Frequent nonreciprocal translocations in the amphidiploid genome of oilseed rape (Brassica napus).

A RFLP map of Brassica napus, consisting of 277 loci arranged in 19 linkage groups, was produced from genetic segregation in a combined population of 174 doubled-haploid microspore-derived lines. The integration of this map with a B. napus map derived from a resynthesized B. napus x oilseed rape cross allowed the 10 linkage groups of the B. napus A genome and the 9 linkage groups of the C genome to be identified. Collinear patterns of marker loci on different linkage groups suggested potential partial homoeologues. RFLP patterns consistent with aberrant chromosomes were observed in 9 of the 174 doubled-haploid lines. At least 4 of these lines carried nonreciprocal, homoeologous translocations. These translocations were probably the result of homoeologous recombination in the amphidiploid genome of oilseed rape, suggesting that domesticated B. napus is unable to control chromosome pairing completely. Evidence for genome homogenization in oilseed rape is presented and its implications on genetic mapping in amphidiploid species is discussed. The level of polymorphism in the A genome was higher than that in the C genome and this might be a general property of oilseed rape crosses.     

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.     

Homoeologous loci control the accumulation of seed glucosinolates in oilseed rape (Brassica napus).

The genetic control of seed glucosinolate content in oilseed rape was investigated using two intervarietal backcross populations. Four QTLs segregating in the population derived from a Brassica napus L. 'Victor' x Brassica napus L. 'Tapidor' cross, together accounting for 76% of the phenotypic variation, were mapped. Three of these loci also appeared to control the accumulation of seed glucosinolates in a Brassica napus L. 'Bienvenu' x 'Tapidor' cross, and accounted for 86% of the phenotypic variation. The three QTLs common to both populations mapped to homoeologous regions of the B. napus genome, suggesting that seed glucosinolate accumulation is controlled by duplicate genes. It was possible to extend the comparative analysis of QTLs controlling seed glucosinolate accumulation by aligning the published genetic maps generated by several research groups. This comparative mapping demonstrated that high-glucosinolate varieties often carry low-glucosinolate alleles at one or more of the loci controlling seed glucosinolate accumulation.     

Biodiversity of nematofauna of oilseed rape (Brassica napus L.)

Few data is available on the nematodes found in Brassicaceae, except for the most important plant parasite. However, studying the structure of nematofauna could be an important database for the soil quality and in order to assess the effects of future disturbance. This is particularly important considering that the diffusion of the canola crop in the world is increasing because of its use as a bio-diesel. Very diffused is also the Bt variety of oil seed rape, and, in this case, the study of the impact on the soil health and on bio-diversity is essential. In this research we have analyzed the nematode community, used as a bio-indicator of the soil condition. The nematofauna found in canola (Brassica napus var. oleifera) fields located in Southern Italy (Metaponto - MT) was investigated. The nematode community was studied considering its abundance, genus composition and trophic structure. Maturity and biodiversity indices were also calculated. A total of 5286 nematodes were extracted. They belong to 14 families and 24 genera. Bacterial and fungal feeders, 50.18% and 42.90% of the total respectively, dominated the trophic structure. Aphelencus is the most abundant genus (23.71%) followed by Acrobeloides (20.49%) and Aphelencoides (19.18%). Among plant feeders (6.59%), Pratylenchus is the dominant genus (2.20%) and Tylenchidae the main family (3.54%). No infestation of Meloidogyne, Heterodera or Naboccus, important plant-parasitic nematodes of canola crops, was recorded. Other important phytophagous were Helycotylenchus (0.5%), Trichotylenchus (0.5%) and Filenchus (0.9%). All of them had an abundance level below injury level. The indices of biodiversity are rather low (H'=0.93, J'=0.67), as is typical for agro ecosystems. However, the nematofauna community is quite well structured (N2=6.31, D=0.16) and the maturity index rather high (EMI=1.94). These values demonstrate that oilseed rape has a lower impact on the soil compared to other crop systems and that it could be taken into consideration for crop rotation programs. Canola could follow other more stressful crops, trying to maintain soil equilibrium.     

