Consequences of gene flow between oilseed rape (Brassica napus) and its relatives

Plant Science 211 (2013) 42–51

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Review

Consequences of gene flow between oilseed rape (Brassica napus) and its relatives Yongbo Liu a,b,c , Wei Wei b , Keping Ma b , Junsheng Li a , Yuyong Liang d , Henri Darmency c,∗ a

State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, 20 Nanxincun, Beijing 100093, China INRA, UMR1347 Agroécologie, 17 rue Sully, Dijon BP 86510, 21065, France d Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, China b c

a r t i c l e

i n f o

Article history: Received 9 November 2012 Received in revised form 4 July 2013 Accepted 6 July 2013 Available online 12 July 2013 Keywords: Gene flow Hybridization Introgression Inter-generic Mitigation Brassica

a b s t r a c t Numerous studies have focused on the probability of occurrence of gene flow between transgenic crops and their wild relatives and the likelihood of transgene escape, which should be assessed before the commercial release of transgenic crops. This review paper focuses on this issue for oilseed rape, Brassica napus L., a species that produces huge numbers of pollen grains and seeds. We analyze separately the distinct steps of gene flow: (1) pollen and seeds as vectors of gene flow; (2) spontaneous hybridization; (3) hybrid behavior, fitness cost due to hybridization and mechanisms of introgression; (4) and fitness benefit due to transgenes (e.g. herbicide resistance and Bt toxin). Some physical, biological and molecular means of transgene containment are also described. Although hybrids and first generation progeny are difficult to identify in fields and non-crop habitats, the literature shows that transgenes could readily introgress into Brassica rapa, Brassica juncea and Brassica oleracea, while introgression is expected to be rare with Brassica nigra, Hirschfeldia incana and Raphanus raphanistrum. The hybrids grow well but produce less seed than their wild parent. The difference declines with increasing generations. However, there is large uncertainty about the evolution of chromosome numbers and recombination, and many parameters of life history traits of hybrids and progeny are not determined with satisfactory confidence to build generic models capable to really cover the wide diversity of situations. We show that more studies are needed to strengthen and organize biological knowledge, which is a necessary prerequisite for model simulations to assess the practical and evolutionary outputs of introgression, and to provide guidelines for gene flow management. © 2013 Elsevier Ireland Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of gene flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Seed movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Pollen dispersal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hybridizations between Brassica napus and its wild relatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Primary gene pool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Other gene pools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consequences of hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Effects of hybridization on the plant genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Effects of hybridization on morphology traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Effects of hybridization on plant growth and reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Introgression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consequences of transgenes flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Herbicide-resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Other transgenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +33 380 693186; fax: +33 380 693262. E-mail addresses: [email protected], [email protected] (H. Darmency). 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.07.002

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Means for reducing gene flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Physical means . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Biological means . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Molecular means . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The risks of introducing genetically engineered organisms into the environment were anticipated before any such release [1]. Gene flow between transgenic crops and wild relatives is considered to be one of the possible undesirable consequences of the commercial release of transgenic crops conferring herbicide, insect or virus-resistance, modified protein or lipid content, different mating system, drought or frost resistance, etc. Commercialized transgenic varieties of many important crop species, such as soybean, rice, wheat, maize, cotton, oilseed rape, alfalfa and sugar beet, are cultivated worldwide [2]. Most of these transgenic species could potentially hybridize with their wild relatives at some time or place [3]. Oilseed rape, Brassica napus L, is a likely candidate for gene flow because it produces a large number of pollen grains and seeds, and there are several closely related species [4]. Transgenic, herbicideresistant varieties are grown in Canada, USA, Chile and Australia, and other transgenic traits are being currently bred (but varieties with male sterility and restoration systems are no longer used) [2]. European regulatory authorities were reluctant to allow cultivation of transgenic oilseed rape because of concerns over gene flow toward both wild relatives and conventional varieties, and it is also not permitted in China. In previous times, gene flow was not identified by botanists and did not have major consequences because the species belonging to the same botanical family shared more or less similar traits, selection limited hybrid success, and the few existing hybrids were considered as natural variants of the species. For instance, before the release of the herbicide isoxaben, there was no selective herbicide of oilseed rape that killed weedy Raphanus raphanistrum: other herbicides were safe for both species, or killed both species, and hybrids had no benefit, just aneuploidy troubles. Now, atrazine, bromoxynil, imidazolinone, glyphosate and glufosinate herbicideresistant varieties could allow interspecific hybrids to express herbicide-resistance in various crops of the crop rotation, thus gaining higher survival value over years and, consequently, serving as bridges between species. Identifying past introgression between the crop and its wild relatives through genetic diversity analysis of weedy populations, and checking if there is any change in the life cycle and behavior of the introgressed populations, is a possible way to assess the impact of gene flow. However, this is a difficult task, because many molecular markers are shared by these species so that it is difficult to distinguish between genes inherited from a common ancestor or through gene flow. As gene flow via seeds or pollen is a basic biological process, the assessment of potential risks of transgenic plants should take into account transgene flow to: (1) wild relatives, because of conservation, biological resource and evolutionary concerns (e.g. for Brassica oleracea that is a protected species in many countries); and (2) weedy relatives, to prevent stacking of beneficial transgenes and crop mimicry in species that are already widespread, troublesome weeds. A recent review and a simulation modeling exercise dismissed the importance of gene flow for agriculture and environment [5,6], although another review pointed out the uncertainty of results of gene flow analyses [7]. We update previous reviews on

