Extraction of DNA and genotyping of SNPs
Genomic DNA obtained was extracted from the peripheral blood of all of the subjects with the QIAamp DNA Blood Mini kit (Qiagen, Valencia, CA, USA). The targeted sequence was amplified by the PCR using appropriate primers(Yu, Liu, Bai, Kijlstra, & Yang, 2014).Genotyping of the nine SNPs was performed using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. A total of 7 μL PCR product was digested with 3 U of restriction enzyme according to the manufacturer’s instructions, involving Pdi-I, DraIII, Rsal, Eco471, BshNI, HindIII, TscAI, Ndel, and BstNI (ThermoFisher Scientific, Waltham, MA, USA) overnight (Table S1). Digestion products were separated on 4% agarose gels added with goldview I nucleic acid dye (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). To confirm the PCR-RFLP genotyping results, we randomly selected a part of the samples to reperform genotyping using direct sequencing.