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.

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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.

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Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

Rapeseed (Brassica napus) is the third-largest source of edible vegetable oil in the world. One of the most crucial techniques to increase rapeseed oil production is to develop and utilize saline-alkali soil. Rapeseed biochemical underpinnings for tolerance to abiotic stress, particularly salt stress, are not entirely established. The tubby-like protein(TLP) gene family is a class of transcription factors originally discovered in obese mice. It plays several roles in the growth and development of plants, particularly in responseto biotic and abiotic stresses. Twenty-nine BnaTLPs members were identified in the B. napus cultivar Zhongshuang 11, which were unevenly distributed on 16 chromosomes of B. napus. Phylogenetic analysis showed that TLPproteins were separated into six groups based on protein homology in 9 plant species. Gene structure analysis showed that the majority of the BnaTLPs members have similar intron/exon and motif structures. Conserved domain analysis showed that BnaTLP8A, BnaTLP8C, and AtTLP8 lacked the N-terminal F-box protein, which may have an impact on how well they function. Collinearity analysis revealed that most BnaTLPsare the products of heterologous polyploidization and segmental duplication events. RNA-seq data showed that the majority of BnaTLPs members are expressed in significant tissues and organs at various developmental stages. Real-time qPCR results showed that BnaTLP9A, BnaTLP3C, BnaTLP2C, and BnaTLP10C.2 are upregulated in response to drought and salt stress in B. napus, while BnaTLP8A is downregulated. This study provides new insight into how TLPs react to drought and salt stresses, in addition to identifying candidate genes for abiotic stress improvement in B. napus.

Rapeseed is an important source of oilseed crop in the world. Achieving genetic improvement has always been the major goal in rapeseed production. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Nowadays, Cas‐base editing acts as an efficient tool to mediate single‐base substitution at the target site. In this study, four adenine base editors (ABE) were modified to achieve adenosine base editing at different genome sites in allotetraploid Brassica napus. We designed 18 small guide RNAs to target phytoene desaturase (PDS), acetolactate synthase (ALS), CLAVATA3 (CLV3), CLV2, TRANSPARENT TESTA12 (TT12), carotenoid isomerase (CRTISO), designated de‐etiolated‐2 (DET2), BRANCHED1 (BRC1), zeaxanthin epoxidase (ZEP) genes, respectively. Among the four ABE systems, pBGE17 had the highest base‐editing efficiency, with an average editing efficiency of 3.51%. Target sequencing results revealed that the editing window ranged from A5 to A8 of the protospacer‐adjacent motif (PAM) sequence. Moreover, the ABEmax‐nCas9NG system with NG PAM was developed, with a base‐editing efficiency of 1.22%. These results revealed that ABE system developed in this study could efficiently induce A to G substitution and the ABE‐nCas9NG system could broaden editing window in oilseed rape.