population formation abilities and individual genetic structures were calculated. Our results show clear breed differentiation, whereby all the considered breeds show reasonable genetic variability despite small population sizes and variable selection schemes. These results provide meaningful data to stakeholders in specific breed and environmental conservation programmes.

A method for single nucleotide polymorphism selection for parentage assessment in goats.

Accurate pedigrees are essential to optimize genetic improvement and conservation of animal genetic resources. In goats, the use of mating groups and kidding management procedures hamper the identification of parentage. Small panels of single nucleotide polymorphisms (SNP) have been proposed in other species to substitute microsatellites for parentage assessment. Using data from the current GoatSNP50 chip, we developed a new 3-step procedure to identify a low-density SNP panel for highly accurate parentage assessment. Methodologies for SNP selection used in other species are less suitable in the goat because of uncertainties in the genome assembly. The procedure developed in this study is based on parent-offspring identification and on estimation of Mendelian errors, followed by canonical discriminant analysis identification and stepwise regression reduction. Starting from a reference sample of 109 Alpine goats with known pedigree relationships, we first identified a panel of 200 SNP that was further reduced to 2 final panels of 130 and 114 SNP with random coincidental match inclusion of 1.51×10(-57) and 2.94×10(-34), respectively. In our reference data set, all panels correctly identified all parent-offspring combinations, revealing a 40% pedigree error rate in the information provided by breeders. All reference trios were confirmed by official tests based on microsatellites. Panels were also tested on Saanen and Teramana breeds. Although the testing on a larger set of breeds in the reference population is still needed to validate these results, our findings suggest that our procedure could identify SNP panels for accurate parentage assessment in goats or in other species with unreliable marker positioning.

Short communication: the unusual genetic trend of αS1-casein in Alpine and Saanen breeds.

Genetic variation at the αS1-casein locus (CSN1S1) is recognized as being crucial in the selection of dairy goats for cheese yield. At this locus, the existence of alleles that have strong, intermediate, weak, and null favorable effects on cheese yield and curd firmness is well known. Selection for alleles that have a strong favorable effect has been deliberately carried out, especially in France. In fact, the importance of αS1-casein in selection was recently confirmed in the selling policies of semen, where bucks are marketed according to their genotypes. We evaluated genotypes and alleles frequencies at the αS1-casein locus in 491 Italian Saanen and Alpine goats and compared them with previous data to investigate their evolution over the past decade. We also estimated soft cheese yield in a subset of the most represented genotypes to quantify the economic importance of considering the genetic trend of αS1-casein genotype frequencies. We found a significant increase in frequency of the allele with the strongest favorable effect, A (+12 and +13%), and of the intermediate allele E (+17 and +7%) in Saanen and Alpine goats, respectively. Surprisingly, the frequency of the strong allele B decreased strikingly over time (-12% in Saanen, -6% in Alpine from 2004 to 2012). This is consistent with the current marketing of semen, in that bucks that are homozygous for strong (AA and BB) and intermediate alleles (EE) and even heterozygous for these alleles (BE and AE) are considered equal. It is worth noting that this practice strongly penalizes the best breeders that have flocks composed almost entirely of goats that are homozygous for strong alleles. For heterozygous goats, we estimated an economic loss of €85 and €215 per goat per lactation, respectively, for AE and BE, compare with AA and BB genotypes. The marketing of buck semen should clearly differentiate these 2 alleles to ensure the best economic genetic progress at this locus.

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.

Associations of acetyl-coenzyme A carboxylase α, stearoyl-coenzyme A desaturase, and lipoprotein lipase genes with dairy traits in Alpine goats.

Milk yield and composition are of great economic importance for the dairy goat industry. The identification of genes associated with phenotypic differences for these traits could allow for the implementation of gene-assisted selection programs in goats. Associations between polymorphisms at 3 candidate genes and milk production traits in Alpine goats farmed in Italy were investigated in the present research. Considered genes were acetyl-coenzyme A carboxylase α (ACACA), the major regulatory enzyme of fatty acid biosynthesis; stearoyl-coenzyme A desaturase (SCD), involved in the biosynthesis of monounsaturated fatty acids in the mammary gland; and lipoprotein lipase (LPL), which plays a central role in plasma triglyceride metabolism. An approach somewhat similar to the granddaughter design for detecting quantitative trait loci in dairy cattle was followed. Effects of genotypes of a sample of 59 Alpine bucks on phenotypes of their 946 daughters raised in 75 flocks were investigated. Data comprised 13,331 daily records for milk yields (L/d), fat and protein yields (kg/d), and fat and protein contents (%) of 2,200 lactations. Population genetics parameters were calculated and associations between milk production traits and 10 single nucleotide polymorphisms (SNP) at the 3 genes were tested. Two markers at the ACACA, 1 for the SCD and 1 at the LPL locus, deviated significantly from the Hardy-Weinberg equilibrium, with an observed heterozygosity lower than expected. Flock, age of the goat, kidding season, and stage of lactation affected all traits considered, except fat percentage. Three SNP were found to be significantly associated with milk production traits. The SNP located on the ACACA gene showed an effect on milk yield, with daughters of TT bucks having an average test-day milk yield of about 0.3 to 0.25 L/d lower than the other 2 genotypes. The marker on the LPL locus was highly associated with milk yield, with the largest values for CC daughters (about 0.50L more than GG). The TGT deletion located on the untranslated region of the SCD gene showed significant effects on average milk and protein yields. The homozygote-deleted genotype had values about 0.5 L/d and 16 g/d lower for milk and protein daily yield, respectively, compared with the TGT/TGT genotype. Differences between genotypes were quite constant across most of the lactation. Associations found in the present study, which should be tested in a larger sample, especially for those markers that show rare genotypes, may offer useful indications for the genetic improvement of dairy traits in goats.

