Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

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 BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, 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.

BnKAT2 Positively Regulates the Main Inflorescence Length and Silique Number in Brassica napus by Regulating the Auxin and Cytokinin Signaling Pathways.

Brassica napus is the dominant oil crop cultivated in China for its high quality and high yield. The length of the main inflorescence and the number of siliques produced are important traits contributing to rapeseed yield. Therefore, studying genes related to main inflorescence and silique number is beneficial to increase rapeseed yield. Herein, we focused on the effects of BnKAT2 on the main inflorescence length and silique number in B. napus. We explored the mechanism of BnKAT2 increasing the effective length of main inflorescence and the number of siliques through bioinformatics analysis, transgenic technology, and transcriptome sequencing analysis. The full BnKAT2(BnaA01g09060D) sequence is 3674 bp, while its open reading frame is 2055 bp, and the encoded protein comprises 684 amino acids. BnKAT2 is predicted to possess two structural domains, namely KHA and CNMP-binding domains. The overexpression of BnKAT2 effectively increased the length of the main inflorescence and the number of siliques in B. napus, as well as in transgenic Arabidopsis thaliana. The type-A Arabidopsis response regulator (A-ARR), negative regulators of the cytokinin, are downregulated in the BnKAT2-overexpressing lines. The Aux/IAA, key genes in auxin signaling pathways, are downregulated in the BnKAT2-overexpressing lines. These results indicate that BnKAT2 might regulate the effective length of the main inflorescence and the number of siliques through the auxin and cytokinin signaling pathways. Our study provides a new potential function gene responsible for improvement of main inflorescence length and silique number, as well as a candidate gene for developing markers used in MAS (marker-assisted selection) breeding to improve rapeseed yield.

SsCox17, a copper chaperone, is required for pathogenic process and oxidative stress tolerance of Sclerotinia sclerotiorum.

Stem rot, caused by Sclerotinia sclerotiorum has emerged as one of the major fungal pathogens of oilseed Brassica across the world. The pathogenic development is exquisitely dependent on reactive oxygen species (ROS) modulation. Cox17 is a crucial factor that shuttles copper ions from the cytosol to the mitochondria for the cytochrome c oxidase (CCO) assembly. Currently, no data is available regarding the impact of Cox17 in fungal pathogenesis. The present research was carried out to functionally characterize the role of Cox17 in S. sclerotiorum pathogenesis. SsCox17 transcripts showed high expression levels during inoculation on rapeseed. Intramitochondrial copper content and CCO activity were decreased in SsCox17 gene-silenced strains. The SsCox17 gene expression was up-regulated in the hyphae under oxidative stress and a deficiency response to oxidative stress was detected in SsCox17 gene-silenced strains. Compared to the S. sclerotiorum wild-type strain, there was a concomitant reduction in the virulence of SsCox17 gene-silenced strains. The SsCox17 overexpression strain was further found to increase copper content, CCO activity, tolerance to oxidative stress and virulence. We also observed a certain correlation of appressoria formation and SsCox17. These results provide evidence that SsCox17 is positively associated with fungal virulence and oxidative detoxification.

Host-Induced Gene Silencing of a Multifunction Gene Sscnd1 Enhances Plant Resistance Against Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic fungal pathogen and has a substantial economic impact on crop production worldwide. Magnaporthe appressoria-specific (MAS) proteins have been suggested to be involved in the appressorium formation in Magnaporthe oryzae. Sscnd1, an MAS homolog gene, is highly induced at the early infection stage of S. sclerotiorum. Knock-down the expression of Sscnd1 gene severely reduced the virulence of S. sclerotiorum on intact rapeseed leaves, and their virulence was partially restored on wounded leaves. The Sscnd1 gene-silenced strains exhibited a defect in compound appressorium formation and cell integrity. The instantaneous silencing of Sscnd1 by tobacco rattle virus (TRV)-mediated host-induced gene silencing (HIGS) resulted in a significant reduction in disease development in tobacco. Three transgenic HIGS Arabidopsis lines displayed high levels of resistance to S. sclerotiorum and decreased Sscnd1 expression. Production of specific Sscnd1 siRNA in transgenic HIGS Arabidopsis lines was confirmed by stem-loop qRT-PCR. This study revealed that the compound appressorium-related gene Sscnd1 is required for cell integrity and full virulence in S. sclerotiorum and that Sclerotinia stem rot can be controlled by expressing the silencing constructs of Sscnd1 in host plants.

Both overlapping and independent loci underlie seed number per pod and seed weight in Brassica napus by comparative quantitative trait loci analysis.

