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Summary Brassica napus L. (B. napus) is a major edible oil crop grown around the southern part of China, which often faces cold stress, posing potential damage to vegetative tissues. To sustain growth and reproduction, a detailed understanding of fundamental regulatory processes in B. napus against long‐term low temperature (LT) stress is necessary for breeders to adjust the level of LT adaption in a given region and is therefore of great economic importance. Till now, studies on microRNAs (miRNAs) in coping with LT adaption in B. napus are limited. Here, we performed an in‐depth analysis on two B. napus varieties with distinct adaptability to LT stress. Through integration of RNA sequencing (RNA‐seq) and small RNA‐sequencing (sRNA‐seq), we identified 106 modules comprising differentially expressed miRNAs and corresponding potential targets based on strong negative correlations between their dynamic expression patterns. Specifically, we demonstrated that Bna‐miR397a post‐transcriptionally regulates a LACCASE (LAC) gene, BnaLAC2, to enhance the adaption to LT stresses in B. napus by reducing the total lignin remodelling and ROS homeostasis. In addition, the miR397–LAC2 module was also proved to improve freezing tolerance of Arabidopsis, indicating a conserved role of miR397–LAC2 in Cruciferae plants. Overall, this work provides the first description of a miRNA‐mediated‐module signature for LT adaption and highlights the prominent role of laccase in future breeding programme of LT tolerant B. napus.

Interspecific hybridization between B. oleracea inbred lines of head cabbage, Brussels sprouts, kale and B. taurica and inbred lines of rapeseed (B. napus L.) were performed aiming at the development of the new sources of genetic variability of vegetable Brassicas. Using conventional crossings and the embryo-rescue techniques the following interspecific hybrids were developed: 11 genotypes of F1 generation, 18 genotypes of F2 and F1 × F2 generations (produced after self- and cross-pollination of interspecific F1 hybrids), 10 plants of the BC1 generation (resulted from crossing head cabbage cytoplasmic male-sterile lines with interspecific hybrids of the F2 and F1 generations) and 8 plants of BC1 × (F1 × F2). No viable seeds of the BC2 generation (B. oleracea) were obtained due to the strong incompatibility and high mortality of embryos. The morphological characteristics during the vegetative and generative stages, pollen characteristics, seed development and propagation, nuclear DNA contents and genome compositions of interspecific hybrids were analyzed. All the interspecific F1 hybrids were male-fertile with a majority of undeveloped and malformed pollen grains. They showed intermediate values for morphological traits and nuclear DNA contents and had nearly triploid chromosomal numbers (27 to 29) compared with parental lines. The F2 generation had a doubled nuclear DNA content, with 52 and 56 chromosomes, indicating their allohexaploid nature. F2 hybrids were characterized by a high heterosis of morphological characteristics, viable pollen and good seed development. F1 × F2 hybrids were male-fertile with a diversified DNA content and intermediate pollen viability. BC1 plants were male-sterile with an intermediate nuclear DNA content between the F2 and head cabbage, having 28 to 38 chromosomes. Plants of the BC1 × (F1 × F2) generation were in majority male-fertile with 38–46 chromosomes, high seed set, high heterosis and intermediate values for morphological traits. The obtained interspecific hybrids are valuable as new germplasm for improving Brassica-breeding programs.

The DABB proteins, which are characterized by stress-responsive dimeric A/B barrel domains, have multiple functions in plant biology. In Arabidopsis thaliana, these proteins play a crucial role in defending against various pathogenic fungi. However, the specific roles of DABB proteins in Brassica napus remain elusive. In this study, 16 DABB encoding genes were identified, distributed across 10 chromosomes of the B. napus genome, which were classified into 5 branches based on phylogenetic analysis. Genes within the same branch exhibited similar structural domains, conserved motifs, and three-dimensional structures, indicative of the conservation of BnaDABB genes (BnaDABBs). Furthermore, the enrichment of numerous cis-acting elements in hormone induction and light response were revealed in the promoters of BnaDABBs. Expression pattern analysis demonstrated the involvement of BnaDABBs, not only in the organ development of B. napus but also in response to abiotic stresses and Sclerotinia sclerotiorum infection. Altogether, these findings imply the significant impacts of BnaDABBs on plant growth and development, as well as stress responses.

