Expression Comparison in Liver

Utilizing independent liver tissue samples, we compared differential expression in liver tissue gene expression among ohnologs of grayling and Atlantic salmon with their ohnolog-tetrad tissue expression evolution categories. The liver samples from four grayling individuals were sampled in the river Gudbrandsdalslågen (61°18′53.09″N 10°18′1.53″E). The samples were from two males (370,375 mm) and two females (330,360 mm). The fish was euthanized and dissected immediately after capture, and the liver was stored in RNAlater. Total RNA was extracted and 100-bp single-end read libraries were generated for two individuals and sequenced using the Illumina HiSeq4000 platform. For the other two individuals, 150-bp paired-end read libraries were generated and sequenced using the Illumina HiSeq2500 platform. RNA-Seq data for an additional four Atlantic salmon liver tissue samples were obtained from a feeding experiment (Gillard et al. 2018). Presmolt salmon were raised on fish oil-based diets under freshwater conditions.

The RNA-Seq read data were quality processed using CutAdapt (Martin 2011) before alignment we aligned the reads to grayling or Atlantic salmon (ICSASG_v2; Lien et al. 2016) genomes, respectively, using STAR (Dobin et al. 2013). RSEM (Li and Dewey 2011) expected counts were generated for gene features. EdgeR (Robinson et al. 2010) was used to generate normalized library sizes of samples (TMM normalization), followed by a differential expression analysis using the exact test method between the gene expression of both the grayling and Atlantic salmon ohnologs in each ohnolog-tetrad. The fold change (log2 scaled) and significance of differential expression (false discovery rate-corrected P-values) were produced for grayling and Atlantic salmon duplicates, as well as relative counts in the form of CPM.