Indagine preliminare sul polimorfismo lattoproteico in diverse popolazioni caprine allevate in Sicilia.

La consistenza del patrimonio caprino in Italia ha subito negliiltimi anni un aumento considerevole. Significativo è stato l’incremento del 6,6% della produzione di latte tra il 2000 e il 2002. Il recente sviluppo dell’allevamento caprino è dovuto in gran parte al crescente interesse dei consumatoru sia verso i prodotti caseari tipici sia verso il consumo diretto del latte. Le caseina caprine sono caratterizzate da un elevato polimorfismo genetico, sia a livello qualitativo che quantitativo, diventato oggetto di un generale interesse scientifico per le importanti relazioni con la qualità e le caratteristiche nutrizionali e tecnologiche del latte. Il patrimonio caprino siciliano è rappresent…

Studio del gene della β-lattoglobulina in razze ovine e caprine autoctone siciliane

Development of multiplex-PCR protocol to amplify 12S and 16S rRNA genes of mtDNA for traceability of Sicilian mono-species dairy products.

Species identification in dairy sector is important not only to safeguard public health but also to verify compliance with the Production Regulations of many typical dairy products (PDO/PGI). The most common fraud in dairy sector is represented by the mixture of milks from different species, resulting in mislabeling of protected designation of origin (PDO) products. For a rapid, specific and sensitive identification of cattle’s, sheep’s and goat’s milk in mono-species Sicilian dairy products, species-specific multiplex-PCR protocol was developed. DNA from blood and experimental cheeses of Sicilian autochthonous breeds was used to amplify the 12S and 16S rRNA genes of the mitochondrial DNA. …

Using of SNP markers to estimate inbreeding, coancestry and effective population size in Sicilian cattle breeds

Maintaining the highest levels of genetic diversity and limiting the increase in inbreeding is the premise of most conservation programs. The aim of this work was to estimate the inbreeding (F), coancestry (f) and effective population size (Ne) in two Sicilian cattle breeds, Cinisara (CIN) and Modicana (MOD). Rate of molecular inbreeding and coancestry were used to estimate the Ne. A total of 144 animals were genotyped using the Illumina Bovine SNP50K v2 BeadChip. The average molecular F and f coefficients were 0.68±0.024 and 0.67±0.03 in CIN and 0.69±0.020 and 0.70±0.03 in MOD cattle breeds, respectively. The results were not unexpected considering the reduced number of reared animals and …

Genome-wide association study reveals the locus responsible for microtia in Valle del Belice sheep breed.

Microtia is a congenital deformity of the outer ear with phenotypes varying from a small auricle to total absence (anotia). The genetic basis is still poorly understood, and very few studies have been performed in sheep. Valle del Belice sheep is a breed showing microtia. The aim of this study was to identify the potential genomic regions involved in microtia in sheep. A total of 40 individuals, 20 with microtia and 20 normal, were genotyped with the Illumina OvineSNP50 BeadChip. The comparison among the results from a genome-wide association study, Fisher's exact test and FST analysis revealed a single strong association signal: rs419889303 on chromosome 1, located within intron 3 of the C…

Development and validation of RP-HPLC method for the quantitative estimation of αs1-genetic variants in goat milk

A high-performance liquid chromatographic (HPLC) method was developed and validated for separation and quantification of the most common genetic variants of as1-casein in goat’s milk, to evaluate the effect of as1-casein polymorphisms on casein content. Chromatography was carried out by binary gradient technique on a reversed-phase C8 Zorbax column and the detection was made at a wavelength of 214 nm. The procedure was developed using individual raw milk samples of Girgentana goats. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes, considering that commercial standards for goat genetic variants were not available.…

The Root Mycobiota of Betula aetnensis Raf., an Endemic Tree Species Colonizing the Lavas of Mt. Etna (Italy)

Betula aetnensis is an endemic tree of high conservation value, which thrives on the nutrient-poor volcanic soils of Mount Etna. Since plant–microbe interactions could play a crucial role in plant growth, resource uptake, and resistance to abiotic stresses, we aimed to characterize the root and rhizosphere microbial communities. Individuals from natural habitat (NAT) and forest nursery (NURS) were surveyed through microscopy observations and molecular tools: bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA), fungal denaturing gradient gel electrophoresis (DGGE). B. aetnensis was found to be simultaneously colonized by arbuscular (AM), ectomycorrhizal (ECM), ericoid…

Genetic variability at αs2-casein gene in Girgentana dairy goat breed

Casein genes are highly polymorphic and the high degree of variability has qualitative and quantitative effects on milk composition thereby affecting chemical, physical and technological properties of goat milk. The aim of this work was to evaluate the genetic polymorphisms of the αs2-casein (CSN1S2) gene in the endangered Girgentana dairy goat breed in order to assess the genotypes distribution, as it is known genotype influences technological and nutritional milk properties. The study was performed on 207 sample of Girgentana goat breed, analysed with different PCR protocols. The most frequent alleles was A (0.722), followed by F (0.225), C (0.051) and E (0.002) while B, D and 0 alleles w…

12S rRNA mitochondrial gene as marker to trace Sicilian mono-species dairy products

Abstract For a rapid, specific and sensitive identification of cows', ewes' and goats' milk in mono-species Sicilian dairy products, species-specific duplex-PCR protocol was applied. DNA samples from blood and experimental cheeses of Sicilian autochthonous breeds were extracted to amplify the 12S rRNA (and part of 16S rRNA in case of Ovis aries ) mitochondrial species-specific gene fragment. The use of species-specific primers for Bos taurus , Capra hircus and Ovis aries species, after electrophoresis on agarose gel, yielded fragments of 256 bp, 326 bp and 172 bp, respectively. Amplification by duplex - PCR of DNA pools from two species showed detection thresholds of 0.1% of “contaminant” D…

Polymorphism and chromosomal localization of the porcine signal transducer and activator of transcription 5B gene (STAT5B).

Signal transducers and activators of transcription (STATs) are a family of transcription factors. STAT5A and 5B are two highly related proteins encoded by two distinct genes. Transgenic knockout mice studies have indicated the importance of STAT5 proteins for the regulation of both lactation and growth performance. Moreover, different studies determine the role of STAT5 proteins in the modulation of adipocyte function. In this study, we sequenced one fragment of STAT5B gene from animals of six breeds (Duroc, Iberian, Landrace, Large White, Pie´train and Meishan) to identify genetic variants. A G/A single nucleotide polymorphism in intron 14 creates a polymorphic PstI restriction site and wa…

Loss in milk yield and related composition changes resulting from different levels of somatic cell count in Valle del Belice dairy sheep

Mastitis is the most common and costly disease in the dairy industry and is responsible for several production effects. Mammary infections cause high somatic cell count (SCC) and severe damage to udder tissue and result in important losses of milk yield and quality, curd and cheese yields in dairy ewes. The aim of this study was to determine the effect of different levels of SCC on milk yield and composition in Valle del Belice dairy sheep. The original data set used for this study included 92,261 records of 6,763 ewes. Data were collected between 1994 and 2006 in 15 flocks. Test-day records of milk yield (MY), fat % (F%), protein % (P%), and SCC were collected following an A4 recording sch…

Proteomics for milk proteins characterization in Girgentana goat breed

Proteomics has been widely used to characterize milk protein content in livestock species and unravel protein biological functions. The aim of this work was to characterize the Girgentana goat breed whole milk and milk fraction proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. Bulk milk samples from two farms locate in Palermo and Agrigento (Sicily) areas were collected during the early stage of lactation. Initially, 2D-PAGE and protein detection protocols were set-up. In particular, 2D-PAGE analyses were carried out on whole milk using 18-cm IPG strips ranging from pH 3-10 and 4-7 and 12.5% polyacrylamide gel for first and the second dimension …

