RNA-seq analysis of barley (Hordeum vulgare) single mutants in genes encoding barley CBC (HvCBP20, HvCBP80) and a double mutant in both genes encoding barley CBC in response to drought stress applied at the booting stage and subsequent rewatering
RNA-seq analysis of barley (Hordeum vulgare) single mutants in genes encoding barley CBC (HvCBP20, HvCBP80) and a double mutant in both genes encoding barley CBC in response to drought stress applied at the booting stage and subsequent rewatering
As the object, we used barley plants (Hordeum vulgare (L.)), cultivar 'Sebastian' and its mutants TILLING hvcbp20.ab, hvcbp80.b and hvcbp20.ab/hvcbp80.b. Plants were grown under drought stress during which soil moisture was reduced to 1.5% volumetric water content. Drought stress lasted 10 days and was followed by a rehydration period. Soil moisture was monitored daily using TDR EasyTest (Institute of Agrophysics PAS, Poland). The tissue was collected from the 3rd barley leaf from its middle part in 4 biological replicates, where each replicate included tissue from 3 independent plants. Tissue collected after drought stress (75 days after planting) and after rehydration (85 days after planting) High-quality reads were aligned to the barley reference transcriptome (BaRTv2.18) with Kallisto v. 0.46.2. Differential gene expression was analyzed using 3DRNAseq within the R software environment. Read counts and transcripts per million (TPM) were derived from Kallisto outputs via the tximport R package. Transcripts and genes with low expression levels were filtered based on the mean-variance relationship, and genes were classified as expressed if they met defined thresholds for counts per million (CPM). The trimmed mean of M-values (TMM) method was applied for count normalization, and principal component analysis (PCA) revealed no notable batch effects. As the object, we used barley plants (Hordeum vulgare (L.)), cultivar 'Sebastian' and its mutants TILLING hvcbp20.ab, hvcbp80.b and hvcbp20.ab/hvcbp80.b. Plants were grown under drought stress during which soil moisture was reduced to 1.5% volumetric water content. Drought stress lasted 10 days and was followed by a rehydration period. Soil moisture was monitored daily using TDR EasyTest (Institute of Agrophysics PAS, Poland). The tissue was collected from the 3rd barley leaf from its middle part in 4 biological replicates, where each replicate included tissue from 3 independent plants. Tissue collected after drought stress (75 days after planting) and after rehydration (85 days after planting) As the object, we used barley plants (Hordeum vulgare (L.)), cultivar 'Sebastian' and its mutants TILLING hvcbp20.ab, hvcbp80.b and hvcbp20.ab/hvcbp80.b. Plants were grown under drought stress during which soil moisture was reduced to 1.5% volumetric water content. Drought stress lasted 10 days and was followed by a rehydration period. Soil moisture was monitored daily using TDR EasyTest (Institute of Agrophysics PAS, Poland). The tissue was collected from the 3rd barley leaf from its middle part in 4 biological replicates, where each replicate included tissue from 3 independent plants. Tissue collected after drought stress (75 days after planting) and after rehydration (85 days after planting) High-quality reads were aligned to the barley reference transcriptome (BaRTv2.18) with Kallisto v. 0.46.2. Differential gene expression was analyzed using 3DRNAseq within the R software environment. Read counts and transcripts per million (TPM) were derived from Kallisto outputs via the tximport R package. Transcripts and genes with low expression levels were filtered based on the mean-variance relationship, and genes were classified as expressed if they met defined thresholds for counts per million (CPM). The trimmed mean of M-values (TMM) method was applied for count normalization, and principal component analysis (PCA) revealed no notable batch effects. Total RNA from leaf tissue was extracted using miRvana isolation kit (ThermoFisher Scientific, USA) according to the manufacturer's guidelines. Concentration and quality were assessed using a NanoDrop spectrophotometer (ND-1000). cDNA libraries generated using the Illumina TruSeq protocol were sequenced on an Illumina NovaSeq 6000 platform by Macrogen, producing paired-end reads of 2 × 150 bp length. Each sequencing lane contained six samples, and an entire flow cell was utilized. 