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Summary: Rapeseed (Brassica napus) silique is the major carbohydrate source for seed development, and the final silique length has attracted great attention from breeders. However, no studies had focused on the dynamic character of silique elongation length (SEL). Here, the dynamic SEL investigation in a natural population including 588 lines over two years indicate that dynamic SEL during 0–20 days after flowering was the most essential stage associated with seed number per silique (SPS) and thousand seed weight (TSW). Then, nine loci were identified to be associated with SEL based on GWAS analysis, among which five SNPs (over 50%) distributed on the A02 chromosome within 6.08 to 6.48 Mb. Subsequently, we screened 5078 differentially expressed genes between two extreme materials. An unknown protein, BnaA02.SE, was identified combining with GWAS and RNA‐Seq analysis. Subcellular localization and expression profiles analysis demonstrated that BnaA02.SE is a chloroplast‐ and nucleus‐localized protein mainly expressed in pericarps and leaves. Furthermore, transgenic verification and dynamic cytological observation reveal that overexpressed BnaA02.SE can promote silique elongation by regulating JA and IAA contents, affecting cell proliferation and expansion, respectively, and finally enhance seed yield by influencing SPS and TSW. Haplotype analysis reveal that the homologs of BnaA02.SE may also be involved in silique elongation regulation. Our findings provided comprehensive insights into a newly SEL trait, and cloned the first gene (BnaA02.SE) controlling silique elongation in B. napus. The identified BnaA02.SE and its homologs can offer a valuable target for improving B. napus yield. [ABSTRACT FROM AUTHOR]

Alfin-like proteins (ALs) form a plant-specific transcription factor (TF) gene family involved in the regulation of plant growth and development, and abiotic stress response. In this study, 30 ALs were identified in Brassica napus ecotype 'Zhongshuang 11' genome (BnaALs), and unevenly distributed on 15 chromosomes. Structural characteristic analysis showed that all of the BnaALs contained two highly conserved domains: the N terminal DUF3594 domain and the C-terminal PHD-finger domain. The BnaALs were classified into four groups (Group I-IV), supported by conserved intron–exon and protein motif structures in each group. The allopolyploid event between B. oleracea and B. rapa ancestors and the small-scale duplication events in B. napus both contributed to the large BnaALs expansion. The promoter regions of BnaALs contained multiple abiotic stress cis-elements. The BnaALs in I-IV groups were mainly expressed in cotyledon, petal, root, silique, and seed tissues, and the duplicated gene pairs shared highly similar expression patterns. RNA-seq and RT-qPCR analysis showed that BnaALs were obviously induced by low nitrogen (LN) and low phosphorus (LP) treatments in roots. Overexpressing BnaAL02 and BnaAL28 in Arabidopsis demonstrated their functions in response to LN and LP stresses. BnaAL28 enhanced primary roots' (PRs) length and lateral roots' (LRs) number under LP and LN conditions, where BnaAL02 can inhibit LR numbers under the two conditions. They can promote root hair (RH) elongation under LP conditions; however, they suppressed RH elongation under LN conditions. Our result provides new insight into the functional dissection of this family in response to nutrient stresses in plants. [ABSTRACT FROM AUTHOR]

B-box (BBX) proteins, a subfamily of zinc-finger transcription factors, are involved in various environmental signaling pathways. In this study, we conducted a comprehensive analysis of BBX family members in Brassica crops. The 482 BBX proteins were divided into five groups based on gene structure, conserved domains, and phylogenetic analysis. An analysis of nonsynonymous substitutions and (Ka)/synonymous substitutions (Ks) revealed that most BBX genes have undergone purifying selection during evolution. An analysis of transcriptome data from rapeseed (Brassica napus) organs suggested that BnaBBX3d might be involved in the development of floral tissue-specific RNA-seq expression. We identified numerous light-responsive elements in the promoter regions of BnaBBX genes, which were suggestive of participation in light signaling pathways. Transcriptomic analysis under shade treatment revealed 77 BnaBBX genes with significant changes in expression before and after shading treatment. Of these, BnaBBX22e showed distinct expression patterns in yellow- vs. black-seeded materials in response to shading. UPLC-HESI-MS/MS analysis revealed that shading influences the accumulation of 54 metabolites, with light response BnaBBX22f expression correlating with the accumulation of the flavonoid metabolites M46 and M51. Additionally, BnaBBX22e and BnaBBX22f interact with BnaA10.HY5. These results suggest that BnaBBXs might function in light-induced pigment accumulation. Overall, our findings elucidate the characteristics of BBX proteins in six Brassica species and reveal a possible connection between light and seed coat color, laying the foundation for further exploring the roles of BnaBBX genes in seed development. [ABSTRACT FROM AUTHOR]

Soil is indispensable for agricultural production but has been seriously polluted by cadmium and salt in recent years. Many crops are suffering from this, including rapeseed, the third largest global oilseed crop. However, genes simultaneously related to both cadmium and salt stress have not been extensively reported yet. In this study, BnaA10.WRKY75 was screened from previous RNA-seq data related to cadmium and salt stress and further analyses including sequence comparison, GUS staining, transformation and qRT-PCR were conducted to confirm its function. GUS staining and qRT-PCR results indicated BnaA10.WRKY75 was induced by CdCl2 and NaCl treatment. Sequence analysis suggested BnaA10.WRKY75 belongs to Group IIc of the WRKY gene family and transient expression assay showed it was a nuclear localized transcription factor. BnaA10.WRKY75-overexpressing Arabidopsis and rapeseed plants accumulated more H2O2 and O2− and were more sensitive to CdCl2 and NaCl treatment compared with untransformed plants, which may be caused by the downregulation of BnaC03.CAT2. Our study reported that BnaA10.WRKY75 increases sensitivity to cadmium and salt stress by disrupting the balance of reactive oxygen species both in Arabidopsis and rapeseed. The results support the further understanding of the mechanisms underlying cadmium and salt tolerance and provide BnaA10.WRKY75 as a valuable gene for rapeseed abiotic stress breeding. [ABSTRACT FROM AUTHOR]

The CCT gene family is present in plants and is involved in biological processes such as flowering, circadian rhythm regulation, plant growth and development, and stress resistance. We identified 87, 62, 46, and 40 CCTs at the whole-genome level in B. napus, B. rapa, B. oleracea, and A. thaliana, respectively. The CCTs can be classified into five groups based on evolutionary relationships, and each of these groups can be further subdivided into three subfamilies (COL, CMF, and PRR) based on function. Our analysis of chromosome localization, gene structure, collinearity, cis-acting elements, and expression patterns in B. napus revealed that the distribution of the 87 BnaCCTs on the chromosomes of B. napus was uneven. Analysis of gene structure and conserved motifs revealed that, with the exception of a few genes that may have lost structural domains, the majority of genes within the same group exhibited similar structures and conserved domains. The gene collinearity analysis identified 72 orthologous genes, indicating gene duplication and expansion during the evolution of BnaCCTs. Analysis of cis-acting elements identified several elements related to abiotic and biotic stress, plant hormone response, and plant growth and development in the promoter regions of BnaCCTs. Expression pattern and protein interaction network analysis showed that BnaCCTs are differentially expressed in various tissues and under stress conditions. The PRR subfamily genes have the highest number of interacting proteins, indicating their significant role in the growth, development, and response to abiotic stress of B. napus. [ABSTRACT FROM AUTHOR]

