Differential expression analysis between experimental groups
Differences in normalized transcript abundance levels were tested using
a generalized linear model (glmQFit and glmQLFTest), with
log2-transformed transcript abundance as the response
variable with a genome-wide false discovery rate (FDR) of 0.05 with the
function “Toptags ”. The results of the gene expression
differences are presented as centred and scaled heatmaps, plotted with
gplots (Warnes et al. 2020). The groups that were compared was:
comparison 1) saltwater control (SwC) vs. saltwater ecotype exposed to
freshwater (SwFw); comparison 2) freshwater control vs. freshwater
ecotype exposed to saltwater (FwSw); comparison 3) controls (FwC- SwC)
and comparison 4) transcripts that are significantly different between
controls, and similarly expressed within ecotype; all freshwater
ecotypes vs. all saltwater ecotypes (FwC_FwSw vs. SwC_SwFw)
(Figure 2b ). Comparison 3 identify transcripts that are
differentially expressed between the control groups, and to a lesser
extent between comparison 1 and comparison 2, but where expression
patterns within ecotype and salinity change do not differsignificantly. Comparison 4 identifies genes that are similarly
expressed within salinity of origin (the ecotype ) independently
of treatment. The transcripts in comparison 4 were extracted from the
transcripts identified in comparison 3, by calculating the average
expression for each transcript within each group (SwC, SwFw, FwC, FwSw)
and extracting the genes that had less than 0.4 CMP differential
expression between the respective control and exposed group. Overlapping
genes in the four comparisons were identified and extracted with the
package Venn-diagrams (Gao 2019).