High gene flow maintains genetic diversity following selection for high EPSPS copy number in the weed kochia (Amaranthaceae).

Kochia, a major weed species, has evolved resistance to four herbicide modes of action. Herbicide resistance appears to spread quickly, which could result in diminished standing genetic variation, reducing the ability of populations to adapt further. Here we used double digest restriction enzyme associated sequencing to determine the level of gene flow among kochia populations and whether selection for glyphosate resistance reduces genetic variation. Canadian Prairie populations show little to no genetic differentiation (FST = 0.01) and no correlation between genetic and geographic distance (r2 = - 0.02 p = 0.56), indicating high gene flow and no population structure. There is some evidence that kochia populations are genetically depauperate compared to other weed species, but genetic diversity did not differ between glyphosate susceptible and resistant populations or individuals. The inbreeding coefficients suggest there are 23% fewer heterozygotes in these populations than expected with random mating, and no variation was found within the chloroplast. These results indicate that any alleles for herbicide resistance can be expected to spread quickly through kochia populations, but there is little evidence this spread will reduce the species' genetic variation or limit the species' ability to respond to further selection pressure.

Hybridization rate and hybrid fitness for Camelina microcarpa Andrz. ex DC (♀) and Camelina sativa (L.) Crantz(Brassicaceae) (♂).

Hybridization between crops and their wild relatives has the potential to introduce novel variation into wild populations. Camelina (Camelina sativa) is a promising oilseed and cultivars with modified seed characteristics and herbicide resistance are in development, prompting a need to evaluate the potential for novel trait introgression into weedy relatives. Little-podded false flax (littlepod; Camelina microcarpa) is a naturalized weed in Canada and the USA. Here we evaluated the hybridization rate between the three cytotypes of littlepod (♀) and camelina (♂), assessed characteristics of hybrids, and evaluated the fitness of hexaploid littlepod and camelina hybrids in the glasshouse and field. In total we conducted, 1,005 manual crosses with diploid littlepod, 1, 172 crosses with tetraploid littlepod, and 896 crosses with hexaploid littlepod. Hybrids were not produced by the diploids, but were produced by the tetraploids and hexaploids at rates of one hybrid for 2,000 ovules pollinated and 24 hybrids for 25 ovules pollinated, respectively. Hybrids between tetraploid littlepod and camelina showed low pollen fertility and produced a small number of seeds. In the glasshouse, hybrids between hexaploid littlepod and camelina also showed significantly lower pollen fertility and seed production than parental lines, but their seeds showed high viability. A similar pattern was observed in field trials, with hybrids showing earlier flowering, reduced biomass, seed production and seed weight. However, seed produced by the hybrids showed greater viability than that produced by hexaploid littlepod and is potentially the result of a shortened lifecycle. The introgression of lifecycle traits into littlepod populations may facilitate range expansion and contribute to crop gene persistence. Consequently, future work should evaluate the hybridization rate in the field, the fitness of advanced generation backcrosses, and the role of time to maturity in limiting hexaploid littlepod's distribution.

Lack of efficacy of transgenic pea (Pisum sativum L.) stably expressing antifungal genes against Fusarium spp. in three years of confined field trials.

Fusarium root rot is a major pea disease in Canada and only partial tolerance exists in germplasm. Transgenic technologies may hold promise but the economic benefits of genetically modified (GM) pea will need to surpass the regulatory costs, time and labor involved in bringing a GM crop to market. European pea (Pisum sativum L.) cultivars expressing four antifungal genes, 1-3 ß glucanase (G), endochitinase (C) (belonging to PR proteins family), polygalacturonase inhibiting proteins (PGIPs) (P) and stilbene synthase (V) have been transformed for disease tolerance and showed disease tolerance under laboratory conditions. Transgenic lines with four antifungal genes inserted either individually or stacked through crossing were tested for their efficacy against Fusarium root rot (Fusarium avenaceum) in confined trials over three years (2013 to 2015) in comparison with two parental German lines and three Canadian lines. Superior emergence, higher fresh weight or lower disease ratings above and below ground, of transgenic lines in presence of disease inoculum were not observed consistently in the three years of field experiments when compared to the parental and Canadian lines in the presence of disease inoculum. No indication of an advantage of stacked genes over single genes was observed. Most transgenic lines had lower relative gene expression in the roots than in the leaves in greenhouse trials suggesting a possible explanation for poor tolerance to Fusarium root rot. Field trials are necessary to verify the agronomic performance and ecological relevance of the promising effects detected under laboratory conditions.

