Overexpression of oilseed rape trehalose-6-phosphate synthesis gene BnaC02.TPS8 confers sensitivity to low nitrogen and high sucrose-induced anthocyanin accumulation in Arabidopsis.

MAIN CONCLUSION: Overexpression of BnaC02.TPS8 increased low N and high sucrose-induced anthocyanin accumulation. Anthocyanin plays a crucial role in safeguarding photosynthetic tissues against high light, UV radiation, and oxidative stress. Their accumulation is triggered by low nitrogen (N) stress and elevated sucrose levels in Arabidopsis. Trehalose-6-phosphate (T6P) serves as a pivotal signaling molecule, sensing sucrose availability, and carbon (C) metabolism. However, the mechanisms governing the regulation of T6P synthase (TPS) genes responsible for anthocyanin accumulation under conditions of low N and high sucrose remain elusive. In a previous study, we demonstrated the positive impact of a cytoplasm-localized class II TPS protein 'BnaC02.TPS8' on photosynthesis and seed yield improvement in Brassica napus. The present research delves into the biological role of BnaC02.TPS8 in response to low N and high sucrose. Ectopic overexpression of BnaC02.TPS8 in Arabidopsis seedlings resulted in elevated shoot T6P levels under N-sufficient conditions, as well as an increased carbon-to-nitrogen (C/N) ratio, sucrose accumulation, and starch storage under low N conditions. Overexpression of BnaC02.TPS8 in Arabidopsis heightened sensitivity to low N stress and high sucrose levels, accompanied by increased anthocyanin accumulation and upregulation of genes involved in flavonoid biosynthesis and regulation. Metabolic profiling revealed increased levels of intermediate products of carbon metabolism, as well as anthocyanin and flavonoid derivatives in BnaC02.TPS8-overexpressing Arabidopsis plants under low N conditions. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses demonstrated that BnaC02.TPS8 interacts with both BnaC08.TPS9 and BnaA01.TPS10. These findings contribute to our understanding of how TPS8-mediated anthocyanin accumulation is modulated under low N and high sucrose conditions.

Functional genomics of Brassica napus: Progresses, challenges, and perspectives.

Brassica napus, commonly known as rapeseed or canola, is a major oil crop contributing over 13% to the stable supply of edible vegetable oil worldwide. Identification and understanding the gene functions in the B. napus genome is crucial for genomic breeding. A group of genes controlling agronomic traits have been successfully cloned through functional genomics studies in B. napus. In this review, we present an overview of the progress made in the functional genomics of B. napus, including the availability of germplasm resources, omics databases and cloned functional genes. Based on the current progress, we also highlight the main challenges and perspectives in this field. The advances in the functional genomics of B. napus contribute to a better understanding of the genetic basis underlying the complex agronomic traits in B. napus and will expedite the breeding of high quality, high resistance and high yield in B. napus varieties.

The story of a decade: Genomics, functional genomics, and molecular breeding in Brassica napus.

Rapeseed (Brassica napus L.) is one of the major global sources of edible vegetable oil and is also used as a feed and pioneer crop and for sightseeing and industrial purposes. Improvements in genome sequencing and molecular marker technology have fueled a boom in functional genomic studies of major agronomic characters such as yield, quality, flowering time, and stress resistance. Moreover, introgression and pyramiding of key functional genes have greatly accelerated the genetic improvement of important traits. Here we summarize recent progress in rapeseed genomics and genetics, and we discuss effective molecular breeding strategies by exploring these findings in rapeseed. These insights will extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process, ultimately contributing to more sustainable agriculture throughout the world.

Trehalose-6-phosphate synthase 8 increases photosynthesis and seed yield in Brassica napus.

Trehalose-6-phosphate (T6P) functions as a vital proxy for assessing carbohydrate status in plants. While class II T6P synthases (TPS) do not exhibit TPS activity, they are believed to play pivotal regulatory roles in trehalose metabolism. However, their precise functions in carbon metabolism and crop yield have remained largely unknown. Here, BnaC02.TPS8, a class II TPS gene, is shown to be specifically expressed in mature leaves and the developing pod walls of Brassica napus. Overexpression of BnaC02.TPS8 increased photosynthesis and the accumulation of sugars, starch, and biomass compared to wild type. Metabolomic analysis of BnaC02.TPS8 overexpressing lines and CRISPR/Cas9 mutants indicated that BnaC02.TPS8 enhanced the partitioning of photoassimilate into starch and sucrose, as opposed to glycolytic intermediates and organic acids, which might be associated with TPS activity. Furthermore, the overexpression of BnaC02.TPS8 not only increased seed yield but also enhanced seed oil accumulation and improved the oil fatty acid composition in B. napus under both high nitrogen (N) and low N conditions in the field. These results highlight the role of class II TPS in impacting photosynthesis and seed yield of B. napus, and BnaC02.TPS8 emerges as a promising target for improving B. napus seed yield.

