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Summary: Rapeseed is a crop of global importance but there is a need to broaden the genetic diversity available to address breeding objectives. Radiation mutagenesis, supported by genomics, has the potential to supersede genome editing for both gene knockout and copy number increase, but detailed knowledge of the molecular outcomes of radiation treatment is lacking. To address this, we produced a genome re‐sequenced panel of 1133 M2 generation rapeseed plants and analysed large‐scale deletions, single nucleotide variants and small insertion–deletion variants affecting gene open reading frames. We show that high radiation doses (2000 Gy) are tolerated, gamma radiation and fast neutron radiation have similar impacts and that segments deleted from the genomes of some plants are inherited as additional copies by their siblings, enabling gene dosage decrease. Of relevance for species with larger genomes, we showed that these large‐scale impacts can also be detected using transcriptome re‐sequencing. To test the utility of the approach for predictive alteration of oil fatty acid composition, we produced lines with both decreased and increased copy numbers of Bna.FAE1 and confirmed the anticipated impacts on erucic acid content. We detected and tested a 21‐base deletion expected to abolish function of Bna.FAD2.A5, for which we confirmed the predicted reduction in seed oil polyunsaturated fatty acid content. Our improved understanding of the molecular effects of radiation mutagenesis will underpin genomics‐led approaches to more efficient introduction of novel genetic variation into the breeding of this crop and provides an exemplar for the predictive improvement of other crops. [ABSTRACT FROM AUTHOR]

Background: Oilseed rape (Brassica napus L.) is known as one of the most important oilseed crops cultivated around the world. However, its production continuously faces a huge challenge of Sclerotinia stem rot (SSR), a destructive disease caused by the fungus Sclerotinia sclerotiorum, resulting in huge yield loss annually. The SSR resistance in B. napus is quantitative and controlled by a set of minor genes. Identification of these genes and pyramiding them into a variety are a major strategy for SSR resistance breeding in B. napus. Results: Here, we performed a genome-wide association study (GWAS) using a natural population of B. napus consisting of 222 accessions to identify BnaA08g25340D (BnMLO2_2) as a candidate gene that regulates the SSR resistance. BnMLO2_2 was a member of seven homolog genes of Arabidopsis Mildew Locus O 2 (MLO2) and the significantly SNPs were mainly distributed in the promoter of BnMLO2_2, suggesting a role of BnMLO2_2 expression level in the regulation of SSR resistance. We expressed BnMLO2_2 in Arabidopsis and the transgenic plants displayed an enhanced SSR resistance. Transcriptome profiling of different tissues of B. napus revealed that BnMLO2_2 had the most expression level in leaf and silique tissues among all the 7 BnMLO2 members and also expressed higher in the SSR resistant accession than in the susceptible accession. In Arabidopsis, mlo2 plants displayed reduced resistance to SSR, whereas overexpression of MLO2 conferred plants an enhanced SSR resistance. Moreover, a higher expression level of MLO2 showed a stronger SSR resistance in the transgenic plants. The regulation of MLO2 in SSR resistance may be associated with the cell death. Collinearity and phylogenetic analysis revealed a large expansion of MLO family in Brassica crops. Conclusion: Our study revealed an important role of BnMLO2 in the regulation of SSR resistance and provided a new gene candidate for future improvement of SSR resistance in B. napus and also new insights into understanding of MLO family evolution in Brassica crops. [ABSTRACT FROM AUTHOR]

Crucial studies have verified that IAA is mainly generated via the two-step pathway in Arabidopsis, in which tryptophan aminotransferase (TAA) and YUCCA (YUC) are the two crucial enzymes. However, the role of the TAA (or TAR) and YUC genes in allotetraploid oilseed rape underlying auxin biosynthesis and development regulation remains elusive. In the present study, all putative TAR and YUC genes were identified in B. napus genome. Most TAR and YUC genes were tissue that were specifically expressed. Most YUC and TAR proteins contained trans-membrane regions and were confirmed to be endoplasmic reticulum localizations. Enzymatic activity revealed that YUC and TAR protein members were involved in the conversion of IPA to IAA and Trp to IPA, respectively. Transgenic plants overexpressing BnaYUC6a in both Arabidopsis and B. napus displayed high auxin production and reduced plant branch angle, together with increased drought resistance. Moreover, mutation in auxin biosynthesis BnaTARs genes by CRISPR/Cas9 caused development defects. All these results suggest the convergent role of BnaYUC and BnaTAR genes in auxin biosynthesis. Different homoeologs of BnaYUC and BnaTAR may be divergent according to sequence and expression variation. Auxin biosynthesis genes in allotetraploid oilseed rape play a pivotal role in coordinating plant development processes and stress resistance. [ABSTRACT FROM AUTHOR]