the occurrence of hybridization and introgression between oilseed rape, Brassica napus L., and wild relatives [3] and the potential consequences of gene flow. For this purpose, we examine separately the successive steps of the gene flow process. Hybridization depends on the crop occurring in the same area with a compatible relative (weedy, wild or crop), with overlapping flowering period, appropriate pollen and seed dispersal, and successful fertilization: these are prerequisites for gene flow, but not all hybridizations result in gene flow. The next step of gene flow is the behavior and fertility of hybrids. Introgression into a receiving species or population depends mainly on the fate of chromosome transmission, the evolution and fate of interspecific progeny, and their relative fitness and competitiveness, all of which are consequences of the hybridization process. The fate of transgenes depends on chromosome location and the potential costs and benefits of transgenes in the progeny. Separating distinct steps of gene flow is essential for imagining different mitigation strategies for gene flow between oilseed rape and relatives. 2. Mechanisms of gene flow Gene flow in B. napus can occur within a given field or across fields through long distance dispersal of pollen or seeds. Seed migration in time (i.e. through dormant seeds in the soil) and in space (Fig. 1) may result in new sources of plants that introduce transgenes in locations and years where oilseed rape fields are not present [8]. Pollen is the ordinary vehicle of the exchange of genetic information amongst related plants, including related species and genera with different genomes. 2.1. Seed movement Seed movement can occur through space and time. Transport and processing of transgenic crop seeds can lead to unwanted gene flow. Transgenic oilseed rape plants were detected at major ports and along roadsides and railways, and probably resulted from imported transgenic oilseed rape seeds where transgenic seeds were not commercially cultivated [5]. Where such varieties were cultivated, up to two thirds of the feral oilseed rape plants sampled along roadsides and railways were transgenic, but their contribution to seed admixture in the harvest of cultivated oilseed rape seems to be low enough to not trigger specific management [5]. Seed banks of dormant seeds can also build up in the soil because of crop seed loss at harvest [4]. Seed banks decrease rapidly with time, especially under no-till or minimum tillage seeding regimes as in Canada and Australia [4,9], but a few seeds can remain dormant over the longer time. Cultivar type, shallow cultivation and conditions of ploughing (date, depth and humidity) are suggested as key factors that control incorporation of seeds into the seed bank [10] and trigger inducible secondary dormancy [11]. The appearance of volunteers may continue for a long time after the oilseed rape crop was grown. In one study, a non-negligible proportion of the volunteers belonged to varieties cultivated 4–17 years earlier [12]. In another, herbicide-tolerant seedlings emerged ten years after a field trial [13]. Extensive transgene flow between oilseed rape varieties generated plants with three different types of stacked

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Fig. 1. Potential gene flow routes among genetically modified (GM) and conventional (Conv.) cultivars, volunteers and feral plants of Brassica napus, and wild or weedy relative species through pollen and seeds over space and time in production fields. Flowers of plants bearing the transgene are colored purple while those without the transgene are yellow. In the first year, GM pollen from transgenic crop plants fertilizes (blue arrows) B. napus in the adjacent field 2 as well as feral populations and related Brassica species nearby in other habitats (roadsides, waste places, natural ecosystems). Seeds are released (black arrows) mainly at harvest in field 1 and propagated by farm tools and trucks to field 2, roadsides and other habitats. In the second year, the transgene (*) is found in fields 1 and 2 (e.g. in wheat), and in roadsides and other habitats, and pollen flow occurs among volunteers, feral and weedy plants. Source: with permission of Nathalie Colbach.

herbicide resistance [14]. Volunteers having multiple-herbicideresistance (i.e. belonging to crosses among herbicide-resistant varieties) were found five to eight years after the last transgenic cultivar was grown in a multi-year, farm-scale study [15]. Feral populations could last up to 9 years [16] and, together with volunteers arising from the seed bank, can serve as noticeable source of pollen for gene flow to wild species even far from areas cultivated with oilseed rape. Hence, gene flow is not only a matter of geographically limited, instantaneous pollen flow, but can also happen in unsuspected locations and delayed times.

3. Hybridizations between Brassica napus and its wild relatives Apart from B. napus and Brassica juncea, all species within the tribe are mainly self-incompatible thanks to a pollen/pistil sporophytic recognition system [4]. Therefore, an isolated wild plant cannot produce seed unless foreign pollen reaches its flowers, which due to the absence of pollen competition is a perfect situation for interspecific crosses.

3.1. Primary gene pool 2.2. Pollen dispersal Oilseed rape is self-fertile, but produces a huge amount of pollen (more than 1012 grains ha−1 ) which are dispersed by both wind and insects [4]. Approximately half of the pollen produced by an individual plant fell within 3 m of the plant [17]. Generally, the regression model that best fit the relationship between pollen flow and the distance between pollen source and recipient population followed a fat-tailed negative power function [18]. However, pollen-mediated gene flow in oilseed rape is affected by a variety of factors, including flowering time, genotype, wind turbulence, wind speed and direction, distance between donor and recipient populations, insect type and movement, opening and position of flowers on plants, plant aggregation, inter-patch distances, size of the source population and pollen contributions of all surrounding plants [19]. No simple value can be given to indicate the risk of pollen flow. Realized pollen flow, i.e. that effectively fertilizes oilseed rape flowers, ranges from 12 to 47% when plants are in close proximity in the field [4]. The majority of cross fertilization occurs between plants less than 10 m apart, while it rarely exceeds 1% at distances farther than 30 m [20]. Random distributions of oilseed rape pollen with isolated pollination events were detected several kilometers away from a pollen source [21], distributions that currently escape model prediction.