Seed number per pod (SNPP) and seed weight (SW) are two components of seed yield in rapeseed (Brassica napus). Here, a natural population of rapeseed was employed for genome-wide association analysis for SNPP and SW across multi-years. A total of 101 and 77 SNPs significantly associated with SNPP and SW with the phenotypic variances (R2) ranging from 1.35 to 29.47% and from 0.78 to 34.58%, respectively. And 43 and 33 homologs of known genes from model plants were located in the 65 and 49 haplotype blocks (HBs) for SNPP and SW, respectively. Notably, we found 5 overlapping loci and 3 sets of loci with collinearity for both SNPP and SW, of which 4 overlapping loci harbored the haplotypes with the same direction of genetic effects on SNPP and SW, indicating high possibility to simultaneously improve SNPP and SW in rapeseed. Our findings revealed both overlapping and independent loci controlling seed number per pod and seed weight in rapeseed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01232-1.

Correction to: Both overlapping and independent loci underlie seed number per pod and seed weight in Brassica napus by comparative quantitative trait loci analysis.

[This corrects the article DOI: 10.1007/s11032-021-01232-1.].

Combining quantitative trait locus and co-expression analysis allowed identification of new candidates for oil accumulation in rapeseed.

In crops there are quantitative trait loci (QTLs) in which some of the causal quantitative trait genes (QTGs) have not been functionally characterized even in the model plant Arabidopsis. We propose an approach to delineate QTGs in rapeseed by coordinating expression of genes located within QTLs and known orthologs related to traits from Arabidopsis. Using this method in developing siliques 15 d after pollination in 71 lines of rapeseed, we established an acyl-lipid metabolism co-expression network with 21 modules composed of 270 known acyl-lipid genes and 3503 new genes. The core module harbored 76 known genes involved in fatty acid and triacylglycerol biosynthesis and 671 new genes involved in sucrose transport, carbon metabolism, amino acid metabolism, seed storage protein processes, seed maturation, and phytohormone metabolism. Moreover, the core module closely associated with the modules of photosynthesis and carbon metabolism. From the co-expression network, we selected 12 hub genes to identify their putative Arabidopsis orthologs. These putative orthologs were functionally analysed using Arabidopsis knockout and overexpression lines. Four knockout mutants exhibited lower seed oil content, while the seed oil content in 10 overexpression lines was significantly increased. Therefore, combining gene co-expression network analysis and QTL mapping, this study provides new insights into the detection of QTGs and into acyl-lipid metabolism in rapeseed.

Whole-genome resequencing reveals Brassica napus origin and genetic loci involved in its improvement.

Brassica napus (2n = 4x = 38, AACC) is an important allopolyploid crop derived from interspecific crosses between Brassica rapa (2n = 2x = 20, AA) and Brassica oleracea (2n = 2x = 18, CC). However, no truly wild B. napus populations are known; its origin and improvement processes remain unclear. Here, we resequence 588 B. napus accessions. We uncover that the A subgenome may evolve from the ancestor of European turnip and the C subgenome may evolve from the common ancestor of kohlrabi, cauliflower, broccoli, and Chinese kale. Additionally, winter oilseed may be the original form of B. napus. Subgenome-specific selection of defense-response genes has contributed to environmental adaptation after formation of the species, whereas asymmetrical subgenomic selection has led to ecotype change. By integrating genome-wide association studies, selection signals, and transcriptome analyses, we identify genes associated with improved stress tolerance, oil content, seed quality, and ecotype improvement. They are candidates for further functional characterization and genetic improvement of B. napus.

Simultaneous Transcriptome Analysis of Host and Pathogen Highlights the Interaction Between Brassica oleracea and Sclerotinia sclerotiorum.

White mold disease caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops. Here, we simultaneously assessed the transcriptome changes from lesions produced by S. sclerotiorum on disease-resistant (R) and -susceptible (S) B. oleracea pools bulked from a resistance-segregating F2 population. Virulence genes of S. sclerotiorum, including polygalacturonans, chitin synthase, secretory proteins, and oxalic acid biosynthesis, were significantly repressed in lesions of R B. oleracea at 12 h postinoculation (hpi) but exhibited similar expression patterns in R and S B. oleracea at 24 hpi. Resistant B. oleracea induced expression of receptors potentially to perceive Sclerotinia signals during 0 to 12 hpi and deployed complex strategies to suppress the pathogen establishment, including the quick accumulation of reactive oxygen species via activating Ca2+ signaling and suppressing pathogen oxalic acid generation in S. sclerotiorum. In addition, cell wall degradation was inhibited in the resistant B. oleracea potentially to prevent the expansion of Sclerotinia hyphae. The transcriptome changes in S. sclerotiorum and host revealed that resistant B. oleracea produces strong responses against S. sclerotiorum during early infection.

Arabidopsis AGDP1 links H3K9me2 to DNA methylation in heterochromatin.