Tocopherols are essential nutrients for human health known as vitamin E. Vitamin E deficiency can have a profound effect on human health, including the central nervous system and cardiovascular and immune protection. Multiple enzymatic steps are involved in the conversion between different forms of tocopherols. Among them, γ-tocopherol methyltransferase encoded by gene VTE4 catalyzes the conversion of γ- to α-tocopherol or δ- to β-tocopherol isoforms. However, the gene copies and their functional contribution of VTE4 homologs in Brassica napus were not elucidated. To this end, different mutation combinations of four putative BnVTE4 homologous copies were generated by using CRISPR/Cas9 genome editing technology. Editing of those BnVTE4 homologs led to a significant change of the α-tocopherol content and the ratio between α- and γ-tocopherol compared with wide-type control. Analysis of the different combinations of BnVTE4 -edited homologs revealed that the contribution of the BnVTE4 individual gene displayed obvious functional differentiation in α-tocopherol biosynthesis. Their contribution could be in order of VTE4.C02-2 (BnaC02G0331100ZS) > VTE4.A02-1 (BnaA02G0247300ZS) > VTE4.A02-2 (BnaA02G0154300ZS). Moreover, the VTE4.A02-1 and VTE4.A02-2 copies might have severe functional redundancies in α-tocopherol biosynthesis. Overall, this study systemically studied the different effects of BnVTE4 homologs, which provided a theoretical basis for breeding high α-tocopherol content oilseed rape. [ABSTRACT FROM AUTHOR]

The CONSTANS-LIKE (COL) genes are important signaling component in the photoperiod pathway and flowering regulation pathway. However, people still know little about their role in Brassica napus. To achieve a better understanding of the members of the BnaCOL gene family, reveal their evolutionary relationship and related functions involved in photoperiod regulation, we systematically analyzed the BnaCOL family members in B. napus genome. A total of 33 BnaCOL genes distributed unevenly on 16 chromosomes were identified in B. napus and could be classified into three subfamilies. The same subfamilies have relatively conservative gene structures, three-dimensional protein structures and promoter motifs such as light-responsive cis -elements. The collinearity analysis detected 37 pairs of repetitive genes in B. napus genome. A 67.7% of the BnaCOL genes were lost after B. napus genome polyploidization. In addition, the BnaCOL genes showed different tissue-specific expression patterns. A 81.8% of the BnaCOL genes were mainly expressed in leaves, indicating that they may play a conservative role in leaves. Subsequently, we tested the circadian expression profiles of nine homologous genes that regulate flowering in Arabidopsis. Most BnaCOL genes exhibit several types of circadian rhythms, indicating that these BnaCOL genes are involved in the photoperiod pathway. As such, our research has laid the foundation for understanding the exact role of the BnaCOL family in the growth and development of rapeseed, especially in flowering. [ABSTRACT FROM AUTHOR]

TCP proteins are plant-specific transcription factors that have multipurpose roles in plant developmental procedures and stress responses. Therefore, a genome-wide analysis was performed to categorize the TCP genes in the rapeseed genome. In this study, a total of 80 BnTCP genes were identified in the rapeseed genome and grouped into two main classes (PCF and CYC/TB1) according to phylogenetic analysis. The universal evolutionary analysis uncovered that BnTCP genes had experienced segmental duplications and positive selection pressure. Gene structure and conserved motif examination presented that Class I and Class II have diverse intron-exon patterns and motifs numbers. Overall, nine conserved motifs were identified and varied from 2 to 7 in all TCP genes; and some of them were gene-specific. Mainly, Class II (PCF and CYC/TB1) possessed diverse structures compared to Class I. We identified four hormone- and four stress-related responsive cis -elements in the promoter regions. Moreover, 32 bna-miRNAs from 14 families were found to be targeting 21 BnTCPs genes. Gene ontology enrichment analysis presented that the BnTCP genes were primarily related to RNA/DNA binding, metabolic processes, transcriptional regulatory activities, etc. Transcriptome-based tissue-specific expression analysis showed that only a few genes (mainly BnTCP9, BnTCP22, BnTCP25, BnTCP48, BnTCP52, BnTCP60, BnTCP66, and BnTCP74) presented higher expression in root, stem, leaf, flower, seeds, and silique among all tested tissues. Likewise, qRT-PCR-based expression analysis exhibited that BnTCP36, BnTCP39, BnTCP53, BnTCP59, and BnTCP60 showed higher expression at certain time points under various hormones and abiotic stress conditions but not by drought and MeJA. Our results opened the new groundwork for future understanding of the intricate mechanisms of BnTCP in various developmental processes and abiotic stress signaling pathways in rapeseed. [ABSTRACT FROM AUTHOR]

