A complex locus regulates highly lobed-leaf formation in Brassica juncea

Lineage-specific evolution of RCO was described in Brassicaceae. BjRCO.1 and BjRCO.2 within the complex locus regulated highly lobed-leaf formation in Brassica juncea. RCO regulates the formation of lobed leaves in Brassicaceae species. RCO originated from the duplication of LMI1-type sequences and evolved through gene duplication and loss within the Brassicaceae. However, the evolutionary process and diversification of RCO in different lineages of Brassicaceae remain unclear. Although the RCO locus in B. juncea has been associated with lobed-leaf formation, its complexity has remained largely unknown. This study involved the identification of 55 LMI1-like genes in 16 species of Brassicaceae through syntenic analysis. We classified these LMI1-like genes into two types, namely LMI1-type and RCO-type, based on their phylogenetic relationship. Additionally, we proposed two independent lineage-specific evolution routes for RCO following the divergence of Aethionema. Our findings revealed that the LMI1-like loci responsible for lobed-leaf formation in Brassica species are located on the LF subgenomes. For B. juncea (T84-66V2), we discovered that the complex locus underwent duplication through segments of nucleic acid sequence containing Exostosin-LMI1-RCO (E-R-L), resulting in the tandem presence of two RCO-type and two LMI1-type genes on chromosome A10. As additional evidence, we successfully mapped the complex locus responsible for highly lobed-leaf formation to chromosome A10 using a B. juncea F₂ population, which corroborated the results of our evolutionary analysis. Furthermore, through transcriptome analysis, we clarified that BjRCO.1 and BjRCO.2 within the complex locus are functional genes involved in the regulation of highly lobed-leaf formation. The findings of this study offer valuable insights into the regulation of leaf morphology for the breeding of Brassica crops.