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.