Fig. 5

Alternative splicing events revealed by Iso-seq. a Number of genes with one or more splicing isoforms in D4h and L4h Iso-seq datasets. b Number, frequency, and types of AS events identified by Iso-seq. altA, alternative acceptor site; altD, alternative donor site; CE, cryptic exon; CI, cryptic intron; ES, exon skipping; IR, intron retention. c–e Illustration and RT-PCR validation of At3g09600 (RVE8) gene models (c), and two novel gene models with At4g32780-At4g32785 fusion transcript (d) and At4g18590-At4g18593 fusion transcript (e). The representative TAIR10 gene model is shown in blue. D4h or L4H Iso-seq gene models are shown in black and orange, respectively. White boxes, black boxes, and lines represent the untranslated regions, CDS, and introns, respectively. RT-PCR was performed with forward and reverse primers marked with red arrowheads. Sizes of the amplified fragments of At3g09600 (RVE8), At4g32780-At4g32785, and At4g18590-At4g18593 fusion transcripts are 823 bp, 1205 bp, and 854 bp, respectively. AS variants confirmed by Sanger sequencing are marked with asterisks (red or black for TAIR10 or Iso-seq gene model, respectively). RT or -RT represents that RT-PCR with or without reverse transcription. -RT as a RT-PCR negative control. UBQ 5 as a RT-PCR control. Scale bar, 500 bp