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Summary Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T0 plants were edited very efficiently (56.30%–67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T1 and T2 progeny. We also obtained transgene‐free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.

Background Seed storage lipids are valuable for human diet and for the sustainable development of mankind. In recent decades, many lipid metabolism genes and pathways have been identified, but the molecular mechanisms that underlie differences in seed oil biosynthesis in species with developed embryo and endosperm are not fully understood. Results We performed comparative genome and transcriptome analyses of castor bean and rapeseed, which have high seed oil contents, and maize, which has a low seed oil content. These results revealed the molecular underpinnings of the low seed oil content in maize. First of all, transcriptome analyses showed that more than 61% of the lipid- and carbohydrate-related genes were regulated in castor bean and rapeseed, but only 20.1% of the lipid-related genes and 22.5% of the carbohydrate-related genes were regulated in maize. Then, compared to castor bean and rapeseed, fewer lipid biosynthesis genes but more lipid metabolism genes were regulated in the maize embryo. More importantly, most maize genes encoding lipid-related transcription factors, triacylglycerol (TAG) biosynthetic enzymes, pentose phosphate pathway (PPP) and Calvin Cycle proteins were not regulated during seed oil synthesis, despite the presence of many homologs in the maize genome. Additionally, we observed differential regulation of vital oil biosynthetic enzymes and extremely high expression levels of oil biosynthetic genes in castor bean, which were consistent with the rapid accumulation of oil in castor bean developing seeds. Conclusions Compared to high-oil seeds (castor bean and rapeseed), less oil biosynthetic genes were regulated during the seed development in low-oil seed (maize). These results shed light on molecular mechanisms of lipid biosynthesis in maize, castor bean, and rapeseed. They can provide information on key target genes that may be useful for future experimental manipulation of oil production in oil plants.

Background Brassica napus provides approximately 13–16% of global vegetable oil for human consumption and biodiesel production. Plant height (PH) is a key trait that affects plant architecture, seed yield and harvest index. However, the genetic mechanism of PH in B. napus is poorly understood. Results A dwarf mutant df59 was isolated from a large-scale screening of an ethyl methanesulphonate-mutagenized rapeseed variety Ningyou 18. A genetic analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped on C9 chromosome by quantitative trait loci sequencing analysis and designated as BnaDwf.C9. To fine-map BnaDwf.C9, two F2 populations were constructed from crosses between conventional rapeseed cultivars (Zhongshuang 11 and Holly) and df59. BnaDwf.C9 was fine-mapped to the region between single-nucleotide polymorphism (SNP) markers M14 and M4, corresponding to a 120.87-kb interval of the B. napus ‘Darmor-bzh’ genome. Within this interval, seven, eight and nine annotated or predicted genes were identified in “Darmor-bzh”, “Ningyou 7” and “Zhongshuang 11” reference genomes, respectively. In addition, a comparative transcriptome analysis was performed using stem tips from Ningyou 18 and df59 at the stem elongation stage. In total, 3995 differentially expressed genes (DEGs) were identified. Among them, 118 DEGs were clustered in plant hormone-related signal transduction pathways, including 81 DEGs were enriched in auxin signal transduction. Combining the results of fine-mapping and transcriptome analyses, BnaC09g20450D was considered a candidate gene for BnaDwf.C9, which contains a SNP that co-segregated in 4746 individuals. Finally, a PCR-based marker was developed based on the SNP in BnaC09g20450D. Conclusions The combination of quantitative trait loci sequencing, fine-mapping and genome-wide transcriptomic analysis revealed one candidate gene located within the confidence interval of 120.87-kb region. This study provides a new genetic resource for semi-dwarf breeding and new insights into understanding the genetic architecture of PH in B. napus.

14-3-3s are general regulatory factors (GF14s or GRFs) involved in a variety of physiological regulations in plants, including the control of flowering time. However, there are poorly relevant reports in rapeseed so far. In this study, we identified a homologous 14-3-3 gene BnGF14-2c (AtGRF2_Like in Brassica napus) in rapeseed based on bioinformatic analysis by using the sequences of the flowering-related 14-3-3s in other plant species. Then, we found that overexpression of BnGF14-2c in the semi-winter rapeseed “93275” promoted flowering without vernalization. Moreover, both yeast two-hybrid and bimolecular fluorescence complementation analysis indicated that BnGF14-2c may interact with two vernalization-related flowering regulators BnFT.A02 and BnFLC.A10., respectively. qPCR analysis showed that the expression of BnFT (AtFT_Like) was increased and the expression of two selected vernalization-related genes were reduced in the overexpression transgenic plants. Further investigation on subcellular localization demonstrated that BnGF14-2c localized in the nucleus and cytoplasm. The results of RNA-seq analysis and GUS staining indicated that BnGF14-2c is ubiquitously expressed except for mature seed coat. In general, the interaction of 14-3-3 and FLC was firstly documented in this study, indicating BnGF14-2c may act as a positive regulator of flowering in rapeseed, which is worthy for more in-depth exploration.