Background: Rapeseed (Brassica napus L.) is the third largest source of vegetable oil in the world, and Sclerotinia sclerotiorum (Lib.) is a major soil-borne fungal plant pathogen that infects more than 400 plant species, including B. napus. Sclerotinia stem rot caused an annual loss of 10 − 20% in rapeseed yield. Exploring the molecular mechanisms in response to S. sclerotiorum infection in B. napus is beneficial for breeding and cultivation of resistant varieties. To gain a better understanding of the mechanisms regarding B. napus tolerance to Sclerotinia stem rot, we employed a miRNAome sequencing approach and comprehensively investigated global miRNA expression profile among five relatively resistant lines and five susceptible lines of oilseed at 0, 24, and 48 h post-inoculation. Results: In this study, a total of 40 known and 1105 novel miRNAs were differentially expressed after S. sclerotiorum infection, including miR156, miR6028, miR394, miR390, miR395, miR166, miR171, miR167, miR164, and miR172. Furthermore, 8,523 genes were predicted as targets for these differentially expressed miRNAs. These target genes were mainly associated with disease resistance (R) genes, signal transduction, transcription factors, and hormones. Constitutively expressing miR156b (OX156b) plants strengthened Arabidopsis resistance against S. sclerotiorum accompanied by smaller necrotic lesions, whereas blocking miR156 expression in Arabidopsis (MIM156) led to greater susceptibility to S. sclerotiorum disease, associated with extensive cell death of necrotic lesions. Conclusions: This study reveals the distinct difference in miRNA profiling between the relatively resistant lines and susceptible lines of B. napus in response to S. sclerotiorum. The identified differentially expressed miRNAs related to sclerotinia stem rot resistance are involved in regulating resistance to S. sclerotiorum in rapeseed by targeting genes related to R genes, signal transduction, transcription factors, and hormones. miR156 positively modulates the resistance to S. sclerotiorum infection by restricting colonization of S. sclerotiorum mycelia. This study provides a broad view of miRNA expression changes after S. sclerotiorum infection in oilseed and is the first to elucidate the function and mechanism underlying the miR156 response to S. sclerotiorum infection in oilseed rape. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus) is a major edible oilseed crop consumed worldwide. However, its yield is seriously affected by infection from the broad-spectrum non-obligate pathogen Sclerotinia sclerotiorum due to a lack of highly resistant germplasm. Here, we identified a Sclerotinia -resistant and light-dependent lesion mimic mutant from an ethyl methanesulfonate-mutagenized population of the rapeseed inbred Zhongshuang 11 (ZS11) named lesion mimic mutant 1 (lmm1). The phenotype of lmm1 is controlled by a single recessive gene, named LESION MIMIC MUTANT 1 (LMM1), which mapped onto chromosome C04 by bulked segregant analysis within a 2.71-Mb interval. Histochemical analysis indicated that H2O2 strongly accumulated and cell death occurred around the lesion mimic spots. Among 877 differentially expressed genes (DEGs) between ZS11 and lmm1 leaves, 188 DEGs were enriched in the defense response, including 95 DEGs involved in systemic acquired resistance, which is consistent with the higher salicylic acid levels in lmm1. Combining bulked segregant analysis and transcriptome analysis, we identified a significantly up-regulated gene, BnaC4.PR2 , which encodes β-1,3-glucanase, as the candidate gene for LMM1. Overexpression of BnaC4.PR2 may induce a reactive oxygen species burst to trigger partial cell death and systemic acquired resistance. Our study provides a new genetic resource for S. sclerotiorum resistance as well as new insights into disease resistance breeding in B. napus. [ABSTRACT FROM AUTHOR]

Trehalose-6-phosphate synthase (TPS) is an important enzyme for the synthesis of Trehalose-6-phosphate (T6P). In addition to being a signaling regulator of carbon allocation that improves crop yields, T6P also plays essential roles in desiccation tolerance. However, comprehensive studies, such as evolutionary analysis, expression analysis, and functional classification of the TPS family in rapeseed (Brassica napus L.) are lacking. Here, we identified 35 BnTPSs, 14 BoTPSs, and 17 BrTPSs in cruciferous plants, which were classified into three subfamilies. Phylogenetic and syntenic analysis of TPS genes in four cruciferous species indicated that only gene elimination occurred during their evolution. Combined phylogenetic, protein property, and expression analysis of the 35 BnTPSs suggested that changes in gene structures might have led to changes in their expression profiles and further functional differentiation during their evolution. In addition, we analyzed one set of transcriptome data from Zhongshuang11 (ZS11) and two sets of data from extreme materials associated with source-/sink-related yield traits and the drought response. The expression levels of four BnTPSs (BnTPS6, BnTPS8, BnTPS9, and BnTPS11) increased sharply after drought stress, and three differentially expressed genes (BnTPS1, BnTPS5, and BnTPS9) exhibited variable expression patterns among source and sink tissues between yield-related materials. Our findings provide a reference for fundamental studies of TPSs in rapeseed and a framework for future functional research of the roles of BnTPSs in both yield and drought resistance. [ABSTRACT FROM AUTHOR]

Glucosinolates (GSLs) are secondary plant metabolites that are enriched in rapeseed and related Brassica species, and they play important roles in defense due to their anti-nutritive and toxic properties. Here, we conducted a genome-wide association study of six glucosinolate metabolites (mGWAS) in rapeseed, including three aliphatic glucosinolates (m145 gluconapin, m150 glucobrassicanapin and m151 progoitrin), one aromatic glucosinolate (m157 gluconasturtiin) and two indole glucosinolates (m165 indolylmethyl glucosinolate and m172 4-hydroxyglucobrassicin), respectively. We identified 113 candidate intervals significantly associated with these six glucosinolate metabolites. In the genomic regions linked to the mGWAS peaks, 187 candidate genes involved in glucosinolate biosynthesis (e.g., BnaMAM1, BnaGGP1, BnaSUR1 and BnaMYB51) and novel genes (e.g., BnaMYB44, BnaERF025, BnaE2FC, BnaNAC102 and BnaDREB1D) were predicted based on the mGWAS, combined with analysis of differentially expressed genes. Our results provide insight into the genetic basis of glucosinolate biosynthesis in rapeseed and should facilitate marker-based breeding for improved seed quality in Brassica species. [ABSTRACT FROM AUTHOR]

Argonautes (AGOs) interact with microRNAs (miRNAs) to form the RNA-induced silencing complex (RISC), which can posttranscriptionally regulate the expression of targeted genes. To date, however, the AGOs and their miRNA triggers remain elusive in rapeseed (Brassica napus). Here, we systematically performed a phylogenetic analysis and examined the collinear relationships of the AGOs among four Brassicaceae species. Their physicochemical properties, gene structures, and expression patterns among 81 tissues from multiple materials and developmental stages were further analyzed. Additionally, their posttranscriptional regulation was analyzed using psRNATarget prediction, miRNA-/mRNA-Seq analyses, and a qRT-PCR verification. We finally identified 10 AtAGOs, 13 BolAGOs, 11 BraAGOs, and 24 BnaAGOs. An expression analysis of the BnaAGOs in the B. napus cultivar ZS11, as well as genotypes with extreme phenotypes in various yield-related traits, revealed the conservation and diversity of these genes. Furthermore, we speculated the posttranscriptional regulation of the B. napus miR168a–AGO1s and miR403–AGO2s modules. Combining miRNA-Seq and mRNA-Seq analyses, we found that the B. napus miR168a–AGO1s module may play an essential role in negatively regulating yield traits, whereas the miR403–AGO2s module positively impacts yield. This is the first attempt to comprehensively analyze the AGOs and their miRNA triggers in B. napus and provides a theoretical basis for breeding high-yielding varieties through the manipulation of the miRNA–AGOs modules. [ABSTRACT FROM AUTHOR]

Key message: A novel mutation in the BnaA03.IAA7 protein reduces plant height and enhances gibberellin signaling in Brassica napus L. Rapeseed (Brassica napus) is an excellent and important source for vegetable oil production, but its production is severely affected by lodging. Lodging hinders mechanization and decreases yield, and an ideal solution is semidwarf breeding. Limited by germplasm resources, semidwarf breeding developed slowly in rapeseed. In the current study, a mutant called sdA03 was isolated from EMS-mutagenized lines of Zhongshuang 11 (ZS11). The inheritance analysis showed that phenotypes of sdA03 were controlled by a single semidominant gene. Genetic mapping, RNA-seq and candidate gene analysis identified BnaA03.IAA7 as a candidate gene, and a function test confirmed that the mutated BnaA03.iaa7 regulates plant architecture in a dose-dependent manner. Yeast two-hybrid and transient expression experiments illustrated the P87L substitution in the GWPPV/I degron motif of BnaA03.iaa7 impaired the interaction between BnaA03.IAA7 and TIR1 proteins, and BnaA03.iaa7 prevented ARF from activating the auxin signaling pathway.The gibberellin (GA) content was higher in sdA03 hypocotyls than in those of ZS11. Further expression analysis showed more active gibberellin signaling in hypocotyl and richer expression of GA synthetic genes in root and cotyledon of sdA03 seedlings. Finally, a marker was developed based on the SNP found in BnaA03.iaa7 and used in molecular breeding. The study enriched our understanding of the architectural regulation of rapeseed and provided germplasm resources for breeding. [ABSTRACT FROM AUTHOR]

