Natural variation and artificial selection at the BnaC2.MYB28 locus modulate Brassica napus seed glucosinolate.

The degradation products of glucosinolates (GSLs) greatly lower the nutritional value of rapeseed (Brassica napus) meal; thus, reduction of seed GSL content (SGC) has become an important objective of rapeseed breeding. In our previous study, we finely mapped a major QTL (qGSL-C2) for SGC to a 49-kb collinear region on B. rapa chromosome A2. Here, we experimentally validated that BnaC2.MYB28, encoding an R2R3-MYB transcription factor, is the causal gene of qGSL-C2. BnaC2.MYB28 is a nucleus-localized protein mainly expressed in vegetative tissues. Knockout of BnaC2.MYB28 in the high-SGC parent G120 reduced SGC to a value lower than that in the low-SGC parent ZY50, while overexpression of BnaC2.MYB28 in both parental lines (G120 and ZY50) led to extremely high SGC, indicating that BnaC2.MYB28 acts as a positive regulator of SGC in both parents. Molecular characterization revealed that BnaC2.MYB28 forms a homodimer and specifically interacts with BnaMYC3. Moreover, BnaC2.MYB28 can directly activate the expression of GSL biosynthesis genes. Differential expression abundance resulting from the polymorphic promoter sequences, in combination with the different capability in activating downstream genes involved in aliphatic GSL biosynthesis, caused the functional divergence of BnaC2.MYB28 in SGC regulation between the parents. Natural variation of BnaC2.MYB28 was highly associated with SGC in natural germplasm and has undergone artificial selection in modern low-GSL breeding. This study provides important insights into the core function of BnaC2.MYB28 in regulating SGC and a promising strategy for manipulating SGC in rapeseed.

Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase: implications for rapeseed resistance to fungal infection.

MAIN CONCLUSION: BnPGIPs interacted with Sclerotinia sclerotiorum PGs to improve rapeseed SSR resistance at different levels; the BnPGIP-overexpression lines did not affect plant morphology or seed quality traits. Plant polygalacturonase-inhibiting proteins (PGIPs) play a crucial role in plant defence against phytopathogenic fungi by inhibiting fungal polygalacturonase (PG) activity. We overexpressed BnPGIP2, BnPGIP5, and BnPGIP10 genes in an inbred line 7492 of rapeseed (Brassica napus). Compared with 7492WT, the overexpression of BnPGIP2 lines significantly increased Sclerotinia sclerotiorum resistance in both seedlings and adult plants. BnPGIP5 overexpression lines exhibited decreased S. sclerotiorum disease symptoms in seedlings only, whereas BnPGIP10 overexpression lines did not improve Sclerotinia resistance for seedlings or adult plants. Quantitative real-time PCR analysis of S. sclerotiorum PG1, SsPG3, SsPG5, and SsPG6 genes in overexpressing BnPGIP lines showed that these pathogenic genes in the Sclerotinia resistance transgenic lines exhibited low expression in stem tissues. Split-luciferase complementation experiments confirmed the following: BnPGIP2 interacts with SsPG1 and SsPG6 but not with SsPG3 or SsPG5; BnPGIP5 interacts with SsPG3 and SsPG6 but not with SsPG1 or SsPG5; and BnPGIP10 interacts with SsPG1 but not SsPG3, SsPG5, or SsPG6. Leaf crude protein extracts from BnPGIP2 and BnPGIP5 transgenic lines displayed high inhibitory activity against the SsPG crude protein. BnPGIP-overexpression lines with Sclerotinia resistance displayed a lower accumulation of H2O2 and higher expression of the H2O2-removing gene BnAPX (ascorbate peroxidase) than 7492WT, as well as elevated expression of defence response genes including jasmonic acid/ethylene and salicylic acid pathways after S. sclerotiorum infection. The plants overexpressing BnPGIP exhibited no difference in either agronomic traits or grain yield from 7492WT. This study provides potential target genes for developing S. sclerotiorum resistance in rapeseed.

Optimizing glyphosate tolerance in rapeseed by CRISPR/Cas9-based geminiviral donor DNA replicon system with Csy4-based single-guide RNA processing.

Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.

A 24,482-bp deletion is associated with increased seed weight in Brassica napus L.

KEY MESSAGE: A major QTL for seed weight was fine-mapped in rapeseed, and a 24,482-bp deletion likely mediates the effect through multiple pathways. Exploration of the genes controlling seed weight is critical to the improvement of crop yield and elucidation of the mechanisms underlying seed formation in rapeseed (Brassica napus L.). We previously identified the quantitative trait locus (QTL) qSW.C9 for the thousand-seed weight (TSW) in a double haploid population constructed from F1 hybrids between the parental accessions HZ396 and Y106. Here, we confirmed the phenotypic effects associated with qSW.C9 in BC3F2 populations and fine-mapped the candidate causal locus to a 266-kb interval. Sequence and expression analyses revealed that a 24,482-bp deletion in HZ396 containing six predicted genes most likely underlies qSW.C9. Differential gene expression analysis and cytological observations suggested that qSW.C9 affects both cell proliferation and cell expansion through multiple signaling pathways. After genotyping of a rapeseed diversity panel to define the haplotype structure, it could be concluded that the selection of germplasm with two specific markers may be effective in improving the seed weight of rapeseed. This study provides a solid foundation for the identification of the causal gene of qSW.C9 and offers a promising target for the breeding of higher-yielding rapeseed.

