Illumina HiSeq 2000 paired end sequencing; RNA-seq of coding RNA of wheat heads from 3, 6, 12, 24, 36, and 48 hours after inoculation of fungal pathogen Fusarium graminearum or mock inoculation
RNA-seq of coding RNA of wheat heads from 3, 6, 12, 24, 36, and 48 hours after inoculation of fungal pathogen Fusarium graminearum or mock inoculation
F. graminearum  conidia spores from strain IFA65 required for inoculation were produced on defined SNA medium under UV-light at 25C. After two weeks conidia were harvested and diluted to 50,000 conidia/mL in water. Aliquots were stored at  80C. The inoculation experiment was conducted under controlled light house conditions in December 2013 as described in (REF). Briefly, twelve florets from 6 central spikelets (the two basal florets) were inoculated at anthesis with 10 ul of spore suspension (500 conidia) or mock. Plant heads were moistened and covered in plastic bags until sampling but no longer than 24 hours to provide favorable humid conditions for the fungus to germinate. Plants were sampled at 3, 6, 12, 24, 36 and 48 hours after inoculation by shock-freezing the inoculated section of the head including the rachis. In total 360 plants were inoculated comprising two NILs, six time points, two treatments and three replicates each including five pooled samples. 20 heads per NIL remained undissected and remained for phenotyping the total number of diseased spikelets per head 21 days after inoculation in the greenhouse. Frozen samples were ground under sterile conditions and pooled to comprise a single sample/data point as described in Kugler et al (2013). 100 mg frozen tissue was used to extract RNA using the RNeasy Plant Mini Kit (Qiagen, Venlo, Netherlands). Quality and quantity was checked on an an automated electrophoresis-system (Experion, #701-7000, Bio-Rad, Hercules, CA, US).  Samples were Illumina sequenced on HiSeq2000/MiSeq machines (Eurofins MGW) yielding at least 20M 100bp paired-end reads per samples. Each sample represents one experimental condition (pathogen/mock; time points; genotype). Each experimental condition has been replicated 3x.
Experimental Factor: mock inoculation
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Experimental Factor: 36
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