A complex RCO locus regulates lobed leaf formation in Brassica juncea

Abstract In Brassicaceae, RCO regulates lobed leaves, an important agronomic trait. RCO evolved through duplication, regulatory diversification, and loss. However, how RCO evolved and diversified in different lineages of Brassicaceae is unclear. The RCO locus in B. juncea had been reported to be responsible for lobed leaf formation, but its complexity has largely remained unknown. In this study, we identified 54 RCO homologues in 16 species of Brassicaceae through syntenic analysis. Based on the phylogenetic relationship, we classified these homologues into two types, LMI1-type and RCO-type. Further, we proposed two independent lineage-specific evolution routes for RCO after the divergence from Aethionema arabicum. In Brassiceae species, we revealed that the RCO loci regulating lobed leaf formation were located on the LF subgenomes. In the case of B. juncea (T84-66), we found that the complex RCO locus was formed through the duplication of a large DNA segment comprising Exostosin- LMI1- RCO (E-R-L), resulting in the tandem presence of four RCO homologues on chromosome A10. As further evidence, we mapped the complex RCO locus regulating lobed leaf formation to chromosome A10 using a B. juncea F2 population, which was consistent with the evolutionary analysis results. We further clarified that BjRCO.1 and BjRCO.2 were functional in regulating lobed leaf formation by transcriptome analysis. This study provides valuable information on the control of leaf morphology in the breeding of Brassiceae crops.