Developmental pleiotropy of SDP1 from seedling to mature stages in B. napus.

Rapeseed, an important oil crop, relies on robust seedling emergence for optimal yields. Seedling emergence in the field is vulnerable to various factors, among which inadequate self-supply of energy is crucial to limiting seedling growth in early stage. SUGAR-DEPENDENT1 (SDP1) initiates triacylglycerol (TAG) degradation, yet its detailed function has not been determined in B. napus. Here, we focused on the effects of plant growth during whole growth stages and energy mobilization during seedling establishment by mutation in BnSDP1. Protein sequence alignment and haplotypic analysis revealed the conservation of SDP1 among species, with a favorable haplotype enhancing oil content. Investigation of agronomic traits indicated bnsdp1 had a minor impact on vegetative growth and no obvious developmental defects when compared with wild type (WT) across growth stages. The seed oil content was improved by 2.0-2.37% in bnsdp1 lines, with slight reductions in silique length and seed number per silique. Furthermore, bnsdp1 resulted in lower seedling emergence, characterized by a shrunken hypocotyl and poor photosynthetic capacity in the early stages. Additionally, impaired seedling growth, especially in yellow seedlings, was not fully rescued in medium supplemented with exogenous sucrose. The limited lipid turnover in bnsdp1 was accompanied by induced amino acid degradation and PPDK-dependent gluconeogenesis pathway. Analysis of the metabolites in cotyledons revealed active amino acid metabolism and suppressed lipid degradation, consistent with the RNA-seq results. Finally, we proposed strategies for applying BnSDP1 in molecular breeding. Our study provides theoretical guidance for understanding trade-off between oil accumulation and seedling energy mobilization in B. napus.

Kinase CIPK9 integrates glucose and abscisic acid signaling to regulate seed oil metabolism in rapeseed.

Rapeseed (Brassica napus), an important oil crop worldwide, provides large amounts of lipids for human requirements. Calcineurin B-like (CBL)-interacting protein kinase 9 (CIPK9) was reported to regulate seed oil content in the plant. Here, we generated gene-silenced lines through RNA interference biotechnology and loss-of-function mutant bnacipk9 using CRISPR/Cas9 to further study BnaCIPK9 functions in the seed oil metabolism of rapeseeds. We discovered that compared with wild-type (WT) lines, gene-silenced and bnacipk9 lines had substantially different oil contents and fatty acid compositions: seed oil content was improved by 3%-5% and 1%-6% in bnacipk9 lines and gene-silenced lines, respectively; both lines were with increased levels of monounsaturated fatty acids and decreased levels of polyunsaturated fatty acids. Additionally, hormone and glucose content analyses revealed that compared with WT lines the bnacipk9 lines showed significant differences: in bnacipk9 seeds, indoleacetic acid and abscisic acid (ABA) levels were higher; glucose and sucrose contents were higher with a higher hexose-to-sucrose ratio in bnacipk9 mid-to-late maturation development seeds. Furthermore, the bnacipk9 was less sensitive to glucose and ABA than the WT according to stomatal aperture regulation assays and the expression levels of genes involved in glucose and ABA regulating pathways in rapeseeds. Notably, in Arabidopsis (Arabidopsis thaliana), exogenous ABA and glucose imposed on developing seeds revealed the effects of ABA and glucose signaling on seed oil accumulation. Altogether, our results strongly suggest a role of CIPK9 in mediating the interaction between glucose flux and ABA hormone signaling to regulate seed oil metabolism in rapeseed.

A gain-of-function mutation in BnaIAA13 disrupts vascular tissue and lateral root development in Brassica napus.

