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Stem lodging resistance is a serious problem impairing crop yield and quality. ZS11 is an adaptable and stable yielding rapeseed variety with excellent resistance to lodging. However, the mechanism regulating lodging resistance in ZS11 remains unclear. Here, we observed that high stem mechanical strength is the main factor determining the superior lodging resistance of ZS11 through a comparative biology study. Compared with 4D122, ZS11 has higher rind penetrometer resistance (RPR) and stem breaking strength (SBS) at flowering and silique stages. Anatomical analysis shows that ZS11 exhibits thicker xylem layers and denser interfascicular fibrocytes. Analysis of cell wall components suggests that ZS11 possessed more lignin and cellulose during stem secondary development. By comparative transcriptome analysis, we reveal a relatively higher expression of genes required for S-adenosylmethionine (SAM) synthesis, and several key genes (4-COUMATATE-CoA LIGASE, CINNAMOYL-CoA REDUCTASE, CAFFEATE O-METHYLTRANSFERASE, PEROXIDASE) involved in lignin synthesis pathway in ZS11, which support an enhanced lignin biosynthesis ability in the ZS11 stem. Moreover, the difference in cellulose may relate to the significant enrichment of DEGs associated with microtubule-related process and cytoskeleton organization at the flowering stage. Protein interaction network analysis indicate that the preferential expression of several genes, such as LONESOME HIGHWAY (LHW), DNA BINDING WITH ONE FINGERS (DOFs), WUSCHEL HOMEOBOX RELATED 4 (WOX4), are related to vascular development and contribute to denser and thicker lignified cell layers in ZS11. Taken together, our results provide insights into the physiological and molecular regulatory basis for the formation of stem lodging resistance in ZS11, which will greatly promote the application of this superior trait in rapeseed breeding.

Plants rely on root systems for nutrient uptake from soils. Marker-assisted selection helps breeders to select desirable root traits for effective nutrient uptake. Here, 12 root and biomass traits were investigated at the seedling stage under low nitrogen (LN), low phosphorus (LP), and low potassium (LK) conditions, respectively, in a recombinant inbred line (RIL) population, which was generated from Brassica napus L. Zhongshuang11 and 4D122 with significant differences in root traits and nutrient efficiency. Significant differences for all the investigated traits were observed among RILs, with high heritabilities (0.43–0.74) and high correlations between the different treatments. Quantitative trait loci (QTL) mapping identified 57, 27, and 36 loci, explaining 4.1–10.9, 4.6–10.8, and 4.9–17.4% phenotypic variances under LN, LP, and LK, respectively. Through QTL-meta analysis, these loci were integrated into 18 significant QTL clusters. Four major QTL clusters involved 25 QTLs that could be repeatedly detected and explained more than 10% phenotypic variances, including two NPK-common and two specific QTL clusters (K and NK-specific), indicating their critical role in cooperative nutrients uptake of N, P, and K. Moreover, 264 genes within the four major QTL clusters having high expressions in roots and SNP/InDel variations between two parents were identified as potential candidate genes. Thirty-eight of them have been reported to be associated with root growth and development and/or nutrient stress tolerance. These key loci and candidate genes lay the foundation for deeper dissection of the NPK starvation response mechanisms in B. napus.

Previous studies have clearly demonstrated that the putative phytohormone melatonin functions directly in many aspects of plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of melatonin in seed oil and anthocyanin accumulation, and corresponding underlying mechanisms, remain unclear. Here, we found that serotonin N-acetyltransferase1 (SNAT1) and caffeic acid O-methyltransferase (COMT) genes were ubiquitously and highly expressed and essential for melatonin biosynthesis in Arabidopsis developing seeds. We demonstrated that blocking endogenous melatonin biosynthesis by knocking out SNAT1 and/or COMT significantly increased oil and anthocyanin content of mature seeds. In contrast, enhancement of melatonin signaling by exogenous application of melatonin led to a significant decrease in levels of seed oil and anthocyanins. Further gene expression analysis through RNA sequencing and reverse-transcription quantitative PCR demonstrated that the expression of a series of important genes involved in fatty acid and anthocyanin accumulation was significantly altered in snat1-1 comt-1 developing seeds during seed maturation. We also discovered that SNAT1 and COMT significantly regulated the accumulation of both mucilage and proanthocyanidins in mature seeds. These results not only help us understand the function of melatonin and provide valuable insights into the complicated regulatory network controlling oil and anthocyanin accumulation in seeds, but also divulge promising gene targets for improvement of both oil and flavonoids in seeds of oil-producing crops and plants.

