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Many major crops are sexually compatible with their weedy types and wild relatives, leading to so-called crop-to-weed or crop-to-wild gene flow in their sympatric regions (Arnold, 1992, 1997; Rieseberg, 1995; Arriola & Ellstrand, 1996). Gene flow from crop taxa can add new genes into wild populations, which then can re-assort into novel combinations and therefore may have a substantial impact on the evolution of wild populations (Arriola & Ellstrand, 1996; Arnold, 1997; Ellstrand et al., 1999; Jarvis & Hodgkin, 1999). This might result in more aggressive weeds (Colwell et al., 1985; Langevin et al., 1990; Arias & Rieseberg, 1994; Arriola & Ellstrand, 1996) and/or lead to extinction of rare and endangered species (Ellstrand & Elam, 1993; Rhymer & Simberloff, 1996). Transgenic crop varieties are now grown worldwide and the area planted to these varieties continues to increase rapidly. Transgenic crops can greatly benefit humanity in terms of world food security, but there are biosafety concerns related to tranegene escape from genetically modified crops to wild populations through crop-to-wild gene flow (Dale, 1992; Gray & Raybould, 1998). A better understanding of crop-to-wild gene flow is therefore essential for ecological risk assessment of transgene escape (Raybould, 1999; Auberson, 2000), in addition to its importance for strategic conservation of irreplaceable genetic resources (Bartsch et al., 1999; Storfer, 1999).
Rice is one of the most important crops in the world, providing staple food for over 50% of the world's population. In order to feed the increasing global population, there is a need for rice varieties with enhanced yield, resistance to pests or pathogens, and tolerance to environmental stress. It is very likely that such varieties will be produced by biotechnology, including genetic engineering, and that these varieties will be widely grown. The common wild rice Oryza rufipogon is the putative ancestor of the Asian cultivated rice (O. sativa), and the most important genetic resources for rice improvement in terms of its accessibility from gene transfer through sexual means (Oka, 1988). It is widely distributed in South and South-east Asian countries, including China where O. sativa is a major staple food and extensively cultivated. Natural hybridization between O. rufipogon and O. sativa has been reported in many locations (Langevin et al., 1990; Majumder et al., 1997; Suh et al., 1997; Lu, 1999; Song et al., 2002), indicating that O. rufipogon has high compatibility with cultivated rice. Therefore, transgenes from the transgenic rice might be captured by this wild rice through outcrossing. The resulting interspecific hybrids might serve as a bridge from which transgenes could further spread to other wild relatives. Little is known about the frequency and range of gene flow between the two species, although several reports mentioned the occurrence of gene flow between the cultivated and wild rice. Understanding of gene flow between the cultivated rice and O. rufipogon would allow us to minimize ecological risks caused by the transgenic escape by properly regulating and managing the transgenic rice by, for example, establishing a safety distance.
Gene flow can be estimated using direct or indirect methods. Pollen and seed, for flowering plants, are the two major vectors of gene flow. In direct methods, observations of pollen flow and/or seed dispersal are used to estimate range of gene flow (Nilsson et al., 1992). Much of the work on gene flow has been conducted using indirect methods, such as allele frequencies or paternity analysis where different isozyme systems or DNA assays were applied (Snow & Parker, 1998). Data of gene flow measured under natural conditions is significant for risk assessment of transgenes escape. However, it is somehow difficult to determine precisely gene flow under field conditions, even using specific molecular markers, because gene flow can be influenced by a number of factors such as timing, population size and background (Bossart & Prowell, 1998; Jarvis & Hodgkin, 1999). A designed experimental system simulating natural conditions can minimize the influence of such factors and allow us to measure the frequency and range of gene flow with the assistance of specific molecular markers easily (Crawley et al., 1993; Messeguer et al., 2001).
This study reports experiments estimating gene flow from cultivated rice to O. rufipogon conducted under typical cultivation conditions in which distribution patterns of wild rice in variable sizes of patches in the vicinity of cultivated rice are commonly found in Asian countries, particularly in regions with moderate scale of rice cultivation by small farmers. The experiments made use of simple sequence repeat (SSR) markers, that have been applied in many other studies of gene flow (Wu & Tanksley, 1993; Chase et al., 1996), to identify the incidence of hybridization. The objectives of the study were to investigate the frequency and range of gene flow from cultivated rice to O. rufipogon under the typical cultivation conditions described above.
