Location: Weed and Insect Biology Research
2023 Annual Report
Objectives
Objective 1: Determine the nature of inter and intraspecific competition and extent of crop yield loss among relay or double cropped agricultural plant species in comparison to natural weed competition.
Sub-objective 1A: Identify, under field conditions, the genes that are differentially regulated by natural weed populations, cover crops, and intra-specific competition in sunflower (Helianthus annuus).
Sub-objective 1B: Examine the impact of intra-specific competition on sunflower and corn (Zea mays L.) yield loss and gene expression under controlled conditions
Objective 2: Identify genetic or biochemical signals associated with interspecific competition and determine the associated biological mechanisms that can be used as targets for genetic manipulation.
Sub-objective 2A: Create constructs from corn promoters to identify the transcription factor(s) binding sites regulating weed- and/or cover crop-inducible genes.
Sub-objective 2B: Test if changes in salicylic acid levels corresponds to weed perception in corn.
Sub-objective 2C: Utilize the weed inducible promoter from corn to suppress the salicylic acid signaling during weed-crop or crop-cover crop interactions under controlled greenhouse conditions.
Objective 3: Functionally characterize specific targets impacting interspecific competition for genetic manipulation of weed tolerance, winter survival, early maturation, and/or response to bioherbicides.
Sub-objective 3A: Identify winter hardy canola and camelina germplasm that also have an early maturity trait for reducing competition between the cover crop and the relay-crop. As a first step, we will map early maturation Quantitative Trait Loci (QTLs) in a segregating Recombinant Inbred Line (RIL) population of Camelina sativa.
Sub-objective 3B: Determine if the freezing tolerance genes identified from winter camelina will increase freezing tolerance in canola (Brassica napus L.).
Sub-objective 3C: Functionally characterize the weed-induced PIF3 genes in soybean (Glycine max (L.) Merr).
Approach
Integrated weed management (IWM) is considered the most effective approach for managing weeds. In the northern Great Plains, incorporation of winter-hardy crops or cover crops as components of IWM systems are gaining popularity as an approach for managing weeds and the spread of herbicide resistant weeds. However, just like weeds, inter-specific competition with winter crops or cover crops, when used in multi-cropping systems, results in yield losses in major commodities. In this project, multi-cropping refers to fall-planting of oilseed cover crops that overwinter and are terminated or harvested prior to planting a primary summer commodity crop (double-cropping) or a primary commodity crop inter-seeded into the cover crop such that their life cycles overlap (relay-cropping). Factors impacting competition-induced yield losses have only been evaluated in a limited number of traditional multi-cropping systems under field conditions and this gap in knowledge needs to be addressed as new cropping and IWM systems suitable for the northern Great Plains are developed. To generate new knowledge for regionally-appropriate IWM approaches, the goals of this project are to: 1) understand how major commodity crops perceive and respond to inter- and intra-specific competition, 2) identify genes regulating winter survival and early maturity that can be manipulated to improve these traits in winter crops and cover crops, and 3) identify targets for mitigating competition-induced yield losses through breeding or genetic manipulation. Being able to multi-crop major commodities with winter-hardy crops or cover crops without resulting in yield loss, or mitigating weed-induced yield losses in general, would provide new IWM options. Thus, the objectives of this project will address gaps in our knowledge that limit the ability to develop sustainable IWM approaches appropriate for agricultural intensification in the northern Great Plains.
Progress Report
Objective 1A: Tissue samples collected from replicated field plots of sunflower grown under inter- or intra-specific competition with alfalfa or natural weed populations in 2022 at Hickson, North Dakota, Red Lake Falls, Minnesota, and Brookings, South Dakota, have been analyzed for nutrient content and used for developing Ribonucleic Acid sequencing (RNAseq) libraries that are at various stages in the sequencing and analysis process. Field studies in Minnesota and North Dakota were planted at two different row spacings (72- and 152- centimeter) and interseeded with or without alfalfa. At the South Dakota location, sunflower was planted at two times the normal planting rate for intra-specific competition studies or was interseeded with natural weeds for inter-specific competition studies. At the R1-R8 (reproductive 1 - 8) stage of development, tissue samples were collected three times from healthy leaf tissue and from roots (including soil associated with the rhizosphere) of two sunflower plants per replicated treatment.
