Scale effect on rice pollen‐mediated gene flow: implications in assessing transgene flow from genetically engineered plants

Transgene flow from genetically engineered (GE) crops to non‐GE varieties and wild relatives via pollen‐mediated gene flow (PMGF) may create food and environmental biosafety concerns. Assessing the level of PMGF from GE crops is required before commercialization. Whether the level of PMGF estimated at relatively small scales can sufficiently represent the actual scenario at large production scales remains unresolved. Here, we estimated average PMGF frequencies from three insect‐resistant GE rice lines to their non‐GE counterparts at four scales ranging from 9 to 576 m², having the number of GE to non‐GE plants constantly at the ratio of 8:1. Based on nearly 1.3 million examined seedlings from non‐GE rice plots, very low frequencies (<0.1%) of transgene flow were detected. The highest frequencies were found in plots at the smallest scales. Scale had a significantly negative effect on the frequency of PMGF in rice, with decreased gene flow at increased scale. An extended PMGF model could well represent the experimental data. Field experiments at relatively small scales combined with mathematical modelling could provide useful prediction on the level of rice transgene flow at large production scales. This is probably applicable for other crop species with wind‐ and self‐pollination.     

Evidence for gene flow via seed dispersal from crop to wild relatives in Beta vulgaris (Chenopodiaceae): consequences for the release of genetically modified crop species with weedy lineages

Gene flow and introgression from cultivated to wild plant populations have important evolutionary and ecological consequences and require detailed investigations for risk assessments of transgene escape into natural ecosystems. Sugar beets (Beta vulgaris ssp. vulgaris) are of particular concern because: (i) they are cross-compatible with their wild relatives (the sea beet, B. vulgaris ssp. maritima); (ii) crop-to-wild gene flow is likely to occur via weedy lineages resulting from hybridization events and locally infesting fields. Using a chloroplastic marker and a set of nuclear microsatellite loci, the occurrence of crop-to-wild gene flow was investigated in the French sugar beet production area within a 'contact-zone' in between coastal wild populations and sugar beet fields. The results did not reveal large pollen dispersal from weed to wild beets. However, several pieces of evidence clearly show an escape of weedy lineages from fields via seed flow. Since most studies involving the assessment of transgene escape from crops to wild outcrossing relatives generally focused only on pollen dispersal, this last result was unexpected: it points out the key role of a long-lived seed bank and highlights support for transgene escape via man-mediated long-distance dispersal events.     

Establishment of a rice transgene flow model for predicting maximum distances of gene flow in southern China

We aimed to establish a rice gene flow model based on (i) the Gaussian plume model, (ii) data from a three-location x 3-yr field experiment on transgene flow to common rice cultivars (Oryza sativa), male sterile (ms) lines (O. sativa) and common wild rice (Oryza rufipogon), and (iii) 32-yr historical meteorological data collected from 38 meteorological stations in southern China during the rice flowering period. The concept of the gene flow coefficient (GFC) is proposed; that is, the ratio of the transgene flow frequency (G%) obtained from field experiments to the aggregated pollen dispersal frequency (P%) calculated based on the pollen dispersal model. The maximum distances of gene flow (MDGF) to traditional rice cultivars, ms lines, and common wild rice at a threshold value of either 1.0 or 0.1% were determined. The MDGF and its spatial distribution in southern China show that the gene flow pattern is significantly affected by the monsoon climate, the topography, and the outcrossing ability of recipients. We believe that the information provided in this study will be useful for the risk assessment of transgenic rice in other rice-growing regions.     

Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor

Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.     

