Flowering time is a key agronomic trait, directly influencing crop yield and quality. Many flowering-time genes have been identified and characterized in the model plant Arabidopsis thaliana; however, these genes remain uncharacterized in many agronomically important Brassica crops. In this study, we identified 1064, 510, and 524 putative orthologs of A. thaliana flowering-time genes from Brassica napus, Brassica rapa, and Brassica oleracea, respectively, and found that genes involved in the aging and ambient temperature pathways were fewer than those in other flowering pathways. Flowering-time genes were distributed mostly on chromosome C03 in B. napus and B. oleracea, and on chromosome A09 in B. rapa. Calculation of non-synonymous (Ka)/synonymous substitution (Ks) ratios suggested that flowering-time genes in vernalization pathways experienced higher selection pressure than those in other pathways. Expression analysis showed that most vernalization-pathway genes were expressed in flowering organs. Approximately 40% of these genes were highly expressed in the anther, whereas flowering-time integrator genes were expressed in a highly organ-specific manner. Evolutionary selection pressures were negatively correlated with the breadth and expression levels of vernalization-pathway genes. These findings provide an integrated framework of flowering-time genes in these three Brassica crops and provide a foundation for deciphering the relationship between gene expression patterns and their evolutionary selection pressures in Brassica napus.

Although rapeseed (Brassica napus L.) mutant forming multiple siliques was morphologically described and considered to increase the silique number per plant, an important agronomic trait in this crop, the molecular mechanism underlying this beneficial trait remains unclear. Here, we combined bulked-segregant analysis (BSA) and whole genome re-sequencing (WGR) to map the genomic regions responsible for the multi-silique trait using two pools of DNA from the near-isogenic lines (NILs) zws-ms (multi-silique) and zws-217 (single-silique). We used the Euclidean Distance (ED) to identify genomic regions associated with this trait based on both SNPs and InDels. We also conducted transcriptome sequencing to identify differentially expressed genes (DEGs) between zws-ms and zws-217. Genetic analysis using the ED algorithm identified three SNP- and two InDel-associated regions for the multi-silique trait. Two highly overlapped parts of the SNP- and InDel-associated regions were identified as important intersecting regions, which are located on chromosomes A09 and C08, respectively, including 2044 genes in 10.20-MB length totally. Transcriptome sequencing revealed 129 DEGs between zws-ms and zws-217 in buds, including 39 DEGs located in the two abovementioned associated regions. We identified candidate genes involved in multi-silique formation in rapeseed based on the results of functional annotation. This study identified the genomic regions and candidate genes related to the multi-silique trait in rapeseed.

Plant height is a vital agronomic trait for crops, including oilseed crops such as rapeseed (Brassica napus L.). It affects the crop yield, oil content, and lodging resistance in rapeseed. In this study, we investigated a dwarf trait controlled by a semi-dominant allele in rapeseed. A dwarf line, YA2016-12, was crossed with a tall line, G184-189, and an F2 population was established. Forty of the tallest plants and 40 of the shortest plants from the F2 population were selected and two DNA pools (tall and dwarf) were constructed by the bulked segregant analysis (BSA) method. The two DNA pools and two parental DNAs were then re-sequenced. A sliding window analysis was used to calculate the Δ(SNP-index) and discover an association region on chromosome A03 with a length of 12.4 Mb. Within this region, we found 1225 genes, including 811 genes with non-synonymous or frameshift mutations between YA2016-12 and G184-189. Alignment to known plant height-related orthologs in Arabidopsis thaliana, as well as KEGG pathway and gene ontology annotations, was used to identify nine candidate genes (BnaA03g31770D, BnaA03g37960D, BnaA03g24740D, BnaA03g40550D, BnaA03g26120D, BnaA03g35130D, BnaA03g42350D, BnaA03g25610D, and BnaA03g39850D) involved in gibberellin or cytokinin signaling. Identification of the causal gene for this trait, and of genetic markers linked to favorable alleles, has potential utility for marker-assisted selection to breed rapeseed varieties with improved height.

The trade-off between growth and defense is common in plants. We previously demonstrated that BnaA03.WRKY28 weakened resistance strength but promoted shoot branching in Brassica napus (rapeseed). However, the molecular mechanism by which WRKY28 promotes branching formation is still obscure. In this study, we found that BnaA01.BRC1, BnaC01.BRC1, and BnaC03.BRC1 are mainly expressed in the leaf axils and contained W-box cis-acting elements in the promoter regions. BnaA03.WRKY28 directly bound to the promoter regions of these three copies and inhibited their expression. The brc1 mutants, the BnaA01.BRC1, BnaC01.BRC1 BnaA03.BRC1 and BnaC03.BRC1 were simultaneously knocked out, mediated by CRISPR/Cas9, and exhibited excessive branching. The expression level of the ABA biosynthesis encoding gene NCED3 was significantly reduced in the mutant compared to that in the WT. Instead, the expression level of the ABA catabolism encoding gene CYP707A3 was significantly higher than that in WT. These results suggest that the excessive branching of the brc1 mutant may be caused by the release of ABA-mediated bud dormancy. This study provides direct evidence for the potential mechanism of the WRKY28-BRC1 transcription factor module contributing to shoot branching in rapeseed.

Based on previous researches, we further investigated the multi-silique trait in rapeseed (Brassica napus L.) line zws-ms. In this study, we used a relatively comprehensive list of flowering related genes in rapeseed and compared them between zws-ms and its near-isogenic line (NIL) zws-217. Genes were studied on genome, transcriptome and proteome levels and then we focused on genes with non-synonymous single nucleotide polymorphism (SNP) or frame-shift insertion-deletion (InDel), finding some genes on the list which changes their sequences. Then, combined with their annotation and the information of their orthologs, certain genes such as BnaA09g05900D, ortholog of AGAMOUS-LIKE 42 (AGL42), which encodes an MADS-box protein, were assumed as probably responsible for the multi-silique trait. Also, we analyzed the Differentially Accumulated Proteins (DAPs) between zws-ms and zws-217, revealing some genes involved in homologous recombination and mismatch repair pathways. Since the development of flowers/siliques is crucial to crops and it influences the yield of rapeseed, this study paved a way to deeply understand the mechanism of the multi-pistil flower formation, which may facilitate researches on rapeseed production in future.

Nitrogen (N) is one of the basic nutrients and signals for plant development and deficiency of it would always limit the productions of crops in the field. Quantitative research on expression of N-stress responsive proteins on a proteome level remains elusive. In order to gain a deep insight into the proteins responding to nitrogen stress in rapeseed (Brassica napus L.), comparative proteomic analysis was performed to investigate changes of protein expression profiles from the root, stem and leaf under different N concentrations, respectively. More than 200 differential abundance proteins (DAPs) were detected and categorized into groups according to annotations, including “binding and catalytic activity”, “involved in primary metabolism and cellular processes”, “stress-response” and so on. Variation in chlorophyll (Chl) content and antioxidant activities further revealed that oxidative stress raised with the increase of N concentration. Bioinformatics analysis based on the expression level of total proteins suggested these DAPs might play important roles in adaptation to N-stress conditions. Generally, these results provides a new aspect into N-stress responding proteins in Brassica plants.