Objective 1B: Samples were collected from medium maturity sunflower hybrid (Falcon) and a corn inbred (B73) planted in pots and grown under greenhouse conditions. Data collected from the second replicated study on intra-specific competition of sunflower have been analyzed and tissue samples collected have been used to develop RNAseq libraries that were sequenced and analyzed. Plant tissue samples collected from intra-specific corn competition studies are being processed for RNAseq analysis. Treatments included 1, 2, or 4 sunflower plants per pot. At 4 weeks of growth, plant height and stem diameter data were collected. At the R4 stage of sunflower development, plant height, stem diameter, and fresh and dry weight of above ground tissue were recorded, and tissue samples were collected from a healthy mid-level leaf and from root tissue of each sunflower replicate per treatment. For corn, a single corn seedling was grown in a 2-gallon pot with four additional corn plants grown in cone-tainers in the same pot to prevent root-to-root contact and with opaque above-ground cones to prevent light quality signaling between plants until corn was at the V6 (vegetative stage 6) stage of development. At that point, root-to-root contact was re-established by removing the corn plants from their cone-tainers and replanting the corn plant back into the soil in half of the pots. The other half were mock treated by removing the corn in cone-tainers from the potted soil, but then replacing the corn and cone-tainers back into the potted soil as they were. Root material was collected at 0, 1, 2, 3, 7, and 14 days from treated and mock-treated plants.
Objective 2A: Transgenic corn plants containing a reporter gene (Red3 - which is easily detected by red fluorescence) turned on by a weed-inducible promoter, isolated from the corn DC1 gene, were grown in a greenhouse for self-pollination and used to identify homozygous lines from T2 seed.
Objective 2B: Initial High-Performance Liquid Chromatography (HPLC) runs to determine levels of salicylic acid (SA) from extracted plant tissue samples revealed inconsistencies in samples spiked with internal standards. Thus, we have sent the corn samples collected from field plots in competition with alfalfa or weeds or under greenhouse conditions in competition with rapeseed to Dr. Nishanth Tharayil at Clemson University for SA analysis. Their laboratory has expertise in analyzing hormones from plant tissues.
Objective 2C: Transgenic corn with NahG (a gene that makes salicylate hydroxylase) under the control of the weed-inducible promoter were obtained. The transgenic plants expressed NahG RNA under control conditions and were confirmed to have enhanced expression under weedy conditions. These studies have been repeated and confirmed the weed-inducibility of the DC1 promoter.
Objective 3A: An F7 Recombinant Inbred Line (RIL) population, consisting of 254 lines of camelina, has been phenotyped and genotyped. The population was phenotyped for flowering time, plant height, biomass, days to maturity, seed yield, and freezing tolerance in response to treatment with or without cold acclimation for 8 weeks. Genotyping of the F7 RIL population identified over 4507 high quality single nucleotide polymorphism (SNP) markers, which have been mapped to the camelina genome and used to identify Quantitative Trait Loci (QTL) on chromosomes associated with flowering time, freezing tolerance, and seed size. QTLs associated with flowering time and freezing tolerance on chromosome 8 and 13 were consistently observed and covered a region of the chromosome containing a gene know as FLOWERING LOCUS C (FLC). FLC is known to regulate flowering and appears to play some role in freezing tolerance as well. Two additional loci on chromosomes 16 and 18 have either FLC-like genes such as MAF3 (MADS AFFECTING FLOWERING 3) or genes known to interact with and facilitate the function of FLC. Finally, mapping also highlighted a region on chromosome 11 that was identified as having a role in freezing tolerance through a homozygosity mapping project. This research is being presented at the 2023 Association for the Advancement of Industrial Crops meeting and several manuscripts from these presentations will be invited for submission to a Special Issue of Industrial Crops and Products.
Objective 3B: Due to a critical vacancy, a Postdoctoral Research Associate and several graduate students have been recruited to join the project. The Postdoc has designed constructs for a canola gene (SENSITIVE TO FREEZING 2; SFR2) identified from Genome-Wide Association Studies using a polymerase chain reaction cloning method. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology and over-expression of target genes will be applied to characterize the roles of SFR2 in transgenic plants. Likewise, a graduate student is working on building constructs to both turn off and over-express VERNALIZATION INDEPENDENCE 3 (VIP3), which is involved in regulating deacclimation processes in canola and arabidopsis. The student has cloned multiple genomic copies of VIP3 from canola varieties with differing deacclimation rates and these clones are currently being sequenced. We are also in the process of confirming the impact of turning off VIP3 on the transcriptome of arabidopsis. We also determined that loss of VIP3 expression resulted in continued expression of COLD-REGULATED genes in arabidopsis following a deacclimation period and we have initiated studies to determine the mechanisms by which VIP3 regulates their expression.
To validate the function of candidate genes in freezing tolerance, we have developed an efficient plant regeneration system using canola hypocotyl segments excised from three different regions (top, medium, and bottom) of 6-, 7-, and 8-day old seedlings. We have confirmed that hypocotyl segments excised from the top region of 8-day old seedlings exhibited the greatest potential for shoot regeneration. A graduate student is working on the development of a genetic transformation systems for canola using the above-mentioned regeneration system.