Rapeseed (Brassica napus) is an important oilseed crop worldwide. Plant vascular tissues are responsible for material transport and provide mechanical support. The lateral roots (LRs) absorb sufficient water and nutrients. The genetic basis of vascular tissues and LRs development in rapeseed remains unknown. This study characterized an EMS-mutagenized rapeseed mutant, T16, which showed dwarf stature, reduced LRs, and leaf wilting. Scanning electron microscopy observations showed that the internode-cell shortened. Observations of the tissue sections revealed defects in the development of vascular bundles in the stems and petioles. Genetic analysis revealed that the phenotypes of T16 were controlled by a single semi-dominant nuclear gene. Map-based cloning and genetic complementarity confirmed that BnaA03.IAA13 is the functional gene, a G-to-A mutation in second exon changed the glycine at the 79th position to glutamic acid, disrupting the conserved degron motif VGWPP. Transcriptome analysis in roots and stems showed that auxin and cytokinin signaling pathways were disordered in T16. Evolutionary analysis showed that AUXIN/INDOLE-3-ACETIC ACID was conserved during plant evolution. The heterozygote of T16 significantly reduced the plant height while maintaining other agronomic traits. Our findings provide novel insights into the regulatory mechanisms of vascular tissues and LRs development, and provide a new germplasm resource for rapeseed breeding.

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.

BnaCRCs with domestication preference positively correlate with the seed-setting rate of canola.

Canola (Brassica napus) is an important oil crop worldwide. The seed-setting rate (SS) is a critical factor in determining its yield, and the development of pistils affects pollination and seed sets. However, research on seed-setting defects has been limited owing to difficulties in the identification of phenotypes, mutations, and complex genetic mechanisms. In this study, we found a stigma defect (sd) mutant in B. napus, which had no nectary. The SS of sd mutants in the field was approximately 93.4% lower than that of the wild type. Scanning and transmission electron microscopy imaging of sd mutants showed a low density of stigma papillary cells and stigma papillary cell vacuoles that disappeared 16 h after flowering. Genetic analysis of segregated populations showed that two recessive nuclear genes are responsible for the mutant phenotype of sd. Based on re-sequencing and map-based cloning, we reduced the candidate sites on ChrA07 (BnaSSA07) and ChrC06 (BnaSSC06) to 30 and 67 kb, including six and eight predicted genes, respectively. Gene analyses showed that a pair of CRABS CLAW (CRC) homeologous genes at BnaSSA07 and BnaSSC06 were associated with the development of carpel and nectary. BnaSSA07.CRC and BnaSSC06.CRC candidate genes were found to be expressed in flower organs only, with significant differences in their expression in the pistils of the near-isogenic lines. DNA sequencing showed transposon insertions in the upstream region and intron of the candidate gene BnaSSA07.crc. We also found that BnaSSC06.crc exists widely in the natural population and we give possible reasons for its widespread existence.

A systematic dissection in oilseed rape provides insights into the genetic architecture and molecular mechanism of yield heterosis.

Heterosis refers to the better performance of cross progeny compared with inbred parents, and its utilization contributes greatly to agricultural production. Several hypotheses have been proposed to explain heterosis mainly including dominance, over-dominance (or pseudo-overdominance) and epistasis. However, systematic dissection and verification of these hypotheses are rarely documented. Here, comparison of heterosis level across different traits showed that the strong heterosis of composite traits (such as yield) could be attributed to the multiplicative effects of moderate heterosis of component traits, whether at the genome or locus level. Yield heterosis was regulated by a complex trait-QTL network that was characterized by obvious centre-periphery structure, hub QTL, complex up/downstream and positive/negative feedback relationships. More importantly, we showed that better-parent heterosis on yield could be produced in a cross of two near-isogenic lines by the pyramiding and complementation of two major heterotic QTL showing partial-dominance on yield components. The causal gene (BnaA9.CYP78A9) of QC14 was identified, and its heterotic effect results from the heterozygous status of a CACTA-like transposable element in its upstream regulatory region, which led to partial dominance at expression and auxin levels, thus resulting in non-additive expression of downstream responsive genes involved in cell cycle and proliferation, eventually leading to the heterosis of cell number. Taken together, the results at the phenotypic, genetic and molecular levels were highly consistent, which demonstrated that the pyramiding effect of heterotic QTL and the multiplicative effect of individual component traits could well explain substantial parts of yield heterosis in oilseed rape. These results provide in-depth insights into the genetic architecture and molecular mechanism of yield heterosis.

