Genetic diversity and population structure of Sicilian sheep breeds using microsatellite markers

Genetic diversity studies in domestic animals aim at evaluating genetic variation within and across breeds mainly for conservation purposes. In Sicily, dairy sheep production represents an important resource for hilly and mountain areas economy. Their milk is used for the production of traditional raw milk cheeses, sometimes protected designation of origin (PDO) cheeses. In some cases, the quality of these products is linked to a specific breed, i.e. mono-breed labelled cheeses and it is therefore important to be able to distinguish the milk of a breed from that of others, in order to guarantee both the consumer and the breed itself. In order to investigate the genetic structure and to perform an assignment test, a total of 331 individuals (Barbaresca, BAR n=57, Comisana, COM n=65, Pinzirita, PIN n=75, Sarda, SAR n=64, and Valle del Belice, VDB n=70) were analysed using a panel of 20 microsatellite markers. A total of 259 alleles were observed with average polymorphic information content equal to 0.76, showing that the microsatellites panel used was highly informative. Estimates of observed heterozygosity ranged from 0.65 in the BAR breed to 0.75 in the COM breed. The low value of genetic differentiation among breeds (Fst=0.049) may indicate that these breeds are little differentiated probably due to common history and breeding practices. The low Fis and Fit values indicated low level of inbreeding within and among breeds. The unrooted neighbor-joining dendrogram obtained from the Reynold's genetic distances, and factorial correspondence analysis revealed a separation between BAR and the other sheep breeds. Recent migration rates were estimated, showing that four out of the five breeds have not received a significant proportion of migrants. Only for the PIN breed a recent introgression rate from the VDB breed (7.2%) was observed. The Bayesian assignment test showed that BAR and SAR breeds had a more definite genetic structure (proportion of assignment of 92% and 86.6%, respectively), whereas the lowest assignment value was found in the PIN breed (67.1%). Our results indicated high genetic variability, low inbreeding and low genetic differentiation, except for BAR breed, and were in accordance with geographical location, history, and breeding practices. The low robustness of the assignment test makes it unfeasible for traceability purposes, due to the high level of admixture, in particular for COM, PIN and VDB

Genetic polymorphism at the CSN1S1 gene in Girgentana dairy goat breed

The aim of this work was to evaluate the variability of the s1-casein locus in the endangered Girgentana dairy goat breed in order to define genetic improvement and 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 CSN1S1 genotype information could be utilized in selection strategies for milk protein content and milk yield, in order to select genetic lines for the production of “drinking milk” using weak and null genotypes, and for niche products using strong genotypes

Effect of hairless gene polymorphism on the breeding values of milk production traits in Valle del Belice 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 genotype produced 1.5 times more daily milk than CC homozygotes. Individuals with CC genotype showed eight times less FAT content and 1.7 times less PRT content than the heterozygous. However, these differences were not statistically different, probably due to the low frequency of the CT genotype. Considering our results, polymorphisms of the hr gene do not directly influence production traits, but if further studies confirm our hypothesis, the hr gene could be used in a marker assisted selection program

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 alti di MNA, Ho e He sono stati trovati in MIX (MNA=7,17, Ho=0,700 e He=0,731), seguito da MAS (MNA=5,94, Ho=0,697 e He=0,703) e GIR (MNA= 5,89, Ho=0,590 e He=0,666). Il più alto valore del Fis, che misura il livello di inbreeding all’interno di ciascuna popolazione, è stato trovato in GIR, mentre MAS ha mostrato il più basso valore. Alleli privati, in particolare in MAS, sono stati trovati con frequenza relativamente alta (13% e 20%). L’analisi delle distanze genetiche di Nei e Reynolds indicano una maggiore distanza tra MAS e GIR. Anche l’ACF mostra una chiara separazione di MAS rispetto agli altri due gruppi. Il test di assegnazione effettuato con STRUCTURE mostra per MAS un cluster meno definito rispetto a quello di GIR, ma più definito rispetto a MIX. Questo studio riporta i primi risultati sulla caratterizzazione genetica della capra MAS. I dati ottenuti mostrano una uniformità genetica confrontabile con quella riportata per altre razze o popolazioni ufficialmente riconosciute. Ulteriori studi verranno condotti al fine di confermare i risultati ottenuti e definire le origini della capra MAS

