Melatonin seed priming imparts terminal heat stress tolerance in Indian bread wheat cv HD2967

Seeds of wheat (Triticum aestivum L.) cv. HD2967 released from Indian Agricultural Research Institute (IARI), New Delhi was used as experimental material. Seed were primed with 50 mg/l melatonin as priming medium and distilled water was used as control and incubated for 24 h at 22degreeC +- 2degreeC under dark conditions at seedling stage in temperature-controlled growth chamber and root and shoot length, germination percentage and vigour index were measured. Primed seeds were raised in season in field and exposed to elevated temperature intermittently for 4 hrs (11am - 3pm) in an indigenously built heat trap chamber at anthesis (Zadoks growth stage 60; i.e., the beginning of pollination) for three consecutive days. Extensive phenotyping of a series of agronomic, biochemical and physiological parameters were recorded and seeds were harvested at maturity. After the treatments tissue samples were collected from developing ear head under both conditions, frozen in liquid nitrogen and stored at -80 degreeC for further RNA isolation and expression analysis.RNA was isolated from the control and the heat-treated ear head samples of HD2967 were quantified by spectrophotometer. Quality of RNA was assessed by visualization on agarose gel electrophoresis and measuring RIN (RNA Integrity Number). cDNA was synthesized from the isolated RNA and observed as smear on agarose gel electrophoresis. The total RNA of HD 2967 was processed further for RNA sequencing using Illumina (NOVASEQ 6000) platform. Further, processing of data for removing the adapters from reads by trim_galore version 0.4.1 tool was carried out and the data passed the quality control analysis. Reference genome used was Triticum_ aestivum_refseq2.1 and the tool used for alignment and assembly were tophat-2.1.1 and cufflinks-2.2.1 respectively. Further, based on FDR, 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. Few DEGs selected on basis of log 2-fold change were validated for differential expression under heat stress by qRT-PCR analysis.