Progress 01/15/02 to 10/02/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is part of National Program 302, Plant Biological and Molecular Processes (relevant to Component 3, Plant Biotechnology Risk Assessment). This project was initiated to characterize the type and frequency of genetic exchange among plants, especially to characterize potential risks associated with genetically engineered (GE) crops. The successful deployment of genetically improved crops, including GE crops, is contingent upon minimizing their ecological impact on the environment while enhancing the economic opportunities for growers to produce a safe and nutritious food supply. Genes are incorporated into crops and maintained by asexual and sexual processes with the intent that they not move into other cultivars of a crop, or into wild species with which a crop can intermate.
Seed producers utilize biological mechanisms, such as sexual incompatibility, and geographic separation to eliminate unintentional outcrossing, but sometimes these systems are ineffective in limiting pollen movement and consequent gene flow. This project examines the level of gene flow and the potential impact of genes in their novel environments. While some data are available to document the movement of genes between cultivated crops and wild plant species occurring in production areas, the extent of gene flow and the impact of new genes are not well known. Furthermore, there is little known about the biological mechanisms that limit or stimulate this process. The deployment of genetically engineered crops brings an urgent need for understanding and manipulating factors affecting and mechanisms underlying these processes. 2. List by year the currently approved milestones (indicators of research progress) The project plan for this project was accepted spring 2006. Milestones for 2006
were taken from last year's annual report. The milestones for the new project plan are for 2007-2011. 1. Complete the data analyses for the study examining the impact of insect pollinators on outcrossing. rate; write up and submit the manuscript. 2. Continue genotyping leaves and seeds from the pollinator exclusion experiment using microsatellite markers. 3. Start examining differences between pollinators in pollen carryover and paternity shadow. 4. Continue genotyping individuals from native plant populations using microsatellites to determine genetic diversity. 5. Continue making appropriate crosses between transgenic cultivated and native species. The new project plan includes the following milestones: 2007 1. Genotype all plants for paternity shadow in the greenhouse. 2. Set up experiment for pollen carryover and paternity shadow in greenhouse. 3. Set up pollen carryover in the field. 4. Set up field experiment for multiple visits in field. 5. Collect seeds and leaves
from experimental plants and leaves from potential fathers within 30 meter radius. 6. Start genotyping leaves of all potential fathers using microsatellites. 7. Complete pollen competition experiment in field. 8. Start growing seedlings in the greenhouse, inoculate and test for presence of transgene. 2008 1. Count pollen dyes for pollen carryover in the greenhouse and in the field. 2. Run microsatellites on seeds for paternity shadow in the greenhouse. 3. Paternity shadow in the field. 4. Multiple visits in field: Genotype 20 seeds from 20 plants within each pollinator treatment using microsatellites. 5. Complete comparative fitness experiment in field. 6. Continue testing seedlings in the greenhouse for presence of transgene. 7. Start locating wild populations and gather leaf samples from each visited population. 8. Test microsatellite loci for variability in wild squash. 2009 1. Data analyses for pollen carryover and paternity shadow in greenhouse. 2. Write up pollen
carryover and paternity shadow in greenhouse. 3. Run microsatellites for paternity shadow in field. 4. Finish genotyping all seeds for multiple visits experiment. 5. Estimate selfing rate for the 20 plants per treatment and determine impact of pollinators on outcrossing rate. 6. Start running paternity analyses on data. 7. Process samples from comparative fitness experiment (pollen count, fruit and seed count). 8. Write manuscript on pollen competition experiments 9. Continue locating wild populations and gathering leaf samples. 10. Gather seed samples from 40 (or all) individuals in one population. 11. Start running microsatellite primers on leaves and seeds. 12. Test leaves for presence of viruses. 2010 1. Data analyses for pollen carryover and paternity shadow in field. 2. Write manuscript on comparative fitness experiments. 3. Gather pollinator data from one wild squash population. 4. Continue locating wild populations and collecting leaf samples. 5. Continue running
