Effects of CRISPR/Cas9 generated drooping leaf (dl) alleles on midrib and carpel formations in Oryza sativa Nipponbare.

Abstract

We generated drooping leaf rice mutants by CRISPR/Cas and identified two novel alleles with specific editing that allow underpinning of the function of the DL protein domain towards midrib and carpel formations. The DROOPING LEAF (DL) gene plays an essential role in regulating midrib formation and carpel specification in rice and other grass species, but the specific function of DL protein domains in different developmental processes is unclear. Analysis of different dl mutant alleles will allow dissecting the function of DL. Here, we generated Nipponbare rice dl mutants using CRISPR/Cas gene editing and identified two novel dl alleles with different effects on midrib formation and carpel development. Phenotypic and genotypic analysis of T0 and segregated T1 edited lines showed that while dl-51S allele (a 3bp deletion and a serine deletion at position 51) reduces midrib sizes and produces normal carpels, the dl-50LS allele (a 6bp deletion and a leucine-serine deletion at position 50-51) causes the lack of midribs and abnormal stigma. This result indicates that the 51-serine is important for midrib formation and the 50-leucine is essential for midrib and carpel development. These dl mutant alleles contribute to the DL gene functional analysis and to gain insights into possible modifications of leaf architecture of rice and other grass species.