Indistinguishable patterns of recombination resulting from male and female meioses in Brassica napus (oilseed rape).

An F1 individual derived from a cross between two distinct lines of spring oilseed rape (Brassica napus) was used to produce a pair of complementary backcross populations, each consisting of 90 individuals. The F1 donated male gametes to the Male population and female gametes to the Female population. Genetic maps were generated from both populations and aligned using 117 common loci to form an integrated genome map of B. napus with 243 RFLP-defined loci. A comparison of the frequency and distribution of crossovers in the two populations of F1 gametes (assayed in the Male and Female populations) detected no differences. The genetic maps derived from the Male and Female populations each consisted of 19 linkage groups spanning 1544 and 1577 cM, respectively. The maps were aligned with other B. napus maps, and all 19 equivalent linkage groups were unambiguously assigned. The genetic size and general organisation of the new maps were comparable with those of pre-existing B. napus maps in most respects, except that the levels of polymorphism in the constituent A and C genomes were unusually similar in the new cross.     

Zygotic embryo cloning in oilseed rape (Brassica napus L.)

When late torpedo- to early cotyledonary-stage zygotic embryos of Brassica napus were cultured on a solidified B5 basal medium with or without 0.05 mg l⁻¹ 6-benzylaminopurine (BAP), they proliferated by direct formation on the hypocotyl surface of structures resembling accessory cotyledons and root poles. When embryos bearing these structures were transferred to slowly rotating liquid culture on the same media, proliferation continued. As the underlying tissues senesced, meristematic structures became detached which produced cotyledonary and root tissues. A random sample of plants raised from these structures by direct transfer to sterilized potting mix all showed normal morphology.     

Cloning, sequencing and salt induced expression of PEAMT and BADH in oilseed rape (Brassica napus).

Agriculture productivity is severely hampered by soil salinity, drought and other environmental stresses. Studies on stress-resistant plants (halophytes, xerophytes, accumulating plants for specific toxic ions) have illuminated some mechanisms of stress tolerance in plants at metabolic or molecular levels, which gave some clues on how to genetically engineer stress-tolerant crops. With the isolation of more stress-responsive genes, genetic engineering with modified expression of stress responsive genes may be an effective way to produce stress-tolerant crops. In the present report, two genes (PEAMT and BADH) encoding the corresponding key enzymes for choline and glycine betaine (an important osmoprotectant) biosynthesis in plants were isolated in oilseed rape, an important oil crop in the world. Effects of salt stress on their expression were studied with quantitative PCR and their potential use in the genetic engineering of oilseed rape was discussed.     

Photosynthesis by developing embryos of oilseed rape (Brassica napus L.)

The aim of this study was to assess the photosynthetic potentialof developing seeds of oilseed rape (Brassica napus L.) andto compare photosynthetic properties of embryo plastids withthose of leaf chloroplasts from the same species. Measurementsof CO₂-dependent O₂ evolution show that developing seeds ofB. napus are photosynthetically active in vitro. Essentially,all of the photosynthetic activity of the developing seed isaccounted for by the embryo. The rate of photosynthesis by developingembryos increased until the onset of desiccation, after whichit declined, so that by maturity embryos were no longer photosyntheticallyactive. Photosynthetic activity was positively correlated withchlorophyll content throughout development. Comparison of thephotosynthetic characteristics of leaf and embryo chloroplastsrevealed that rates of uncoupled electron transport were 2.5-foldgreater in those from the embryo. Light-saturated rates of CO₂-dependentO₂ evolution, per unit chlorophyll, and CO₂ saturation pointswere similar for chloroplasts from both tissues. However, light-saturationpoints and chlorophyll a/b ratios were lower for embryo thanfor leaf choroplasts. Embryos and embryo chloroplasts also containedconsiderably less ribulose 1,5-bisphosphate carboxylase/oxygenaseprotein per unit total protein, than leaves. Although excisedembryos were capable of high rates of CO₂-dependent O₂ evolution(90–100 mol mg^–1 chlorophyll h^–1) under asaturating photosynthetic photon flux density (PPFD), low transmittanceof light through the silique wall (30%), together with the highPPFD required to achieve light compensation points in developingseeds (500 mol m^–2 s^–1), suggests that photosynthesisin vivo is unlikely to make a net contribution to carbon economyunder normal environmental conditions. Key words: Embryo, development, photosynthesis, chloroplast, Brassica napus L.     

