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

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

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 most likely responsible for different level of gene expression. The aim of this work was to study the genetic polymorphism at DNA level of b-lg gene in Girgentana goat breed. A total of 238 genomic DNA samples of Girgentana breed were genotyped. A fragment of 709 bp, including 588 bp of proximal promoter region and 121 bp of exon 1, was amplified using primers GOAPF3 and GoatE1R2. PCR-RFLP procedure was used for fast detection of two single nucleotide substitutions as described by Graziano et al. (2003). The base substitutions originating the polymorphic sites consist of: 1. a transition T›C at position -341 and 2. a transition C›T at position -60. A FspBI PCR-RFLP protocol was used to detect the mutation -341 (T/C) and a SmaI PCR-RFLP protocol for the mutation -60 (C/T) of the proximal promoter region. The allelic frequencies and the Hardy-Weinberg equilibrium were estimated using the GENEPOP software. Girgentana goat breed shows no significant deviation from Hardy- Weinberg equilibrium for the allele frequencies found in both polymorphic sites considered. The genotypic frequencies for both mutations resulted in 0.65 (T/T), 0.33 (T/C) and 0.02 (C/C) for the position -341, and 0.82 (C/C), 0.17 (C/T) and 0.01 (T/T) for the position -60. These results are in agreement with the previous obtained by Graziano et al. (2003) in the same breed. Further analysis are in progress to investigate the possible effect of these variants on the expression of b-lg gene, on the milk protein composition and on milk production traits

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

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 showed high variability within breeds. Analysis of genetic diversity indexes showed that the Sarda breed presented the lowest nucleotide diversity, whereas the Comisana breed presented the highest one. Comparing the nucleotide diversity among breeds, the highest value was obtained between Valle del Belice and Pinzirita breeds, whereas the lowest one was between Valle del Belice and Sarda breeds. Considering that polymorphisms in the promoter region of BLG gene could have a functional role associated with milk composition, the lowest value of nucleotide diversity between Valle del Belice and Sarda breeds may be related to a higher similarity of milk composition of these two breeds compared to the others. Further analyses will be conducted in order to evaluate the possible correlation between the genetic diversity indexes and the BLG content in milk of our breeds