Search

The objectives of the present work were to evaluate polymorphism of microsatellite markers, develop a parentage test and estimate misidentification rate on Valle del Belice Sicilian dairy sheep breed. Polymorphism was evaluated from 184 randomly selected animals from 5 different flocks. A total of 21 microsatellite markers including CSRD247, ILST011, McM527, APO010, INRA132, DU194351, DU323541, FCB128, McM16, OarCP49, ILST087, DU223896, ILST005, TCRVB06, SPS113A, INRA063A, SPS115, DU206192, DU216028, BM827 and McM54 were amplified using three PCR reactions and fragment lengths determined in a single multiplex electrophoresis run. Markers were easily genotyped, very polymorphic and in Hardy-Weinberg equilibrium (except McM527 and ILST_005) thus suitable for paternity test. Statistical analysis was performed using Cervus 3.0. The mean number of alleles per locus = 9.29, expected heterozygosity = 0.749 and PIC= 0.70. The combined non-exclusion probability = 5.5 x10-5, 5 x10-8 and 3.2 x10-13 for the first, second and the parent pair respectively. The paternity test was performed on 64 families generated with multiple sires mating. The results indicated errors in 20.3% and 28.7% of mothers and fathers respectively. In 8 cases, out of 15 maternal exclusions, the real mother was identified among the other females. Misidentification rate indicates the necessity of keeping more efficient control of genealogical records.

β-lactoglobulin (β-lg) is the major whey protein in milk of ruminants and several non ruminant species, but it is lacking in rodents, lagomorphs and human. The aim of this work was sequencing the promoter region of β-lg gene in Sicilian sheep and goat dairy breeds, in order to identify polymorphisms. In these species, the promoter region was aligned using the sequences available on database. A common set of primers was designed to amplify and sequence a fragment of approximately 2.1 kb. Within this region, 12 single nucleotide polymorphisms (SNPs) were identified in goat breeds: 7 out of them were not yet reported in literature. Moreover, in sheep breeds 15 SNPs were newly identified. Using TRANSFAC database, binding sites for transcription factors (TFs) were found within the β-lg promoter region, in sheep and goat. Multiple binding sites to MPBFs, NF-1, C/EBP, and Sp1 are present within conserved regions, both in sheep and goat. Some SNPs found in the promoter region were detected within TFs sites in both species. Further studies are required to investigate the effect of these point mutations on binding affinity of TFs, their relationship with β-lg gene expression and milk production.

processo civile

The aim of this work was to develop and evaluate a PCR based microsatellite markers multiplex system for parentage verification of Sicilian Valle del Belice dairy sheep. A total of 85 samples of blood and hair were collected and genotyped for 24 microsatellite markers in multiplex electrophoresis runs. A total of 269 alleles were detected across the 24 loci investigated. The PIC considering all loci was equal to 0.736, showing that this microsatellite panel was very polymorphic and highly informative. A parentage test was performed on 64 families generated with multiple sires. Results indicated 20.3% and 29.7% misidentification rates for females and males, respectively. In 8 cases, out of 13 maternal exclusions, the real mother was identified among other females within the flock. The observed misidentification rates indicated the necessity of keeping more efficient collection of genealogical records, in order to properly control inbreeding or implement a breeding program. The parentage test presented here could be a helpful tool on verifying or even reconstructing the current pedigree data of Valle del Belice dairy sheep breed.

Several beta-lactoglobulin (BLG) polymorphisms have been described within the proximal promoter region and coding region of the caprine gene, although no genetic variants affecting the protein amino acid composition and/or expression level have been characterized so far. Binding sites for several transcription factors (TFs) are present in the BLG promoter region. The aims of this work were to sequence the full-length promoter region of three Sicilian goat breeds in order to identify polymorphisms, analyze the identified haplotypes, search for differences between breeds for the presence of polymorphisms in this gene region, search for putative TFs binding sites, and check if polymorphisms lay within the identified TFs binding sites. The promoter region of BLG gene in Sicilian goat breeds showed high level of polymorphism due to the presence of 36 single nucleotide polymorphisms (SNPs). Association between polymorphic sites was computed within the whole sample analyzed and 18 haplotypes were inferred. Binding sites for three milk protein binding factors (MPBFs) and four nuclear factor-I (NF-I) were found within BLG promoter region based on the ovine sequence. The identification of some SNPs within TFs binding sites allowed hypothesizing the loss of TFs. Further studies are in progress to evaluate the effect of these mutations on binding affinity of TFs, the functional interaction of the TFs with the goat BLG promoter, and the relationship of the polymorphisms with BLG gene expression and milk production and composition.