PMC

Genes predicted based on RNA-Seq analysis for three floral developmental stages. (A) Number of highly confident genes expressed in each stage. (B) Unique and shared genes among three developmental stages. IM represents inflorescent meristem, F1–9 for flower development stage 1–9, F12 for flower development stage 12.

Analysis of novel identified transcripts. (A) Display of a novel transcribed region identified by RNA-Seq analysis. It includes three exons, supported by short reads (in red bars). Detailed alignment information (first exon-intron junction) is given in the lower panel. (B) Comparison of the expression values of novel transcripts and annotated genes. Comparison of gene lengths is given in the upper right panel. (C) RT-PCR validation of 18 randomly selected novel transcribed regions.

Hierarchical clustering and function enrichment analysis of 1716 differentially expressed isoforms among three stages. (A) Differentially expressed isoforms were clustered into four clusters: 359 isoforms in cluster I (with expression values as F1–9 > IM > F12), 460 isoforms in cluster II (F12 > F1–9 > IM), 401 isoforms in cluster III (IM > F1–9 > F12), and 526 isoforms in cluster IV (the others). Genes expressed higher than average are colored in red while lower in blue. (B) A list of enriched GO terms for genes in Cluster III. Red bars represent ratio of total annotated genes for each function, blue bars for differentially expressed genes within cluster III.

The details of reads mapping and reproducibility of RNA-Seq data. (A) Distribution of aligned reads, which are mapped either uniquely or to multiple positions on the genome. (B) Percentage of reads distributed to exons, introns, and untranslated or intergenic regions. (C) Classification of detected genes by annotated genomic features (percentage: mRNA 0.95; ncRNA 0.01; snoRNA 0.002; tRNA 0.009; miRNA 0.003; snRNA 0.0004; rRNA 0.0005; pesudogene 0.021). (D) Gene expression comparison between the two technical replicates for the inflorescent meristem (IM) stage. (E) Same comparison for flower development stage 1 to stage 9 (F1–9).

An example of alternative spliced isoforms (of AT2G30940) expressed specifically at F12 stage. The fourth exons of the two isoforms end at different sites (pointed out in a red triangle) according to gene annotations in TAIR10, and this alternative donor event was further confirmed by PCR validation on flower tissues. Each bar (middle panel) represents a short read mapped to the reference genome. Two ends of a split read, indicting a cleavage of an intron, are connected by a gray line. One of the two transcripts, AT2G30940.1, is only expressed in F12, revealed both by alignment details of reads mapped to an exon-exon junction and by RT-PCR validation (relative ratio approximately 220.5, consistent with that of FPKM).