Rnase free dnasei

RNAs were extracted from immature maize seeds collected 6, 9, 14, and 20 DAP. Genomic DNA was digested using RNase-free DNaseⅠ(Fermentas, Ontario, Canada). cDNA synthesis and quantitative real-time PCR (qRT-PCR) of miRNAs were performed using the All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, Maryland, USA). U6 was used as an internal control. For qRT-PCR of coding genes, PrimeScript^TM Ⅱ1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) and SYBR Green FS Universal SYBR Green Master (Roche Applied Science, Indiana, USA) were used. PCR was carried out on the CFX96^TM Real-Time PCR Detection System (Bio-Rad, California, USA). The thermal cycling program consists of an initial denaturation step at 95°C for 10 min, then 40 cycles at 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s. Target gene abundance in each sample was normalized according to the U6 expression (for miRNAs) or TUBULIN expression (for coding genes) levels using the formula ΔCt = Ct (target gene)–Ct (U6) or Ct (TUBULIN). The experiment was performed with three biological repeats, each with three technical repeats. The primers used for qRT-PCR are listed in S4 Table.

Total RNAs were extracted from two-staged seeds of the three materials using TRIzol reagent (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s protocol and treated with RNase-free DNase Ⅰ (Fermentas, Burlington, Canada). Purified RNAs were used to construct the RNA-seq library. In this study, the RNA sequencing library was sequenced by Illumina HiSeq^TM 4000. Adapter sequences and low-quality sequences were filtered from the raw reads by SOAPnuke, and clean reads were mapped to the B. rapa genome v1.5 sequences (http://brassicadb.org/brad/datasets/pub/BrassicaceaeGenome/Brassica_rapa/Bra_Chromosome_V1.5/) using HISAT (http://www.ccb.jhu.edu/software/hisat). After alignment with the reference genome, CPC software was used to predict the potential of coding for new genes.