Flower color is a key trait influencing insect pollination and ornamental value, yet the molecular mechanisms underlying heterozygous flower color remain unclear. In this study, we identified the creation of a yellow-white chimeric flower (cf) mutation in Brassica napus, characterized as the coexistence of yellow and white colors on petals of the same flower. Genetic analysis revealed that chimeric flower formation is controlled by a completely dominant gene. Map-based cloning, transgenic complementation, and CRISPR/Cas9 experiments consistently confirmed that BnaC05G0385300ZS on chromosome C05 is the causal gene of CF, which encodes a plastid DNA polymerase IB (BnaC05.POLIB). A G-to-A mutation in the seventh exon results in a D742N substitution, which disrupts Mg2+ binding and impairs polymerase activity. This leads to a reduced plastid genome copy number, decreased chromoplast formation, and aberrant carotenoid accumulation, ultimately resulting in the chimeric phenotype in a dosage-dependent manner. These findings reveal a novel role for BnaC05.POLIB in petal color patterning and provide a strategy for breeding ornamental plants with heterozygous flowers.

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