Subgenome dominance and differentiation in homoeolog expression patterns
Unequal expression of homoeologous genes in allopolyploids can be an important feature and consequence of polyploidization. We investigated homoeolog expression bias in five different tissues and under three stress treatments. Of 11,861 identified homoeologous gene pairs between subgenomes A and B, 11,409 had homoeologous expression bias (HEB) in at least one tissue or treatment, and 1,160 had biased expression in all sampled tissues and treatments. Pairwise comparisons between syntenic gene pairs support a slight bias in expression toward the B subgenome across all five tissues and three treatments (Figure S10). Roughly 26.6% (3,155) of the 11,861 pairwise comparisons across the five tissues and three stress treatments showed biased expression toward homoeologs in the B subgenome. Each tissue and treatment have approximately 6,000 homoeologous gene pairs with significant differential expression, including 52.16% biased toward the B subgenome (Figure S10).
To investigate the differentiation in homoelog expression patterns between O. kokonorica and C. songorica , we first performed transcriptomic analysis under the same stress treatments (i.e., light drought, cold, heat) in both shoots and roots of O. kokonorica as conducted for C. songorica . Hierarchical cluster analysis of transcriptomic data showed clustering of the four repeats for the shoot or root at each treatment, except for one root and shoot sample in the cold treatment group and one shoot sample in control group (Figure S11), which were discarded for downstream analysis. Differentially expressed genes (DEGs) were identified under different treatments by comparison with the control group. A total of 1,246 and 631 DEGs in shoots and roots, respectively, were identified under cold stress with 2-fold changes compared with the control sample. 2,316 DEGs in shoots and 338 DEGs in roots were identified under heat stress. However, under light drought stress, much lower DEGs (11 in shoots; 420 in roots) were detected compared to the other two treatments (Figure S12). The DEGs in roots were about 57 times greater than in shoots, indicating that drought stress in early stages does not considerably affect the shoots of O. kokonorica .
Next, we downloaded transcriptomic data under the same stress treatments for C. songorica and identified DEGs using the same method as described for O. kokonorica . To reduce bias resulting from the two experiments, we only focused on the orthologous genes that were both DEGs in the two species (either in shoots, roots, or both) under each treatment. A total of 533, 99, 350 orthologous DEG pairs were identified under cold, drought, and heat treatments, respectively (Figure 3B; Tables S15-S17). We examined the differential expression pattern of two orthologous copies in each pair. We found that 53% (52) of orthologous genes had conserved functions in the two species under light drought treatment, consistent with the fact that both are drought resistance species. However, only ~2% and ~11% of orthologous genes under cold and heat treatments, respectively, had similar expression patterns in the two species (Figure 3B, C). Most orthologous genes were differentially expressed either in different tissues or in different ways (i.e. up- vs. down-regulation) or both, which may be related to the significant distinct altitudinal distributions of the two species.