DOI: 10.5061/dryad.m905qfv80
title: Data from: Gene expression differences between western redcedar seedlings resistant and susceptible to cedar leaf blight

description: Western redcedar (T. plicata) is an important Cupressaceae both at
 economic and cultural levels in the Pacific Northwest of North America. In
 adult trees, the species produces one of the most weathering-resistant
 heartwoods among conifers, making it one of the preferred species for
 outdoor applications. However, young T. plicata plants are susceptible to
 infection with cedar leaf blight (D. thujina), an important foliar
 pathogen that can be devastating in nurseries and small-spaced
 plantations. Despite that, variability in the resistance against D.
 thujina in T. plicata has been documented, and such a variability can be
 used to breed T. plicata for resistance against the pathogen. This
 investigation aimed to discern the phenotypic and gene expression
 differences between resistant and susceptible T. plicata seedlings to shed
 light on the potential constitutive resistance mechanisms against cedar
 leaf blight in western redcedar. The study consisted of two parts. First,
 the histological differences between four resistant and four susceptible
 families that were never infected with the pathogen were investigated. And
 second, the differences between one resistant and one susceptible family
 that were infected and not infected with the pathogen were analyzed at the
 chemical (C, N, mineral nutrients, lignin, fiber, starch, and terpenes)
 and gene expression (RNA-Seq) levels. The histological part showed that T.
 plicata seedlings resistant to D. thujina had constitutively thicker
 cuticles and lower stomata densities than susceptible plants. The chemical
 analyses revealed that, regardless of their infection status, resistant
 plants had higher foliar concentrations of sabinene and α-thujene, and
 higher levels of expression of transcripts that code for leucine-rich
 repeat receptor-like protein kinases and for bark storage proteins. In
 conclusion, the data collected in this study shows that constitutive
 differences at the phenotypic (histological and chemical) and gene
 expression level exist between T. plicata seedlings susceptible and
 resistant to D. thujina. Such differences have potential use for
 marker-assisted selection and breeding for resistance against cedar leaf
 blight in western redcedar in the future.
description: This dataset includes transcriptomic and gene expression data
 from an investigation on the impact of <em>Didymascella
 thujina</em> (cedar leaf blight) on <em>Thuja
 plicata</em> (western redcedar). The datasets included in this
 submission were collected and produced as follows below. The information
 shown here was extracted from the manuscript:
 <em>RNA extraction, mRNA enrichment, library production and
 sequencing</em> RNA<em>
 </em>extraction from foliage of three CLB<sup>+
 </sup>and three CLB<sup>- </sup>seedlings was done using
 a modified version of the protocol of Rajakani et al. (2013) [...].
 m<em>RNA </em>enrichment was done using protocol C of the
 Thermo Scientific™ MagJET mRNA<em> </em>Enrichment Kit (Life
 Technologies Inc., Burlington ON, Canada). Libraries were made using the
 NEB Next<sup>® </sup>Ultra™ RNA<em> </em>Library
 Prep Kit for Illumina<sup>® </sup>v. 1.2. (New England
 BioLabs<sup>® </sup>Inc., Ipswich MA, USA). DNA<em>
 </em>was purified as required using the Thermo Scientific GeneJET
 NGS Cleanup Kit (Life Technologies Inc.), and size selection (∼450 bp
 fragment size) was completed with the Thermo Scientific MagJET NGS Cleanup
 and Size Selection Kit (Life Technologies Inc.). Libraries were barcoded
 using the NEB Next<sup>® </sup>Multiplex Oligos for
 Illumina<sup>® </sup>- Index Primers Set 1 (New England
 BioLabs<sup>® </sup>Inc.). Quality control and quantification
 of the individual libraries was done with a DNA<em> </em>1K
 Analysis Kit (Bio-Rad Laboratories, Mississauga ON, Canada) in an
 Experion™ Automated Electrophoresis Station (Bio-Rad Laboratories). The
 final pool consisted of 40 ng of DNA<em> </em>per library.
 Pair-ended 100 base sequencing was completed in a single lane of an
 Illumina<sup>® </sup>HiSeq 2000 sequencer at Genome Quebec
 Innovation Centre (Montreal QC, Canada).
