Costs of transgenic herbicide resistance introgressed from Brassica napus into weedy B. rapa

Wild relatives of genetically engineered crops can acquire transgenic traits such as herbicide resistance via spontaneous crop–wild hybridization. In agricultural weeds, resistance to herbicides is often a beneficial trait, but little is known about possible costs that could affect the persistence of this trait when herbicides are not used. We tested for costs associated with transgenic resistance to glufosinate when introgressed into weedy Brassica rapa . Crosses were made between transgenic B. napus and wild B. rapa from Denmark. F₁ progeny were backcrossed to B. rapa and BC₁ plants were selected for chromosome numbers similar to B. rapa . Further backcrossing resulted in a BC₂ generation that was hemizygous for herbicide resistance. We quantified the reproductive success of 457 BC₃ progeny representing six full-sib families raised in growth rooms (plants were pollinated by captive bumblebees). Pollen fertility and seed production of BC₃ plants were as great as those of B. rapa raised in the same growth rooms. Segregation for herbicide resistance in BC₃ plants was 1:1 overall, but the frequency of resistant progeny was lower than expected in one family and higher than expected in another. There were no significant differences between transgenic and nontransgenic plants in survival or the number of seeds per plant, indicating that costs associated with the transgene are probably negligible. Results from this growth-chamber study suggest that transgenic resistance to glufosinate is capable of introgressing into populations of B. rapa and persisting, even in the absence of selection due to herbicide application.     

Gene transferability from transgenic Brassica napus L. to various subspecies and varieties of Brassica rapa

Gene transferability from transgenic rapeseed to various subspecies and varieties of Brassica rapa was assessed in this study. Artificial crossability was studied in 118 cultivars of 7 B. rapa subspecies and varieties with the transgenic rapeseed GT73 (Brassica napus) as the pollen donor. On average 5.7 seeds were obtained per pollination, with a range from 0.05 to 19.4. The heading type of B. rapa L. showed significantly higher crossability than non-heading types of B. rapa. The spontaneous outcrossing rate between B. rapa (female) and the transgenic rapeseed Ms8 × Rf3 (B. napus) (male) ranged from 0.039 to 0.406%, with an average of 0.19%. The fertilization process and the development of the hybrid seeds as shown by fluorescent staining techniques indicated that the number of adhered pollens on the stigma was reduced by 80%, the number of pollen tubes in the style was reduced by 2/3 and the fertilization time was delayed by over 20 h when pollinated with the transgenic rapeseed Ms8 × Rf3 in comparison with the bud self-pollination of B. rapa as control. About 10–70% of the interspecific hybrid embryos were aborted in the course of development. Some seeds looked cracked in mature pods, which showed germination abilities lower than 10%. The spontaneous outcrossing rates were much lower than the artificial crossability, and their survival fitness of the interspecific hybrid was very low, indicating that it should be possible to keep the adventitious presence of the off-plants under the allowed threshold, if proper measures are taken.     

Slug herbivory on hybrids of the crop Brassica napus and its wild relative B. rapa

Some plant species can cross with each other but stay nevertheless distinct with little gene flow between them. Selective herbivory could explain this pattern when hybrids are more susceptible or intermediate between their parents. We performed choice and no-choice experiments with the slug Arion lusitanicus to test this hypothesis for the crop Brassica napus, wild B. rapa and their backcross hybrids. In both experiments slugs greatly preferred B. napus over B. rapa while average herbivory on backcross hybrids was intermediate. Concentrations of aliphatic glucosinolates in the hybrids were intermediate between the parental species. Slug herbivory potentially reduces establishment of backcross hybrids. This hypothesis needs to be tested in the field.     

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.     

芸薹属(Brassica napus,B.rapa,B.oleracea) CAMTA3基因家族的鉴定及生物信息学分析

芸薹属(Brassica)植物(甘蓝型油菜,白菜,甘蓝)是重要的经济作物,它们之间的关系可用禹氏三角表示.CAMTA是在进化过程中高度保守的一类转录因子家族,该家族成员在芸薹属植物抗逆境胁迫过程中具有重要作用.为了探讨芸薹属CAMTA3基因家族的功能,本研究利用生物信息学手段,鉴定了芸薹属(白菜,甘蓝和甘蓝型油菜)CAMTA3基因家族的8个基因,并从蛋白质的理化性质、家族进化与基因结构和上游启动子等方面进行了分析研究.分析表明,芸薹属CAMTA3基因家族8个成员在进化过程中具有较高的保守性,其亲缘关系与禹氏三角理论相符,且各成员在植物响应抗胁迫过程中占重要地位.研究结果为芸薹属CAMTA3基因家族的功能研究提供了一定的信息基础.     