Amongst the Brassicaceae, consisting of over 3000 species in 370 genera, the species most subjected to gene flow belong to the primary gene pool of oilseed rape. Three species, B. napus (AACC, 2n= 38), B. juncea (AABB, 2n= 36), and Brassica carinata (BBCC, 2n= 34), are allotetraploids derived from three diploid species Brassica nigra (BB, 2n= 16), Brassica oleracea (CC, 2n= 18), and Brassica rapa (AA, 2n= 20). Spontaneous hybrids among B. napus and the five other species have occurred, albeit with varying difficulty. Spontaneous crosses frequently occur between B. napus and B. rapa [22] or B. juncea [23]. A variable frequency of hybrids was obtained according to plant distances and relative proportions of the species [24], which reflects the composition of the available local pollen cloud, a property that can be anticipated by model simulation [18]. From 1 to 17% of individuals were observed to be hybrids in natural populations of B. rapa in oilseed rape fields [25], close to oilseed rape fields [26], and in arable fields with a presumed history of oilseed rape cultivation [27]. Spontaneous hybrids between B. napus and B. oleracea were detected using flow cytometry and crop-specific microsatellite markers in B. oleracea wild populations [28]. No natural crosses have been reported between the three allotetraploid species and B. nigra [29], but male sterile B. napus produced spontaneous hybrids when placed in a B. nigra stand [30]: this last cross could become of greater importance with the increasing

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number of hybrid varieties because a part of their progeny, which could grow as volunteers, is male sterile. No spontaneous crosses have been reported for the other combinations, although it is likely to be possible as hybridization of B. napus × B. carinata and B. juncea × B. carinata have been achieved by hand pollination [31], and hybrids between B. rapa and B. carinata were only obtained when B. carinata was used as the female parent [32]. To date, hybrids between B. rapa and B. nigra were obtained by ovule culture only [33]. 3.2. Other gene pools The necessity of using in vitro embryo rescue techniques to obtain interspecific hybrids even in the primary gene pool [34] was considered by breeders sufficient evidence to sustain an initial belief that natural crosses outside the primary gene pool were highly unlikely. Indeed, other interspecific hybrids can be obtained with other species, but they generally require artificial means as no such hybrids or hybrid descendants have been reported to have occurred spontaneously [35]. In particular, no natural crosses occurred in field experiments between the three allotetraploid Brassica species and Sinapis arvensis (SarSar, 2n = 18) [29], indicating that direct transgene escape from B. napus to S. arvensis appears very unlikely [36]. However, spontaneous hybridization between B. napus and wild radish (R. raphanistrum, RrRr, 2n = 18) can occur in the field at low frequency on R. raphanistrum as the female parent [37], and numerous hybrids were easily produced on male sterile B. napus as the female parent [38]. Genetic polymorphism was observed for pre-zygotic barriers to interspecific hybridization, probably incompatibility genes [39]: first, it was found that oilseed rape pollen could not germinate on the stigmas of some R. raphanistrum plants, but do germinate on other plants from the same population; second, in some R. raphanistrum plants, the pollen tubes entering the ovary cannot target any ovule, while in other plants the rate of fertilization is the same as between R. raphanistrum congeners [39]. A variable capacity for interspecific fertilization was also observed among oilseed rape varieties [40]. This could explain why detection or frequency of hybrids varied among studies and populations [41], precluding estimation of actual hybridization potential. Similarly, reciprocal spontaneous hybridizations were found between B. napus and hoary mustard (Hirschfeldia incana, AdAd, 2n = 14) [42], but depended on the genotype of the wild plants [43]. It is likely that plants having weak interspecific incompatibility genes should be more prone to producing hybrids so that, in the case of a transgene providing high selective value to hybrids, the frequency of these genes would increase in wild populations subjected to repeated successful crosses with B. napus; thus increasing the potential for successful interspecific crosses.

4. Consequences of hybridization Successful hybridization results in viable F1 hybrid plants, whose subsequent survival and reproduction are essential for gene flow. The fate of the resulting plants depends on the segregation of parental traits and chromosome transmission, which are generally independent of the transgene, except in the case of linkage with genes encoding selectively advantageous traits. Introgression results from the sum of these processes which incorporate crop genes in the gene pool of a wild species. 4.1. Effects of hybridization on the plant genome The plant breeding literature reports many cases of artificial hybridization and introgression but focuses only on crop lineages,

