Identification and functional characterization of BrcLL1, a candidate gene controlling leaf lobing in non-heading Chinese cabbage (Brassica rapa subsp. chinensis)

Leaf morphology is a key agronomic trait in non-heading Chinese cabbage (Brassica rapa subsp. chinensis), influencing both its commercial value and adaptability. The lobed leaf phenotype is of particular interest due to its implications for stress tolerance and breeding selection. In this study, a recombinant inbred line (RIL) population derived from a cross between the round-leaf line 14XS39B and the lobed-leaf line 14XS33H was used to fine-map the genetic basis of leaf lobing. A candidate gene, BrcLL1 (lobed-leaf 1), was identified within a 13.5 kb interval on chromosome A10, encodes a homeodomain leucine zipper class I (HD-ZIP I) transcription factor. Quantitative real-time PCR analysis showed that BrcLL1 expression was significantly elevated in the lobed-leaf parent 14XS33H, particularly at the cotyledon stage. Gene copy number analysis revealed that both 14XS39B and 14XS33H carry two copies of BrcLL1 (BrcLL1-A, BrcLL1-B). Sequence of BrcLL1-A showed no difference between the two parents, while the sequence of BrcLL1-B in 14XS33H has a 245 bp deletion in the intron region and four SNPs in the coding region. Transformation validation of the CDS sequences in Arabidopsis thaliana revealed distinct roles for the two alleles: overexpression of BrcLL1-A caused mildly serrated leaf margins, whereas overexpressing BrcLL1-B induced deeply lobed and highly dissected leaves with altered morphology. Furthermore, RNAi-mediated knockdown of BrcLL1 resulted in enlarged leaves, supporting the possibility of a positive regulatory role in the production of lobed leaves. These results indicate that the four SNPs in the coding region of the BrcLL1-B gene copy in the lobed-leaf parent, which lead to elevated expression of the B copy, serve as the primary driver enhancing the lobed-leaf phenotype, whereas the A copy likely plays only a minor role. Collectively, our work provides valuable insights into the molecular basis of lobed leaf formation and marker-assisted breeding in Brassica crops.