Inheritance of rapeseed (Brassica napus)-specific RAPD markers and a transgene in the cross B. juncea x (B. juncea x B. napus)

We have examined the inheritance of 20 rapeseed (Brassica napus)-specific RAPD (randomly amplified polymorphic DNA) markers from transgenic, herbicide-tolerant rapeseed in 54 plants of the BC₁ generation from the cross B. junceax(B. junceaxB. napus). Hybridization between B. juncea and B. napus, with B. juncea as the female parent, was successful both in controlled crosses and spontaneously in the field. The controlled backcrossing of selected hybrids to B. juncea, again with B. juncea as the female parent, also resulted in many seeds. The BC₁ plants contained from 0 to 20 of the rapeseed RAPD markers, and the frequency of inheritance of individual RAPD markers ranged from 19% to 93%. The transgene was found in 52% of the plants analyzed. Five synteny groups of RAPD markers were identified. In the hybrids pollen fertility was 0–28%. The hybrids with the highest pollen fertility were selected as male parents for backcrossing, and pollen fertility in the BC₁ plants was improved (24–90%) compared to that of the hybrids.     

Frequency-dependent fitness of hybrids between oilseed rape (Brassica napus) and weedy B. rapa (Brassicaceae)

Fitness of interspecific hybrids is sometimes high relative to their parents, despite the conventional belief that they are mostly unfit. F(1) hybrids between oilseed rape (Brassica napus) and weedy B. rapa can be significantly more fit than their weedy parents under some conditions; however, under other conditions they are less fit. To understand the reasons, we measured the seed production of B. napus, B. rapa, and different generations of hybrid plants at three different densities and in mixtures of different frequencies (including pure stands). Brassica napus, B. rapa, and backcross plants (F(1) ♀ × B. rapa) produced many more seeds per plant in pure plots than in mixtures and more seeds in plots when each was present at high frequency. The opposite was true for F(1) plants that produced many more seeds than B. rapa in mixtures, but fewer in pure stands. Both vegetative and reproductive interactions may be responsible for these effects. Our results show that the fitness of both parents and hybrids is strongly frequency-dependent and that the likelihood of introgression of genes between the species thus may depend on the numbers and densities of parents and their various hybrid offspring in the population.     

Transfer of resistance to clubroot (Plasmodiophora brassicae) to swedes {Brassica napus L. var. napobrassica Peterm) from B. rapa

In 1975, tests with UK populations of Plasmodiophora brassicae not only revealed a lack of effective clubroot resistance in swedes (Brassica napus), but also the outstanding resistance of the European Clubroot Differential (ECD)04 (B. rapa). It was, therefore, decided to transfer the resistance genes from ECD04 to swedes, using the most pathogenic UK population of clubroot (C56) available for screening purposes. An autotetraploid form of ECD04 was crossed with tetraploid kale (B. oleracea) using the latter as female parent. One of the euploid, 2n = 38, hybrids secured by embryo rescue in 1976 was crossed to the swede cultivars Marian and Ruta Øtofte. Three further backcrosses of clubroot resistant plants to lines derived from modern swede cultivars were made over the period 1980 to 1982. Selfing commenced in 1983 to produce F₂ populations. From F₃ to F₅ there was family selection for yield and agronomic characters, as well as single plant selection for clubroot resistance. In 1991, the six most promising F₅ families were multiplied for subsequent evaluation in replicated yield trials in Dundee. The most promising family entered official trials at the beginning of 1993 and, 2 years later, was added to the National List as cv. Invitation and granted Plant Breeders' Rights. The first certified seed was sold in 1996, 20 years after the original synthetic B. napus was produced. The breeding programme provided evidence for only one of the three postulated dominant genes in ECD04 being required for resistance to C56 and also good evidence of differential resistance from tests with other clubroot populations. Hence, whilst the differential resistance in cv. Invitation should prove useful in the UK in the immediate future, it may not be durable in the longer term. It is, therefore, argued that the next and more difficult goal to achieve should be to introduce high levels of non-differential resistance from B. oleracea.     