Objective 3C: A visiting postdoc created a series of constructs designed to turn off the two weed-inducible PHYTOCHROME INTERACTING FACTOR (PIF3) genes from soybean, and another set of constructs that would turn off all six of the PIF3 genes from soybean. These constructs were confirmed by sequencing and are ready to be transferred into soybean.
Accomplishments
1. Winter hardy oilseed cash cover crops for integrated weed management approaches. The lack of freezing tolerance among oilseed cash cover crops limits their ability to overwinter in colder climates and to suppress early season weeds. ARS scientists in Fargo, North Dakota, and Morris, Minnesota, conducted field studies to determine relationships between winter hardy oilseed crops and weed suppression in colder climates and soils. The field research, including 621 accessions of winter canola/rapeseed and a winter camelina check, revealed that overwinter survival of camelina or canola/rapeseed had a direct correlation on suppression of early season weeds. However, fall planting date of winter oilseed crops affected winter survival in both North Dakota and Minnesota. Through this research we identified nine freezing tolerant canola/rapeseed lines that survived field conditions in both North Dakota and Minnesota. These winter hardy canola/rapeseed accessions are good candidates for breeding commercial canola cultivars adaptable to colder climates and soils.
2. Identifying signaling mechanisms involved in weed-induced crop yield loss. Crops perceive weed-generated signals through changes in light quality, as well as through volatile and soil-soluble chemicals. Understanding how genes are impacted by these signals is needed to manipulate crop responses to weeds and mitigate weed-induced yield losses. ARS scientists in Fargo, North Dakota, conducted time course studies to examine weed-induced changes in corn growth and gene expression when exposed to soil soluble and light quality signals separately or in combination. Soil soluble signals had a greater impact on corn growth than light quality signals and impacted photosynthesis in leaves and cell wall production in the roots. The light quality signal had no consistent impact on leaves, but it did impact protein turnover and cell growth in roots. The research implicated certain gene networks and protein complexes known to regulate the balance between defense and growth in plants in the response of corn to weeds. This work will help breeders create more weed-tolerant crops by suggesting ways to manipulate crop responses to weed-generated signals.
3. Identifying regions of the Camelina sativa genome associated with important agronomic traits. Breeding for desired agronomic traits is critical for improving cropping systems. ARS scientists in Fargo, North Dakota, Clay Center, Nebraska, and Stoneville, Mississippi, and scientists at North Dakota State University collaborated to sequence and identify regions of the genome associated with freezing tolerance, flowering time, and seed size in a population of camelina developed from crossing a winter biotype with a summer biotype. Sequencing of the camelina genomes allowed for the identification of specific markers within the 20 assembled chromosomes associated with these important agronomic traits. This work improved on the assembly of the camelina genome and helped identify candidate genes associated with differences observed for freezing tolerance, flowering time, and seed size. These outcomes will assist breeders to improve important agronomic traits in oilseed cash crops used for developing cropping systems suitable for colder climates and soils.
4. Identifying molecular mechanisms for improving freezing tolerance in canola. Acclimating canola plants to low temperatures improves freezing tolerance but short periods of warm temperatures in late fall and early spring can cause deacclimation and loss of freezing tolerance. ARS scientists in Fargo, North Dakota, identified a canola gene known as VERNALIZATION INDEPENDENCE 3 (VIP3) that, when mutated in the model plant Arabidopsis thaliana, prevents deacclimation. This research furthers our understanding of the deacclimation process and provides breeders with knowledge for improving freezing tolerance in crops suitable for colder climates and soils and for combating the impact of climate change.
5. Herbicide-resistant camelina and canola provides good weed suppression. A lack of approved herbicides in camelina limits its integration into cropping systems. ARS scientists in Fargo, North Dakota, collaborated with scientists at North Dakota State University to evaluate weed suppression, nutrient retention, and seed yield in non-transgenic sulfonylurea-resistant camelina and canola treated with or without sulfonylurea herbicide. Sulfonylurea-resistant camelina and canola alone reduced late-season dry weight biomass of weeds by > 75% and > 60%, respectively, compared with fallow plots. Mid-season treatment with sulfonylurea herbicide did not significantly reduce weed pressure over that of the untreated crop but, in some cases, herbicide treatment had an additive effect on further reducing weed pressure. This work suggests that sulfonylurea-resistant camelina or canola can provide additional options for improving integrated weed management approaches, and for reducing nutrient leaching and herbicide application rates in the upper Midwest and northern Great Plains of the U.S.