Antifungal genes expressed in transgenic pea (Pisum sativum L.) do not affect root colonization of arbuscular mycorrhizae fungi.

Genetically modified crops have raised concerns about unintended consequences on non-target organisms including beneficial soil associates. Pea transformed with four antifungal genes 1-3 ß glucanase, endochitinase, polygalacturonase-inhibiting proteins, and stilbene synthase is currently under field-testing for efficacy against fungal diseases in Canada. Transgenes had lower expression in the roots than leaves in greenhouse experiment. To determine the impact of disease-tolerant pea or gene products on colonization by non-target arbuscular mycorrhizae and nodulation by rhizobium, a field trial was established. Transgene insertion, as single gene or stacked genes, did not alter root colonization by arbuscular mycorrhiza fungus (AMF) or root nodulation by rhizobium inoculation in the field. We found no effect of transgenes on the plant growth and performance although, having a dual inoculant with both AMF and rhizobium yielded higher fresh weight shoot-to-root ratio in all the lines tested. This initial risk assessment of transgenic peas expressing antifungal genes showed no deleterious effect on non-target organisms.

Glyphosate resistance reduces kochia fitness: Comparison of segregating resistant and susceptible F2 populations.

Glyphosate is considered the world's most important herbicide, but widespread and continual use has resulted in the evolution of resistance. Kochia scoparia (kochia) has evolved resistance via tandem gene amplification of glyphosate's target, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and resistant populations have been reported from the Canadian Prairies and the Northern Great Plains. Here, we evaluated the fitness costs of EPSPS amplification in kochia by comparing susceptible and resistant full siblings from segregating F2 populations generated from within six populations. Kochia was expected to be highly diverse because of strong gene flow; however, six of the seven field-collected parents with higher EPSPS copy number were homozygous. Under competitive greenhouse conditions, the EPSPS type of the line's maternal parent showed persistent effects: delayed emergence, delayed flowering, and reductions in viable seed count and weight overall. High EPSPS copy number individuals had reduced seed count and weight, reduced competitive ability, and reduced final height in mixed stands, but better germination of the F3. However, all characteristics were highly variable and fitness costs were not constant across genetic backgrounds. In the absence of selection from glyphosate, kochia with increased EPSPS copy number will be at a competitive disadvantage in some genetic backgrounds.

Molecular markers as a complementary tool in risk assessments: quantifying interspecific gene flow from triticale to spring wheat and durum wheat.

Triticale is being considered as a bioindustrial crop in Canada using genetic modification. Because related spring wheat (Triticum aestivum) and durum wheat (T. durum) may exhibit synchronous flowering and grow in proximity, determination of interspecific gene flow when triticale is the pollen donor is necessary to evaluate potential risk. Pollen-mediated gene flow risk assessments generally rely on phenotypic markers to detect hybridization but DNA markers could be powerful and less ambiguous in quantifying rare interspecific gene flow. Six cultivars representing four species [spring wheat, durum wheat, triticale and rye (Secale cereale)] were screened with 235 spring wheat and 27 rye SSR markers to evaluate transferability and polymorphism. Fifty-five polymorphic markers were used in conjunction with morphological characterization to quantify interspecific gene flow from a blue aleurone (BA) triticale line to two spring wheat cultivars (AC Barrie and AC Crystal) and one durum wheat cultivar (AC Avonlea). Approximately 1.9 Million seeds from small plot experiments were visually screened in comparison with known hybrid seed. In total 2031 putative hybrids were identified and 448 germinated. Morphological analysis of putative hybrid plants identified five hybrids while molecular analysis identified 11 hybrids and two were common to both. Combined, 14 hybrids were confirmed: 10 spring wheat × triticale (0.0008 % of harvested seed): seven AC Barrie × BA triticale (0.001 %) and three AC Crystal × BA triticale (0.0005 %); and four durum wheat × triticale (0.0006 %). The occurrence of rare hybrids does not present a substantial risk to the development of GM triticale.

