WGBS for wheat seeds
After genomic DNAs were extracted from the samples, DNA concentration and integrity were detected by NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and Agarose Gel Electrophoresis respectively. Then the DNA libraries for bisulfite sequencing were prepared. Briefly, genomic DNAs were fragmented into 100-300bp by Sonication(Covaris,Massachusetts,USA) and purified with MiniElute PCR Purification Kit(QIAGEN,MD,USA). The fragmented DNAs were end repaired and a single A nucleotide was added to the 3 end of the blunt fragments. Then the genomic fragments were ligated to methylated sequencing adapters. Fragments with adapters were bisulfite converted using Methylation-Gold kit(ZYMO,CA,USA), unmethylated cytosine is converted to uracil during sodium bisulfite treatment. Finally, the converted DNA fragments were PCR amplified and sequenced using Illumina HiSeqTM 2504 by Gene Denovo Biotechnology Co.