Gene Introgression in Weeds Depends on Initial Gene Location in the Crop: Brassica napus-Raphanus raphanistrum Model.

The effect of gene location within a crop genome on its transfer to a weed genome remains an open question for gene flow assessment. To elucidate this question, we analyzed advanced generations of intergeneric hybrids, derived from an initial pollination of known oilseed rape varieties (Brassica napus, AACC, 2n = 38) by a local population of wild radish (Raphanus raphanistrum, RrRr, 2n = 18). After five generations of recurrent pollination, 307 G5 plants with a chromosome number similar to wild radish were genotyped using 105 B. napus specific markers well distributed along the chromosomes. They revealed that 49.8% of G5 plants carried at least one B. napus genomic region. According to the frequency of B. napus markers (0-28%), four classes were defined: Class 1 (near zero frequency), with 75 markers covering ∼70% of oilseed rape genome; Class 2 (low frequency), with 20 markers located on 11 genomic regions; Class 3 (high frequency), with eight markers on three genomic regions; and Class 4 (higher frequency), with two adjacent markers detected on A10. Therefore, some regions of the oilseed rape genome are more prone than others to be introgressed into wild radish. Inheritance and growth of plant progeny revealed that genomic regions of oilseed rape could be stably introduced into wild radish and variably impact the plant fitness (plant height and seed number). Our results pinpoint that novel technologies enabling the targeted insertion of transgenes should select genomic regions that are less likely to be introgressed into the weed genome, thereby reducing gene flow.

Relationship between weed dormancy and herbicide rotations: implications in resistance evolution.

It is suggested that selection for late germinating seed cohorts is significantly associated with herbicide resistance in some cropping systems. In turn, it is conceivable that rotating herbicide modes of action selects for populations with mutations for increased secondary dormancy, thus partially overcoming the delaying effect of rotation on resistance evolution. Modified seed dormancy could affect management strategies - like herbicide rotation - that are used to prevent or control herbicide resistance. Here, we review the literature for data on seed dormancy and germination dynamics of herbicide-resistant versus susceptible plants. Few studies use plant material with similar genetic backgrounds, so there are few really comparative data. Increased dormancy and delayed germination may co-occur with resistance to ACCase inhibitors, but there is no clear-cut link with resistance to other herbicide classes. Population shifts are due in part to pleiotropic effects of the resistance genes, but interaction with the cropping system is also possible. We provide an example of a model simulation that accounts for genetic diversity in the dormancy trait, and subsequent consequences for various cropping systems. We strongly recommend adding more accurate and detailed mechanistic modelling to the current tools used today to predict the efficiency of prevention and management of herbicide resistance. These models should be validated through long-term experimental designs including mono-herbicide versus chemical rotation in the field. © 2017 Society of Chemical Industry.

Simulating changes in cropping practices in conventional and glyphosate-resistant maize. II. Weed impacts on crop production and biodiversity.

Overreliance on the same herbicide mode of action leads to the spread of resistant weeds, which cancels the advantages of herbicide-tolerant (HT) crops. Here, the objective was to quantify, with simulations, the impact of glyphosate-resistant (GR) weeds on crop production and weed-related wild biodiversity in HT maize-based cropping systems differing in terms of management practices. We (1) simulated current conventional and probable HT cropping systems in two European regions, Aquitaine and Catalonia, with the weed dynamics model FLORSYS; (2) quantified how much the presence of GR weeds contributed to weed impacts on crop production and biodiversity; (3) determined the effect of cultural practices on the impact of GR weeds and (4) identified which species traits most influence weed-impact indicators. The simulation study showed that during the analysed 28 years, the advent of glyphosate resistance had little effect on plant biodiversity. Glyphosate-susceptible populations and species were replaced by GR ones. Including GR weeds only affected functional biodiversity (food offer for birds, bees and carabids) and weed harmfulness when weed effect was initially low; when weed effect was initially high, including GR weeds had little effect. The GR effect also depended on cultural practices, e.g. GR weeds were most detrimental for species equitability when maize was sown late. Species traits most harmful for crop production and most beneficial for biodiversity were identified, using RLQ analyses. None of the species presenting these traits belonged to a family for which glyphosate resistance was reported. An advice table was built; the effects of cultural practices on crop production and biodiversity were synthesized, explained, quantified and ranked, and the optimal choices for each management technique were identified.