Clubroot disease, caused by Plasmodiophora brassicae, is a threat to Brassica crops; therefore, understanding of host-resistance is important for developing clubroot-resistant cultivars. Using multi-omics analysis of clubroot-resistant (CR) and -susceptible (CS) near-isogenic lines (NILs) of B. napus, carrying the resistance of turnip (B. rapa var. rapifera), we characterized the host resistance mechanisms. Through proteome analysis, we identified 6626 differentially abundant proteins (DAPs) (2353 in CR-NILs, 4273 in CS-NILs) (q < 0.05), of which 50 in CR- and 62 in CS-NILs were detected across the disease developmental stages. Notable proteins included those involved in reactive oxygen species scavenging (BnaA09T0647200WE)], cell-wall modifications (BnaA04T0244300WE) and glucosinolate biosynthesis (BnaA01T0266700WE) in the CR-NILs. Additionally, disease-resistance proteins like ENHANCED DISEASE RESISTANCE 2-like (BnaA03T0055600WE) and hairpin-induced family protein YLS9 (BnaA08T0237900WE) showed increased abundance in CR-NILs. In contrast, CS-NILs exhibited decreased abundance of defense-related proteins, including proteins containing CUPIN domain (BnaA09T0578800WE) and LACCASE (BnaA02T0019200WE). Integration of proteome data with transcriptome data revealed 33 genes in CR- and 32 in CS-NILs showing a consistent pattern, including the genes related to PLANT INVERTASE/PECTIN METHYLESTERASE INHIBITOR (BnaC04T0003100WE), KELCH MOTIF (BnaC02T0374800WE), LACCASE (BnaA02T0019200WE), and antioxidant-related transcripts [GLUTATHIONE S-TRANSFERASES (BnaA03T0280900WE) and 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (BnaA09T0641500WE)]. Our findings offer valuable new targets for breeding clubroot-resistant B. napus.

Oilseed rape (Brassica napus L.) is a major oil crop which is grown worldwide. Adaptation to different environments and regional climatic conditions involves variation in the regulation of flowering time. Winter types have a strong vernalization requirement whereas semi-winter and spring types have a low vernalization requirement or flower without exposure to cold, respectively. In Arabidopsis thaliana, FRIGIDA (FRI) is a key regulator which inhibits floral transition through activation of FLOWERING LOCUS C (FLC), a central repressor of flowering which controls vernalization requirement and response. Here, four FRI homologues in B. napus were identified by BAC library screening and PCR-based cloning. While all homologues are expressed, two genes were found to be differentially expressed in aerial plant organs. One of these, BnaA.FRI.a, was mapped to a region on chromosome A03 which co-localizes with a major flowering time quantitative trait locus in multiple environments in a doubled-haploid mapping population. Association analysis of BnaA.FRI.a revealed that six SNPs, including at least one at a putative functional site, and one haplotype block, respectively, are associated with flowering time variation in 248 accessions, with flowering times differing by 13–19 d between extreme haplotypes. The results from both linkage analysis and association mapping indicate that BnaA.FRI.a is a major determinant of flowering time in oilseed rape, and suggest further that this gene also contributes to the differentiation between growth types. The putative functional polymorphisms identified here may facilitate adaptation of this crop to specific environments through marker-assisted breeding.

Rapeseed ( L.) is a very important edible oil crop in the world, and the production is inhibited by abiotic stresses, such as salinity. Plant hormones can alleviate the stress by regulating the physiological processes and gene expression. To study the plant responses to salinity in combination with GR24, a synthesized strigolactone, the oilseed rape variety (Zhongshuang 11) replications were grown in the pots in a controlled growth chamber under three levels of salinity (0, 100, and 200 mM NaCl) and 0.18 μM GR24 treatments at the seedling stage for 7 days. The results showed that salinity depressed the shoots and roots growth, whereas GR24 improved the growth under salt stress. Leaf chlorophyll contents and gas exchange parameters (net photosynthetic rates, stomatal conductance, intercellular CO concentration, and transpiration rate) were also reduced significantly with increasing salinity, and these effects could be partially reversed by GR24 application. Additionally, GR24 treatment significantly increased and decreased the photosystem II quantum yield and non-photochemical quenching, respectively, under salinity stress conditions. The activities of peroxidase and superoxide dismutase increased, and lipid peroxidation measured by the level of malondialdehyde reduced due to GR24 application. The transcriptome analysis of root and shoot was conducted. Three hundred and forty-two common differentially expressed genes (DEGs) after GR24 treatment and 166 special DEGs after GR24 treatment under salinity stress were identified in root and shoot. The DEGs in root were significantly more than that in shoot. Quantitative PCR validated that the stress alleviation was mainly related to the gene expression of tryptophan metabolism, plant hormone signal transduction, and photosynthesis.