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so that it is of little value for predicting the behavior of introgression in wild species. Most of the interspecific hybrids described above with B. napus (AACC) have half of the genome of each parent, which makes, in the case of a cross with a diploid species, a triploid structure ACX (with X possibly A, B, C, Sar, Rr, Ad), which is clearly seen using genomic in situ hybridization techniques [44]. However, some hybrid plants can result spontaneously from unreduced gametes with AACCX and AACCXX structures, which could eventually generate new polyploid species because the more balanced chromosome formula reduces the possibility of reproductive problems. Spontaneous polyploidization of triploid hybrids may also contribute to successful gene flow. Although stable polyploids have seldom been observed in recent experiments with transgenic material [22], they could have dramatic impact on new species formation and plant invasion [45]. B. napus itself is a relatively young species with a wide diversity of forms, and Brassicoraphanus (AARrRr, 4n = 36) was selected less than a century ago [46]. The direct effect of these structures on plant growth has been poorly studied, except for pollen fertility, which was much higher in amphiploid than triploid hybrids. In triploids, although non homologous, the three genomes show chromosome pairing, sometimes with tri- and quadrivalents, which indicates possible gene recombination. The presence of molecular markers of the Cgenome in 40% of the plants of a natural B. rapa population [27] could therefore result from recombination or represent a transient stage of the purge of C-chromosomes after hybridization. Inheritance studies of molecular markers of the different chromosomes show evidence of recombination between genomes of B. napus with B. rapa in experimental populations [47], with B. juncea [48], and with R. raphanistrum [49]. However, in the case of RAPD and AFLP markers, it is not possible to exclude the erroneous, specific attribution of some bands, which could lead to exaggerated observed recombination rates and gene flow potential. In triploid hybrids, meiosis is far from regular and the chromosome distribution in the progeny has various patterns according to the parent to which the hybrid was backcrossed, the generation, and the chromosome [27,44,47–49]. The stability of the hybrid progeny chromosome formula is a very important issue for successful versus poor (or absence of) gene introgression to wild relatives, according to the difference in genome type between B. napus and its wild relatives. The presence of a closely parented homologous genome is favorable to recover a stable XX formula. In the case of B. napus × B. rapa triploid hybrids, the presence of the A genome in both parent species results in almost 100% transmission of the A chromosomes in the progeny. In contrast, C chromosomes can be variably transmitted in backcross to B. rapa at rates less than 50%, resulting in extra, unpaired C chromosomes in the progeny [47]. Thereafter, the number of C chromosomes had to decrease to eventually conform to that of a genuine B. rapa with 2n = 20, AA. There are very few data on counting chromosomes of series of backcrossed plants, and this is missing to get a clear picture of what occurs in a lineage. In B. rapa, a unique set of data from BC1 to BC6 was used for modeling the presence of these extra chromosomes in the progeny [50]. Models have value in understanding how many generations could the C chromosomes be stable in hybrid progenies and in wild populations under various types and intensities of environmental selection. However, their weakness stems from the fact that the same data set that is always used for parameterization [6]. In the case of R. raphanistrum, again a unique set of data was modeled to examine those parameters which influence the rate of decrease of the chromosome number and its relationship to fertility [49]. There is clearly a lack of independent data on progeny series to account for the diversity and the evolution of the chromosome structure along with the generations after interspecific hybridization.

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4.2. Effects of hybridization on morphology traits The first measurable trait is seed size of the hybrid seed which is most often smaller than the average value recorded for oilseed rape when male sterile B. napus was used, except in a cross with B. rapa that showed a range of sizes [29,30]. This indicates problems in embryo development often due to different ploidy levels. There are seldom data when the wild species was used as the female, so that this precludes any inference about the importance of seed trait on the seed survival and seedling establishment of hybrids. Hybrids of B. napus and H. incana frequently had an intermediate leaf hairiness [43], while those formed between B. carinata and B. rapa had intermediate leaf and flower morphology [32]. However, the morphology of hybrids can often fall within the range of variability occurring amongst crop cultivars or within wild species, which could explain why such hybrids are seldom identified in floras and cannot be used as a proof of the absence of gene flow. Obviously, the evolution of morphological traits in a population depends on natural selection, which can eliminate unfit traits while preserving neutral traits, such as small B. napus-like flowers in purportedly introgressed R. raphanistrum populations which was not detrimental to pollination by bees (Liu, unpublished). Nuclear-cytoplasmic interaction could also affect the morphology of the hybrids and their progeny. For example, after seven backcrosses of B. carinata to B. napus, “plants with the cytoplasm of B. napus flowered later, had shorter filaments and longer pistils, lower pollen amount, lower seed set, lower petal length and width and different petal color than the corresponding euplasmic sibs” [31]. Lack of chlorophyll production was found in BC6 progeny of B. napus × R. raphanistrum hybrids while those with R. raphanistrum cytoplasm were a normal green color [51]. In the progeny of natural crosses with B. rapa, the B. napus chloroplast apparently confers higher selective advantage in riverside populations, but not in weedy populations [52]. Taken together, hybrids formed on B. napus as females seem to have lower spread potential than those formed on the reciprocal cross, which is of interest to breeders of oilseed rape with the transgene located in the chloroplast. 4.3. Effects of hybridization on plant growth and reproduction The first measurable trait is seed size of the hybrid seed which is most often smaller than the average value recorded for oilseed rape in the case male sterile B. napus was used, except for the cross with B. rapa that showed a range of sizes [29,30]. This indicates problems in embryo development often due to different ploidy levels. When the wild plant was used as the female, there was seldom data on seed size, which precludes any inference about seed size importance on hybrid seed survival and seedling establishment. Hybridization between B. napus and B. rapa, with or without Bttransgene introgression, resulted in first generation hybrid plants with less vegetative growth than the B. rapa parent [53]. Seed production by the hybrids ranged from being smaller than that of B. rapa, intermediary between the two parent species, to even higher than that of B. rapa [54]. In one study, late and longer-flowering weedy B. rapa plants had a higher probability of producing hybrids than early flowering plants, but they also had the lowest fecundity; a trait which may result in counter selection of hybrid progeny [55]. Under natural riverside conditions, the effective pollen contribution of spontaneous hybrids, in terms of sired seeds, was only 17% of that of B. rapa because of lower pollen viability, and their seed production as females was reduced by 50% [26]. Similar reductions in male function, in terms of the ability to fertilize B. rapa, was observed in garden studies [24,56], showing that the female function rather than the pollen is of major importance for the spread of hybrid progeny. Crop alleles or wild alleles can be differentially advantageous according to the conditions of the habitat

