Association mapping of quantitative resistance for <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Stem canker caused by the fungus Leptosphaeria maculans is a major disease of Brassica napus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.     

Interaction of silicon and cadmium in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

The objective of this study was to determine the effect of silicon (Si) and cadmium (Cd) on root and shoot growth and Cd uptake in two hydroponically cultivated Brassica species (B. juncea (L.) Czern. cv. Vitasso and B. napus L. cv. Atlantic). Both species are potentially usable for phytoextraction. Inhibitory effects of Cd on root elongation were diminished by the impact of Si. Primary roots elongation in the presence of Cd + Si compared with Cd was stronger and the number of lateral roots was lower in B. juncea than in B. napus. Cd content per plant was higher in B. napus roots and shoots compared with B. juncea. Suberin lamellae were formed closer to the root apex in Cd + Si than in Cd treated plants and this effect was stronger in B. napus than in B. juncea. Accelerated maturation of endodermis was associated with reduced Cd uptake. Cd decreased the content of chlorophylls and carotenoids in both species, but Si addition positively influenced the content of photosynthetic pigments which was higher in B. napus than in B. juncea. Si enhanced more substantially translocation of Cd into the shoot of B. napus than of B. juncea. Based on our results B. napus seems to be more suitable for Cd phytoextraction than B. juncea because these plants produce more biomass and accumulate higher amount of Cd. The protective effect of Si on Cd treated Brassica plants could be attributed to more extensive development of suberin lamellae in endodermis.     

Pollen-mediated intraspecific gene flow from herbicide resistant oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The cultivation of genetically modified (GM) herbicide resistant oilseed rape (Brassica napus) has increased over the past few years. The transfer of herbicide resistance genes via pollen (gene flow) from GM crops to non-GM crops is of relevance for the realisation of co-existence of different agricultural cultivation forms as well as for weed management. Therefore the likelihood of pollen-mediated gene flow has been investigated in numerous studies. Despite the difficulty to compare different experiments with varying levels of outcrossing, we performed a literature search for world-wide studies on cross-fertilisation in fully fertile oilseed rape. The occurrence and frequency of pollen-mediated intraspecific gene flow (outcrossing rate) can vary according to cultivar, experimental design, local topography and environmental conditions. The outcrossing rate from one field to another depends also on the size and arrangement of donor and recipient populations and on the ratio between donor and recipient plot size. The outcrossing levels specified in the presented studies are derived mostly from experiments where the recipient field is either surrounding the donor field (continuous design) or is located as a patch at different distances from the donor field (discontinuous design). Reports of gene flow in Brassica napus generally show that the amount of cross-fertilisation decreases as the distance from the pollen source increases. The evidence given in various studies reveals that the bulk of GM cross-fertilisation occurs within the first 10 m of the recipient field. The removal of the first 10 m of a non-transgenic field facing a GM crop might therefore be more efficient for reducing the total level of cross-fertilisation in a recipient sink population than to recommend separation distances. Future experiments should investigate cross-fertilisation with multiple adjacent donor fields at the landscape level under different spatial distributions of rapeseed cultivars and different cropping systems. The level of cross-fertilisation occurring over the whole field is mainly important for co-existence and has not been investigated in agricultural scale experiments until now. Potential problems with herbicide resistant oilseed rape volunteers arising from intraspecific gene flow can be largely solved by the choice of suitable cultivars and herbicides as well as by soil management.     

