title: Ion Torrent Proton sequencing; Transcription analysis by RNAseq of adult wheat after inoculation with Puccinia graminis f. sp. tritici
design: Transcription analysis by RNAseq of adult wheat after inoculation with Puccinia graminis f. sp. tritici
library_name: B_W6979_72hpi_s
library_construction_protocol: Samples were taken from Puccinia graminis f. sp. tritici inoculated adult wheat plants at indicated hours post inoculation. The stem of inoculated wheat was cut just below the flag leaf and above the flag-1 leaf. The last internode was retained while the leaves, head and peduncle were discarded. The flag leaf sheath was removed from the last internode and the stem discarded. Leaf sheath samples were immediately frozen in liquid nitrogen. Tissue was ground to a fine powder in liquid nitrogen using a mortar and pestle and thereafter stored at -80°C. Wheat was grown in a greenhouse under natural light conditions at 18-25°C (night-day cycle). Three seeds were planted in a sterilised soil-peat moss mixture in plastic pots each measuring 16 cm x 15 cm. Seedlings were thinned to two plants per pot at two weeks old and then grown until eight weeks old. Plants were continually fertilised with Multifeed Classic (N:P:K ratio 19:8:16 plus micronutrients) at a concentration of 8 g/l twice a week. Adult wheat plants were inoculated at the milk to dough stage (Zadoks scale 77-85; Zadoks et al., 1974). After inoculation with Puccinia graminis f. sp. tritici, wheat plants were maintained under greenhouse conditions as described above and samples taken.   Zadoks, JC, Chang, TT and Konzak, CF. 1974. A decimal code for the growth stages of cereals. Weed Research 14 (6): 415-421. Puccinia graminis f. sp. tritici urediniospores of the race PTKST (University of the Free State isolate UVPgt60) were used for inoculation. Adult wheat plants were inoculated according to the method developed by Pretorius et al. (2007), and modified by Bender et al. (2016). Freshly collected urediniospores were suspended in distilled water containing 0.003% (v/v) Tween 20 (polyoxyethylene (20) sorbitan monolaurate) to a final concentration of 0.7 mg/ml. A spore suspension of 80 ml was used to inoculate plants in seven randomised pots. A compressed air sprayer (spray-gun and compressor) at 250 kPa was used to apply the spore suspension uniformly on the adult plants. Inoculated plants were incubated in chambers measuring 100 cm x 85 cm x 60 cm that were constructed from an aluminium frame covered with 200 μm plastic sheeting. The chambers were submerged in 5 cm water to assure high humidity. Pots were randomly placed on a metal grid raised above the water line with 28 pots in each chamber. Plants within the chambers were kept under greenhouse conditions and natural light for 20 hours. During incubation, the temperature fluctuated between 20 and 28˚C within the chambers.  Pretorius, ZA, Pienaar, L and Prins, R. 2007. Greenhouse and field assessment of adult plant resistance in wheat to Puccinia striiformis f. sp. tritici. Australasian Plant Pathology 36: 552-559.  Bender, CM, Prins, R and Pretorius, ZA. 2016. Development of a greenhouse screening method for adult plant response in wheat to stem rust. Plant Disease 100 (8): 1627-1633. Total RNA was extracted from ground wheat leaf sheath tissue using the TRIzol reagent (Thermo Fisher Scientific) and following the supplied protocol. Total RNA samples were further purified using the RNAeasy Mini kit (Qiagen) according to the manufacturer's instructions. An on-column DNase I (Thermo Fisher Scientific) treatment was included. Nucleic acid library construction was done at the Central Analytical Facility (University of Stellenbosch, South Africa). mRNA enrichment was done using the Dynabeads mRNA purification kit (Invitrogen) according to the manufacturer's instructions. The cDNA libraries were constructed with the Ion Total RNA-Seq Kit v2 (Thermo Fischer Scientific) using the guidelines for whole transcriptome libraries.