Male fitness of oilseed rape (Brassica napus), weedy B. rapa and their F(1) hybrids when pollinating B. rapa seeds

The likelihood that two species hybridise and backcross may depend strongly on environmental conditions, and possibly on competitive interactions between parents and hybrids. We studied the paternity of seeds produced by weedy Brassica rapa growing in mixtures with oilseed rape (B. napus) and their F(1) hybrids at different frequencies and densities. Paternity was determined by the presence of a transgene, morphology, and AFLP markers. In addition, observations of flower and pollen production, and published data on pollen fertilisation success, zygote survival, and seed germination, allowed us to estimate an expected paternity. The frequency and density of B. napus, B. rapa, and F(1) plants had a strong influence on flower, pollen, and seed production, and on the paternity of B. rapa seeds. Hybridisation and backcrossing mostly occurred at low densities and at high frequencies of B. napus and F(1), respectively. F(1) and backcross offspring were produced mainly by a few B. rapa mother plants. The observed hybridisation and backcrossing frequencies were much lower than expected from our compilation of fitness components. Our results show that the male fitness of B. rapa, B. napus, and F(1) hybrids is strongly influenced by their local frequencies, and that male fitness of F(1)hybrids, when pollinating B. rapa seeds, is low even when their female fitness (seed set) is high.     

Spontaneous gene flow from rapeseed (Brassica napus) to wild Brassica oleracea

Research on the environmental risks of gene flow from genetically modified (GM) crops to wild relatives has traditionally emphasized recipients yielding most hybrids. For GM rapeseed (Brassica napus), interest has centred on the 'frequently hybridizing' Brassica rapa over relatives such as Brassica oleracea, where spontaneous hybrids are unreported in the wild. In two sites, where rapeseed and wild B. oleracea grow together, we used flow cytometry and crop-specific microsatellite markers to identify one triploid F1 hybrid, together with nine diploid and two near triploid introgressants. Given the newly discovered capacity for spontaneous introgression into B. oleracea, we then surveyed associated flora and fauna to evaluate the capacity of both recipients to harm cohabitant species with acknowledged conservational importance. Only B. oleracea occupies rich communities containing species afforded legislative protection; these include one rare micromoth species that feeds on B. oleracea and warrants further assessment. We conclude that increased attention should now focus on B. oleracea and similar species that yield few crop-hybrids, but possess scope to affect rare or endangered associates.     

Integration of Brassica A genome genetic linkage map between Brassica napus and B. rapa.

An integrated linkage map between B. napus and B. rapa was constructed based on a total of 44 common markers comprising 41 SSR (33 BRMS, 6 Saskatoon, and 2 BBSRC) and 3 SNP/indel markers. Between 3 and 7 common markers were mapped onto each of the linkage groups A1 to A10. The position and order of most common markers revealed a high level of colinearity between species, although two small regions on A4, A5, and A10 revealed apparent local inversions between them. These results indicate that the A genome of Brassica has retained a high degree of colinearity between species, despite each species having evolved independently after the integration of the A and C genomes in the amphidiploid state. Our results provide a genetic integration of the Brassica A genome between B. napus and B. rapa. As the analysis employed sequence-based molecular markers, the information will accelerate the exploitation of the B. rapa genome sequence for the improvement of oilseed rape.     

