Flower color is a key trait influencing insect pollination and ornamental value, yet the molecular mechanisms underlying heterozygous flower color remain unclear. In this study, we identified the creation of a yellow-white chimeric flower (cf) mutation in Brassica napus, characterized as the coexistence of yellow and white colors on petals of the same flower. Genetic analysis revealed that chimeric flower formation is controlled by a completely dominant gene. Map-based cloning, transgenic complementation, and CRISPR/Cas9 experiments consistently confirmed that BnaC05G0385300ZS on chromosome C05 is the causal gene of CF, which encodes a plastid DNA polymerase IB (BnaC05.POLIB). A G-to-A mutation in the seventh exon results in a D742N substitution, which disrupts Mg2+ binding and impairs polymerase activity. This leads to a reduced plastid genome copy number, decreased chromoplast formation, and aberrant carotenoid accumulation, ultimately resulting in the chimeric phenotype in a dosage-dependent manner. These findings reveal a novel role for BnaC05.POLIB in petal color patterning and provide a strategy for breeding ornamental plants with heterozygous flowers.

Thousand-seed weight (TSW) is a critical target for genetic improvement in rapeseed (Brassica napus L.). However, phenotypic selection for this trait remains challenging due to its polygenic regulation by multiple quantitative trait loci (QTL). Here, six favorable TSW QTL alleles from two donor parents were introgress into an elite restorer line, 621R, using an integrated strategy combining marker-assisted backcrossing and speed breeding protocols. Through six rounds of backcrossing and convergent crossing followed by two generations of selfing strategies, we developed 13 advanced lines with diverse TSW QTL combinations within 24 months. Field evaluations across three environments revealed that all lines exhibited significantly increased TSW in spring conditions (Minle, Gansu) and winter environments (Wuhan and Jiangling, Hubei) except for two lines which only showed increase in the spring environment. Hybridization assays using these lines as male parents crossed with two male-sterile lines (RG430A and 616A) demonstrated transgressive segregation for TSW: For RG430A-derived hybrids, all crosses significantly outperformed the original control (RG430A×621R) in Wuhan, with 8/13 and 9/13 crosses showing significant TSW increases in Minle and Jiangling, respectively. For 616A-derived hybrids, 11/13 and 10/13 crosses exhibited significant TSW enhancement in Minle and Jiangling, compared to 3/13 in Wuhan. Notably, two top-performing hybrids achieved 13.0% and 6.8% higher plot yields, respectively. Our results demonstrate that strategic pyramiding of complementary TSW QTL alleles effectively enhances seed weight in rapeseed, and these improved lines represent valuable genetic resources for developing high-yield hybrids.

Background Improving the quality and frying stability of rapeseed oil has long been an important goal in rapeseed quality breeding. This goal can be achieved through molecular marker-assisted selection (MAS) combined with speed breeding strategies. In this study, genomic data from the high-oleic-acid, low-linolenic-acid material L3 and the recessive genic male sterile (RGMS) line RG430A were used to identify functional variation sites underlying key quality traits: one governing oleic acid content and two controlling linolenic acid content. Additionally, Kompetitive Allele-Specific PCR markers were developed to efficiently genotype these oleic and linolenic acid-related variants. Results By integrating speed breeding with MAS and phenotypic screening, a new RGMS line was developed with high oleic acid, low linolenic acid content, and favorable agronomic performance. Compared with the original RG430A, the new line shows an approximately 15.0% increase in oleic acid content (reaching 78.0%) and an approximately 52.6% reduction in linolenic acid content (decreasing to 3.6%), with no significant alterations in major agronomic traits. Conclusions Our study offers strategic insights for rapeseed quality breeding, and the developed RGMS line represents a promising germplasm resource for future high-quality hybrid breeding.

Seed weight is a pivotal yield-determining trait in crops, and yet, the genetic and molecular mechanisms underlying its regulation in polyploid species remain underexplored. In a previous study, we identified cqSW.A03-2, a QTL that regulates thousand seed weight (TSW) in rapeseed (Brassica napus). Here, we identify BnaA3.AHK2, encoding a histidine kinase, as the causal gene of cqSW.A03-2. BnaA3.AHK2 enhances TSW through maternal control of seed coat cell expansion without significantly compromising other yield-related traits. Protein sequence divergence between parental haplotypes caused functional differentiation, with only the ZY50 allele showing functional kinase activity and rescuing developmental defects in Arabidopsis cytokinin receptor mutants. Strikingly, BnaA3.AHK2 seems to be a cytokinin-independent operator, contrasting with the canonical cytokinin signaling pathway. Transcriptome and protein interaction analyses reveal a signaling module where BnaA3.AHK2 engages BnaAHP-BnaARR phosphorelay components to regulate downstream targets. Notably, the favorable cqSW.A03-2 haplotype has been historically selected in modern breeding, and its introgression into elite hybrids boosted TSW by 3.6%-9.1%, demonstrating its breeding value. Our findings unveil a non-canonical signaling pathway for seed size regulation, providing a strategic genetic target to break yield trade-offs in polyploid crops.