Gene flow, invasiveness, and ecological impact of genetically modified crops.

The main environmental concerns about genetically modified (GM) crops are the potential weediness or invasiveness in the crop itself or in its wild or weedy relatives as a result of transgene movement. Here we briefly review evidence for pollen- and seed-mediated gene flow from GM crops to non-GM or other GM crops and to wild relatives. The report focuses on the effect of abiotic and biotic stress-tolerance traits on plant fitness and their potential to increase weedy or invasive tendencies. An evaluation of weediness and invasive traits that contribute to the success of agricultural weeds and invasive plants was of limited value in predicting the effect of biotic and abiotic stress-tolerance GM traits, suggesting context-specific evaluation rather than generalizations. Fitness data on herbicide, insect, and disease resistance, as well as cold-, drought-, and salinity-tolerance traits, are reviewed. We describe useful ecological models predicting the effects of gene flow and altered fitness in GM crops and wild/weedy relatives, as well as suitable mitigation measures. A better understanding of factors controlling population size, dynamics, and range limits in weedy volunteer GM crop and related host or target weed populations is necessary before the effect of biotic and abiotic stress-tolerance GM traits can be fully assessed.

Pollen-mediated gene flow from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming to conventional safflower.

Field experiments were conducted in Chile and western Canada to measure short-distance (0 to 100 m) outcrossing from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming to non-transgenic commodity safflower of the same variety. The transgenic safflower used as the pollen source was transformed with a construct for seed-specific expression of a high-value protein and constitutive expression of a gene conferring resistance to the broad-spectrum herbicide glufosinate. Progeny of non-transgenic plants grown in plots adjacent to the transgenic pollen source were screened for glufosinate resistance to measure outcrossing frequency. Outcrossing frequency differed among locations: values closest to the transgenic pollen source (0 to 3 m) ranged from 0.48 to 1.67% and rapidly declined to between 0.0024 to 0.03% at distances of 50 to 100 m. At each location, outcrossing frequency was spatially heterogeneous, indicating insects or wind moved pollen asymmetrically. A power analysis assuming a binomial distribution and a range of alpha values (type 1 error) was conducted to estimate an upper and lower confidence interval for the probable transgenic seed frequency in each sample. This facilitated interpretation when large numbers of seeds were screened from the outcrossing experiments and no transgenic seeds were found. This study should aid regulators and the plant molecular farming industry in developing confinement strategies to mitigate pollen mediated gene flow from transgenic to non-transgenic safflower.

Potential for seed-mediated gene flow in agroecosystems from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming.

Safflower has been transformed for field scale molecular farming of high-value proteins including several pharmaceuticals. Viable safflower seed remaining in the soil seed bank after harvest could facilitate seed and pollen-mediated gene flow. Seeds may germinate in subsequent years and volunteer plants may flower and potentially outcross with commodity safflower and/or produce seed. Seeds from volunteers could become admixed with conventional crops at harvest, and/or replenish the seed bank. Seed in following crops could be transported locally and internationally and facilitate gene flow in locations where regulatory thresholds and public acceptance differ from Canada. Seed-mediated gene flow was examined in three studies. Safflower seed loss and viability following harvest of commercial fields of a non-transgenic cultivar were determined. We assessed seed longevity of transgenic and non-transgenic safflower, on the soil surface and buried at two depths. Finally, we surveyed commercial safflower fields at different sites and measured density and growth stage of safflower volunteers, in other crops the following year and documented volunteer survival and viable seed production. Total seed loss at harvest in commercial fields, ranged from 231 to 1,069 seeds m(-2) and the number of viable seeds ranged from 81 to 518 seeds m(-2). Safflower has a relatively short longevity in the seed bank and no viable seeds were found after 2 years. Based on the seed burial studies it is predicted that winter conditions would reduce safflower seed viability on the soil surface by >50%, leaving between 40 and 260 viable seeds m(-2). The density of safflower volunteers emerging in the early spring of the following year ranged from 3 to 11 seedlings m(-2). Safflower volunteers did not survive in fields under chemical fallow, but in some cereal fields small numbers of volunteers did survive and generate viable seed. Results will be used to make recommendations for best management practices to reduce seed-mediated gene flow from commercial production of plant molecular farming with safflower.

