Identification and characterization of the KNOTTED1-like homeobox as an active regulator of leaf development in chickpea

Leaf architecture is a key factor influencing plant productivity. In chickpea ( Cicer arietinum L.), leaf type varies from the typical compound form to simple-leaf mutants. Previous studies have suggested a monogenic inheritance pattern for leaf type, although the causal genes remain unidentified and functionally uncharacterized. Despite advances in understanding leaf development in other legumes, the molecular basis of leaf morphology in chickpea remains poorly explored. Using an integrative strategy combining phenotyping, genetic analysis, and high-throughput sequencing, we developed near-isogenic lines (NILs) segregating for leaf type to refine the genomic region underlying this trait. Variant discovery revealed a strong candidate gene, LOC101500499, annotated as a homeotic knotted-1-like ( KNOX ) gene on chromosome 8. A single-nucleotide deletion within the open reading frame was identified exclusively in simple-leaf genotypes, generating a frameshift and premature stop codon. Transcriptomic comparisons between compound- and simple-leaf plants showed significant differences in KNOX expression levels consistent with the mutation’s predicted functional impact. To validate the association, we designed a KASP marker targeting the deletion, which accounted for 90% of phenotypic variation in leaf type across diverse materials. Conserved domain analysis and phylogenetic classification indicated that the encoded protein belongs to the M subclass of the KNOX gene family, lacking the ELK and homeodomain regions typical of classes I and II. The discovery of a causal deletion in the knotted-1-like KNOX gene linked to the simple-leaf phenotype, together with its robust validation through KASP genotyping, provides a valuable molecular tool for breeding and deepens our understanding of leaf morphology in the species. Further studies on the regulatory networks interacting with this gene will help elucidate the mechanisms governing compound leaf development in chickpea.