UNDERSTANDING AND DIAGNOSING HERBICIDE RESISTANCE IN WEEDS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016207
• Project Start Date: Jul 1, 2018
• Project End Date: Jun 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Agricultural Biology

Non Technical Summary
Herbicide resistance poses a major threat to current and future food production. To meet future demands for food and fiber without dramatic increases in land clearing, agricultural production must continue to improve its efficiency and sustainability. As fewer farmers manage larger areas in industrialized nations, weed management is critical to production efficiency. Currently, herbicides are the best available solution for weed control. However, over-reliance on single or relatively few herbicide modes of action strongly selects for any and all genetic mechanisms that enable survival and reproduction. In many cases, herbicide resistance can no longer be prevented; it must be managed. Management will involve good integrated weed management practices, such as preventing seed set, reducing the seed bank, growing competitive crops, diversifying crop rotations, and diversifying control methods. Management is improved with timely diagnosis of resistance. This project applies molecular genetics, molecular biology, and genomics approaches to the economically important problem of herbicide resistance, with the goals of developing more effective and less expensive diagnostic molecular markers, and studying the genetic basis of herbicide resistance and other weedy traits.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
213	2300	1140	60%
201	2300	1080	40%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms; 213 - Weeds Affecting Plants;

Subject Of Investigation
2300 - Weeds;

Field Of Science
1080 - Genetics; 1140 - Weed science;

Keywords

weedy traits

herbicide resistance

genomics

controlling weedy plants

plant molecular biology

Goals / Objectives
Identify the molecular and genetic basis of economically important herbicide resistance traits in weeds and develop diagnostic molecular markers to improve speed and reduce cost of herbicide resistance diagnosis.Utilize genomics to study herbicide resistance mechanisms and study the genetic basis of other weedy traits.