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The present study showed that gene flow from cultivated rice to its wild relative O. rufipogon occurred at a significant level, which confirms that occurrence of gene flow from cultivated to wild rice is beyond question (Ellstrand et al., 1999; Jarvis & Hodgkin, 1999). This study was carried out under experimental conditions where the factors that influenced gene exchange were optimized. It was fortunate that the weather during anthesis in this study was also suitable for fertilization and seed development, resulting in a high seed set that allowed more viable hybrid seeds to be produced. According to previous studies, O. rufipogon populations showed significant differences both in seed set and outcrossing rate due to geographical differentiation or introgression with cultivated rice, and introgression of cultivated rice increases the outcrossing rate of O. rufipogon (Morishima et al., 1961; Oka & Chang, 1961). This would increase the possibility of gene exchange between cultivated and wild rice species. The sampled Chaling O. rufipogon population was suggested to be an introgressed population (Song et al., 2001b), and under natural conditions, introgression between cultivated rice and O. rufipogon was frequently observed (Z.P. Song, personal observation). It was therefore not surprising that relatively high seed set and high hybridization rate were observed in this study. Although cultivated rice and O. rufipogon often occur sympatrically, the flowering time of cultivated rice does not always perfectly match that of O. rufipogon, and the two species do not always grow immediately adjacent to each other. These factors will reduce interspecific gene flow in many natural populations (Arriola & Ellstrand, 1997; Messeguer et al., 2001). The results of this study might represent the maximum frequency of gene flow between the two species under cultivation conditions.
The maximum frequency of gene flow from cultivated rice to O. rufipogon in this study (2.94%) is much higher than that reported by Messeguer et al. (2001) between cultivated rice varieties (0.5%) under similar conditions. This is mainly attributable to the differences of breeding systems between these species involved. Cultivated rice is a self-pollinated species and its floral structure, such as short stigmas, limits the occurrence of outcrossing (Messeguer et al., 2001). In contrast, O. rufipogon has a significantly variable outcrossing rate from 5% to nearly 60% (Oka & Morishima, 1967). Its floral structure includes long and outstretched stigmas, increasing the opportunity of capturing foreign pollen grains. The frequency of gene flow from cultivated rice to O. rufipogon is significantly lower than that reported for the weedy rice (red rice) (52.18%) by Langevin et al. (1990). Genetic distance of the cultivated rice with O. rufipogon and weed rice, resulting in differences on sexual compatibility, perhaps account for this difference. Notably, Oka & Chang (1961) once reported the hybridization rate in the wild rice (O. perennis or O. sativa f. spontanea) from Taiwan population was up to 30.7% when the wild relative was grown surrounded by a glutinous cultivated variety. The used wild rice plants by Oka & Chang might be O. sativa f. spontanea, that is, weedy rice rather than true O. rufipogon. It is also possible that this result might not be representative because of the small sample size (45/114 seeds) and lack of replication.
This study indicated that gene flow was not randomly distributed across the recipient range, but was significantly influenced by wind. This observation is similar to that reported by Messeguer et al. (2001). It is obvious that wind can significantly affect pollen flow and that downwind recipient plants may capture more foreign pollen grains, thus enhancing gene flow (Timmon et al., 1995). However, this study also showed that, besides the prevailing wind direction some hybridization occurred at other directions including the direction opposite of that of the wind. This pattern of gene flow could be attributable to the influence of microclimate, which is influenced by the microlandscape. The experimental site in this study was surrounded by hills running in a north–south direction. The wind was mainly from north to south and but occasionally was reversed, and whirlwinds also occurred occasionally. The whirlwind might result in randomly horizontal distribution of gene flow and therefore have slight effects on gene flow being found (Rognli et al., 2000; Staniland et al., 2000), but have significant effects of spatial distribution of pollen flow (Jackson & Lyford, 1999). The pollen flow of Minghui-63 directly measured in the same experimental system also showed the same pattern (Z.P. Song, unpublished data).
