Development and validation of RP-HPLC method for the quantitative estimation of as1-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 data obtained for Girgentana goat breed showed that A, B, F variants were alleles associated with a content of as1-casein in milk of 3.2 ± 0.4, 5.4 ± 0.5 and 0.7 ± 0.1 g/L, respectively, whereas N variant was a ‘null’ allele associated with the absence of as1-casein in milk

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)

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

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” DNA in each mixture. Finally, duplex-PCR assay was applied to experimental cheeses in order to detect the minimum threshold of DNA belonging to one species in cheese made with milk of two species. The results showed a sensitive threshold of 0.1% of ewes' milk in cows' and goats' cheeses, 0.1% of cows' milk in ewes' and goats' cheeses, and finally 0.1% of goats' milk in cows' and ewes' cheeses. The proposed assay represents a rapid and straightforward method of species traceability for the detections of adulteration in Sicilian mono-species dairy products

Genetic characterisation of CSN2 gene in Girgentana goat breed

Among the calcium sensitive caseins, the B-casein is the most abundant in milk, representing up to 50% of total casein content. The goat B-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 genetic polymorphisms of the B-casein gene in the Girgentana dairy goat breed, in order to assess the genotype distribution and to evaluate how the frequencies have changed during the last 10 years, as it is known genotype influences technological and nutritional milk properties. Sequencing analysis and alignment of the obtained sequences of B-casein exon 7, showed the presence of A, C, and C1 strong alleles, and 0' null allele, with frequencies of 0.597, 0.326, 0.023, and 0.054, respectively. Seven genotypic classes were found in Girgentana goat breed and the most frequent genotype was CC1 (0.423) followed by CC (0.327), C1C1 (0.107), and C0' (0.097). No AA and 0'0' homozygous individuals were found. The presence of strong alleles at CSN2 gene in Girgentana goat breed could be useful for the production of milk with high protein content and good cheese-making properties. Moreover, food business operators should consider the possibility of reviving interest in Girgentana goat milk using weak and null genotypes at CSN2 locus to make peculiar food products, such as drinking milk

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 total of 20 microsatellite markers were analyzed on 338 individual samples from Girgentana, Maltese, and Derivata di Siria goat breeds. Specific microsatellite markers useful for traceability of dairy products were identified. Eight microsatellite markers showed alleles present at the same time in Maltese and Derivata di Siria and absent in Girgentana and, therefore, they were tested on DNA pools of the three breeds. Considering the electropherograms' results, only FCB20, SRCRSP5, and TGLA122 markers were tested on DNA samples extracted from cheeses of Girgentana goat breed. These three microsatellite markers could be applied in a breed genetic traceability system of Girgentana dairy products in order to detect adulteration due to Maltese and Derivata di Siria goat breeds

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. T583→C583. Comparing the amino acid sequences of N and A alleles, the first two SNPs caused no amino acid change, whereas the other SNPs produced changes (Val65/Ile65, Val119/Ile119, and Ser159/Pro159, respectively). Comparison of N allele with C revealed theamino acid change Val156→Ala156. The most frequent allele was A (0.480) followed by B (0.363), D (0.112), and N (0.034). The D’ and G alleles were identified only in two animals and in heterozygous conditions with a very low frequency (0.005). The most common genotype was AB (39.5%) followed by AA (19.5%), AD (12.7%), and BB (11.7%). Homozygous D’D’, GG, and NN individuals were not found. Further analysis will be performed in order to establish associations among genotypes and quantitative and qualitative milk traits