Xanthophyll esterases in association with fibrillins control the stable storage of carotenoids in yellow flowers of rapeseed (Brassica juncea).

Biosynthesis, stabilization, and storage of carotenoids are vital processes in plants that collectively contribute to the vibrant colors observed in flowers and fruits. Despite its importance, the carotenoid storage pathway remains poorly understood and lacks thorough characterization. We identified two homologous genes, BjA02.PC1 and BjB04.PC2, belonging to the esterase/lipase/thioesterase (ELT) family of acyltransferases. We showed that BjPCs in association with fibrillin gene BjFBN1b control the stable storage of carotenoids in yellow flowers of Brassica juncea. Through genetic, high-resolution mass spectrometry and transmission electron microscopy analyses, we demonstrated that both BjA02.PC1 and BjB04.PC2 can promote the accumulation of esterified xanthophylls, facilitating the formation of carotenoid-enriched plastoglobules (PGs) and ultimately producing yellow pigments in flowers. The elimination of BjPCs led to the redirection of metabolic flux from xanthophyll ester biosynthesis to lipid biosynthesis, resulting in white flowers for B. juncea. Moreover, we genetically verified the function of two fibrillin genes, BjA01.FBN1b and BjB05.FBN1b, in mediating PG formation and demonstrated that xanthophyll esters must be deposited in PGs for stable storage. These findings identified a previously unknown carotenoid storage pathway that is regulated by BjPCs and BjFBN1b, while offering unique opportunities for improving the stability, deposition, and bioavailability of carotenoids.

Transcription factor WRKY28 curbs WRKY33-mediated resistance to Sclerotinia sclerotiorum in Brassica napus.

Sclerotinia sclerotiorum causes substantial damage and loss of yield in oilseed rape (Brassica napus). The molecular mechanisms of oilseed rape defense against Sclerotinia remain elusive. In this study, we found that in the early stages of B. napus infection a conserved mitogen-activated protein kinase (MAPK) cascade mediated by BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 module phosphorylates the substrate BnWRKY33, enhancing its transcriptional activity. The activated BnWRKY33 binds to its own promoter and triggers a transcriptional burst of BnWRKY33, thus helping plants effectively resist the pathogenic fungi by enhancing the expression of phytoalexin synthesis-related genes. The expression of BnWRKY33 is fine-tuned during defense. Ongoing Sclerotinia infection induces BnaA03.WRKY28 and BnaA09.VQ12 expression. BnaA09.VQ12 interacts physically with BnaA03.WRKY28 to form a protein complex, causing BnaA03.WRKY28 to outcompete BnWRKY33 and bind to the BnWRKY33 promoter. BnaA03.WRKY28 induction suppresses BnWRKY33 expression in the later stages of infection but promotes branch formation in the leaf axils by regulating the expression of branching-related genes such as BnBRC1. BnaA03.WRKY28 participates in the trade-off between defense and growth. These findings suggest that oilseed rape plants may modulate defense-response strength and develop alternative reproduction and survival strategies in the face of lethal pathogens.

The transcription factor BnaWRKY10 regulates cytokinin dehydrogenase BnaCKX2 to control cytokinin distribution and seed size in Brassica napus.

