Illumina HiSeq 2500 paired end sequencing; Transcriptomic analysis of cocoa (Theobroma cacao L.) response to water deficit.
Transcriptomic analysis of cocoa (Theobroma cacao L.) response to water deficit.
Third or fourth leaf of each individual plant (3 biological replicates by genotype and each water condition) was collected the day 52 of treatment (without water - termed drought environment) and it was frozen in liquid nitrogen. The samples were transported to the laboratory and they were frozen at 80 °C until RNA extraction  For the physiological evaluation of the selected cocoa genotypes, a water stress assay was established in a greenhouse. 5 months after grafting, plants of T. cacao of the genotypes EET 8 and TSH565 were tested for water deficit trial. From the moment of establishment, plants were irrigated to field capacity, to guarantee their emergence and growth until the application of the treatments. A divided plots experimental design was applied: the water condition of the two genotypes were evaluated with two levels: field capacity (C) and water deficit-Drought (D) by irrigation´s suspension during a period that generated severe stress (Leaf Water Potential of -3.0 Mpa). The experiment included three replicates consisting of an experimental unit of 12 individuals for each genotype tested. These genotypes were chosen by their tolerance level to drought as the most tolerant (TSH) and the most susceptible (EET8) based on physiological parameters (water content, gas exchange, chlorophyll fluorescence, growth and recovery from wilting). Total RNA extraction was carried out follow Chang et al., 1993 protocol with some modifications. Like the use of Phase Lock gel tubes in the chloroform extraction steps and the quantities was scaled to 2 ml tubes. Libraries were constructed by Novogene C.A in Sacramento USA. They used NEB eukaryotic mRNA library kit for plants. The library type was 250 - 300 bp insert cDNA. These libraries are unstranded and with paired end