Sclerotinia stem rot (SSR) is an important disease in rapeseed production caused by Sclerotinia sclerotiorum. Zhongshuang11(ZS11) is an elite double-low (low glucosinolate and low erucic acid) rapeseed cultivar with moderate SSR resistance. Identifying SSR resistance loci in ZS11 is of great significance for improving the SSR resistance of new double-low rapeseed varieties. In this study, 12 quantitative trait loci were mapped in the doubled haploid (DH) population, constructed using the resistant line ZS11 and susceptible line 19514 A, through linkage analysis. In the same population, seven intervals were identified using bulked segregant analysis sequencing. Among these, qDIA3-1, qDIA4-1, and qDIC4-1 could be identified by both methods and were considered as stable loci. Transcriptome analysis results revealed that a series of identical response pathways were induced in both ZS11 and 19514 A after infection. Furthermore, ZS11 produced a more pronounced response in the microtubule-associated and ribosome-associated pathways, which may partially explain the stronger resistance of ZS11 over 19514 A. Finally, a list of candidate genes of qDIA3-1, qDIA4-1, and qDIC4-1 were predicted by putative functions and expression levels. This research contributes to the advancement of knowledge regarding the genetic underpinnings and regulatory pathways of resistance to SSR in rapeseed, especially the explanation of the high resistance of ZS11, and provides a reference for the application of ZS11 for SSR resistance improvement.

Cadmium (Cd) is one of the heavy metal pollutants in soil. Cd stress affects the growth and development of rapeseed and significantly reduces its yield and quality. Here, we conducted a comprehensive analysis combining transcriptomic and metabolomic analyses to explore the molecular mechanisms of rapeseed response to Cd stress. The rapeseed seedlings were treated with 5 concentrations (0, 100, 200,300, and 400 mu M) of Cd. Seedlings exposed to Cd (0-400 mu M) exhibited dose-dependent growth inhibition alongside increased proline accumulation and elevated superoxide dismutase (SOD) and peroxidase (POD) activities. Integrated analysis revealed that differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in roots were predominantly enriched in glutathione (GSH) metabolism, glucosinolate (GSL) biosynthesis, ABC transporters, and phenylpropanoid biosynthesis pathways. In shoots, DEGs and DAMs were primarily associated with MAPK signaling, GSH metabolism, and photosynthesis. Further analysis indicated that Cd stress redirected sulfur flux toward GSH metabolism at the expense of GSL biosynthesis. Moreover, genetic transformation experiments demonstrated that overexpression of BnaMYB28 enhanced Cd tolerance by promoting GSH and sulfur metabolism. Our findings elucidate the molecular response mechanism of rapeseed to Cd stress and provide a foundation for improving Cd tolerance.

Rapeseed (Brassica napus L.) has a high sulfur requirement for optimal growth, development, and pathogen resistance. In this study, we identified zinc finger transcription factors, BnaSTOP2s, that play key roles in sulfur metabolism and Sclerotinia sclerotiorum resistance. First, our results suggested that BnaSTOP2s are involved in sulfur as evidenced from extensive protein interaction screening. Knockout of BnaSTOP2s reduced the response sensitivity in both sulfur-deficient and sulfur-excessive conditions by promoting the elongation of primary roots of seedlings. Furthermore, the content of essential sulfur-containing metabolites, including glucosinolate and glutathione, were substantially down-regulated in roots and leaves of Bnastop2 mutants, which is consistent with the significantly lowered transcriptional levels of key players of GSL synthesis and transportation, BnaMYB28s and BnaGTR2s, respectively. Through comprehensive RNA-seq analysis, we revealed the substantial effect of BnaSTOP2s on sulfur metabolism from source to sink. Additionally, we observed a significant decrease while increase in leaf lesion sizes of the BnaSTOP2-OE and Bnastop2 mutants, respectively, when compared to the wild type during Sclerotinia sclerotiorum infection, suggesting the vital role of BnaSTOP2 in plant defense response. Overall, our findings highlight that BnaSTOP2s seems to be global regulators of sulfur metabolism and confer resistance to Sclerotinia sclerotiorum infection in B. napus.### Competing Interest StatementThe authors have declared no competing interest.

