Using primers designed on bubaline genome (GenBank accession no. NC_037551.1, from 32020000 to 32040337 complement) and bubaline mRNA sequence (GenBank accession nos. FM865618.1, FM865619.1) (Supplementary Table 1), the DNA regions of the CSN1S2 gene spanning from the 5′- to the 3′-UTR of two Mediterranean river buffalo homozygotes for the alleles A and B were amplified by iCycler (BioRad, CA, USA). A typical 50-μl PCR reaction mix including 100 ng of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 3 mM MgCl2, 200 nmol of each primer, dNTPs each at 400 μM, 2.5 U of Taq DNA Polymerase (Promega, Madison, WI), and 0.04% BSA. The thermal condition for the amplification consisted of an initial denaturation at 95°C for 4 min, followed by 35 cycles at 94°C for 45 s, 54.0–57.4°C for 45 s (according to the amplicon) and 72°C for 2 min. A final extension of 10 min was accomplished to end the reaction. All PCR products were analyzed directly by electrophoresis in 1.5% TBE agarose gel (Bio-Rad, CA, USA) in 0.5X TBE buffer and stained with SYBR® green nucleic acid stain (Lonza Rockland, Inc., USA). PCR products were sequenced on both strands at CEINGE–Biotecnologie Avanzate (Naples, Italy) using Sanger DNA sequencing technology.

The entire panel of 747 Mediterranean river buffalo DNA samples was genotyped in outsourcing (KBiosciences, Herts, UK, for the SNPs g.7539G>C (FM865620:g.773G>C) and g.14067A>G.

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