Location: Sunflower and Plant Biology Research
2017 Annual Report
Objectives
Objective 1: Identify, at the genome and physiological levels, plant-plant interactions that impact plant growth and lead to crop yield losses, especially crop-weed interactions that occur during the critical weed-free period, and interactions that occur between the different crops inter-planted in relay cropping systems, such as corn, soybeans, or sunflowers relayed with camelina, ryegrass, or canola. [NP304, Component 2, Problem Statement 2A3]
Sub-objective 1.A: Determine the parameters for evaluating the impacts of winter annual cover crops on corn, sunflower, and Amaranthus spp. productivity.
Sub-objective 1.B: Identify physiological and molecular mechanisms that control interactions between cover crops and corn, sunflower, and Amaranthus spp.
Sub-objective 1.C: Evaluate impacts of candidate genes on cover crop-relay crop and cover crop-weed interactions.
Objective 2: Determine the molecular and physiological mechanisms by which winter annual cover crops suppress weeds in northern temperate agroecosystems, and identify genes that will enhance weed suppression in these crops, such as genes associated with weed-tolerance, cover-crop tolerance, and cold hardiness. [NP304, Component 2, Problem Statement 2A3]
Sub-objective 2.A: Identify genetic markers for improving the weed-suppressing trait of winter hardiness in winter canola and/or camelina varieties.
Sub-objective 2.B: Evaluate the weed-suppressing traits of winter-hardy canola and camelina in the field.
Approach
Weeds are major pests of agro-ecosystems that reduce production of the nation’s food, feed, fiber and fuel crops. The industry-adopted practice of rotating crops with engineered tolerance to a limited set of herbicides continues to put selection pressure on the evolution of herbicide-resistant weeds. As part of a holistic and sustainable approach to managing weeds in temperate agro-ecosystems, we propose to identify cover crop-relay crop interactions to enhance relay crop productivity, cover crop-weed interactions to enhance weed suppression, and identify winter-hardy annual cover crops that suppress weeds in relay cropping systems. In this proposal, winter canola will serve a dual purpose as both a cover crop for evaluating weed suppression, and as a surrogate weed for weed-relay crop interactions. Our model relay cropping system consists of inter-seeding a commodity crop (corn or sunflower) into an established cover crop (winter canola) such that their lifecycles overlap. Currently, no winter-hardy annual broadleaf cover crops are economically suited for weed suppression in relay cropping systems in the upper Midwest (UMW) and Northern Great Plains (NGP). Consequently, the objectives of this project are: (1) elucidating regulatory signals and pathways associated with cover crop-relay crop and cover crop-weed interactions that impact plant productivity, and (2) identifying economically suited winter-hardy broadleaf cover crops that suppress weed establishment in inter-seeded relay crops. These objectives will be accomplished through the use of physiological, molecular, and genomic approaches, with the long-term goals of revolutionizing weed management practices.
Progress Report
Preliminary experiments for year 2 of sub-objective 1.A indicated that winter canola, when planted in combination with corn, sunflower, or Amaranthus, significantly reduced biomass, leaf area, height, and stem diameter in all three species. Additionally, the response of different cultivars of corn, sunflower and Amaranthus to different cultivars of winter canola were statistically similar for the measured parameters. Although corn and sunflower were repressed when Amaranthus cultivars were used as competitors, most of these measured parameters were not significantly different. These preliminary experiments indicate that: 1) crop responses to weeds/competitors appears to be species specific, 2) crop responses to weeds do not appear to be cultivar specific, and 3) the data provided sufficient statistical power to distinguish differences between treatments with as few as 4 blocks in our experimental design.
Several milestones for sub-objective 1.B were substantially accomplished. First, we demonstrating that, under field conditions, weed-induced developmental (i.e., yield, height, leaf area) and physiological (i.e., photosynthetic activity, reactive oxygen species balance) responses in corn occur through phytochrome and general biotic stress signaling mechanisms (oxidative stress and salicylic acid). These signals were manifested later in crop development - even if weeds were removed early in the critical period for weed control in corn. A manuscript describing these results has been submitted for peer-review. Second, our preliminary research [aimed at determining the nature of the signal(s) controlling crop-weed interactions] suggested that signal(s) controlling the response of corn, sunflowers, and arabidopsis to weeds acts more like a switch than a rheostat. These results help to support the theory that weeds alter crop yield by mechanisms other than direct competition for resources. The experiments conducted using arabidopsis suggested that mutations to genes (phytochrome interacting factor 4/5) controlling the shade avoidance response had no significant impact on the response of arabidopsis to weed pressure. Thus, at least in the model plant arabidopsis, these observations suggest that signals other than those controlling shade avoidance are involved in reducing yield (biomass) and plant architecture (branching) in response to weeds.
Progress on initiating phenotyping and genotyping studies for winter hardiness under sub-objective 2.A was fully completed in year 2. Protocols established in FY16, for conducting high throughput vernalization and freezing treatments, were used in FY17 to screen and rate the 413 accessions within a winter canola diversity panel. Preliminary analysis of phenotyping data from one replicated study, using 3 plants/accession, identified 51 lines that scored a visual rating of 3 (all three plants survived) for freezing tolerance. Additionally, DNA collected from 429 accessions of canola (including some spring-types) was successfully genotyped using genotyping-by-sequencing (GBS) through an agreement with Cornell University. Analysis of the GBS data identified 251,575 high quality single-nucleotide polymorphisms (SNPs) after quality and frequency filtering and imputation. Average distance between these SNPs is 3377.3 bases, with a median distance of 53 bases, and with more than three quarters of the SNPs closer than 1877 bases apart. The largest gap between any two markers was less than 200 kilobases. This data is already being used to accomplish components of milestones for year 3 of sub-objective 2.A [initiate Genome Wide Association Studies (GWAS) analysis on winter canola diversity panel]. As an example, genotyping data for our winter canola diversity panel has already been used to perform GWAS on the initial freezing resistance dataset obtained using environmental chambers, and on field phenotyping data from the winter canola diversity panel (field phenotyping data obtained from Dr. Stamm at Kansas State University). Preliminary results obtained from these analyses have identified several markers associated with freezing tolerance and identified 5 candidate genes that may play a role in altering freezing tolerance in winter canola. The phenotyping data on freezing tolerance and the GBS data will be helpful for identifying genetic markers (SNPs) linked to the winter hardiness traits based on GWAS listed under sub-objective 2.A in years 3 and 4.