Evaluation of crossability between triticale (X Triticosecale Wittmack) and common wheat, durum wheat and rye.

Development of transgenic triticale as a platform for novel bio-industrial products is predicated on an environmental biosafety assessment that quantifies the potential risks associated with its release. Pollen-mediated gene flow to related species and conventional triticale varieties is one pathway for transgene movement. A tier 1 quantification of triticale hybridization was conducted by emasculating and hand pollinating flowers under greenhouse conditions. Approximately 2000 manual pollinations were conducted for each cross and its reciprocal between two triticale genotypes: a modern triticale cultivar (AC Alta) and primary triticale (89TT108), and common wheat, durum wheat and rye. The frequency of outcrossing, hybrid seed appearance and weight, and F(1) emergence and fertility were recorded. Outcrossing, F(1) emergence and fertility rates were high from crosses between triticale genotypes. Outcrossing in inter-specific crosses was influenced by the species, and the genotype and gender of the triticale parent. In crosses to common and durum wheat where triticale was the male parent, outcrossing was > or =73.0% and > or =69.5%, respectively, but < or =23.9% and < or =3.0% when triticale was the female parent. Overall, outcrossing with rye was lower than with common and durum wheat. F(1) hybrid emergence was greater when triticale was the female parent. With the exception of a single seed, all wheat-triticale F(1) hybrid seeds were non-viable when triticale was the male parent in the cross. Only seven durum wheat-triticale F(1) hybrids emerged from 163 seeds sown, and all were produced with triticale 89TT108 as female parent. With rye, 8 F(1) hybrids emerged from 38 seeds sown, and all were produced from crosses to AC Alta; five with AC Alta as the female parent and three as the male. Interspecific F(1) hybrids were self-sterile, with the exception of those produced in crosses between common wheat and triticale where triticale was the female parent. Tier 2 hybridization quantification will be conducted under field conditions.

Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster.

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (NPFR76F, also called GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62-66% similarity and 32-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila.

tipE regulates Na+-dependent repetitive firing in Drosophila neurons.

The tipE gene, originally identified by a temperature-sensitive paralytic mutation in Drosophila, encodes a transmembrane protein that dramatically influences sodium channel expression in Xenopus oocytes. There is evidence that tipE also modulates sodium channel expression in the fly; however, its role in regulating neuronal excitability remains unclear. Here we report that the majority of neurons in both wild-type and tipE mutant (tipE-) embryo cultures fire sodium-dependent action potentials in response to depolarizing current injection. However, the percentage of tipE- neurons capable of firing repetitively during a sustained depolarization is significantly reduced. Expression of a tipE+ transgene, in tipE- neurons, restores repetitive firing to wild-type levels. Analysis of underlying currents reveals a slower rate of repolarization-dependent recovery of voltage-gated sodium currents during repeated activation in tipE- neurons. This phenotype is also rescued by expression of the tipE+ transgene. These data demonstrate that tipE regulates sodium-dependent repetitive firing and recovery of sodium currents during repeated activation. Furthermore, the duration of the interstimulus interval necessary to fire a second full-sized action potential is significantly longer in single- versus multiple-spiking transgenic neurons, suggesting that a slow rate of recovery of sodium currents contributes to the decrease in repetitive firing in tipE- neurons.