Comparative transcriptome profiling reveals molecular mechanisms of freezing stress responses in cultivated and wild Cicer species.

Chickpea is a globally important food legume, but its productivity is significantly constrained by low-temperature stress, particularly during autumn and winter sowing, as well as by sudden temperature fluctuations in late spring. Although wild relatives of chickpea exhibit enhanced cold tolerance, their molecular responses to freezing stress remain poorly understood. In this study, we compared cold-tolerant and cold-sensitive cultivated chickpea genotypes ILC8262 and ILC533 along with three wild relatives, Cicer bijugum, Cicer reticulatum, and Cicer echinospermum, using de novo transcriptome analysis through RNA sequencing. A total of 1.38 billion raw reads were generated, resulting in the assembly of 181,756 transcripts and 56,556 unigenes. Between 9,921 and 11,149 DEGs were identified per genotype under freezing stress. Functional annotation identified 453 unigenes related to cold response, while 519 DEGs were associated with cold or freezing tolerance based on gene ontology enrichment. Wild Cicer species showed a broader and more dynamic transcriptional response to freezing stress, including the activation of key calcium signaling components such as glutamate receptor-like channels, calmodulin-like proteins, CBL-CIPK modules, and calcium-dependent protein kinases, as well as MAPK cascades and genes in the ICE-CBF-COR pathway. Among the wild species, C. bijugum exhibited the highest number of species-specific DEGs and TFs, suggesting a distinct and potentially powerful cold adaptation strategy. In addition, we identified 8659 SSRs and 232,271 SNPs, with a subset validated as polymorphic markers. This study provides novel and comprehensive insights into the molecular mechanisms of freezing tolerance in chickpea and offers valuable genomic resources for breeding freezing-tolerant varieties.