The two parent lines NILqknr6 and NILqKNR6 DNA methylation sequencing

The genomic DNA was separately extracted from the 2-5 mm ears of both parent lines . DNA sample was fragmented by sonication to 200–300 bp. After 3′-A addition and adaptor ligation, the DNA fragments were subjected to sodium bisulfite conversion using the ZYMO EZ DNA Methylation-Gold kit (ZYMO Research, Orange County, CA, USA) according to manufacturer instructions. The sequencing was performed by Wuhan Genoseq Technology with Illumina Hiseq2500 (Illumina Inc., SanDiego, CA, USA). Each library was sequenced approximately 554 million raw reads. And clean and high quality reads were then generated by filtering out the adapters and low-quality reads using Trimmomatic-0.33. The clean reads were aligned to the maize B73 reference genome (www.maizeGDB.org) using Bismark . Only perfect matches were filtered in for methylation analysis. To calculate the methylation density of cytosine, the total number of nucleotides cytosine and thymidine that overlap with each genomic cytosine site across the whole genome was calculated. The methylation level for each cytosine site was calculated by the sequencing depth divided by the number of unconverted cytosine.