Development of a dCAPS marker to genotype the polymorphism at the G181A SNP

Degenerated CAPS (dCAPS) was developed to genotype the G181A SNP detected by sequencing. The primers (mos_Pst1_F1 and mos_dcaps_R1; Supplementary Table 2) were designed using dCAPS Finder 2.0⁴⁰. PCR reactions were performed in a volume of 15 µl with 10 ng genomic DNA, 1 X Green GoTaq^® Flexi buffer, 0.125 mM dNTPs, 2 mM MgCl2, 0.2 µM of each primer, and 0.1 U GoTaq^® Flexi DNA polymerase (Promega Corporation, Madison, WI, USA), with the following program: 95 °C for 3 min, 40 cycles (95 °C for 30 s, 60 °C for 40 s, and 72 °C for 30 s), and 72 °C for 5 min.

Amplified PCR fragments were then digested using PstI restriction enzyme (#ER0615, Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. The digestion led to the production of three DNA fragments for the allele, with a guanine at position 181, RoKSN^G181 (37, 50 and 64 bp), and two DNA fragments for the allele with an adenine at the same position, RoKSN^A181 (64 and 87 bp). DNA fragments were separated on a Resophor gel (4.5% w/v), stained with ethidium bromide.