Different ecological types of rapeseed (Brassica napus L.), including winter, spring, and semi-winter cultivars, exhibit varying flowering times and cannot be planted in the same cultivation areas. FLOWERING LOCUS T (FT) plays a key role in regulating flowering. In allotetraploid B. napus six copies of FT (BnaFT) have been reported. However, there is uncertainty about how the translated products of each paralog, as well as cis-allelic variations at each locus, contribute functionally to flowering time and define specific crop types. In this study, we confirm that BnaFT exhibit distinct expression patterns in different crop types of rapeseed. Using the CRISPR/Cas9 gene editing system, we provide functional evidence that the mutants between Bnaft paralogues affects the regulation of flowering time. Furthermore, we identify a new haplotype of BnaFT.A2 that is associated with early flowering time, although this appears necessary but not sufficient to confer a spring type phenotype. Three haplotypes of BnaFT.C6 were further identified and associated with both flowering time and crop types. We speculate that variations in both BnaFT.A2 and BnaFT.C6 may have undergone diversifying selection during the divergence of seasonal crop types in rapeseed.

Rapeseed (Brassica napus L.) exhibits high-sulfur requirements to achieve optimal growth, development, and pathogen resistance. Despite the importance of sulfur, the mechanisms regulating its metabolism and disease resistance are not fully understood. In this study, we found that the zinc finger transcription factors BnaSTOP2s play a pivotal role in sulfur metabolism and Sclerotinia sclerotiorum resistance. Our findings indicate that BnaSTOP2s are involved in sulfur metabolism, as evidenced by extensive protein interaction screening. BnaSTOP2s knockout reduced the content of essential sulfur-containing metabolites, including glucosinolate and glutathione, which is consistent with the significantly lowered transcriptional levels of BnaMYB28s and BnaGTR2s, key factors involved in glucosinolate synthesis and transportation, respectively. Comprehensive RNA-seq analysis revealed the substantial effect of BnaSTOP2s on sulfur metabolism from roots to siliques, which serve as pivotal sources and sinks for sulfur metabolism, respectively. Furthermore, we found that leaf lesion size significantly decreased and increased in the BnaSTOP2-OE and Bnastop2 mutants, respectively, compared with the wild-type during S. sclerotiorum infection, suggesting a vital role of BnaSTOP2s in plant defense response. In conclusion, BnaSTOP2s act as global regulators of sulfur metabolism and confer resistance to S. sclerotiorum infection in B. napus. Thus, they have potential implications for improving crop resilience.

Brassica napus is an important oil crop and cold stress severely limits its productivity. To date, several studies have reported the regulatory genes and pathways involved in cold-stress responses in B. napus. However, transcriptome-scale identification of the regulatory genes is still lacking. In this study, we performed comparative transcriptome analysis of cold-tolerant C18 (CT - C18) and cold-sensitive C6 (CS - C6) Brassica napus genotypes under cold stress for 7 days, with the primary purpose of identifying cold-responsive transcription in B. napus. A total of 6061 TFs belonging to 58 families were annotated in the B. napus genome, of which 3870 were expressed under cold stress in both genotypes. Among these, 451 TFs were differentially expressed (DE), with 21 TF genes expressed in both genotypes. Most TF members of the MYB (26), bHLH (23), and NAC (17) families were significantly expressed in the CT - C18 genotype compared with the CS - C6 B. napus genotype. GO classification showed a significant role in transcription regulation, DNA-binding transcription factor activity, response to chitin, and the ethylene-activated signaling pathway. KEGG pathway annotation revealed these TFs are involved in regulating more pathways, resulting in more tolerance. In conclusion, the results provide insights into the molecular regulation mechanisms of B. napus in response to freezing treatment, expanding our understanding of the complex molecular mechanisms in plants' response to freezing stress.

Dear Editor, Brassica napus L.,commonly known as rapeseed,canola,or oilseed rape,is the world's third oilseed crop and accounts for～12％of major worldwide oil production(FAO,2022).Rapeseed provides not only healthy and nutritionally balanced edible oil for humans but also protein-rich fodder for animals and renewable materials for biodiesel and industrial ap-plications.

Brassica napus, commonly known as rapeseed or canola, is a major oil crop contributing over 13% to the stable supply of edible vegetable oil worldwide. Identification and understanding the gene functions in the B. napus genome is crucial for genomic breeding. A group of genes controlling agronomic traits have been successfully cloned through functional genomics studies in B. napus. In this review, we present an overview of the progress made in the functional genomics of B. napus, including the availability of germplasm resources, omics databases and cloned functional genes. Based on the current progress, we also highlight the main challenges and perspectives in this field. The advances in the functional genomics of B. napus contribute to a better understanding of the genetic basis underlying the complex agronomic traits in B. napus and will expedite the breeding of high quality, high resistance and high yield in B. napus varieties.