ANALYSIS OF MICROSATELLITE MARKERS IN SICILIAN GOATS BREED FOR TRACEABILITY OF GIRGENTANA TYPICAL DAIRY PRODUCTS

The establishment of useful analytical methods able to ensure the origin of the products, including the breed used, are important in maintaining the reliability of these products in order to develop a market segment. Traceability, obtained by molecular analysis, could be a reliable proposal for the authentication and valorization of animal products. In Sicily, the three most important dairy goat breeds are Girgentana (GR), Maltese (ML) and Derivata di Siria (DS). The GR is an endangered autochthonous goat breed. Preservation of endangered breeds could be achieved by establishing economic reasons for their survival. The aim of this work was to verify the use of microsatellite markers to asse…

Analysis of Illumina BovineSNP50 BeadChip data in different Italian cattle breeds to identify useful markers for breed authentication of dairy and beef products

The identification of the breed of origin of cattle derived products has recently assumed particular relevance since the increasing interest in marketing mono-breed labelled cheese and beef. These products are usually sold at a higher price creating financial incentives to substituting them with lower value products coming from undifferentiated or common breeds or crossbred animals. Frauds can damage these valuable niche markets that, indirectly, may help to preserve genetic diversity through the economic incentive on raising local breeds, derived by the high value products, that are usually less productive. DNA analysis can help to identify fraudulent behaviour as we already demonstrated f…

Genetic characterisation of CSN2 gene in Girgentana goat breed

Among calcium sensitive caseins, β-casein is the most abundant in goat milk, representing up to 50% of total casein content. The goat β-casein locus has been widely investigated and at least ten alleles have been identified in different goat breeds. The aim of this work was to investigate the polymorphisms of β-casein gene in Girgentana dairy goat breed in order to assess the genotype distribution and evaluate how frequencies have changed during the last 10 years, as genotype is known to influence technological and nutritional milk properties. Sequencing analysis and alignment of the obtained sequences of β-casein exon 7, showed the presence of C, C1, and A strong alleles, and 0' null allel…

High-density single nucleotide polymorphism markers reveal the population structure of 2 local chicken genetic resources

Italy counts a large number of local chicken populations, some without a recognized genetic structure, such as Val Platani (VPL) and Cornuta (COS), which represent noteworthy local genetic resources. In this study, the genotype data of 34 COS and 42 VPL, obtained with the Affymetrix Axiom600KChicken Genotyping Array, were used with the aim to investigate the genetic diversity, the runs of homozygosity (ROH) pattern, as well as the population structure and relationship within the framework of other local Italian and commercial chickens. The genetic diversity indices, estimated using different approaches, displayed moderate levels of genetic diversity in both populations. The identified ROH h…

Phenotypic and genetic analysis of udder health using SCC in Valle del Belice dairy sheep

Intramammary infections (IMI) are a complex of inflammatory diseases which are defined as an inflammation of the mammary gland resulting from the introduction and multiplication of pathogenic micro-organisms.

Genomic Tools for the Characterization of Local Animal Genetic Resources: Application in Mascaruna Goat

Italy contains a large number of local goat populations, some of which do not have a recognized genetic structure. The “Mascaruna” is a goat population reared for milk production in Sicily. In this study, a total of 72 individuals were genotyped with the Illumina Goat_IGGC_65K_v2 BeadChip with the aim to characterize the genetic diversity, population structure and relatedness with another 31 Italian goat populations. The results displayed a moderate level of genetic variability for Mascaruna, in concordance with the estimated values for Italian goats. Runs of homozygosity islands are linked to genes involved in milk production, immune response and local adaptation. Population structure anal…

Two-dimensional difference in gel electrophoresis (2D-DIGE) for milk proteins characherization in Girgentana goat breed

Milk proteomics has rapidly become an eligible approach, since the protein fraction constitutes the most biologically relevant component in milk. Proteomic applications can vary greatly from straightforward protein identification to complex characterization of post-translational modifications and protein-protein interactions. The aim of this work was to compare protein profile of Girgentana whole milk samples between lactation periods and geographical areas by two dimensional difference in gel electrophoresis (2D-DIGE). Milk samples were collected in two flocks located in two different areas of Sicily at three different time points from calving: 30, 55, and 120 days, respectively. Protein p…

Parentage verification of Valle del Belice dairy sheep using microsatellite markers

The objectives of the present work were to evaluate polymorphism of microsatellite markers, develop a parentage test and estimate misidentification rate on Valle del Belice Sicilian dairy sheep breed. Polymorphism was evaluated from 184 randomly selected animals from 5 different flocks. A total of 21 microsatellite markers including CSRD247, ILST011, McM527, APO010, INRA132, DU194351, DU323541, FCB128, McM16, OarCP49, ILST087, DU223896, ILST005, TCRVB06, SPS113A, INRA063A, SPS115, DU206192, DU216028, BM827 and McM54 were amplified using three PCR reactions and fragment lengths determined in a single multiplex electrophoresis run. Markers were easily genotyped, very polymorphic and in Hardy-…

Genome-wide analysis in endangered populations: a case study in Barbaresca sheep

Analysis of genomic data is becoming increasingly common in the livestock industry and the findings have been an invaluable resource for effective management of breeding programs in small and endangered populations. In this paper, with the goal of highlighting the potential of genomic analysis for small and endangered populations, genome-wide levels of linkage disequilibrium, measured as the squared correlation coefficient of allele frequencies at a pair of loci, effective population size, runs of homozygosity (ROH) and genetic diversity parameters, were estimated in Barbaresca sheep using Illumina OvineSNP50K array data. Moreover, the breed's genetic structure and its relationship with oth…

Determination of milk production losses and variations of fat and protein percentages according to different levels of somatic cell count in Valle del Belice dairy sheep

The somatic cell count (SCC) of milk is widely used to monitor udder health and the milk quality and because of its positive genetic correlation with mastitis this trait was included in breeding schemes of dairy sheep. The aim of this study was to estimate the loss in milk yield (MY) and related composition resulting from different levels of somatic cell count in Valle del Belice dairy sheep. Data were collected between 2006 and 2016 in 15 flocks following an A4 recording scheme. Somatic cell count (SCC), fat and protein percentage (F% and P%) were determined using mid-infrared spectroscopy. To evaluate loss in test day MY, F% and P%, five different classes of SCC were arbitrarily defined: …

Genome-wide assessment of diversity and differentiation between original and modern Brown cattle populations

Identifying genomic regions involved in the differences between breeds can provide information on genes that are under the influence of both artificial and natural selection. The aim of this study was to assess the genetic diversity and differentiation among four different Brown cattle populations (two original vs. two modern populations) and to characterize the distribution of runs of homozygosity (ROH) islands using the Illumina Bovine SNP50 BeadChip genotyping data. After quality control, 34 735 SNPs and 106 animals were retained for the analyses. Larger heterogeneity was highlighted for the original populations. Patterns of genetic differentiation, multidimensional scaling, and the neig…

Energy intake effects at pasture on milk production and coagulation properties in girgentana goats with different as1-cn genotypes

The aim of this study was to investigate whether proerties of milk from Girgentana goats of different as1-CN genotypes were affected by the energy intake at pasture. Thirty six goats were genotyped at as1-CN locus using several genomic techniques and milk protein analysis. Eight genotypes associted with low (FF, 2 goats), medium (AF, AN, BF, BN, 21 goats) and high (AA, AB, BB, 13 goats) as1-CN level have beed typed. The increase in energy intake (E) of goats from low (2.0 Mcal/d) corresponded to an increasing dry matter intake, diet CP percentage, milk yield and clotting parameters, and a reduction in milk fat. Depending on genotypes linked to increasign as1-CN, milk was higher in casein, c…

Population genetic structure and milk production traits in Girgentana goat breed

The aim of this work was to evaluate the genetic status of the Girgentana goat, an endangered breed from Sicily (Italy), using microsatellite markers. Furthermore, as the main purpose of the Girgentana breed is milk production, quantitative milk traits were investigated, including fatty acid profile. Molecular data from CSN1S1, CSN2, CSN1S2, and CSN3 casein genes were also used to infer haplotypes. A total of 264 individuals were collected. Samples of Maltese (n = 41) and Derivata di Siria (n = 33) goat breeds were also used to understand the genetic relationship among breeds. Test-day records for milk production were collected to determine daily milk yield, fat, protein, casein, lactose, a…

Genetic diversity and phylogenetic relationships among four breeds reared in Sicily using β-lactoglobulin promoter region polymorphisms

The aim of this work was to analyze the genetic diversity and the phylogenetic relationships among the Valle del Belice (VDB) breed and the other sheep breeds considered as ancestors, using -lactoglobulin (BLG) promoter region polymorphisms. The BLG promoter region of 50 randomly chosen animals from the four breeds (VDB n=20; Comisana (COM) n=10; Pinzirita (PIN) n=10; Sarda (SAR) n=10) were sequenced and aligned, and results showed the presence of 36 single nucleotide polymorphisms (SNPs) and one deletion. The number of polymorphic sites identified showed high variability within breeds. Nucleotide (π) and haplotype (Hd) diversity were estimated with DnaSP software, whereas PHASE software w…

Haplotypes of Single Nucleotide Polymorphisms (SNPs) in casein loci of Girgentana goats explain isoelectrofocusing results.