Simulating changes in cropping practises in conventional and glyphosate-tolerant maize. I. Effects on weeds.

Herbicide-tolerant (HT) crops such as those tolerant to glyphosate simplify weed management and make it more efficient, at least at short-term. Overreliance on the same herbicide though leads to the spread of resistant weeds. Here, the objective was to evaluate, with simulations, the impact on the advent of glyphosate resistance in weeds of modifications in agricultural practises resulting from introducing HT maize into cropping systems. First, we included a single-gene herbicide resistance submodel in the existing multispecific FLORSYS model. Then, we (1) simulated current conventional and probable HT cropping systems in two European regions, Aquitaine and Catalonia, (2) compared these systems in terms of glyphosate resistance, (3) identified pertinent cultural practises influencing glyphosate resistance, and (4) investigated correlations between cultural practises and species traits, using RLQ analyses. The simulation study showed that, during the analysed 28 years, (1) glyphosate spraying only results in glyphosate resistance in weeds when combined with other cultural factors favouring weed infestation, particularly no till; (2) pre-sowing glyphosate applications select more for herbicide resistance than post-sowing applications on HT crops; and (3) glyphosate spraying selects more for species traits avoiding exposure to the herbicide (e.g. delayed early growth, small leaf area) or compensating for fitness costs (e.g. high harvest index) than for actual resistance to glyphosate, (4) actual resistance is most frequent in species that do not avoid glyphosate, either via plant size or timing, and/or in less competitive species, (5) in case of efficient weed control measures, actual resistance proliferates best in outcrossing species. An advice table was built, with the quantitative, synthetic ranking of the crop management effects in terms of glyphosate-resistance management, identifying the optimal choices for each management technique.

Choosing the best cropping systems to target pleiotropic effects when managing single-gene herbicide resistance in grass weeds. A blackgrass simulation study.

BACKGROUND: Managing herbicide-resistant weeds is becoming increasingly difficult. Here we adapted the weed dynamics model AlomySys to account for experimentally measured fitness costs linked to mutants of target-site resistance to acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides in Alopecurus myosuroides. We ran simulations to test how effectively cultural practices manage resistance. RESULTS: Simulations of an oilseed rape/winter wheat/winter barley rotation showed that, when replacing one of the seven applied herbicides with an ACCase-inhibiting one, resistant mutants exceeded 1 plant m(-2) , with a probability of 40%, after an average of 18 years. This threshold was always exceeded when three or four ACCase-inhibiting herbicides were used, after an average of 8 and 6 years respectively. With reduced herbicide rates or suboptimal spraying conditions, resistance occurred 1-3 years earlier in 50% of simulations. Adding spring pea to the rotation or yearly mouldboard ploughing delayed resistance indefinitely in 90 and 60% of simulations respectively. Ploughing also modified the genetic composition of the resistant population by selecting a previously rare mutant that presented improved pre-emergent growth. The prevalence of the mutations was influenced more by their associated fitness cost or benefit than by the number of ACCase-inhibiting herbicides to which they conferred resistance. CONCLUSION: Simulations allowed us to rank weed management practices and suggest that pleiotropic effects are extremely important for understanding the frequency of herbicide resistance in the population. © 2016 Society of Chemical Industry.

Fitness cost due to herbicide resistance may trigger genetic background evolution.

This article investigates the possible existence of mechanisms counterbalancing the negative pleiotropic effects on development and reproduction that are conferred by alleles responsible for herbicide resistance in the weed Alopecurus myosuroides. We considered three herbicide-resistant, mutant acetyl-coenzyme A carboxylase (ACCase) alleles, Leu1781, Asn2041, and Gly2078, found in eight resistant populations. Of these, Gly2078 is the only allele with a known fitness cost. We compared plants homozygous for wild-type ACCase alleles that were siblings of plants carrying a given mutant resistant ACCase allele with plants from three populations where resistance did not evolve. In each of two series of experiments, we measured germination dynamics, seedling vigor, plant height, vegetative biomass, and seed production. The wild-type siblings of plants carrying Gly2078 performed better in the field, on average, than wild-type plants that were sibling of plants carrying other mutant ACCase alleles, and particularly those carrying Leu1781. We propose that rapid evolution of the genetic background of plants from the populations where the Gly2078 allele originally arose could partially counterbalance Gly2078 fitness cost, enhancing the spread of the resistant genotypes.