Hypersensitive-induced response protein (HIR) is a class of plant immune proteins that play pivotal roles in Sclerotinia sclerotiorum (Lib.) de Bary resistance. However, there has been no systematic investigation and identification of HIR genes in rapeseed (Brassica napus L.). Hence, we identified 50 BnHIR genes and classified them into four groups. Subcellular localization prediction suggested that HIR proteins are mainly localized in the mitochondria. Cis-acting elements involved in light and diverse abiotic stress were found in the promoter regions of BnHIR. The majority of BnHIR genes in Groups 1/3/4 were expressed in most examined tissues, especially in leaves and siliques pericarp, while the BnHIR genes in Group 2 were not or had low expression in all detected tissues. In the case of S. sclerotiorum inoculation, HIR genes in Groups 1/3/4 were strongly induced, especially homologous genes in Group 1, which exhibited different expression patterns. Moreover, overexpression of BnHIR2.7 in Arabidopsis thaliana illustrated its prominent resistance to S. sclerotiorum. Our study provides insight into the evolutionary relationships of the HIR family genes in B. napus and lays the foundation for their resistance to S. sclerotiorum in B. napus. [ABSTRACT FROM AUTHOR]

SUMMARY: Brassica napus is an important oil crop and an allotetraploid species. However, the detailed analysis of gene function and homoeologous gene expression in all tissues at different developmental stages was not explored. In this study, we performed a global transcriptome analysis of 24 vegetative and reproductive tissues at six developmental stages (totally 111 tissues). These samples were clustered into eight groups. The gene functions of silique pericarp were similar to roots, stems and leaves. In particular, glucosinolate metabolic process was associated with root and silique pericarp. Genes involved in protein phosphorylation were often associated with stamen, anther and the early developmental stage of seeds. Transcription factor (TF) genes were more specific than structural genes. A total of 17 100 genes that were preferentially expressed in one tissue (tissue‐preferred genes, TPGs), including 889 TFs (5.2%), were identified in the 24 tissues. Some TPGs were identified as hub genes in the co‐expression network analysis, and some TPGs in different tissues were involved in different hormone pathways. About 67.0% of the homoeologs showed balanced expression, whereas biased expression of homoeologs was associated with structural divergence. In addition, the spatiotemporal expression of homoeologs was related to the presence of transposable elements (TEs) and regulatory elements (REs); more TEs and fewer REs in the promoters resulted in divergent expression in different tissues. This study provides a valuable transcriptional map for understanding the growth and development of B. napus, for identifying important genes for future crop improvement, and for exploring gene expression patterns in the B. napus. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus) is the third-largest source of vegetable oil in the world with an edible, medicinal, and ornamental value. However, insufficient light or high planting density directly affects its growth, development, yield, and quality. Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that play key roles in regulating the responses to biotic and abiotic stresses in plants. In this study, we found that the promoter of BnaMAPK1 contained several light-responsive elements (including the AT1-motif, G-Box, and TCT-motif), consistent with its shading stress-induced upregulation. Compared with the wild type under shading stress, BnaMAPK1 -overexpressing plants showed higher light capture efficiency and carbon assimilation capacity, enhancing their shading tolerance. Using RNA sequencing, we systematically investigated the function of BnaMAPK1 in shading stress on photosynthetic structure, Calvin cycle, and light-driven electron transport. Notably, numerous genes encoding light-harvesting chlorophyll a/b-binding proteins (BnaLHCBs) in photosystem II-light-harvesting complex (LHC) II supercomplex were significantly downregulated in the BnaMAPK1 -overexpressing lines relative to the wild type under shading stress. Combining RNA sequencing and yeast library screening, a candidate interaction partner of BnaMAPK1 regulating in shading stress, BnaLHCB3, was obtained. Moreover, yeast two-hybrid and split-luciferase complementation assays confirmed the physical interaction relationship between BnaLHCB3 and BnaMAPK1, suggesting that BnaMAPK1 may involve in stabilizing the photosystem II–LHC II supercomplex. Taken together, our results demonstrate that BnaMAPK1 positively regulates photosynthesis capability to respond to shading stress in rapeseed, possibly by controlling antenna proteins complex in photosystem II, and could provide valuable information for further breeding for rapeseed stress tolerance. [ABSTRACT FROM AUTHOR]

Background: Leaf color mutants have reduced photosynthetic efficiency, which has severely negative impacts on crop growth and economic product yield. There are different chlorophyll mutants in Arabidopsis and crops that can be used for genetic control and molecular mechanism studies of chlorophyll biosynthesis, chloroplast development and photoefficiency. Chlorophyll mutants in Brassica napus are mostly used for mapping and location research but are rarely used for physiological research. The chlorophyll-deficient mutant in this experiment were both genetically mapped and physiologically analyzed. Results: In this study, yellow leaf mutant of Brassica napus L. mutated by ethyl methyl sulfone (EMS) had significantly lower chlorophyll a, b and carotenoid contents than the wild type, and the net photosynthetic efficiency, stomatal conductance and transpiration rate were all significantly reduced. The mutant had sparse chloroplast distribution and weak autofluorescence. The granule stacks were reduced, and the shape was extremely irregular, with more broken stromal lamella. Transcriptome data analysis enriched the differentially expressed genes mainly in phenylpropane and sugar metabolism. The mutant was mapped to a 2.72 Mb region on A01 by using BSA-Seq, and the region was validated by SSR markers. Conclusions: The mutant chlorophyll content and photosynthetic efficiency were significantly reduced compared with those of the wild type. Abnormal chloroplasts and thylakoids less connected to the stroma lamella appeared in the mutant. This work on the mutant will facilitate the process of cloning the BnaA01.cd gene and provide more genetic and physiological information concerning chloroplast development in Brassica napus. [ABSTRACT FROM AUTHOR]

Harvest index (HI), the ratio of harvested seed weight to total aboveground biomass weight, is an economically critical value reflecting the convergence of complex agronomic traits. HI values in rapeseed (Brassica napus) remain much lower than in other major crops, and the underlying regulatory network is largely unknown. In this study, we performed mRNA and small RNA sequencing to reveal the mechanisms shaping HI in B. napus during the seed-filling stage. A total of 8,410 differentially expressed genes (DEGs) between high-HI and low-HI accessions in four tissues (silique pericarp, seed, leaves, and stem) were identified. Combining with co-expression network, 72 gene modules were identified, and a key gene BnaSTY46 was found to participate in retarded establishment of photosynthetic capacity to influence HI. Further research found that the genes involved in circadian rhythms and response to stimulus may play important roles in HI and that their transcript levels were modulated by differentially expressed microRNAs (DEMs), and we identified 903 microRNAs (miRNAs), including 46 known miRNAs and 857 novel miRNAs. Furthermore, transporter activity-related genes were critical to enhancing HI in good cultivation environments. Of 903 miRNAs, we found that the bna-miR396–Bna.A06SRp34a/Bna.A01EMB3119 pair may control the seed development and the accumulation of storage compounds, thus contributing to higher HI. Our findings uncovered the underlying complex regulatory network behind HI and offer potential approaches to rapeseed improvement. [ABSTRACT FROM AUTHOR]