Heterochromatin is a tightly packed form of chromatin that is associated with DNA methylation and histone 3 lysine 9 methylation (H3K9me). Here, we identify an H3K9me2-binding protein, Agenet domain (AGD)-containing p1 (AGDP1), in Arabidopsis thaliana. Here we find that AGDP1 can specifically recognize the H3K9me2 mark by its three pairs of tandem AGDs. We determine the crystal structure of the Agenet domain 1 and 2 cassette (AGD12) of Raphanus sativus AGDP1 in complex with an H3K9me2 peptide. In the complex, the histone peptide adopts a unique helical conformation. AGD12 specifically recognizes the H3K4me0 and H3K9me2 marks by hydrogen bonding and hydrophobic interactions. In addition, we find that AGDP1 is required for transcriptional silencing, non-CG DNA methylation, and H3K9 dimethylation at some loci. ChIP-seq data show that AGDP1 preferentially occupies long transposons and is associated with heterochromatin marks. Our findings suggest that, as a heterochromatin-binding protein, AGDP1 links H3K9me2 to DNA methylation in heterochromatin regions.

Genome-Wide Association Study Reveals Both Overlapping and Independent Genetic Loci to Control Seed Weight and Silique Length in Brassica napus.

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.

Transcriptomic comparison between Brassica oleracea and rice (Oryza sativa) reveals diverse modulations on cell death in response to Sclerotinia sclerotiorum.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops, but not in rice. The leaves of a rice line, a partial resistant (R) and a susceptible (S) Brassica oleracea pool that bulked from a resistance-segregating F2 population were employed for transcriptome sequencing before and after inoculation by S. sclerotiorum for 6 and 12 h. Distinct transcriptome profiles were revealed between B. oleracea and rice in response to S. sclerotiorum. Enrichment analyses of GO and KEGG indicated an enhancement of antioxidant activity in the R B. oleracea and rice, and histochemical staining exhibited obvious lighter reactive oxygen species (ROS) accumulation and cell death in rice and the R B. oleracea as compared to that in the S B. oleracea. Significant enhancement of Ca(2+) signalling, a positive regulator of ROS and cell death, were detected in S B. oleracea after inoculation, while it was significantly repressed in the R B. oleracea group. Obvious difference was detected between two B. oleracea groups for WRKY transcription factors, particularly for those regulating cell death. These findings suggest diverse modulations on cell death in host in response to S. sclerotiorum. Our study provides useful insight into the resistant mechanism to S. sclerotiorum.

Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola.

Understanding the regulation of lipid metabolism is vital for genetic engineering of canola (Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

Comparative quantitative trait loci for silique length and seed weight in Brassica napus.

Silique length (SL) and seed weight (SW) are important yield-associated traits in rapeseed (Brassica napus). Although many quantitative trait loci (QTL) for SL and SW have been identified in B. napus, comparative analysis for those QTL is seldom performed. In the present study, 20 and 21 QTL for SL and SW were identified in doubled haploid (DH) and DH-derived reconstructed F2 populations in rapeseed, explaining 55.1-74.3% and 24.4-62.9% of the phenotypic variation across three years, respectively. Of which, 17 QTL with partially or completely overlapped confidence interval on chromosome A09, were homologous with two overlapped QTL on chromosome C08 by aligning QTL confidence intervals with the reference genomes of Brassica crops. By high density selective genotyping of DH lines with extreme phenotypes, using a Brassica single-nucleotide polymorphism (SNP) array, the QTL on chromosome A09 was narrowed, and aligned into 1.14-Mb region from 30.84 to 31.98 Mb on chromosome R09 of B. rapa and 1.05-Mb region from 27.21 to 28.26 Mb on chromosome A09 of B. napus. The alignment of QTL with Brassica reference genomes revealed homologous QTL on A09 and C08 for SL. The narrowed QTL region provides clues for gene cloning and breeding cultivars by marker-assisted selection.

Transfer of sclerotinia resistance from wild relative of Brassica oleracea into Brassica napus using a hexaploidy step.

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. 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.

Differential accumulation of phenolic compounds and expression of related genes in black- and yellow-seeded Brassica napus.

Developing yellow-seeded Brassica napus (rapeseed) with improved qualities is a major breeding goal. The intermediate and final metabolites of the phenylpropanoid and flavonoid pathways affect not only oil quality but also seed coat colour of B. napus. Here, the accumulation of phenolic compounds was analysed in the seed coats of black-seeded (ZY821) and yellow-seeded (GH06) B. napus. Using toluidine blue O staining and liquid chromatography-mass spectrometry, histochemical and biochemical differences were identified in the accumulation of phenolic compounds between ZY821 and GH06. Two and 13 unique flavonol derivatives were detected in ZY821 and GH06, respectively. Quantitative real-time PCR analysis revealed significant differences between ZY821 and GH06 in the expression of common phenylpropanoid biosynthetic genes (BnPAL and BnC4H), common flavonoid biosynthetic genes (BnTT4 and BnTT6), anthocyanin- and proanthocyandin-specific genes (BnTT3 and BnTT18), proanthocyandin-specific genes (BnTT12, BnTT10, and BnUGT2) and three transcription factor genes (BnTTG1, BnTTG2, and BnTT8) that function in the flavonoid biosynthetic pathway. These data provide insight into pigment accumulation in B. napus, and serve as a useful resource for researchers analysing the formation of seed coat colour and the underlying regulatory mechanisms in B. napus.