Single molecule RNA sequencing (Iso-seq) experiment and data analysis
For gene annotation of the genome, transcriptome sequencing was performed with mixed tissues of a young seedling (14 day after imbibition). RNA was extracted with the TRIzol Reagent (Invitrogen, USA). The RNA quality was checked by a spectrophotometer (LabTech, USA) and a 2100 Bioanalyzer (Agilent Technologies, USA). The verified RNA was used for transcriptome sequencing library construction. Briefly, the mRNA was reversely transcribed using a Clontech SMARTer cDNA synthesis kit. A BluePippin Size Selection System (Pacific Biosciences of California, Menlo Park, CA, USA) was used to perform the size selection for the two libraries, sized 0–3 kb and 2–6 kb, respectively, after cDNA amplification and purification. The SMRTbell libraries were constructed according to the manufacturer’s protocol, and sequenced on the PacBio SEQUEL II platform (Pacific Biosciences of California, Menlo Park, CA, USA). Last, we used SMRTLink 7.0 (https://www.pacb.com/support/software-downloads/) to produce all the mRNA sequences for genome annotation.