Glucosinolates (GSLs) are naturally occurring secondary metabolites found in the Brassicaceae family, which mainly synthesize in the siliques with a wide range of functions. In this study, we investigated the effects of lights on metabolites in siliques of rapeseed through ultra high-performance liquid chromatography (UPLC)—heated electrospray ionization (HESI)–tandem mass spectrometry (MS/MS). A total of 249 metabolites, including 29 phenolic acids, 38 flavonoids, 22 GSLs, 93 uncalculated and 67 unknown compounds, were identified in siliques of rapeseed. Meanwhile, 62 metabolites showed significant differences after shading treatment, which were mainly GSLs and unknown compounds. Interestingly, the amounts of 10 GSLs had high accumulation levels in siliques, while the expression levels of their corresponding biosynthetic genes (AOP , GSL-OH , IGMT , and ST5a) were obviously reduced after shading treatment. Further evidence showed that the amounts of GSLs were significantly reduced in seeds, in accordance with the expression profiles of transporter genes (BnaGTRs). Our findings indicated that lights could affect the accumulation and transportation of GSLs from siliques to seeds in rapeseed. Therefore, this study facilitates a better understanding of metabolic characteristics of siliques and provides insight into the importance of light for GSLs accumulation and transportation in siliques and seeds of rapeseed. [ABSTRACT FROM AUTHOR]

The petal colour of rapeseed plants is usually yellow but can range from milky white to orange or pink, making petal colour a popular target in rapeseed breeding programmes. In this study, metabolites and RNA were extracted from the yellow (Bn‐Y) and orange (Bn‐O) petal colour lines in rapeseed. Chromatography analysis, RNA‐Seq and quantitative RT‐PCR analysis were conducted. A total of 89 differential metabolites including 64 down‐regulated and 25 up‐regulated metabolites were identified in the petals of orange versus yellow rapeseed by UHPLC‐ESI‐MS/MS. A total of 168 differentially expressed genes (DEGs) were detected in the petals of Bn‐O versus Bn‐Y plants. KEGG functional annotation and quantitative RT‐PCR analysis showed that BnaC08g39510D and BnaA09g10680D expression was significantly higher in orange petals than in yellow and that BnaC09g07880D and BnaC09g08690D were strongly expressed in yellow petals but were not expressed in orange petals. These results will be useful for further understanding the molecular mechanisms underlying petal colour and provide the theoretical and practical basis for breeding varieties with colourful petals. [ABSTRACT FROM AUTHOR]

Ferulate‐5‐hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate‐5‐hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D‐NMR profiling in KO‐7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength. The oilseed rape production is unremittingly affected by Sclerotinia sclerotiorum, which causes stem and leaf rot and reduces the production of rapeseed oil. Lignin is an established physical barrier against pathogens, and the different lignin monomer G and S content can have different lignin structures. In this study, four dominantly expressed BnF5H genes were knocked out by CRISPR/Cas9 simultaneously in B. napus, and the f5h mutants were generated. The S‐/G‐lignin composition ratio was decreased compared to that of the wild type (WT) based on the results of Mӓule staining and 2D‐NMR profiling in the KO‐7 line. The resistance to S. sclerotiorum in stems and leaves increased in knock‐out lines. Furthermore, we found that the stem strength of mutants was significantly increased compared to that of the WT. Collectively, for the first time, we demonstrate that knockout of the lignin pathway gene F5H decreases the S/G ratio, improves S. sclerotiorum resistance in B. napus, and increases stem strength. [ABSTRACT FROM AUTHOR]

Background: Rapeseed is the third-largest oilseed crop after soybeans and palm that produces vegetable oil for human consumption and biofuel for industrial production. Silique length (SL) is an important trait that is strongly related to seed yield in rapeseed. Although many studies related to SL have been reported in rapeseed, only a few candidate genes have been found and cloned, and the genetic mechanisms regulating SL in rapeseed remain unclear. Here, we dissected the genetic basis of SL by genome-wide association studies (GWAS) combined with transcriptome analysis. Results: We identified quantitative trait locus (QTL) for SL using a recombinant inbred line (RIL) population and two independent GWAS populations. Major QTLs on chromosomes A07, A09, and C08 were stably detected in all environments from all populations. Several candidate genes related to starch and sucrose metabolism, plant hormone signal transmission and phenylpropanoid biosynthesis were detected in the main QTL intervals, such as BnaA9.CP12-2, BnaA9.NST2, BnaA7.MYB63, and BnaA7.ARF17. In addition, the results of RNA-seq and weighted gene co-expression network analysis (WGCNA) showed that starch and sucrose metabolism, photosynthesis, and secondary cell wall biosynthesis play an important role in the development of siliques. Conclusions: We propose that photosynthesis, sucrose and starch metabolism, plant hormones, and lignin content play important roles in the development of rapeseed siliques. [ABSTRACT FROM AUTHOR]

Background: In the oilseed crop Brassica napus (rapeseed), various metabolic processes influence seed oil content, oil quality, and biological yield. However, the role of plastid membrane proteins in these traits has not been explored. Results: Our genome-wide association study (GWAS) of 520 B. napus accessions identified the chloroplast membrane protein-localized FATTY ACID EXPORTER 1-1 (FAX1-1) as a candidate associated with biological yield. Seed transcript levels of BnaFAX1-1 were higher in a cultivar with high seed oil content relative to a low-oil cultivar. BnaFAX1-1 was localized to the plastid envelope. When expressed in Arabidopsis thaliana, BnaFAX1-1 enhanced biological yield (total plant dry matter), seed yield and seed oil content per plant. Likewise, in the field, B. napus BnaFAX1-1 overexpression lines (BnaFAX1-1-OE) displayed significantly enhanced biological yield, seed yield, and seed oil content compared with the wild type. BnaFAX1-1 overexpression also up-regulated gibberellic acid 4 (GA4) biosynthesis, which may contribute to biological yield improvement. Furthermore, oleic acid (C18:1) significantly increased in BnaFAX1-1 overexpression seeds. Conclusion: Our results indicated that the putative fatty acid exporter BnaFAX1-1 may simultaneously improve seed oil content, oil quality and biological yield in B. napus, providing new approaches for future molecular breeding. [ABSTRACT FROM AUTHOR]

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum , is a devastating disease in rapeseed. The objective of this study was to investigate the role and the mechanism of silicon (Si) in alleviating the disease severity of S. sclerotiorum in rapeseed. In the absorption assays, the rapeseed that absorbed 10 mM of K2SiO3 exhibited an 86% decrease in lesion size on infected leaves as compared with controls. In the spray assay, the lesion length on rapeseed stems was reduced by 30.5–32.9% with the use of 100 mM of a foliar Si fertilizer as compared with controls. In the pot assay, the lesion length on rapeseed stems was reduced by 34.9–38.3% when using the Si fertilizer as basal fertilizer. In the field assay, both the disease incidence and disease index of sclerotinia stem rot were significantly reduced with the usage of a solid Si fertilizer, Si foliar fertilizer, and the application of both, without negative affection on the main agronomic traits and seed quality of rapeseed. The transcriptome sequencing, quantitative reverse transcription PCR (qRT-PCR), and biochemical assays between K2SO4- and K2SiO3- treated rapeseed leaves revealed that Si promoted the biosynthesis of defense-related substances and enhanced the antioxidation and detoxification abilities of rapeseed after infection. Thus, this study concluded that Si can alleviate the disease severity of S. sclerotiorum in rapeseeds, partially due to the induced defense responses. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus L.) is the second most important oilseed crop in edible vegetable oil and bioenergy; however, drought stress generally causes a decrease in rapeseed yield and oil content, especially during the reproductive stage. In our study, we measured the oil and protein contents and gibberellic acid (GA) and abscisic acid (ABA) levels in seeds that were acquired on the 30th, 40th, and 50th days after flowering under control and drought treatments. RNA and protein libraries were constructed from the stressed seeds to perform transcriptome and proteome analyses, respectively. Our results demonstrated that the oil content decreased due to four primary mechanisms: downregulation of fatty acid biosynthesis‐associated genes and proteins; upregulation of fatty acid degradation‐associated genes and proteins; enhancement of protein storage due to changes in the abundances of relevant genes and proteins; and upregulation of Gly‐Asp‐Ser‐Leu (GDSL) gene expression, potentially as the result of upregulating the GA biosynthesis gene GA20ox3 and downregulating the GA inactivating gene GA2ox3 and thus an increase in GA content. During seed maturation, oil storage change may also relate to increasing ABA content as the upregulation of two members of NCED6 (9‐cis‐epoxycarotenoid dioxygenase) gene family involved in ABA biosynthesis, and the upregulation of genes involved in ABA signal transduction. These results will help to establish a foundation for breeding excellent varieties of rapeseed with high oil content for areas with frequent droughts to promote the supply of edible vegetable oil and biofuel. [ABSTRACT FROM AUTHOR]