Competitive interactions and the effect of herbivory on Bt-Brassica napus, Brassica rapa and Lolium perenne

1.  The probability of a transgenic crop establishing a feral population outside cultivated areas and possibly outcompeting naturally occurring species needs to be assessed to make an ecological risk assessment of the transgenic crop.
2.  The interaction between herbivory and competition is thought to determine the ecological success of insect-resistant plants, and this interaction was investigated in a competition experiment with transgenic insect-resistant Bt- Brassica napus , Brassica rapa , Lolium perenne , and herbivory from the large white butterfly Pieris brassicae .
3.  As expected, herbivory had a negative effect on the biomass of B. rapa at high plant densities. The competitive ability of L. perenne , when growing with B. rapa , increased significantly with the level of herbivory on B. rapa .
4.  To predict the effect of herbivory in a natural ecosystem, plant competition between the two annual Brassica species was analysed in a population ecological model. It was concluded that it is probable that transgenic Bt- B. napus plants may invade a natural habitat if herbivory is sufficiently high and the habitat is suitable for B. napus .
5.   Synthesis and applications . The results indicate that it is important to study the interaction between herbivory and competition when assessing the ecological risk of insect-resistant genetically modified crops. Furthermore, combining ecological data from manipulated experiments with population ecological modelling is a fruitful approach when conducting environmental risk assessments.     

Low probability of chloroplast movement from oilseed rape (Brassica napus) into wild Brassica rapa

Pollen-mediated movement of transgenes from transplastomic oilseed rape (Brassica napus) into wild relatives will be avoided if chloroplasts are maternally transmitted. We assess the probability of chloroplast exchange between conventional oilseed rape and wild Brassica rapa to model the future behavior of transplastomic cultivars. Primers specific to cpDNA were used to demonstrate maternal inheritance of chloroplasts in 47 natural hybrids between cultivated B. napus and wild B. rapa. We conclude that there will be no or negligible pollen-mediated chloroplast dispersal from oilseed rape. Transgene introgression could still occur in mixed populations, however, if B. napus acted as the recurrent female parent. Rate of transfer would then depend on the abundance of mixed populations, their persistence as mixtures, and hybridization frequency within stands. A low incidence of sympatry (0.6-0.7%) between wild B. rapa and cultivated B. napus along the river Thames, UK, in 1997 and 1998, suggests mixed stands will form only rarely. Eighteen feral populations of B. napus also showed a strong tendency toward rapid decline in plant number, seed return, and ultimately, extinction within 3 years. Conversely, hybrid production is significant in mixed stands, and the absence of control practices means that oilseed rape will have slightly greater persistence. We infer that some introgression from transplastomic B. napus into B. rapa is inevitable in mixed populations even though such populations will occur infrequently and will tend to lose B. napus plants relatively quickly. Chloroplast exchange will be extremely rare and scattered.     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

转基因油菜的基因流及生态风险

综合评述了转基因油菜的基因流及其生态风险.油菜作为最早的转基因作物之一目前已在加拿大和澳大利亚大面积商业化应用.(常)异花授粉作物油菜的天然异交率可达30%左右,也易与其它芸苔属作物杂交,因此转基因油菜的生态风险已引起各国科学家的高度重视.转基因油菜主要通过与其野生近缘种的花粉交换和与非转基因油菜的花粉交换两种方式进行花粉的输出.基因可能逃逸到相关野生近缘种,但在大田环境下能够得到杂种的可能性很小;由于基因的漂流在油菜田块间确实存在,因此在种植转基因油菜的过程中必须考虑其间隔距离.     

GM risk assessment: Pollen carriage from Brassica napus to B. rapa varies widely between pollinators

Characterizing insect pollen carriage between closely related plant species is especially challenging where source species possess morphologically identical pollen and share many pollinators in common. Here, we use an SNP-based assay using the plant DNA barcoding locus matK to characterize pollen carriage between cultivated Brassica napus and wild Brassica rapa in three sites across southern England. The assay differentiated B. napus and B. rapa pollen carried by honey bees (Apis melifera), bumblebees (Bombus spp.), mining bees (Andrena spp.) and hoverflies (Syrphidae) captured on B. napus plants 1–2 m from wild B. rapa, and on B. rapa plants at various distances from the crop . Apis individuals foraging on B. rapa and carrying B. napus pollen were rarely found beyond 10 m from the crop. However, Bombus and Andrena individuals captured on B. rapa occasionally carried crop pollen up to 300 m from the source field. Hoverflies carried less pollen overall but featured high proportions of B. napus pollen even at the most distant capture points. We predict that different pollinator species will evoke markedly different patterns of interspecific hybrid formation. We conclude that more exhaustive surveys of this kind will help parameterize future mechanistic models to predict the distribution of hybrids between Genetically Modified B. napus and B. rapa on a landscape scale.     