Development of INDELs markers in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) using re-sequencing data

Insertions/deletions (INDELs), a type of abundant length polymorphisms in the plant genomes, combine the characteristics of both simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP), and thus can be developed as desired molecular markers for genetic studies and crop breeding. There has been no large-scale characterization of INDELs variations in Brassica napus yet. In this study, we identified a total of 538,691 INDELs in size range of 1–10 bp by aligning whole-genome re-sequencing data of 23 B. napus inbred lines (ILs) to the B. napus genome sequence of ‘Darmor-bzh.’ Of these, 104,190 INDELs were uniquely mapped on the pseudochromosomes of the reference genome. A set of 595 unique INDELs of 2–5 bp in length was selected for experimental validation in the 23 ILs. Of these INDELs, 530 (89.01 %) produced a single PCR product and were single locus. A total of 523 (87.9 %) INDELs were found polymorphic among the 23 ILs. A genetic linkage map containing 108 single-locus INDELs and 89 anchor SSR markers was constructed using 188 recombinant ILs. The majority of INDELs markers on the linkage map showed consistency with the pseudochromosomes of the B. napus cultivar ‘Darmor-bzh.’ The INDELs variations and markers reported here will be valuable resources in future for genetic studies and molecular breeding in oilseed rape.     

Defence reactions in the apoplastic proteome of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> var. <Emphasis Type="Italic">napus</Emphasis>) attenuate <Emphasis Type="Italic">Verticillium longisporum</Emphasis>growth but not disease symptoms

Background

Verticillium longisporum is one of the most important pathogens of Brassicaceae that remains strictly in the xylem during most stages of its development. It has been suggested that disease symptoms are associated with clogging of xylem vessels. The aim of our study was to investigate extracellular defence reactions induced by V. longisporum in the xylem sap and leaf apoplast of Brassica napus var. napus in relation to the development of disease symptoms, photosynthesis and nutrient status.

Results

V. longisporum (strain VL43) did not overcome the hypocotyl barrier until 3 weeks after infection although the plants showed massive stunting of the stem and mild leaf chlorosis. During this initial infection phase photosynthetic carbon assimilation, transpiration rate and nutrient elements in leaves were not affected in VL43-infected compared to non-infected plants. Proteome analysis of the leaf apoplast revealed 170 spots after 2-D-protein separation, of which 12 were significantly enhanced in response to VL43-infection. LS-MS/MS analysis and data base searches revealed matches of VL43-responsive proteins to an endochitinase, a peroxidase, a PR-4 protein and a β-1,3-glucanase. In xylem sap three up-regulated proteins were found of which two were identified as PR-4 and β-1,3-glucanase. Xylem sap of infected plants inhibited the growth of V. longisporum.

Conclusion

V. longisporum infection did not result in drought stress or nutrient limitations. Stunting and mild chlorosis were, therefore, not consequences of insufficient water and nutrient supply due to VL43-caused xylem obstruction. A distinct array of extracellular PR-proteins was activated that might have limited Verticillium spreading above the hypocotyl. In silico analysis suggested that ethylene was involved in up-regulating VL43-responsive proteins.

     

Metabolically engineered male sterility in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Male sterility is of special interest as a mechanism allowing hybrid breeding, especially in important crops such as rapeseed (Brassica napus). Male sterile plants are also suggested to be used as a biological safety method to prevent the spread of transgenes, a risk that is high in the case of rapeseed due to the mode of pollination, out-crossing by wind or insects, and the presence of related, cross-pollinating species in the surrounding ecosystem in Europe. Different natural occurring male sterilities and alloplasmic forms have been tried to be used in rapeseed with more or less success. Due to the difficulties and limitations with these systems, we present a biotechnological alternative: a metabolically engineered male sterility caused by interference with anther-specific cell wall-bound invertase. This is an essential enzyme for carbohydrate supply of the symplastically isolated pollen. The activity of this enzyme is reduced either by antisense interference or by expressing an invertase inhibitor under control of the anther-specific promoter of the invertase with the consequence of a strong decrease of pollen germination ability.     

The effect of <Emphasis Type="Italic">INDEHISCENT</Emphasis> point mutations on silique shatter resistance in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>)

Key message

This study elucidates the influence of indehiscent mutations on rapeseed silique shatter resistance. A phenotype with enlarged replum-valve joint area and altered cell dimensions in the dehiscence zone is described.

