Computational protocol: Transcriptome Profiling of Buffalograss Challenged with the Leaf Spot Pathogen Curvularia inaequalis

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[…] Approximately 100 mg of leaf tissue of each sample (95-55, NE-BFG-7-3459-17, Prestige, and NE-BFG-7-3453-50) was homogenized in liquid nitrogen using a mortar and pestle and RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA samples were qualitatively analyzed by agarose gel electrophoresis, and quantified using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). Total RNA from 24 samples [4 buffalograss lines × 3 replicates × 2 treatments (inoculated/uninoculated)] was sent to the High-Throughput DNA Sequencing and Genotyping Core Facility located at the University of Nebraska Medical Center, Omaha, Nebraska for transcriptome sequencing. The cDNA libraries were prepared and then sequenced using a HiSeq 2000 sequencing platform (Illumina, San Diego, CA, USA) according to the manufacturer’s RNA-seq protocol. The 24 samples were separately barcoded and run on three lanes of the HighSeq 2000 to obtain 100 bp single-end reads. Quality filtering of the reads was done by the Genotyping Core Facility. FastQC was used to visualize the quality of the reads using default parameters. Since FastQC showed several overrepresented reads consisting of Illumina adapter and primer sequences, Trimmomatic-0.30 () was used to remove those contaminants. The reads were trimmed to a uniform length of 80 bp prior to downstream analysis. A fastq file containing the sequencing reads and quality data was used for down-stream analysis. The sequencing reads were mapped with Bowtie2-2.1.0 () to the B. dactyloides cv. Prestige transcriptome (). Reads that did not map to the reference were retained and assembled using Trinity-r2013-02-25 (). The Trinity assembled contigs and the Prestige reference transcriptome were merged and cd-hit-est version 4.5.4 () was used to remove redundancy with a 100% identity threshold to create the buffalograss transcriptome. Single-end raw sequencing reads of each individual were mapped with Bowtie2 to the buffalograss transcriptome to allow for the estimation of transcript abundance per individual relative to the buffalograss transcriptome. A read count table was produced using SAMtools () and Perl. To account for the variability of total initial Illumina sequencing results among samples, mapped read counts were subjected to normalization and then analyzed for differential expression using the DESeq2 Bioconductor package () in R program (version 3.0.2).Read counts of the two inoculated resistant (R) lines were compared separately to each inoculated susceptible (S) line. We used a final adjusted P-value of < 0.01 to select transcripts that showed a difference in expression between inoculated resistant and susceptible lines. The differentially up-regulated transcripts of 95-55 (R) vs. NE-BFG-7-3453-50 (S) were compared with 95-55 (R) vs. Prestige (S) and common transcripts were identified. Similarly, common up-regulated transcripts between inoculated resistant line NE-BFG-7-3459-17 (R) vs. inoculated susceptible lines were selected. Then the two sets of up-regulated genes (i.e., 95–55 vs. susceptible lines and NE-BFG-7-3459-17 vs. susceptible lines) were compared to each other and common transcripts were identified for annotation. By this filtering procedure, we identified transcripts that are common and differentially up-regulated in both inoculated resistant lines compared to the susceptible lines (up-regulated in resistant inoculated; URI). In the same way, we compared each uninoculated resistant line with each uninoculated susceptible line and used the same filtering procedure to identify genes in common that have different levels of expression between both resistant and susceptible lines (basal up-regulated expressions; BUE). Then we identified down-regulated transcripts in resistant inoculated and uninoculated cultivars compared to inoculated and uninoculated susceptible lines, respectively (down-regulated in resistant inoculated: DRI; basal down-regulated expressions: BDE).The four sets of transcripts were pooled and annotated with Blast2GO using default settings (). The annotated transcripts were analyzed separately to identify genes responsible for induced resistance (transcripts of URI and DRI) and innate immunity (transcripts of BUE and BDE) in buffalograss. Blast2GO was used to prepare graphs of biological, cellular and molecular processes at level two gene ontologies. Gene ontology IDs resulting from the analysis were mined for disease resistance related terms. [...] Ten differentially expressed transcripts were selected for validation by reverse transcription PCR (RT-PCR). These transcripts showed up-regulation either in inoculated or uninoculated resistant buffalograss lines compared to inoculated or uninoculated susceptible lines, respectively. Some transcripts were chosen because they did not have any read counts for either inoculated or uninoculated susceptible lines (Supplementary Table ). The primers (Table ) were synthesized for each transcript using Primer3web version 4.0.0. The primers for ubiquitin conjugating enzyme (UCE) were used as a positive control (Table ). RNA was extracted from new plants of inoculated and uninoculated 95–55, NE-BFG-7-3459-17, Prestige, and NE-BFG-7-3453-50 as described previously. cDNA was synthesized using an InvitrogenTM SuperScript® III First-Strand Synthesis System (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. Using a standard PCR protocol (each 25 μl reaction mixture contained 1x Taq DNA polymerase buffer, 0.2 μM forward and reverse primers, 0.2 mM each dNTPs, 1 U Taq polymerase) each primer pair was used to amplify 100 ng of cDNA template in a Mastercycler®Pro thermalcycler (Eppendorf, Hamburg, Germany) with the following conditions: Initial denaturation at 94°C for 3 min, was followed by 35 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 10 min. Thereafter the reaction was stopped by reducing the temperature to 4°C and PCR products were stored at -20°C. Aliquots (5 μl) of amplified products were separated by electrophoresis on a gel containing 1.7% (w/v) agarose and 1x TAE, at 100 V for 1 h. The presence and size of the DNA fragments were verified by staining the gel with ethidium bromide and observing under UV light. […]

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