Lobed leaves are advantageous for gas exchange, canopy architecture, and high-density planting; however, the genetic mechanisms of leaf lobe formation in Brassica crops remains poorly understood. Here, lob10.1, our previously identified major QTL controlling the presence/absence of leaf lobes in B. rapa (AA), was fine mapped to a confidence interval of 69.8 kb. REDUCED COMPLEXITY ORGAN (BrRCO, BraA10g032440.3c), a homeodomain leucine zipper class I (HD ZIP I) transcription factor, was predicted to be the most likely candidate gene underlying lob10.1. Null mutations of BrRCO by CRISPR/Cas9 in the lobed-leaf parent RcBr and over-expression in the counter-part near isogenic lines (NILRcBr) lead to entire and lobed leaves, respectively. Analysis of the gene evolution revealed that A10. RCO functions as a core gene and was generally negatively selected in B. rapa. Moreover, BrRCO function as a negative regulator by directly binding to promoters of BrACP5 and repressing its expression. The function of ACID PHOSPHATASE TYPE 5 (BrACP5) was subsequently confirmed as VIGS-BrACP5 produced entire leaves in RcBr. This study identified the core gene BrRCO to be involved in the development of leaf lobes in B. rapa and elucidated a new pathway for leaf lobe formation by the BrRCO-BrACP5 module. These findings provide a theoretical basis for the formation of leaf lobes in Brassica crops.

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