Identification of a Donkey CSN1S2I Allele Resulting from a Non-Constitutive Splicing Event

The αs2-casein is a phosphoprotein secreted in the milk of most mammals, and it is the most hydrophilic of all caseins. Contrary to ruminants, in donkeys, two different encoding genes (CSN1S2I and CSN1S2II) have been identified. The first, spanning over a cDNA fragment of 1016 nt, is made of 19 exons and encodes for the αs2-I protein of 221 amino acids. Nonetheless, while ruminants have been extensively studied in this regard, detailed characterization of the variability at this locus in donkeys has been lacking so far. Therefore, this study aimed to identify and analyze the variability of the CSN1S2I locus in Ragusana and Amiatina donkeys reared in Italy. For this purpose, the transcripts and genomic DNAs of eight subjects for each breed were sequenced. The sequence comparison revealed a transition G > A at the splice acceptor site of exon 17 that results in a skipping of the first 15 nt of this exon encoding for the peptide 176NKINQ180 and the recognition of an in-frame cryptic splicing acceptor site: arAACAAAATCAACCAG. The comparison of the sequences available in GeneBank showed that this peptide is constitutively spliced in all species belonging to the Perissodactyla order, in contrast to what is observed in species belonging to the Cetartiodactyla and Carnivora orders. Furthermore, the contemporary presence of the canonical and cryptic acceptor sites of the 17th exon is observed only for species belonging to the sub-order Ruminantia. It is interesting to note that the 176NKINQ180 sequence is a perfect duplication of the pentapeptide encoded by the first 15 nucleotides of exon 12 (92NKINQ96), which is a trait of two major IgE-binding epitopes of the bovine αs2-CN. Therefore, the absence of duplication could be related to the demonstrated low allergenic properties of donkey’s milk. The transition G > A alters an XbaI restriction site. Thus, a PCR-RFLP protocol was set up for the quick genotyping of 105 Ragusana and 14 Amiatina donkeys. Out of the total investigated population, the G allele has a frequency of 0.7563 with no evidence of departure from the Hardy-Weinberg equilibrium. Results indicate that donkeys, similar to buffalo