Cytokinins (CKs) are phytohormones that promote cell division and differentiation. However, the regulation of CK distribution and homeostasis in Brassica napus is poorly understood. Here, the endogenous CKs were first quantified by LC-ESI-MS/MS in rapeseed tissues and visualized by TCSn::GUS reporter lines. Interestingly, the cytokinin oxidase/dehydrogenase BnaCKX2 homologs were mainly expressed in reproductive organs. Subsequently, the quadruple mutants of the four BnaCKX2 homologs were generated. Endogenous CKs were increased in the seeds of the BnaCKX2 quadruple mutants, resulting in a significantly reduced seed size. In contrast, overexpression of BnaA9.CKX2 resulted in larger seeds, probably by delaying endosperm cellularization. Furthermore, the transcription factor BnaC6.WRKY10b, but not BnaC6.WRKY10a, positively regulated BnaA9.CKX2 expression by binding directly to its promoter region. Overexpression of BnaC6.WRKY10b rather than BnaC6.WRKY10a resulted in lower concentration of CKs and larger seeds by activating BnaA9.CKX2 expression, indicating that the functional differentiation of BnaWRKY10 homologs might have occurred during B. napus evolution or domestication. Notably, the haploid types of BnaA9.CKX2 were associated with 1000-seed weight in the natural B. napus population. Overall, the study reveals the distribution of CKs in B. napus tissues, and shows that BnaWRKY10-mediated BnaCKX2 expression is essential for seed size regulation, providing promising targets for oil crop improvement.

Fine mapping of BnDM1-the gene regulating indeterminate inflorescence in Brassica napus.

KEY MESSAGE: A candidate gene Bndm1 related to determinate inflorescence was mapped to a 128-kb interval on C02 in Brassica napus. Brassica napus plants with determinate inflorescence exhibit improved traits in field production, such as lower plant height, improved lodging resistance, and consistent maturity. Compared to plants with indeterminate inflorescence, such features are favorable for mechanized harvesting techniques. Here, using a natural mutant 6138 with determinate inflorescence, it is demonstrated that determinate inflorescence reduces plant height significantly without affecting thousand-grain weight and yield per plant. Determinacy was regulated by a single recessive gene, Bndm1. Using a combination of SNP arrays and map-based cloning, we mapped the locus of determinacy to a 128-kb region on C02. Based on sequence comparisons and the reported functions of candidate genes in this region, we predicted BnaC02.knu (a homolog of KNU in Arabidopsis) as a possible candidate gene of Bndm1 for controlling determinate inflorescence. We found a 623-bp deletion in a region upstream of the KNU promoter in the mutant. This deletion led to the significant overexpression of BnaC02.knu in the mutant compared to that in the ZS11 line. The correlation between this deletion and determinate inflorescence was examined in natural populations. The results indicated that the deletion affected the normal transcription of BnaC02.knu in the plants with determinate inflorescence and played an important role in maintaining flower development. This study presents as a new material for optimizing plant architecture and breeding novel canola varieties suitable for mechanized production. Moreover, our findings provide a theoretical basis for analyzing the molecular mechanisms underlying the formation of determinate inflorescence in B. napus.

The dynamics of lncRNAs transcription in interspecific F1 allotriploid hybrids between Brassica species.

Interspecific hybridization is the intrinsic forces behind genome evolution. Long non-coding RNAs (lncRNAs) are important for plant biological processes regulation. However, it is unclear that these non-coding fractions are impacted by interspecific hybridization. Here we examined the profiles of lncRNAs by comparing them with coding genes in Brassica napus, three accessions of Brassica rapa, and their F1 hybrids. 6206 high-confidential lncRNAs were identified in F 1 hybrids and their parentals, and the lncRNAs transcriptome in the F1 hybrids was reprogrammed by the genome shock. Notably, genome-wide unbalanced of lncRNAs were observed between An and Ar subgenomes, ELD (Expression Level Dominance) was biased toward the An -genome in F1 hybrids, and ELD of non-conserved lncRNAs was more than conserved lncRNAs. Our findings demonstrate that the reprogramed lncRNAs acts as important role in enhancing plant plasticity, leading to the acquisition of desirable traits in polyploid Brassica species.

Comparative transcriptomic analysis reveals the molecular mechanism underlying seedling biomass heterosis in Brassica napus.

