Gene expression responses to heat stress in early- and late-flowering chickpea cultivars
Gene expression responses to heat stress in early- and late-flowering chickpea cultivars
Leaf tissues of five selected chickpea lines (309E, 309L, 224E, 224L, 4958) were collected before heat stress, after heat stress, and during the recovery, immediately frozen in liquid nitrogen and stored at −80 °C. Three biological replicates were carried out for each genotype and treatment. Frozen leaves were ground to a fine powder with a previously sterilized mortar and pestle using liquid nitrogen to prevent RNA degradation. Seeds were sowed in 4L pots (one plant/pot) containing a mixture of tuff, coarse vermiculite and coarse peat (1:1:1, v/v/v), on 26.1.2022. The pots were supplemented with 10gr of slow-release fertilizer (Osmocote, ICL, Israel) and drip irrigated with tap water. The plants were grown in Beersheva, Israel, in two chambers within a controlled greenhouse under a temperature of 28/18oC (day/night). Heat stress treatment was achieved in one of the chambers by applying a regime of 38/28oC (day/night) for 4 days during the pod set (from 29.5.2022 to 2.6.2022). Frozen leaves (100mg) have been ground to a fine powder with a previously sterilized mortar and pestle using liquid nitrogen to prevent RNA degradation. RNA extraction was performed using 100 mg of ground material using Zymo Quick RNA miniprep (Zymo Research) with DNAse treatment, following the protocol. RNA libraries were prepared using Illumina mRNA Prep kit, following the manufacturer instructions and a unique dual index combinations were used for each sample for barcoding. The concentration of each of the libraries was determined using Qubit™ 4 Fluorometer (dsDNA High Sensitivity Kit - Invitrogen).