title: VS061
organism: Triticum aestivum
description: Protocols: Growth condition and heat stress: Present study comprised of three genotypes viz., Banks, EGA Gregory and Fang-60 with former two are the most prominent cultivars grown in Australia and known for its grain quality; while the latter one is reported to be heat tolerant cultivar. Seeds of these three genotypes were received from Australian Genebank. Hence procured seeds were sown in pots approximately containing four litre of UQ23 potting mix. Experiment was conducted using three biological replicates. Hence sown pots were maintained in a temperature controlled growth cabinet at 20C day temperature and 18C nnight temperature with a 12-hour cycle with an relative humidity of 40-50%. An automated irrigation setup was made to irrigate twice a day. Plants were subjected to heat stress by transferring the potted plants to adjacent cubicle with temperature maintained at 38C / 20C respective day / night cycle with a 12-hour cycle with 40-50% relative humidity and watering was done thrice a day manually to avoid drought stress. Heat stress was given in two stages of grain filling viz., early and late in a mutually exclusive fashion. For early grain filling heat stress, potted plants were shifted to high temperature cubicle when the spikes are at 11 days-post-anthesis (dpa) stage and maintained under same condition for three days and at 14dpa, developing spikes (middle 50% with top and bottom 25% being discarded) were harvested and snap frozen using liquid nitrogen. After which, the potted plants were shifted back to control conditions to observe the recovery phase and grains harvested at maturity to study the physical characteristics. Similarly, tagged spikes from control conditions were also harvested at same stage. Also, same set of procedure was followed for late grain filling heat stress with the difference being the plants shifted to high temperature cubicle at 27dpa, and spikes sampled at 30dpa from heat stressed and control environment. Potted plants that are subjected to heat stress at early grain filling and the ones at late grain filling are mutually exclusive. Sample collection: Middle 50% of the spikes excluding awns were selected and snap frozen using liquid nitrogen in the growth cabinet and approximately eight spikes were harvested for transcriptome studies under each stage and heat stress / control conditions per replicate per genotype. They are immediately taken to lab and stored at -80C for further processing. Grains from spikes were removed manually under liquid nitrogen and pulverized using TissueLyser II (Qiagen) at low temperature with the help of liquid nitrogen and stored at -80C till RNA extraction. For further details, readers can refer to the Article reporting this study Approximately 500mg of pulverized samples were used for RNA extraction using Trizol and the supernatant was subjected to column purification using Qiagen RNA extraction kit. In-column DNA digestion was performed to avoid DNA contamination and total RNA was eluted. RNA was tested for its quality and quantity using Agilent Bioanalyzer and Nanodrop 8000 spectrophotometer. Samples range from RIN values of 7.4 to 10.0 with most of the samples are around 9.0 with RNA concentration ranging from 363 to 4849 ng per uL. Approximately 10ug of total RNA for all the 36 samples (VS01 to VS36) was sent to TRI, UQ, Australia for library preparation and sequencing. Indexed TruSeq libraries were prepared and subjected for sequencing. TruSeq RNA Library Preparation Kit