Mutating BnEOD1 via CRISPR-Cas9 increases the seed size and weight in Brassica napus

Seed weight, which is highly positively correlated to seed size, considered as a critical agronomic trait that determines the plant yield of Brassica napus. However, there have been limited researches on the genes involved in regulating seed size. In Arabidopsis thaliana, ENHANCER OF DA1 (EOD1), an E3 ubiquitin ligase gene, has been identified as a significant negative regulator in controlling organ size, but the function of its homologues in rapeseed remains unknown. Only two homologous of EOD1 have been found In B.napus. In this study, these two homologous of EOD1, BnaEOD1.A04 and BnaEOD1.C04, were mutated by the CRISPR-Cas9 system. Three T-DNA-free lines, T₂-157-1-C8, T₂-390-2-B8 and T₂-397-2-E2, were identified from the homozygous T₂ mutant lines. The BnaEOD1.A04 showed a similar type of editing in these mutants, whereas the BnaEOD1.C04 in T₂-397-2-E2 was only missing 26 amino acids and the translation was not prematurely terminated, which was a difference with other two mutants. In parallel, the mutation in BnaEOD1s resulted in a noteworthy augmentation of both seed size and seed weight in the three editing lines. Additionally, there was a significant decline in the SPS and SL in T₂-157-1-C8 and T₂-390-2-B8, however, T₂-397-2-E2 did not show any significant changes in the SPS and SL, possibly due to distinct types of editing in the three lines. The above results indicate the conserved function of EOD1 homologues and provides promising germplasm for breeding novel high-yield rapeseed varieties by improving seed size and thousand-seed weight.