Background: Brassica napus L. (2n = 38, AACC) is one of the most important oil crops and sources of protein for animal feed worldwide. Lignin is a large molecule aromatic polymer and a major cell wall component. However, lignin in the seed coat reduces the availability and restricts the development of rapeseed cake. Therefore, it is critical to reduce the lignin content of the seed coat. Here, high-lignin (H-lignin) and low-lignin (L-lignin) content recombinant inbred lines (RILs) were selected from an RIL population for analysis. Results: The cross-section results indicated that the seed coat of the H-lignin lines was thicker than that of the L-lignin lines, especially the palisade layer. The seed coats and embryos at 35, 40 and 46 days after flowering (DAF) were subjected to RNA sequencing (RNA-Seq), and the expression of the BnPAL and BnC4H gene families in the lignin pathway was significantly higher in the H-lignin seed coat than in the L-lignin seed coat. The Bn4CL gene family also showed this trend. In addition, among the genes related to plant hormone synthesis, BnaC02g01710D was upregulated and BnaA07g11700D and BnaC09g00190D were downregulated in H-lignin lines. Some transcription factors were upregulated, such as BnNAC080, BnNAC083, BnMYB9, BnMYB9-1, BnMYB60 and BnMYB60-1, while BnMYB91 was downregulated in H-lignin lines. Moreover, most genes of the flavonoid pathway, such as BnCHS and BnDFR, were strongly expressed in H-lignin seed coat. Conclusions: In Our study, some key genes such as hormone synthesis genes, transcription factors and miRNAs related to lignin and flavonoid biosynthesis were identified. A regulatory model of B. napus seed coat lignin was proposed. These results provide new insight into lignin and flavonoid biosynthesis in B. napus. [ABSTRACT FROM AUTHOR]

Petal size determines the value of ornamental plants, and thus their economic value. However, the molecular mechanisms controlling petal size remain unclear in most non-model species. To identify quantitative trait loci and candidate genes controlling petal size in rapeseed (Brassica napus), we performed a genome-wide association study (GWAS) using data from 588 accessions over three consecutive years. We detected 16 significant single nucleotide polymorphisms (SNPs) associated with petal size, with the most significant SNPs located on chromosomes A05 and C06. A combination of GWAS and transcriptomic sequencing based on two accessions with contrasting differences in petal size identified 52 differentially expressed genes (DEGs) that may control petal size variation in rapeseed. In particular, the rapeseed gene BnaA05.RAP2.2, homologous to Arabidopsis RAP2.2, may be critical to the negative control of petal size through the ethylene signaling pathway. In addition, a comparison of petal epidermal cells indicated that petal size differences between the two contrasting accessions were determined mainly by differences in cell number. Finally, we propose a model for the control of petal size in rapeseed through ethylene and cytokinin signaling pathways. Our results provide insights into the genetic mechanisms regulating petal size in flowering plants. [ABSTRACT FROM AUTHOR]

Background: Brassica napus is one of the most important oilseed crops, and also an important biofuel plant due to its low air pollution and renewability. Growth period are important traits that affect yield and are crucial for its adaptation to different environments in B. napus. Results: To elucidate the genetic basis of growth period traits, genome-wide association analysis (GWAS) and linkage mapping were employed to detect the quantitative trait loci (QTL) for days to initial flowering (DIF), days to final flowering (DFF), flowering period (FP), maturity time (MT), and whole growth period (GP). A total of 146 SNPs were identified by association mapping, and 83 QTLs were identified by linkage mapping using the RIL population. Among these QTLs, 19 were pleiotropic SNPs related to multiple traits, and six (q18DFF.A03-2, q18MT.A03-2, q17DFF.A05-1, q18FP.C04, q17DIF.C05 and q17GP.C09) were consistently detected using both mapping methods. Additionally, we performed RNA sequencing to analyze the differential expression of gene (DEG) transcripts between early- and late-flowering lines selected from the RIL population, and the DEGs were integrated with association mapping and linkage analysis to confirm their roles in the growth period. Consequently, 12 candidate genes associated with growth period traits were identified in B. napus. Among these genes, seven have polymorphic sites in the coding sequence and the upstream 2-kb sequence based on the resequencing data. The haplotype BnaSOC1.A05-Haplb and BnaLNK2.C06-Hapla showed more favorable phenotypic traits. Conclusions: The candidate genes identified in this study will contribute to our genetic understanding of growth period traits and can be used as targets for target mutations or marker-assisted breeding for rapeseed adapted to different environments. [ABSTRACT FROM AUTHOR]

The AINTEGUMENTA-like (AIL) proteins, which belong to the AP2 family, play important roles in regulating the growth and development of plant organs. The AIL family has not yet been comprehensively studied in rapeseed (Brassica napus), an allotetraploid and model organism for the study of polyploid evolution. In the present study, 99 AIL family genes were identified and characterized from B. rapa, B. oleracea, B. napus, B. juncea, and B. nigra using a comprehensive genome-wide study, including analyses of phylogeny, gene structure, chromosomal localization, and expression pattern. Using a phylogenetic analysis, the AIL genes were divided into eight groups, which were closely related to the eight AtAIL genes, and which shared highly conserved structural features within the same subfamily. The non-synonymous/synonymous substitution ratios of the paralogs and orthologs were less than 1, suggesting that the AIL genes mainly experienced purifying selection during evolution. In addition, the RNA sequencing data and qRT-PCR analysis revealed that the B. napus AIL genes exhibited organ- and developmental stage-specific expression patterns. Certain genes were highly expressed in the developing seeds (BnaAIL1, BnaAIL2, BnaAIL5, and BnaAIL6), the roots (BnaANT, BnaAIL5, and BnaAIL6), and the stem (BnaAIL7B). Our results provide valuable information for further functional analysis of the AIL family in B. napus and related Brassica species. [ABSTRACT FROM AUTHOR]

Background: Brassica rapa is an important oilseed and vegetable crop species and is the A subgenome donor of two important oilseed Brassica crops, Brassica napus and Brassica juncea. Although seed size (SZ), seed color (SC), and oil content (OC) substantially affect seed yield and quality, the mechanisms regulating these traits in Brassica crops remain unclear. Results: We collected seeds from a pair of B. rapa accessions with significantly different SZ, SC, and OC at seven seed developmental stages (every 7 days from 7 to 49 days after pollination), and identified 28,954 differentially expressed genes (DEGs) from seven pairwise comparisons between accessions at each developmental stage. K-means clustering identified a group of cell cycle-related genes closely connected to variation in SZ of B. rapa. A weighted correlation analysis using the WGCNA package in R revealed two important co-expression modules comprising genes whose expression was positively correlated with SZ increase and negatively correlated with seed yellowness, respectively. Upregulated expression of cell cycle-related genes in one module was important for the G2/M cell cycle transition, and the transcription factor Bra.A05TSO1 seemed to positively stimulate the expression of two CYCB1;2 genes to promote seed development. In the second module, a conserved complex regulated by the transcription factor TT8 appear to determine SC through downregulation of TT8 and its target genes TT3, TT18, and ANR. In the third module, WRI1 and FUS3 were conserved to increase the seed OC, and Bra.A03GRF5 was revealed as a key transcription factor on lipid biosynthesis. Further, upregulation of genes involved in triacylglycerol biosynthesis and storage in the seed oil body may increase OC. We further validated the accuracy of the transcriptome data by quantitative real-time PCR of 15 DEGs. Finally, we used our results to construct detailed models to clarify the regulatory mechanisms underlying variations in SZ, SC, and OC in B. rapa. Conclusions: This study provides insight into the regulatory mechanisms underlying the variations of SZ, SC, and OC in plants based on transcriptome comparison. The findings hold great promise for improving seed yield, quality and OC through genetic engineering of critical genes in future molecular breeding. [ABSTRACT FROM AUTHOR]