Rapeseed is the third largest oil crop in the world and the largest oil crop in China. The multi-functional development and utilization of rapeseed is an effective measure for the high-quality development of rapeseed industry in China. In this study, several basic nutrients of eight rapeseed sprouts and five bean sprouts (3–5 varieties each) were determined, including sugar, crude protein, crude fiber, vitamin E, minerals, fatty acids, amino acids, and glucosinolates. Data analysis revealed that compared with bean sprouts, rapeseed sprouts were nutritionally balanced and were richer in active nutrients such as glucose, magnesium, selenium, vitamin E, and glucosinolate. Moreover, rapeseed sprouts exhibited reasonable amino acid composition and abundant unsaturated fatty acids (accounting for 90.32% of the total fatty acids). All these results indicated the potential of rapeseed sprout as a functional vegetable. Subsequently, three dominant nutrients including vitamin E, glucosinolate, and selenium were investigated in seeds and sprouts of 44 B. napus L. varieties. The results showed that germination raised the ratio of α-tocopherol/γ-tocopherol from 0.53 in seeds to 9.65 in sprouts, greatly increasing the content of α-tocopherol with the strongest antioxidant activity among the eight isomers of vitamin E. Furthermore, germination promoted the conversion and accumulation of glucosinolate components, especially, glucoraphanin with strong anti-cancer activity with its proportion increased from 1.06% in seeds to 1.62% in sprouts. In addition, the contents of selenium, vitamin E, and glucosinolate in rapeseed sprouts were highly correlated with those in seeds. Furthermore, these three dominant nutrients varied greatly within B. napus varieties, indicating the great potential of rapeseed sprouts to be further bio-enhanced. Our findings provide reference for the multi-purpose development and utilization of rapeseed, lay a theoretical foundation for the development of rapeseed sprout into a functional vegetable, and provide a novel breeding direction.

Roots are complicated quantitative characteristics that play an essential role in absorbing water and nutrients. To uncover the genetic variations for root-related traits in rapeseed, twelve mature root traits of a Brassica napus association panel were investigated in the field within three environments. All traits showed significant phenotypic variation among genotypes, with heritabilities ranging from 55.18% to 79.68%. Genome-wide association studies (GWAS) using 20,131 SNPs discovered 172 marker-trait associations, including 103 significant SNPs (−log10 (p) > 4.30) that explained 5.24–20.31% of the phenotypic variance. With the linkage disequilibrium r2 > 0.2, these significant associations were binned into 40 quantitative trait loci (QTL) clusters. Among them, 14 important QTL clusters were discovered in two environments and/or with phenotypic contributions greater than 10%. By analyzing the genomic regions within 100 kb upstream and downstream of the peak SNPs within the 14 loci, 334 annotated genes were found. Among these, 32 genes were potentially associated with root development according to their expression analysis. Furthermore, the protein interaction network using the 334 annotated genes gave nine genes involved in a substantial number of interactions, including a key gene associated with root development, BnaC09g36350D. This research provides the groundwork for deciphering B. napus’ genetic variations and improving its root system architecture.

A preliminary study suggested that BrTT1, which encodes a WIP zinc finger transcription factor, was responsible for seed coat color formation in Dahuang. In this study, cloning and functional analyses of the BrTT1 gene were performed to verify this, where no difference was detected between the upstream/downstream sequences of the target gene. The full-length genomic sequence of BrTT1 gene (1804 bp), together with the upstream (1796 bp) and downstream sequences of the target gene (986 bp), was transformed into the A. thalianatt1 mutant, tt1-1, and the seed coat color was completely restored to brown. The CDS from yellow- and brown-seeded plants was also transformed into tt1-1 under control of the 35S promoter. Only the coding sequence from brown-seeded plants could restore the seed coat color of tt1-1, suggesting that mutations in the coding sequence affected the function of Brtt1 and led to the yellow-seeded trait in Dahuang. In addition, BrTT1 was localized in the nucleus. The aberrant expression of BrTT1 in the yellow-seeded material of Brassica rapa may cause the aberrant or absent expression of several important BrTT genes of the flavonoid biosynthetic pathway, then blocked proanthocyanidin accumulation and resulted in a transparent testa in Dahuang.

