Lf2 is a knotted homeobox regulator that modulates leaflet number in soybean

Variation in leaf complexity modulates light capture and is a target for crop enhancement. Soybean typically has compound leaves with three leaflets each, but a spontaneous mutation, designatedlf2,possesses seven leaflets, offering a means to dissect the molecular mechanisms specifying leaflet number and assess its potential for soybean improvement. However, the developmental and genetic bases of thelf2mutation remain unknown. Here, we characterize the seven-leaflet phenotype and identify the mutation responsible for the phenotypic changes. Microscopic examination of leaf emergence sites revealed that the seven-leaflet phenotype arises in a two-step process: five leaflets form initially followed by secondary leaflet initiation at the margins of the central leaflet. Genetic mapping delineatedlf2to a ∼2.5 Mb region at the start of chromosome 11. Fortuitously, integration of pedigree analysis with comparative analysis of genomic sequences from the region pinpointed a 2-bp deletion in the coding sequence of a gene, which is homologous to the ArabidopsisKNAT7encoding aKNOTTED1-LIKE HOMEOBOX 2transcription factor, as the sole candidate forLf2.The deletion is predicted to result in disruption of the putative DNA-binding homeodomain. Expression of the wild-type allele of the candidate gene in the seven-leafletlf2mutant restored the three-leaflet phenotype, while disruption of the wild-type allele through CRISPR-Cas9 editing induced extra leaflet formation. This study advances our understanding of leaflet formation in legumes and provides a template for utilizing compound leaf architecture to optimize photosynthetic efficiency and yield in soybean.