Identification of genes involved in drought tolerance in seedlings of the desert grass, Psammochloa villosa (Poaceae), based on full-length isoform sequencing and de novo assembly from short reads

Psammochloa villosa is a perennial herbaceous plant that is dominant within arid regions of the Inner Mongolian Plateau and the Qinghai-Tibet Plateau in China, where it is an endemic species and exhibits strong drought tolerance and wind resistance. To study drought tolerance in P. villosa and determine its molecular basis, we simulated high and moderate drought stress in a controlled environment and then analyzed transcriptome sequences by combining long-read sequences from a representative, wild-grown individual with short reads from the treatment groups. We obtained 184,076 high-quality isoforms as a reference and 168,650 genes (91.6%), which we were able to annotate according to public databases. Ultimately, we obtained 119,005 unigenes representing the transcriptome of P. villosa under drought stress and, among these, we identified 3089 differentially expressed genes and 1484 transcription factors. Physiologically, P. villosa that was exposed to high and moderate drought stress had reduced germination rates and shorter buds but generated more chlorophyll, which is atypical under drought stress and possibly reflects an adaptation of these plants to their arid environment. We inferred that significantly upregulated genes were annotated as 'Chlorophyll a-b binding protein' and 'Light-harvesting chlorophyll-protein' among drought and control groups. Broadly, our analyses revealed that drought stress triggered many genome-level responses, especially related to mitigation of radical oxygen species (ROS), which increase in concentration under drought stress. In particular, in the high drought stress group compared with the control, GO enrichment analysis revealed a significant enrichment of upregulated genes (n = 10) involved in mitigation of oxidative stress. Similarly, using KEGG we found significant enrichment of genes in the phenylpropanoid biosynthesis pathway (11 genes), which yields phenols that scavenge ROS. We also inferred that many genes involved in metabolism of arginine and proline, which may serve as both scavengers of ROS and osmoprotectants that interact with stress response genes based on our protein-protein interaction network analysis. We verified the relative expression levels of eight genes associated with mitigation of ROS, DNA repair, and transmembrane transporter activity using qRT-PCR, and the results were consistent with our inferences from transcriptomes. This study provides insights into the genomic and physiological basis of drought tolerance in P. villosa and represents a resource for development of the species as a forage crop via molecular breeding within arid lands.