One of the most important breeding goals for rapeseed (Brassica napus L.) is improving the oil content (OC) to increase edible or industrial oil quality. Using backbone parents as research materials to excavate OC genetic loci is not only helpful in enhancing the understanding of the genetic basis of OC in rapeseed but also offers a promising approach to improve backbone parents. In this study, a doubled haploid population constructed using the backbone parent 19514 A (with low OC) and the inbred line ZY50 (with high OC) were investigated in eight environments. A total of 30 quantitative trait loci (QTLs) were identified, among which qOC.A10 was detected in six environments. Local QTL scanning for the BC4F2 population showed that qOC.A10 accounted for 22.77% of the phenotypic variance and 0.97% of the additive effect. Further, we improved the parent 19514 A on qOC.A10 by marker-assisted selection, and the background response rate of the improved line A10-19514A reached 97.5%. The phenotypic identification results showed that the OC of A10-19514A and its hybrid significantly increased compared with that of the control, with no negative effects on other agronomic traits. Transcriptome analysis and sequence difference analysis facilitated the prediction of potential functional genes of qOC.A10. This study increases the knowledge regarding the genetic foundation of OC in rapeseed. Furthermore, it identified and confirmed a stable QTL that can be detected in various studies and possesses practical breeding significance.

•The SD-method is developed to quantify the silique parameters of rapeseed (length, width and thickness).•Two ImageJ macros were created to make the processing steps automated for high-throughput analysis.•Using a medium dataset, the SD-method is demonstrated to be precise, robust and efficient.•The SD-method performs well in other crops (Maize, wheat, soybean, chili pepper and cucumber).

The degradation products of glucosinolates (GSLs) greatly lower the nutritional value of rapeseed (Brassica napus) meal; thus, reduction of seed GSL content (SGC) has become an important objective of rapeseed breeding. In our previous study, we finely mapped a major QTL (qGSL-C2) for SGC to a 49-kb collinear region on B. rapa chromosome A2. Here, we experimentally validated that BnaC2.MYB28, encoding an R2R3-MYB transcription factor, is the causal gene of qGSL-C2. BnaC2.MYB28 is a nucleus-localized protein mainly expressed in vegetative tissues. Knockout of BnaC2.MYB28 in the high-SGC parent G120 reduced SGC to a value lower than that in the low-SGC parent ZY50, while overexpression of BnaC2.MYB28 in both parental lines (G120 and ZY50) led to extremely high SGC, indicating that BnaC2.MYB28 acts as a positive regulator of SGC in both parents. Molecular characterization revealed that BnaC2.MYB28 forms a homodimer and specifically interacts with BnaMYC3. Moreover, BnaC2.MYB28 can directly activate the expression of GSL biosynthesis genes. Differential expression abundance resulting from the polymorphic promoter sequences, in combination with the different capability in activating downstream genes involved in aliphatic GSL biosynthesis, caused the functional divergence of BnaC2.MYB28 in SGC regulation between the parents. Natural variation of BnaC2.MYB28 was highly associated with SGC in natural germplasm and has undergone artificial selection in modern low-GSL breeding. This study provides important insights into the core function of BnaC2.MYB28 in regulating SGC and a promising strategy for manipulating SGC in rapeseed.

Drought stress poses a persistent threat to field crops and significantly limits global agricultural productivity. Plants employ ubiquitin-dependent degradation as a crucial post-translational regulatory mechanism to swiftly adapt to changing environmental conditions. JUL1 is a RING-type E3 ligase related to drought stress in Arabidopsis. In this study, we explored the function of BnaJUL1 (a homologous gene of JUL1 in Brassica napus) and discovered a novel gene BnaTBCC1 participating in drought tolerance. First, we utilised BnaJUL1-cri materials through the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 system. Second, we confirmed that BnaJUL1 regulated drought tolerance through the drought tolerance assay and transcriptome analysis. Then, we identified a series of proteins interacting with BnaJUL1 through yeast library screening, including BnaTBCC1 (a tubulin binding cofactor C domain-containing protein); whose homologous gene TBCC1 knockdown mutants (tbcc1-1) exhibited ABA-sensitive germination in Arabidopsis, we then confirmed the involvement of BnaTBCC1 in drought tolerance in both Arabidopsis and Brassica. Finally, we established that BnaJUL1 could ubiquitinate and degrade BnaTBCC1 to regulate drought tolerance. Consequently, our study unveils BnaJUL1 as a novel regulator that ubiquitinates and degrades BnaTBCC1 to modulate drought tolerance and provided desirable germplasm for further breeding of drought tolerance in rapeseed.

Dear Editor, Rapeseed(Brassica napus)provides quality edible plant oil and industrial raw materials,as well as protein-rich animal feed;how-ever,molecular characterization of functional genes in rapeseed remains challenging,largely owing to its complex polyploid genome,relatively long generation time,and environmentally sensitive traits.To accelerate functional genomics research on rapeseed,especially in an artificially controlled,stable environ-ment like that of comprehensive speed breeding(CSB;Song et al.,2022),we screened out the representative B.napus accession Xiaoyun(小芸),with a rapid cycling time and scaled-down but normal plant architecture.