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).