Biased retention and functional diversification of CESA superfamily genes in Medicago mediating development and abiotic stress responses

Medicago sativa is a globally cultivated forage crop, and improving its digestibility and stress tolerance remains a central breeding goal. The Cellulose synthase (CESA) superfamily is central to cell wall biosynthesis and influences plant development, stress responses, and forage quality; however, its evolutionary trajectory in Medicago is poorly resolved. Here, we identified 431 CESA superfamily genes from five Medicago species and four outgroup taxa. Phylogenetic analyses classified these genes into eight clades, including CesA and CslA/B/C/D/E/G/M subfamilies. Gene duplication analyses revealed that both tandem duplication and whole genome duplication (WGD) drove superfamily expansion, with a WGD event occurring in the common ancestor of Papilionooideae (PWGD). Tandem duplication plays a more prominent role than the PWGD-derived duplication. Transcriptomic analyses demonstrated that CESA genes in M. sativa subsp. sativa are broadly involved in development and respond strongly to salt and drought stresses. Notably, loss of CesA gene copies and biased retention of duplicated genes were observed in the MsCslB, MsCslA, MsCslE, and MsCslD subfamilies. Among tandem duplicates, MsCslB5.1 and MsCslB10.1 exhibited pronounced stress-responsive expression. These patterns may reflect domestication-related selection for improved forage quality and stress tolerance. Together, our findings establish a comprehensive evolutionary framework for the CESA superfamily and provide candidate genes with potential value for molecular breeding in Medicago forage crops.