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.: 1006620
• Project Start Date: Jul 1, 2015
• Project End Date: Jun 30, 2018
• 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. 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

(N/A)

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
A. 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.B. 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. 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. An alternative marker based on sequence polymorphisms is possible. Sequence analysis showed that two EPSPS alleles were present in kochia, differentiated by seven single nucleotide polymorphisms (SNPs) (Wiersma et al. 2015). These SNPs were present at roughly equal proportions in glyphosate-susceptible kochia, but glyphosate-resistant kochia only had one nucleotide at each position. This sequence difference was due to the expression of the duplicated EPSPS allele.A potential diagnostic marker for glyphosate resistance in kochia (and likely in other species with glyphosate resistance due to EPSPS gene duplication) is therefore to measure for the proportion of the allele-diagnostic SNPs in a genomic DNA sample. The EPSPS sequence from a glyphosate-resistant individual with EPSPS gene duplication will contain primarily the SNPs from the duplicated allele, while the EPSPS sequence from a glyphosate susceptible individual will have roughly equal proportions of SNPs from both EPSPS alleles. This SNP genotyping approach has the potential to cost less per sample than biological assays in the greenhouse and well-established qPCR methods for copy number measurement. The SNP genotyping approach also has the potential to be more rapid and provide an in-season response to stakeholders desiring to diagnose glyphosate resistance in kochia and other weeds.Multiple SNP genotyping approaches are available, including CAPS (cleaved amplified polymorphic sequence), SNP-specific PCR primers, pyrosequencing, and KASP markers. These methods will be evaluated for their utility and the most efficient SNP marker system will be developed for a rapid genotyping assay. Once the genotyping assay is developed, multiple kochia samples from different geographic locations will be genotyped. These results will be used to assess the utility and accuracy of the SNP marker system for diagnosing glyphosate resistance.Additional research into the molecular and genetic basis of herbicide resistance mechanisms will include assessing additional glyphosate resistance mechanisms in kochia. To date, two kochia samples have been identified that have 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. Similarly, glyphosate resistant species from South America will be assessed for resistance mechanisms, including glyphosate metabolism in Chloris truncatula.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 and Dr. Pat Tranel from Illinois. Our current genomic assembly includes over 200 million base pairs of genomic sequence (out of the estimated 1.0-1.3 Gb kochia genome) In addition, Dr. Phil Westra and Andrew Wiersma have assembled 38.6 million base pairs of transcriptome data including 34,969 unigenes of 1,324 bp average length (Wiersma et al. 2015). Using annotation tools such as Trinnotate and protein databases including UniProt and Pfam, we will annotate the assembled transcriptome and use the transcript data to identify gene coding sequences in our genome. 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 resistance to sugar beet pathogens. Kochia and sugar beet are closely related, and similar pathogens infect both species, such as Rhizoctonia solani. Dr. Kimberley Webb of the USDA-ARS is an expert in Rhizoctonia and sugar beet. The two inbred kochia lines will be assessed for seedling and adult plant reponse to Rhizoctonia, with the goal of identifying genetic variation in response to pathogens. The hypothesis is that kochia is well-adapted to growing in sugar beet fields, and genetic variation for disease resistance may be present in some kochia populations. This may represent new sources of resistance that can be assessed for utility in sugar beet. If such disease resistance is identified in kochia, the genomics resources will be used to study the molecular and genetic basis of the trait(s). Alternatively, kochia may be an alternate host for Rhizoctonia and provide a source of inoculum to infect sugar beet. With the increasing frequency of glyphosate-resistant kochia in sugar beet fields that primarily depend on glyphosate for weed control, the interaction of pathogens with kochia needs to be characterized.

Progress 07/01/15 to 06/30/18

Outputs
Target Audience:The target audiences focused upon 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? Nothing Reported

Impacts
What was accomplished under these goals? Progress towards the major goals of the project was made during the reporting period. Under objective A, a survey of glyphosate-resistance in kochia and Palmer amaranth in both sugarbeet and corn was completed (Colorado Corn and Western Sugar). A manuscript was published regarding population genetics of glyphosate resistance in Palmer amaranth. A molecular marker was utilzied in stakeholder submitted samples to identify Palmer amaranth in a mixture of other Amaranthus seeds. Under objective B, a revised funding application was submitted to the AFRI Pests and Beneficial Species in Agricultural Production Systems program to study the fitness cost of 2,4-D resistance in waterhemp, and the proposal is currently under consideration. A draft genome assembly and annotation for kochia has been completed. A manuscript is in preparation to report the kochia genome, and a proposal to the NSF Plant Genome Research Program is being prepared.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Beckie, H., R. Blackshaw, J. Leeson, P. Stahlman, T. Gaines, and E. Johnson. 2018. Seed bank persistence, germination and early growth of glyphosate-resistant Kochia scoparia. Weed Research. 58: 177-187. DOI: 10.1111/wre.12294
• Type: Journal Articles Status: Published Year Published: 2018 Citation: K�pper, A., H. Manmathan, D. Giacomini, E. Patterson, W. McCloskey, and T. Gaines*. 2018. Population genetic structure in glyphosate-resistant and -susceptible Palmer amaranth (Amaranthus palmeri) populations using genotyping-by-sequencing (GBS). Frontiers in Plant Science. 9: 29. DOI: 10.3389/fpls.2018.00029.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Schroeder, J., M. Barrett, D. Shaw, A. Asmus, H. Coble, D. Ervin, R Jussaume, M. Owen, I. Burke, C. Creech, A.S. Culpepper, W. Curran, D. Dodds, T. Gaines, J. Gunsolus, B. Hanson, P. Jha, A. Klodd, A. Kniss, R. Leon, S. McDonald, D. Morishita, B. Schutte, C. Sprague, P. Stahlman, L. Steckel, and M VanGessel. al. 2018. Managing wicked herbicideresistance: Lessons from the field. Weed Technology. 32: 475-488. DOI: 10.1017/wet.2018.49.

