## 4 Materials and Methods
### PacBio Iso-Seq Library Construction, Sequencing, and Gene Functional Annotation  
Total RNA was isolated from each sample with the polysaccharide polyphenol plant total RNA extraction kit (Tiangen, Beijing, China). The quality and quantity of total RNA were determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and ND-2000 (NanoDrop Thermo Scientific, Wilmington, DE, USA), respectively. Total RNA from six independent samples (high-yield and common-yield with three biological replicates) was mixed at equal ratios. One mixed Iso-Seq library was constructed according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences (PN 100-092-800-03). Sequence data of PacBio Sequel was processed using the SMRTlink V7.0 software ([[https://www.pacb.com/support/software-downloads/](https://www.pacb.com/support/software-downloads/)]([https://www.pacb.com/support/software-downloads/](https://www.pacb.com/support/software-downloads/)) (accessed on 6 December 2019)). Circular consensus sequences (CCSs) were generated from subread BAM files by setting the parameters to min_length 50, max_drop_fraction 0.8, no_polish TRUE, min_zscore -9999.0, min_passes 1, min_predicted_accuracy 0.8, max_length 15,000. CCS.BAM files were output, which were then classified into full-length and non-full-length reads using lima, removing polyA by refinement. Full-length fasta files were fed into the cluster step, corrected by isoform-level clustering (ICE), and then final arrow polishing was conducted, parameters setting as: h_quiver_min_accuracy 0.99, bin_by_primer false, bin_size_kb 1, qv_trim_5p 100, qv_trim_3p 30. Additional nucleotide errors in consensus reads were corrected using LoRDEC software ([[http://atgc.lirmm.fr/lordec](http://atgc.lirmm.fr/lordec)]([http://atgc.lirmm.fr/lordec](http://atgc.lirmm.fr/lordec)) (accessed on 15 December 2019)) based on the Illumina RNA-Seq data. The redundancy in corrected consensus reads was removed by CD-HIT software ([[https://github.com/weizhongli/cdhit](https://github.com/weizhongli/cdhit)]([https://github.com/weizhongli/cdhit](https://github.com/weizhongli/cdhit)), -c 0.95 -T 6 -G 0 -aL 0.00 -aS 0.99 -AS 30 (accessed on 28 December 2019)) to obtain final transcripts for the further analysis.  
Gene function was annotated based on the following databases: NCBI Non-redundant Protein (Nr, cut-off E value \(\leq 1\times 10^{-10}\)), NCBI Non-redundant Nucleotide (Nt, cut-off E value \(\leq 1\times 10^{-10}\)), protein family (Pfam, cut-off E-value \(\leq 0.01\)), eukaryotic Ortholog Groups (KOG, cut-off E value \(\leq 1\times 10^{-10}\)), Swiss-Prot protein (cut-off E value \(\leq 1\times 10^{-10}\)), Kyoto Encyclopedia of Genes and Genomes (LEGG, cut-off E value \(\leq 1\times 10^{-10}\)), and Gene Ontology (GO, cut-off E value \(\leq 1\times 10^{-10}\)). Protein coding sequences from cDNA were identified using the ANGEL pipeline (a long-read implementation of ANGLE). The closely related species were used to ensure that the protein sequences were ANGEL-trained and then performed the ANGEL prediction for given sequences.