Summary: CRISPR/Cas‐base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A‐PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base‐editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base‐edited plants of ALS conferred high herbicide resistance, while base‐edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T0 to T1 generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off‐target sites, large‐scale SNP variations were determined through whole‐genome sequencing between some base‐edited and wild‐type plants. These results revealed that A3A‐PBE base‐editing system could effectively convert C to T substitution with high‐editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting. [ABSTRACT FROM AUTHOR]

Seed loss resulting from pod shattering is a major constraint in production of oilseed rape (Brassica napus L.). However, the molecular mechanisms underlying pod shatter resistance are not well understood. Here, we show that the pod shatter resistance at quantitative trait locus qSRI.A9.1 is controlled by one of the B. napus SHATTERPROOF1 homologs, BnSHP1.A9 , in a doubled haploid population generated from parents designated R1 and R2 as well as in a diverse panel of oilseed rape. The R1 maternal parental line of the doubled haploid population carried the allele for shattering at qSRI.A9.1 , while the R2 parental line carried the allele for shattering resistance. Quantitative RT-PCR showed that BnSHP1.A9 was expressed specifically in flower buds, flowers, and developing siliques in R1, while it was not expressed in any tissue of R2. Transgenic plants constitutively expressing either of the BnSHP1.A9 alleles from the R1 and R2 parental lines showed that both alleles are responsible for pod shattering, via a mechanism that promotes lignification of the en b layer. These findings indicated that the allelic differences in the BnSHP1.A9 gene per se are not the causal factor for quantitative variation in shattering resistance at qSRI.A9.1. Instead, a highly methylated copia -like long terminal repeat retrotransposon insertion (4803 bp) in the promotor region of the R2 allele of BnSHP1.A9 repressed the expression of BnSHP1.A9 , and thus contributed to pod shatter resistance. Finally, we showed a copia -like retrotransposon-based marker, BnSHP1.A9 R2, can be used for marker-assisted breeding targeting the pod shatter resistance trait in oilseed rape. [ABSTRACT FROM AUTHOR]

To understand rapeseed (Brassica napus L.) heterosis on salt tolerance, hybrid cultivar Ningza 21 was used to investigate genes differentially expression compared with its parental lines. Through the next-generation high-throughput transcriptome sequencing technology with no salt treatments as negative control, 1078 differentially expressed genes (DEGs) were obtained, which were recognized to be closely related to heterosis of salt tolerance in oilseed rape at early seedling stage. These DEGs were annotated by released salt stress related QTLs, and expression of some DEGs were verified by qPCR, indicating the reliability of these DEGs. Additionally, the hybrid had more up-regulation candidate DEGs in shoot than those in root, and the expression pattern of DEGs was much closer to that of the maternal line than the paternal line in hybrid Ningza 21. More DEGs in hybrid similar to female parent showed positive regulation, and their function tended to be assimilation of biosynthesis. In contrast, DEGs similar to male parent were much related to redox and other regulatory functions. It was predicted that SNP loci were specific for heterosis of salt tolerance. All the results were expected to be helpful for salt tolerance improvement of hybrid rapeseed. [ABSTRACT FROM AUTHOR]

