Transcriptome Profiling of Rust Resistance in Switchgrass Using RNA‐Seq Analysis

Abstract

Switchgrass rust caused by Puccinia emaculata is a major limiting factor for switchgrass (Panicum virgatum L.) production, especially in monoculture. Natural populations of switchgrass displayed diverse reactions to P. emaculata when evaluated in an Ardmore, OK, field. To identify the differentially expressed genes during the rust infection process and the mechanisms of switchgrass rust resistance, transcriptome analysis using RNA‐Seq was conducted in two pseudo‐F1 parents (‘PV281’ and ‘NFGA472’), and three moderately resistant and three susceptible progenies selected from a three‐generation, four‐founder switchgrass population (K5 × A4) × (AP13 × VS16). On average, 23.5 million reads per sample (leaf tissue was collected at 0, 24, and 60 h post‐inoculation (hpi)) were obtained from paired‐end (2 × 100 bp) sequencing on the Illumina HiSeq2000 platform. Mapping of the RNA‐Seq reads to the switchgrass reference genome (AP13 ver. 1.1 assembly) constructed a total of 84,209 transcripts from 98,007 gene loci among all of the samples. Further analysis revealed that host defense‐related genes, including the nucleotide binding site–leucine‐rich repeat domain containing disease resistance gene analogs, play an important role in resistance to rust infection. Rust‐induced gene (RIG) transcripts inherited across generations were identified. The rust‐resistant gene transcripts can be a valuable resource for developing molecular markers for rust resistance. Furthermore, the rust‐resistant genotypes and gene transcripts identified in this study can expedite rust‐resistant cultivar development in switchgrass.

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass

“…While other studies have aimed to understand the molecular mechanisms controlling switchgrass resistance to switchgrass rust [13, 30], none of these studies have mined the currently available draft switchgrass genome (v 1.1) for potential NB-LRR resistance gene homologs. In this research, a homology-based computational approach was used to identify 1011 unique RGHs in the switchgrass genome.…”

“…A recent report found that many genes were differentially expressed during switchgrass rust infection and that some of these genes belonged to the NB-LRR gene family, which is the largest family of plant disease resistance ( R ) genes [13]. NB-LRR genes encode proteins that contain a C-terminal leucine rich repeat (LRR) domain, a highly conserved central nucleotide binding (NB) domain, and a variable N-terminal region [14].…”

Transcriptome analysis in switchgrass discloses ecotype difference in photosynthetic efficiency

“…The RNA was extracted using TRIzol® Reagent (total RNA isolation reagent) following the manufacturer’s instructions (Life Technologies, Grand Island, NY, USA) as previously described [44]. The RNA was pooled from three independent pots of each genotype and for each of the three biological replicates.…”

“…RNA was eluted in RNase-free water and quantified using a NanoDrop1000 spectrophotometer (Thermo Scientific, DE, USA) and RNA integrity was evaluated with RNA6000 n Assay using the Agilent 2100 Bioanalyzer™ (Agilent Technologies, Palo Alto, CA, USA) according to manufacturer’s instructions. The TruSeq stranded total RNA library preparation kit (Illumina, San Diego, CA, USA) was used for cDNA libraries construction as detailed in Serba et al [44]. In brief, polyA containing messenger RNA was first purified from total RNA using poly-T oligo-attached magnetic beads and then chemically fragmented and primed with random hexamer priming for single-stranded cDNA synthesis.…”

“…Quality evaluation and trimming was as described in Serba et al [44]. Briefly, in-house program was used to trim out low-quality bases (<Q15) from both the 5’- and 3’- ends of the reads to ensure two or more consecutive bases obtained a Phred score of 30 (99.90% base call accuracy) or more.…”