As the major determinant for nutrient uptake, root system architecture (RSA) has a massive impact on nitrogen use efficiency (NUE). However, little is known the molecular control of RSA as related to NUE in rapeseed. Here, a rapeseed recombinant inbred line population (BnaZNRIL) was used to investigate root morphology (RM, an important component for RSA) and NUE-related traits under high-nitrogen (HN) and low-nitrogen (LN) conditions by hydroponics. Data analysis suggested that RM-related traits, particularly root size had significantly phenotypic correlations with plant dry biomass and N uptake irrespective of N levels, but no or little correlation with N utilization efficiency (NUtE), providing the potential to identify QTLs with pleiotropy or specificity for RM- and NUE-related traits. A total of 129 QTLs (including 23 stable QTLs, which were repeatedly detected at least two environments or different N levels) were identified and 83 of them were integrated into 22 pleiotropic QTL clusters. Five RM-NUE, ten RM-specific and three NUE-specific QTL clusters with same directions of additive-effect implied two NUE-improving approaches (RM-based and N utilization-based directly) and provided valuable genomic regions for NUE improvement in rapeseed. Importantly, all of four major QTLs and most of stable QTLs (20 out of 23) detected here were related to RM traits under HN and/or LN levels, suggested that regulating RM to improve NUE would be more feasible than regulating N efficiency directly. These results provided the promising genomic regions for marker-assisted selection on RM-based NUE improvement in rapeseed.

Gene duplication followed by functional divergence in the event of polyploidization is a major contributor to evolutionary novelties. The Brassica genus evolved from a common ancestor after whole-genome triplication. Here, we studied the evolutionary and functional features of Brassica spp. homologs to Tic40 (for translocon at the inner membrane of chloroplasts with 40 kDa). Four Tic40 loci were identified in allotetraploid Brassica napus and two loci in each of three basic diploid Brassica spp. Although these Tic40 homologs share high sequence identities and similar expression patterns, they exhibit altered functional features. Complementation assays conducted on Arabidopsis thaliana tic40 and the B. napus male-sterile line 7365A suggested that all Brassica spp. Tic40 homologs retain an ancestral function similar to that of AtTic40, whereas BolC9.Tic40 in Brassica oleracea and its ortholog in B. napus, BnaC9.Tic40, in addition, evolved a novel function that can rescue the fertility of 7365A. A homologous chromosomal rearrangement placed bnac9.tic40 originating from the A genome (BraA10.Tic40) as an allele of BnaC9.Tic40 in the C genome, resulting in phenotypic variation for male sterility in the B. napus near-isogenic two-type line 7365AB. Assessment of the complementation activity of chimeric B. napus Tic40 domain-swapping constructs in 7365A suggested that amino acid replacements in the carboxyl terminus of BnaC9.Tic40 cause this functional divergence. The distribution of these amino acid replacements in 59 diverse Brassica spp. accessions demonstrated that the neofunctionalization of Tic40 is restricted to B. oleracea and its derivatives and thus occurred after the divergence of the Brassica spp. A, B, and C genomes.

Primary root (PR) development is a crucial developmental process that is essential for plant survival. The elucidation of the PR transcriptome provides insight into the genetic mechanism controlling PR development in crops. In this study, we performed a comparative transcriptome analysis to investigate the genome-wide gene expression profiles of the seedling PRs of four Brassica napus genotypes that were divided into two groups, short group (D43 and D61), and long group (D69 and D72), according to their extremely different primary root lengths (PRLs). The results generated 55,341,366–64,631,336 clean reads aligned to 62,562 genes (61.9% of the current annotated genes) in the B. napus genome. We provide evidence that at least 44,986 genes are actively expressed in the B. napus PR. The majority of the genes that were expressed during seedling PR development were associated with metabolism, cellular processes, response to stimulus, biological regulation, and signaling. Using a pairwise comparison approach, 509 differentially expressed genes (DEGs; absolute value of log2 fold-change ≥1 and p ≤ 0.05) between the long and short groups were revealed, including phytohormone-related genes, protein kinases and phosphatases, oxygenase, cytochrome P450 proteins, etc. Combining GO functional category, KEGG, and MapMan pathway analyses indicated that the DEGs involved in cell wall metabolism, carbohydrate metabolism, lipid metabolism, secondary metabolism, protein modification and degradation, hormone pathways and signaling pathways were the main causes of the observed PRL differences. We also identified 16 differentially expressed transcription factors (TFs) involved in PR development. Taken together, these transcriptomic datasets may serve as a foundation for the identification of candidate genes and may provide valuable information for understanding the molecular and cellular events related to PR development.