RING-type E3 ligase BnaJUL1 ubiquitinates and degrades BnaTBCC1 to regulate drought tolerance in Brassica napus L.

Drought stress poses a persistent threat to field crops and significantly limits global agricultural productivity. Plants employ ubiquitin-dependent degradation as a crucial post-translational regulatory mechanism to swiftly adapt to changing environmental conditions. JUL1 is a RING-type E3 ligase related to drought stress in Arabidopsis. In this study, we explored the function of BnaJUL1 (a homologous gene of JUL1 in Brassica napus) and discovered a novel gene BnaTBCC1 participating in drought tolerance. First, we utilised BnaJUL1-cri materials through the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 system. Second, we confirmed that BnaJUL1 regulated drought tolerance through the drought tolerance assay and transcriptome analysis. Then, we identified a series of proteins interacting with BnaJUL1 through yeast library screening, including BnaTBCC1 (a tubulin binding cofactor C domain-containing protein); whose homologous gene TBCC1 knockdown mutants (tbcc1-1) exhibited ABA-sensitive germination in Arabidopsis, we then confirmed the involvement of BnaTBCC1 in drought tolerance in both Arabidopsis and Brassica. Finally, we established that BnaJUL1 could ubiquitinate and degrade BnaTBCC1 to regulate drought tolerance. Consequently, our study unveils BnaJUL1 as a novel regulator that ubiquitinates and degrades BnaTBCC1 to modulate drought tolerance and provided desirable germplasm for further breeding of drought tolerance in rapeseed.

Mutating BnEOD1s via CRISPR-Cas9 increases the seed size and weight in Brassica napus.

Seed weight, which is highly correlated to seed size, is a critical agronomic trait that determines the yield of Brassica napus. However, there have been limited researches on the genes involved in regulating seed size. In Arabidopsis thaliana, ENHANCER OF DA1 (EOD1), an E3 ubiquitin ligase gene, has been identified as a significant negative regulator in controlling organ size, but the function of its homologs in rapeseed remains unknown. Only two homologous of EOD1, BnaEOD1.A04 and BnaEOD1.C04, have been found in B. napus and were mutated using the CRISPR-Cas9 system. Three T-DNA-free lines, T2-157-1-C8, T2-390-2-B8, and T2-397-2-E2, were identified from the homozygous T2 mutant lines. The BnaEOD1.A04 showed a similar type of editing in these mutants, whereas the BnaEOD1.C04 in T2-397-2-E2 was only missing 26 amino acids, and the translation was not prematurely terminated, which was different from the other two mutants. In parallel, mutation of BnaEOD1s resulted in a noteworthy increase in both seed size and seed weight in the three editing lines. Additionally, there was a significant decline in the number of seeds per silique (SPS) and silique length (SL) in T2-157-1-C8 and T2-390-2-B8, but T2-397-2-E2 did not show any significant changes in the SPS and SL, possibly due to distinct types of editing in the three lines. The above results indicate the conserved function of EOD1 homologs and provides promising germplasm for breeding novel high-yield rapeseed varieties by improving seed size and thousand-seed weight. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01430-z.

Linkage and association mapping of ovule number per ovary (ON) in oilseed rape (Brassica napus L.).

Ovule number (ON) produced during flower development determines the maximum number of seeds per silique and thereby affects crop productivity; however, the genetic basis of ON remains poorly understood in oilseed rape (Brassica napus). In this study, we genetically dissected the ON variations in a double haploid (DH) population and in natural population (NP) by linkage mapping and genome-wide association analysis. Phenotypic analysis showed that ON displayed normal distribution in both populations with the broad-sense heritability of 0.861 (DH population) and 0.930 (natural population). Linkage mapping identified 5 QTLs related to ON, including qON-A03, qON-A07, qON-A07-2, qON-A10, and qON-C06. Genome-wide association studies (GWAS) revealed 214, 48, and 40 significant single-nucleotide polymorphisms (SNPs) by individually using the single-locus model GLM and the multiple-locus model MrMLM and FASTMrMLM. The phenotypic variation explained (PVE) by these QTLs and SNPs ranged from 2.00-17.40% to 5.03-7.33%, respectively. Integration of the results from both strategies identified four consensus genomic regions associated with ON from the chromosomes A03, A07, and A10. Our results preliminarily resolved the genetic basis of ON and provides useful molecular markers for plant yield improvement in B. napus. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01355-7.