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 strumento utile per la tutela e la valorizzazione dei prodotti tipici, che possono portare allo sviluppo di aree marginali, favorendo la conservazione della biodiversità e di conseguenza la tutela delle razze locali, è rappresentato dalla tracciabilità molecolare. L’applicazione di marcatori molecolari, quali i microsatelliti e il DNA mitocondriale, è uno strumento importante per studiare la diversità genetica delle razze caprine, bovine e ovine autoctone siciliane e per la caratterizzazione e la valorizzazione delle loro produzioni lattiero-casearie. In questa tesi, sono stati utilizzati i marcatori microsatelliti per analizzare non solo gli individui di razza caprina Girgentana ma anche le produzioni lattiero-casearie da essa derivate. Inoltre, è stato preso in considerazione il DNA mitocondriale, grazie alla sua specificità, per sviluppare protocolli molecolari utili per autenticare e tracciare le produzioni lattiero-casearie siciliane mono-specie. L’obiettivo generale di questa tesi è l’applicazione di tecnologie molecolari per la caratterizzazione e valorizzazione delle razze autoctone siciliane e per la tracciabilità genetica delle produzioni lattiero-casearie tipiche siciliane da esse derivate. Nel capitolo 2, sono stati analizzati 20 marcatori microsatelliti in 388 campioni individuali appartenenti alle razze caprine autoctone Siciliane: Girgentana, Maltese e Derivata di Siria al fine di tracciare i prodotti lattiero-caseari di razza Girgentana. In ogni razza analizzata sono stati rilevati alleli privati, ma la nostra attenzione si è focalizzata principalmente sugli alleli presenti contemporaneamente nelle razze caprine Maltese e Derivata di Siria e assenti nella razza Girgentana. All’interno del pannello analizzato, solo 8 marcatori microsatelliti hanno mostrato questi alleli e, pertanto, sono stati testati su pool di DNA delle tre razze. In particolare, 3 marcatori (FCB20, SRCRSP5 e TGLA122) presentavano alleli utili per il sistema di tracciabilità dei prodotti lattiero-casearie pertanto sono stati testati su campioni di DNA estratti da formaggio. Considerando i risultati ottenuti, questi marcatori microsatelliti potrebbero essere applicati in un sistema di tracciabilità genetica dei prodotti lattiero-caseari di razza Girgentana al fine di rilevare eventuali adulterazioni causate dall’aggiunta di latte caprino di razza Maltese e Derivata di Siria. Nel capitolo 3, sono stati riportati i protocolli di multiplex-PCR per identificare la presenza di latte di individui appartenenti alle specie bovina, ovina e caprina in prodotti lattiero-caseari Siciliani mono-specie. Dal sangue di individui appartenenti alle principali razze autoctone Siciliane e dai formaggi sperimentali è stato estratto il DNA per l’amplificazione dei geni 12S e 16S rRNA del DNA mitocondriale. L’uso di primers specie-specifici ha permesso l’amplificazione di frammenti di diversa lunghezza per le specie ovina, bovina e caprina, rispettivamente di 172 bp, 256 bp e 326 bp. Nella fase successiva, il protocollo di multiplex-PCR è stato applicato a pools di DNA ed i risultati hanno evidenziato una soglia di detezione dello 0,1% per le miscele di pools di DNA bovino/caprino e ovino/caprino, mentre una soglia di detezione dello 0,5% per le miscele di pools di DNA bovino/ovino e ovino/bovino. Infine lo stesso protocollo è stato applicato ai campioni di DNA estratto dai formaggi sperimentali al fine di rilevare la soglia minima di adulterazione nel latte. I risultati fin qui ottenuti hanno mostrato una soglia di detezione dello 0,1% di latte ovino in formaggi bovini e dello 0,5% di latte bovino in formaggi ovini. Il test proposto rappresenta un metodo rapido e semplice per rilevare eventuali adulterazioni dei prodotti lattiero-caseari mono-specie.The Sicilian cattle, sheep and goat breeds and their dairy products are an important source for the economy of livestock sector. Some of these dairy products are “mono-breed” and are important elements for the conservation and valorization of animal populations, territories and traditions. Valorization, authentication, and traceability of dairy products require wide knowledge on breeds genetic structure, morphological and attitudinal traits, geographic distribution/environment, and genetic diversity within and among breeds. Molecular traceability is a useful tool for preservation and valorization of typical products that can lead to develop marginal areas, to promote the conservation of biodiversity and, therefore, the protection of local breeds. The application of molecular markers, such as microsatellites and mitochondrial DNA, is a very important tool to study the genetic diversity of caprine, bovine and ovine Sicilian autochthonous breeds and for characterization and valorization of their dairy products. In this thesis, microsatellite markers were used for analysis of Girgentana goat breed individuals and dairy products. Moreover, mitochondrial DNA was considered for its species specificity in order to develop molecular protocols for authentication and traceability of mono-species Sicilian dairy products. The overall aim of this thesis was the application of molecular technologies for the characterization and valorization of Sicilian autochthonous breeds for genetic traceability of typical dairy products. In Chapter 2, a total of 388 individual samples of Girgentana, Maltese and Derivata di Siria Sicilian autochthonous goat breeds were analyzed by 20 microsatellite markers in order to identify breed specific microsatellite markers that can be used for traceability of Girgentana dairy products. Private alleles were found in each analyzed breed but we focused our attention mainly on alleles present at the same time in Maltese and Derivata di Siria and absent in Girgentana. Only eight microsatellite markers within the analyzed panel showed these alleles and, therefore, they were tested on DNA pools of each breed. Three markers, FCB20, SRCRSP5, and TGLA122 presented alleles useful for traceability purpose of Girgentana dairy products, and they were tested on DNA samples extracted from cheeses. Considering our results, these microsatellite markers could be applied in a genetic traceability system of Girgentana dairy products in order to detect adulteration due to Maltese and Derivata di Siria goat milk. In Chapter 3, species-specific multiplex-PCR protocols for identification of cattle’s, sheep’s and goat’s milk in mono-species Sicilian dairy products were reported. DNA from blood and experimental cheeses of Sicilian autochthonous breeds was extracted to amplify the 12S and 16S rRNA genes of mitochondrial DNA. Fragments of different length were obtained using specific primers for ovine, bovine and caprine species (172 bp, 256 bp, and 326 bp, respectively). In next step, multiplex-PCR protocols were applied on mixtures of DNA pools and results showed a sensitive threshold of 0.1% for bovine/caprine and ovine/caprine DNA mixtures, and 0.5% for bovine/ovine and ovine/bovine DNA mixtures. Finally, the same assay was applied to bovine/ovine experimental cheeses to detect the minimum threshold of milk adulteration. The results showed a sensitive threshold of 0.1% for bovine/ovine cheeses and 0.5% for ovine/bovine ones. The proposed assay represents a rapid and straightforward method for the detections of adulteration in mono-species dairy products

