CRISPR/Cas9-mediated mutagenesis of BnaAOG1s reveals functional divergence in silique and seed development in Brassica napus L

Abstract

Oilseed rape (Brassica napus L.) is a major oil crop, and both silique and seed size are critical determinants of yield. In Arabidopsis thaliana, mutation of the ABORTED GAMETOPHYTE 1 (AOG1) gene leads to severe defects in gametophyte development and a pronounced reduction in silique length. However, the function of AOG1 homologs in rapeseed remains uncharacterized. In this study, four homologous copies of AOG1 were identified in B. napus: BnaAOG1.A03, BnaAOG1.A10, BnaAOG1.C03, and BnaAOG1.C09. Then a systematic analysis that considered the physiochemical properties, evolution, conserved motifs, gene structure, and cis-regulatory elements of BnaAOG1s family members was conducted. Utilizing the CRISPR/Cas9 system, two of these copies, BnaAOG1.A03 and BnaAOG1.C03, were targeted, and homozygous double mutants were generated. Unlike the Arabidopsis ortholog, the BnaAOG1s were specifically expressed during seed development. Phenotypic evaluation revealed that the double mutants did not exhibit significant changes in silique length, seed number per silique, or thousand-seed weight compared to the wild type. These results indicate that BnaAOG1.A03 and BnaAOG1.C03 are not individually essential for silique and seed development in B. napus. Their functions may be compensated by other homologous copies or have undergone divergence during polyploidization. This investigation provides a valuable case for functional analysis of homologous genes in polyploid crops.