microsatellite primers on leaf and seed samples. 6. Collect stigmas and count pollen grains. 2011 1. Write up data on pollen carryover and paternity shadow in field. 2. Write manuscript on impact of insect pollinator on gene dispersal. 3. Finish microsatellites on all samples. 4. Prepare manuscript on gene flow and outcrossing rate in wild squash. 4a List the single most significant research accomplishment during FY 2006. This research is relevant to Component 3, Plant Biotechnology Risk Assessment, of the Plant Biological and Molecular Processes Action Plan (NP 302). Customers include scientists working on diverse crops species and wild plant species closely related to the cultivars; conventional farmers (crop production and seed industry) interested in reducing the escape of transgenes and new alleles they are deploying from their field to other fields or to wild plant species; organic farmers who want to keep transgenic crops away from their field and regulatory agencies
interested in scientific data on the risk of transgene escape and the fate of transgenes or other crops genes following a recent escape in their new environment. To date, it has been determined that distinct insect pollinators can differentially affect outcrossing rate (short title: impact of distinct insect pollinators on outcrossing rate). These findings suggest that distinct insect pollinators can differentially affect gene flow, hence the risk of transgene escape and level of seed purity of a crop. This research will lead to better predictions of the potential for gene escape from transgenic crops, which will allow better estimates of risks. 5. Describe the major accomplishments to date and their predicted or actual impact. This research is relevant to Component 3, Plant Biotechnology Risk Assessment, of the Plant Biological and Molecular Processes Action Plan (NP 302). Customers include scientists working on diverse crops species and wild plant species closely related to the
cultivars; conventional farmers (crop production and seed industry) interested in reducing the escape of transgenes and new alleles they are deploying from their field to other fields or to wild plant species; organic farmers who want to keep transgenic crops away from their field and regulatory agencies interested in scientific data on the risk of transgene escape and the fate of transgenes or other crops genes following a recent escape in their new environment. This project was initiated in January 2002 as a result of the FY 2002 Appropriations Bill passed by Congress to characterize potential risks associated with genetically engineered (GE) crops, and other gene flow in agriculture. An Ecologist was hired in June 2003 and established a new laboratory to start examining these questions. The ecologist set up a cooperative agreement with Oregon State University in 2003 to examine the potential of the blue columbine, Aquilegia coerulea as a model system to study the impact of distinct
insect pollinators on gene flow. The ecologist examined the efficiency of various microsatellite primers to pursue paternity analysis in our model system. It has been determined that distinct insect pollinators can differentially affect outcrossing rate. These findings suggest that distinct insect pollinators can differentially affect gene flow, and hence the risk of transgene escape and the level of seed purity of a crop. The laboratory has also made crosses between wild squash and a transgenic squash cultivar that is resistant to three viral pathogens. Theses crosses will be used to examine the potential risk of introgression of a transgene for disease resistance in wild populations. This research will lead to better predictions of the potential for gene escape from transgenic crops and the potential impact of escaped transgenes, which will allow better estimates of risks. The findings will be important to how transgenic crops will be deployed in the future and what bioconfinement
methods might be necessary. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The information obtained from our experiments has been presented at the meetings of the Botanical Society of America and at invited seminars at two Universities in 2006. Our data are the first to document that distinct insect pollinators differentially affect outcrossing rate and gene flow via pollen.
Impacts
(N/A)
Publications
- Brunet, J., Sweet, H. 2006. Impact of insect pollinator group and floral display size on outcrossing rate. Evolution. 60:234-246.
- Brunet, J., Sweet, H.R. 2006. The maintenance of selfing in a population of the Rocky Mountain Columbine. International Journal of Plant Science. 167:213-219.
