RsRCO and RsLMI1 co-regulate the formation of lobed leaf in radish (Raphanus sativus L.).

Key message: Adjacent two homeobox genes RsRCO and RsLMI1 co-regulate lobed leaf development in radish and their co-segregating markers are used to breed new lobed-leaf radish varieties. Leaf shape is an important agronomic trait affecting photosynthesis in plants. However, the genetic mechanism of leaf shape in radish (Raphanus sativus L.) remains largely unknown. In this study, an F₂ segregating population was constructed by crossing a lobed-leaf radish 'QZ-16' and an entire-leaf radish '55.' Using bulk segregant analysis and genomic resequencing, we identified Leaf Shape (LS) gene controlling the lobed leaf phenotype in radish. Linkage analysis fine mapped LS locus to a 52.3 kb region on chromosome Rs7. Adjacent two homeobox genes RsRCO and RsLMI1 in this region were determined as the candidate genes, and overexpression of these two genes in Arabidopsis thaliana revealed that they co-regulated the formation of lobed leaf. Furthermore, two co-segregating structure variation markers derived from RsRCO and RsLMI1 genes were developed to genotype the 174 radish accessions, and approximately 80% leaf phenotypes can be explained by the RsLMI1/RsRCO haplotype. GO and KEGG enrichment analyses revealed that the differentially expressed genes were significantly enriched in photosynthesis and phytohormone pathways. Additionally, the contents of cytokinins and auxins were significantly higher in 'QZ-16' than in '55.' Our constructed near-isogenic lobed-leaf NIL-55 line exhibited higher photosynthetic efficiency and biomass than entire-leaf '55.' Finally, a new lobed-leaf variety, 'Chuyu No.15,' was bred from the lobed-leaf cytoplasmic male sterility line 'CLA' using a combination of marker-assisted selection and traditional hybrid breeding methods. Our findings clarified the inheritance regularity of lobed leaf formation in radish and laid a theoretical foundation for further exploring molecular mechanism of leaf development.