PCR primers were done with a touchdown PCR strategy but the annealing temperatures were locus specific. Final annealing temperature was 50°C. All PCR products were checked on a 1.5% agarose gel to ensure amplification and specificity prior to running the pyrosequencing reactions. The Pyrosequencing reactions were done as described by the manufacturer (Qiagen). Briefly, the resulting biotinylated PCR product was diluted in binding buffer (10 mM Tris-HCL, 2 M NaCl, 1 mM EDTA, 0.1% Tween 20) and bound to sepharose-streptavidin (SA) beads (GE Healthcare). The dsDNA-SA-beads complex was washed in 70% ethanol, denatured in 0.2N NaOH and washed in 10mM Tris-Acetate pH 7.6. The beads were then placed in annealing buffer (20 mM Tris-Acetate, 2 mM MgAc2) containing the appropriate sequencing primer (0.3 mM final), heated to 80°C for 2 min and allowed to cool to 25°C. Pyrosequencing was done in the PyroMark HS-96 pyrosequencing machine (Qiagen) as per the manufacturer’s instructions. The genotyping call rate was 96.4%.