- Brunet, J. 2005. The impact of ecological factors on outcrossing rate in the blue columbine, Aquilegia coerulea (Ranunculaceae)[abstract]. Ecological Society of America Abstracts. Paper No. 78-7.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This project was initiated to characterize the type and frequency of genetic exchange among plants, especially to characterize potential risks associated with genetically engineered (GE) crops. The successful deployment of genetically improved crops, including GE crops, is contingent upon minimizing their ecological impact on the environment while enhancing the economic opportunities for growers to produce a safe and nutritious food supply. Genes are incorporated into crops and maintained by asexual and sexual processes with the intent that they not move into other cultivars of a crop, or into wild species with which a crop can intermate. Seed producers utilize biological mechanisms, such as sexual incompatibility, and geographic separation to eliminate unintentional outcrossing, but sometimes these
systems are ineffective in limiting pollen movement and consequent gene flow. This project examines the level of gene flow and the potential impact of genes in their novel environments. While some data are available to document the movement of genes between cultivated crops and wild plant species occurring in production areas, the extent of gene flow and the impact of new genes are not well known. Furthermore, there is little known about the biological mechanisms that limit or stimulate this process. The deployment of genetically engineered crops brings an urgent need for understanding and manipulating factors affecting and mechanisms underlying these processes. 2. List the milestones (indicators of progress) from your Project Plan. The project plan is currently in OSQR review, and has been submitted on September 16. The following milestones include milestones that were presented in the 2004 annual report for years 2004 and 2005 and milestones from the recently submitted project plan
for years 2006-2008. 1. Set up the laboratory. (2004) 2. Collect plant leaf and seed samples to develop appropriate genetic markers to study genetic diversity, population structure, and gene flow. (2004) 3. Set up field experiments to measure the impact of pollinator species on outcrossing rate and gene flow. (2004) 4. Genotype seeds using microsatellite markers from the experiment to measure outcrossing rate in different pollinator treatments. 5. Using microsatellite markers, start genotypic seeds from pollinator exclusion experiment to determine probability of paternal exclusion for the experiment. 6. Start running microsatellites and/or AFLPs to characterize gentic diversity within and among each of 12 Aquilegia populations. 7. Make crosses between species to create hybrids between transgenic cultivatarand wild squash species. 8. Genotype all plants for paternity shadow in the greenhouse. 9. Set up experiment for pollen carryover and paternity shadow in the greenhouse. 10. Set up
pollen carryover in the field. 11. Set up field experiment for multiple visits in the field. 12. Collect seeds and leaves from experimental plants and leaves from potential fathers within 30 meter radius. 13. Start genotyping leaves of all potential fathers using microsatellites. 14. Complete comparative fitness experiment. 15. Count pollen dyes for pollen carryover in the greenhouse and in the field. 16. Run microsatellites on seeds for paternity shadow in the greenhouse. 17. Do paternity shadow in the field. 18. Genotype 20 seeds from 20 plants within each pollinator treatment using microsatellites for impact of pollinator. 19. Process samples and perform data analyses from comparative fitness experiment. 20. Start pollen competition experiments. 21. Gather first year of data on disease incidence, population size, density, and reproductive output in wild squash populations. 22. Data analyses for pollen carryover and paternity shadow in the greenhouse. 23. Write up pollen carryover
and paternity shadow in the greenhouse. 24. Run microsatellites for paternity shadow in the field. 25. Finish genotyping all seeds for multiple visits experiment. 26. Estimate selfing rate for 20 plants per treatment and determine impact of insec pollinators on outcrossing rate. 27. Start running paternity analyses on data. 28. Process samples from pollen competition experiments in squash. 29. Complete pollen competition experiments. 30. Write manuscript for comparative fitness experiment in squash. 31. Gather second year of data on disease incidence, population size, and density in wild squash populations. 32. Start model development (to examine spread of transgene conferring disease resistance in wild squash populations). 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Genotype seeds using microsatellite markers from the experiment to measure outcrossing
rate in different pollinator treatments. Milestone Substantially Met 2. Using microsatellite markers, start genotyping seeds from pollinator exclusion experiment to determine probability of paternal exclusion for the experiment. Milestone Fully Met 3. Start running microsatellites and/or AFLPs to characterize gentic diversity within and among each of 12 Aquilegia populations. Milestone Substantially Met 4. Make crosses between species to create hybrids between transgenic cultivator and wild squash species. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The Project Plan is currently in OSQR review and the proposed milestones are listed below. FY2006 Milestone: Genotype all plants for paternity shadow in the greenhouse. Milestone: Set up experiment for pollen carryover and paternity shadow in the greenhouse. Milestone: Set
up pollen carryover in the field. Milestone: Set up field experiment for multiple visits in the field. Milestone: Collect seeds and leaves from experimental plants and leaves from potential fathers within 30 meter radius. Milestone: Start genotyping leaves of all potential fathers using microsatellites. Milestone: Complete comparative fitness experiment. FY2007 Milestone: Count pollen dyes for pollen carryover in the greenhouse and in the field. Milestone: Run microsatellites on seeds for paternity shadow in the greenhouse. Milestone: Do paternity shadow in the field. Milestone: Genotype 20 seeds from 20 plants within each pollinator treatment using microsatellites for impact of pollinator. Milestone: Process samples and perform data analyses from comparative fitness experiment. Milestone: Start pollen competition experiments. Milestone: Gather first year of data on disease incidence, population size, density, and reproductive output in wild squash populations. FY2008 Milestone: Data
