Genome-Wide Analysis of KNOX Genes in Brassicaceae: Evolution, Comparative Genomics, and Expression Dynamics in B. napus Floral and Silique Development

Background Knotted-like homeobox ( KNOX ) genes, belonging to a subfamily of the homeobox gene family, play crucial roles in cell fate determination and body plan specification during early embryogenesis in higher organisms. Brassica rapa ( B. rapa , AA), Brassica oleracea ( B. oleracea , CC) and their natural hybridization named Brassica napus ( B. napus , AACC) are excellent models for the study of polyploidy genes because they undego genome triplication events after Arabidopsis-Brassiceae divergence. Moreover, the specific gene structure and functional differentiation of KNOXs in B. napus is still unclear. Methods and results KNOX homologs from the three Brassica species, namely, B. rapa , B. oleracea and B. napus were downloaded from the Brassica database. Their evolutionary conservation and classification were analyzed with bioinformatics tools. This study identified 32, 15 and 14 KNOX genes in the genome of B. napus , B. rapa and B. oleracea , respectively. Phylogenetic analysis revealed that KNOXs can be classified into three classes based on their structural characteristics. The KNOX homologue proteins across the three Brassica species consistently share a highly conserved domain organization. Synteny analysis indicated that the KNOX gene family of B. napus expanded during allopolyploidization, with whole-gene duplication and segmental duplication being the primary contributors to the majority of KNOX gene duplications. Further analyses of the cis -elements, gene structures and expression patterns of KNOX genes in B. napus showed high conservation among members within the same group. RNA-seq data clearly divided BnKNOXs into three classes: Class I exhibited moderate and specific expression in buds and inflorescence tips; Class III showed specific low expression in seeds and stamens; while the second class shows expression in most tissues. qRT-PCR results indicated widespread involvement of KNOX genes in reproductive organ development. Conclusions The evolutionary conservation and diversification of KNOX proteins are significant in plant evolution and species formation, providing a robust foundation following the Arabidopsis-Brassiceae divergence. The gene structure, cis -elements, and tissue-specific expression patterns underscore the sequence and functional differences among BnKNOX genes. The distinct roles of BnKNOX genes in reproductive development are highlighted. These findings lay the groundwork for further functional studies of BnKNOX genes in B. napus .