Goat milk, cheese and yoghurt offer an alternative to cow milk products. In Sicily one of the important goat breeds reared is the Girgentana. Traditionally the milk from Girgentana goats was used for nourishment of infants and elderly. Recently, the Girgentana breed has been in decline. This of concern, as the Girgentana may carry unique milk protein genetic variants. If health benefits of Girgentana goat milk is proven, then increasing production from this population will be of great interest. The most abundant proteins in goat milk, as in other milks, are the caseins, αS1-, β-, αS2- and κ-casein, coded by the loci CSN1S1, CSN2, CSN1S2 and CSN3 respectively. In other goat breeds, the casei…

Genome-wide scan for runs of homozygosity identifies potential candidate genes associated with local adaptation in Valle del Belice sheep

Background Because very large numbers of single nucleotide polymorphisms (SNPs) are now available throughout the genome, they are particularly suitable for the detection of genomic regions where a reduction in heterozygosity has occurred and they offer new opportunities to improve the accuracy of inbreeding (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F$$\end{document}F) estimates. Runs of homozygosity (ROH) are contiguous lengths of homozygous segments of the genome where the two haplotypes inherited from t…

Microbial Activation of Wooden Vats Used for Traditional Cheese Production and Evolution of Neoformed Biofilms.

ABSTRACT Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differenc…

Antibacterial activity of Borago officinalis and Brassica juncea aqueous extracts evaluated in vitro and in situ using different food model systems

The present study was undertaken to characterize the antibacterial activity of the aqueous extracts (AEs) obtained from the leaves of Borago officinalis L. and Brassica juncea L. The antagonistic activity was evaluated against several bacteria (42 strains of Listeria monocytogenes, 35 strains of Staphylococcus aureus, 38 strains of Enterobacter spp. and 18 strains of Salmonella enterica) commonly associated with foodborne diseases by paper disc diffusion method. The susceptibility to the plant extracts was strain specific. Thirty-five strains (7 L. monocytogenes, 11 S. aureus, 1 S. Enteritidis, 1 S. Veneziana, 7 Enterobacter hormaechei, 5 Enterobacter cloacae, 1 Enterobacter sakazakii and 2…

Identificazione di una nuova variante alla κ-caseina nella razza caprina Girgentana

La κ-caseina è la lattoproteina che determina la grandezza e la funzione specifica delle micelle nel latte e la sua idrolisi, chimosina dipendente, è responsabile della coagulazione del latte stesso. Il gene della κ-caseina comprende 5 esoni. Ad oggi sono state identificate 16 varianti alleliche, di cui 13 sono varianti proteiche e 3 mutazioni silenti, per un totale di 15 siti polimorfici.Lo scopo di questo lavoro è stato la caratterizzazione dell’esone 4 del gene della κ-caseina nella razza caprina Girgentana. Un nuova variante alleliche, denominata X, è stata riscontrata con una frequenza relativamente bassa (0,04).

Caratterizzazione genetica mediante microsatelliti di una popolazione caprina siciliana

I microsatelliti sono ad oggi i marcatori molecolari maggiormente utilizzati per la caratterizzazione genetica nei caprini. Lo scopo del presente lavoro è stato quello di caratterizzare la struttura genetica della capra Mascaruna per verificare se può essere definita come una popolazione. L’analisi è stata condotta utilizzando un pannello di 18 microsatelliti. Il DNA è stato estratto da 60 individui di cui 20 Mascaruna (MAS), 20 Girgentana (GIR) e 20 animali derivanti da diversi incroci (MIX). Un totale di 148 alleli sono stati osservati di cui 106 in GIR, 107 in MAS e 129 in MIX; il valore del PIC è di 0,69 e tutti i marcatori hanno mostrato un numero di alleli superiori a 4. Valori più al…

LINKAGE DISEQUILIBRIUM AND GENETIC DIVERSITY IN TWO SICILIAN CATTLE BREEDS ASSESSED BY BOVINE SNP CHIP

The Modicana (MOD) and Cinisara (CIN) are two Sicilian cattle breeds farmed in extensive systems and their economic importance lies on the traditional making of two typical ‘pasta filata’ cheeses. The aim of this study was to explore the genetic structure and the extent of Linkage Disequilibrium (LD) of MOD and CIN cattle breeds. A total of 144 animals were genotyped, using the Bovine SNP50K v2 BeadChip. The squared correlation coefficient between two loci (r2) was used as a measure of LD. Principal components analysis (PCA), molecular inbreeding (F) and Bayesian clustering algorithm (Pritchard et al., 2000) were used to explore the relationship between individuals and populations. The r2 r…

Valorization of indigenous dairy cattle breed through salami production

The aim of the research was to produce salami manufactured with meat of three different commercial categories of bovine breed: cow on retirement, beef and young bull. A total of six experimental productions, at small-scale plant, were carried out with and without starter culture inoculums. The evolution of physico-chemical parameters in all trials followed the trend already registered for other fermented meat products. Several LAB species were found during process with different levels of species diversity and frequency of isolation among inoculated (mainly Pediococcus pentosaceus and Staphylococcus xylosus) and uninoculated (mainly Enterococcus devriesei, Lactobacillus curvatus and Lactoba…

Genome-Wide Patterns of Homozygosity Reveal the Conservation Status in Five Italian Goat Populations.

The application of genomic technologies has facilitated the assessment of genomic inbreeding based on single nucleotide polymorphisms (SNPs). In this study, we computed several runs of homozygosity (ROH) parameters to investigate the patterns of homozygosity using Illumina Goat SNP50 in five Italian local populations: Argentata dell’Etna (N = 48), Derivata di Siria (N = 32), Girgentana (N = 59), Maltese (N = 16) and Messinese (N = 22). The ROH results showed well-defined differences among the populations. A total of 3687 ROH segments &gt

Genome-wide association study between CNVs and milk production traits in Valle del Belice sheep.

Copy number variation (CNV) is a major source of genomic structural variation. The aim of this study was to detect genomic CNV regions (CNVR) in Valle del Belice dairy sheep population and to identify those affecting milk production traits. The GO analysis identified possible candidate genes and pathways related to the selected traits. We identified CNVs in 416 individuals genotyped using the Illumina OvineSNP50 BeadChip array. The CNV association using a correlation-trend test model was examined with the Golden Helix SVS 8.7.0 tool. Significant CNVs were detected when their adjusted p-value was <0.01 after false discovery rate (FDR) correction. We identified 7,208 CNVs, which gave 365 C…

Genome-wide association studies for milk production traits in Valle del Belice sheep using repeated measures

Genome-wide association studies (GWASes) have become a powerful tool for identifying genomic regions associated with important traits in livestock. Milk production traits in dairy sheep are measured at different time points during their life span. Using phenotypic data generated from longitudinal traits could improve the power of association studies but until now have received less attention in GWASes as a methodology and has not been implemented. The aim of this study was to carry out a GWAS for milk production traits in Valle del Belice sheep using repeated measures. After quality control, 469 ewes and 37 228 SNPs were retained for the analysis, and phenotypic data included 5586 test-day …

