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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.

Background In terms of global demand, rapeseed is the third-largest oilseed crop after soybeans and palm, which produces vegetable oil for human consumption and biofuel for industrial production. Roots are vital organs for plant to absorb water and attain mineral nutrients, thus they are of great importance to plant productivity. However, the genetic mechanisms regulating root development in rapeseed remain unclear. In the present study, seven root-related traits and shoot biomass traits in 280 Brassica napus accessions at five continuous vegetative stages were measured to establish the genetic basis of root growth in rapeseed. Results The persistent and stage-specific genetic mechanisms were revealed by root dynamic analysis. Sixteen persistent and 32 stage-specific quantitative trait loci (QTL) clusters were identified through genome-wide association study (GWAS). Root samples with contrasting (slow and fast) growth rates throughout the investigated stages and those with obvious stage-specific changes in growth rates were subjected to transcriptome analysis. A total of 367 differentially expressed genes (DEGs) with persistent differential expressions throughout root development were identified, and these DEGs were significantly enriched in GO terms, such as energy metabolism and response to biotic or abiotic stress. Totally, 485 stage-specific DEGs with different expressions at specific stage were identified, and these DEGs were enriched in GO terms, such as nitrogen metabolism. Four candidate genes were identified as key persistent genetic factors and eight as stage-specific ones by integrating GWAS, weighted gene co-expression network analysis (WGCNA), and differential expression analysis. These candidate genes were speculated to regulate root system development, and they were less than 100 kb away from peak SNPs of QTL clusters. The homologs of three genes (BnaA03g52990D, BnaA06g37280D, and BnaA09g07580D) out of 12 candidate genes have been reported to regulate root development in previous studies. Conclusions Sixteen QTL clusters and four candidate genes controlling persistently root development, and 32 QTL clusters and eight candidate genes stage-specifically regulating root growth in rapeseed were detected in this study. Our results provide new insights into the temporal genetic mechanisms of root growth by identifying key candidate QTL/genes in rapeseed.

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.

Application of a low-cost and high-efficiency hydroponic system in a rapeseed population verified two types of genetic factors (“persistent” and “stage-specific”) that control root development. The root system is a vital plant component for nutrient and water acquisition and is targeted to enhance plant productivity. Genetic dissection of the root system generally focuses on a single stage, but roots grow continuously during plant development. To reveal the temporal genetic patterns of root development, we measured nine root-related traits in a rapeseed recombinant inbred line population at six continuous stages during vegetative growth, using a modified hydroponic system with low-cost and high-efficiency features that could synchronize plant growth under controlled conditions. Phenotypic correlation and growth dynamic analysis suggested the existence of two types of genetic factors (“persistent” and “stage-specific”) that control root development. Dynamic (unconditional and conditional) quantitative trait loci (QTL) mapping detected 28 stage-specific and 23 persistent QTLs related to root growth. Among them, 13 early stage-specific, 19 persistent and 8 later stage-specific QTLs were detected at 7 DAS (days after sowing), 16 DAS and 5 EL (expanding leaf stage), respectively, providing efficient and adaptable stages for QTL identification. The effective prediction of biomass accumulation using root morphological traits (up to 96.6% or 92.64% at a specific stage or the final stage, respectively) verified that root growth allocation with maximum root uptake area facilitated biomass accumulation. Furthermore, marker-assistant selection, which combined the “persistent” and “stage-specific” QTLs, proved their effectiveness for root improvement with an excellent uptake area. Our results highlight the potential of high-throughput and precise phenotyping to assess the dynamic genetics of root growth and provide new insights into ideotype root system-based biomass breeding.

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.

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.

A recessive epistatic genic male sterile two-type line, 7365AB (Bnms3ms3ms4msRrfRf/BnMs3ms3ms4ms4RfRf), combined with the fertile interim-maintainer 7365C (Bnms3ms3ms4ms4rfrf) is an effective pollination control system in hybrid rapeseed production. We report an effective strategy used to fine map BnMs4 and BnRf. The two genes were both defined to a common microsyntenic region with Arabidopsis chromosome 3 using intron polymorphism (IP) markers developed according to Arabidopsis genome information and published genome organization of the A genome. The near-isogenic lines 7365AC (Bnms3ms3ms4ms4Rfrf/Bnms3ms3ms4ms4rfrf) of BnRf and 736512AB (Bnms3ms3Ms4ms4RfRf/Bnms3ms3ms4ms4RfRf) of BnMs4 were constructed to screen developed markers and create genetic linkage maps. Nine polymorphic IP markers (P1–P9) were identified. Of these, P2, P3, P4, and P6 were linked to both BnMs4 and BnRf with genetic distances