Progressive introgression between Brassica napus (oilseed rape) and B. rapa

We have earlier shown extensive introgression between oilseed rape (Brassica napus) and B. rapa in a weedy population using AFLP markers specific for the nuclear genomes. In order to describe the progress of this introgression, we examined 117 offspring from 12 maternal plants from the introgressed population with the same AFLP-markers; AFLP data were supported by chromosome counting. We also analysed the offspring with a species-specific chloroplast marker and finally evaluated the reproductive system in selected maternal plants. Our results indicated a high outcrossing rate of the introgressed maternal plants. It seemed that B. rapa most often functioned as the maternal plant in the introgression process and that the amount of oilseed rape DNA was highly diminished in the offspring compared to their introgressed maternal plants. However, our analysis of plants from the weedy population indicated that introgression can lead to both (1) exchange of chloroplast DNA between species producing B. rapa-like plants with B. napus chloroplasts and (2) incorporation of B. napus C-genome DNA into the B. rapa genome. Therefore, we question whether it can be regarded as containment to position transgenes in the chloroplast or in specific parts of the nuclear genome of B. napus.     

Viruses infecting winter oilseed rape (Brassica napus ssp. oleifera)

Turnip mosaic virus (TuMV) and cauliflower mosaic virus (CaMV) have been found infecting field crops of winter oilseed rape (Brassica napus ssp. oleifera) in South Warwickshire. Other viruses found include broccoli necrotic yellows virus (BNYV) and a member of the beet western yellows virus group. Systemic leaf symptoms caused by TuMV varied within and between cultivars; the three predominant reaction types were classified as necrotic, mosaic and immune. Some recently introduced cultivars of oilseed rape were more severely affected by TuMV infection than older cultivars. Reactions to CaMV were less varied and immunity was not found. The seed yield from TuMV and CaMV-infected plants was less than that of healthy control plants. This effect was due to infected plants producing either fewer seeds, smaller seeds or both. Germination of seeds from infected plants was unaffected if sown soon after harvest. After storage for one year the germination of seed from a virus infected plant was significantly less than that of seed from a virus-free plant. All commercial cultivars tested were experimentally susceptible to turnip yellow mosaic virus (TYMV) and some American strains of cucumber mosaic virus (CMV).     

Persistence of oilseed rape (Brassica napus L.) outside of cultivated fields

It is widely assumed that most cultivated plants cannot persist in natural or semi-natural habitats. Most people thus assume that the plants growing outside of fields (in particular oilseed rape along roadsides) find their origins in the current or previous year’s cultivation of that crop. One consequence of this assumption is that the identity of plants growing on road verges is thought to reflect one of the cultivars currently or recently cultivated, while another consequence is the widespread belief that transgenic plants can be simply managed and controlled by stopping their cultivation. Our work shows that this assumption is false. We identify relict plants of a now unmarketable cultivar type of oilseed rape which have persisted in a semi-natural habitat (road verges) for at least 8 years according to farmer surveys in the studied area. More generally, we confirm that the dynamics of the feral oilseed rape plants of road verges is more complex than those resulting from spillage from agricultural machines or from neighbouring arable fields cultivated the previous year. Within the scope of transgenic oilseed rape cultivation, these results suggest that more studies on the dynamics of feral oilseed rape are needed in order to assess more precisely the risks of its invasiveness and its potential impact on genetic pollution between GM fields and non-GM fields. Received: 15 June 2000 / Accepted 31 July 2000     

Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.)

Summary Microspore-derived embryos from Brassica napus L. were dried to less than 15% moisture and stored dry for a minimum of 7 days. Successful plant regeneration was observed when embryos at the cotyledonary stage of development were treated with 50 uM ABA for 7 days prior to desiccation. Solid agar or liquid medium gave similar results. The rate of drying of embryos after ABA pretreatment had only minor effects on embryo survival, but for untreated embryos, slow drying gave a small degree of survival. These results are very comparable to those with alfalfa somatic embryos, suggesting that the ABA treatment of cotyledonary stage embryos may be broadly used as a pretreatment for inducing the expression of desiccation tolerance in plant embryos.     