Beta-lactoglobulin polymorphism in Girgentana goat breed

Beta-lactoglobulin (b-lg) is a globular protein belonging to the lipocalin family. It is the major whey protein in the milk of ruminants. It is also present in the milk of most mammals but is lacking in rodents, lagomorphs and humans. A large number of variants have been reported for cow and sheep milk. Several studies have shown association between b-lg variants and milk production and composition, even if the results are not always concordant. In goat, no b-lg variants related with amino acid change have been characterized at DNA level, but some authors described the presence of polymorphisms in the 3’UTR and in the proximal promoter region. Mutations in the promoter region could be those…

Identification of two new alleles at k-casein (CSN3) gene in Girgetana goat breed

In the milk of ruminants, more than 95% of proteins are synthesized by six structural genes, four caseins and two whey proteins. The κ-casein fraction plays an important role in the formation, stabilization and aggregation on casein micelles and thus affects technological and nutritional properties of milk. Goat κ-casein (CSN3) gene comprises five exons with the mRNA coding region for mature protein spanning from exon 3 to exon 4. The κ-casein locus has been widely investigated and several genetic variants have been found in different goat breeds. At protein level, AIEF and BIEF groups corresponding to isoelectric point 5.53 and 5.78, respectively, have been identified. In this study, exon …

Whole mitochondrial genomes unveil the impact of domestication on goat matrilineal variability

Background The current extensive use of the domestic goat (Capra hircus) is the result of its medium size and high adaptability as multiple breeds. The extent to which its genetic variability was influenced by early domestication practices is largely unknown. A common standard by which to analyze maternally-inherited variability of livestock species is through complete sequencing of the entire mitogenome (mitochondrial DNA, mtDNA). Results We present the first extensive survey of goat mitogenomic variability based on 84 complete sequences selected from an initial collection of 758 samples that represent 60 different breeds of C. hircus, as well as its wild sister species, bezoar (Capra aega…

Biological diversity of the microbial film associated to the central-Mediterranean Dendropoma cristatum (Biondi, 1859) reefs.

Microbial films may provide physical and bio-chemical cues which positively affect the settlement dynamic of a variety of benthic marine organisms, driving the development of ecosystems. Also for the Mediterranean intertidal reef-builder Dendropoma cristatum (Biondi, 1859), biofilm maturity has been found to enhance the settlement pattern. However, the microbial diversity associated with these bioconstructions has never been described. This study investigates the D. cristatum reef bacterial assemblage composition and temporal evolution in two localities in the northwest of Sicily. Biological diversity of the reef-associated biofilm and of 3 progressively older biofims obtained on artificial…

Genomic structural diversity in local goats: Analysis of copy-number variations

Copy-number variations (CNVs) are one of the widely dispersed forms of structural variations in mammalian genomes, and are present as deletions, insertions, or duplications. Only few studies have been conducted in goats on CNVs derived from SNP array data, and many local breeds still remain uncharacterized, e.g., the Sicilian goat dairy breeds. In this study, CNV detection was performed, starting from the genotypic data of 120 individuals, belonging to four local breeds (Argentata dell&rsquo

Polymorphisms of beta-lactoglobulin promoter region in three Sicilian goat breeds

Several beta-lactoglobulin (BLG) polymorphisms have been described within the proximal promoter region and coding region of the caprine gene, although no genetic variants affecting the protein amino acid composition and/or expression level have been characterized so far. Binding sites for several transcription factors (TFs) are present in the BLG promoter region. The aims of this work were to sequence the full-length promoter region of three Sicilian goat breeds in order to identify polymorphisms, analyze the identified haplotypes, search for differences between breeds for the presence of polymorphisms in this gene region, search for putative TFs binding sites, and check if polymorphisms la…

Identification of Copy Number Variations and Genetic Diversity in Italian Insular Sheep Breeds

Simple Summary The aim of this work is to identify CNVs and perform a CNV-based population genetics analysis in five Italian sheep breeds (Barbaresca, Comisana, Pinzirita, Sarda, and Valle del Belìce). We identified 10,207 CNVs and 365 CNV regions (CNVRs). The different approaches used to disclose the genetic relationship showed that the five breeds tend to cluster in distinct groups. Gene enrichment was described for the 37 CNVRs selected considering the top 10%. Gene Ontology analysis showed that several of these genes are involved in lipid metabolism, immune response, and the olfactory pathway. CNVs represent valuable molecular resources to provide useful information for separating the p…

Genetic characterization of the Mascaruna goat, a Sicilian autochthonous population, using molecular markers

The aim of this work was to characterize a Sicilian autochthonous goat population using microsatellite markers and genetic polymorphisms at the casein genes. In order to investigate the genetic structure of the Mascaruna goat, a total of 60 (20 Girgentana, 20 mixed populations, and 20 Mascaruna) individuals were analyzed, using a panel of 18 microsatellite markers. Moreover, the Mascaruna goats were genotyped at casein loci using several molecular techniques. Based on the genetic structure at casein genes, the Mascaruna goat was similar to most goat breeds from the Mediterranean area, which are characterized by the predominance of strong alleles. The low value of genetic differentiation amo…

Use of microsatellite markers for genetic traceability of Girgentana dairy products

Genetic traceability is based on the identification of both animals and their products through the study of DNA. With the goal of developing a genetic traceability system for dairy products, the aim of this study was to identify specific microsatellite markers able to discriminate among the most important Sicilian dairy goat breeds, in order to detect possible adulteration in Girgentana dairy products. A total of 20 microsatellite markers were analyzed on a total of 338 individual samples from Girgentana (GIR), Maltese (MAL) and Derivate di Siria (DdS) goat breeds. The first step was to identify breed specific microsatellite markers that can be used for the traceability of dairy products. A…

A pipeline for variants discovery using next-generation DNA sequencing data

Recent advances in next generation sequencing (NGS) technology provide a cost-effective approach to large-scale resequencing of livestock samples in order to study several biological phenomena. NGS produces millions of short DNA sequences that require an unbiased way to make possible comprehensive searches for variation to identify putative causative mutations for economically important traits. The aim of this work was to present a bioinformatics pipeline analysis for variants discovery in ovine genome. A total of 30 individuals belonging to Valle del Belice dairy ewes was used for whole genome sequencing of pooled libraries prepared using Illumina Nextera Kit. Paired-end sequencing was car…

Genome structure in Sicilian cattle breeds

Genomic technologies provide background information concerning genome structure in domestic animals. The aim of this work was to investigate the genetic structure and the patterns of linkage disequilibrium (LD) in two Sicilian local cattle breeds, Cinisara and Modicana. Genotypes from animals of Italian Holstein breed were also used to investigate the relationship among breeds. Structure software was used to analyze the genetic structure and assign the individuals to each cluster. The genetic relationship between individuals was estimated by Principal Components Analysis (PCA) of genetic distance. A standard descriptive LD parameter (r2) was obtained between adjacent SNPs and for all pairwi…

Genome-wide detection of copy-number variations in local cattle breeds

The aim of the present study was to identify copy-number variations (CNVs) in Cinisara (CIN) and Modicana (MOD) cattle breeds on the basis of signal intensity (logR ratio) and B allele frequency of each marker, using Illumina’s BovineSNP50K Genotyping BeadChip. The CNVs were detected with the PennCNV and SVS 8.7.0 software and were aggregated into CNV regions (CNVRs). PennCNV identified 487 CNVs in CIN that aggregated into 86 CNVRs, and 424 CNVs in MOD that aggregated into 81 CNVRs. SVS identified a total of 207 CNVs in CIN that aggregated into 39 CNVRs, and 181 CNVs in MOD that aggregated into 41 CNVRs. The CNVRs identified with the two softwares contained 29 common CNVRs in CIN and 17 co…

Molecular characterization of the Sicilian goat populations

The Sicilian goats are characterized by a strong population admixture structure, caused by geographical location of the farms, influences of natural mating and traditional breeding systems where flock represents an important breeding unit. Nowadays, several local populations are reared in Sicily, some of which do not have a recognized defined genetic structure, having taken origin from several crosses between animals of different breeds/populations sharing the same environment. The aim of this work was to characterize the Sicilian autochthonous goat populations using microsatellite markers and genetic polymorphisms of casein genes

