Transcriptome sequencing
For transcriptome sequencing, total RNA was extracted from the grains at
5 and 10 DAF, using the RNAprep Pure Plant kit (Tiangen). RNA integrity
and quantity were determined with an Agilent 2100 Bioanalyzer according
to the manufacturer’s protocol. Enrichment of mRNA from total RNA,
complementary DNA synthesis, and construction of the library were
performed at Vazyme Biotech. The total libraries were sequenced using an
Illumina HiSeq X Ten platform. The raw reads were filtered by removing
reads with adapters, reads in which unknown bases were >
5%, and low-quality reads. The FASTQC program was used to assess the
quality of the clean reads, which were then aligned to the rice
reference genome Release 7 of the MSU Rice Genome Annotation Project
using TOPHAT v.2.1.0 (Trapnell et al., 2012). CUFFLINKS v.2.1.1 was then
used to normalize and estimate the gene expression level according to
fragments per kilobase of transcript per million reads (FPKM, Mortazavi
et al., 2008). The differentially expressed genes (DEGs) were also
calculated using CUFFLINKS v.2.1.1 at a significance level of q
< 0.05 and the absolute value of log2 ratio ≥ 1.