6. Identifying the time and temperature thresholds for deacclimation in canola. Predicting loss of winter hardiness (deacclimation) due to sporadic warm periods in late fall and early spring is critical for canola growers. ARS scientists in Fargo, North Dakota, collaborated with scientists at North Dakota State University to determine the critical temperature and timing for deacclimation in eight winter canola varieties. Deacclimation occurred with warm periods of between seven and ten degrees Celsius (C) and maximal loss of freezing tolerance occurred within two to three days, although some varietal differences were observed. In several varieties, freezing tolerance was recovered when plants were treated at seven or ten C for longer than seven days. This information provides breeders with baseline data needed to select for deacclimation-resistant varieties of canola. It will allow growers to better predict likely damage to winter canola crops and when to take preemptive actions to mitigate crop losses.
7. Development of an efficient method for doubled haploid induction in sunflower. Doubled haploid (DH) technology offers a fast way to develop pure lines for breeding purposes. ARS scientists in Fargo, North Dakota, used irradiation of sunflower pollen with gamma rays to successfully stimulate the development of DH plants. The optimal gamma ray dose for pollen grain irradiation and DH plant production was determined to be 100 gray. In addition, two sunflower lines were identified as common male plants for induction of DH in desired female sunflower lines using gamma-irradiated pollen grains. This work provides geneticists and breeders with new approaches for developing DH sunflower plants for advancing breeding pipelines.
Review Publications
Horvath, D.P., Doherty, C.J., Desai, J., Clark, N.M., Anderson, J.V., Chao, W.S. 2023. Weed-induced changes in the maize root transcriptome reveal transcription factors and physiological processes impacted early in crop-weed interactions. AoB Plants. https://doi.org/10.1093/aobpla/plad013.
Zhao, X., Niu, Y., Chao, W.S., Lu, P., Bai, X., Mao, T. 2023. Genetic variation, DIMBOA accumulation, and candidate genes identification in maize multiple insect-resistance. International Journal of Molecular Sciences. 24. Article 2138. https://doi.org/10.3390/ijms24032138.
Chao, W.S., Anderson, J.V., Li, X., Gesch, R.W., Berti, M., Horvath, D.P. 2023. Overwintering camelina and canola/rapeseed show promise for improving integrated weed management approaches in the upper Midwestern U.S. Plants. 12(6). Article 1329. https://doi.org/10.3390/plants12061329.
Horvath, D.P., Clay, S., Swanton, C., Anderson, J.V., Chao, W.S. 2023. Weed-induced crop yield loss: A new paradigm and new challenges. Trends in Plant Science. 28(5):567-582. https://doi.org/10.1016/j.tplants.2022.12.014.
Wang, H., Hou, H., Jan, C., Chao, W.S. 2023. Irradiated pollen-induced parthenogenesis for doubled haploid production in sunflower (Helianthus spp.). Plants. 12. Article 2430. https://doi.org/10.3390/plants12132430.
Anderson, J.V., Bigger, B., Howatt, K., Mettler, J., Berti, M.T. 2022. Weed pressure, nutrient content, and seed yield in field grown sulfonylurea-resistant Camelina sativa and Brassica napus. Agronomy. 12(11). Article 2622. https://doi.org/10.3390/agronomy12112622.
Shaikh, T., Rahman, M., Horvath, D.P., Smith, T.P., Anderson, J.V., Chao, W.S. 2023. Homozygosity mapping identified loci and candidate genes responsible for freezing tolerance in Camelina sativa. The Plant Genome. 16(2). Article e20318. https://doi.org/10.1002/tpg2.20318.
Young, S.L., Anderson, J.V., Baerson, S.R., Bajsa Hirschel, J.N., Blumenthal, D.M., Boyd, C.S., Boyette, C.D., Brennan, E.B., Cantrell, C.L., Chao, W.S., Chee Sanford, J.C., Clements, D.D., Dray Jr, F.A., Duke, S.O., Porter, K.M., Fletcher, R.S., Fulcher, M.R., Gaskin, J., Grewell, B.J., Hamerlynck, E.P., Hoagland, R.E., Horvath, D.P., Law, E.P., Madsen, J., Martin, D.E., Mattox, C.M., Mirsky, S.B., Molin, W.T., Moran, P.J., Mueller, R.C., Nandula, V.K., Newingham, B.A., Pan, Z., Porensky, L.M., Pratt, P.D., Price, A.J., Rector, B.G., Reddy, K.N., Sheley, R.L., Smith, L., Smith, M., Snyder, K.A., Tancos, M.A., West, N.M., Wheeler, G.S., Williams, M., Wolf, J.E., Wonkka, C.L., Wright, A.A., Xi, J., Ziska, L.H. 2023. Agricultural Research Service weed science research: past, present, and future. Weed Science. 71(4):312-327. https://doi.org/10.1017/wsc.2023.31.
