Yungu Zhai

Yellow seed is a desirable trait with great potential for improving seed quality in Brassica crops. Unfortunately, no natural or induced yellow seed germplasms have been found in Brassica napus, an important oil crop, which likely reflects its genome complexity and the difficulty of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we demonstrate the first application of CRISPR/Cas9 for creating yellow-seeded mutants in rapeseed. The targeted mutations of the BnTT8 gene were stably transmitted to successive generations, and a range of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The yellow-seeded phenotype could be recovered only in targeted mutants of both BnTT8 functional copies, indicating that the redundant roles of BnA09.TT8 and BnC09.TT8b are vital for seed colour. The BnTT8 double mutants produced seeds with elevated seed oil and protein content and altered fatty acid (FA) composition without any serious defects in the yield-related traits, making it a valuable resource for rapeseed breeding programmes. Chemical staining and histological analysis showed that the targeted mutations of BnTT8 completely blocked the proanthocyanidin (PA)-specific deposition in the seed coat. Further, transcriptomic profiling revealed that the targeted mutations of BnTT8 resulted in the broad suppression of phenylpropanoid/flavonoid biosynthesis genes, which indicated a much more complex molecular mechanism underlying seed colour formation in rapeseed than in Arabidopsis and other Brassica species. In addition, gene expression analysis revealed the possible mechanism through which BnTT8 altered the oil content and fatty acid composition in seeds.

Abstract Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T 1 and T 2 generations and a large collection of homozygous mutants with combined loss‐of‐function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T 1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.

Manipulation of flowering time to develop cultivars with desired maturity dates is fundamental in plant breeding. It is desirable to generate polyploid rapeseed (Brassica napus L.) germplasm with varying flowering time controlled by a few genes. In the present study, BnaSVP, a rapeseed homolog of the Arabidopsis SVP (Short Vegetative Phase) gene, was characterized and a set of mutants was developed using a CRISPR/Cas9-based gene-editing tool. A single construct targeting multiple sites was successfully applied to precisely mutate four copies of BnaSVP. The induced mutations in these copies were stably transmitted to subsequent generations. Homozygous mutants with loss-of-function alleles and free transgenic elements were generated across the four BnaSVP homologs. All mutant T1 lines tested in two environments (summer and winter growing seasons) showed early-flowering phenotypes. The decrease in flowering time was correlated with the number of mutated BnaSVP alleles. The quadruple mutants showed the shortest flowering time, with a mean decrease of 40.6%–50.7% in length relative to the wild type under the two growth conditions. Our study demonstrates the quantitative involvement of BnaSVP copies in the regulation of flowering time and provides valuable resources for rapeseed breeding.