Pollen flow of cultivated rice measured under experimental conditions

The pollen flow pattern of a cultivated rice variety, Minghui-63, was studied at horizontal and vertical levels under experimental conditions. Data obtained from pollen traps for six designed populations (as pollen sources) at different intervals showed that the dispersal of rice pollen decreased with the increase of distance from pollen sources and that the rice pollen flow was significantly influenced by weather conditions, particularly by wind direction and speed. For a mean wind speed of 2.52 m/s in a downwind direction, the observed distance of rice pollen dispersal was 38.4 m, indicating that rice pollen grains normally disperse at a relatively small range. However, the maximum distance of rice pollen flow could be up to 110 m, using regression analysis of pollen flow and wind speed, when the wind speed reached 10 m/s in this study. The frequency of pollen flow was positively correlated with pollen source size within a given range, suggesting that pollen flow will occur effectively at a considerable rate in rice fields with sufficiently large pollen sources. In addition, many more pollen grains were detected at the height of 1.0–1.5 m than at 2.0 m, indicating that rice pollen mainly disperses at relatively low heights. Results from this study are useful both for minimizing transgene escape from transgenic rice and in situ conservation of wild relatives of rice, as well as for hybrid seed production, where an effective isolation buffer zone needs to be established.     

High-Resolution Gene Flow Model for Assessing Environmental Impacts of Transgene Escape Based on Biological Parameters and Wind Speed

Environmental impacts caused by transgene flow from genetically engineered (GE) crops to their wild relatives mediated by pollination are longstanding biosafety concerns worldwide. Mathematical modeling provides a useful tool for estimating frequencies of pollen-mediated gene flow (PMGF) that are critical for assessing such environmental impacts. However, most PMGF models are impractical for this purpose because their parameterization requires actual data from field experiments. In addition, most of these models are usually too general and ignored the important biological characteristics of concerned plant species; and therefore cannot provide accurate prediction for PMGF frequencies. It is necessary to develop more accurate PMGF models based on biological and climatic parameters that can be easily measured in situ. Here, we present a quasi-mechanistic PMGF model that only requires the input of biological and wind speed parameters without actual data from field experiments. Validation of the quasi-mechanistic model based on five sets of published data from field experiments showed significant correlations between the model-simulated and field experimental-generated PMGF frequencies. These results suggest accurate prediction for PMGF frequencies using this model, provided that the necessary biological parameters and wind speed data are available. This model can largely facilitate the assessment and management of environmental impacts caused by transgene flow, such as determining transgene flow frequencies at a particular spatial distance, and establishing spatial isolation between a GE crop and its coexisting non-GE counterparts and wild relatives.     

Gene flow from genetically modified rice to its wild relatives: Assessing potential ecological consequences

Pollen-mediated gene flow is the major pathway for transgene escape from GM rice to its wild relatives. Transgene escape to wild Oryza species having AA-genome will occur if GM rice is released to environments with these wild Oryza species. Transgenes may persist to and spread in wild populations after gene flow, resulting unwanted ecological consequences. For assessing the potential consequences caused by transgene escape, it is important to understand the actual gene flow frequencies from GM rice to wild relatives, transgene expression and inheritance in the wild relatives, as well as fitness changes that brought to wild relatives by the transgenes. This article reviews studies on transgene escape from rice to its wild relatives via gene flow and its ecological consequences. A framework for assessing potential ecological consequences caused by transgene escape from GM rice to its wild relatives is discussed based on studies of gene flow and fitness changes.     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.     

Population genetics of transgene containment

Several strategies have been proposed for creating transgenic cultivars from which transgene escape to wild relatives would seem unlikely; for example, to impede escape through pollen, a transgene could be inserted into chloroplast DNA (cpDNA), which in many crops is rarely transmitted through pollen. None of these strategies would be failsafe; for example, the rate of cpDNA transmission through pollen may be low but non‐zero in many crops. Here, we study how the probability distribution of escape time depends on the rates of pollen and seed flow from the crop to wild populations, the number and sizes of the wild populations, the selection coefficient for the transgene, and a leakage parameter characteristic of the strategy, for example, the rate of cpDNA transmission through pollen. We find that even with a leakage parameter as small as 10^?3, the probability of escape within as few as 10 generations could be appreciable.     