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.

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.

A simple and efficient method to quantify the cell parameters of the seed coat, embryo and silique wall in rapeseed.

BACKGROUND: Researchers interested in the seed size of rapeseed need to quantify the cell size and number of cells in the seed coat, embryo and silique wall. Scanning electron microscope-based methods have been demonstrated to be feasible but laborious and costly. After image preparation, the cell parameters are generally evaluated manually, which is time consuming and a major bottleneck for large-scale analysis. Recently, two machine learning-based algorithms, Trainable Weka Segmentation (TWS) and Cellpose, were released to overcome this long-standing problem. Moreover, the MorphoLibJ and LabelsToROIs plugins in Fiji provide user-friendly tools to deal with cell segmentation files. We attempted to verify the practicability and efficiency of these advanced tools for various types of cells in rapeseed. RESULTS: We simplified the current image preparation procedure by skipping the fixation step and demonstrated the feasibility of the simplified procedure. We developed three methods to automatically process multicellular images of various tissues in rapeseed. The TWS-Fiji (TF) method combines cell detection with TWS and cell measurement with Fiji, enabling the accurate quantification of seed coat cells. The Cellpose-Fiji (CF) method, based on cell segmentation with Cellpose and quantification with Fiji, achieves good performance but exhibits systematic error. By removing border labels with MorphoLibJ and detecting regions of interest (ROIs) with LabelsToROIs, the Cellpose-MorphoLibJ-LabelsToROIs (CML) method achieves human-level performance on bright-field images of seed coat cells. Intriguingly, the CML method needs very little manual calibration, a property that makes it suitable for massive-scale image processing. Through a large-scale quantitative evaluation of seed coat cells, we demonstrated the robustness and high efficiency of the CML method at both the single-cell level and the sample level. Furthermore, we extended the application of the CML method to developing seed coat, embryo and silique wall cells and acquired highly precise and reliable results, indicating the versatility of this method for use in multiple scenarios. CONCLUSIONS: The CML method is highly accurate and free of the need for manual correction. Hence, it can be applied for the low-cost, high-throughput quantification of diverse cell types in rapeseed with high efficiency. We envision that this method will facilitate the functional genomics and microphenomics studies of rapeseed and other crops.

BnaC7.ROT3, the causal gene of cqSL-C7, mediates silique length by affecting cell elongation in Brassica napus.

Siliques are a major carbohydrate source of energy for later seed development in rapeseed (Brassica napus). Thus, silique length has received great attention from breeders. We previously detected a novel quantitative trait locus cqSL-C7 that controls silique length in B. napus. Here, we further validated the cqSL-C7 locus and isolated its causal gene (BnaC7.ROT3) by map-based cloning. In 'Zhongshuang11' (parent line with long siliques), BnaC7.ROT3 encodes the potential cytochrome P450 monooxygenase CYP90C1, whereas in 'G120' (parent line with short siliques), a single nucleotide deletion in the fifth exon of BnaC7.ROT3 results in a loss-of-function truncated protein. Sub-cellular localization and expression pattern analysis revealed that BnaC7.ROT3 is a membrane-localized protein mainly expressed in leaves, flowers and siliques. Cytological observations showed that the cells in silique walls of BnaC7.ROT3-transformed positive plants were longer than those of transgene-negative plants in the background of 'G120', suggesting that BnaC7.ROT3 affects cell elongation. Haplotype analysis demonstrated that most alleles of BnaC7.ROT3 are favorable in B. napus germplasms, and its homologs may also be involved in silique length regulation. Our findings provide novel insights into the regulatory mechanisms of natural silique length variations and valuable genetic resources for the improvement of silique length in rapeseed.

Genome- and transcriptome-wide association studies reveal the genetic basis and the breeding history of seed glucosinolate content in Brassica napus.

A high content of seed glucosinolates and their degradation products imposes anti-nutritional effects on livestock; therefore, persistent efforts are made to reduce the seed GSL content to increase the commercial value of rapeseed meal. Here, we dissected the genetic structure of SGC by genome-wide association studies (GWAS) combined with transcriptome-wide association studies (TWAS). Fifteen reliable quantitative trait loci (QTLs) were identified to be associated with the reduced SGC in modern B. napus cultivars by GWAS. Analysis of the selection strength and haplotypes at these QTLs revealed that low SGC was predominantly generated by the co-selection of qGSL.A02.2, qGSL.C02.1, qGSL.A09.2, and qGSL.C09.1. Integration of the results from TWAS, comprehensive bioinformatics, and POCKET algorithm analyses indicated that BnaC02.GTR2 (BnaC02g42260D) is a candidate gene underlying qGSL.C02.1. Using CRISPR/Cas9-derived Bna.gtr2s knockout mutants, we experimentally verified that both BnaC02.GTR2 and its three paralogs positively regulate seed GSL accumulation but negatively regulated vegetative tissue GSL contents. In addition, we observed smaller seeds with higher seed oil content in these Bna.gtr2 mutants. Furthermore, both RNA-seq and correlation analyses suggested that Bna.GTR2s might play a comprehensive role in seed development, such as amino acid accumulation, GSL synthesis, sugar assimilation, and oil accumulation. This study unravels the breeding selection history of low-SGC improvement and provides new insights into the molecular function of Bna.GTR2s in both seed GSL accumulation and seed development in B. napus.