On the way to functional agro biodiversity: coat colour gene variability in goats.

Functional agro biodiversity defines the exploitation of biodiversity to provide ecosystem services, support sustainable agricultural production and benefit the regional and global environment and the public at large (ELN-FAB, 2009; www.eln_fab.eu). Tracking of animal products back to the breed of origin based on their genetic make-up undoubtedly falls in this category. The aim of this paper was to identify and validate a set of single nucleotide polymorphisms (SNPs) in goat coat colour genes, most of which have not been investigated before, to trace five goat populations of the Italian Alps and their product. Several regions of 28 genes influencing coat colour pathways were amplified in eight animals (two per breed). Sequence comparison revealed 48 SNPs and three INDEL (INsertion DELetion). No breed-specific alleles were detected; however, several SNPs showed an uneven frequency distribution between breeds. In BIO, the genotype frequency distribution of a non-synonymous SNP suggested a possible role of TYRP1 in brown eumelanic goat coat colour. A total of 29 independent SNPs in 20 genes were selected and used to allocate 159 minimally related goat samples using STRUCTURE 2.2 and GeneClass 2 software. STRUCTURE 2.2 assigns 99% of individuals to the correct breed considering the prior information on putative breed of origin for each sample and 81% using only the genotypic data. The three algorithms available in GeneClass 2 performed with nearly equal efficiency, with 86% and 87% correct allocations. All the methods yielded an average probability of assignment >0.92 and a specificity index >0.86. Despite their coat colour variability, individuals belonging to ORO were fully assigned, showing that, in the absence of a breed-specific allele tied to coat colour, the best assignment resulted for the most genetically distinct breed. The lowest rate of correct assignment was observed in Verzaschese (73%), not ascertained in the breed panel used in the SNP discovery phase.

Molecular cloning, promoter analysis and SNP identification of Italian Nicastrese and Saanen lactoferrin gene.

Lactoferrin (Lf) is an iron-binding glycoprotein found in exocrine secretions including milk. High levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. In this report we sequenced and characterized goat lactoferrin cDNA and its promoter region in two different breeds of goat. The complete cDNA comprised 2356 nucleotides, including 38 bp at the 5'-UTR and 194 bp at the 3'-UTR. The open reading frame is 2127 bp long and it encodes a mature protein of 689 aminoacids. A total of 19 nucleotide differences, 11 of them being responsible for 8 aminoacid changes, were identified through the comparison with French, Korean and Tibetan goat lactoferrin cDNAs. About 1700 bp of the lactoferrin gene promoter were sequenced. Sequence analysis revealed a non-canonical TATA box, multiple SP1/GC elements, and other putative binding sites for transcription factors, such as NF-kappaB, STAT3 and AP2. Two SNPs were identified, one of which would seem to create a new putative AP2 consensus sequence. The presence of an additional AP2 binding site could be associated with quantitative differences of such protein fraction, which could enhance all the activities related to such protein, and improve mammary gland defence against bacterial infections.

Assessing SNP markers for assigning individuals to cattle populations.

The effectiveness of single nucleotide polymorphisms (SNPs) for the assignment of cattle to their source breeds was investigated by analysing a panel of 90 SNPs assayed on 24 European breeds. Breed assignment was performed by comparing the Bayesian and frequentist methods implemented in the STRUCTURE 2.2 and GENECLASS 2 software programs. The use of SNPs for the reallocation of known individuals to their breeds of origin and the assignment of unknown individuals was tested. In the reallocation tests, the methods implemented in STRUCTURE 2.2 performed better than those in GENECLASS 2, with 96% vs. 85% correct assignments respectively. In contrast, the methods implemented in GENECLASS 2 showed a greater correct assignment rate in allocating animals treated as unknowns to a reference dataset (62% vs. 51% and 80% vs. 65% in field tests 1 and 2 respectively). These results demonstrate that SNPs are suitable for the assignment of individuals to reference breeds. The results also indicate that STRUCTURE 2.2 and GENECLASS 2 can be complementary tools to assess breed integrity and assignment. Our findings also stress the importance of a high-quality reference dataset in allocation studies.

Stearoyl CoA desaturase (SCD) gene polymorphisms in Italian cattle breeds.