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

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.

Pleiotropic effects of herbicide-resistance genes on crop yield: a review.

The rapid adoption of genetically engineered herbicide-resistant crop varieties (HRCVs)-encompassing 83% of all GM crops and nearly 8% of the worldwide arable area-is due to technical efficiency and higher returns. Other herbicide-resistant varieties obtained from genetic resources and mutagenesis have also been successfully released. Although the benefit for weed control is the main criteria for choosing HRCVs, the pleiotropic costs of genes endowing resistance have rarely been investigated in crops. Here the available data of comparisons between isogenic resistant and susceptible varieties are reviewed. Pleiotropic harmful effects on yield are reported in half of the cases, mostly with resistance mechanisms that originate from genetic resources and mutagenesis (atrazine in oilseed rape and millet, trifluralin in millet, imazamox in cotton) rather than genetic engineering (chlorsulfuron and glufosinate in some oilseed rape varieties, glyphosate in soybean). No effect was found for sethoxydim and bromoxynil resistance. Variable minor effects were found for imazamox, chlorsulfuron, glufosinate and glyphosate resistance. The importance of the breeding plan and the genetic background on the emergence of these effects is pointed out. Breeders' efforts to produce better varieties could compensate for the yield loss, which eliminates any possibility of formulating generic conclusions on pleiotropic effects that can be applied to all resistant crops.

Spread of introgressed insect-resistance genes in wild populations of Brassica juncea: a simulated in-vivo approach.

Introgression between transgenic, insect-resistant crops and their wild relatives could lead to a progressive increase of the frequency of resistant plants in wild populations. However, few studies help predict the impact on the population dynamics. To simulate the performance of introgressed insect-resistant plants of wild Brassica juncea, independently from the interspecific hybridization cost, healthy plants were cultivated in pure and mixed stands with damaged plants through cutting leaves in field experiments over two field seasons. As expected, resistant (healthy) plants held a competitive advantage when in competition with susceptible (damaged) plants. Individual biomass and seed production of both types of plants decreased as the percentage of resistant plants increased, so that the relative advantage of resistant plants increased. The combined effects of defoliation and competition on the individual performance of B. juncea were additive. Replacement series experiments confirmed this trend but did not show different seed output in pure stand of susceptible versus resistant plots. The total vegetative and reproductive production of mixed populations was not significantly different of that of pure populations. These results suggest that if a transgene for insect-resistance were to colonize wild populations, high herbivory of susceptible plant and low resource availability would facilitate the spread of resistant individuals. However, at the population level, the shift from an insect-susceptible to a predominantly resistant population would not result in exacerbated habitat colonization.

The effects of seed size on hybrids formed between oilseed rape (Brassica napus) and wild brown mustard (B. juncea).

BACKGROUND: Seed size has significant implications in ecology, because of its effects on plant fitness. The hybrid seeds that result from crosses between crops and their wild relatives are often small, and the consequences of this have been poorly investigated. Here we report on plant performance of hybrid and its parental transgenic oilseed rape (Brassica napus) and wild B. juncea, all grown from seeds sorted into three seed-size categories. METHODOLOGY/PRINCIPAL FINDINGS: Three seed-size categories were sorted by seed diameter for transgenic B. napus, wild B. juncea and their transgenic and non-transgenic hybrids. The seeds were sown in a field at various plant densities. Globally, small-seeded plants had delayed flowering, lower biomass, fewer flowers and seeds, and a lower thousand-seed weight. The seed-size effect varied among plant types but was not affected by plant density. There was no negative effect of seed size in hybrids, but it was correlated with reduced growth for both parents. CONCLUSIONS: Our results imply that the risk of further gene flow would probably not be mitigated by the small size of transgenic hybrid seeds. No fitness cost was detected to be associated with the Bt-transgene in this study.