Project Methods
Objective AGlyphosate-resistant kochia has duplication of the EPSPS gene, and the increase in gene copy number has been used successfully as a marker for glyphosate resistance in kochia, Palmer amaranth, and waterhemp (Gaines et al. 2016; Gaines et al. 2010; Lorentz et al. 2014; Wiersma et al. 2015). This marker diagnoses glyphosate resistance and requires relatively expensive reagents for quantitative PCR, making the typical cost to analyze one sample about $12-15. I offer this diagnostic assay to Colorado growers through my lab, using funding from commodity groups such as Western Sugar and Colorado Wheat. A less expensive assay will be based on the presence of a transposon insertion next to the EPSPS duplicated genes, discovered by my recently-completed PhD student Eric Patterson. This PCR-based assay will be developed into the standard test for glyphosate resistance in kochia. Additional research into the molecular and genetic basis of herbicide resistance mechanisms will include assessing additional glyphosate resistance mechanisms in kochia. To date, a kochia population has been identified that has low level glyphosate resistance, but no duplication of the EPSPS gene (Gaines, Westra, and Kniss, 2015, unpublished). These samples will be evaluated for other resistance mechanisms, including altered translocation and metabolism. Synthetic auxin herbicide resistance will be investigated, including enhanced metabolism and mutations in auxin receptors and co-receptors.Objective BBecause high-throughput gene-sequencing technology continues to improve in scale and affordability, a data-intensive approach will be used to investigate the complex traits that make weeds successful, specifically Kochia scoparia, the most important dicotyledonous weed in the U.S. central Great Plains. The overall goal is to annotate the Kochia genome and build the bioinformatics resources necessary to eventually begin to study the genetic basis of complex traits including drought and salt tolerance.A project is currently in progress to assemble the genome of Kochia, in collaboration with Eric Patterson, Dr. Pat Tranel from Illinois, and Dr. Chris Saski from Clemson. Our current genomic assembly includes over 700 million base pairs of genomic sequence (out of the estimated 1.0 Gb kochia genome). Using annotation tools such as Trinnotate and protein databases including UniProt and Pfam, we have annotated the assembled genome and found that it includes at least 80% of the expected gene content. We will continue to improve this assembly so that it can be used in genetic mapping experiments. Dr. Phil Westra has two inbred lines of Kochia, produced by 12 generations of single-seed descent (Preston et al. 2009). These two inbred lines will be used to study the genetic basis of herbicide resistance mechanisms and disease response in kochia. The annotated transcriptomic and genomic resources will be used to study herbicide resistance mechanisms in kochia, through whole-transcriptome gene expression profiling and SNP discovery. Candidate genes, whether due to differences in gene expression regulation or sequence polymorphisms, will be validated using forward genetics (testing co-segregation of candidate resistance genes with the resistance phenotype in segregating F2 populations) and population validation (association of candidate genes with the resistance phenotype in diverse populations). By discovering the genetic basis of herbicide resistance mechanisms in kochia, this project will assist with identification and more rapid diagnosis of herbicide resistance.Merging bioinformatics and biology will enable new insights into complex traits in kochia. A long-term goal of this genomics research is to identify the genetic basis of complex traits in weeds that may be useful in crops. Initial research towards this goal will begin by evaluating kochia for cold tolerance both at seed germination and at early seedling stages. We have identified candidate lines with this genetic variation. The outline of the project is to verify and characterize the divergent cold tolerance phenotypes, make initial F1 crosses, produce a segregating F2 and then advance by single seed descent to F4, and use genome-wide genotype-by-sequencing and F2 phenotyping to map the quantitative trait loci contributing to the cold tolerance traits.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audiences focused upon in this reporting period include the Western Sugar Joint Research Committee, the Colorado Wheat Administrative Committee, and the Western IPM Center Kochia Work Group. Interactions with these groups were conducted to facilitate research progress and identify critical research focus areas. A two-day meeting of 45 stakeholders with interests in kochia management was conducted in October 2019 using Western IPM Center funding. Stakeholders including sugar beet and wheat growers and crop consultants, along with crop protection industry stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities for undergraduate and graduate students in laboratory methods and publication writing have been provided. How have the results been disseminated to communities of interest?Outreach activities to the Colorado Wheat Administrative Committee and the Western Sugar Joint Research Committee have been conducted, including written reports, oral presentations, and a wheat field day one-page written report. Outreach to the public through CSU Extension Crop Production Clinics and Colorado Weed Management Association has been conducted, as well as to private pesticide applicator groups and independent crop consultants. A two-day meeting of stakeholders with interests in kochia management was conducted in October 2019 using Western IPM Center funding. The meeting developed priority areas, including further development of molecular markers for herbicide resistance testing. What do you plan to do during the next reporting period to accomplish the goals?I will continue research activities towards developing molecular markers to diagnose herbicide resistance, researching the multiple origins of glyphosate resistance in kochia, and utilizing the new kochia genome assembly.

