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- Material and methods
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Many crops contain domestication genes that are generally considered to lower fitness of crop–wild hybrids in the wild environment. Transgenes placed in close linkage with such genes would be less likely to spread into a wild population. Therefore, for environmental risk assessment of GM crops, it is important to know whether genomic regions with such genes exist, and how they affect fitness. We performed quantitative trait loci (QTL) analyses on fitness(-related) traits in two different field environments employing recombinant inbred lines from a cross between cultivated Lactuca sativa and its wild relative Lactuca serriola. We identified a region on linkage group 5 where the crop allele consistently conferred a selective advantage (increasing fitness to 212% and 214%), whereas on linkage group 7, a region conferred a selective disadvantage (reducing fitness to 26% and 5%), mainly through delaying flowering. The probability for a putative transgene spreading would therefore depend strongly on the insertion location. Comparison of these field results with greenhouse data from a previous study using the same lines showed considerable differences in QTL patterns. This indicates that care should be taken when extrapolating experiments from the greenhouse, and that the impact of domestication genes has to be assessed under field conditions.
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- Material and methods
- Literature cited
- Supporting Information
Hybridization between crop and wild relatives occurs for many crops in at least part of their geographic range. Molecular evidence for transfer of nontransgenic crop alleles to wild relatives has been found for a variety of crop species (Kwit et al. 2011). This includes crops that were beforehand thought to be of very low introgression risk, such as soybean and common bean (Stewart et al. 2003; Kwit et al. 2011), suggesting that hybridization between crops and their wild relatives is a more common phenomenon than previously considered (Ellstrand 2003). In addition, escape of transgenes for herbicide resistance from commercially grown crops into wild relatives is reported for at least 14 individual events in North America (Ellstrand in press), for example in oilseed rape (Warwick et al. 2008).
At present, there are no studies showing evidence for any potential negative ecological consequences of gene flow from transgenic crops to wild relatives (Kwit et al. 2011), such as increased invasiveness of the wild relative. Nevertheless, the approval of new transgenic crops is very stringent (EFSA 2011), and scientists and crop breeders are searching for methods to minimize the likelihood of transgene escape. Several model studies have addressed which factors are most important to the spread of crop alleles after a hybridization event. These studies suggest that hybrid fitness is one of the most important factors and that a selectively advantageous gene can spread rapidly in spite of very low gene flow pressure (Huxel 1999; Haygood et al. 2004). If so, the fitness of a transgene and natural selection acting upon it could be more important than rates of gene flow (Chapman and Burke 2006).
It has therefore been suggested that transgenes placed in close linkage with an allele that is selected against in the wild are more likely to be purged from the wild population (Gressel 1999; Stewart et al. 2003); we will refer to this mitigation strategy as a ‘purging strategy’. The basis for such a purging strategy is the fact that chances for introgression of transgenes into a wild relative depend, on the one hand, on gene flow and/or propagule pressure, but even more so, on the fitness of initial hybrids and the fitness effect of transgenes in the wild genomic background (Ellstrand 2003).
Consequently, the fate of a transgene does not only depend on the fitness effect of the transgene itself, but also on the genes around it. If a transgene is linked to a crop allele that is positively selected for in the wild habitat, genetic hitchhiking could cause the transgene to spread even if the transgene is selectively neutral or even mildly deleterious (Stewart et al. 2003). Alternatively, if a transgene is placed in close linkage with a gene or genomic block that causes a lower fitness in the wild habitat compared to the wild relative, it will have a smaller chance to introgress (Gressel 1999; Stewart et al. 2003). A purging mitigation strategy was already experimentally tested in tobacco (Al-Ahmad et al. 2004) and oilseed rape hybrids (Rose et al. 2009), where a transgene was placed in close linkage with a dwarfing gene. In both cases, there was a dramatic reduction in the survival of transgenic hybrid individuals carrying the dwarfing gene. This confirms that the location where a transgene is placed within the crop genome can be of vital importance to the probabilities of introgression.
Many studies on hybrid fitness are conducted in the greenhouse or solely in an agricultural setting as opposed to realistic field conditions for the wild species (Hails and Morley 2005). Conclusions based on these experiments might be misleading because Genotype × Environment (G × E) interactions can cause different selection pressures between a controlled greenhouse setting and variable field conditions (Weinig et al. 2002; Martin et al. 2006; Latta et al. 2007). For example, crop alleles might be favored in a greenhouse pot experiment, whereas in more competitive environments, wild alleles could be favored.
Moreover, there is an overall lack of information regarding genes or genomic blocks under selection in the field (Hails and Morley 2005). It would be valuable for risk assessment, as proposed by EFSA (2011), to know in which crop–wild systems, there are regions in the crop genome that are more or are less likely to introgress, to assess the effectiveness of a purging strategy. Quantitative Trait Loci (QTL) analysis allows pinpointing the location of regions under selection, and the traits associated with these regions. To our knowledge, only a few studies on crop–wild hybrids have used QTL analysis for this purpose (Baack et al. 2008; Dechaine et al. 2009).
We use the crop lettuce (Lactuca sativa L.), a leafy vegetable, and its wild relative prickly lettuce (Lactuca serriola L.) as a crop–wild model system. In the past 50–60 years, L. serriola has expanded its range dramatically in Western Europe (Hooftman et al. 2006; D’Andrea et al. 2009). In a series of field experiments, Hooftman et al. (2005, 2007, 2009) showed that at least four generations of lettuce crop–wild hybrids had higher germination and survival rates than the wild parent. Further genetic analysis showed that crop alleles were favorable at some loci, but disfavored at others, suggesting the possibility for genetic hitchhiking as well as purging (Hooftman et al. 2009, 2011). Lettuce might be a good candidate for transgene mitigation strategies, because it is a predominantly selfing species. This means the initial linkage disequilibrium (LD) in first-generation hybrids decays slowly, and selection will effectively act on large genomic blocks rather than on individual loci (Flint-Garcia et al. 2003).
In this study, we use recombinant inbred lines (RILs) from a cross between the cultivated Iceberg lettuce (L. sativa cv. Salinas) and L. serriola (UC96US23) (Johnson et al. 2000) to analyze the effects of selective field conditions on hybrid fitness and QTL analysis to identify genomic regions under selection. We identified QTL in field experiments for a broad set of fitness and fitness-related traits at different life stages relevant to the success of Lactuca hybrids in the field. In addition, we compare these field results with domestication-related QTL from the same RIL population grown in the greenhouse (Y. Hartman, D.A.P. Hooftman, M.E. Schranz and P.H. van Tienderen, unpublished data).
Because the genomic location of crop (trans)genes can be of vital importance for the chance and rate of introgression, we studied the selection on genomic regions in different environments. Specifically, we addressed the following questions: (i) Which traits are important for fitness in the field and do crop alleles confer a selective (dis-)advantage? (ii) Are there regions where crop alleles provide such negative fitness effects that they could be effective in a purging strategy? (iii) How important is G × E? In particular, how do field QTL compare to greenhouse QTL and can small-scale contained greenhouse experiments be used to assess potential ecological consequences? The results are a first step in establishing whether the genomic location of a transgene in the crop is important for predicting its fate if outcrossing occurs to wild relatives.