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Sample GSM6958178

Query DataSets for GSM6958178

Status

Public on Sep 15, 2023

Title

Grace, drought, F.culmorum, 96hpi, rep2

Sample type

SRA

Source name

spike

Organism

Hordeum vulgare

Characteristics

cultivar: Grace
tissue: spike
abiotic stress: drought
inoculation: Fusarium culmorum
harvest time point [hpi]: 96

Treatment protocol

Fugal inoculum was cultured and propagated according to Linkmeyer et al. (2013). Therefore, three isolates of F. culmorum (Fc002, Fc03, Fc06 – culture collection, Chair of Phytopathology, Technical University of Munich) and three isolates of F. avenaceum (Fa002, Fa02, Fa04 – culture collection, Chair of Phytopathology, Technical University of Munich) known to strongly infect barley spikes and to produce DON (Linkmeyer et al., 2013; Hofer et. al., 2016, Hoheneder et al., 2022) were used in equal amounts. Spore solution was adjusted to 50.000 conidia per ml tap water and contained 1 ml/L Tween 80 to improve wetting of the spike tissue. The respective mock solution contained the same amount of Tween 80. The spikes of the plants were sprayed with handheld spray flasks till run-off either with F. culmorum spore solution or mock solution. To maintain optimal moist conditions for infection (99 % relative air humidity), spikes were covered and sealed with transparent polythene bags for two days. Respective mock sprayed plants were similarly treated. 48h and 96h after inoculation, individual spikes were cut from each pot and flash frozen in liquid nitrogen.

Growth protocol

The pots were placed on flooding tables for daily automatic watering and with additional lightning for 16 h per day. Plants were held upright with support devices and were randomized twice a week to assure uniform growth. Temperature was set to 18 °C (day) and 16 °C (night) with a relative air humidity of 60 %. Drought conditions were set on by a stop of automatic watering on separate flooding tables. To prevent plants from early and premature plant death due to fast desiccation of the substrate, each pot received little amounts of water (50 - 100 ml) in the first three days after stopping daily irrigation.

Extracted molecule

total RNA

Extraction protocol

Each individual spike sample of each biological triplicate per treatment variation was divided into two pieces using one part for individual RNA extraction. After separation, the spike samples were immediately ground in liquid nitrogen and stored at minus 70 °C. RNA extraction was performed with Direct-zol RNA Miniprep Plus Kit (ZymoResearch, USA) according to manufacturer’s protocol.
Preparation of libraries for 3’mRNA sequencing was carried out with Lexogen QuantSeq 3'mRNA-Seq Library Prep Kit (FWD) (Lexogen, Austria) for Illumina sequencing according to manufacturer’s protocol. Quantification of input RNA was conducted with Qubit Fluorometer 2.0 (Invitrogen, USA) and Qubit RNA BR (broad range) Assay Kit (Invitrogen, USA) according to manufacturer’s protocol in a range of 10 to 1200 ng total RNA. Quantification of library size was performed with Qubit Fluorometer 2.0 und Qubit DNA High Sensitivity Assay Kit (Invitrogen, USA). A check for distribution of mRNA fragment size of each library was performed with am Agilent 2100 Electrophoresis Bioanalyzer (Agilent Technologies, USA). Final quantification of each library was determined according to Illumina qPCR guide with a KAPA SYBR Fast Mastermix Low ROX (Peqlab, Germany) in a QuantStudio 5 real-time PCR system (Applied Biosystems, USA). Final libraries were normalized with elution buffer (Qiagen, Germany) to a final concentration to 2 nM and pooled with an equal amount of each sample library. Denaturation and dilution of libraries for HiSeq Clustering was performed according to protocol A of user guide (Illumina) with a concentration for clustering of 10 pM.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Grace_D_Fc_96_2

Data processing

Data processing steps for the CPM_Horvu_Morex_V2
The quality of the raw RNAseq data was analyzed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
The trimming step was done with Trimmomatic using the parameters ILLUMINACLIP:Illumina-SE.fasta:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:40.
We used Hisat2 with parameters -U --rna-strandness FR to map the trimmed data to the Morex v2 assembly. In order to add an artificial 3’UTR because of the 3’-RNAseq reads, we used a custom script to elongate the last exon either by 3 kb or until the next gene in the gff file of Morex v2.
The counts of the generated bam files were assigned to genes using featureCounts with the parameters -t gene -s 1 -M -O.
CPM values were calculated using edgeR with "calcNormFactors" and "cpm( log = FALSE, normalized.lib.sizes = TRUE)"

Data processing steps for the CPM_Horvu_Morex_V3 and CPM_Fusarium_culmorum_UK99
The RNAseq data was analyzed using the nf-core/rnaseq pipeline version 3.9 with default settings. An artificially merged genome was used for mapping consisting of MorexV3 and Fc-UK99
The gene counts were split again into the two species and CPM values were calculated separately for each species using edgeR with "calcNormFactors" and "cpm( log = FALSE, normalized.lib.sizes = TRUE)"

Assembly: MorexV2 (Mascher et al., 2019), MorexV3 (Mascher et al., 2020), Fc UK99 (Urban et al., 2016)

Supplementary files format and content: CPM_Horvu_Morex_V2.csv is a comma-separated file containing the counts per million values for all samples, mapped against MorexV2
Supplementary files format and content: CPM_Horvu_Morex_V3.csv is a comma-separated file containing the counts per million values for all samples, mapped against MorexV3
Supplementary files format and content: CPM_Fusarium_culmorum_UK99.csv is a comma-separated file containing the counts per million values for all samples, mapped against Fusarium culmorum UK99

Submission date

Jan 23, 2023

Last update date

Sep 15, 2023

Contact name

Ralph Hückelhoven

E-mail(s)

[email protected]

Organization name

Technische Universität München

Department

Chair of Phytopathology