analyses for pollen carryover and paternity shadow in the greenhouse. Milestone: Write up pollen carryover and paternity shadow in the greenhouse. Milestone: Run microsatellites for paternity shadow in the field. Milestone: Finish genotyping all seeds for multiple visits experiment. Milestone: Estimate selfing rate for 20 plants per treatment and determine impact of insec pollinators on outcrossing rate. Milestone: Start running paternity analyses on data. Milestone: Process samples from pollen competition experiments in squash. Milestone: Complete pollen competition experiments. Milestone: Write manuscript for comparative fitness experiment in squash. Milestone: Gather second year of data on disease incidence, population size, and density in wild squash populations. Milestone: Start model development (to examine spread of transgene conferring disease resistance in wild squash populations). 4a What was the single most significant accomplishment this past year? The impact of different
insect pollinators on outcrossing rate As transgenes are being introduced into vegetables and ornamentals and these plants often have close relatives in native populations, gene flow from vegetables and ornamentals to native plants is likely to have a major impact on native populations. A long-range goal of our studies is to examine and quantify the impact of different ecological factors (pollinators, mating system, disease, herbivory, and competition) on gene flow and on the spread of transgenes in their new environment. We have almost completed the microsatellite analysis to determine the impact of two different insect pollinators on the outcrossing rate of a plant species. As both pollinators and outcrossing rate influence gene flow a better understanding of the impact of pollinators on outcrossing rate will increase our ability to predict gene flow in various crop-crop and crop-native plants systems. 5. Describe the major accomplishments over the life of the project, including
their predicted or actual impact. This research will lead to better modeling of the potential for gene escape from transgenic crops, which will allow better estimates of risks. The research will also examine the potential ecological impact of transgenes providing disease resistance. The findings will be important to how transgenic crops will be deployed in the future and what bioconfinement methods might be necessary. This research is relevant to Component 3, Plant Biotechnology Risk Assessment, of the Plant Biological and Molecular Processes Action Plan (NP 302). The project plan for this research is going for review this Fall. Customers include scientists working on diverse crops species and wild plant species closely related to the cultivars; conventional farmers (crop production and seed industry) interested in reducing the escape of transgenes and new alleles they are deploying from their field to other fields or to wild plant species; organic farmers who want to keep transgenic
crops away from their field and regulatory agencies interested in scientific data on the risk of transgene escape and the fate of transgenes or other crops genes following a recent escape in their new environment. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The information obtained from our experiments will be transmitted to scientists at regional and international meetings. Meetings with the general public and farmers can be set up to discuss results of interest. Meetings with vegetable seed producers have been initiated. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Brunet, J. 2005 Plant-Pollinator
Interactions and Pollen Dispersal. In Field methods in Pollination Ecology", A. Dafni, P. Kevan, and B. Husband (eds.), Enviroquest, Cambridge, Canada.
Impacts
(N/A)
Publications
- Brunet, J. 2003. The safety of genetically modified crops. Plant Science Bulletin. 49(3):114.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This project was initiated to characterize the type and frequency of genetic exchange among plants, especially to characterize potential risks associated with genetically engineered (GE) crops. The successful deployment of genetically improved crops, including GE crops, is contingent upon minimizing their ecological impact on the environment while enhancing the economic opportunities for growers to produce a safe and nutritious food supply. Genes are incorporated into crops and maintained by asexual and sexual processes with the intent that they not move into other cultivars of a crop, or into wild species with which a crop can intermate. Seed producers utilize biological mechanisms, such as sexual incompatibility, and geographic separation to eliminate unintentional outcrossing, but sometimes these
systems are ineffective in limiting pollen movement and consequent gene flow. This project examines the level of gene flow and the potential impact of genes in their novel environments. While some data are available to document the movement of genes between cultivated crops and wild plant species occurring in production areas, the extent of gene flow and the impact of new genes are not well known. Furthermore, there is little known about the biological mechanisms that limit or stimulate this process. The deployment of genetically engineered crops brings an urgent need for understanding and manipulating factors affecting and mechanisms underlying these processes. 2. List the milestones (indicators of progress) from your Project Plan. The project plan is currently being developed. The following milestones include general milestones for the next five years. - Set up the laboratory. - Collect plant leaf and seed samples to develop appropriate genetic markers to study genetic diversity,