Compared with the mutation efficiency (less than 10% edited offspring) generated by maize haploid inducer-mediated genome-editing methods (Kelliher I et al i ., 2019; Wang I et al i ., 2019), all doubled haploid lines induced by Y3380-CRISPR/Cas9 were detected with targeted mutagenesis. Four out of five Y3380-15 T SB 1 sb plants, five out of nine Y3380-62 T SB 1 sb plants, two out of seven Y3380-112 T SB 1 sb plants are octaploid/tetraploid mosaics (Figure 1e and 1f), indicating that a certain percentage of tetraploid cells may be separated from octaploid Y3380 donor material during tissue culture process. Keywords: doubled haploid inducer; genome editing; Brassica oleracea; Brassica napus EN doubled haploid inducer genome editing Brassica oleracea Brassica napus 1889 1891 3 10/05/21 20211001 NES 211001 The diploid I Brassica oleracea i and allopolyploidy I Brassica napus i are predominant members of commonly consumed vegetables and plant oil, respectively. Direct modification of multiple gene homoeologs in Brassica oleracea and Brassica napus using doubled haploid inducer-mediated genome-editing system. [Extracted from the article]

Improvement of pod shatter resistance is the basic target for rapeseed breeding suitable for mechanized harvesting. To better understand the inheritance of pod shatter resistance in Brassica napus, a doubled haploid (DH) population was used in the study. Two parents of the population had contrasting pod shatter resistance levels. Pod shatter resistance index was determined using random impact test method in two years, and the inheritance was analyzed by major gene plus polygene genetic model for plant quantitative traits as well as the analysis of skewness and kurtosis. Results showed that the pod shatter resistance was controlled by 3 genes with major effects and 5 - 7 polygenes with minor effects. There were major genes additive effects and complementary gene interactions. The heritability of major genes was greater than 85. 00% in both years, indicating pod shatter resistance was mainly controlled by major genes and less influenced by environments. Therefore, in breeding process for pod shatter resistance of rapeseed, pyramiding major genes from lines containing different major genes by hybridization and selection in early generation should be considered, ad the influence of environmental factors can be ignored. [ABSTRACT FROM AUTHOR]

To understand the genetic effects of silique related traits on silique shatter resistance of oilseed rape, an incomplete diallel cross with 11 parental lines and 30 F1 crosses were made for multivariable conditional analysis at Xinzhou and Hanchuan counties of Hubei Province. Results showed that the largest contribution rate of phenotypic (28.2% ) and additive (56.8% ) effect to silique shatter resistance index (SRI) variance was from silique wall weight, the second was from silique length, with contribution rates of 17. 5% and 33. 4% for phenotypic and additive effects to SRI variance respectively. The investigated silique related traits had low contribution rates of dominant effect to SRI variance. The contribution rate of dominant x environment interaction effect of silique number per centimeter to SRI variance was the highest (44. 7% ) among silique related traits. The silique trait with the highest contribution rate of additive effect to silique SRI variance varied depending on different parental lines, indicating that the parental lines, especially those with relatively high silique SRI, possess diferent genetic backgrounds. It suggested that the strategies for improving shatter resistance included utilizing wild backgrounds of parents with large additive effects, and hybrids from multiple crosses with high contribution traits to increase resistance. [ABSTRACT FROM AUTHOR]

According to pentatricopeptide repeat (PPR) protein sequence encoded by restorer gene from cytoplasmic male sterile rapeseed plant, a pair of degenerate primers was obtained after PCR screening of Nsa CMS (hybrid of Brassica napus and Sinapis arvensis). PCR results showed expected products specific to fertility restored plants and S. arvensis parent. The fragment was 309bp which was 69% identical to fertility restorer gene of Radish CMS, 70% identical to PPR gene cluster from the first chromosome of Arabidopsis thaliana, and 85% identical to a B. rapa accession clone which restores the fertility of pol CMS. Further analysis revealed that the amino acid sequence encoded by this fragment contained two adjacent PPR motifs with 35 amino acids each. Results indicated this fragment could be part of the candidate Rƒ gene for Nsa CMS. [ABSTRACT FROM AUTHOR]