The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and -transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus ( , and ) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the and genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 μg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into and protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated plants without the use of transgenesis.

Background The BES1 gene family, an important class of plant-specific transcription factors, play key roles in the BR signal pathway in plants, regulating various development processes. Until now, there has been no comprehensive analysis of the BES1 gene family in Brassica napus , and a cross-genome exploration of their origin, copy number changes, and functional innovation in plants was also not available. Results We identified 28 BES1 genes in B. napus from its two subgenomes (AA and CC). We found that 71.43% of them were duplicated in the tetraploidization, and their gene expression showed a prominent subgenome bias in the roots. Additionally, we identified 104 BES1 genes in another 18 representative angiosperms and performed a comparative analysis with B. napus , including evolutionary trajectory, gene duplication, positive selection, and expression pattern. Exploiting the available genome datasets, we performed a large-scale analysis across plants and algae suggested that the BES1 gene family could have originated from group F, expanding to form other groups (A to E) by duplicating or alternatively deleting some domains. We detected an additional domain containing M4 to M8 in exclusively groups F1 and F2. We found evidence that whole-genome duplication (WGD) contributed the most to the expansion of this gene family among examined dicots, while dispersed duplication contributed the most to expansion in certain monocots. Moreover, we inferred that positive selection might have occurred on major phylogenetic nodes during the evolution of plants. Conclusions Grossly, a cross-genome comparative analysis of the BES1 genes in B. napus and other species sheds light on understanding its copy number expansion, natural selection, and functional innovation.

Summary Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down‐regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down‐regulated in B. napus by over expressed of AtDA1R358K, which is a functional deficiency of DA1 with an arginine‐to‐lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.

The introduction of high-yielding and hybrid cultivars and the opening of new markets in the food and feed sector have steadily increased rapeseed production since the 1980s in the main production regions, Canada, Europe, China, India, and Australia. Since the 1990s, however, the average growth rate of yields has declined in Europe and Australia, which has been associated with a less effective control of biotic stresses. A global survey including the knowledge of 22 experts from 10 countries revealed a total of 16 diseases, 37 insect pests, several species of nematodes, and slugs currently affecting rapeseed production globally. A ranking of the top 10 most important biotic stresses in the four global regions where Brassica napus is grown (Canada, China, Europe, Australia) indicated an increase in several important stresses and distinct regional differences in the priority of prevailing diseases and pests. A stronger overlap exists among diseases, with Sclerotinia stem rot, Phoma stem canker, and clubroot occurring in all the four global regions on the top 10 list, while the range of prevailing insect pests was more diverse among the regions, with no top 10 insect playing an equally important role worldwide. Management options are substantially broader in disease than in pest control, making the latter the larger challenge. Since common integrated pest management (IPM) tools such as crop rotation, soil management, resistant cultivars or biocontrol are ineffective or not available, insect control largely relies on insecticides. Increasing restrictions on insecticide use, particularly in Europe, and losses in insecticide efficacy threaten the profitability of oilseed rape production and its role as an important break crop in cereal dominated cropping systems. Since the survival time of insects in the absence of their main host is relatively short (<1 year), a regional synchronization of cropping schemes resulting in one or more years without the crop could lead to a substantial disruption of regional insect populations. If rotation schemes were implemented on the landscape instead the farm level, by coordination among growers in zones covering the range distances of insect pests, an efficient and chemical low management strategy could be established and enable a more sustainable rapeseed production in the future.