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

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 simultaneously investigated by PCR-RFLP (2). AS-PCR was used for the detection of the CSN1S1 E (3) and 01 alleles (4). Milk samples were prepared following the method proposed by Bobe et al. (5) and analyzed by RP-HPLC method (6). A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5µm,300Å, 150×4.6 I.D.) was used 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 allelic variants were not available. In particular, were used animals with AA, BB, FF and NN homozygous genotypes. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas

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. Average values of r2 estimated for all pairwise combinations of SNPs pooled over all autosomes were 0.058±0.023 for Valle del Belice, 0.056±0.021 for Comisana, and 0.037±0.017 for Pinzirita breeds. The LD declined as a function of distance and average r2 was lower than the values observed in other sheep breeds. Consistency of results among the several used approaches (Principal component analysis, Bayesian clustering, FST, Neighbor networks) showed that while Valle del Belice and Pinzirita breeds formed a unique cluster, Comisana breed showed the presence of substructure. In Valle del Belice breed, the high level of genetic differentiation within breed, the heterogeneous cluster in Admixture analysis, but at the same time the highest inbreeding coefficient, suggested that the breed had a wide genetic base with inbred individuals belonging to the same flock. The Sicilian breeds were characterized by low genetic differentiation and high level of admixture. Pinzirita breed displayed the highest genetic diversity (He, Ne) whereas the lowest value was found in Valle del Belice breed. Conclusions This study has reported for the first time estimates of LD and genetic diversity from a genome-wide perspective in Sicilian dairy sheep breeds. Our results indicate that breeds formed non-overlapping clusters and are clearly separated populations and that Comisana sheep breed does not constitute a homogenous population. The information generated from this study has important implications for the design and applications of association studies as well as for development of conservation and/or selection breeding programs