Marker Based Estimation of Gene Flow in Tropical Rice Fields and Its Ecological Consequences

Pollen mediated gene flow is the most widely debated biosafety issue in case of genetically modified crop plants and is a primary determining factor for permitting their field release, particularly major food crops like rice. The alleged consequences of gene escape into wild/weedy relatives that coexist together with cultivated forms in rice fields in several countries is perceived to be a major concern. In an effort to estimate the gene flow in rice in a tropical environment, rice varieties stacked with three bacterial blight (BB) resistance alleles and a cytoplasmic male sterile (CMS) line were used as donors and receiver of pollen respectively and the pollen flow was tracked with molecular markers that are closely linked to BB resistance genes. The study could detect gene flow up to 20?m distance with environmental factors like temperature and humidity influencing it. The gene flow observed at longer distances than the 10?m distance suggested earlier advocate caution and suggests that all the relevant biosafety issues need to be addressed prior to release of GM rice in tropical countries where sympatric association of wild relatives with cultivated rice with frequent crop-weedy gene flow both ways is quite common.     

转基因逃逸及其环境生物安全评价研究进展——抗虫水稻案例分析

转基因技术研发为提高我国水稻产量和减少劳动力投入提供了巨大机遇。我国对转基因水稻研发进行了大量的投入,目前已培育了具有不同新性状的转基因水稻品系,许多品系已进入生物安全评价阶段。风险评价对转基因水稻的安全生产至关重要,是其商品化生产之前必须解决的问题,其中包括转基因逃逸及其潜在环境影响。对水稻抗虫转基因逃逸及其潜在环境风险的评价包括3个重要环节：（1）通过田间试验和模型模拟检测转基因漂移到非转基因栽培稻及其野生近缘种的频率;（2）检测转基因在栽培稻和野生近缘种后代中的表达;（3）确定转基因对野生近缘种群体适合度和进化潜力的影响。大量研究表明,在近距离的空间范围内栽培稻品种之间的基因漂移频率很低（〉0.1%）,但栽培稻与其野生近缘种的基因漂移频率变异很大。进一步研究还表明,Bt抗虫转基因在栽培稻与普通野生稻后代中均能正常表达,但在其不同生长阶段,表达量有很大变异。在有较高水平的害虫虫压下,含有抗虫转基因的栽培稻及野生近缘种杂交后代与不含转基因的对照相比,抗虫性显著提高且适合度利益明显;但是,在虫害发生水平较低时,含有抗虫转基因的群体与不含抗虫转基因的群体相比没有显著的适合度优势。综上,转基因逃逸到非转基因水稻的频率极低,并且可以通过空间隔离阻断其逃逸。虽然抗虫转基因向杂草稻以及与栽培稻距离较近的野生稻群体的逃逸无法避免,但是野生稻和杂草稻群体周围环境中的总体虫压较低,所以基因漂移带来的环境影响应十分有限。     

Deficiency of very long chain alkanes biosynthesis causes humidity‐sensitive male sterility via affecting pollen adhesion and hydration in rice

Pollen adhesion and hydration are the earliest events of the pollen–stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect‐pollinated plants. Here, we characterized the role of OsGL1‐4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1‐4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1‐4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography–mass spectrometry analysis suggested that the humidity‐sensitive male sterility of osgl1‐4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.     

Transgene escape: what potential for crop–wild hybridization?

To date, regional surveys assessing the risk of transgene escape from GM crops have focused on records of spontaneous hybridization to infer the likelihood of crop transgene escape. However, reliable observations of spontaneous hybridization are lacking for most floras, particularly outside Europe. Here, we argue that evidence of interspecific reproductive compatibility derived from experimental crosses is an important component of risk assessment, and a useful first step especially where data from field observations are unavailable. We used this approach to assess the potential for transgene escape via hybridization for 123 widely grown temperate crops and their indigenous and naturalized relatives present in the New Zealand flora. We found that 66 crops (54%) are reproductively compatible with at least one other indigenous or naturalized species in the flora. Limited reproductive compatibility with wild relatives was evident for a further 12 crops (10%). Twenty-five crops (20%) were found to be reproductively isolated from all their wild relatives in New Zealand. For the remaining 20 crops (16%), insufficient information was available to determine levels of reproductive compatibility with wild relatives. Our approach may be useful in other regions where spontaneous crop-wild hybridization has yet to be well documented.     