To ensure the hybrid oilseed rape (OSR, Brassica napus) seed production, two important things are necessary, the stamen sterility on the female OSR plants and the effective pollen spread onto the pistil from the OSR male plants to the OSR female plants. The unmanned agricultural aerial system (UAAS) has developed rapidly in China. It has been used on supplementary pollination and aerial spraying during the hybrid OSR seed production. This study developed a new method to rapidly recognize the male OSR plants and extract the row center line for supporting the UAAS navigation. A male OSR plant recognition model was constructed based on the convolutional neural network (CNN). The sequence images of male OSR plants were extracted, the feature regions and points were obtained from the images through morphological and boundary process methods and horizontal segmentation, respectively. The male OSR plant image recognition accuracies of different CNN structures and segmentation sizes were discussed. The male OSR plant row center lines were fitted using the least-squares method (LSM) and Hough transform. The results showed that the segmentation algorithm could segment the male OSR plants from the complex background. The highest average recognition accuracy was 93.54%, and the minimum loss function value was 0.2059 with three convolutional layers, one fully connected layer, and a segmentation size of 40 pix × 40 pix. The LSM is better for center line fitting. The average recognition model accuracies of original input images were 98% and 94%, and the average root mean square errors (RMSE) of angle were 3.22° and 1.36° under cloudy day and sunny day lighting conditions, respectively. The results demonstrate the potential of using digital imaging technology to recognize the male OSR plant row for UAAS visual navigation on the applications of hybrid OSR supplementary pollination and aerial spraying, which would be a meaningful supplement in precision agriculture. [ABSTRACT FROM AUTHOR]

Lodging is one of the important reasons for rapeseed yield reduction. Increasing the lignin content in aerial part has become an effective way to improve rapeseed lodging resistance. However, knowledge of the genetic basis underlying lignin content in rapeseed is limited. Therefore, we undertook association mapping to identify quantitative trait nucleotides (QTNs) associated with lignin content in the stalk and taproot of 125 natural rapeseed accessions across three independent environments by BLUP mixed line model. Among the natural population, lignin content ranged from 6.05% and 12.76% for the stalk, and 3.21% and 12.33% for the taproot with massive phenotypic variation. Four QTL regions were identified for total lignin content in stalk and taproot, qBnADL.C09.1 and qBnADL.C09.2 with phenotypic variance ranging from 6.05% to 12.76% in stalk and BnADL.A05.1 and qBnADL.A09.1 from 3.21% to 12.33% in taproot. Lignin content significantly differed between superior and alternative alleles, with 4 haplotype groups. We identified 2 putative candidate genes based on the haplotype QTNs for lignin content, e.g., BnLI.C09 and BnRPS17. Combining with the RNA-seq data, identified 3 candidate genes with lignin pathway, e.g., BnLAC17 , BnF8H and BnDIR20. This study provides novel insights into the lodging improvement of high lignin content and also facilitates the MAS for low lignin straw selection of rapeseed. • The recycling and reuse of agricultural waste, such as rapeseed straw, requires fundamental work to understand genetic control of lignin. • In this paper, we used the GWAS and RNA-seq methods to discover superior alleles and candidate genes for lignin content in rapeseed. • This knowledge can improve the economic value by regulating lignin content by genes and superior alleles, increasing the development of biomass energy and lodging resistance of rapeseed. [ABSTRACT FROM AUTHOR]

Yellow seed is one desirable trait with great potential to improve seed oil quality and yield. The present study surveys the redundant role of BnTTG1 genes in the proanthocyanidins (PA) biosynthesis, oil content and abiotic stress resistance. Stable yellow seed mutants were generated after mutating BnTTG1 by CRISPR/Cas9 genome editing system. Yellow seed phenotype could be obtained only when both functional homologues of BnTTG1 were simultaneously knocked out. Homozygous mutants of BnTTG1 homologues showed decreased thickness and PA accumulation in seed coat. Transcriptome and qRT-PCR analysis indicated that BnTTG1 mutation inhibited the expression of genes involved in phenylpropanoid and flavonoid biosynthetic pathways. Increased seed oil content and alteration of fatty acid (FA) composition were observed in homozygous mutants of BnTTG1 with enriched expression of genes involved in FA biosynthesis pathway. In addition, target mutation of BnTTG1 accelerated seed germination rate under salt and cold stresses. Enhanced seed germination capacity in BnTTG1 mutants was correlated with the change of expression level of ABA responsive genes. Overall, this study elucidated the redundant role of BnTTG1 in regulating seed coat color and established an efficient approach for generating yellow-seeded oilseed rape genetic resources with increase oil content, modified FA composition and resistance to multiple abiotic stresses. • Yellow seed mutants were generated after mutation of two BnTTG1 copies by CRISPR/Cas9. • BnTTG1 mutants Increased seed oil content and modified FA composition. • BnTTG1 accelerated seed germination rate under salt and cold stresses. • BnTTG1 mutants increase abiotic stresses was correlated with the ABA signaling. [ABSTRACT FROM AUTHOR]