Cellule somatiche in latte ovino: analisi e applicazione delle curve ROC

Negli ovini da latte le mastiti costituiscono una delle principali cause di perdite economiche per la ridotta produzione e soprattutto per la scarsa qualità tecnologica del latte. Diversi studi hanno dimostrato che il contenuto in cellule somatiche (SCC) del latte può essere impiegato sia come metodo diagnostico sia come criterio di selezione nei programmi di miglioramento genetico per la resistenza alle mastiti. Obiettivo di questo lavoro è stato applicare la metodologia delle Receiver Operating Characteristics curves (ROC) per valutare la capacità diagnostica del SCC nel discriminare le mammelle infette da quelle sane e per individuare un valore soglia discriminante in ovini da latte. In totale sono stati analizzati 14.072 campioni di latte individuale da 720 pecore di razza Valle del Belice in 5 allevamenti. L’isolamento e l’identificazione batterica sono stati effettuati tramite semina di 10 μl di latte su Agar Sangue incubato a 37°C per 24 - 48h in aerofilia e, se necessario, in microaerofilia e la determinazione delle SCC mediante citometria di flusso. In base al risultato dell’esame batteriologico sono stati identificati 9 gruppi. La media aritmetica del SCS per i test negativi e positivi è risultata rispettivamente di 5.17 ± 0.008 e 5.80 ± 0.010, la media geometrica del SCC per i test negativi e positivi è risultata rispettivamente di 146.96 x 103 e 630.85 x 103 cells/ml. Complessivamente sono risultati positivi all’esame batteriologico 6207 campioni con una prevalenza pari al 44,1%. L’AUC è risultata pari a 0.73 ± 0.004 (P 0.0001). Il POO è risultato essere >427x103 cells/ml, in corrispondenza di tale punto la sensibilità è pari al 59,2% mentre la specificità si attesta all’80,9%. In corrispondenza di tale valore il 71% dei soggetti positivi ed il 72% dei soggetti negativi sono classificati correttamente. L’approccio di tipo non parametrico per la stima delle ROC ha dimostrato che il SCC può essere validamente utilizzato per discriminare gli animali infetti da quelli sani. Tuttavia ulteriori studi si ritengono necessari per una più precisa valutazione economica dei costi delle mastiti in considerazione del fatto che il POO sulle ROC tende a spostarsi verso valori con più alta Se e più bassa Sp man mano che i costi dei falsi positivi si riducono rispetto a quelli dei falsi negativi o quando aumenta il valore della prevalenza

Cellule somatiche ed isolamento di agenti mastidogeni in ovini di razza Valle del Belice

Il conteggio delle cellule somatiche nel latte ovino si è dimostrato un valido strumento per la determinazione dello stato sanitario della mammella. Oggetto del presente studio è stato valutare eventuali relazioni fra il contenuto in cellule somatiche (SCC) e le infezioni mammarie (IMI) negli ovini. L’indagine è stata condotta nel periodo 2006-2012 su n.14072 campioni di latte individuale. In base al tenore i campioni sono state inseriti in 4 classi: 300.000; da 301.000 a 500.000; da 501.000 a 1.000.000 e ≥1.001.000 cellule/ml. Per l’isolamento colturale sono stati seminati 10 μl di latte su piastre di Agar Sangue incubate per 24-48 h a 37 °C in aerofilia e, se necessario, in microaerofilia. Sono stati distinti 10 gruppi: nessun isolamento, isolamento di stafilococchi coagulasi positivi (S. aureus, S. intermedius e S. hyicus), stafilococchi coagulasi negativi (CNS), streptococchi patogeni (S. agalactiae, S. dysgalactiae e S. uberis), altri streptococchi o enterococchi (S. acidominimus,S. canis, S. viridans, S. mitis, S. mitior ed E. faecalis), Corynebacterium, Mannheimia haemolithyca, Bacillus spp, altri germi (Proteus spp., E.coli e Pseudomonas spp.) ed associazione di CNS ed altri streptococchi. Per ogni gruppo è stata calcolata la media geometrica del SCC. Il 44% dei campioni analizzati mostrava positività all’esame microbiologico con percentuali crescenti in funzione della classe di SCC d’appartenenza. Si riportano i microrganismi isolati e le relative prevalenze: CNS 80%, stafilococchi coagulasi positivi 11,76% di cui 677 S. aureus (10,9%), 43 S. intermedius (0,69%) e 10 S. hyicus (0,16%), Streptococcus spp. 3,09%, streptococchi patogeni 2,64%, M. haemolytica 0.63%, Corynebacterium 0,18%, Pseudomonas 0,10%, Bacillus 0,68%, E. coli 0,06% e Proteus 0,34%. La media geometrica del SCC mostra differenze statisticamente significative fra i vari gruppi di batteri. Nell’allevamento ovino gli CNS si confermano i principali agenti di mastite subclinica con tendenza alla cronicizzazione pertanto ulteriori approfondimenti sono necessari per indagare il loro ruolo patogenetic

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 among populations (Fst=0.027) could indicate that these populations were differentiated little probably due to gene flow and breeding practices. The analysis of genetic distances between groups indicated that the Mascaruna goat was the most distanced group, and this result was confirmed by the unrooted neighbor-joining dendrogram, the factorial correspondence analysis, the presence of several private alleles and the Bayesian assignment test. However, the Mascaruna group, despite the influences from other populations, presents a certain degree of uniqueness and could be considered as a population with particular genetic background

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 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 among populations (Fst=0.027) could indicate that these populations were differentiated little probably due to gene flow and breeding practices. The analysis of genetic distances between groups indicated that the Mascaruna goat was the most distanced group, and this result was confirmed by the unrooted neighbor-joining dendrogram, the factorial correspondence analysis, the presence of several private alleles and the Bayesian assignment test. However, the Mascaruna group, despite the influences from other populations, presents a certain degree of uniqueness and could be considered as a population with particular genetic background.Keywords: Local goat population, genetic characterization, microsatellite markers, casein gene clusterAfrican Journal of BiotechnologyVol. 12(24), pp. 3758-376