Parentage verification of Valle del Belice dairy sheep using multiplex microsatellite panel

Abstract The aim of this work was to develop and evaluate a PCR based microsatellite markers multiplex system for parentage verification of Sicilian Valle del Belice dairy sheep. A total of 85 samples of blood and hair were collected and genotyped for 24 microsatellite markers in multiplex electrophoresis runs. A total of 269 alleles were detected across the 24 loci investigated. The PIC considering all loci was equal to 0.736, showing that this microsatellite panel was very polymorphic and highly informative. A parentage test was performed on 64 families generated with multiple sires. Results indicated 20.3% and 29.7% misidentification rates for females and males, respectively. In 8 cases,…

La Messinese risorsa autoctona tutta siciliana

Scopo del presente lavoro è la presentazione della situazione attuale e delle prospettive future di sviluppo e valorizzazione della capra Messinese, che tra le popolazioni autoctone siciliane, presenta buone caratteristiche produttive. La capra Messinese è largamente diffusa nella zone dei monti Nebrodi dove prevale l’allevamento brado. Il fabbisogno alimentare è dato da pascoli naturali con integrazione di fave e fieno polifita. La mungitura è manuale. La stagione di monta ha inizio a giugno quando i becchi vengono immessi nelle greggi. Dal punto di vista fenotipico, gli individui mostrano un mantello che nella maggior parte dei casi è nero ma può anche presentarsi grigio, marrone e rossic…

Genetic variability of Girgentana goat breed using molecular markers

Assessing genetic diversity of local breeds through the information provided by neutral molecular markers, allows determination of their extinction risk and to design strategies for their management and conservation. The aim of this work was to quantify the levels of genetic variability in Girgentana goat breed using microsatellite markers and genetic polymorphisms at the casein genes. A total of 264 individuals were genotyped for 20 microsatellites, selected as suggested by ISAG and FAO. Moreover, the same individuals were characterized for the casein loci (CSN1S1, CSN2, CSN1S2, and CSN3) using PCR and sequencing protocols. Several genetic diversity indexes were estimated. A total of 130 a…

Assignment of signal transducer and activator of transcription 5A (STAT5A) to porcine chromosome 12p13→p11 by radiation hybrid panel mapping

A combined genome-wide approach identifies a new potential candidate marker associated with the coat color sidedness in cattle

Abstract Coat color is one of the most important phenotypic features in livestock breeds. Cinisara is a local cattle breed generally of uniform black color which occasionally presents a particular phenotype, with animals typically display a white band along their spine, from the head to the tail, and on the ventral line (color sidedness). Therefore, this breed provides an ideal model to study the genetic components underlying phenotypic variation in coat color. A total of 63 animals, ten with sidedness phenotype and 53 with uniform black color were genotyped with Illumina Bovine 50 K. The comparison among genome-wide association study and FST analysis revealed a single nucleotide polymorphi…

Quantitative determination of casein genetic variants in goat milk: Application in Girgentana dairy goat breed

The study was conducted to develop a high-performance liquid chromatographic (HPLC) method to quantify casein genetic variants (αs2-, β-, and κ-casein) in milk of homozygous individuals of Girgentana goat breed. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes. The described HPLC approach was precise, accurate and highly suitable for quantification of goat casein genetic variants of homozygous individuals. The amount of each casein per allele was: αs2-casein A = 2.9 ± 0.8 g/L and F = 1.8 ± 0.4 g/L; β-casein C = 3.0 ± 0.8 g/L and C1 = 2.0 ± 0.7 g/L and κ-casein A = 1.6 ± 0.3 g/L and B = 1.1 ± 0.2 g/L. A good correl…

Detection of genomic regions underlying milk production traits in Valle del Belice dairy sheep using regional heritability mapping

The aim of this study was to identify genomic regions underlying milk production traits in the Valle del Belice dairy sheep using regional heritability mapping (RHM). Repeated measurements for milk yield (MY), fat percentage and yield (F% and FY) and protein percentage and yield (P% and PY), collected over a period of six years (2006-2012) on 481 Valle del Belice ewes, were used for the analysis. Animals were genotyped with the Illumina 50k SNP chip. Variance components, heritabilities, and repeatability within and across lactations were estimated, fitting parity, litter size, season of lambing, and fortnights in milk, as fixed; and additive genetic, permanent environment within and across …

Weighted Single-Step Genome-Wide Association Study Uncovers Known and Novel Candidate Genomic Regions for Milk Production Traits and Somatic Cell Score in Valle del Belice Dairy Sheep

The objective of this study was to uncover genomic regions explaining a substantial proportion of the genetic variance in milk production traits and somatic cell score in a Valle del Belice dairy sheep. Weighted single-step genome-wide association studies (WssGWAS) were conducted for milk yield (MY), fat yield (FY), fat percentage (FAT%), protein yield (PY), protein percentage (PROT%), and somatic cell score (SCS). In addition, our aim was also to identify candidate genes within genomic regions that explained the highest proportions of genetic variance. Overall, the full pedigree consists of 5534 animals, of which 1813 ewes had milk data (15,008 records), and 481 ewes were genotyped with a …

The genome-wide structure of two economically important indigenous Sicilian cattle breeds1

Genomic technologies, such as high- throughput genotyping based on SNP arrays, provided background information concerning genome structure in domestic animals. The aim of this work was to inves- tigate the genetic structure, the genome-wide estimates of inbreeding, coancestry, effective population size (N e ), and the patterns of linkage disequilibrium (LD) in 2 eco- nomically important Sicilian local cattle breeds, Cinisara (CIN) and Modicana (MOD), using the Illumina Bovine SNP50K v2 BeadChip. To understand the genetic rela- tionship and to place both Sicilian breeds in a global context, genotypes from 134 other domesticated bovid breeds were used. Principal component analysis showed that…

Genome-wide analyses reveal the regions involved in the phenotypic diversity in Sicilian pigs.

Nero Siciliano (Sicilian Black, SB) is a local pig breed generally of uniform black color. In addition to this officially recognized breed, there are animals showing morphological characteristics resembling the SB but with gray hair (Sicilian Grey, SG). The SG, compared with the SB, also shows a more compact structure with greater transverse diameters, higher average daily gains and lower thickness of the back fat. In this study, using the Illumina PorcineSNP60 BeadChip, we run genome-wide analyses to identify regions that may explain the phenotypic differences between SB (n = 21) and SG (n = 27) individuals. Combining the results of the two case–control approaches (GWAS and FST), we identi…

β-lactoglobulin gene promoter: new SNPs in sheep and goat

β-lactoglobulin (β-lg) is the major whey protein in milk of ruminants and several non ruminant species, but it is lacking in rodents, lagomorphs and human. The aim of this work was sequencing the promoter region of β-lg gene in Sicilian sheep and goat dairy breeds, in order to identify polymorphisms. In these species, the promoter region was aligned using the sequences available on database. A common set of primers was designed to amplify and sequence a fragment of approximately 2.1 kb. Within this region, 12 single nucleotide polymorphisms (SNPs) were identified in goat breeds: 7 out of them were not yet reported in literature. Moreover, in sheep breeds 15 SNPs were newly identified. Using…

Variation of proteomic profile during lactation in Girgentana goat milk: a preliminary study

The knowledge of milk proteome has been greatly enhanced by technological advances in the proteomics field as the use of the two-dimensional differential in-gel electrophoresis, a gel-based approach which allowed the analysis of proteins from complex mixtures and the comparing of several protein samples in the same experiment. The aim of this study was to characterise the whole milk proteomic profile in Girgentana dairy goat breed by two-dimensional differential in-gel elecrophoresis. The obtained representative 2D whole milk proteomic map showed a general picture of the protein distributions over the pH 3–10 NL including about 100 spots, most of them organised like a spot train. Among diff…

Full-length sequencing and identification of novel polymorphisms in the ACACA gene of Valle del Belice sheep breed

