Sequence variation and detection of a functional promoter polymorphism in the lysozyme c-type gene from Ragusano and Grigio Siciliano donkeys

Aim of the study was to report the full characterization of donkey lysozyme c-type gene (Lyz) and to detect functional polymorphisms. Using the genomic DNA as template, we sequenced the whole Lyz gene (4 exons), plus 342 nucleotides at the promoter and 1017 nucleotides at the 3’ flanking region, in 6 donkeys belonging to two different Italian breeds: Ragusano and Grigio Siciliano. The comparison of the obtained sequences showed a high degree of genetic variability at this locus. In particular, the transition g.4443G>A, detected at exon level, is responsible for the amino acid change p.Ser147Phe. This nonsynonymous substitution represents the first example of coding alleles (Lyz A and Lyz B, respectively) in the donkey lysozyme c-type gene. At promoter level, we focused on the SNP g.203C>T that alters a putative binding site of the transcription factor NF-1 (Nuclear Factor 1). The set-up of a luciferase assay confirmed that the transition g.203C>T influences the promoter activity. The reduced activity of homozygote TT suggests an effective role of this promoter-binding site in the expression of the Lyz gene also in donkey milk. A PCR-RFLP based method by AvaI was developed for the identification of the carriers of this mutation point. The results of this study suggest that the SNP g.203C>T might be useful to promote association studies with all the traits affected by the quali-quantitative variability of donkey lysozyme c-type.