Divergent gene expression levels between diploid and autotetraploid Tolmiea relative to the total transcriptome, the cell, and biomass

Premise of the Study Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea , we present an integrated approach for cross‐ploidy comparisons that account for differences in transcriptome size and cell density and make multiple comparisons of transcript abundance. Methods We use RNA spike‐in standards in concert with cell size and density to identify and correct for differences in transcriptome size and compare levels of gene expression across multiple scales: per transcriptome, per cell, and per biomass. Key Results In total, ~17% of all loci were identified as differentially expressed ( DEG s) between the diploid and autopolyploid species. The per‐transcriptome normalization, the method researchers typically use, captured the fewest DEG s (58% of total DEG s) and failed to detect any DEG s not found by the alternative normalizations. When transcript abundance was normalized per biomass and per cell, ~66% and ~82% of the total DEG s were recovered, respectively. The discrepancy between per‐transcriptome and per‐cell recovery of DEG s occurs because per‐transcriptome normalizations are concentration‐based and therefore blind to differences in transcriptome size. Conclusions While each normalization enables valid comparisons at biologically relevant scales, a holistic comparison of multiple normalizations provides additional explanatory power not available from any single approach. Notably, autotetraploid loci tend to conserve diploid‐like transcript abundance per biomass through increased gene expression per cell, and these loci are enriched for photosynthesis‐related functions.