An in-silico approach to establish evolutionary relationship among the cold-regulated genes (CORs) for understanding cold stress response in Eucalyptus

Cold stress is one of the most severe abiotic stressors of Eucalyptus, limiting its spread, productivity, and yield globally. Eucalyptus is a commercially significant forestry species, hence developing cold tolerance is essential. Different signaling cascades are known to be involved during the response to cold stress in this forest tree. In the present investigation, 24 cold-regulated genes (CORs) from the genus Eucalyptus were identified. Based on the presence of signature motifs and conserved domains in the sequences, they were classified into three gene families, i.e. ICE, CBF, and DHN. These sequences were subsequently examined using a variety of bioinformatic tools to determine the evolutionary links among them. Phylogenetic analysis of these genes revealed that the genus contains a cluster of CBF genes with significant similarity, indicating a substantial duplication event throughout its evolutionary trajectory. Protein-Protein interaction exhibited identified 9 distinct local network clusters which emphasized on the cross-talk among the them. Gene co-occurrence revealed evolutionary interaction of CORs occurring in several taxa beyond the genus Eucalyptus. The CBF genes exhibited master regulatory function in cold stress sensing pathways as several CBF genes (EgrCBF3, EgrCBF5, EgrCBF7, EgrCBF10 and EgrCBF11) were observed to be involved in most of the KEGG pathways during gene ontology study. Majority of the CORs were overexpressed in shoot tips and young leaves during in-silico expression profiling indicating their role in cold-acclimation process of Eucalyptus. Particularly, CBF genes of E. gunnii (EguCBF1-4) were expressed significantly higher manifesting its higher cold-tolerance level. The findings of this study will help researchers better understand the cold acclimatization process and may prove to foundational for developing genetically improved cold-tolerant Eucalyptus varieties.