Abstract

Silique shattering is a major factor reducing the yield stability of oilseed rape (Brassica napus). Attempts to improve shatter resistance often include the use of mutations in target genes identified from Arabidopsis (Arabidopsis thaliana). A variety of phenotyping methods assessing the level of shatter resistance were previously described. However, a comparative and comprehensive evaluation of the methods has not yet been undertaken. We verified the increase of shatter resistance in indehiscent double knock-down mutants obtained by TILLING with a systematic approach comparing three independent phenotyping methods. A positive correlation of silique length and shatter resistance was observed and accounted for in the analyses. Microscopic studies ruled out the influence of different lignification patterns. Instead, we propose a model to explain increased shattering resistance of indehiscent rapeseed mutants by altered cell shapes and sizes within the contact surfaces of replum and valves.

     

Phosphinothricin-resistant <Emphasis Type="Italic">Brassica napus</Emphasis> + <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> somatic hybrids

Hybrid plants resistant to phosphinothricin (PPT) are obtained as a result of experiments with somatic hybridization between Brassica napus L. cv. Kalinins’kyy and Orychophragmus violaceus L. O.E. Shulz. The hybrids inherited PPT resistance from O. violaceus plants that had been previously transformed by a vector containing the maize transposon system Spm/dSPm with bar gene located within the nonautonomous transposon. The morphologically obtained plants occupy an intermediate position between the initial forms, which is in agreement with the results of isoenzyme analyses (analysis of multiple forms of amylase and esterase) and PCR analysis (presence of the genes bar, gus, and SpmTPase). Inheritance of the plastome occurs from oilseed rape, while that of the mitochondrion, from O. violaceus, which is proved by means of PCR-RFLP analysis. The plant hybrids may be utilized for further selection research with oilseed rape following determination of the edible quality of its oil as well as in experiments with chloroplast transformation, a topic which is of critical importance for oilseed rape.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Twinned microspore-derived embryos of canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) are genetically identical

Microspore culture is used extensively in several crop species to generate diverse populations of homozygous, doubled haploid lines for breeding and genetic analyses. In our canola (Brassica napus L.) doubled haploid breeding programme we regularly observe conjoined microspore-derived embryos, most commonly twins, joined either at the base of the hypocotyl or along the length of the hypocotyl axis. The aim of this study was to determine if twinned embryos were genetically identical or non-identical in order to gauge their value for breeding and linkage analysis. Microsatellite marker fingerprinting of 12 pairs of twinned embryos produced by microspore culture of heterozygous F₁ lines revealed that pairs of twins were genetically identical. Based on this finding, we recommend breeders and geneticists using microspore culture technology to retain only one embryo from each pair of twinned embryos.     

Antioxidant defense system in leaves of Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) under cadmium stress

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from environment. Using two brassica species (Brassica juncea and Brassica napus), nutrient solution experiments were conducted to study variation in tolerance to cadmium (Cd) toxicity based on (1) lipid peroxidation and (2) changes in antioxidative defense system in leaves of both plants (i.e., superoxide dismutase (SOD EC 1.15.1.1), catalase (CAT EC 1.11.1.6), ascorbate peroxidase (APX EC 1.11.1.11), guaiacol peroxidase (GPX EC 1.11.1.7), glutathione reductase (GR EC 1.6.4.2), levels of phytochelatins (PCs), non-protein thiols (NP-SH), and glutathione. Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 10, 25 and 50 μM) for 15 days. Results showed marked differences between both species. Brassica napus under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde (MDA) content in leaves. However, in Brassica juncea treated plants, MDA content remained unchanged. In Brassica napus, with the exception of GPX, activity levels of some antioxidant enzymes involved in detoxification of reactive oxygen species (ROS), including SOD, CAT, GR, and APX, decreased drastically at high Cd concentrations. By contrast, in leaves of Brassica juncea treated plants, there was either only slight or no change in the activities of the antioxidative enzymes. Analysis of the profile of anionic isoenzymes of GPX revealed qualitative changes occurring during Cd exposure for both species. Moreover, levels of NP-SH and PCs, monitored as metal detoxifying responses, were much increased in leaves of Brassica juncea by increasing Cd supply, but did not change in Brassica napus. These results indicate that Brassica juncea plants possess the greater potential for Cd accumulation and tolerance than Brassica napus.     

