Pan-genomic dissection of the CCT gene family in wheat reveals evolutionary dynamics and regulatory divergence

Abstract

The CCT (CONSTANS, CO-LIKE, and TIMING OF CAB EXPRESSION 1) gene family regulates flowering time, circadian rhythm maintenance, and abiotic stress responses. However, previous investigations in wheat have relied on single-reference genomes, limiting the understanding of inter-varietal structural variation. Here, we performed a pan-genomic analysis of the CCT gene family across 16 wheat accessions. Copy numbers varied from 27 in diploid Triticum urartu to 69–103 in modern common wheat cultivars, with subgenomic contributions displaying an A > D > B gradient. Orthogroup classification based on the 12 T. aestivum cultivars identified 22 Core, 9 Softcore, 2 Dispensable, and 2 predicted Private orthogroups, all under strong purifying selection; the Private orthogroups require assembly-level validation because most cultivars used projected gene models. Protein-protein interaction network analysis identified a small set of highly connected nodes, with enrichment in circadian rhythm and photoperiodic flowering pathways, though this is largely expected given the query sequences; DeepGOPlus annotation further supported their roles as transcriptional regulators. Hexaploid wheat possessed longer mRNA lengths than tetraploids and diploids, but this may partly reflect annotation gaps. Promoter cis -elements were conserved across subgenomes, while paired analysis of homoeologous triads revealed significant expression divergence among subgenome copies. Tissue-specific profiling in Chinese Spring identified grain-preferential genes enriched for seed-specific elements, and stress treatments revealed candidate cis-elements, with WUN-motif and CGTCA-motif remaining significant after FDR correction. These findings provide a comprehensive view of CCT gene family evolution and regulatory diversification, offering candidate genes for stress tolerance and yield improvement in wheat.