Overexpression of OsPGIP2 confers Sclerotinia sclerotiorum resistance in Brassica napus through increased activation of defense mechanisms.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is the most serious disease affecting the yield of the agriculturally and economically important crop Brassica napus (rapeseed). In this study, Oryza sativa polygalacturonase-inhibiting protein 2 (OsPGIP2) was found to effectively enhanced rapeseed immunity against S. sclerotiorum infection. Leaf extracts of B. napus plants overexpressing OsPGIP2 showed enhanced S. sclerotiorum resistance by delaying pathogen infection. The constitutive expression of OsPGIP2 in rapeseed plants provided a rapid and effective defense response, which included the production of reactive oxygen species, interactions with S. sclerotiorum polygalacturonases (SsPG3 and SsPG6), and effects on the expression of defense genes. RNA sequencing analysis revealed that the pathogen induced many differentially expressed genes associated with pathogen recognition, redox homeostasis, mitogen-activated protein kinase signaling cascades, hormone signaling pathways, pathogen-/defense-related genes, and cell wall-related genes. The overexpression of OsPGIP2 also led to constitutively increased cell wall cellulose and hemicellulose contents in stems without compromising seed quality. The results demonstrate that OsPGIP2 plays a major role in rapeseed defense mechanisms, and we propose a model for OsPGIP2-conferred resistance to S. sclerotiorum in these plants.

Optimised Agrobacterium-Mediated Transformation and Application of Developmental Regulators Improve Regeneration Efficiency in Melons.

Melon (Cucumis melo L.) is a protected crop in China with high economic value. Agrobacterium-mediated genetic transformation is a powerful tool to improve agronomic traits and obtain elite germplasm. However, current transformation protocols in melons are inefficient and highly genotype-dependent. To improve transformation in melon, we tested different infiltration methods for Agrobacterium-mediated transformation. Among these methods, micro-brushing and sonication for 20 s, followed by vacuum infiltration at -1.0 kPa for 90 s, resulted in the strongest green fluorescent protein signal and increased the proportion of infected explants. We transformed melon with developmental regulatory genes AtGRF5, AtPLT5, AtBBM, AtWUS, AtWOX5, and AtWIND1 from Arabidopsis and estimated regeneration frequencies as the number of regenerating shoots/total number of inoculated explants in the selection medium. The overexpression of AtGRF5 and AtPLT5 in melon resulted in transformation efficiencies of 42.3% and 33% in ZHF and 45.6% and 32.9% in Z12, respectively, which were significantly higher than those of the control. AtGRF5 and AtPLT5 expression cassettes were added to CRISPR/Cas9 genome-editing vectors to obtain transgenic phytoene desaturase CmPDS knockout mutants. Using AtGRF5 or AtPLT5, multi-allelic mutations were observed at CmPDS target sites in recalcitrant melon genotypes. This strategy enables genotype-flexible transformation and promotes precise genome modification technologies in melons.

Quantitative trait locus mapping and improved resistance to sclerotinia stem rot in a backbone parent of rapeseed (Brassica napus L.).

There are three main challenges to improving sclerotinia stem rot (SSR) resistance in rapeseed (Brassica napus L.). First, breeding materials such as the backbone parents have not been extensively investigated, making the findings of previous studies difficult to directly implement. Second, SSR resistance and flowering time (FT) loci are typically linked; thus, use of these loci requires sacrifice of the rapeseed growth period. Third, the SSR resistance loci in susceptible materials are often neglected, thereby reducing the richness of resistant resources. This study was conducted to investigate the stem resistance, disease index, and FT of a doubled haploid population consisting of 151 lines constructed from the backbone parent 19514A and conventional rapeseed cultivar ZY50 within multiple environments. Quantitative trait locus (QTL) mapping revealed 13 stem resistance QTLs, 9 disease index QTLs, and 20 FT QTLs. QTL meta-analysis showed that uqA04, uqC03.1, and uqC03.2 were repeatable SSR resistance QTLs derived from different parents but not affected by the FT. Based on these three QTLs, we proposed a strategy for improving the SSR resistance of 19514A and ZY50. This study improves the understanding of the resistance to rapeseed SSR and genetic basis of FT and demonstrates that SSR resistance QTLs can be mined from parents with a minimal resistance level difference, thereby supporting the application of backbone parents in related research and resistance improvement.