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

2013

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.…

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

2016

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…

Genetic characterisation of CSN2 gene in Girgentana goat breed

2014

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…

Population genetic structure and milk production traits in Girgentana goat breed

2017

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…

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

2017

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 …

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

2015

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…

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

2015

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.…

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

2015

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. …

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

2013

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…

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

2013

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).

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

2013

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 …

PRELIMINARY STUDY ON QUANTIFICATION OF aS1-CASEIN VARIANTS IN GIRGENTANA GOAT BREED BY DIRECT CHROMATOGRAPHIC ANALYSIS OF MILK

2012

Goat αs1-casein is a highly polymorphic protein, coded by CSN1S1 gene. Nowadays, several alleles were identified and associated with different levels of αs1-casein in goat milk. Polymorphisms at αs1-casein locus have been shown to affect not only the quantity of this casein in goat milk, but also the structural and nutritional characteristics (hypoallergenic properties) and technological properties of the milk (1). The aim of this work was to separate and quantify the most common allelic variants of αs1-casein in milk of Girgentana goat breed, a Sicilian autochthonous breed, and to evaluate the effect of αs1-casein polymorphisms on casein content. The CSN1S1 A/01, B/E, F, and N alleles were…

APPLICATION OF MOLECULAR MARKERS FOR GENETIC TRACEABILITY OF SICILIAN AUTOCHTHONOUS BREEDS AND TYPICAL DAIRY PRODUCTS

In Sicilia, le razze bovine, ovine e caprine e le loro produzioni lattiero-casearie rappresentano una risorsa importante per l’economia del settore zootecnico. Alcuni di questi prodotti di origine animale sono “prodotti monorazza” e rappresentano elementi importanti per la conservazione e lo sviluppo di queste popolazioni, dei territori e delle tradizioni locali. Il processo di valorizzazione, autenticazione e tracciabilità delle produzioni lattiero-casearie richiede una conoscenza approfondita sulla struttura genetica delle razze, sulle caratteristiche morfologiche e attitudinali, sulla distribuzione geografica/ambiente di produzione e sulla diversità genetica entro e tra razze. Uno strume…

Use of microsatellite markers for genetic traceability of Girgentana dairy products

2015

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…

Genetic variability of Girgentana goat breed using molecular markers

2014

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…

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

2015

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…

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

2014

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…