The SNP g.133A>C in SCD promoter affects gene expression and quali-quantitative properties of river buffalo milk.

The stearolyl-CoA desaturase (SCD) gene has been deeply investigated in ruminants because of its effect on milk fat composition. In river buffalo, the SNP g.133A>C at the gene promoter has been associated with milk quality and yield. However, the biological reason for such effects remained unexplored. In this study we have combined mRNA profile analysis, electromobility shift assay and q-PCR to elucidate the role of this SNP on gene transcription and its effects on milk fat traits. A preliminary genotyping of the SNP g.133A>C was carried out on a sample of 303 river buffaloes for the choosing individuals for the downstream applications. Allele frequencies showed an increase of the minor allele C (0.25) compared with previous findings (0.16). Six animals (two for each genotype) were chosen for cloning and a total of 216 positive cDNA recombinant clones for SCD (72 per genotype) were analyzed by PCR. All clones showed the same length on agarose gel, therefore random clones were chosen for the sequencing. No qualitative differences were found and all gene transcripts resulted correctly assembled. Electrophoretic mobility shift assay was performed to evaluate the binding of the transcription factor Sp1 to DNA sequences including the SNP g.133A>C. The genotype CC showed a stronger intensity binding than the genotype AA in two different conditions as result of Sp1 motif cluster. The following q-PCR confirmed an up-regulation of the CC compared with AA and AC genotypes. The association study with milk fat traits revealed a favourable effect of the allele C. The heterozygous genotype had the highest values for MUFAs, oleic acid (C18:1 c9), PUFAs and odd branched-chain fatty acids (OBCFAs), and the lowest values for SFAs, atherogenic and thrombogenic indices, and significantly differed from the AA genotype. The AC genotype had been previously associated with higher milk yield. Therefore, the SNP g.133A>C is a marker with a dual impact and it is an interesting candidate for assisted selection programs in river buffalo. These data clarified the biological role of the SNP g.133A>C in the SCD promoter and how it affects the gene function, providing important knowledge on the genetic background of lipid metabolism, including the future possibility of selecting 1 alleles with quantitative or qualitative favourable effects.