 <em>Assembling and annotation of the reference
 transcriptome</em> [...] All of the processes
 described below were completed on the WestGrid Hermes cluster
 (https://www.westgrid.ca/) hosted at the University of Victoria using
 customized shell, Python and R scripts. HPC GridRunner was used to enhance
 annotation searches such as BLAST and HMMER. Paired-end
 FASTQ Illumina<sup>® </sup>1.9 (Phred-33 ASCII) compressed
 files were produced for each sample after sequencing. Each file was
 checked for quality before and after trimming using FastQC v. 0.11.2
 (Andrews, 2014). Trimming was done in Trimmomatic v. 0.33 (Bolger et al.
 2014; [...]). The reference transcriptome was built using Trinity v. 2.0.6
 (Grabherr et al., 2011) with the default settings for paired-end data, and
 its statistics were calculated in PRINSEQ v. 0.20.1 (Schmieder and
 Edwards, 2011). Annotation was completed using Trinotate v. 2.0.2
 (http://trinotate.github.io; [...]).
 <em>Differential expression analyses</em>
 The downstream analyses (Haas et al., 2013) were conducted using
 the assembled contigs and contig variants from Trinity v. 2.0.6 (Grabherr
 et al., 2011) instead of the smaller number of corresponding deduced
 genes. Trinity refers to the contigs and variants as “transcripts” [...].
 Reads were mapped to the assembly with RSEM v. 1.2.20 (Li and Dewey, 2011)
 and fragments per kilobase of transcript per million mapped (FPKMs,
 Trapnell et al. 2010) were calculated. Normalization was achieved in edgeR
 (Robinson et al., 2010) by computing the trimmed mean of
 <em>M</em>-values (TMM; Dillies et al. 2013, Robinson and
 Oshlack 2010). The differential expression (DE) analysis was completed by
 comparing all samples in pairs using the default settings in edgeR, and
 then extracting and merging the sequences that had a minimum fold-change
 of four and a maximum false discovery rate of 0.001 from all the samples
 into a single matrix.
description: # Gene expression differences between Western Redcedar seedlings resistant
 and susceptible to Cedar Leaf Blight
 The sets deposited here
 include 1) the transcriptomic assembly, and 2) the differential expression
 data produced from an investigation that aimed to detect constitutive
 differences between *Thuja plicata* (western redcedar) seedlings that were
 susceptible or resistant to *Didymascella thujina* (cedar leaf blight,
 CLB). The study also included histological and foliar chemical composition
 components, but those data are part of the manuscript itself. The
 assembled transcriptome can be considered a metagenome as sequences
 annotated as belonging to organisms other than *T. plicata* or *D.
 thujina* were not removed unless they did not meet the minimum nucleotide
 length. That approach was taken in case that organisms other than *T.
 plicata* could account for resistance against *D. thujina* in this
 pathosystem (e.g. due to endosymbionts), which could have been overlooked
 if just a *T. plicata* genome was used. The experimental design used in
 this part of the study was as follows (see the manuscript as well):
 Twenty, one-year-old seedlings from each resistant category were exposed
 to natural *D. thujina* inoculum in an infected 10-year-old progeny trial
 in Jordan River (British Columbia, Canada). These CLB+ plants (see table
 below) were placed in a greenhouse and maintained after inoculation until
 symptoms developed. A similar number of seedlings per resistance category
 that had never been exposed to *D. thujina* (i.e. the CLB- treatment, se
 below) were maintained at a different facility while the CLB+ seedlings
 were being inoculated and then they were moved to the same greenhouse as
 the infected ones, but kept in different rooms for the same length of
 time. Only three randomly selected seedlings per resistance category and
 infection condition were used for RNA extraction and RNA-Seq analysis (for
 a total of 12 RNA-Seq samples). Here is a summary of the experimental
 design: | **Item** | **Details** | |