Key message: Resistant rapeseed lines pyramided with multiple resistant QTLs derived from Brassica oleracea were developed via a hexaploidy strategy. Rapeseed (Brassica napus L.) suffers heavily from Sclerotinia stem rot, but the breeding of Sclerotinia-resistant rapeseed cultivar has been unsuccessful. During the study, interspecific hexaploids were generated between rapeseed variety 'Zhongshuang 9' and a wild B. oleracea which was highly resistant to S. sclerotiorum, followed by backcrossing with Zhongshuang 9 and successive selfing. By molecular marker-assisted selection, three major resistant QTLs were transferred and pyramided from B. oleracea into two BC1F8 lines which exhibited ~ 35% higher resistance level than Zhongshuang 9 and produced good seed yield and seed quality. It is the first report on successful development of Sclerotinia-resistant rapeseed lines by introducing multiple resistant loci from wild B. oleracea. This study revealed the effectiveness of pyramiding multiple QTLs in improving Sclerotinia resistance in rapeseed and provided a novel breeding strategy on utilization of B. oleracea in rapeseed improvement. [ABSTRACT FROM AUTHOR]

Glucosinolates are amino acid‐derived secondary metabolites that act as chemical defense agents against pests. However, the presence of high levels of glucosinolates severely diminishes the nutritional value of seed meals made from rapeseed (Brassica napus L.). To identify the loci affecting seed glucosinolate content (SGC), we conducted genome‐wide resequencing in a population of 307 diverse B. napus accessions from the three B. napus ecotype groups, namely, spring, winter, and semi‐winter. These resequencing data were used for a genome‐wide association study (GWAS) to identify the loci affecting SGC. In the three ecotype groups, four common and four ecotype‐specific haplotype blocks (HBs) were significantly associated with SGC. To identify candidate genes controlling SGC, transcriptome analysis was carried out in 36 accessions showing extreme SGC values. Analyses of haplotypes, genomic variation, and candidate gene expression pointed to five and three candidate genes in the common and spring group‐specific HBs, respectively. Our expression analyses demonstrated that additive effects of the three candidate genes in the spring group‐specific HB play important roles in the SGC of B. napus. [ABSTRACT FROM AUTHOR]

Brassica napus L. is a widely cultivated oil crop and provides important resources of edible vegetable oil, and its quality is determined by fatty acid composition and content. To explain the genetic basis and identify more minor loci for fatty acid content, the multi-locus random-SNP-effect mixed linear model (mrMLM) was used to identify genomic regions associated with fatty acid content in a genetically diverse population of 435 rapeseed accessions, including 77 winter-type, 55 spring-type, and 303 semi-winter-type accessions grown in different environments. A total of 149 quantitative trait nucleotides (QTNs) were found to be associated with fatty acid content and composition, including 34 QTNs that overlapped with the previously reported loci, and 115 novel QTNs. Of these, 35 novel QTNs, located on chromosome A01, A02, A03, A05, A06, A09, A10, and C02, respectively, were repeatedly detected across different environments. Subsequently, we annotated 95 putative candidate genes by BlastP analysis using sequences from Arabidopsis thaliana homologs of the identified regions. The candidate genes included 34 environmentally-insensitive genes (e.g., CER4, DGK2, KCS17, KCS18, MYB4 , and TT16) and 61 environment-sensitive genes (e.g., FAB1, FAD6, FAD7, KCR1, KCS9, KCS12 , and TT1) as well as genes invloved in the fatty acid biosynthesis. Among these, BnaA08g08280D and BnaC03g60080D differed in genomic sequence between the high- and low-oleic acid lines, and might thus be the novel alleles regulating oleic acid content. Furthermore, RT-qPCR analysis of these genes showed differential expression levels during seed development. Our results highlight the practical and scientific value of mrMLM or QTN detection and the accuracy of linking specific QTNs to fatty acid content, and suggest a useful strategy to improve the fatty acid content of B. napus seeds by molecular marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Seed weight (SW) is one of three determinants of seed yield, which positively correlates with silique length (SL) in Brassica napus (rapeseed). However, the genetic mechanism underlying the relationship between seed weight (SW) and silique length (SL) is largely unknown at present. A natural population comprising 157 inbred lines in rapeseed was genotyped by whole-genome re-sequencing and investigated for SW and SL over four years. The genome-wide association study identified 20 SNPs in significant association with SW on A01, A04, A09, C02, and C06 chromosomes and the phenotypic variation explained by a single locus ranged from 11.85% to 34.58% with an average of 25.43%. Meanwhile, 742 SNPs significantly associated with SL on A02, A03, A04, A07, A08, A09, C01, C03, C04, C06, C07, and C08 chromosomes were also detected and the phenotypic variation explained by a single locus ranged from 4.01 to 48.02% with an average of 33.33%, out of which, more than half of the loci had not been reported in the previous studies. There were 320 overlapping or linked SNPs for both SW and SL on A04, A09, and C06 chromosomes. It indicated that both overlapping and independent genetic loci controlled both SW and SL in B. napus. On the haplotype block on A09 chromosome, the allele variants of a known gene BnaA.ARF18.a controlling both SW and SL were identified in the natural population by developing derived cleaved amplified polymorphic sequence (dCAPS) markers. These findings are valuable for understanding the genetic mechanism of SW and SL and also for rapeseed molecular breeding programs. [ABSTRACT FROM AUTHOR]

For many plants, regulating lignin content and composition to improve lodging resistance is a crucial issue. Caffeic acid O-methyltransferase (COMT) is a lignin monomer-specific enzyme that controls S subunit synthesis in plant vascular cell walls. Here, we identified 12 BnCOMT1 gene homologues, namely BnCOMT1-1 to BnCOMT1-12. Ten of 12 genes were composed of four highly conserved exons and three weakly conserved introns. The length of intron I, in particular, showed enormous diversification. Intron I of homologous BnCOMT1 genes showed high identity with counterpart genes in Brassica rapa and Brassica oleracea, and intron I from positional close genes in the same chromosome were relatively highly conserved. A phylogenetic analysis suggested that COMT genes experience considerable diversification and conservation in Brassicaceae species, and some COMT1 genes are unique in the Brassica genus. Our expression studies indicated that BnCOMT1 genes were differentially expressed in different tissues, with BnCOMT1-4, BnCOMT1-5, BnCOMT1-8, and BnCOMT1-10 exhibiting stem specificity. These four BnCOMT1 genes were expressed at all developmental periods (the bud, early flowering, late flowering and mature stages) and their expression level peaked in the early flowering stage in the stem. Drought stress augmented and accelerated lignin accumulation in high-lignin plants but delayed it in low-lignin plants. The expression levels of BnCOMT1s were generally reduced in water deficit condition. The desynchrony of the accumulation processes of total lignin and BnCOMT1s transcripts in most growth stages indicated that BnCOMT1s could be responsible for the synthesis of a specific subunit of lignin or that they participate in other pathways such as the melatonin biosynthesis pathway. [ABSTRACT FROM AUTHOR]

