Transcriptome analysis
Total RNA was extracted from samples pulverized in liquid nitrogen, using a cetyltrimethylammonium bromide (CTAB) and LiCl method (Chang et al., 1993). RNA was treated with DNase I to remove genomic DNA (Yanget al., 2016). RNA quality and integrity was determined using an Agilent 2100 Bioanalyzer with Plant RNA Nano chip assay (Agilent; http://www.agilent.com). For RNA sequencing (RNA-seq) library synthesis, three biological replicates per P treatment were sequenced using an Illumina HiSeq 2500 instrument, as described previously (Serba et al., 2015). Gene expression levels were normalized by calculating reads per kilo base of transcript per million fragments mapped (RPKM). HISAT2 2.0.5 and Stringtie 1.2.4 were used to identify gene transcripts after mapping to the reference genome (Phytozome Panicum virgatum v4.1) (Kim et al., 2015; Pertea et al., 2015). Differentially expressed gene transcripts (DEGs) were analyzed using Cuffdiff software (Trapnell et al., 2013) and filtered with log2FC (fold change) ≥ 1 or ≤ -1 and settled on 2-fold changes as cut-off, P adj ≤ 0.05 and RPKM ≥ 3. DEGs were also employed for gene ontology (GO) enrichment analysis using the PlantRegMap database with default parameters (Jin et al., 2016).