Genetic variability at the FASN locus in the Italian Mediterranean river buffalo (bubalus bubalis, 2n=50).

Fatty acid synthase (FASN) is a enzyme complex that plays a central role in de novo synthesis of long-chain satured fatty acids (SFA). The structural and functional characterization of the FASN gene was reported in several species and genetic diversity has been found in cattle. For instance, the SNP g.763G>C has been associated with milk fat content in dairy cattle, whereas other SNPs have been associated with the FAs composition of adipose and milk fat. Despite these information in cattle, no studies have been carried out in a closely related ruminant, the river buffalo. In Italy this species is important in economic terms, being connected to the production of Mozzarella PDO. Although the buffalo genome project indicated the FASN sequence (EMBL ID AWWX01575345.1), this is not annotated and no data on genetic diversity are available so far. Therefore, the aim of this study was to explore the natural genetic variability existing at FASN. Genomic DNA was isolated from 17 individual blood samples collected in different farms of Campania region. A preliminary investigation was carried out by PCR on the region spanning the exons 6-9 by using the following primers (For: 5’-CTGACCAAGAAGTCCCTG-3’ and Rev: 5’-AAGGAGTTGATGCCCACG- 3’) designed on the homologous bovine gene (EMBL ID AF285607.2). PCR products were analysed in 1.5% TBE agarose gel, purified and sequenced. Sequence analysis was performed by DNAsis software. A DNA fragment of 1345 bp including the exons 6-9 of the buffalo FASN gene was amplified and sequenced. The analysis of the sequences showed 7 SNPs. Five polymorphisms were found at intronic level, whereas 2 SNPs were exonic and both responsible of amino acid change. In particular, the SNP g.5976A>C at the 99th nt of the exon 6 is responsible for a Asn256>His replacement, whereas the SNP g.6100A>T at the 15th nt of the exon 7 is responsible for the change Thr265>Ser (numbering is relative to the homologous gene in cattle). These SNPs are the first polymorphisms reported at FASN locus for the Italian river buffalo. Considering the influence of the FASN on the FA composition and given that the average fat content in buffalo milk was 8.16% in 2013, the observed variability is interesting because it offers potential tools for the set up of future association studies with fat traits and for the application of marker assisted selection programmes to improve the productivity of this species.