2.4.3 Transcriptome analysis
For gene expressions of individuals from the same region, drought adaptation and response mechanisms were analyzed. First, Venn’s diagram of the cross-soil moisture treatment comparison of gene expressions based on RNA-seq data from each region identified through using Venny 2.0 (Oliveros, 2015). Second, KEGG functional enrichment analysis was carried out based on gene expressions obtained for each treatment (seed source of region × soil moisture treatment) using KEGG Orthology Based Annotation System software (KOBAS, v2.0) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Significant pathways were identified with a cut-off of corrected P -value lower than 0.05 (Mao et al., 2005). Third, a heatmap of inter-sample correlation was generated from the variance stabilized and transformed counts based on the gene expressions of each treatment using pheatmap R package (Kolde, 2019). Fourth, DEGs between drought-treated seedlings compared to the non-drought control (light drought treatment vs control, heavy drought treatment vs control) were identified (|log2FC|≧1 and FDR<0.05) using the DESeq2 (Love et al., 2014) for individuals from the same region, and the gene expressions level of each DEG was calculated according to the transcripts per million reads (TPM) method using RSEM (Li & Dewey, 2011). Venn’s diagram of the cross-seed source of region comparison of DEGs was identified through using Venny 2.0 (Oliveros, 2015), obtaining specific DEGs for each region. KEGG functional enrichment analysis was carried out based on the DEGs between heavy drought treatment vs control for each region.
To explore the drought response mechanisms of S. krylovii , KEGG functional annotation analysis were carried out for DCGs between heavy drought treatment vs control within the three regions.