This study revealed that cultivated rice pollen could move downwind at a distance over 43 m, which is much wider than the isolation distance of 10 m generally recommended for hybrid seed production (Khush, 1993). On the other hand, the distance of pollen flow of cultivated rice found in this study was significantly shorter than that from investigation of rice pollen flow using male sterile cultivated variety as pollen recipients (43.2 m vs 100 m) (Y.G. Zhu, unpublished data). These disagreements were mainly due to differences in specific features between the cultivated varieties used by different researchers, and might also be attributable to different approaches applied for study. Gene flow is usually lower than pollen flow including the frequency and range due to the factors that influence the success of hybridization (Snow & Parker, 1998). Theoretically, the true range of pollen flow of Minghui-63 should be wider than 43 m because pollen may move further if the wind speed is strong enough (Jackson & Lyford, 1999). Pollen flow of Minghui-63 was observed up to 110 m when wind velocity was 10 m s−1 (Z.P. Song, unpublished data). The flowering pattern of rice and short longevity of rice pollen might be accounted for the limited distance of 43 m observed for hybridization in the present study (Messeguer et al., 2001; Song et al., 2001a). Consequently, as compared with the similar studies of other crops and their relatives (Klinger et al., 1991; Amand et al., 2000; Rognli et al., 2000; Saeglitz et al., 2000; Wilkinson et al., 2000), the gene flow between cultivated and wild rice occurred over a relatively short distance. These differences indicate that pollen flow or gene flow is associated with specific floral syndromes and pollination systems, in addition to climate conditions.
The frequency of gene flow significantly decreased with the increasing of distance away from the pollen source (Fig. 4), as has been found in other studies (Klinger et al., 1991; Rognli et al., 2000; Messeguer et al., 2001). From the relationship between gene flow and distance, the predictable distance of gene flow with a certain frequency or the frequency of gene flow at a certain distance can be easily obtained. The highest gene flow examined in this study was not at zero distance but instead at a distance of 3.6 m. One possible explanation for this observation is that O. rufipogon plants with an average height of 194.37 ± 5.65 cm are taller than Minghui-63 (64.27 ± 1.87 cm). The significant differences in height between the cultivated and wild rice species resulted in spatial isolation between flowers even though the two species were grown side by side.
Although the data from this study did not show significant influence of gene flow by the size of pollen sources, the scale-dependent effect of donor plot size on the frequency of gene flow (Amand et al., 2000) cannot be neglected. The inconsistent results might due to the difference of density between pollen donor (Minghui-63) and pollen recipient plants (O. rufipogon). Even in the CPC-1 design which had the smallest pollen source (20 Minghui-63 individuals), each individual had an average of eight flowering panicles and more than 20 spikelets per day discharged pollen grains. On the recipient plants, planted at a lower density, each individual had only about five panicles per individual and fewer spikelets flowered (4–9 flowering spikelets per day). One pollen grain is theoretically sufficient to fertilize one O. rufipogon spikelet, which has only a single ovule. Consequently, the large number of pollen grains of Minghui-63 could in principle fertilize all of the O. rufipogon spikelets. The relative inadequate data from the CPC-1–3 may influence the analytical results because the recipient plants did not have sufficient distance from the pollen sources. This suggests that further experiments with a larger scale are needed to address this question.
This study clearly showed that genes of cultivated rice could move to its sympatric wild relative O. rufipogon at a relatively high frequency within a certain distance. This suggests that some O. rufipogon populations might have introgressed genes from cultivated rice, because under natural conditions these populations co-occur with cultivated rice with a distance of less than 100 m. A sufficient isolation distance should be established for in situ conservation of O. rufipogon to minimize the contamination of the wild rice by cultivated rice pollen flow. This study also demonstrates a high probability that transgenes from genetically engineered rice varieties will escape to O. rufipogon populations in the areas where the wild and cultivated rice coexist. For containment of transgenic rice, spatial isolation between transgenic rice and their wild relatives should be seriously considered, in addition to other effective approaches. Importantly, a close monitoring of transgene escape and persist in environment should be implemented. It seems to be very difficult to completely avoid transgenes escape through gene flow, thus the safest strategy for management of transgenic rice is to avoid planting such rice varieties in regions where wild rice occurs. Moreover, a larger scale experiment to determine gene flow frequencies under natural conditions should provide more predictive results for the biosafety assessment, but this study clearly indicates that transgene escape will occur if transgenic rice and its close wild relatives are grown together. Therefore, questions of ecological consequences of such crop-to-wild gene flow need to be urgently addressed both for effective conservation of genetic resources and appropriate assessment of the risk caused by gene escape from transgenic crops. The experimental systems established in this study will be useful tools for such studies, especially for wind- and self-pollinated crop species.