Performing Department
Plant Science
Non Technical Summary
The evolution of herbicide resistance is one of the greatest challenges to sustainably perform agricultural activities. In addition to economic losses due to competition, herbicide resistance can impact the adoption of conservation agriculture practices such as no-tillage, reduce the price of the final product, and impact export markets due to seed contamination. The US leads the ranking of herbicide resistance evolution worldwide.Italian ryegrass (Lolium perenne L. ssp. multiflorum) is a winter annual grass weed, and herbicide resistance in this species hashas been identified in many US states. This project will assess whether the herbicide resistant populations identified throughout the US are genetically related, or whether they evolved independently. We will also identify the genes involved in herbicide resistance. Results from this project will elucidate whether the widespread resistance in Italian ryegrass is the results of widespread gene flow due to agricultural activities, or whether it is the result of local weed management practices. In addition, we will generate new knowledge in terms of how weeds can evolve under strong selection pressure from herbicides. Ultimately, our results could help minimize gene flow or local selection pressure on weed populations, as well as provide a starting point for the development of quick herbicide resistance diagnostics.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
The goal of this research-only project is to generate foundational knowledge of glyphosate resistance dispersal and evolution. This is a collaborative work among scientists from 12 institutions. We will analyze Italian ryegrass populations from 13 US states to dissect their ancestry using population genomics. A novel glyphosate resistance gene (based on preliminary QTL data) will be fine mapped and functionally validated. This work will help minimize the spread of glyphosate resistance. This project will lay the foundation for PD Brunharo's research program by supporting future grant applications, forging new collaborative efforts, and attracting outstanding personnel.
Project Methods
Objective 1.Investigate the genetic relationship among glyphosate-resistant Italian ryegrass populations collected from 13 US states.We obtained a comprehensive germplasm of glyphosate-resistant Italian ryegrass populations to study the continental gene flow of glyphosate resistance in Italian ryegrass. We will conductdose-response experiments to quantify glyphosate resistance levels under common environmental conditions. Next, we will perform a reduced-representation DNA sequencing with the NextRAD technique, which will identify SNPs throughout the genome for population genomics analyses. Leaf tissue will be collected for DNA extraction, and a lethal dose of glyphosate (approximately 2,200 g active ingredient ha-1, or 2× field rate) will be applied to 2-tiller plants to phenotype for glyphosate resistance. DNA from 10 individuals per population will be extracted, and samples will be prepared forpaired-end short-read sequencingusing state-of-the-art sequencing technologies. Reads will be mapped to the reference genome, and population genomics estimates obtained. The SNP dataset will be used to perform a principal component analysis (PCA) to obtain an overall picture of the genetic background of the populations. To further dissect the historical demographic events, we will infer patterns of ancestry. Isolation-by-distance will be assessed with geographicalcoordinates of origin and pairwise genetic distance. A phylogenetic tree will also be constructed. Genetic diversity within and between populations will be estimated with the expected heterozygosity, nucleotide diversity, inbreeding coefficient, and Tajima's D.Objective 2.Fine mapping and functional validation of a novel glyphosate resistance gene.A fine mapping experiment will be performed with an F2 mapping population. A total of 1200 F2 individuals will be used for fine mapping. After tissue collection for DNA extraction, plants will be phenotyped for glyphosate resistance. We will utilize KASP markers for genotyping. SNPs will be identified from QTL mapping experiments, and 15 markers will be developed that surround the QTL region to identify recombination breakpoints. At this point, the genomic region associated with glyphosate resistance will be narrower, with a few promising candidate genes, and genomic features will be identified and compared to the susceptible genes. After the fine mapping experiment is complete, we will perform a functional characterization experiment. We will use rice as a model system, as this is a species of choice for function characterization of genes from grasses. Full length cDNA of the top 1-3 candidate genes will be amplified from Italian ryegrass and cloned into a binary vector, plasmid sequence checked by long-read Nanopore sequencing, and transformed into Agrobacterium tumefaciensbefore Nipponbare rice transformation. Hygromycin-resistant calli will be transferred to differentiation and rooting media to obtain transformed and wild-type control (empty vector) seedlings. A total of 10 events (T1) will be grown in growth chamber, and controlled crossings performed because T1 will likely be hemizygous (i.e., single-copy resistance allele). T1 will be self-pollinated to generate T2 populations. T2 seed will be assessed with hygromycin in Petri dish for their segregation ratio, expecting a 3:1 (resistant:susceptible). An additional selection with hygromycin will be performed to create T3 generations that are homozygous for the construct. Frequency of hygromycin resistance in the T3 generation will be 100%. We will advance to T4 homozygous to increase seed, and T4 line will be used for glyphosate resistance characterization. All lines will be grown in a growth chamber to shorten the life cycle to ~80 d.At each generation, PCR and Sanger sequencing will be performed to confirm presence of construct and genes. Transformed lines will be subjected to dose-response experiments to quantify the resistance level compared to controls, and considered resistant if the resistance index (herbicide dose required to reduce growth by 50% of the resistant compared to the susceptible) is greater than 2, as modeled by log-logistic regressions. A shikimate accumulation experiment (a proxy for response to glyphosate) in 96-well plate format, and a root elongation assay in agarose, will also be performed to complement dose-response studies.