Genomic DNA (500 ng) was bisulfite converted with the EZ DNA methylation-Gold kit (D5005; Zymo Research, Orange, CA, USA) according to the manufacturer's recommendations. Modified DNA was amplified with the primers listed in Table S1 . The PCRs were carried out with 25 ng bisulfite-treated DNA, 1 ml of HotStart Taq DNA polymerase (Qiagen), 106 supplied HotStart buffer, 4 ml of dNTP (25 mM), and 3 ml of PCR primers (5 mM). PCR conditions were 94uC for 15 min, then 40 cycles of 94uC for 30 s, 57uC for 30 s, and 72uC for 30 s. The PCR products were subjected to purification using the PCR cleanup kit (28160; Qiagen) following the manufacturer's instructions. The purified PCR products (150 ng) were then subcloned into a TA cloning vector (50 ng) (pGEM-T Easy vector; Promega, Madison, WI, USA). Several clones of each sample were verified by sequencing with the T7 universal primer.