BACKGROUND: Heterosis is an important biological phenomenon in which the hybrids exceed the parents in many traits. However, the molecular mechanism underlying seedling heterosis remains unclear. RESULTS: In the present study, we analyzed the leaf transcriptomes of strong hybrids (AM, HM) and weak hybrids (CM, HW) and their parents (A, C, H, M, and W) at two periods. Phenotypically, hybrids had obvious biomass heterosis at the seedling stage, with statistically significant differences between the strong and weak hybrids. The transcriptomic analysis demonstrated that the number of differentially expressed genes (DEGs) between parents was the highest. Further analysis showed that most DEGs were biased toward parental expression. The biological processes of the two periods were significantly enriched in the plant hormone signal transduction and photosynthetic pathways. In the plant hormone signaling pathway, DEG expression was high in hybrids, with expression differences between strong and weak hybrids. In addition, DEGs related to cell size were identified. Similar changes were observed during photosynthesis. The enhanced leaf area of hybrids generated an increase in photosynthetic products, which was consistent with the phenotype of the biomass. Weighted gene co-expression network analysis of different hybrids and parents revealed that hub genes in vigorous hybrid were mainly enriched in the plant hormone signal transduction and regulation of plant hormones. CONCLUSION: Plant hormone signaling and photosynthesis pathways, as well as differential expression of plant cell size-related genes, jointly regulate the dynamic changes between strong and weak hybrids and the generation of seedling-stage heterosis. This study may elucidate the molecular mechanism underlying early biomass heterosis and help enhance canola yield.

Transcriptome shock in interspecific F1 allotriploid hybrids between Brassica species.

Interspecific hybridization drives the evolution of angiosperms and can be used to introduce novel alleles for important traits or to activate heterosis in crop breeding. Hybridization brings together gene expression networks from two different species, potentially causing global alterations of gene expression in the F1 plants which is called 'transcriptome shock'. Here, we explored such a transcriptome shock in allotriploid Brassica hybrids. We generated interspecific F1 allotriploid hybrids between the allotetraploid species Brassica napus and three accessions of the diploid species Brassica rapa. RNA-seq of the F1 hybrids and the parental plants revealed that 26.34-30.89% of genes were differentially expressed between the parents. We also analyzed expression level dominance and homoeolog expression bias between the parents and the F1 hybrids. The expression-level dominance biases of the Ar, An, and Cn subgenomes was genotype and stage dependent, whereas significant homoeolog expression bias was observed among three subgenomes from different parents. Furthermore, more genes were involved in trans regulation than in cis regulation in allotriploid F1 hybrids. Our findings provide new insights into the transcriptomic responses of cross-species hybrids and hybrids showing heterosis, as well as a new method for promoting the breeding of desirable traits in polyploid Brassica species.

Genetic and multi-omics analyses reveal BnaA07.PAP2In-184-317 as the key gene conferring anthocyanin-based color in Brassica napus flowers.

The molecular mechanisms underlying anthocyanin-based flower coloration remain unknown in Brassica napus. To identify the key genes and metabolites associated with apricot and pink flower colors, metabolome, BSA-seq, and RNA-seq analyses were conducted on apricot-, pink-, yellow-, and white-flowered F2B. napus. Yellow carotenoids and red anthocyanins were abundant in apricot petals, while colorless carotenoids and red anthocyanins accumulated in pink petals. Most carotenoid genes were not differentially regulated between apricot and yellow or between pink and white petals. Three regulator genes, BnaMYBL2, BnaA07.PAP2, and BnaTT8, and structural genes in anthocyanin biosynthesis were dramatically enhanced in apricot and pink petals in comparison with yellow and white petals. Map-based cloning revealed that BnaA07.PAP2 is responsible for anthocyanin-based flower color and encodes a nucleus-localized protein predominantly expressed in apricot and pink flowers. Two insertions in the promoter region are responsible for the transcriptional activation of BnaA07.PAP2 in flowers. Introducing the BnaA07.PAP2In-184-317 allele broadly activated the expression of anthocyanin-related genes and promoted anthocyanin accumulation in flowers, yielding color change from yellow to apricot. These findings illustrate the genetic basis of anthocyanin-based flower coloration and provide a valuable genetic resource for breeding varieties with novel flower colors in B. napus.

