GO enrichment analysis and Blast2Go analysis of T. pratense genomes

To further explore the digital gene expression results and to find more candidate genes/ to identify differentially expressed gene clusters, an enrichment analysis with Gene Ontology (GO) terms [80–82] was performed. For each pairwise comparison, the up-regulated genes were screened for enriched and depleted GO terms using the GOSeq package [83] separately for each treatment. Identified GO terms for each pairwise comparison were then also compared in a mown vs. non-mown manner to show treatment specific GO terms. The results of this analysis were visualized with the program GOplot [84] implemented in RStudio [85] with the program R [86].

Two local BLAST searches [87] with word-size of 3, e-value of 1.0e-3 and HSP length cutoff of 33 were performed against the PlnTFDB using Blast2GO [88]. Only the blast hits with the highest similarity were used for further comparisons (number of BLAST hits = 1), sequences with a similarity below 50% and an e-value higher than 1.0e-4 were omitted. The Blast2GO output was compared with an in-house python3-script utilizing NumPy (https://numpy.org/), Pandas (https://pandas.pydata.org/) and Seaborn (https://seaborn.pydata.org/) applying the list of transcription factors (TF) downloaded from PlnTFDB. We searched the Uniprot database hits for development and phytohormone related genes. Subsequently, we searched for gene IDs of gibberellin genes in our annotated T. pratense transcriptomes. Matches were filtered based on TPM values and classified based on biosynthesis and its regulation, catabolism, activation/repression or signaling/response, and the corresponding expression patterns within the transcriptome were identified.

To assess the effect of gibberellin during the regrowth reaction of T. pratense, 14 red clover plants were mown as described in [24]. Of these plants, seven were used as control plants and seven plants were sprayed with 100 μM GA₃ (Duchefa Biochemie B. V, Haarlem, The Netherlands) once per week as described in [89]. Different morphological characters (leaf number, length/width of leaflets, petiole length, number of inflorescences, and number of main shoots) were measured every second day for 4 weeks.