Heat stress responsive transcriptome analysis at post anthesis stage in Indian bread wheat Raj 3765

India bread wheat genotype Raj 3765 used in this study was obtained from the Division of Genetics, IARI, New Delhi, India. The seeds of the genotype were pre-vernalized, germinated on petri plates and transferred to twelve inches pots containing soilrite and grown under greenhouse conditions at the National Phytotron Facility, IARI, New Delhi. The growth conditions were maintained at temperature of 24 degree C, light intensity of 350 micro mol/m/s with photoperiod for 16/8 h and 60% humidity. The plants were regularly watered.Triticum aestivum cv Raj3765 plants at the post-anthesis stage (Feekes scale-10.53) were given heat stress (HS) at 37 degree C and 42 degree C for six (6) continuous hours. Heat stress treatment was carried out in an incubator chamber by increasing the temperature at 1 degree C per 10 min until it reached the desired HS temperature. After heat stress treatment the controlled conditions were brought down in same manner. The plants grown without HS treatment (24 degree C) were used as control. Flagleaf samples (three biological replicates) were collected from HS and control plants and were immediately frozen in liquid nitrogen and stored at -80 degree C for further molecular biology experimentation.RNA was isolated from the control and the heat-treated flag leaf samples of Raj 3765 (heat tolerant wheat variety) and quantified by spectrophotometer. Quality of RNA was assessed by visualization on agarose gel electrophoresis and measuring RIN (RNA Integrity Number). RNA samples were observed to have RIN (RNA Integrity Number) value in the range of 7.2 to 9.0 cDNA was synthesized from the isolated RNA and observed as smear on agarose gel electrophoresis. The total RNA Raj 3765 was processed further for RNA sequencing using Illumina HiSeq 4000 platform. The data passed the quality control analysis done using FastQC v0.11.5 software. Further, processing of data for removing the adapters from reads by Trimmomatic software v.0.36 tool was carried out and DEGs (Differentially Expressed Genes) were identified. The samples were given different codes to find out DEGs expressed under heat stress conditions. Heat map, Volcano plot, MA plot, distribution of DEGs, GO (Gene ontology) based classification of DEGs under heat stress conditions were identified. The SSRs (Simple Sequence Repeat) under heat stress conditions in flag leaf were identified. Few DEGs selected on basis of log 2-fold change were validated for differential expression under heat stress by qRT-PCR analysis.