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ARS Home » Midwest Area » Madison, Wisconsin » Vegetable Crops Research » Research » Research Project #434431

Research Project: Pollinators and Gene Flow

Location: Vegetable Crops Research

2019 Annual Report

Objective 1: Identify pollinator behaviors, pollinator management strategies, and crop production strategies that together mitigate unintended gene flow. Sub-objective 1.1: Pollinator behavior and plant reproductive strategies affect gene flow risk. Sub-objective 1.2: Visual and Olfactory cues that attract pollinators can guide the development of pollinator or crop management strategies that reduce gene flow and increase yield. Objective 2: Determine the impacts of cultivated carrot genes on the genomic landscape of wild carrot.

Objective 1. This objective is divided into two sub-objectives, each with three hypotheses to be tested. Sub-Objective 1.1. We will use a combination of field and greenhouse experiments to test the hypotheses within this subobjective. For example, the rules bees use when moving between patches or fields will be tested using patches of distinct sizes and isolation distances and measuring the number of transitions made by bees from a center glyphosate-resistant patch to the different conventional patches. The number of gene flow events in the different conventional patches, identified by the presence of glyphosate-resistant seeds, will also be used to test the decision making process of bumble bees. Greenhouse experiments will examine the pattern of seed deposition on flowers visited in succession by three bee species, honey bees, leafcutting bees and bumble bees. We will use glyphosate-resistant pollen donor and conventional pollen recipients and examine the number and proportion of glyphosate-resistant seeds on flowers visited in succession to determine the seed curve for each bee species. Sub-Objective 1.2. To determine the preference of each of three bee species to visual and/or olfactory cues, we will perform greenhouse experiments and quantify approaches and landings to different visual and/or olfactory cues. To identify a blend derived from nest cells that attract leafcutting bees, we will capture and identify the chemicals present in the bee cell using Gas Chromatography-Mass Spectrometry (GC-MS); determine whether there is a behavioral response and then use couple gas chromatography – electroantennographic detection (GC-EAD) to identify physiological responses. Finally, the electrophysiologically active constituents will be tested using a behavioral assay. Objective 2. We will use genotyping by sequencing on both cultivated carrots used in a breeding program and wild carrots in close proximity to the breeding area and far away to detect the presence of cultivated carrot genes in wild carrot populations. The presence of cultivated genes in wild populations represents introgression. We will determine the extent of introgression of cultivar genes in wild carrot populations.

Progress Report
Objective 1, Sub-objective 1.1. ARS and University of Wisconsin scientists and other collaborators at Madison, Wisconsin have collected transition data for bumble bees. These data indicate the number of times a bee flies from the center patch in the experiment to each of four peripheral patches of two different sizes and at two distances from the center patch. At the end of the flowering season, seeds were collected from each of the four peripheral patches. A phenotypic test was developed to help identify seeds that originated from the center patch and 1,000 seeds were checked from each of the four peripheral patches. Objective 1, Sub-objective 1.2. ARS and University of Wisconsin scientists at Madison, Wisconsin reared leafcutting bees in the greenhouse and examined their behavior while foraging on alfalfa flowers. Objective 2. ARS and other collaborators at Madison, Wisconsin obtained genotyping by sequencing data on four wild carrot populations close to a carrot cultivar breeding area and four wild carrot populations far away from the carrot breeding area. The wild carrot single-nucleotide polymorphism (SNP) data were combined with the SNP data from cultivars bred in the carrot breeding area. Population genetic structure analyses of these populations indicated differences between cultivars and wild carrots and between wild carrots from near and far populations. Genetic diversity was greater in near relative to far wild carrot populations which suggest the introgression of cultivar genes in near wild carrot populations. University of Wisconsin scientists completed the experiments comparing trapping methods for bees and are performing statistical analyses of these data. ARS scientists have genotyped all seeds and leaves collected from 32 alfalfa seed-production fields at ten microsatellite loci. Selfing rates of these 32 fields have been estimated using the genotype data of progeny arrays and maternal plants. Genetic diversity analyses have been performed and analyses of genetic structure of the seed production fields are in progress. Management practices are being compiled from these fields by collaborators so ARS scientists can examine the factors that most impact selfing rate. Georgia collaborators are growing alfalfa plants and performing self and outcross crosses in order to phenotypically quantify inbreeding depression in alfalfa. ARS and University of Wisconsin scientists have obtained a permit to grow colonies of Lygus hesperus and maintain colonies for behavioral experiments. Wild plant species, host and non-host of Lygus hesperus are growing in the greenhouse to perform floral scent extraction and behavioral experiments.

1. Bee foraging behavior that most affect gene flow. Identifying bee behaviors that most impact gene flow will help us predict differences in gene flow risk among bee species. Such information would benefit farmers and industry concerned about coexistence of different seed production markets in alfalfa seed production fields. We identified tripping rate as a behavior that strongly impact gene flow risk. Our results dictate limiting the use of honey bees as alfalfa pollinators to warm areas where tripping rate is higher in order to reduce gene flow risk. Results identify selection to increase ease of tripping in alfalfa varieties as a method to increase seed set and reduce gene flow risk. Results of this study are important because they can guide the development of management and breeding practices to reduce gene flow. They also provide mechanisms to explain observed differences in gene flow risks among bee species.

2. Introgression of cultivar genes into wild carrot populations. Wild and cultivated carrots belong to the same species and can easily hybridize. Cultivar genes can infiltrate and spread in wild carrot populations in a process called introgression. The deployment of new gene editing technologies has increased the likelihood that genetically modified (GM) carrot cultivars will be released in the future. To anticipate the potential impact of these GM carrot genes, it is important and timely to quantify the extent of introgression of cultivar genes into wild carrot populations. It is also important to identify if some areas of the wild carrot genome are free of introgression from cultivar genes. This information is important to biotechnology regulators, scientists, and the public interested in introgression from cultivated plants to wild populations. We detected genetic differences between cultivated and wild carrots and between near and far wild populations with greater genetic diversity in near populations suggestive of introgression of cultivar genes. We identified genetic markers capable of detecting introgression but these markers were spread over the nine carrot chromosomes. While there were no chromosomes free of introgression, such regions could serve as target for transgene introduction as they would prevent the spread of these transgenes into natural populations.

Review Publications
Minahan, D.F., Brunet, J. 2018. Strong interspecific differences in foraging activity observed between honey bees and bumble bees using miniaturized radio frequency identification (RFID). Frontiers in Ecology and Evolution.
Brunet, J., Ziobro, R., Osvatic, J., Clayton, M.K. 2019. The effects of time, temperature and plant variety on pollen viability and its implications for gene flow risk. Plant Biology. 21(4):715-722.
Brunet, J., Zhao, Y., Clayton, M.K. 2019. Linking the foraging behavior of three bee species to pollen dispersal and gene flow. PLoS One. 14(2):e0212561.
Mandel, J.R., Brunet, J. 2019. Gene flow in carrot. In: P. W. Simon, M. Iorizzo, D. Grzebelus and R. Baranski, editors. The Carrot Genome, Compendium of Plant Genomes. Springer Nature: Cham, Switzerland. p. 59-76.