Fig. 1

Flowchart of the pipeline for the A. donax leaf transcriptome sequencing, de novo assembly, annotation, and analysis. The pipeline performs multiple operations from sampling and preparation to sequencing and de novo assembly to functional annotation and analysis. First, mRNA extraction from the first fully expanded leaf (5th from the top) was carried out followed by cDNA preparation and library construction (gray). Sequencing was performed using an Illumina HiSeq platform. The sequenced reads were then subjected to quality control and filtering, and identical sequences were removed (blue). Next, de novo assemblies of transcripts were generated using a two-step approach: first, multi-k-mer (Trans-ABySS and rnaSPAdes) and single-k-mer (Trinity) methods were used to generate the pre-assemblies (pink); second, pre-assemblies were then concatenated and redundant transcripts were removed using CD-HIT and the EvidentialGene tr2aadcs pipeline (purple). The quality of the de novo assembled leaf transcriptome was then assessed (green). Finally, the non-redundant (NR) transcript dataset was functionally annotated by homology and gene ontology (GO), and metabolic pathways were analyzed (mint blue). Simple sequence repeats (SSRs) and polymorphic SSRs (PolySSRs) were also identified (orange)