Stearoyl CoA desaturase (SCD) is the key enzyme involved in the endogenous synthesis of conjugated linoleic acid (CLA) in ruminants. Changes in the enzymatic activity as a result of SCD gene polymorphism and regulation have been hypothesized to cause diet-independent variations of CLA content in milk. Evidences for the direct influence of SCD polymorphism on fatty acid composition of milk and beef have also been reported. To evaluate genetic differences because of breed and/or selection goal, we investigated the polymorphism of three previously reported single nucleotide polymorphisms located in exon 5 of the SCD gene in 11 cattle breeds raised in Italy and selected for different production goals. Results obtained: (i) evidenced a high variability in the allele frequencies across breeds; (ii) detected three novel haplotypes, one of which is private to indigenous beef breeds, and (iii) showed a significant association between haplotypes and selective goal.

Traceability of four European Protected Geographic Indication (PGI) beef products using Single Nucleotide Polymorphisms (SNP) and Bayesian statistics.

The use of SNPs in combination with Bayesian statistics for the geographic traceability of cattle was evaluated using a dataset comprising 24 breeds from Italy, France, Spain, Denmark, the Netherlands, Switzerland and UK genotyped with 90 polymorphic markers. The percentage of correct assignment of the individuals to their Country of origin was 90%, with an average assignment probability of 93% and an average specificity of 92%. The higher value was observed for UK breeds (97% of correct assignment) while Swiss animals were the most difficult to allocate (77% of correct assignment). Tracing of Protected Geographic Indication (PGI) products, the approach correctly assigned 100% of Guaranteed Pure Highland Beef; 97% of "Vitellone dell'Appennino Centrale" breeds; 84% of Ternera de Navarra, and 80% of Boeuf de Chalosse. Methods to verify Products of Designated Origin (PDO) and Protected Geographic Indication (PGI) products will help to protect regional foods and promote the economic growth of marginal rural areas by encouraging the production of high quality niche market foods.

Breed assignment of Italian cattle using biallelic AFLP markers.

The verification of the breed origin of animal products is relevant for food safety and authenticity. We assessed the suitability of AFLP molecular markers in the assignment of cattle individuals to their breed of origin. Three hundred and ninety-six animals belonging to 16 cattle breeds genotyped with 141 AFLP markers were used as reference data set. Assignment was performed with likelihood (aflpop) and Bayesian (structure) methods. The Bayesian approach was superior to the likelihood algorithm with respect to (i) the correct assignment of simulated individuals to their breed of origin (93% vs. 81% respectively), (ii) the correct assignment of 44 sampled Romagnola animals (91% vs. 45% respectively) and (iii) the correct classification of animals belonging to a breed that was not included within the reference dataset. Thus, AFLP profiling in combination with the Bayesian approach seems a useful tool for breed assignment.

Fertility of cryopreserved sperm in three bulls with different Robertsonian translocations.

The fertility of three bulls carrying different Robertsonian translocations (rob(1;29), rob(14;17) and rob(26;29)) was evaluated. Oocytes-cumulus complexes obtained from slaughterhouse-derived ovaries were matured and then fertilised in vitro with frozen/thawed seminal material from the above mentioned subjects, and from control bulls with normal karyotype. An assessment was first made of the concentration, vitality and acrosome integrity of the seminal material to be sure that possible differences in the results of the in vitro fertilisation experiments were not due to seminal material quality. The results of the experiments, evaluated by the percentage of cleaved embryos and blastocysts per cleaved embryo, indicated that the three bulls carrying Robertsonian translocations had similar fertilising power and semen qualitative parameters to the controls. These data suggest that neither gametogenesys impairment nor decreased spermatozoa fertilising capacity is responsible for the reduced fertility in bulls with Robertsonian translocations. What the data do confirm is that the observed in vivo hypofertility for karyologically abnormal bulls is mainly due to early embryonic mortality.

Assessing genetic diversity in Italian goat populations using AFLP markers.

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variation in a sample of seven goat (Capra hircus) populations. A total of 210 individuals (30 per population) were analysed using seven selected AFLP primer combinations that produced 219 clear polymorphisms. Four autochthonous goat breeds (Bionda dell'Adamello, Frisa, Orobica and Verzaschese), two primary populations, one from the Lombardy Alps (Val di Livo) and the other from Sardinia island (Sarda) and a reference cosmopolitan breed (Saanen) were included in the analysis. The expected heterozygosity (Het) did not differ significantly among breeds (range 0.21-0.24). No breed specific markers were identified. The variability at AFLP loci was largely maintained within breeds, as indicated by the coefficient of genetic differentiation (Gst) value (0.11). Dice similarities calculated between pairs of individuals belonging to the same or to different breeds largely overlapped. Bootstrapping on markers indicated that the coefficient of variation (CV) of the genetic indexes tested decreases only marginally by adding markers over 100 AFLPs. Cluster analysis based on standard genetic distance between breeds indicates that Sarda is the most distant population, while Bionda, Frisa, Verzaschese and Val di Livo seem to be highly related populations. Interestingly, Saanen is closer than Orobica to the other four goat populations of the Lombardy Alps. Principal co-ordinates analysis based on Dice similarities confirms these observations. Genetic diversity of the goat populations investigated confirms what is expected on the basis of their geographical location. Results from Orobica are not correlated with geographical distances and may reflect undocumented migrations and gene flows and identify an original genetic resource.