 :--------------------------------------------- |
 :----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Resistance categories and infection conditions | 2 Resistance categories (resistant, susceptible); 2 Treatments (with and without disease symptoms; CLB+, CLB-); ·3 Replicates per resistance category × treatment combination. | | Seedlings' age | Older than a year when infected. Close to 2 years old when sampled (symptoms had developed in the exposed). | | Inoculation technique | ·Exposure to *Didymascella thujina* in a *Thuja plicata* progeny trial site in Jordan River (British Columbia, Canada). | | *RNA*-Seq analyses | Illumina HiSeq 100bp paired-end technology. *In house* made libraries per sample, outsourced to Génome Québec. | RNA-Seq data from the 12 seedlings sampled (i.e. 2 × 2 × 3 in item "Resistance categories and infection conditions" in the above table) were used to assemble the reference transcriptome that was uploaded here. The RNA-Seq data of those 12 seedlings can be found at the Sequence Read Archive of the National Center for Biotechnology Information (NCBI) (BioProject PRJNA994939, entries SRR25280989-SRR25281000). ## Description of the data and file structure The data uploaded here are as follows: * *Transcriptome_SRA_PRJNA994939.fasta*: This is a *de novo* transcriptomic (RNA-Seq based) assembly produced in Trinity to analyze the gene expression data produced in this study. The sequences' names are as those found in the manuscript that this assembly is associated with. * *edgeR_DE-SRA_PRJNA994939.matrix*: This matrix contains the differential expression data produced after reads from the RNA-Seq samples were mapped to the transcriptomic assembly (notice that these are the raw count data, which had not been log2-centered). File *edgeR_DE-SRA_PRJNA994939.matrix* is the result of the following procedure: 1\) Mapping of reads in BioProject PRJNA99493 (*NCBI entries SRR25280989-SRR25281000*) to *Transcriptome_SRA_PRJNA994939.fasta* using RSEM v. 1.2.20. 2\) Calculation of the fragments per kilobase of transcript per million mapped (FPKMs). 3\) Normalization of the FPKMs by calculating their trimmed mean of M-values (TMM). 4\) Pairwise differential expression analyses using edgeR, and compilation of such data from sequences that had a minimum fold-change of four and a maximum false discovery rate (FDR) of 0.001. The names of the sequences in file *edgeR_DE-SRA_PRJNA994939.matrix* match those in the transcriptome (*Transcriptome_SRA_PRJNA994939.fasta*). Refer to the following table to identify the respective sample in the columns of file *edgeR_DE-SRA_PRJNA994939.matrix*: | Column | Seedling ID | CLB resistance class | Condition | | :-------------------------------------- | :---------- | :------------------- | :--------- | | HI.2078.008.Index\_2.U\_Filtered.RSEM | 583-23 | Susceptible | Uninfected | | HI.2078.008.Index\_3.V\_Filtered.RSEM | 583-27 | Susceptible | Uninfected | | HI.2078.008.Index\_4.W\_Filtered.RSEM | 583-33 | Susceptible | Uninfected | | HI.2533.001.Index\_7.Z\_Filtered.RSEM | 685-24 | Resistant | Uninfected | | HI.2078.008.Index\_6.Y\_Filtered.RSEM | 685-27 | Resistant | Uninfected | | HI.2078.008.Index\_5.X\_Filtered.RSEM | 685-34 | Resistant | Uninfected | | HI.2078.008.Index\_1.T\_Filtered.RSEM | 583-2 | Susceptible | Infected | | HI.2533.001.Index\_12.Q\_Filtered.RSEM | 583-12 | Susceptible | Infected | | HI.2533.001.Index\_10.CC\_Filtered.RSEM | 583-17 | Susceptible | Infected | | HI.2533.001.Index\_8.AA\_Filtered.RSEM | 685-1 | Resistant | Infected | | HI.2533.001.Index\_9.BB\_Filtered.RSEM | 685-4 | Resistant | Infected | | HI.2533.001.Index\_11.R\_Filtered.RSEM | 685-18 | Resistant | Infected | ## Sharing/Access information The transcriptomic assembly and differential expression data shared here is linked to NCBI’s BioProject PRJNA994939 (entries SRR25280989-SRR25281000). Refer to NCBI’s Sequence Read Archive () to access the raw RNA-Seq data produced from the biological material sampled in this investigation.