Pangenome and Pan‐Transcriptome Analysis of the WOX Gene Family Reveals Evolutionary Conservation and Diversification in Alfalfa

ABSTRACT

The WUSCHEL‐related homeobox ( WOX ) gene family encodes plant‐specific transcription factors that play pivotal roles in meristem maintenance, organogenesis, regeneration, and developmental phase transitions. Despite their importance, the pangenome‐scale composition, expansion dynamics, and evolutionary constraints of WOX genes in alfalfa ( Medicago sativa ) remain poorly understood. Here, we performed an integrated pangenome and pan‐transcriptome analysis of the WOX gene family using 24 high‐quality alfalfa genomes. A total of 432 WOX genes were identified and clustered into 24 orthologous gene groups (OGGs). Pangenome profiling revealed that the WOX family is highly conserved across alfalfa accessions, with members of the Ancient clade showing greater conservation than those of the Intermediate and WUS clades. Duplication pattern analysis indicated that dispersed duplication was the primary force driving WOX family expansion, whereas whole‐genome duplication (WGD) events predominantly contributed to the long‐term retention of core and softcore members. Pairwise Ka/Ks analysis indicated strong purifying selection on most WOX gene pairs, whereas wild‐germplasm genes and Intermediate‐clade members showed elevated selective pressures. Notably, MsWOX9‐related homologs had exceptionally high Ka/Ks values, suggesting potential functional divergence linked to agronomically relevant traits. Furthermore, pan‐transcriptome analysis revealed that WOX genes could be classified into three distinct expression patterns during leaf senescence. Collectively, this study presents the first comprehensive pangenome‐ and pan‐transcriptome‐based characterization of the WOX family in alfalfa, providing new insights into its evolutionary dynamics and expression divergence.