Generation of the SAD1 -overexpressing transgenic plants (SAD1-OE) and the splicing variants of SAD1 in the wild type, sad1 and SAD1-OE. (A) Morphology of the wild type, sad1 and SAD1-OE seedlings in soil. (B) Genotype analysis of plants shown in (A). The upper and lower bands of PCR products represent the endogenous SAD1 gene and the transgenic cDNA, respectively. (C) RNA-seq reads were visualized by the Integrative Genomics Viewer (IGV) browser across the SAD1 gene. Exon-intron structure was given at the bottom of each panel. The arcs generated by IGV browser indicate splice junction reads that support the splice junctions. The grey peaks indicate RNA-seq read density across the gene. The upper panel depicts the mutation of sad1 that changed the wild-type invariant dinucleotide AG to AA at the 3′ splicing acceptor recognition site of the first intron. The middle panel shows transcripts with two aberrant 3′ splice sites (3′SSs) that respectively occurred at the 20 bp (enlarged and marked by 3) and 2 bp (enlarged and marked by 2) downstream of the mutated splice site and transcripts with the retention of the first intron (marked by 1) in sad1. Also shown are SAD1 transcripts in the wild type where they were normally spliced. (D) Three variants of SAD1 transcripts discovered in sad1 by RNA-seq were validated by RT-PCR using junction-flanking primers. The three bands in the sad1 mutant from top to bottom represent transcripts with the first intron retained, the first aberrant 3′SSs and the second aberrant 3′SSs, respectively. Note in the wild-type and SAD1-OE plants, only one wild-type SAD1 band was detected. (E) SAD1 expression levels were shown using the reads per kilobase per million value and quantitative RT-PCR. bp, base pairs; RPKM, reads per kilobase per million; sad1, sad1 mutant; SAD1-OE, plants over-expressing wild-type SAD1 in the sad1 mutant background; WT, wild type.