Testing coexistence and genetic containment for an autogamous crop.

Is there any risk that the threshold for admixture of genetically modified seeds in the harvest of a conventional cultivar, 0.9% in Europe, will be exceeded in the case of inbreeder crops? Using herbicide-resistant foxtail millet, Setaria italica, as a model of a preferentially autogamous crop, such as wheat and rice, field experiments show that genotype admixture due to pollen flow between adjacent fields is about 0.03% on average for the 10 adjacent meters, and 10 times less in the next 20-m lane. In the case of a maternally inherited resistance gene, the admixture rate is at least 100 times lower. Recessive herbicide resistance has also been tested but would be efficient only if the agreed coexistence rules were based on phenotype detection.

Pollen dispersal in sugar beet production fields.

Pollen-mediated gene flow has important implications for biodiversity conservation and for breeders and farmers' activities. In sugar beet production fields, a few sugar beet bolters can produce pollen as well as be fertilized by wild and weed beet. Since the crop, the wild beets, and the weed beets are the same species and intercross freely, the question of pollen flow is an important issue to determine the potential dispersal of transgenes from field to field and to wild habitats. We report here an experiment to describe pollen dispersal from a small herbicide-resistant sugar beet source towards male sterile target plants located along radiating lines up to 1,200 m away. Individual dispersal functions were inferred from statistical analyses and compared. Pollen limitation, as expected in root-production fields, was confirmed at all the distances from the pollen source. The number of resistant seeds produced by bait plants best fitted a fat-tailed probability distribution curve of pollen grains (power-law) dependent on the distance from the pollen source. A literature survey confirmed that power-law function could fit in most cases. The b coefficient was lower than 2. The number of fertilized flowers by background (herbicide-susceptible) pollen grains was uniform across the whole field. Airborne pollen had a fertilization impact equivalent to that of one adjacent bolter. The individual dispersal function from different pollen sources can be integrated to provide the pollen cloud composition for a given target plant, thus allowing modeling of gene flow in a field, inter-fields in a small region, and also in seed-production area. Long-distance pollen flow is not negligible and could play an important role in rapid transgene dispersal from crop to wild and weed beets in the landscape. The removing of any bolting, herbicide-resistant sugar beet should be compulsory to prevent the occurrence of herbicide-resistant weed beet, thus preventing gene flow to wild populations and preserving the sustainable utility of the resistant varieties. Whether such a goal is attainable remains an open question and certainly would be worth a large scale experimental study.

Impact of transgene inheritance on the mitigation of gene flow between crops and their wild relatives: the example of foxtail millet.

Developing genetically modified crop plants that are biologically contained could reduce significantly the potential spread of transgenes to conventional and organic crop plants and to wild or weedy relatives. Among several strategies, the hereditary mode of transmission of transgenes, whether dominant, recessive, or maternal, could play a major role in interspecific gene flow. Here we report on the gene flow between foxtail millet (Setaria italica), an autogamous crop, and its weedy relative, S. viridis, growing within or beside fields containing the three kinds of inherited herbicide resistance. Over the 6-year study, in the absence of herbicide selection, the maternal chloroplast-inherited resistance was observed at a 2 x 10(-6) frequency in the weed populations. Resistant weed plants were observed 60 times as often, at 1.2 x 10(-4) in the case of the nuclear recessive resistance, and 190 times as often, at 3.9 x 10(-4) in the case of the dominant resistance. Because the recessive gene was not expressed in the first-generation hybrids, it should be more effective than dominant genes in reducing gene flow under normal agricultural conditions where herbicides are sprayed because interspecific hybrids cannot gain from beneficial genes.

Transgene escape in sugar beet production fields: data from six years farm scale monitoring.