Larger bees facilitate the deposition of oilseed rape pollen (Brassica napus L.)

Oilseed rape is the third-largest source of vegetable oil and the second-largest source of protein meal in the world. Pollinators are vital in the production of oilseed rape. However, pollination efficiency could be influenced by the morphology and foraging behaviors of pollinators. To evaluate the effect of pollinator size and foraging behavior on pollination efficiency, the intertegular distances (ITDs) of five bumble bees (Bombus pyrosoma, B. lantschouensis, B. melanurus, B. sichelii and B. sibiricus) and two honey bees (Apis mellifera and A. cerana) were evaluated. Their foraging activities and single visit depositions (SVDs) on oilseed rape were observed and counted. The ITDs of bumble bees were significantly larger than honey bees (P < 0.05). The single-visit duration of A. mellifera (2.43 ± 0.06 s) was significantly longer than other bees (P < 0.05). The interval time of the A. cerana was longer than other bees (P < 0.05). Larger bees tended to deposit more pollen on stigmas at each visit than smaller bees. The interspecific ITD of bees had a positive effect on SVD (P < 0.001). There was no correlation between ITD and SVD among individuals of the same species. In conclusion, the size of bees had a positive impact on pollination efficiency. Bees with a greater ITD deposited substantially more pollen and had a significantly shorter interval between flowers. Understanding the foraging behavior and pollination efficiency of these bee species and developing habitats to support them should enable growers to achieve resilient production.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Sources of genetic diversity in feral oilseed rape (Brassica napus) populations

Feral oilseed rape populations on roadsides and along other semi-natural habitats contribute to the uncertainty regarding the potential risk of transgene escape from genetically modified (GM) oilseed rape to wild or weedy populations. Knowledge about the underlying mechanism of establishment, persistence, and the evolutionary potential of feral populations is a prerequisite for a successful risk assessment and of high policy relevance. We analysed the origin, persistence, and genetic variation of feral oilseed rape populations in northwest Germany based on a field survey (2004–2007) and nuclear microsatellite markers. A maximum-likelihood approach was used to allocate individuals of feral populations to their source varieties and to hybrids between these varieties. The majority (72%) of the analysed sites was inhabited by oilseed rape for at least 2 years. The proportion of feral populations setting seeds varied between years (30–48%) and was higher than in comparable studies. Within-population genetic diversity of feral oilseed rape populations was higher than that of the common grown varieties studied. Repeated escapes of different varieties and hybridisation between these varieties were identified as the most important sources of genetic variation in feral oilseed rape populations. Up to four different source varieties were identified in feral populations. The detection of three hybrids between different varieties indicates that feral oilseed rape populations may persist via self-recruitment. Our results highlight the evolutionary potential of feral oilseed rape populations and have consequences for approaches to regulate the coexistence of genetically modified and non-GM oilseed rape varieties in the same region. Feral populations may serve as stepping stones for intraspecific and interspecific gene flow and can promote persistence of transgenes outside cultivation. The likelihood of introgressive interspecific hybridisation depends on the abundance of crossing partners, which may vary strongly from region to region.     

Genetic control of oil content in oilseed rape (Brassica napus L.)