WRKY transcription factors play important roles in responses to environmental stress stimuli. Using a genome-wide domain analysis, we identified 287 WRKY genes with 343 WRKY domains in the sequenced genome of Brassica napus, 139 in the A sub-genome and 148 in the C sub-genome. These genes were classified into eight groups based on phylogenetic analysis. In the 343 WRKY domains, a total of 26 members showed divergence in the WRKY domain, and 21 belonged to group I. This finding suggested that WRKY genes in group I are more active and variable compared with genes in other groups. Using genome-wide identification and analysis of the WRKY gene family in Brassica napus, we observed genome duplication, chromosomal/segmental duplications and tandem duplication. All of these duplications contributed to the expansion of the WRKY gene family. The duplicate segments that were detected indicated that genome duplication events occurred in the two diploid progenitors B. rapa and B. olearecea before they combined to form B. napus. Analysis of the public microarray database and EST database for B. napus indicated that 74 WRKY genes were induced or preferentially expressed under stress conditions. According to the public QTL data, we identified 77 WRKY genes in 31 QTL regions related to various stress tolerance. We further evaluated the expression of 26 BnaWRKY genes under multiple stresses by qRT-PCR. Most of the genes were induced by low temperature, salinity and drought stress, indicating that the WRKYs play important roles in B. napus stress responses. Further, three BnaWRKY genes were strongly responsive to the three multiple stresses simultaneously, which suggests that these 3 WRKY may have multi-functional roles in stress tolerance and can potentially be used in breeding new rapeseed cultivars. We also found six tandem repeat pairs exhibiting similar expression profiles under the various stress conditions, and three pairs were mapped in the stress related QTL regions, indicating tandem duplicate WRKYs in the adaptive responses to environmental stimuli during the evolution process. Our results provide a framework for future studies regarding the function of WRKY genes in response to stress in B. napus. [ABSTRACT FROM AUTHOR]

A stable yellow-seeded variety is the breeding goal for obtaining the ideal rapeseed (Brassica napus L.) plant, and the amount of acid detergent lignin (ADL) in the seeds and the hull content (HC) are often used as yellow-seeded rapeseed screening indices. In this study, a genome-wide association analysis of 520 accessions was performed using the Q + K model with a total of 31,839 single-nucleotide polymorphism (SNP) sites. As a result, three significant associations on the B. napus chromosomes A05, A09, and C05 were detected for seed ADL content. The peak SNPs were within 9.27, 14.22, and 20.86 kb of the key genes BnaA.PAL4, BnaA.CAD2/BnaA.CAD3, and BnaC.CCR1, respectively. Further analyses were performed on the major locus of A05, which was also detected in the seed HC examination. A comparison of our genome-wide association study (GWAS) results and previous linkage mappings revealed a common chromosomal region on A09, which indicates that GWAS can be used as a powerful complementary strategy for dissecting complex traits in B. napus. Genomic selection (GS) utilizing the significant SNP markers based on the GWAS results exhibited increased predictive ability, indicating that the predictive ability of a given model can be substantially improved by using GWAS and GS. [ABSTRACT FROM AUTHOR]

Key message: Sclerotinia resistance was transferred into rapeseed from a wild relative of Brassica oleracea ( B. incana ) using hexaploids derived from crosses between B. incana and rapeseed as a bridge. Abstract: A high level of resistance against Sclerotinia sclerotiorum has been documented in wild Brassica oleracea, but not in cultivated rapeseed ( Brassica napus). To transfer sclerotinia resistance from a wild relative into rapeseed, a strategy was proposed using hexaploids (AACCCC) derived from crosses between the wild B. oleracea-related B. incana genotype 'C01' and the Chinese rapeseed variety 'Zhongshuang 9' as a bridge. Progenies (BC1F1) generated by backcrossing the hexaploid to 'Zhongshuang 9' could be generated with a high crossability (average 18.3 seeds per pod). Seventy-three individuals in BC1F1 were firstly screened for resistance with five molecular markers linked to the major resistance QTL on chromosome C09 in 'C01', and 11 individuals harboring resistance loci were selected to develop vegetative clones. Of these, five exhibited significantly higher resistance than 'Zhongshuang 9' and the most resistant individual was chosen to develop the BC1F2 progeny. Finally, five individual genotypes with nearly twofold higher resistance than 'Zhongshuang 9' were found among 100 BC1F2 individuals by using marker-assisted selection and resistance evaluation. Hereof, one rapeseed-type individual with 38 chromosomes and good self-fertility (15.0 ± 3.56 seeds/pod) was identified. Our results indicate that the proposed strategy is effective for transferring sclerotinia resistance from a wild relative of B. oleracea into rapeseed. [ABSTRACT FROM AUTHOR]

Sclerotinia stem rot of rapeseed ( Brassica napus) has strong negative impacts on seed yield and quality. To elucidate the link between Sclerotinia disease resistance and flowering time in rapeseed, quantitative trait loci (QTL) analyses for resistance against Sclerotinia sclerotiorum and flowering time were carried out in a doubled haploid population derived from a cross between European winter and Chinese semi-winter rapeseed. Six and five QTL for resistance in the field (FR) and under controlled condition (SR) were identified, while 17 QTL were found for flowering time (FT) over 2 years. Among these, four FR QTL, two SR QTL, and three FT QTL clustered within an 8.1-cM region on linkage group C02, which was found to be homeologous with a region on A02 carrying one SR QTL and two FT QTL. The association between Sclerotinia resistance and FT was discussed. [ABSTRACT FROM AUTHOR]

The TIFY gene family plays important roles in various plant biological processes and responses to stress and hormones. The chromosome-level genome of the Brassiceae species has been released, but knowledge concerning the TIFY family is lacking in the Brassiceae species. The current study performed a bioinformatics analysis on the TIFY family comparing three diploid (B. rapa, B. nigra, and B. oleracea) and two derived allotetraploid species (B. juncea, and B. napus). A total of 237 putative TIFY proteins were identified from five Brassiceae species, and classified into ten subfamilies (six JAZ types, one PPD type, two TIFY types, and one ZML type) based on their phylogenetic relationships with TIFY proteins in A. thaliana and Brassiceae species. Duplication and synteny analysis revealed that segmental and tandem duplications led to the expansion of the TIFY family genes during the process of polyploidization, and most of these TIFY family genes (TIFYs) were subjected to purifying selection after duplication based on Ka/Ks values. The spatial and temporal expression patterns indicated that different groups of BnaTIFYs have distinct spatiotemporal expression patterns under normal conditions and heavy metal stresses. Most of the JAZIII subfamily members were highest in all tissues, but JAZ subfamily members were strongly induced by heavy metal stresses. BnaTIFY34, BnaTIFY59, BnaTIFY21 and BnaTIFY68 were significantly upregulated mostly under As3+ and Cd2+ treatment, indicating that they could be actively induced by heavy metal stress. Our results may contribute to further exploration of TIFYs, and provided valuable information for further studies of TIFYs in plant tolerance to heavy metal stress. [ABSTRACT FROM AUTHOR]

The YABBY family of plant-specific transcription factors play important regulatory roles during the development of leaves and floral organs, but their functions in Brassica species are incompletely understood. Here, we identified 79 YABBY genes from Arabidopsis thaliana and five Brassica species (B. rapa, B. nigra, B. oleracea, B. juncea, and B. napus). A phylogenetic analysis of YABBY proteins separated them into five clusters (YAB1–YAB5) with representatives from all five Brassica species, suggesting a high degree of conservation and similar functions within each subfamily. We determined the gene structure, chromosomal location, and expression patterns of the 21 BnaYAB genes identified, revealing extensive duplication events and gene loss following polyploidization. Changes in exon–intron structure during evolution may have driven differentiation in expression patterns and functions, combined with purifying selection, as evidenced by Ka/Ks values below 1. Based on transcriptome sequencing data, we selected nine genes with high expression at the flowering stage. qRT-PCR analysis further indicated that most BnaYAB family members are tissue-specific and exhibit different expression patterns in various tissues and organs of B. napus. This preliminary study of the characteristics of the YABBY gene family in the Brassica napus genome provides theoretical support and reference for the later functional identification of the family genes. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) cascades are common and conserved signal transduction pathways and play important roles in various biotic and abiotic stress responses and growth and developmental processes in plants. With the advancement of sequencing technology, more systematic genetic information is being explored. The work presented here focuses on two protein families in Brassica species: MAPK kinases (MKKs) and their phosphorylation substrates MAPKs. Forty-seven MKKs and ninety-two MAPKs were identified and extensively analyzed from two tetraploid (B. juncea and B. napus) and three diploid (B. nigra, B. oleracea, and B. rapa) Brassica species. Phylogenetic relationships clearly distinguished both MKK and MAPK families into four groups, labeled A–D, which were also supported by gene structure and conserved protein motif analysis. Furthermore, their spatial and temporal expression patterns and response to stresses (cold, drought, heat, and shading) were analyzed, indicating that BnaMKK and BnaMAPK transcript levels were generally modulated by growth, development, and stress signals. In addition, several protein interaction pairs between BnaMKKs and C group BnaMAPKs were detected by yeast two-hybrid assays, in which BnaMKK3 and BnaMKK9 showed strong interactions with BnaMAPK1/2/7, suggesting that interaction between BnaMKKs and C group BnaMAPKs play key roles in the crosstalk between growth and development processes and abiotic stresses. Taken together, our data provide a deeper foundation for the evolutionary and functional characterization of MKK and MAPK gene families in Brassica species, paving the way for unraveling the biological roles of these important signaling molecules in plants. [ABSTRACT FROM AUTHOR]