(e.g. waste place vs. arable field, tall vs. dwarf crop), and thus the progeny of hybrids may be selected more quickly than the progeny of normal B. rapa plants, as observed under experimental conditions [57]. Advanced backcross generations generally recover the regular chromosome number and fitness equivalent to B. rapa, whether or not a transgene is present [58], but some studies report on reduced seed production and less competitive population with respect to the parents [53]. Such descendants of introgression can be detected by monitoring wild populations a long time after planting of oilseed rape is abandoned [27,59]. In the case of B. napus × B. juncea cross, the reduced seed production observed in early backcross generations disappears in subsequent generations, which suggests rapid incorporation of hybrid progeny into wild populations [60]. The backcross to B. napus has higher vegetative performance than B. napus so that it could readily establish as volunteer in the crop or as feral population [23]. In the case of B. napus × R. raphanistrum cross, F1 hybrids are mainly triploids with low fertility (1% of that of R. raphanistrum) [37], significantly lower and delayed seedling emergence rates, lower survival rates, and decreased dry matter compared with their parents [61]. However, some of the F2 and BC1 plants grew well and produced viable seeds [37], and their fertility increased with repeated backcrosses to wild radish, and showed no more difference in BC6 [51]. In the case of B. napus × H. incana cross, hybrids were as vigorous as the wild parent in an oilseed rape stand, and more vigorous than H. incana in a H. incana stand, which indicates a great potential for plant establishment [62]. However, although the hybrids produced a very large number of flowers, they had less than one seed per plant on average, and no progeny of these hybrids survived to the 5th generation [42]. 4.4. Introgression On the whole, the rise of stabilized, introgressed plants can take several generations during which selection strongly reduces the frequency of hybrid progeny within populations. The fitness cost due to hybridization and the uneven number of chromosomes, accumulated over generations, and the recombination or the loss of the chromosome supporting a transgene, would make it unlikely to spread within and among populations. Quantitative estimates of gene flow velocity and intensity are premature. The discovery of a single introgressed herbicide-resistant B. rapa plant in Canada [59] shows evidence that introgression does occur and can make that a transgene persists in the fields at low frequency, even in absence of favorable selection pressure. 5. Consequences of transgenes flow If a transgene transferred from oilseed rape to a related plant is expressed, it is likely to provide the same direct effect in that plant as in the crop, and thus similarly express herbicide resistance, insect resistance, modified lipid synthesis, male sterility, etc., because most transgenes are fundamentally dominant. This can either bring benefit to the plant, such as resistance in the presence of herbicides or insects, or be costly for some plant functions, especially when there is no selection pressure for the transgenic trait. High selective value of hybrids and their offspring could counterbalance intrinsic hybrid problems and allow the progeny to spread. Conversely, if the transgene encodes for a selective disadvantage, the progeny, if any, collapses quickly. There are several fitness studies that consider the effect of the transgene, but very few studies have addressed the persistence of transgenes in the field; most were carried out on hybrids or early generations after hybridization so that the effects of the transgene are confounded by the consequences of hybridization as discussed above. This has been done for transgenes

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conferring herbicide-resistance only because they are the only ones commercialized up to now for that crop. 5.1. Herbicide-resistance There was no fitness cost associated with the expression of a glufosinate-resistance transgene after introgressing the transgene from oilseed rape into B. rapa [58], but it was associated with reduced seed production in backcross progeny of B. napus × B. juncea crosses; a difference that is perhaps due to genomic location of the transgene [60]. No difference in glyphosate-resistance transgene expression at the mRNA level was found in hybrids compared to their transgenic oilseed rape parents [63]. This indicates this particular transgene expression was not impacted by genotypic background, so that hybrids can survive the same herbicide dose as the crop. In the case one introgressed diploid individual of B. rapa was detected as having resistance to glyphosate six years after the last transgenic oilseed rape was grown on the field, in absence of the glyphosate selection pressure, it is suggested that fitness cost of the transgene, if any, could not prevent introgression [59]. In that study, the frequency of glyphosate-resistant hybrids and hybrid descendants decreased with time, but it was likely due to intermediate ploidy and consequences of the hybridization itself as reported above. In contrast, use of herbicides, or herbicide drift from nearby treated areas, provides high selective value to the plants expressing the transgene [64]. In the experimental study of the progeny of B napus × R. raphanistrum hybrids, the glufosinate resistance gene never introgressed into the genome of the wild species, but was rather inherited as a supplementary chromosome with non-Mendelian inheritance pattern and transmitted in only 10% of gametes; it probably would have disappeared if the artificial populations had not been sprayed each generation with the herbicide [65]. Models are now available for volunteers [66] or progeny of interspecific hybrids [67] that simulate various scenarios of crop rotation, management of roadsides, use of herbicides and conventional weed management techniques, and can suggest the agronomic strategies that delay or reduce the occurrence of the transgene spread. In the case of B. rapa, field infestation by herbicide-resistant progeny of hybrids could quickly occur, thus challenging the use of the herbicide to control the other weeds. In contrast, in the case of more distant related species, as R. raphanistrum, there could be several generations before forming a troublesome herbicide-resistant population. In addition, uncertainty analysis for parameter confidence intervals in the stochastic cellular model available to date predicted low certainty in the fate of the transgene in R. raphanistrum populations (Garnier, unpublished). 5.2. Other transgenes The introgression of transgenes conferring insect and disease resistance, frost and drought resistance, or improved photosynthetic efficiency may result in increased weediness, competitiveness or colonization ability of wild species [68]. Prediction of the consequences of other transgenes, such as those modifying seed quality or reproduction, is neither straightforward nor easy. For instance, one may ask whether hybrid progeny inheriting the RNase transgene that destroys the pollen sac tapetum in transgenic male sterile varieties could allocate more resources to female organs, thus contributing more to their offspring than normal self-incompatible hermaphrodite plants. This question has not been experimentally tested, but similar tests of other hypotheses have been documented. Seeds of hybrids between B rapa and lipid-modified oilseed rape could have slow and reduced germination, which is perhaps detrimental in the field, but they may have higher enforced dormancy than parental seeds under