Identification and Fine Mapping of the Candidate Gene Controlling Multi-Inflorescence in Brassica napus.

As a desirable agricultural trait, multi-inflorescence (MI) fulfills the requirement of mechanized harvesting and yield increase in rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait remain poorly understood. We previously identified a difference of one pair of dominant genes between the two mapping parental materials. In this study, phenotype and expression analysis indicated that the imbalance of the CLAVATA (CLV)-WUSCHEL (WUS) feedback loop may contribute to the abnormal development of the shoot apical meristem (SAM). BnaMI was fine-mapped to a 55 kb genomic region combining with genotype and phenotype of 5768 BCF1 individuals using a traditional mapping approach. Through comparative and expression analyses, combined with the annotation in Arabidopsis, five genes in this interval were identified as candidate genes. The present findings may provide assistance in functional analysis of the mechanism associated with multi-inflorescence and yield increase in rapeseed.

CRISPR/Cas9-Mediated Targeted Mutagenesis of BnaCOL9 Advances the Flowering Time of Brassica napus L.

Rapeseed (Brassica napus L.) is one of the most important oil crops in the world. The planting area and output of rapeseed are affected by the flowering time, which is a critical agronomic feature. COL9 controls growth and development in many different plant species as a member of the zinc finger transcription factor family. However, BnaCOL9 in rapeseed has not been documented. The aim of this study was to apply CRISPR/Cas9 technology to create an early-flowering germplasm resource to provide useful material for improving the early-maturing breeding of rapeseed. We identified four COL9 homologs in rapeseed that were distributed on chromosomes A05, C05, A03, and C03. We successfully created quadruple BnaCOL9 mutations in rapeseed using the CRISPR/Cas9 platform. The quadruple mutants of BnaCOL9 flowered earlier than the wild-type. On the other hand, the flowering time of the BnaCOL9 overexpression lines was delayed. An analysis of the expression patterns revealed that these genes were substantially expressed in the leaves and flowers. A subcellular localization experiment demonstrated that BnaCOL9 was in the nucleus. Furthermore, we discovered that two key flowering-related genes, BnaCO and BnaFT, were highly elevated in the BnaCOL9 mutants, but dramatically downregulated in the BnaCOL9 overexpression lines. Our findings demonstrate that BnaCOL9 is a significant flowering inhibitor in rapeseed and may be employed as a crucial gene for early-maturing breeding.

Combined BSA-Seq Based Mapping and RNA-Seq Profiling Reveal Candidate Genes Associated with Plant Architecture in Brassica napus.

Plant architecture involves important agronomic traits affecting crop yield, resistance to lodging, and fitness for mechanical harvesting in Brassica napus. Breeding high-yield varieties with plant architecture suitable for mechanical harvesting is the main goal of rapeseed breeders. Here, we report an accession of B. napus (4942C-5), which has a dwarf and compact plant architecture in contrast to cultivated varieties. A BC8 population was constructed by crossing a normal plant architecture line, 8008, with the recurrent parent 4942C-5. To investigate the molecular mechanisms underlying plant architecture, we performed phytohormone profiling, bulk segregant analysis sequencing (BSA-Seq), and RNA sequencing (RNA-Seq) in BC8 plants with contrasting plant architecture. Genetic analysis indicated the plant architecture traits of 4942C-5 were recessive traits controlled by multiple genes. The content of auxin (IAA), gibberellin (GA), and abscisic acid (ABA) differed significantly between plants with contrasting plant architecture in the BC8 population. Based on BSA-Seq analysis, we identified five candidate intervals on chromosome A01, namely those of 0 to 6.33 Mb, 6.45 to 6.48 Mb, 6.51 to 6.53 Mb, 6.77 to 6.79 Mb, and 7 to 7.01 Mb regions. The RNA-Seq analysis revealed a total of 4378 differentially expressed genes (DEGs), of which 2801 were up-regulated and 1577 were down-regulated. There, further analysis showed that genes involved in plant hormone biosynthesis and signal transduction, cell structure, and the phenylpropanoid pathway might play a pivotal role in the morphogenesis of plant architecture. Association analysis of BSA-Seq and RNA-Seq suggested that seven DEGs involved in plant hormone signal transduction and a WUSCHEL-related homeobox (WOX) gene (BnaA01g01910D) might be candidate genes responsible for the dwarf and compact phenotype in 4942C-5. These findings provide a foundation for elucidating the mechanisms underlying rapeseed plant architecture and should contribute to breed new varieties suitable for mechanization.

