CONTRIBUTION OF SARDINIAN MOUFLON TO DOMESTIC SHEEP MILK PROTEINS

In ruminants, milk protein genetic variability is well known and it has been shown to affect milk composition and technological quality (Pirisi et al. 1999). Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk (Ceriotti et al. 2004). Since European mouflon was found closely related to one of the five haplogroups identified within domestic sheep species (Sanna et al. 2015), the aim of this work was to assess milk protein pattern differences between sheep and Sardinian mouflon and its genetic contribution to current sheep milk proteins. Raw milk from sheep and mouflon was skimmed after centrifugation at 3500 g for 10 minutes. The skimmed milk proteins were submitted to 12% SDS Poly Acrylamide gel electrophoresis (PAGE) and 2-D PAGE using a 4-6 pH gradient IPG followed by 12% SDS-PAGE electrophoresis. Gels were stained by brilliant blu G-250 and silver stain. Genomic DNA was extracted from 3 ml of seven mouflon blood samples by the 5Prime kit. The complete CSN1S1 gene was directly amplified by PCRs using 27 pairs of primers. We also analyzed a 520 base pair (bp) region of CSN3 gene, including the complete sequence of the exon 4 where all the polymorphisms described so far were identified by using the KF/KR primers. Sequence reactions were performed by means of the BigDye Terminator kit (v1.1, Applied Biosystem) and products were sequenced using an ABI PRISM 3130xL Genetic Analyzer. High quality profiles were aligned by the Lasergene SeqMan software with Reference Sequences in GeneBank. Results showed a different 2D electrophoretic pattern between proteins of sheep and Sardinian mouflon. Differences in weight and isoelectric points has been evidenced in the ?-s1 and ?-s2 and in the ?-casein clusters but not in the k-casein. To characterize coding DNA sequence (CDS) of the casein genes in Sardinian mouflons we sequenced 17 exons of the CSN1 (?-s1 casein) and CSN3 (k-casein) compared it with sheep and European mouflon homologue sequences. We identified a SNP within the CSN1 gene located in the position 137 of the exon 17 where C in sheep and European mouflon sequences was replaced by T in the 7 Sardinian mouflons. This substitution (ACT-->ATT) resulted in an aminoacid change from Threonine, a polar aminoacid, to Isoleucine, an hydrophobic apolar aminoacid. The Sardinian mouflon CSN3 gene evidenced a polymorphism in the nucleotide 78 of the exon 4 where a C was changed with T. The codon 58 changed from TAC in sheep to TAT which are both codifying for the aminoacid Tyrosine, and then do not alter the primary structure of the k-casein. Overall, our data evidenced different electrophoretic protein patterns between Sardinian mouflon and domestic sheep milk. Some of these differences could be due to genetic differences as showed for ?-s1 casein polymorphism which modify the isoelectric point of this protein. Based on these results Sardinian mouflon could have contribute to the polymorphism of sheep milk proteins in accordance with its role of possible ancestor of domestic sheep carrying the B HPG. Further studies on other milk protein genetic sequences such as ?, ?-s2 casein, lactoglobulins or lactalbumins are needed to improve and confirm these outcomes.