Hybridisation and introgression between Brassica napus and B. rapa in the Netherlands

We used flow cytometry, chromosome counting and AFLP markers to investigate gene flow from the crop plant oilseed rape, Brassica napus (AACC) to wild B. rapa (AA) in the Netherlands. From 89 B. napus source populations investigated, all near cropping fields or at transhipment sites, only 19 contained a B. rapa population within a 2.5‐km radius. During our survey we found only three populations with F1 hybrids (AAC), as recognized by their nine extra chromosomes and by flow cytometry. These hybrids were all collected in mixed populations where the two species grew in close proximity. Populations with F1 hybrids were not close to crops, but instead were located on road verges with highly disturbed soils, in which both species were probably recruited from the soil seed bank. Many plants in the F2, BC1 or higher backcrosses are expected to carry one to eight C chromosomes. However, these plants were not observed among the hybrids. We further investigated introgression with molecular markers (AFLP) and compared sympatric B. rapa populations (near populations of B. napus) with control populations of B. rapa (no B. napus within at least 7 km). We found no difference between sympatric and control populations in the number of C markers in B. rapa, nor did we find that these sympatric populations closely resembled B. napus. Our data show that hybrids occur but also suggest no recent introgression of alleles from the crop plant B. napus into wild B. rapa in the Dutch populations studied.     

Interspecific hybrids between a transgenic rapeseed (Brassica napus) and related species: cytogenetical characterization and detection of the transgene.

In interspecific hybrids produced between a transgenic rapeseed, an allotetraploid species, resistant to herbicide, phosphinotricin, and five diploid related species, the risk for gene introgression in weed genomes was explored through cytogenetic and bar gene characterizations. Among the 75 hybrids studied, most had the expected triploid structure, with the exception of B. napus - B. oleracea amphidiploid plants and one B. napus - S. arvensis amphidiploid plant. In triploid hybrid plants, the reciprocal hybrids did not exhibit any difference in their meiotic behavior. The comparison of the percentage of chromosome pairing in the hybrids with that of haploid rapeseed permit to conclude that allosyndesis between AC genomes and related species genomes took place. This possibility of recombination was confirmed by the presence of multivalent associations in all the interspecific hybrids. Nevertheless, in B. napus - B. adpressa hybrids a control of chromosome pairing seemed to exist. The possibility of amphidiploid plant production directly obtained in the F1 generation increased the risk of gene dispersal. The B. napus - B. oleracea amphidiploid plant presented a meiotic behavior more regular than that of the B. napus - S. arvensis amphidiploid plant. Concerning the herbicide bar gene characterization, the presence of the gene detected by DNA amplification was correlated with herbicide resistance, except for two plants. Different hypotheses were proposed to explain these results. A classification of the diploid species was established regarding their gene dispersal risk based on the rate of allosyndesis between chromosomes of AC genomes of rapeseed and the genomes of the related species.     

RFLP和AFLP分析白菜型油菜和甘蓝型油菜遗传多样性及其在油菜改良中的应用价值

采用RFLP和AFLP标记对来自中国和欧美的7份甘蓝型油菜和22份白菜型油菜进行了遗传多样性分析。在这29份材料中,166个酶-探针组合和2对AFLP引物共检测到1477个RFLP标记和183个AFLP标记。RFLP数据显示以拟南芥EST克隆作探针比用油菜基因组克隆做探针能检测到更多的多态性位点,且采用EcoR Ⅰ或BamH Ⅰ酶切比HindⅢ酶切多态性好,白菜型油菜和甘蓝型油菜中基因的拷贝数平均都为3个左右。UPGMA聚类分析表明中国白菜型油菜的遗传多样性比甘蓝型油菜和欧美白菜型油菜丰富,欧美甘蓝型油菜与欧美白菜型油菜聚为一类,而与中国甘蓝型油菜差异更大。中国白菜型油菜丰富的遗传多样性为中国甘蓝型油菜的改良提供了宝贵的资源,揭示了利用白菜型油菜A基因组和甘蓝型油菜A基因组间亚基因组杂种优势的可能性。