An in planta haploid induction system in Brassica napus.

Doubled haploid technology is widely used to accelerate plant breeding, but its use in the important oilseed crop Brassica napus L. is limited because B. napus haploids could only be obtained through in vitro anther or microspore cultures. Recently, maize (Zea mays) lines containing mutations in Domain of unknown function 679 membrane protein (DMP) were used as haploid inducer lines. This new haploid induction mechanism has been extended to several other plants, including the dicots Arabidopsis thaliana, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). Here, we knocked out four BnaDMP genes in the B. napus cultivar Westar using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 vector with an enhanced green fluorescent protein expression cassette. Plants with DMP mutations in B. napus in the T0 , T1 , and T2 generations exhibited a haploid induction rate up to 2.53%. These results suggest that targeting BnaDMP could be useful for haploid induction in B. napus.

Gene silencing of BnaA09.ZEP and BnaC09.ZEP confers orange color in Brassica napus flowers.

Brassica napus is currently cultivated as an important ornamental crop in China. Flower color has attracted much attention in rapeseed genetics and breeding. Here, we characterize an orange-flowered mutant of B. napus that exhibits an altered carotenoid profile in its petals. As revealed by map-based cloning, the change in color from yellow to orange is attributed to the loss of BnaC09.ZEP (zeaxanthin epoxidase) and a 1695-bp deletion in BnaA09.ZEP. HPLC analysis, genetic complementation and CRISPR/Cas9 experiments demonstrated that BnaA09.ZEP and BnaC09.ZEP have similar functions, and the abolishment of both genes led to a substantial increase in lutein content and a sharp decline in violaxanthin content in petals but not leaves. BnaA09.ZEP and BnaC09.ZEP are predominantly expressed in floral tissues, whereas their homologs, BnaA07.ZEP and BnaC07.ZEP, mainly function in leaves, indicating redundancy and tissue-specific diversification of BnaZEP function. Transcriptome analysis in petals revealed differences in the expression of carotenoid and flavonoid biosynthesis-related genes between the mutant and its complementary lines. Flavonoid profiles in the petals of complementary lines were greatly altered compared to the mutant, indicating potential cross-talk between the regulatory networks underlying the carotenoid and flavonoid pathways. Additionally, our results indicate that there is functional compensation by BnaA07.ZEP and BnaC07.ZEP in the absence of BnaA09.ZEP and BnaC09.ZEP. Cloning and characterization of BnaZEPs provide insights into the molecular mechanisms underlying flower pigmentation in B. napus and would facilitate breeding of B. napus varieties with higher ornamental value.

High-generation near-isogenic lines combined with multi-omics to study the mechanism of polima cytoplasmic male sterility.

BACKGROUND: Cytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima (pol) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the pol CMS restorer gene Rfp and the sterility-inducing gene orf224 in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration. RESULT: In this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene orf224 and the restorer gene Rfp of pol CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven Rfp interaction proteins, with orf224 protein covering five interaction proteins. CONCLUSIONS: We propose that Rfp and its interacting protein cleave the transcript of atp6/orf224, causing the infertility gene to lose its function and restore fertility. When Rfp is not cleaved, orf224 poisons the tapetum cells and anther development-related proteins, resulting in pol CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS's potential molecular mechanism and will help breed B. napus hybrids.