Investigation of rice transgene flow in compass sectors by using male sterile line as a pollen detector

Rice is the most important staple food in the world. The rapid development of transgenic rice and its future commercialization have raised concerns regarding transgene flow and its potential environmental risk. It is known that rice is a self-pollinated crop; the outcrossing rate between common cultivars is generally less than 1%. In order to improve the detection sensitivity of rice transgene flow, a male sterile (ms) line BoA with a high outcrossing rate was used as a pollen detector in this study. A concentric circle design was adopted, in which the transgenic rice B2 containing bar gene as a pollen donor was planted in the center circle and the recipient BoA was planted in eight compass sectors. The frequency of transgene flow in compass sectors was analyzed by continuous sampling to generate cumulative data. The results of two years with sound reproducibility demonstrated that the rice gene flow was closely associated with the wind direction. According to the mean frequency of transgene flow, the eight sectors can be divided into two groups: a higher frequency group downstream of the prevailing wind (DPW) with a mean frequency ranging from 6.47 to 26.24%, and a lower frequency group lateral to or upstream of the prevailing wind (UPW) with a mean frequency of 0.39 to 3.03%. On the basis of the cumulative data, 90–96% of the cumulative gene flow events occurred in the four DPW sectors, while it was 4–10% in the four UPW sectors. By using these systematic data, simulation models and isograms of transgene flow in the eight compass sectors were calculated and drawn, respectively. Q. H. Yuan, L. Shi and S. R. Jia contributed equally to the work.     

一种等分作物花粉扩散圆形源区的方法

【背景】转基因作物花粉在大气中扩散会引起基因漂流,从而可导致不可预知的环境风险,运用模型预测可评估其花粉扩散状况、定量确定可靠的安全扩散距离。为了应用高斯烟羽模型应用于模拟转基因作物花粉在大气中的扩散浓度,提出了一种如何将半径为R的圆形花粉源区划分成许多等面积小面元的方法。【方法】通过数学推导,首次提出了各等面积面元中心点坐标的计算公式。【结果】根据在中国东北地区吉林省公主岭玉米花粉扩散和基因漂流的试验观测资料,将该公式应用到高斯烟羽扩散模型中,模拟了玉米花粉扩散到源区外不同距离处的浓度,对比花粉扩散模拟值与实测值,两者具有较好的一致性,表明应用该公式模拟花粉扩散能取得令人满意的效果。【结论与意义】分析证明这种推导划分半径为R的圆形花粉源区各面元中心点坐标的计算公式是可靠的,该方法可为应用高斯烟羽模型计算花粉源区内不同位置点单个源强对源区外某一距离处的花粉浓度的贡献提供便利。     

Modelling pollen sedimentation in Danish lakes at c.ad 1800: an attempt to validate the POLLSCAPE model

Aims To validate the POLLSCAPE simulation model of pollen dispersal and deposition, and evaluate the effect of factors such as pollen productivity, wind speed and regional plant abundance, using a data set of ad 1800 pollen assemblages and historical land cover data. Location Denmark. Methods ad 1800 land cover from historical maps is digitized for 2000 m radii around 30 Danish lakes (3.5–33 ha). The simulation model POLLSCAPE is used to predict sedimentary pollen assemblages in the lakes from the plant abundance data inferred from these maps, with different model parameter settings for wind speed, pollen productivity, regional pollen loading, etc. The model predictions are compared with observed ad 1800 pollen assemblages from the lake sediment records. Furthermore, pollen productivity is estimated from the ad 1800 pollen and vegetation data using the Extended R‐value model. Results Generally the model reproduces the patterns in the observed pollen assemblages, and for most pollen types there are significant correlations between observed and predicted pollen proportions. The pollen proportions predicted by the POLLSCAPE model are sensitive to the pollen productivity estimates used, the regional background pollen loading and average wind speed. There is a difference in background pollen loading between eastern and western Denmark, especially of Calluna pollen. The fit between predicted and observed pollen assemblages is best at wind speeds around 2.5 m s^?1, and decreases rapidly at lower wind speeds. The pollen productivity estimates from the ad 1800 data set are comparable with estimates from moss polsters in modern analogues of traditional cultural landscapes in Sweden and Norway. Main conclusions The POLLSCAPE model reproduces the patterns in the observed pollen assemblages from the lakes well, considering the uncertainty in the historical plant abundance data. This study indicates that the simulation model can be a useful tool for investigating relationships between vegetation and pollen composition, but also that the simulated pollen proportions are sensitive to the pollen productivity estimates, the regional background and to wind speed.     