In oilseed rape (Brassica napus L.) like in most oleaginous crops, seed oil content is the main qualitative determinant that confers its economic value to the harvest. Increasing seed oil content is then still an important objective in oilseed rape breeding. In the objective to get better knowledge on the genetic determinism of seed oil content, a genetic study was undertaken in two genetic backgrounds. Two populations of 445 and a 242 doubled haploids (DH) derived from the crosses “Darmor-bzh” × “Yudal” (DY) and “Rapid” × “NSL96/25” (RNSL), respectively, were genotyped and evaluated for oil content in different trials. QTL mapping in the two populations indicate that additive effects are the main factors contributing to variation in oil content. A total of 14 and 10 genomic regions were involved in seed oil content in DY and RNSL populations, respectively, of which five and two were consistently revealed across the three trials performed for each population. Most of the QTL detected were not colocalised to QTL involved in flowering time. Few epistatic QTL involved regions that carry additive QTL in one or the other population. Only one QTL located on linkage group N3 was potentially common to the two populations. The comparisons of the QTL location in this study and in the literature showed that: (i) some of the QTL were more consistently revealed across different genetic backgrounds. The QTL on N3 was revealed in all the studies and the QTL on N1, N8 and N13 were revealed in three studies out of five, (ii) some of the QTL were specific to one genetic background with potentially some original alleles, (iii) some QTL were located in homeologous regions, and (iv) some of the regions carrying QTL for oil content in oilseed rape and in Arabidopsis could be collinear. These results show the possibility to combine favourable alleles at different QTL to increase seed oil content and to use Arabidopsis genomic data to derive markers for oilseed rape QTL and identify candidate genes, as well as the interest to combine information from different segregating populations in order to build a consolidated map of QTL involved in a specific trait.     

Frequency and distance of pollen dispersal from transgenic oilseed rape (Brassica napus)

The objective of this study was to evaluate pollen dispersal inBrassica napus (oilseed rape). The selectable marker, used to follow pollen movement, was a dominant transgene (bar) conferring resistance to the herbicide glufosinate-ammonium. Transgenic and non-transgenic plants of the cultivar Westar were planted in a 1.1 ha field trial, with the transgenic plants in a 9 m diameter circle at the centre, surrounded by non-transgenic plants to a distance of at least 47 m in all directions. A 1 m circle of non-transgenic plants was sown in the centre of the transgenic area to allow estimation of the level of pollen dispersal when plants were in close contact. Honeybee hives were placed at the trial site to optimize the opportunity for cross-pollination. During the flowering period, regular observations were made of the number of plants flowering and the number and type of insects present in 60 1 m² areas. These areas were located uniformly around the plot at distances of 1, 3, 6, 12, 24, 36 and 47 m from the edge of the 9 m circle of transgenic plants. Seed samples were harvested from each of the 7 distances so that approximately 20% of the circumference of the plot was sampled at each distance. The centre non-transgenic circle was also sampled. Plants were grown from the seed samples and sprayed with glufosinate to estimate the frequency of pollen dispersal at each distance. In order to screen enough samples to detect low frequency cross-pollination events, seed samples were tested in the greenhouse and on a larger scale in the field. Results were confirmed by testing progeny for glufosinate resistance and by Southern blot analysis. The estimated percentage of pollen dispersal in the non-transgenic centre circle was 4.8%. The frequency was estimated to be 1.5% at a distance of 1 m and 0.4% at 3 m. The frequency decreased sharply to 0.02% at 12 m and was only 0.00033% at 47 m. No obvious directional effects were detected that could be ascribed to wind or insect activity.     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Molecular markers for low-glucosinolate alleles in oilseed rape (Brassica napus L.)

Two recent studies have mapped QTLs associated with the level of seed glucosinolates in oilseed rape (Brassica napus L.). It was likely that the two most significant QTLs identified in each study were the same, as they were linked to RFLP alleles identified by common DNA probes. To investigate the utility of these probes in breeding programmes, they were used to study RFLPs in a range of low- and high-glucosinolate cultivars and breeding lines. It was shown that all low glucosinolate spring and winter cultivars possessed a specific RFLP fragment identified by probe wg3f7 which is linked to theGSL-1 QTL, and all high-glucosinolate cultivars possessed a specific RFLP fragment identified by probe wg7a8, which is linked to theGSL-2 QTL. Cultivar Ariana, which has intermediate levels of glucosinolates possessed the low-glucosinolate fragment atGSL-1 but the high-glucosinolate fragment atGSL-2. A similar result was found with the cvs. Martina and Bronowski which have intermediate and variable levels of glucosinolates. There were no other RFLP fragments identified by other DNA probes which were specific to either the low- or high-glucosinolate phenotypes. The use of probes wg3f7 and wg7a8 in selection of low-glucosinolate lines in breeding programmes is discussed.