The essential role of the acetyl-CoA carboxylase (ACACA) enzyme in milk fatty acid (FA) synthesis suggests that it may be responsible for the phenotypic variability observed in milk. Before attempting association analyses between this gene and/or enzyme and phenotypic traits, a study on the genetic variability within this locus is required. The aim of this work was to sequence the entire coding region of ACACA gene in Valle del Belice sheep breed to identify polymorphic sites. A total of 51 coding exons of ACACA gene were sequenced in 32 individuals of Valle del Belice sheep breed. Sequencing analysis and alignment of obtained sequences showed the presence of 23 polymorphic sites. The most …

Genomic inbreeding estimation in small populations: evaluation of runs of homozygosity in three local dairy cattle breeds

In the local breeds with small population size, one of the most important problems is the increase of inbreeding coefficient (F). High levels of inbreeding lead to reduced genetic diversity and inbreeding depression. The availability of high-density single nucleotide polymorphism (SNP) arrays has facilitated the quantification of F by genomic markers in farm animals. Runs of homozygosity (ROH) are contiguous lengths of homozygous genotypes and represent an estimate of the degree of autozygosity at genome-wide level. The current study aims to quantify the genomic F derived from ROH (F-ROH) in three local dairy cattle breeds. F-ROH values were compared with F estimated from the genomic relati…

Application of microsatellite markers as potential tools for traceability of Girgentana goat breed dairy products

In livestock, breed assignment may play a key role in the certification of products linked to specific breeds. Traceability of farm animals and authentication of their products can contribute to improve breed profitability and sustainability of animal productions with significant impact on the rural economy of particular geographic areas and on breed and biodiversity conservation. With the goal of developing a breed genetic traceability system for Girgentana dairy products, the aim of this study was to identify specific microsatellite markers able to discriminate among the most important Sicilian dairy goat breeds, in order to detect possible adulteration in Girgentana dairy products. A tot…

Genome-wide association study for milk production traits in Valle del Belice sheep

Over the last years, high-throughput technologies have provided the opportunity to explore the genomes of livestock species to identify regions influencing traits of economic interest. Genome wide association studies (GWAS) have been widely used to disentangle the genetic variation in complex phenotypes, such as milk production traits in sheep. The study presented here investigated the possible association of genome-wide SNPs and breeding values for milk production traits in the Valle del Belice sheep. The studied population consisted of a total of 481 ewes belonging to 9 half-sib families with available records for milk yield (MY), protein and fat percentage (P% and F%, respectively). All …

Penalized classification for optimal statistical selection of markers from high-throughput genotyping: application in sheep breeds

The identification of individuals’ breed of origin has several practical applications in livestock and is useful in different biological contexts such as conservation genetics, breeding and authentication of animal products. In this paper, penalized multinomial regression was applied to identify the minimum number of single nucleotide polymorphisms (SNPs) from high-throughput genotyping data for individual assignment to dairy sheep breeds reared in Sicily. The combined use of penalized multinomial regression and stability selection reduced the number of SNPs required to 48. A final validation step on an independent population was carried out obtaining 100% correctly classified individuals. …

Association study between beta-defensin gene polymorphisms and mastitis resistance in Valle del Belice dairy sheep breed

Abstract Mastitis is generally caused by bacteria, and it is the most common disease in livestock species. Defensins are peptides with a broad spectrum of antimicrobial activity and β-defensin genes have been studied in several livestock species due to their important role in the innate immune response. The aim of this study was to establish an association between polymorphisms in the β-defensin 1 and 2 genes and mastitis resistance in the Valle del Belice dairy sheep. Data consisted of 1855 and 2804 observations for case and control group, respectively. Six single nucleotide polymorphisms and seven haplotypes were selected for association studies with mastitis. In particular, polymorphism …

Runs of homozygosity reveal genome-wide autozygosity in Italian sheep breeds

The availability of dense single nucleotide polymorphism (SNP) assays allows for the determination of autozygous segments based on runs of consecutive homozygous genotypes (ROH). The aim of the present study was to investigate the occurrence and distribution of ROH in 21 Italian sheep breeds using medium-density SNP genotypes in order to characterize autozygosity and identify genomic regions that frequently appeared in ROH within individuals, namely ROH islands. After filtering, the final number of animals and SNPs retained for analyses were 502 and 46 277 respectively. A total of 12 302 ROH were identified. The mean number of ROH per breed ranged from 10.58 (Comisana) to 44.54 (Valle del B…

Genomic inbreeding estimation in small populations: evaluation of runs of homozygosity in local cattle breeds

The availability of high throughput genotyping has facilitated the quantification of inbreeding by genomic markers in farm animals. Run of homozygosity (ROH) are contiguous lengths of homozygous genotypes and represent an estimate of the degree of autozygosity at genome-wide level. The current study aims to quantify the genomic inbreeding derived from ROH (FROH) in three Italian local cattle breeds. Individuals of Cinisara (71), Modicana (72), Reggiana (168) were genotyped with the 50K v2 Illumina BeadChip. Genotypes from 96 animals of the Italian Holstein cattle breed were included in the analysis. The following criteria were used to define a ROH: two missing SNPs; one heterozygous SNP; mi…

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 b-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 s…

Genome wide Copy Number Variation (CNV) detection in Cinisara cattle breed

Copy Number Variations (CNVs) are classes of polymorphic genomic regions including deletions, duplications and insertions of DNA fragments from at least 0.5 kb up to several Mb. CNV represents an important source of genetic variability that provides genomics structural information complementary to the single nucleotide polymorphism (SNP) data. Some CNVs have been shown to be important in both normal phenotypic variability and disease susceptibility in livestock. Several approaches to identify CNVs including FISH, aCGH, SNP array or NGS, were proposed and among these SNP genotyping is relatively low cost, high-throughput and high coverage method. The aim of this study was to identify the CNV…

Short communication: casein haplotype variability in sicilian dairy goat breeds.

In the Mediterranean region, goat milk production is an important economic activity. In the present study, 4 casein genes were genotyped in 5 Sicilian goat breeds to 1) identify casein haplotypes present in the Argentata dell'Etna, Girgentana, Messinese, Derivata di Siria, and Maltese goat breeds; and 2) describe the structure of the Sicilian goat breeds based on casein haplotypes and allele frequencies. In a sample of 540 dairy goats, 67 different haplotypes with frequency >or=0.01 and 27 with frequency >or=0.03 were observed. The most common CSN1S1-CSN2-CSN1S2-CSN3 haplotype for Derivata di Siria and Maltese was FCFB (0.17 and 0.22, respectively), whereas for Argentata dell'Etna, Girgenta…

Effect of heat stress on production in Mediterranean dairy sheep

European Mediterranean countries are characterized by exposure to considerable heat between three and six months annually. High ambient temperature, solar radiation, wind speed and relative humidity, cause the effective temperature of the environment to be above the thermo-neutral zone of the animals (5 to 25ºC; McDowell, 1972) and therefore heat stress occurs (Bianca, 1962).

Genetic and protein polymorphism at CSN1S1 locus in two goat breeds.

