Comparative genomics and expression analysis of the CIPK gene family in rice (Oryza sativa) and foxtail millet (Setaria italica)

The <i>CIPK</i> (CBL-interacting protein kinase) gene family serves as a crucial component of calcium-mediated signaling pathways in plants, playing vital roles in abiotic stress responses and developmental regulation. Despite their functional importance, systematic comparative analyses of <i>CIPK</i> gene families between cereal species with distinct physiological adaptations remain limited. To address this knowledge gap, we present a comprehensive comparative genomic analysis of <i>CIPK</i> gene families in two ecologically and physiologically divergent cereal crops - rice (<i>Oryza sativa</i>, a C₃ species adapted to aquatic environments) and foxtail millet (<i>Setaria italica</i>, a C₄ species with superior drought tolerance). We identified 33 and 35 <i>CIPK</i> genes in rice and foxtail millet, respectively, revealing conserved synteny but distinct evolutionary trajectories. Chromosomal mapping showed uneven distributions of <i>CIPK</i> genes in both species. Segmental duplications may have significantly contributed to family expansion (20/33 genes in rice; 19/35 in foxtail millet). Phylogenetic analysis classified members into six clades in foxtail millet and rice. Structural analysis revealed clade-specific exon-intron patterns, with complex architectures (12-20 exons) in Clade I versus simplified structures (1-3 exons) in other clades. Comparative genomics identified 26 orthologous pairs, though some genes (e.g., <i>OsCIPK27</i>) lacked detectable orthologs, indicating species-specific gene loss. Notably, foxtail millet CIPK exhibited lower instability indices, which may indicate potential stability differences. In addition, foxtail millet showed complete conservation of calcium-sensing NAF domains, whereas rice showed one NAF-deficient member (OsCIPK4). Promoter analysis identified species-specific <i>cis</i>-element enrichment: rice <i>CIPK</i> were enriched in drought/ABA-responsive elements, whereas foxtail millet showed greater light-responsive motif diversity. Expression profiling revealed tissue-specific patterns, with foxtail millet displaying more leaf-preferential expression (10 genes vs. 7 in rice), potentially linked to C₄ photosynthesis. Under abiotic stress conditions, rice <i>CIPK</i> genes exhibited strong responses to both salt and cold stressors, whereas foxtail millet <i>CIPK</i> showed greater responsiveness to drought, mirroring their ecological adaptations. These findings provide novel insight into the potential function and evolutionary diversification of calcium signaling components (<i>CIPK</i>) in C₃ and C₄ cereals. They also provide important potential targets for improving the stress resistance of cereal crops.