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

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 (s2-, 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: s2-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 correlation was found between the quantities of s2-casein genetic variants A and F, and β-casein C and C1 with other previously described method. The main important result was obtained for κ-casein because, till now, no data were available on quantification of single genetic variants for this protein

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

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

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)

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

Identification of Copy Number Variants in Braque Français type Pyrénées”dog using CanineHD array

Copy number variants (CNVs) are an important source of genetic variation complementary to single nucleotide polymorphisms (SNPs). Only a few studies have been conducted in dogs on CNVs derived from high-density SNP array data, and many canine breeds still remain uncharacterised, e.g. the Braque Français, type Pyrénées breed (BRA). Therefore, in an effort to more comprehensively investigate the canine genome for CNVs, we used a high-density genome-wide array (170 K) to discover additional CNVs in BRA. A total of 48 BRA individuals (27 females, 21 males) were sampled. After excluding SNPs which were unmapped or mapped to sex chromosomes, a total of 167,183 markers were used. A total of 1047 CNVs were detected using PennCNV, with an average length of 107.123 kb and an average number of 40.3 CNVs per sample. The CNAM univariate segmentation of SVS identified 1638 CNVs with an average length of 110.41 kb and the average number of 63 CNVs per sample. By aggregating the overlapping CNVs from PennCNV, a total of 181 CNVRs were identified. The CNVs identified with SVS were aggregated into 280 CNVRs. Intersecting the two CNVR datasets, a total of 45 CNVRs, ranging from 3.5 kb to 458,716 kb in length were detected in 26 dog samples. Among the stringent CNVRs, 42 CNVRs were defined as pure deletions, and only 3 as loss/gain, meaning that both deletions and duplications were observed. Results overlap moderately in comparison with previous studies on CNVs in dogs, leading to the identification of 16 novel CNVRs. A total of 159 genes were annotated in the CNVRs. Some of these genes are associated with well-known phenotypes in dogs, such as: SOX8 involved in sex determination, SLC38A2 related to hypoxia adaptation, MYOG associated with the development of functional skeletal muscle. Moreover, the gene ontology enrichment analysis provided information on biological processes and cellular components related with muscle structure development and muscle cell differentiation. These are interesting results considering that BRA is a dog breed used for tracking, hunting, pointing and retrieving feathered game. These are hardy dogs, strong, adequately muscled but without heaviness. We can hypothesise that selection for such hunting behaviour, for which particular anatomical features are required, could have shaped, at least in part, the genetic background of this breed and, consequently, the frequency/presence of the detected CNVRs in these genes

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

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 relationship matrix (F GRM), based on the difference between observed v. expected number of homozygous genotypes (F HOM) and the genomic homozygosity of individual i (F MOL i). The molecular coancestry coefficient (f MOL ij) between individuals i and j was also estimated. Individuals of Cinisara (71), Modicana (72) and Reggiana (168) were genotyped with the 50K v2 Illumina BeadChip. Genotypes from 96 animals of Italian Holstein cattle breed were also included in the analysis. We used a definition of ROH as tracts of homozygous genotypes that were >4 Mb. Among breeds, 3661 ROH were identified. Modicana showed the highest mean number of ROH per individual and the highest value of F ROH, whereas Reggiana showed the lowest ones. Differences among breeds existed for the ROH lengths. The individuals of Italian Holstein showed high number of short ROH segments, related to ancient consanguinity. Similar results showed the Reggiana with some extreme animals with segments covering 400 Mb and more of genome. Modicana and Cinisara showed similar results between them with the total length of ROH characterized by the presence of large segments. High correlation was found between F HOM and F ROH ranged from 0.83 in Reggiana to 0.95 in Cinisara and Modicana. The correlations among F ROH and other estimated F coefficients were generally lower ranged from 0.45 (F MOL i-F ROH) in Cinisara to 0.17 (F GRM-F ROH) in Modicana. On the basis of our results, recent inbreeding was observed in local breeds, considering that 16 Mb segments are expected to present inbreeding up to three generations ago. Our results showed the necessity of implementing conservation programs to control the rise of inbreeding and coancestry in the three Italian local dairy cattle 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