Effect of boron on microspore embryogenesis and direct embryo to plant conversion in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.)

The NLN-medium has been successfully used, since 1982, for microspore culture in Brassica napus and other Brassica species. Changes to the media composition were restricted to carbohydrate and nitrogen sources and growth regulators while micro-nutrients have not been optimized. The NLN-medium contains boron at a concentration of 162 µM. Boron is required for diverse physiological and metabolic processes in the cell. This study investigated the effect of seven- and 13-fold increased boron concentration on the induction of embryos in microspore cultures of four genotypes of B. napus. A significant improvement of microspore embryogenesis was achieved by both enhanced boron concentrations in the NLN medium. No effect on the regeneration of embryo to plant conversion was observed.     

Genetic analysis of loci associated with partial resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Sclerotinia stem rot is the most devastating disease of rapeseed (Brassica napus L.) in China. Quantitative trait loci (QTLs) involved in resistance to Sclerotinia sclerotiorum were detected in a rapeseed population of 128-F(2:3) families derived from a cross between the male sterility restorer line H5200 and a partial resistant line Ning RS-1. A total of 107 molecular markers including 72 RFLPs, 30 AFLPs, 3 SSRs and 2 RAPDs were employed to construct a genetic linkage map with 23 linkage groups covering 1,625.7 cM with an average space of 15.2 cM. Resistance was assessed empirically at two developmental stages: with a detached leaf inoculation at the seedling stage and in vivo stem inoculation at the mature plant stage. The observed resistance was scored for each plant as leaf resistance at the seedling stage (LRS) and stem resistance at the mature plant stage (SRM). A total of 13 loci were identified by one-way ANOVA and six QTLs were detected with MapMaker-QTL. We found that three of the six QTLs were associated with leaf resistance at the seedling stage and collectively accounted for 40.7% of the total phenotypic variation, each accounting for 23.2%, 16.6% and 13.6% respectively. Three QTLs were found corresponding to the disease resistance at the mature plant stage, explaining 49.0% of the phenotypic variation. Epistasis was observed for the resistance and the additive by additive interactions were the predominant type of epistasis. It was concluded that both single-locus QTLs and epistatic interactions played important roles in Sclerotinia resistance in rapeseed.     

Increased <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and rooting efficiencies in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) from hypocotyl segment explants

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.     

Characterization of Defense Signaling Pathways of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Brassica carinata</Emphasis> in Response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> Challenge

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.     

Proteomic alterations of <Emphasis Type="Italic">Brassica napus</Emphasis> root in response to boron deficiency

Boron (B) deficiency is a worldwide problem, and Brassica napus is one of the most sensitive crops to B deficiency. To better understand the B starvation response of Brassica napus, we conducted a comparative proteomic analysis of seedling stage Brassica napus root between B-sufficient and B-limited conditions: 45 differentially expressed proteins were successfully identified by 2-DE coupled with MALDI-TOF/TOF-MS and LTQ-ESI-MS/MS analysis. Among these proteins, 10 were down-regulated and 35 were up-regulated under B-limited condition. Combining GO and KEGG analyses with data from previous reports, proteins were categorized into several functional groups, including antioxidant and detoxification, defense-related proteins, signaling and regulation, carbohydrate and energy metabolism, amino acid and fatty acid metabolism, protein translation and degradation, cell wall structure, and transporter. The genes of selected proteins were analyzed by quantitative RT-PCR. Our results provide novel information for better understanding the physiological and biochemical responses to B deficiency in plants.     

Efficient regeneration of <Emphasis Type="Italic">Brassica napus</Emphasis> L. hypocotyls and genetic transformation by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm^–3) and thidiazuron (0.0, 0.15 and 0.30 mg dm^–3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm^–3 benzyladenine and 0.3 mg dm^–3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm^–3 of indolebutyric acid and 10 mg dm^–3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.