Diverse gene regulatory strategies in response to heavy metal stress
To investigate whether the selective genes function stably in response to abiotic stress across different hybrid populations, we treated two genotypes with a series of cadmium and conducted a comprehensive transcriptomic analysis. PCA of the RNAseq dataset showed the first principal component (PC1) accounted for 72% of the variance, while the second principal component (PC2) accounted for 20% of the variations. The transcriptomes of the Heze and Panjin genotypes were clearly separated along PC1. Minimal variation was observed among the Panjin individuals, regardless of Cd treatments, suggesting this genotype is less sensitive to cadmium exposure. In contrast, PC2 separated the gene expression profiles of the Heze control group from those of the treatment groups. One sample treated with a high cadmium concentration grouped with the control genotypes. Although most samples had more than 92% of reads mapped to the reference genome, this particular sample showed a lower mapping rate of 86%, indicating that the individual may not have been in optimal condition during sampling (Fig. 3a ). As a result, we excluded this genotype from subsequent analyses. In the Panjin genotype, compared to the Heze genotype, 2,078 genes were upregulated and 1,492 genes were downregulated (seeSupplementary Fig. S2 ). Among these, 14 selective genes were found to be upregulated, while nine selective genes were downregulated (Supplementary Table S5 ).
The upregulated genes are predominantly involved in biological processes such as responses to external biotic stimuli, defense against bacteria and oomycetes, cell surface receptor signaling pathways, responses to salicylic acid, intracellular water homeostasis, and phosphorylation. Regarding cellular components, these genes mainly impact the plasma membrane and contribute to protein serine/threonine kinase activity, ADP binding, phosphotransferase activity involving alcohol group acceptors, cyclase activity, and transmembrane signaling receptor activity. In contrast, the downregulated genes did not show any significantly enriched Gene Ontology (GO) terms.
The coefficient of variation (CVa) analysis is essential for assessing the variability in gene expression across different treatment conditions relative to the mean expression levels. The Heze genotype under treatment conditions exhibited the highest median CVa among all groups, with a value of 1.53 (Fig. 3b ; Supplementary Table S6 ). In contrast, the control groups of the Heze and Panjin genotypes displayed similar CVa values, with 0.983 and 0.920, respectively. The adaptive genes that were upregulated in the Panjin C vs Heze C comparison, showed the highest CVa values in Panjin HL treatment group. Conversely, downregulated genes in the Panjin C vs Heze C comparison exhibited the highest CVa values in the Heze HL cadmium groups (Supplementary Figure S3, S4 ). Nonetheless, all the selective genes showed higher CVa values than the median CVa of all genes (Supplementary Table S7 ). This suggests that Heze genotype showed higher response to the cadmium stress, and the adaptive genes have played a special role with independent regulatory pathways in each genotype.