A single-base insertion in BoDFR1 results in loss of anthocyanins in green-leaved ornamental kale.

key messageA CRISPR/Cas9-based knockout assay verified that BoDFR1 drives anthocyanin accumulation in ornamental kale and that BoDFR2, an ortholog of BoDFR1, is redundant. Anthocyanins are widely distributed in nature and give plants their brilliant colors. Leaf color is an important trait for ornamental kale. In this study, we measured anthocyanin contents and performed transcriptome deep sequencing (RNA-seq) of leaves from pink and green ornamental kale. We observed substantial differences in the expression levels of the two DIHYDROFLAVONOL 4-REDUCTASE-encoding genes BoDFR1 (Bo9g058630) and its ortholog BoDFR2 (Bo2g116380) between green-leaved and pink-leaved kale by RNA-seq and RT-qPCR. We cloned and sequenced BoDFR1 and BoDFR2 from both types of kale. We identified a 1-bp insertion in BoDFR1 and a 2-bp insertion in BoDFR2 in green-leaved kale compared to the sequences obtained from pink-leaved kale, both mapping to the second exon of their corresponding gene and leading to premature termination of translation. To confirm the genetic basis of the absence of anthocyanins in green kale, we used CRISPR/Cas9 genome editing to separately knock out BoDFR1 or BoDFR2 in the pink-leaved ornamental kale inbred line P23. We detected very low accumulation of anthocyanins in the resulting mutants Bodfr1-1 and Bodfr1-2, while Bodfr2-1 and Bodfr2-2 had anthocyanin levels comparable to those of the wild-type. We conclude that the insertion in BoDFR1, rather than that in BoDFR2, underlies the lack of anthocyanins in green-leaved ornamental kale. This work provides insight into the function of DFR and will contribute to germplasm improvement of ornamental plants.