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.

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.

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.

Heterosis Derived From Nonadditive Effects of the BnFLC Homologs Coordinates Early Flowering and High Yield in Rapeseed (Brassica napus L.).

Early flowering facilitates crops to adapt multiple cropping systems or growing regions with a short frost-free season; however, it usually brings an obvious yield loss. In this study, we identified that the three genes, namely, BnFLC.A2, BnFLC.C2, and BnFLC.A3b, are the major determinants for the flowering time (FT) variation of two elite rapeseed (Brassica napus L.) accessions, i.e., 616A and R11. The early-flowering alleles (i.e., Bnflc.a2 and Bnflc.c2) and late-flowering allele (i.e., BnFLC.A3b) from R11 were introgressed into the recipient parent 616A through a breeding strategy of marker-assisted backcross, giving rise to eight homozygous near-isogenic lines (NILs) associated with these three loci and 19 NIL hybrids produced by the mutual crossing of these NILs. Phenotypic investigations showed that NILs displayed significant variations in both FT and plant yield (PY). Notably, genetic analysis indicated that BnFLC.A2, BnFLC.C2, and BnFLC.A3b have additive effects of 1.446, 1.365, and 1.361 g on PY, respectively, while their dominant effects reached 3.504, 2.991, and 3.284 g, respectively, indicating that the yield loss caused by early flowering can be successfully compensated by exploring the heterosis of FT genes in the hybrid NILs. Moreover, we further validated that the heterosis of FT genes in PY was also effective in non-NIL hybrids. The results demonstrate that the exploration of the potential heterosis underlying the FT genes can coordinate early flowering (maturation) and high yield in rapeseed (B. napus L.), providing an effective strategy for early flowering breeding in crops.

Structural analysis of the meiosis-related protein MS5 reveals non-canonical papain enhancement by cystatin-like folds.

MS5 is a meiosis-related protein belonging to the Brassicaceae-specific domain of unknown function family and characterized by the MS5 superfamily domain (MSD). In this study, we elucidated the three-dimensional crystal structure and potential biochemical function of the MSD. It was observed that the MSD adopts a cystatin-like fold, mainly consisting of a central α-helix and four- or five-stranded antiparallel ß-sheets that wrap around it. However, unlike cystatins, which inhibit cysteine proteases, the MSD displayed allosteric activation of papain. We believe that our study provides insight into novel mechanisms of proteolytic enzyme regulation and may serve as a basis for functional studies of the MS5 family proteins in plants.

Molecular mechanisms underpinning the multiallelic inheritance of MS5 in Brassica napus.

The Brassica-specific gene MS5 mediates early meiotic progression, and its allelic variants contribute to a valuable genic male sterility three-line hybrid production system in rapeseed (Brassica napus L.). However, the underlying mechanisms of its triallelic inheritance are poorly understood. Herein, we show that the restorer allele MS5a and the maintainer allele MS5c are both necessary for male fertility in B. napus. The functional divergence of MS5a and MS5c is strongly related to sequence variations in their coding regions and less strongly to their promoter regions. The male-sterile allele MS5b encodes a chimeric protein containing only the complete MS5 coiled-coil (CC) domain, having lost the MS5 superfamily domain. Both MS5a and MS5c can form homodimers in the nucleus via the CC domain. MS5b can interact competitively with MS5a or MS5c to form non-functional heterodimers. Owing to the close transcript levels of MS5b and MS5c in MS5b MS5c , these heterodimers induced a dominant-negative effect of MS5b on MS5c , resulting in a male-sterile phenotype. The extremely high transcript abundance of MS5a maintains sufficient MS5a homodimers in MS5a MS5b , causing the recovery of male sterility. These findings provide substantial genetic and molecular evidence to improve our understanding of the mechanisms underlying the multiallelic inheritance of MS5, and enable the construction of a solid foundation for improved use of the MS5-controlled GMS system in Brassica species.