Alternative oxidases (AOXs) are the terminal oxidase in the cyanide-resistant respiration pathway in plant mitochondria, which play an important role in abiotic stress and are proposed as a functional marker for high tolerant breeding. In this study, ten AOX genes (BnaAOXs) were identified, and CysI and CysII of AOX isoforms were highly conserved in rapeseed. Among them, Bna.AOX1b was mainly expressed in the ovule and displayed varying expression between rapeseed cultivars which showed different salt resistance in seed germination. We identified its mitochondrial localization of this gene. To investigate the function of BnaAOX1b in rapeseed, transgenic rapeseed lines with overexpressed BnaAOX1b were created and seed germination and seedling establishment assays were performed under osmotic, salt, and ABA treatment. The results indicated that overexpression of BnaAOX1b significantly improved seed germination under osmotic and salt stress and weakened ABA sensitivity. In addition, post-germination seedling growth was improved under high salt condition, but showed hypersensitivity to ABA. RNA-sequencing analysis indicated that the genes involved in electron transport or energy pathway were induced and a number of gene responses to salt stress and ABA were regulated in Bna.AOX1b overexpressing seeds. Taken together, our results imply that Bna.AOX1b confers tolerance to osmotic and salt stress in terms of seed germination and seedling establishment by regulating stress responsive genes and the response to ABA, and could be utilized as a candidate gene in transgenic breeding.

In conventional approaches to plastid and mitochondrial genome sequencing, the sequencing steps are performed separately; thus, plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) should be prepared independently. However, it is difficult to extract pure ptDNA and mtDNA from plant tissue. Following the development of high-throughput sequencing technology, many researchers have attempted to obtain plastid genomes or mitochondrial genomes using high-throughput sequencing data from total DNA. Unfortunately, the huge datasets generated consume massive computing and storage resources and cost a great deal, and even more importantly, excessive pollution reads affect the accuracy of the assembly. Therefore, it is necessary to develop an effective method that can generate base sequences from plant tissue and that is suitable for all plant species. Here, we describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously. First, we obtained high-quality DNA employing Partial Concentration Extraction. Second, we evaluated the purity of the DNA sample and determined the sequencing dataset size employing Vector Control Quantitative Analysis. Third, paired-end reads were obtained using a high-throughput sequencing platform. Fourth, we obtained scaffolds employing Two-step Assembly. Finally, we filled in gaps using specific methods and obtained complete plastid and mitochondrial genomes. To ensure the accuracy of plastid and mitochondrial genomes, we validated the assembly using PCR and Sanger sequencing. Using this method,we obtained complete plastid and mitochondrial genomes with lengths of 153,533 nt and 223,412 nt separately. A simple method for extracting, evaluating, sequencing and assembling plastid and mitochondrial genomes was developed. This method has many advantages: it is timesaving, inexpensive and reproducible and produces high-quality sequence. Furthermore, this method can produce plastid and mitochondrial genomes simultaneously and be used for other plant species. Due to its simplicity and extensive applicability, this method will support research on plant cytoplasmic genomes.

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.

Key messageBnaIAA7 crosstalk with BR signaling is mediated by the interaction between BnaARF8 and BnaBZR1 to regulate rapeseed plant morphogenesis.Auxin (indole-3-acetic acid, IAA) and brassinosteroids (BRs) are essential regulators of plant morphogenesis. However, their roles in rapeseed have not been reported. Here, we identified an extremely dwarf1 (ed1) mutant of rapeseed that displays reduced stature, short hypocotyls, as well as wavy and curled leaves. We isolated ED1 by map-based cloning, and found that it encodes a protein homologous to AtIAA7. ED1 acts as a repressor of IAA signaling, and IAA induces its degradation through its degron motif. A genomic-synteny analysis revealed that ED1 has four homologs in rapeseed, but two were not expressed. Analyses of transcriptomes and of various mutant BnaIAA7s in transgenic plants revealed that the three expressed BnaIAA7 homologs had diverse expression patterns. ED1/BnaC05.IAA7 predominantly functioned in stem elongation, BnaA05.IAA7 was essential for reproduction, while BnaA03.IAA7 had the potential to reduce plant height. Physical interaction assays revealed that the three BnaIAA7 homologs interacted in different ways with BnaTIRs/AFBs and BnaARFs, which may regulate the development of specific organs. Furthermore, BnaARF8 could directly interact with the BnaIAA7s and BnaBZR1. We propose that BnaIAA7s interact with BR signaling via BnaARF8 and BnaBZR1 to regulate stem elongation in rapeseed.

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed Bna MAX 1 genes had redundant functions resembling those of Arabidopsis MAX 1 , which regulates plant height and axillary bud outgrowth. Therefore, we designed two sg RNA s to edit these Bna MAX 1 homologs using the CRISPR /Cas9 system. The T 0 plants were edited very efficiently (56.30%–67.38%) at the Bna MAX 1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T 1 and T 2 progeny. We also obtained transgene‐free lines created by the CRISPR /Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four Bna MAX 1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.