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certain conditions, which could favor the formation of a longerlived seed-bank enriched with the transgenic seeds [69]. Outside agroecosystems, concerns focuses on consequences on the food web by changing species abundances within plant communities, or opening new habitats for invasive species (e.g. salt and drought tolerance). Although this issue has been expressed early before the first commercial release of transgenic plants [1], it is still to be documented. Curiously, one of the few papers dealing with the plant community changes dealt with herbicide drift, which is not a typical trait modifying functional roles of plants in a receiving community, and showed that increased presence of resistant Brassica in simulated roadside plant communities may indirectly negatively impact beneficial ecosystem services [70]. A particularly unique case is insect-resistant oilseed rape because, although this plant has not been commercially released, it has been used as a model system to investigate the fitness of hybrid/backcross plants under variable habitat conditions and natural selection. In general, herbivory has a negative effect on plant fitness, but it depends on many factors such as resource level, damage time, plant species and herbivores [71], so that just one unique, generic scenario cannot be predicted. The Bacillus thuringiensis (Bt) transgene is one of most important transgenes employed in transgenic crops to protect against insect damages. No fitness cost in the absence of the selection pressure (herbivory) was detected in the engineered crop B. napus as well as in engineered weedy B. rapa [72] and B. napus × B. rapa hybrids [73], although there are some indications of lower composite fitness [74]. In contrast, in response to high insect herbivory, insect-resistant B. rapa plants were superior competitors in mixed stands with susceptible individuals [75], and higher biomass and seed yield were observed in transgenic than in non-transgenic B. rapa × B. napus hybrids [73,76]. However, the insect-resistant trait did not result in a fitness increase in the presence of low herbivory [73,75]. Taken together, these studies suggest that the Bt-transgene increases plant fitness under moderate to high herbivore damage, a condition that is found in cultivated fields but probably not in more biodiverse plant communities with a low density of Brassica plants. In addition, the presence of transgenic plants could protect the growth and reproduction of non-transgenic plants in mixed populations through a trap or “halo” effect [77,78] that we have experimentally validated with Bt B. napus × B. juncea hybrid progeny (Liu, unpublished). Thus, the population could either reach equilibrium where resistant and susceptible plants co-occur in a stable proportion or susceptible plants will be completely replaced by resistant plants. In conditions of limited resources, beneficial genes could invade populations partially or totally with no or weak impact on seed production, as has been observed with B. juncea [79]. 6. Means for reducing gene flow Several strategies, based on breeding, agronomic or molecular methods, have been proposed in the literature to contain, or at least mitigate transgene flow from crops to their wild relatives [e.g. 80, 81]. However, only a few of them have been tested with Brassica species to determine whether they reduce the spread and persistence of transgenes in wild and cultivated populations. 6.1. Physical means Physical means are usually considered in policies of environmental risk evaluation and management for transgenic crops. They are designed to control seed movement (cleaning the harvest machines and transport trucks) and pollen dispersal. It is clear from Section 2.2 that designing isolation distances is not an easy task because pollen dispersal depends on too many factors. Generic

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Table 1 Selected references for confirmed gene flow stages to wild relatives of B. napus (AACC). Species