Concerns have been raised in Europe about the efficiency, sustainability, and environmental impact of the first genetically modified crops. The committees and regulators in charge of approving procedures have encouraged a field trial approach for safety assessment studies under current agronomic conditions. We describe the gene flow from sugar beet (Beta vulgaris L.) in a multi-year and multi-crop monitoring study on farmers' fields at two locations that has been carried out since 1995. We analyzed two sugar beet lines that have been genetically transformed for herbicide resistance. One sugar beet has resistance to glufosinate and the other to glyphosate. Large differences among lines, years and locations were observed. These differences provided a broad range of situations to estimate the risks. Sugar beet bolters produced the majority (86%) of the herbicide-resistant seeds harvested in the field. Direct pollen flow from sugar beet bolters to weed beets that were growing within the same field as well as in a neighboring field that was left fallow accounted for only 0.4% of the resistant seeds released over the years and locations. Descendants of the hybrids between the sugar beet and the weed beet produced the remaining 13.6% of resistant seeds. Herbicide-resistant seeds from the progeny of the weed beet were recorded up to 112 m away from the closest transgenic pollen donor. Indications were observed of non-randomness of the weed beet producing resistant progeny. We also analyzed pollen flow to male-sterile bait plants located within and outside of the sugar beet field. Herbicide-resistant pollen flow was recorded up to 277 m, and fitted with an inverse power regression. Using sugar beet varieties with no, or very low, sensitivity to bolting and destroying bolters are two necessary measures that could delay gene flow.

Rapid bidirectional allele-specific PCR identification for triazine resistance in higher plants.

Triazine resistance is reported to be due to chloroplast herbicide target insensitivity in most species, and this is most often caused by a Ser(264)-Gly mutation at the D1 protein. In order to ascertain whether this mutation is really predominant amongst resistant plants, and also for gene flow studies, a rapid test is needed that allows the testing of large quantities of plants. Here a bidirectional allele-specific PCR (polymerase chain reaction) identification is proposed. The designed primers were shown to be universal in the three grass and three broadleaf species examined.

Molecular bases for sensitivity to tubulin-binding herbicides in green foxtail.

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two alpha- and two beta-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding alpha2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional alpha-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.

Lack of stable inheritance of introgressed transgene from oilseed rape in wild radish.

Hybridization of Brassica napus L. (oilseed rape) and Raphanus raphanistrum L. (wild radish) has been demonstrated, and may be the first step towards introgression of transgenes in this wild relative. If wild radish were to display a new adaptive advantage by expressing the transgene, this could modify the ecological balance of species within the agro-ecosystem. To determine if transgenes remained stable in the hybrid, the frequency of herbicide resistance was studied over four advanced generations of hybrid progeny (G8 to G11) that were subjected to herbicide selection pressure. It is expected that hemizygous resistant plants containing an herbicide resistance transgene back-crossed to wild radish would have 50% resistant progeny. In each of the G8 to G11 generations, only 18% of the progeny from resistant plants were resistant. The chromosome complement of herbicide-susceptible progenies, analyzed at G9, was not different from that of wild populations of wild radish. Herbicide-resistant G9 progeny showed higher chromosome instability, and one third of the progeny contained a supernumerary chromosome. These results suggest that in the presence of herbicide selection pressure, the transgene for herbicide resistance would be maintained despite a lack of stabilized introgression. In the absence of selection, the frequency of resistance in the population is expected to decline.

An isoleucine-leucine substitution in chloroplastic acetyl-CoA carboxylase from green foxtail (Setaria viridis L. Beauv.) is responsible for resistance to the cyclohexanedione herbicide sethoxydim.

The cDNAs encoding chloroplastic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) from three lines of Setaria viridis (L. Beauv.) resistant or sensitive to sethoxydim, and from one sethoxydim-sensitive line of Setaria italica (L. Beauv.) were cloned and sequenced. Sequence comparison revealed that a single isoleucine-leucine substitution discriminated ACCases from sensitive and resistant lines. Using near-isogenic lines of S. italica derived from interspecific hybridisation, we demonstrated that the transfer of the S. viridis mutant ACCase allele into a sethoxydim-sensitive S. italica line conferred resistance to this herbicide. We confirmed this result using allele-specific polymerase chain reaction and showed that a single copy of the mutant allele is sufficient to confer resistance to sethoxydim. We conclude that a mutant allele of chloroplastic ACCase encoding a leucine residue instead of an isoleucine residue at position 1780 is a major gene of resistance to sethoxydim.