Progress 10/01/16 to 09/30/17

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 CSUExtension 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 new arrival of Palmer amaranth in Colorado, and utilizing the new kochia genome assembly.

Impacts
What was accomplished under these goals? Progress towards the major goals of the project was made during the reporting period. Under objective A, a survey of glyphosate-resistance inkochia and Palmer amaranth in both sugarbeet and corn was funded (Colorado Corn and Western Sugar) and is currently in progress. A manuscript was published regarding additional molecular investigations of glyphosate resistance in Palmer amaranth. A molecular marker was developed to identify Palmer amaranth in a mixture of other Amaranthus seeds. A molecular marker was also developed to identify Eurasian watermilfoil, northern watermilfoil, and their herbicide-resistant hybrid. Under objective B, an application was submittedto the AFRI Pests and Beneficial Species in Agricultural Production Systems program to study the fitness cost of 2,4-D resistance in waterhemp, and the proposal is currently under consideration. A draft genome assembly and annotation for kochia has been completed. A manuscript is in preparation to report the kochia genome, and a proposal to the NSF Plant Genome Research Program is being prepared.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fern�ndez-Escalada, M., A. Zulet-Gonz�lez, M. Gil-Monreal, A. Zabalza, K. Ravet, T. Gaines, and M. Royuela. 2017. Effects of EPSPS copy number variation (CNV) and glyphosate application on the aromatic and branched chain amino acid synthesis pathways in Amaranthus palmeri. Frontiers in Plant Science. 8: 1970. DOI: 10.3389/fpls.2017.01970.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences focused upon in this reporting period include the Western Sugar Joint Research Committee and the Colorado Wheat Administrative Committee. Interactions with both 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 new arrival of Palmer amaranth in Colorado, and utilizing the new kochia genome assembly.

Impacts
What was accomplished under these goals? Progress towards the major goals of the project was made during the reporting period. Under objective A, a survey of glyphosate-resistant kochia in sugarbeet was completed and published, using a molecular marker to diagnose resistance. Additionally, glyphosate-resistant Palmer amaranth was documented for the first time in Colorado in 2016. Anew platform for droplet digital PCR (ddPCR) was acquired at CSU in 2015 and this ddPCR platform has been used to measure gene amplification in weeds more accurately than before. Under objective B, an application to the AFRI Pests and Beneficial Species in Agricultural Production Systems program was submitted and it received good evaluations, but was not funded. Currently two funding applications to sugar beet funding panels have been submitted study the interaction between glyphosate-resistant kochia and Palmer amaranth in sugarbeet, and a proposal has been submitted to Colorado Corn to study glyphosate-resistant Palmer amaranth. Additionally, a draft genome assembly for kochia has been completed and annotation is underway.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Gaines TA, Barker AL, Patterson EL, Westra P, Westra EP, Wilson RG, Jha P, Kumar V, Kniss AR (2016) EPSPS gene copy number and whole-plant glyphosate resistance level in Kochia scoparia. PLOS ONE 11:e0168295.

Progress 07/01/15 to 09/30/15

Outputs
Target Audience:The target audiences focused upon in this reporting period include the Western Sugar Joint Research Committee and the Colorado Wheat Administrative Committee. Interactions with both 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?This project has provided training opportunities for one post-doctoral scholar (Dr. Margaret Fleming) in designing and evaluating molecular markers for glyphosate resistance and in designing ddPCR approaches. In addition, 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 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, evaluating Rhizoctonia solani resistance in kochia, and assembling the kochia genome.

Impacts
What was accomplished under these goals? Progress towards the major goals of the project was initiated during the reporting period. Under objective A, a potential molecular marker system for diagnosing glyphosate resistance in Kochia scoparia was evaluated using single nucleotide polymorphism (SNP) markers. A post-doctoral research associate conducted the research. It was determined that the hypothesized SNP marker would not be applicable for diagnosing herbicide resistance. However, a new platform for droplet digital PCR (ddPCR) was acquired at CSU and development work was completed to enable the use of this ddPCR platform to measure gene amplification in weeds more accurately than before. Additionally, a survey of kochia from sugar beet production fields was completed, and the gene amplification molecular marker was used to diagnose glyphosate-resistant populations. Under objective B, an application to the NSF Plant Genome program was submitted and it received good evaluations, but was not funded. Currently three funding applications to sugar beet funding panels have been submitted in collaboration with Dr. Kimberly Webb (USDA-ARS) to study the potential for novel Rhizoctonia solani resistance in kochia and to evaluate interactions between the pathogen and glyphosate-resistant kochia. Additionally, substantial progress has been made towards assembling the kochia genome, as recently long-read sequencing data has been obtained. The stated goal of genome assembly has not yet been met, but positive progress has been made.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: 3. Gaines, T., A. Barker, E. Patterson, P. Westra, S. Nissen, E. Westra, R. Wilson, and A. Kniss. 2015. Correlation of glyphosate resistance and EPSPS gene copy number in Kochia scoparia from sugar beet fields. In review, Pest Management Science.