Preliminary experiments are also ahead of schedule to meet future milestones under sub-objectives 2.B. A few freezing tolerant accessions (ARS203, ARS205, ARS206, and ARS208), based on preliminary freezing tests from a subset of 100 winter canola accessions in FY16, were field tested for overwintering survival at the USDA-ARS in Fargo, North Dakota. Two rectangular plots (one located on the Northwest and one located on the Southeast side of the field plot) were used, and each plot was divided into fifteen 1 m2 blocks with 1 m2 spacing. Each accession/cultivar was planted in three replicate blocks per plot and each block had 18 plants. The combined overwintering survival rates were 34.3%, 18.5%, 15.7%, and 10.2% for ARS203, ARS208, ARS206, ARS205, respectively. The results indicated that more canola accessions survived in the Southeast plot. However, the survival rates among three replicate blocks were not consistent, except for ARS203 in the Southeast plot. These preliminary experiments will help establish protocols and overcome pitfalls associated with meeting future milestones listed under sub-objective 2.B.
To meet future milestones listed under sub-objective 1.C, protocols have been established for using CRISPR technology to modify several flowering- and vernalization-related genes, such as FLOWERING LOCUS C (FLC) and MADS-AFFECTING FLOWERING 2 (MAF2) in two camelina cultivars, Joelle and CO46. Partial genomic sequences of FLC and MAF2 were obtained by sequencing PCR-amplified DNA from Joelle and CO46, which were then used to design target sequences for CRISPR gene modification. The backbone vector (psk-AtU626) provided by Dr. Steven H. Strauss, Oregon State University, was used for cloning the target sequences. Six different constructs containing a single guide RNA or two guide RNAs were generated using standard cloning procedures. The six types of guide RNAs were then purified after restriction digestion and ligated into a binary vector (2×35S-AtU626-Nos, also obtained from Strauss lab) containing the Cas9 nuclease. Preliminary experiments are underway to determine how genetic transformation with these six binary vector constructs, using Agrobacterium strain GV3101 for transformation into Joelle and CO46, will alter phenotypic responses in flowering and vernalization.
Accomplishments
1. Winter-hardy oilseed cover crops help suppress establishment of weeds in northern climates. To establish winter canola as an oilseed cover crop in U.S. cropping systems, there is a need to increase freezing-tolerance. To identify genetic traits associated with freezing tolerance in winter canola, ARS scientists in Fargo, North Dakota, in collaboration with scientists from Cornell and Kansas State University, genotyped 429 accessions within a primarily winter canola diversity panel and identified 251,575 single-nucleotide polymorphisms (SNPs). The genotyping data in combination with greenhouse and field phenotyping data for freezing tolerance within the diversity panel are being used to conduct Genome Wide Association Studies. Outcomes from this accomplishment will help identify markers useful in breeding programs aimed at developing winter canola germplasm with increased freezing tolerance.
Review Publications
Berti, M., Gesch, R., Eynck, C., Anderson, J., Cermak, S. 2016. Camelina uses, genetics, genomics, production and management. Industrial Crops and Products. 94:690-710.
Shi, G., Zhang, Z., Friesen, T.L., Raats, D., Fahima, T., Brueggeman, R.S., Lu, S., Trick, H.N., Liu, Z., Chao, W., Frenkel, Z., Xu, S.S., Rasmussen, J.B., Faris, J.D. 2016. The hijacking of a receptor kinase-driven pathway by a wheat fungal pathogen leads to disease. Science Advances. 2:e1600822.
Hao, X., Yang, Y., Yue, C., Wang, L., Horvath, D.P., Wang, X. 2017. Comprehensive transcriptome analyses reveal differential gene expression profiles of Camellia sinensis axillary buds at para-, endo-, ecodormancy, and bud flush stages. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2017.00553.
Chao, W.S., Dogramaci, M., Horvath, D.P., Anderson, J.V., Foley, M.E. 2017. Comparison of phytohormone levels and transcript profiles during seasonal dormancy transitions in underground adventitious buds of leafy spurge. Plant Molecular Biology. 94(3):281-302.
Sintim, H.Y., Zheljazkov, V.D., Foley, M.E., Evangelista, R.L. 2017. Coal-bed methane water: effects on soil properties and camelina productivity. Journal of Environmental Quality. 46(3):641-648.
Long, Y.M., Chao, W.S., Ma, G.J., Xu, S.S., Qi, L.L. 2017. An innovative SNP genotyping method adapting to multiple platforms and throughputs. Theoretical and Applied Genetics. 130(3):597-607.
Dogramaci, M., Anderson, J.V., Chao, W.S., Horvath, D.P., Hernandez, A.G., Mikel, M.A., Foley, M.E. 2017. Foliar glyphosate treatment alters transcript and hormone profiles in crown buds of leafy spurge and induces dwarfed and bushy phenotypes throughout its perennial life cycle. The Plant Genome. https://doi.org/10.3835/plantgenome2016.09.0098.