Genetic polymorphisms of casein have received a considerable research effort for many years because of its potential effect on milk composition. Milk composition, specifically protein content, differs quantitatively and qualitatively among species and also among breeds and individuals. Sicilian goat breeds seem to be an interesting model since they have never been under a selection program, and therefore may carry unique casein polymorphisms. Moreover, the importance of goat milk in infant diets is growing probably because goat milk in some cases is less allergenic than cow milk. It is important to evaluate the genetic polymorphisms at the CSN1S1 locus in the populations of interest, like t…

A genome‐wide comparison between selected and unselected Valle del Belice sheep reveals differences in population structure and footprints of recent selection

About three decades of breeding and selection in the Valle del Belìce sheep are expected to have left several genomic footprints related to milk production traits. In this study, we have assembled a dataset with 451 individuals of the Valle del Belìce sheep breed: 184 animals that underwent directional selection for milk production and 267 unselected animals, genotyped for 40,660 single-nucleotide polymorphisms (SNPs). Three different statistical approaches, both within (iHS and ROH) and between (Rsb) groups, were used to identify genomic regions potentially under selection. Population structure analyses separated all individuals according to their belonging to the two groups. A total of fo…

Genome-Wide Association Study Identifies New Candidate Markers for Somatic Cells Score in a Local Dairy Sheep

In the Mediterranean basin countries, the dairy sheep production is usually based on local breeds, which are very well-adapted to their production systems and environments and can indeed guarantee income, employment, and economic viability in areas where production alternatives are scarce or non-existent. Mastitis is still one of the greatest problems affecting commercial milk production. However, genetic evaluation of mastitis is particularly difficult because of its low heritability and the categorical nature of the trait. The aim of this study was to identify genomic regions putatively associated with somatic cells count (SCC) in the local economically important Valle del Belice sheep br…

Identification of SNPs in the promoter of β-lactoglobulin gene in three Sicilian goat breeds

The aim of this work was to sequence the full-length promoter region of the caprine β-lactoglobulin (β-lg) gene in three Sicilian goat breeds (Girgentana, Maltese, and Derivata di Siria), in order to identify polymorphisms, to search for transcription factors (TFs) sites, and to check if polymorphisms found lay within TFs binding sites. The promoter region of β-lg gene in Sicilian goat breeds showed high level of polymorphism due to the presence of 31 SNPs. Binding sites for several TFs were found within the goat β-lg promoter and within regions conserved between ovine and caprine species. Two SNPs were detected within TFs binding sites, such as MPBF and NF-I. Further st…

Analysis of acetyl-CoA carboxylase gene polymorphisms in Valle del Belice dairy breeds

Acetyl-CoA carboxylase is the flux-determining enzyme in the regulation of fatty acid synthesis within animal tissues. The expression of the mammary gland isoform of this enzyme, during lactation, is regulated by the acetyl-CoA carboxylase-α (ACACA) gene. The ovine ACACA gene, with 51 coding exons, is responsible for phenotypic variability observed in milk fat content and milk fatty acids (FAs) composition. However, before attempting association analyses between this enzyme and phenotypic traits of interest, a study on the genetic variability within this locus is required. The aim of this work was to sequence the encoding region of ACACA gene in Valle del Belice dairy ewes in order to ident…

Casein haplotype variability in Sicilian dairy goat breeds

In the Mediterranean region, goat milk production is an important economic activity. In the present study, 4 casein genes were genotyped in 5 Sicilian goat breeds to 1) identify casein haplotypes present in the Argentata dell'Etna, Girgentana, Messinese, Derivata di Siria, and Maltese goat breeds; and 2) describe the structure of the Sicilian goat breeds based on casein haplotypes and allele frequencies. In a sample of 540 dairy goats, 67 different haplotypes with frequency >or=0.01 and 27 with frequency >or=0.03 were observed. The most common CSN1S1-CSN2-CSN1S2-CSN3 haplotype for Derivata di Siria and Maltese was FCFB (0.17 and 0.22, respectively), whereas for Argentata dell'Etna, Girgenta…

Polymorphisms at β-defensin genes and mastitis in Valle del Belice dairy sheep: a case control study

Mastitis is the most common and costly pathology affecting dairy animals. The identification of genetic markers that allow the inclusion of mastitis resistance in selection programs would help to reduce the costs due to the disease and the use of antibiotics. In recent years, β-defensin genes have been studied in several domestic species due to their important role in the innate immune response. The aim of this study was to determine whether β-defensin genes variability, β-defensin 1 (SBD1) and β-defensin 2 (SBD2), could distinguish between infected cases and healthy controls in Valle del Belice dairy sheep. Somatic cell count (SCC) is a powerful tool to identify animals with intramammary i…

Potencialidades del consorcio microbiano Curvularia kusanoi -Trichoderma pleuroticola como pretratamiento biológico para la degradación de fuentes fibrosas

Objective. To evaluate the potentiality of the microbial consortium Curvularia kusanoi L7- Trichoderma pleuroticola as biological pretreatment of high fiber sources destined for animal production. Materials and methods. The Strains used where Curvularia kusanoi L7 and Trichoderma pleuroticola. The degradative potential was evaluated through the production kinetics of cellulolytic (endo-1,4-β-glucanase and exo-1,4-β-glucanase) and ligninolytics enzymes (laccase and peroxidase) in solid submerged fermentation of bran wheat and sugarcane bagasse. The growth of the co culture in plates was analyzed. The effect of the consortium on the carbon mineralization of raw wheat straw was determined and …

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

Background The 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. Results Average 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. Ave…

Genome-wide identification of runs of homozygosity islands and associated genes in local dairy cattle breeds

Runs of homozygosity (ROH) are widely used as predictors of whole-genome inbreeding levels in cattle. They identify regions that have an unfavorable effect on a phenotype when homozygous, but also identify the genes associated with traits of economic interest present in these regions. Here, the distribution of ROH islands and enriched genes within these regions in four dairy cattle breeds were investigated. Cinisara (71), Modicana (72), Reggiana (168) and Italian Holstein (96) individuals were genotyped using the 50K v2 Illumina BeadChip. The genomic regions most commonly associated with ROHs were identified by selecting the top 1% of the single nucleotide polymorphisms (SNPs) most commonly…

Assessment of genetic variation for pathogen-specific mastitis resistance in Valle del Belice dairy sheep

Background: Mastitis resistance is a complex and multifactorial trait, and its expression depends on both genetic and environmental factors, including infection pressure. The objective of this research was to determine the genetic basis of mastitis resistance to specific pathogens using a repeatability threshold probit animal model. Results: The most prevalent isolated pathogens were coagulase-negative staphylococci (CNS); 39 % of records and 77 % of the animals infected at least one time in the whole period of study. There was significant genetic variation only for Streptococci (STR). In addition, there was a positive genetic correlation between STR and all pathogens together (ALL) (0.36 ±…

Evaluation of microbiological and physico‐chemical parameters of retail ready‐to‐eat mono‐varietal salads

An integrated microbiological and physico-chemical approach was applied to evaluate the decay of mono-varietal ready-to-eat escarole (Cichorium endivia var. latifolium) and red chicory (Cichorium intybus L. var. foliosum Hegi) during refrigeration. Total mesophilic microorganisms, including pseudomonads, and total psychrotrophic microorganisms were detected at high numbers in all samples just after packaging and at the expiry date. The dominant microbial populations analyzed by classical culture-dependent methods belonged to Pseudomonas and yeasts. Illumina sequencing identified Janthinobacterium lividum and Pseudomonas veronii as main species. Regarding the physico-chemical quality between…

A comprehensive analysis of the genetic diversity and environmental adaptability in worldwide Merino and Merino-derived sheep breeds

Abstract Background To enhance and extend the knowledge about the global historical and phylogenetic relationships between Merino and Merino-derived breeds, 19 populations were genotyped with the OvineSNP50 BeadChip specifically for this study, while an additional 23 populations from the publicly available genotypes were retrieved. Three complementary statistical tests, Rsb (extended haplotype homozygosity between-populations), XP-EHH (cross-population extended haplotype homozygosity), and runs of homozygosity (ROH) islands were applied to identify genomic variants with potential impact on the adaptability of Merino genetic type in two contrasting climate zones. Results The results indicate…

Distribution of functional variants within Runs of Homozygosity in four Italian cattle breeds

Several methods have been used to identify genomic regions subjected to selective sweeps. An alternative method can make use of runs of homozygosity (ROH), defined as stretches of homozygous regions in a genome. The aim of the present study was to detect selective sweeps using ROH in four cattle breeds. Individuals of Cinisara (71), Modicana (72),Reggiana (168) and Italian Holstein (96) were genotyped with the Illumina Bovine50SNP v2 BeadChip. To identify genomic regions that were most commonly associated with ROH within each breed, the percentage of occurrences of a single nucleotide polymorphism (SNP) in ROH was calculated across animals. The genomic regions most commonly associated with …

Linkage disequilibrium and genetic diversity estimation in three Sicilian autochthonous dairy sheep breeds

Understanding genetic structure is essential for achieving genetic improvement through genome-wide association studies, genomic selection and the dissection of quantitative traits. In the present study, we used the OvineSNP50K BeadChip to characterize LD, identify haplotype blocks and to analyze genetic diversity in the Valle del Belice (VDB), Comisana (COM) and Pinzirita (PIN) dairy sheep breeds. LD between adjacent SNPs and for all pairwise combinations of SNPs on each chromosome was measured using r2. Haplotype blocks were estimated using D’ based method. Genetic diversity and Principal Component Analysis (PCA) were calculated using PLINK. Small differences in average LD value for adjace…

Phylogenetic analysis of Sicilian goats reveals a new mtDNA lineage.