7365AB, a recessive genetic male sterility system, is controlled by BnMs3 in Brassica napus, which encodes a Tic40 protein required for tapetum development. However, the role of BnMs3 in rapeseed anther development is still largely unclear. In this research, cytological analysis revealed that anther development of a Bnms3 mutant has defects in the transition of the tapetum to the secretory type, callose degradation, and pollen-wall formation. A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis. Reverse genetics was applied by means of Arabidopsis insertional mutant lines to characterize the function of these unigenes and revealed that MSR02 is only required for transport of sporopollenin precursors through the plasma membrane of the tapetum. The real-time PCR data have further verified that BnMs3 plays a primary role in tapetal differentiation by affecting the expression of a few key transcription factors, participates in tapetal degradation by modulating the expression of cysteine protease genes, and influences microspore separation by manipulating the expression of BnA6 and BnMSR66 related to callose degradation and of BnQRT1 and BnQRT3 required for the primary cell-wall degradation of the pollen mother cell. Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors. All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus.

Both the pollination control system and genetic distance are major factors in the utilization of crop heterosis. The recessive genic male sterile line (RGMS) 7-7365A (Bnms3ms3ms4ms4) has been widely applied to hybrid seed production because it can generate a completely male sterile population by crossing with the 7-7365C temporary line (Bnms3ms3rfrf). In this study, the sterile genes of 7-7365A were transferred to the new Brassica napus lines 7-749 and 7-750 with a high content of subgenomes by backcross breeding. We used the amplified fragment length polymorphism (AFLP) technique combined with bulk segregant analysis (BSA) to identify markers linked to the BnMs4 gene. Twelve AFLP markers linked to the BnMs4 gene were identified. Of them, SA06MG09 and P08MG16 were the closest makers, which were on either side of the gene at a distance of 0.9 and 0.8 cM, respectively. Twenty AFLP primer combinations were used to screen the F2, BC1F3, and BC2F4 populations from the breeding program, and the markers linked to the BnMs3 and BnMs4 genes were used to screen the BC2F4 populations. As a result, we obtained two types of improved sterile lines, 7-749A and 7-750A, and their indexes of subgenomic components (ISG) were 44.2–49.8 and 20.2–26.6%, respectively. The combining ability analyses of seed yield character were conducted in the crosses from the three sterile lines and ten restorers within a random block design in three environments for two successive years. The general combining ability (GCA) of the two improved sterile lines were significantly higher than the GCA of 7-7365A in every environment tested. The two improved sterile lines had stability in seed yield, and they will be used in the future for hybrid seed production.

Male sterility is an important contributor to heterosis in Brassica napus L. The B. napus line 7-7365ABC is a recessive epistatic genic male sterile (REGMS) three-line system. The 7-7365A line with the genotype Bnms3ms3ms4ms4RfRf is male-sterile, while the 7-7365B line with the genotype BnMs3ms3ms4ms4RfRf is male-fertile, and 7-7365C with homozygous recessive genotypes at the three loci shows male fertility because the loss function of Bnrf gene causes the inhibition of the genetic trait of the double mutant Bnms3 Bnms4. Histological studies addressing male sterility, transcriptional regulation pathways and the role of abscisic acid (ABA) in the anther development of REGMS plants are reported here. In the male-sterile line 7-7365A, tapetum cell and microspore mother cell separation were affected, and this led to failure of microspore release. The activity of polygalacturonase and the expression of the pectin methylesterase gene (AT3g06830) were significantly downregulated. Nine genes were downregulated in 7-7365A compared to 7-7365B and 7-7365C, including genes specifically expressed in tapetum (A3, A9, MS1) and the ABA-responsive gene KIN1. ABA concentration in 7-7365B was significantly higher than in 7-7365A and 7-7365C in young flower buds. Furthermore, temperature treatment made some sterile 7-7365A flowers become fertile. The stamens in these flowers produced viable pollen, and filament elongation was restored to its level in 7-7365C. We propose that ABA might control the expression of genes involved in cell separation during early anther development. The REGMS phenotype could be controlled by a primary pathway of male sterile metabolism positively regulated by the BnMs3 gene and a supplementary pathway negatively regulated by the BnRf gene.