Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47a^ZY821, but not the yellow-seed allele BnA09MYB47a^GH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

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.

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.

4-Coumarate: CoA ligase (4CL) is a key branch point enzyme at the end of the phenylpropanoid metabolic pathway. It regulates the synthesis of various metabolites and participates in plant growth and development by catalyzing the formation of CoA ester compounds. However, 4CL family members have not been identified and analyzed among U’s triangle species in Brassica. In this study, 53 4CL genes were identified in Brassica U’s triangle species and divided into 4 groups (group I, II, III and IV) according to phylogenetic relationship. Based on phylogenetics, gene structure, conserved motifs, chromosome localization and collinearity analysis, 4CLs were relatively conserved in the evolution of Brassica U’s triangle species. The promoter region contains a large number of cis-acting elements, implying the functional diversity of 4CLs. Further combining transcriptome data and reverse transcription quantitative PCR (RT-qPCR), we found that Bna4CLs have tissue specificity and can not only respond to exogenous phytohormone changes but also regulate the flavonoid biosynthetic pathway in the yellow- and black-seeded B. napus. Our results complement the lack of research on the 4CL gene family in Brassica, clarify the sequence characteristics and functional diversity of these genes and lay a foundation for further exploration of 4CL genes in response to abiotic stress and regulation of seed coat flavonoid accumulation.

Rapeseed (Brassica napus) is an important oilseed crop and yellow-seeded and black-seeded varieties have different metabolite profiles, which determines the quality and edibility of their oil. In this study, we performed a non-targeted metabolomics analysis of seeds from four rapeseed varieties at eight developmental stages. This analysis identified 4540 features, of which 366 were annotated as known metabolites. The content of these metabolites was closely related to seed developmental stage, with the critical period for seed metabolite accumulation being between 10 and 20 days after pollination. Through a comparative analysis, we identified 18 differentially abundant flavonoid features between yellow-seeded and black-seeded rapeseed varieties. By combining the flavonoid data with transcriptome data, we constructed a gene regulatory network that may reflect the accumulation of differentially abundant flavonoid features. Finally, we predicted 38 unknown features as being flavonoid features through molecular networking. These results provide valuable metabolomics information for the breeding of yellow-seeded rapeseed varieties.

Members of the high-affinity potassium transporter (HKT) protein family regulate the uptake and homeostasis of sodium and potassium ions, but little research describes their roles in response to abiotic stresses in rapeseed (Brassica napus L.). In this study, we identified and characterized a total of 36 HKT genes from the species comprising the triangle of U model (U-triangle species): B. rapa, B. nigra, B. oleracea, B. juncea, B. napus, and B. carinata. We analyzed the phylogenetic relationships, gene structures, motif compositions, and chromosomal distributions of the HKT family members of rapeseed. Based on their phylogenetic relationships and assemblage of functional domains, we classified the HKT members into four subgroups, HKT1;1 to HKT1;4. Analysis of the nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios of HKT gene pairs suggested that these genes have experienced strong purifying selective pressure after duplication, with their evolutionary relationships supporting the U-triangle theory. Furthermore, the expression profiles of BnaHKT genes varies among potassium, phytohormone and heavy-metal treatment. Their repression provides resistance to heavy-metal stress, possibly by limiting uptake. Our results systematically reveal the characteristics of HKT family proteins and their encoding genes in six Brassica species and lay a foundation for further exploration of the role of HKT family genes in heavy-metal tolerance.