6 protocols
AccessionType
nucleic acid sequencing protocol
Illumina HiSeq2000, 100 bp single end
treatment protocol
Single seeds were sown and put at 4 degrees C for one week. After vernalization, seeds were germinated at 20 degrees C for two weeks and each two plants were potted into 3 L pots. The plants were initially kept in a 18 degrees C/12 degrees C day/night cycle (12 hours light), which was gradually changed until anthesis to 21 degrees C/18 degrees C at 55 % relative humidity (16 hours light). At least 12 heads per experimental condition (three replications, F. graminearum/mock treatment, two time points) were subjected to further experiments. At anthesis, the plants were inoculated either with a F. graminearum spore suspensions or water: 10 ? of the respective inoculum was pipetted into one floret between palea and lemma, directly onto the generative part. Per head two outer floret of six spikelets in three central rows were treated in this way, while the central florets were not inoculated (in total 12 florets). The inoculated heads were moistened with distilled water and bagged into a plastic bag for 24 hours to provide favourable conditions for disease development. Tissues comprising only the initially inoculated spikelets were sampled 30 or 50 hours after inoculation (hai) and from these the generative parts (stigma, anthers, ovary) as well as the glumes and the central floret were discarded. Only palea, lemma and rachis were frozen immediately in liquid nitrogen and stored at -80 degrees C until use.
growth protocol
Single seeds were sown and put at 4 C for one week. After vernalization, seeds were germinated at 20 degrees C for two weeks and each two plants were potted into 3 L pots. The plants were initially kept in a 18 degrees C/12 degrees C day/night cycle (12 hours light), which was gradually changed until anthesis to 21 degrees C/18 degrees C at 55 % relative humidity (16 hours light). At least 12 heads per experimental condition (three replications, F. graminearum/mock treatment, two time points) were subjected to further experiments. At anthesis, the plants were inoculated either with a F. graminearum spore suspensions or water: 10 ul of the respective inoculum was pipetted into one floret between palea and lemma, directly onto the generative part. Per head two outer floret of six spikelets in three central rows were treated in this way, while the central florets were not inoculated (in total 12 florets). The inoculated heads were moistened with distilled water and bagged into a plastic bag for 24 hours to provide favourable conditions for disease development. Tissues comprising only the initially inoculated spikelets were sampled 30 or 50 hours after inoculation (hai) and from these the generative parts (stigma, anthers, ovary) as well as the glumes and the central floret were discarded. Only palea, lemma and rachis were frozen immediately in liquid nitrogen and stored at -80 degrees C until use.
nucleic acid library construction protocol
Each sample represents one experimental condition (pathogen/mock; time points; genotype) and comprises pools of 12 treated wheat heads. Each experimental condition has been replicated 3x. cDNA was sent to a service provider (GATC konstanz germany) who have performed the library construction following the Illumina TruSeq RNA sample preparation guide (version 2, http://support.illumina.com/sequencing/sequencing_kits/truseq_rna_sample_prep_kit_v2/documentation.ilmn)
growth protocol
We utilized four early isogenic lines (NILs) previously generated from a cross of the resistant spring wheat line CM-82036 and Remus, a susceptible Austrian spring wheat cultivar. The NILs have Remus as the recurrent parent (5 backcrosses) and contain the resistance alleles from CM-82036 of both Fhb1 and Qfhs.ifa-5A (NIL1), or either Fhb1 (NIL2) or Qfhs.ifa-5A (NIL3) or none (NIL4). Additionally, CM-82036 was used in the subsequent experiments. In preparation for fungal innoculation, F. graminearum spores required for inoculation were produced on defined SNA medium under UV-light at 25 degrees C. After two weeks conidia were harvested and diluted to 50.000 conidia/mL in water. Aliquots were stored at -80 degrees C.
nucleic acid extraction protocol
To eliminate RNases, metal jars with inherent metal spheres for Retsch-mill (MM 301, Haan, Germany) were sterilized at 180 degrees C for 3 h and then stored at -80 degrees C. All tissue belonging to one sample was pooled in one precooled jar and clamped in Retsch-mill. Grinding was performed for 30 s at full speed to obtain a fine tissue powder and immediately put back at -80 degrees C. Total-RNA was extracted from 100 ul of frozen tissue powder using the RNeasy Plant Mini Kit (#74903, Qiagen, Venlo, Netherlands) according to manufacturers instructions. The extracted RNA was checked for quality and quantity on an automated electrophoresis-system (Experion, #701-7000, Bio-Rad, Hercules, CA, US). Sequencing was performed on an Illumina HiSeq2000 machine using 8x multiplexing, theoretically generating 22 M reads per sample by an external sequencing-provider GATC (Konstanz, Germany).