Background: Diacylglycerol kinases (DGKs) are signaling enzymes that play pivotal roles in response to abiotic and biotic stresses by phosphorylating diacylglycerol (DAG) to form phosphatidic acid (PA). However, no comprehensive analysis of the DGK gene family had previously been reported in B. napus and its diploid progenitors (B. rapa and B. oleracea). Results: In present study, we identified 21, 10, and 11 DGK genes from B. napus, B. rapa, and B. oleracea, respectively, which all contained conserved catalytic domain and were further divided into three clusters. Molecular evolutionary analysis showed that speciation and whole-genome triplication (WGT) was critical for the divergence of duplicated DGK genes. RNA-seq transcriptome data revealed that, with the exception of BnaDGK4 and BnaDGK6, BnaDGK genes have divergent expression patterns in most tissues. Furthermore, some DGK genes were upregulated or downregulated in response to hormone treatment and metal ion (arsenic and cadmium) stress. Quantitative real-time PCR analysis revealed that different BnaDGK genes contribute to seed oil content. Conclusions: Together, our results indicate that DGK genes have diverse roles in plant growth and development, hormone response, and metal ion stress, and in determining seed oil content, and lay a foundation for further elucidating the roles of DGKs in Brassica species. [ABSTRACT FROM AUTHOR]

Background: Brassica napus L. is one of the most important oil crops in the world. However, climate-change-induced environmental stresses negatively impact on its yield and quality. Cuticular waxes are known to protect plants from various abiotic/biotic stresses. Dissecting the genetic and biochemical basis underlying cuticular waxes is important to breed cultivars with improved stress tolerance. Results: Here a genome-wide association study (GWAS) of 192 B. napus cultivars and inbred lines was used to identify single-nucleotide polymorphisms (SNPs) associated with leaf waxes. A total of 202 SNPs was found to be significantly associated with 31 wax traits including total wax coverage and the amounts of wax classes and wax compounds. Next, epidermal peels from leaves of both high-wax load (HW) and low-wax load (LW) lines were isolated and used to analyze transcript profiles of all GWAS-identified genes. Consequently, 147 SNPs were revealed to have differential expressions between HW and LW lines, among which 344 SNP corresponding genes exhibited up-regulated while 448 exhibited down-regulated expressions in LW when compared to those in HW. According to the gene annotation information, some differentially expressed genes were classified into plant acyl lipid metabolism, including fatty acid-related pathways, wax and cutin biosynthesis pathway and wax secretion. Some genes involved in cell wall formation and stress responses have also been identified. Conclusions: Combination of GWAS with transcriptomic analysis revealed a number of directly or indirectly wax-related genes and their associated SNPs. These results could provide clues for further validation of SNPs for marker-assisted breeding and provide new insights into the genetic control of wax biosynthesis and improving stress tolerance of B. napus. [ABSTRACT FROM AUTHOR]

The multidrug and toxic compound extrusion (MATE) protein family is important in the export of toxins and other substrates, but detailed information on this family in the Brassicaceae has not yet been reported compared to Arabidopsis thaliana. In this study, we identified 57, 124, 81, 85, 130, and 79 MATE genes in A. thaliana, Brassica napus, Brassica oleracea, Brassica rapa, Brassica juncea, and Brassica nigra, respectively, which were unevenly distributed on chromosomes owing to both tandem and segmental duplication events. Phylogenetic analysis showed that these genes could be classified into four subgroups, shared high similarity and conservation within each group, and have evolved mainly through purifying selection. Furthermore, numerous B. napusMATE genes showed differential expression between tissues and developmental stages and between plants treated with heavy metals or hormones and untreated control plants. This differential expression was especially pronounced for the Group 2 and 3 BnaMATE genes, indicating that they may play important roles in stress tolerance and hormone induction. Our results provide a valuable foundation for the functional dissection of the different BnaMATE homologs in B. napus and its parental lines, as well as for the breeding of more stress-tolerant B. napus genotypes. [ABSTRACT FROM AUTHOR]

The genus Brassica contains several economically important crops, including rapeseed (Brassica napus, 2n = 38, AACC), the second largest source of seed oil and protein meal worldwide. However, research in rapeseed is hampered because it is complicated and time-consuming for researchers to access different types of expression data. We therefore developed the Brassica Expression Database (BrassicaEDB) for the research community. In the current BrassicaEDB, we only focused on the transcriptome level in rapeseed. We conducted RNA sequencing (RNA-Seq) of 103 tissues from rapeseed cultivar ZhongShuang11 (ZS11) at seven developmental stages (seed germination, seedling, bolting, initial flowering, full-bloom, podding, and maturation). We determined the expression patterns of 101,040 genes via FPKM analysis and displayed the results using the eFP browser. We also analyzed transcriptome data for rapeseed from 70 BioProjects in the SRA database and obtained three types of expression level data (FPKM, TPM, and read counts). We used this information to develop the BrassicaEDB, including "eFP", "Treatment", "Coexpression", and "SRA Project" modules based on gene expression profiles and "Gene Feature", "qPCR Primer", and "BLAST" modules based on gene sequences. The BrassicaEDB provides comprehensive gene expression profile information and a user-friendly visualization interface for rapeseed researchers. Using this database, researchers can quickly retrieve the expression level data for target genes in different tissues and in response to different treatments to elucidate gene functions and explore the biology of rapeseed at the transcriptome level. [ABSTRACT FROM AUTHOR]

Yellow seed coat color is a desirable characteristic in rapeseed (Brassica napus), as it is associated with higher oil content and higher quality of meal. Alternative splicing (AS) is a vital post-transcriptional regulatory process contributing to plant cell differentiation and organ development. To identify novel transcripts and differences at the isoform level that are associated with seed color in B. napus, we compared 31 RNA-seq libraries of yellow- and black-seeded B. napus at five different developmental stages. AS events in the different samples were highly similar, and intron retention accounted for a large proportion of the observed AS pattern. AS mainly occurred in the early and middle stage of seed development. Weighted gene co-expression network analysis (WGCNA) identified 23 co-expression modules composed of differentially spliced genes, and we picked out two of the modules whose functions were highly associated with seed color. In the two modules, we found candidate DAS (differentially alternative splicing) genes related to the flavonoid pathway, such as TT8 (BnaC09g24870D), TT5 (BnaA09g34840D and BnaC08g26020D), TT12 (BnaC06g17050D and BnaA07g18120D), AHA10 (BnaA08g23220D and BnaC08g17280D), CHI (BnaC09g50050D), BAN (BnaA03g60670D) and DFR (BnaC09g17150D). Gene BnaC03g23650D, encoding RNA-binding family protein, was also identified. The splicing of the candidate genes identified in this study might be used to develop stable, yellow-seeded B. napus. This study provides insight into the formation of seed coat color in B. napus. [ABSTRACT FROM AUTHOR]