Genome

Spontaneous hybridization

Hybrid fitness

Hybrid fertility

Progeny fitness

Ability to introgress

B. rapa B. oleracea B. nigra B. juncea R. raphanistrum H. incana Others

AA CC BB AABB RrRr AdAd

22–24–25–26–27–29 28 30 23–31 22–37-41 42–43 None

26–30–54–56–63–76 28 30 23 61 62

24–27–50

27–50–53–58–59–69

23–48 37–49 42–43 35

23 51–65 42

Likely Likely Difficult Likely Difficult Difficult Unlikely

models can help estimating the minimum inter-field separation distances required for a given seed admixture level under wind pollination, regardless of landscape structure [82]. Planting rows of taller plants or permanent bushes around the field are often used in nurseries, but they are ineffective in large scale farming practices, in particular because weedy, wild and volunteer plants are everywhere in the field and non-field habitats, and insects and wind can transport pollen. Modeling pollen dispersal according to landscape can indicate the potential of hedgerows, bushes and trees to modify dispersal distances [83,84]. Agricultural practice plays an important role in reducing gene flow through seeds, including crop rotation, rotating mode of action of herbicides (in case the transgene confers resistance to a given herbicide), effective harvesting and transport tools to reduce transgenic seed loss on soil and roadsides, delayed soil cultivation and false sowing to destroy volunteers, regular tillage to reduce transgenic seed bank in soil, and adjacent or regional farming system organization (e.g. for transgenic versus conventional varieties co-existence). These agricultural means can be guided by simulation studies modeling the behavior of pollen and seeds according to different farming systems [66]. Earlier or delayed crop sowing can help separating the flowering time of the crop to that of the wild relative. 6.2. Biological means Biological means mainly include recessive inheritance, male sterility, seed sterility, apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), maternal inheritance (e.g. genetic engineering of chloroplast) [80] and earliness [36]. However, all of these tools, although reducing primary gene flow, have their weakness. For instance, male sterility can prevent the production and dispersal of pollen but leaves stigmas to be fertilized by foreign pollen, hence making isolated volunteer plants good candidates for producing many hybrids, as shown in field experiments [85]. As for cleistogamy, models suggest that it is an efficient tool [83], but practical trials show that bees can force the petals open and reach the center of the flower and effect some pollination [86,87]. Finally, selecting cultivars for flowering periods that do not overlap with those of wild relatives is not a clear-cut tool and widely depends on climatic condition, especially if the wild species have long, nonsynchronized flowering time [36,43]. Maternal inheritance of transgenes, i.e. incorporating the transgene into the chloroplast genome instead of the nuclear genome, was proposed to prevent the flow of transgenes through pollen grains [88,89]. Plastid genetic transformation was first developed for tobacco [90], and then the technology has been used to transfer genes into Arabidopsis, rice, tomato, and potato. In the case of maternal inheritance of transgenes, it was once thought that crop pollen cannot transfer chloroplast traits. However, there is about 0.4% possibility of pollen transferring the chloroplast traits [91]. In addition, if such an exceptional transfer through pollen would occur, it would result in a plant line with a fixed transgene without any segregation possibility. Indeed, wild introgressed plants with the crop chloroplast were found in natural mixed populations of

B. rapa and B. napus. In that case, the B. napus cytoplasm seems to have conferred a selective advantage to those introgressed B. rapa, which could lead to a greater dispersal of transgenes [52]. 6.3. Molecular means Of possible molecular containment strategies, genome incompatibility, chemical induction/deletion of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid) are the most frequently cited ones. Since B. napus has multiple genomes (AACC genomes), transgenes located on the Cchromosome might not be transmitted through introgression to B. rapa (AA genomes) because that recipient species does not have the C-genome [92]. However, the C-genome DNA could be incorporated into the B. rapa genome through recombination among homeologous A and C chromosomes [93], suggesting that “safe-integration” sites in B. napus are unlikely [94]. Integration of recombination rate in simulation models could be a next step to better predict if some strategy could help delaying the appearance of stable, transgenic B. rapa introgressed plants [6]. However, it would not matter which genome carried the gene in the case of creation of allopolyploids. Deletion of transgenes in crop pollen is now possible through the Cre/Lox strategy. Chemically inducible site-specific gene excision has been used to remove marker genes from crops using recombinases. Placing the recombinase production under the control of a pollen-specific promoter could result in the excision of a transgene, thus generating non-transgenic pollen and preventing transgene flow through the pollen [95]. It has been applied to develop “an effective gene switch system for spatial and temporal control of gene expression in Arabidopsis thaliana and B. juncea” [96]. Transgenic mitigation strategies aim at decreasing fitness of hybrid to decrease transgene persistence. Among the transgenic strategies, anti-shattering, dwarfing and uniform germination genes could be typical mitigating genes [81]. These mitigator genes could be transformed into crops in a tandem construct containing the beneficial transgene and thus inherited together. Mitigating genes would confer traits that are either positive or neutral for the crop and detrimental to the related weedy or wild species. A gene (SHATTERPROOF) that prevents seed shattering has been isolated from Arabidopsis: it delays the valve opening on the silique, which would be an ideal mitigation strategy for B. napus by preventing seed dispersal if it is transferred to a wild relative [97]. A fitness-mitigating dwarfing gene delta-gai (gibberellic acid insensitive) was inserted in transgenic B. napus × B. rapa hybrid populations. The mitigation construct effectively limits transgene persistence because it confers lower competitiveness, and thus limits the impact of gene flow from transgenic crops to their wild relatives [57,98]. However, dwarfing could be beneficial in some non-competitive habitats, as discussed from plant height records of cultivated and feral oilseed rape populations [99], or in crop with short habit to escape management practices efficient against tall plants, which shows that careful and extensive assessment of the strategy must be implemented before generalization.

