DNA Extraction and Genotyping Protocol

Genomic DNA was extracted using the TIANamp Blood DNA Kit (TianGen Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. TaqMan real-time polymerase chain reaction (PCR) was applied to acquire the genotypes by using the ABI QuantStudio DX system [15 (link),16 (link)]. Briefly, the final volume was 10 μl and consisted of 5 μl TaqMan universal PCR Master Mix, 0.5 μl Primer/probe mix and 10 ng genomic DNA. The PCR program had an initial denaturation step at 95°C for 10 min and 40 cycles at 94°C for 15 s and 60°C for 45 s. Allele frequencies were analyzed by using ABI SDS software. For quality control, genotyping was repeated on a random 10% of the samples, and 100% concordance rate was observed for the results.