Transcriptomic analysis
Transcriptomic analysis was performed comparing the tillers that
flowered in the next season (17Hot) and tillers that remained vegetative
(17Control) as described in Samarth et al. (2019). 150 bp generated
paired-end reads were assembled into a single reference transcriptome
using the Trinity pipeline (Haas et al., 2013). Reads from each of the
samples were aligned to the reference assembly to generate a count
matrix using Bowtie2.0 (Langmead & Salzberg, 2012) followed by the
differential expression analysis using the DESeq2 package in R. To
accurately identify the significantly differentially expressed genes
(adj. p-value<0.05), stringent parameters including an E-value
of 10-5, 70 % query coverage and 50% protein
identity were used for annotation based on orthologous proteins
identified in B. distachyon using blastp. Gene ontology and
pathway enrichment analysis were performed based on the PANTHER server
(Mi et al., 2016). Validation of the RNA-seq results was performed using
RT-qPCR with a random selection of four genes from the transcriptomic
data (Appendix S2).