Impacts
What was accomplished under these goals? Under objective A, PCR primers for detecting glyphosate resistance in kochia and Palmer amaranth have been further optimized, including identification of unique evolutionary origins of glyphosate resistance in kochia using PCR markers (publication in review). Considerable progress has been made in developing diagnostic PCR markers to identify Palmer amaranth seeds from other Amaranthus spp. seeds, to be used in the seed inspection industry. This technology is being deployed in plant diagnostic clinics. Under objective B, the kochia reference genome has been completed and published. TheInternational Weed Genomics Consortium has fully formed and is launching in 2021, an industry and USDA-FFAR sponsored consortium that is administered at CSU and is responsible for completing new reference genome assemblies and training weed scientists in genomics.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Westra, E., S. Nissen, T. Getts, P. Westra, and T. Gaines. Survey reveals frequency of multiple resistance to glyphosate and dicamba in kochia (Bassia scoparia). 2019. Weed Technology. 33: 664-672. DOI: 10.1017/wet.2019.54
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Todd, O., M. Figueiredo, S. Morran, N. Soni, C. Preston, M. Kubea, R. Napier, and T. Gaines. 2020. Synthetic auxin herbicides: finding the lock and key to weed resistance. Plant Science. 300:110631.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audiences focused upon in this reporting period include the Western Sugar Joint Research Committee,the Colorado Wheat Administrative Committee, and the Western IPM Center Kochia Work Group. Interactions with these groups were conducted to facilitate research progress and identify critical research focus areas. A two-day meeting of stakeholders with interests in kochia management was conducted in October 2019 using Western IPM Center funding. Stakeholders including sugarbeet and wheat growers and crop consultants, along with crop protection industry stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities for undergraduate and graduate students in laboratory methods and publication writing have been provided. How have the results been disseminated to communities of interest?Outreach activities to the Colorado Wheat Administrative Committee and the Western Sugar Joint Research Committee have been conducted, including written reports, oral presentations, and a wheat field day one-page written report. Outreach to the public through CSU Extension Crop Production Clinics has been conducted, as well as to private pesticide applicator groups and independent crop consultants.A two-day meeting of stakeholders with interests in kochia management was conducted in October 2019 using Western IPM Center funding. The meeting developed priority areas, including further development of molecular markers for herbicide resistance testing. What do you plan to do during the next reporting period to accomplish the goals?I will continue research activities towards developing molecular markers to diagnose herbicide resistance, researching the multiple origins of glyphosate resistance in kochia, and utilizing the new kochia genome assembly.

Impacts
What was accomplished under these goals? Under objective A, PCR primers for detecting glyphosate resistance in kochia and Palmer amaranth have been further optimized. Considerable progress has been made in developing diagnostic PCR markers to identify Palmer amaranth seeds from other Amaranthus spp. seeds, to be used in the seed inspection industry. Under objective B, the kochia reference genome has been completed and published. Considerable progress has been made towards forming the International Weed Genomics Consortium, an industry-sponsored consortium that will be administered at CSU and be responsible for completing new reference genome assemblies and training weed scientists in genomics.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Takano, H., E. Patterson, S. Nissen, F. Dayan, and T. Gaines*. 2019. Predicting herbicide movement across semi-permeable membranes using three phase partitioning. Pesticide Biochemistry and Physiology, in press. DOI: 10.1016/j.pestbp.2019.05.009.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Patterson, E., C. Saski, D. Sloan, P. Tranel, P. Westra, and T. Gaines*. 2019. The draft genome of Kochia scoparia and the mechanism of glyphosate resistance via transposon-mediated EPSPS tandem gene duplication. Genome Biology and Evolution, in press.

Progress 07/01/18 to 09/30/18

Outputs
Target Audience:The target audiences focused upon in this reporting period include the Western Sugar Joint Research Committee, the Colorado Corn Adminstrative Committee, and the Colorado Wheat Administrative Committee. Interactions with these groups were conducted to facilitate research progress and identify critical research focus areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities for undergraduate and graduate students in laboratory methods and publication writing have been provided. How have the results been disseminated to communities of interest?Outreach activities to the Colorado Wheat Administrative Committee, Western Sugar Joint Research Committee, and Colorado Corn have been conducted, including written reports and oral presentations. Outreach to the public through CSU Extension Crop Production Clinics has been conducted, as well as to private pesticide applicator groups and independent crop consultants. What do you plan to do during the next reporting period to accomplish the goals?I will continue research activities towards developing molecular markers to diagnose herbicide resistance, researching the multiple origins of glyphosate resistance in kochia, and utilizing the new kochia genome assembly

Impacts
What was accomplished under these goals? Under objective A, a new PCR marker to determine multiple, independent origins of glyphosate resistance in kochia has been developed and used to show that Great Plains and Intermountain West kochia populations likely have indepedent origins. Under objective B, the kochia reference genome is complete and nearly ready for publication.

Publications