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7. Conclusions The agricultural consequences of gene flow depend on its frequency as well as on the fitness of hybrids and the trait endowed by the transgene. However, taking into account the large body of information reported above (Table 1), it is difficult to draw general conclusions on the impact of gene flow in Brassica. The story with B. rapa is the most documented and models are underway that include as many as possible of the influential elements: transgenes, genotypes, farming practices, selection pressure, target plant community, landscape, etc. However, there are still great gaps in the knowledge to be filled, such as to account for the effect of genetic variability on successful hybridization, hybrid fertility, correlation between chromosome number and fertility, for the effect of selection pressure and environmental variability on hybrids and transgene success, and for describing population demography and spread in a plant community. A large number of the biological traits and parameters of the life cycle of wild and weedy species are poorly understood and/or highly variable between biogeographic regions and farming systems, which results in models with low predictive power and low certainty in the fate of the transgene in populations. Gathering more precise knowledge on life history traits and life cycle is necessary to feed models and assess the risk of transgene spread and magnitude (see Box 1). In addition, long term monitoring of transgenes or transgene-modified traits in wild and weed populations, necessary to validate models, are still missing. Further evaluation of the consequences and the efficiency of mitigation strategies also need to be conducted to make general conclusions. Some of the containment approaches, previously mentioned, may be effective in protecting some species but not for others, and a combination of these approaches will be necessary for effective transgene containment. North America began cultivating herbicide-resistant varieties of oilseed rape sixteen years ago. While no major adverse effects due to gene flow have been detected, it remains a valid question “is this really long enough of a time span to reach a definitive conclusion?”. Only one occurrence of a persistent, herbicide-resistant introgressed B. rapa has been documented to date, but there is no evidence of any invasive spread or any trouble for the farmer or the environment. First, no other cases have been reported in Canada in fields where herbicide-resistant varieties are repeatedly cultivated. Perhaps, a short-lived and weak seed bank allied to alternate herbicides in the crop rotation and good farming practices against herbicide-resistance could have delayed or controlled the onset of resistant populations. Second, it did not represent a threat for the farmer because he was not using the corresponding herbicide. As the likelihood of the establishment of introgressed progeny would appear to be very low, one could wonder if the risk of appearance of herbicide-resistant weeds is higher with gene flow or with spontaneous mutation? For instance, mutants resistant to carotenoid, acetolactate synthase and photosystem II inhibitors or synthetic auxins have already been reported for R. raphanistrum [100]. The concern over the ecological consequences of gene flow, i.e. in non-crop situations, is still less documented than in crop fields. Beneficial genes could invade populations partially or totally; they could also help plants to better exploit their ecological niche and displace some plant species that cannot afford enhanced concurrence, possibly resulting in plant community changes. It is not necessary that introgressed populations become highly invasive to provoke such changes, not to mention the unpredictable occurrence and impact of a new amphiploid species. Again, more basic biological knowledge is needed to understand and simulate the dynamics of a plant community, and this represents a tantalizing field of evolutionary research.

Box 1: Strengths and weaknesses of gene flow models Gene flow and its consequences are very complex phenomena, depending on many independent and interrelated biological, genetic, ecological, climatic and human factors which, when combined, are impossible to experimentally assess. Thus, it is necessary to synthesize existing knowledge on the fate of transgenes by simulating many diverse scenarios in simulation models. Such models of transgene dispersal can be useful tools to develop public policy about the deployment of transgenes. Separate models have been proposed to describe: pollen dispersal [18], chromosome behavior [6,49], the complete plant life-cycle [66,67,101], as well as the impact of cropping systems [66]. It is uncertain whether these models reflect reality and whether their predictions are robust [102]. Models include many parameters whose values are uncertain, especially when they encompass a wide number of biological processes and interactions with environmental conditions. The uncertainty on the parameters is a major source of output uncertainty not typically discussed with the model predictions. Few of the existing gene flow models have been evaluated by comparing their simulations to independent field observations [66] because the necessary long-term and long-distance data are difficult to collect. Model evaluation requires identifying the most important parameters whose variation results in the greatest output variations, and then to estimate these parameters with the necessary accuracy and their range of variation. Sensitivity analyses are essential, either varying individual parameters (“local sensitivity analysis”) or simultaneously (“global sensitivity analysis”) [103], but have rarely been performed for transgene flow models. Sensitivity analysis of life history traits and field practices allows ranking key parameters, focusing for instance on the fitness of stabilized introgressed plants [67] or on the cropping system [66]. Even complex models are often based on a number of a priori, never tested, simplifying assumptions. They are generally limited to the simulation of the dispersal of a single transgene; they cannot predict its establishment in unintended and new genomic and ecological backgrounds. In particular, the plant and animal composition of the recipient community is not taken into account, and mitigation strategies are not evaluated. Such incomplete models cannot address the consequences to agriculture or environment. Only a limited set of disparate data is available to date to estimate parameters and feed models. They are scattered over many studies using different plant materials in field or greenhouse conditions (Table 1), and are not replicated. More data are needed to estimate the key parameters at each step of gene flow and establishment. A posteriori validations using new, independent sets of data are necessary. With such little knowledge on the behavior and effects of transgenes facing variable meteorological and biological characteristics, prediction of field and environmental impacts is particularly challenging and premature, unless the transgenes inserted are highly unfit outside of crops.

Acknowledgements The authors would like to thank David A. Bohan for provided comments to improve the manuscript. This project was supported by the China Natural Science Foundation (Grant No. 31200288), French National Research Agency (ANR-07-POGM001-01, NATORA), Special Program for New Transgenic Variety Breeding of the Ministry of Science and Technology, China (No. 2012ZX08011002), and General Research Project of CRAES, China (No. 2011YSKY-08). References [1] J.M. Tiedje, et al., The planned introduction of genetically engineered organisms- ecological considerations and recommendations, Ecology 70 (1989) 298–315.

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