The mitochondrial hypervariable region 1 (HVR1) sequence of 67 goats belonging to the Girgentana, Maltese and Derivata di Siria breeds was partially sequenced in order to present the first phylogenetic characterization of Sicilian goat breeds. These sequences were compared with published sequences of Indian and Pakistani domestic goats and wild goats. Mitochondrial lineage A was observed in most of the Sicilian goats. However, three Girgentana haplotypes were highly divergent from the Capra hircus clade, indicating that a new mtDNA lineage in domestic goats was found.

Genome-wide association study for milk production traits in an economically important local dairy sheep breed

In this study, we conducted a genome-wide association study (GWAS) for five milk production traits in the Valle del Belice sheep. Repeated measurements for milk yield (MY), fat percentage and yield (F% and FY) and protein percentage and yield (P% and PY) on 481 ewes, were available for the analysis. The animals were genotyped using the Illumina Ovine 50k BeadChip. Weighted deregressed breeding values (DEBVw) were used as phenotypes for GWAS analysis. A total of 23 genome-wide significant SNPs were identified: 3 associated with MY, 9 with FY, and 11 with P%. Several SNPs mapped within known candidate genes or previously reported QTL for milk production traits in livestock species. Additional…

Whole genome semiconductor based sequencing of farmed European sea bass (Dicentrarchus labrax) Mediterranean genetic stocks using a DNA pooling approach

European sea bass (Dicentrarchus labrax) is an important marine species for commercial and sport fisheries and aquaculture production. Recently, the European sea bass genome has been sequenced and assembled. This resource can open new opportunities to evaluate and monitor variability and identify variants that could contribute to the adaptation to farming conditions. In this work, two DNA pools constructed from cultivated European sea bass were sequenced using a next generation semiconductor sequencing approach based on Ion Proton sequencer. Using the first draft version of the D. labrax genome as reference, sequenced reads obtained a total of about 1.6 million of single nucleotide polymorp…

Comparative selection signature analyses identify genomic footprints in Reggiana cattle, the traditional breed of the Parmigiano-Reggiano cheese production system

Reggiana is an autochthonous cattle breed reared mainly in the province of Reggio Emilia, located in the North of Italy. Reggiana cattle (originally a triple-purpose population largely diffused in the North of Italy) are characterised by a typical solid red coat colour. About 2500 cows of this breed are currently registered to its herd book. Reggiana is now considered a dual-purpose breed even if it is almost completely dedicated to the production of a mono-breed branded Protected Designation of Origin Parmigiano-Reggiano cheese, which is the main driver of the sustainable conservation of this local genetic resource. In this study, we provided the first overview of genomic footprints that c…

Development of "Quadrello di Ovino", a Novel Fresh Ewe's Cheese.

This work was performed to produce a new soft ewe’s milk cheese, namely “Quadrello di ovino” (QdO) cheese, to enlarge ewe’s dairy product portfolio of South Italy, barely limited to Pecorino cheese typology. Cheese making was performed applying the technology for “Crescenza” cheese typology with some modifications. In particular, pasteurized ewes’ milk was inoculated with two commercial starter formulations (SF1 and SF2) of Streptococcus thermophilus to obtain two different productions (QdO-P1 and QdO-P2, respectively). Plate counts demonstrated the ability of both starter formulations to drive the fermentation process, since S. thermophilus counts reached 109 CFU/g in both productions. Gen…

Molecular characterisation of k-casein gene in Girgentana dairy goat breed and identification of two new alleles

The k-casein fraction plays an important role in the formation, stabilisation and aggregation on casein micelles and thus affects technological and nutritional properties of milk. In this study, exon 4 of k-casein (CSN3) gene was sequenced and analysed in Girgentana goat breed. Analyses of the obtained sequences showed the presence of A, B, D, and G known alleles and two new genetic variants, named D’ and N. The new D’ allele differs from D in one transition, G284→A284, which did not cause amino acid change. The new N allele differs from A in five single nucleotide polymorphisms (SNPs): T245/C245, G284/A284, G309/A309, G471/A471 and T591/C591, while it differs from C in one transition, i.e.…

Effect of hairless gene polymorphism on the breeding values of milk production traits in Valle del Belice dairy sheep

The aim of this work was to assess the association between the hairless genotypes and estimated breeding values (EBVs) for milk yield (MY), fat (FAT) and protein (PRT) content in Valle del Belice dairy sheep breed. A data set from 465 randomly chosen unrelated individuals was analyzed. EBV for MY, FAT and PRT contents were estimated by REML analysis of a single trait repeatability animal model. The genotype effect on EBV was assessed by ANOVA and by the Tukey–Kramer multiple comparison test. The PCR-SSCP test showed the presence of CC and CT genotypes in Valle del Belice individuals. Some differences in milk production traits between the genotypes were found. For MY, individuals with CT gen…

Genetic polymorphism at the CSN1S1 gene in Girgentana dairy goat breed

The aim of this work was to evaluate the variability of the αs1-casein locus in the endangered Girgentana dairy goat breed in order to define genetic improvement and a conservation program for this breed. The study was performed on 200 dairy goats by means of different PCR protocols. The most frequent alleles were A (0.590) and F (0.290) followed by B (0.065) and N (0.047). CSN1S1 E allele was identified with a very low frequency (0.008). The most common genotype was AF (0.365) followed by AA (0.340). The high frequency of the strong genotypes is associated with the production of milk with high fat and protein content and with optimal technological properties. In Girgentana goat breed, the …

The Sicilian cattle breeds in a global context: genome-wide relationship with other worldwide cattle

Genomic technologies, such as high-throughput genotyping based on Single Nucleotide Polymorphism (SNP) arrays, provide background information concerning genome structure in domestic animals. The aims of this study were to cluster animals, to explore the relationships among and within breeds, and to place the Sicilian breeds, Cinisara and Modicana, in a global context. The Illumina Bovine SNP50K v2 BeadChip genotyping data from 144 animals of Sicilian breeds and from other 1,543 animals belonging to 134 domesticated bovid breeds (DRYAD) were used. These breeds arose from 3 domesticated (sub)species: Bos javanicus, B. taurus indicus, and B. t. taurus. Principal Component Analysis (PCA) genera…

Analyses of udder health in Valle del Belice dairy sheep using SCC

Intramammary infections (IMI) are a complex of inflammatory diseases of the mammary gland. Mastitis is one of the most frequent IMI affecting small dairy ruminants. Direct selection against clinical mastitis is difficult because mastitis in not widely recorded; therefore, somatic cell count (SCC) is promoted as selection criterion for mastitis resistance. A dataset containing 2,457 first- lactation Valle del Belice ewes from 14 flocks recorded from 1998 to 2003 was analysed. In total 116 sires with at least four daughters with a record were included in the pedigree file. Test-day IMI events were coded as a binary trait using a SCC cut-off value of 750,000 cells/ml. Analyses of risk factors …

Variation of proteomic profile during lactation in Girgentana goat milk: a preliminary study

The knowledge of milk proteome has been greatly enhanced by technological advances in the proteomics field as the use of the two-dimensional differential in-gel electrophoresis, a gel-based approach which allowed the analysis of proteins from complex mixtures and the comparing of several protein samples in the same experiment. The aim of this study was to characterise the whole milk proteomic profile in Girgentana dairy goat breed by two-dimensional differential in-gel elecrophoresis. The obtained representative 2D whole milk proteomic map showed a general picture of the protein distributions over the pH 3–10 NL including about 100 spots, most of them organised like a spot train. Among diff…