Realized gene flow within mixed stands of Quercus robur L. and Q. petraea (Matt.) L. revealed at the stage of naturally established seedling

The estimates of contemporary gene flow assessed based on naturally established seedlings provide information much needed for understanding the abilities of forest tree populations to persist under global changes through migration and/or adaptation facilitated by gene exchange among populations. Here, we investigated pollen‐ and seed‐mediated gene flow in two mixed‐oak forest stands (consisting of Quercus robur L. and Q. petraea [Matt.] Liebl.). The gene flow parameters were estimated based on microsatellite multilocus genotypes of seedlings and adults and their spatial locations within the sample plots using models that attempt to reconstruct the genealogy of the seedling cohorts. Pollen and seed dispersal were modelled using the standard seedling neighbourhood model and a modification—the 2‐component seedling neighbourhood model, with the later allowing separation of the dispersal process into local and long‐distance components. The 2‐component model fitted the data substantially better than the standard model and provided estimates of mean seed and pollen dispersal distances accounting for long‐distance propagule dispersal. The mean distance of effective pollen dispersal was found to be 298 and 463 m, depending on the stand, while the mean distance of effective seed dispersal was only 8.8 and 15.6 m, which is consistent with wind pollination and primarily seed dispersal by gravity in Quercus. Some differences observed between the two stands could be attributed to the differences in the stand structure of the adult populations and the existing understory vegetation. Such a mixture of relatively limited seed dispersal with occasional long distance gene flow seems to be an efficient strategy for colonizing new habitats with subsequent local adaptation, while maintaining genetic diversity within populations.     

A model to predict the frequency of integration of fitness-related QTLs from cultivated to wild soybean

With the proliferation of genetically modified (GM) products and the almost exponential growth of land use for GM crops, there is a growing need to develop quantitative approaches to estimating the risk of escape of transgenes into wild populations of crop relatives by natural hybridization. We assessed the risk of transgene escape by constructing a population genetic model based on information on fitness-related QTLs obtained from an F ₂ population of wild soybean G. soja × cultivated soybean Glycine max. Simulation started with ten F ₁ and 990 wild soybeans reproducing by selfing or outcrossing. Seed production was determined from the genetic effects of two QTLs for number of seeds (SN). Each seed survived winter according to the maternal genotype at three QTLs for winter survival (WS). We assumed that one neutral transgene was inserted at various sites and calculated its extinction rate. The presence of G. max alleles at SN and WS QTLs significantly decreased the probability of introgression of the neutral transgene at all insertion sites equally. The presence of G. max alleles at WS QTLs lowered the risk more than their presence at SN QTLs. Although most model studies have concentrated only on genotypic effects of transgenes, we show that the presence of fitness-related domestication genes has a large effect on the risk of transgene escape. Our model offers the advantage of considering the effects of both domestication genes and a transgene, and they can be widely applied to other wild × crop relative complexes.     

A model of pollen-mediated gene flow for oilseed rape

The development of genetically modified (GM) crops has precipitated the need for risk assessment and regulation of pollen-mediated gene flow. In response to this need we present a mathematical model to predict the spatial distribution of outcrossing between progenitor populations of oilseed rape. The model combines the processes of pollen dispersal and pollination, resulting from wind and insect activity. It includes the effects of post-pollination reproductive processes by relating the number of progeny to both pollen deposition and competition at the stigma. Predictions compare well with a range of experimental results for different-sized GM source crops (i.e. 0.0064-0.8 ha) and non-GM target crops with different fertilities (i.e. self-fertile to 80% male-sterile). For these comparisons, we represent the variation caused by wind and insect exposure as a constrained set of random functions and limit the range of insect transport to typical plant-scale distances. In addition, the model is used to examine the relative sensitivity to the factors that determine gene flow. Target-crop fertility and source-crop size are shown to be more important than other factors, including background pollen and the natural range of insect activity. The concept of isolation distance to regulate gene flow is most effective for self-fertile target crops, but is ineffective for male-sterile target crops with low background pollen.