population structure, and gene flow. (Objectives 1 & 2). - Set up field experiments to measure the impact of pollinator species on outcrossing rate and gene flow.(Objective 1). - Measure the impact of pollinator species on outcrossing rate using microsatellite markers(Objective 1). - Measure gene flow via paternity analysis using microsatellite markers (Objective 1). - Measure genetic diversity, population structure, and gene flow via F statistics (Fst)(Objective 2). - Make crosses between species to create hybrids between transgenic cultivated and native species.(Objective 2). - Set up experiments using hybrids to quantify the impact of different factors (such as disease, herbivory, and/or competition) on gene flow and on fitness of parental species and hybrids (Objective 2). 3. Milestones: A. List the milestones (from list in Question # 2) that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were
not fully or substantially met, briefly explain why not, and your plans to do so. - Set up the laboratory. The laboratory is up and running. - Plant leaf and seed samples will be collected to develop appropriate genetic markers. We have 36 pairs of microsatellite markers to measure outcrossing rate and pursue paternity analyses in our model system. - Set up field experiments to measure the impact of pollinator species on outcrossing rate and gene flow measured via paternity analyses. These experiments were successfully completed and leaf, fruit, and seed samples were collected for further genetic analyses. B. List the milestones (from the list in Question # 2) that you expect to address over the next 3 years (FY 2005, 2006 and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2005: Milestone: Measure the impact of pollinator species on outcrossing rate (Objective 1). - Genotype seeds using microsatellite markers from the experiment
to measure outcrossing rate in different pollinator treatments. - Analyse results. Milestone: Measure the impact of pollinator species on gene flow(Objective 1). - Using microsatellite markers, start genotyping seeds from pollinator exclusion experiment to determine probability of paternal exclusion for the experiment. Milestone: Measure genetic diversity, population structure, and gene flow via F statistics (Fst) (Objective 2). - Collect leaf samples from various native populations. - Start running microsatellites and/or AFLPs to characterize genetic diversity within each population. Milestone: Make crosses between species to create hybrids between cultivated and native species (Objective 2). - Start making crosses between species to create F1 hybrids and first generation backcrosses between cultivated and native species. 2006 Milestone: Measure the impact of pollinator species on outcrossing rate (Objective 1). - Complete the data analyses for this study, write up and submit
the manuscript. Milestone: Measure the impact of pollinator species on gene flow (Objective 1). - Continue genotyping leaves and seeds from the pollinator exclusion experiment with microsatellite markers. Milestone: Measure genetic diversity, population structure and gene flow via F statistics (Fst) (Objective 2). - Continue genotyping individuals from native plant populations using microsatellites and /or AFLPs to determine genetic diversity. Milestone: Make crosses between species to create hybrids between cultivated and native species Objective 2). Continue making appropriate crosses between transgenic cultivated and native species. 2007 Milestone: Measure the impact of pollinator species on gene flow. - Complete paternity analyses to measure impact of pollinators on gene flow; write up results. Milestone: Measure genetic diversity, population structure and gene flow via F statistics (Fst). - Compile results for available populations for genetic diversity and measure of gene
flow using Fst and write up results. Milestone: Set up first year of experiments using F1 and F2 hybrids and first- generation backcrosses to quantify the impact of disease, herbivory, and/or competition on fitness and potentially on gene flow via pollen. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: As transgenes are being introduced into vegetables and ornamentals and these plants often have close relatives in native populations, gene flow from vegetables and ornamentals to native plants is likely to have a major impact on native populations. A long-range goal of our studies is to examine and quantify the impact of different ecological factors (pollinators, mating system, disease, herbivory, and competition) on gene flow and on the spread of transgenes in their new environment. We initiated studies to examine the role of pollinator species on outcrossing rate and gene flow. We are testing the efficiency of
thirty six pairs of microsatellite markers to pursue paternity analysis in our model system. We use paternity analysis to quantify gene flow. A better understanding of the factors that influence gene flow will increase our ability to predict gene flow in various crop-crop and crop-native plants systems. B. Other significant accomplishment(s), if any: None. C. Significant activities that support special target populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The ecologist for this position was hired in June 2003. This first year the scientist has equipped the new laboratory, hired a technician, and is currently looking for a postdoctoral associate. Pollinator exclusion experiments were set up to examine the impact of pollinator species on outcrossing rate and on gene flow. Thirty six pairs of microsatellite primers are being tested for variability and usefulness to measure outcrossing rate and gene flow
via paternity analysis. Understanding the impact of different ecological factors, such as pollinator species, on gene flow increases our ability to predict transgene flow in different crop-crop and crop-native plant species. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The information obtained from our experiments will be transmitted to scientists at regional and international meetings. Meetings with the general public and farmers can be set up to discuss results of interest. Meetings with vegetable seed producers have been initiated.
Impacts
(N/A)
Publications
- Brunet, J. 2003. The safety of genetically modified crops. Plant Science Bulletin. 49(3):114.
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