Source: UNIV OF MINNESOTA submitted to NRP
ADVANCING FIELD PENNYCRESS AS A NEW OILSEED BIODIESEL FEEDSTOCK THAT DOES NOT REQUIRE NEW LAND COMMITMENTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1004021
Grant No.
2014-67009-22305
Cumulative Award Amt.
$1,000,000.00
Proposal No.
2014-07664
Multistate No.
(N/A)
Project Start Date
Sep 1, 2014
Project End Date
Aug 31, 2018
Grant Year
2014
Program Code
[A6151]- Sustainable Bioenergy: Plant Feedstock Genomics for Bioenergy
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Plant Biology
Non Technical Summary
This is a collaborative project between researchers in Minnesota and Illinois to genetically improve the agronomic traits of Field Pennycress (Thlaspi arvense L.; pennycress) for its use as a new winter annual oilseed/meal/cover crop in the Upper Midwest. Pennycress can be double cropped on the same land during the time between the traditional corn harvest and subsequent planting of soybeans the following spring. Pennycress has only recently been isolated from its natural environments throughout the Midwest. Field trials with current isolates have demonstrated that pennycress can be seeded in upper Midwest cornfields in the late summer and fall, at which time the plants begin to grow then overwinter, producing mature seed in the spring that can be harvested without disrupting soybean planting or yields. 2,200 kg/hectare seeds can be produced by wild pennycress varieties, which at 33% by weight oils content, yields 840 liters/hectare oils for biofuels and 1,470 kg/hectare press-cake to be used as a high-protein/high-energy animal feed meal. It has been estimated that pennycress can add approximately $100.00 per acre to a farmer's income. While pennycress holds much agronomic promise, improved domesticated varieties remain to be developed. Wild pennycress varieties are hampered by inconsistent germination and stand establishment, un-optimized maturity for a given growth zone, suboptimal oils quality for biodiesel production, high seed glucosinolate content, and significant harvest loss due to pod shatter. Domestication of many weeds that are now crops often involved the unintended selection of naturally occurring mutants with improved agronomic characteristics. We will use modern mutation-based breeding strategies to identify lines of pennycress that harbor superior agronomic traits. The lines will be incorporated into our ongoing traditional breeding programs.This research will aid in the generation of elite pennycress varieties having consistent stand establishment, higher yields, and other superior traits, allowing for widespread adoption of pennycress as a winter oilseed/meal/cover crop integrated within corn-soybean rotations throughout the Midwest. Elite pennycress varieties will positively impact the profitability of production agriculture and enhance livestock sector returns. The adoption of pennycress will provided additional ecological benefits such as decreased soil erosion and nutrient runoff, reduced herbicide use for weed control, reduced carbon emissions by replacing fossil fuels with biofuels. Finally, pennycress can increase energy security by diversifying the nation's energy portfolio, and contribute to the economic health of rural communities.
Animal Health Component
0%
Research Effort Categories
Basic
10%
Applied
0%
Developmental
90%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20218991080100%
Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
1899 - Oilseed and oil crops, general/other;

Field Of Science
1080 - Genetics;
Goals / Objectives
Project Objectives:Generate DNA pools derived from a population of 1,536 ethyl methanesulfonate (EMS) treated spring pennycress individuals.Generate DNA pools derived from a population of 15,000 fast neutron (FN) treated winter pennycress individuals.Use the TILLING procedure to conduct a reverse genetic screen on DNA pools from EMS treated individuals to identify lines with mutations in genes that may aid in the domestication of pennycress.Use the DeTILLING procedure to conduct a reverse genetic screen on DNA pools from FN treated individuals to identify lines with mutations in genes that may aid in the domestication of pennycressScreen 200,000 M2 plants derived from 10,000 mutagenized M1 plants for plants with desirable phenotypes.Screen 10,000 lines with rapid NIRS and NMR for their oil content/composition and glucosinolate content.Characterize mutants from forward genetic screens and the reverse genetic TILLING and DeTILLING screens in terms of germination efficiency, flowering time, pod shatter, oil quality, glucosinolate content, seed yield, and seed size by growing them in the lab and in the field along side wild type individuals over multiple generations.Outcross mutants to elite selected lines and bulk up seed from mutant lines for further testing.Given that we will likely identify a large number of mutants exhibiting the targeted phenotypes of interest, priority will be given to characterizing in detail ~10 lines having the highest agronomic value (e.g. those lines having the largest trait improvements without compromised fitness).Timetable of Activities: Year one· Isolate DNA and make pools. IL and MN· Subject seeds from individual lines to NIRS screening. IL· Screen for and characterize germination mutants. IL· Screen mutagenized populations in the field. MN· Begin work on any pod mutants. MN· Replant any mutants selected from the field. MN and IL· Replant any individuals showing alterations in the NIRS scan. MN and IL Year two· Begin TILLING and DeTILLING reactions. IL and MN, respectively· Identify homozygous mutants from the TILLING and DeTILLING populations and screen in the lab for phenotypes of interest IL and MN· Make crosses with mutants of interest in the lab. IL and MN· Collect seed from individual M2 plants that showed an altered NIRS spectra and retest. MN· Biochemically analyze lines that retest positive for altered NIRS spectra. IL · Publish interesting findings from TILLING and DeTILLING. IL and MN Year three· Characterize mutants identified in the various screens and outcross those with traits of interest to wild lines with superior characteristics. IL and MN· Publish characterizations of interesting mutants IL and MN
Project Methods
NGS and TILLING of spring EMS population In Arabidopsis, EMS mutagenesis has been shown to generate between 4 and 11 mutations per Mb DNA 1-3. Our goal will be to collect M2 seed from 1,536 EMS treated M1 spring type individuals. Taken together, this population should harbor at least 10 mutations per kb of DNA. We will use the protocol described in Tsai et al., 2011 as a guide for TILLING by NGS sequencing 4. Pools of DNA representing 8 EMS treated M1s will be loaded into single wells of 2 96-well plates. In all, DNA derived from 1,536 M1 individuals will be distributed into 192 wells. Each plate will be used to generate 12 column pools and 8 row pools. Using the above scheme for pooling, we will generate 20 pools of DNA for each 96-well plate. Illumina sequencing of these pooled DNA sample will be used to identify mutations in genes of interestDeTILLING of FN population Mutations in the FN population are most likely to be loss of function deletions. A strategy called DeTILLING has been developed for identifying targeted gene deletions in DNA pools derived from large populations of FN-treated plants 5. Using this protocol, it was shown that a deletion fragment could be detected when genomic DNA containing a known deletion was diluted 24,000 fold with wild type DNA. Candidate gene targets for TILLING and DeTILLINGSeed Dormancy: Our highest priority will be to identify gene variants that remove layers of seed dormancy, thereby allowing for the development of pennycress varieties with more uniform germination and stand establishment. An attractive gene target is DOG1 (DELAY OF GERMINATION1; 6-9). DOG1, which was first cloned in Arabidopsis, is a major quantitative trait locus specific for controlling seed dormancy in Brassica species. In addition to DOG1, other targeted genes will include genes such as ABA INSENSITIVE 3, 4, and 5, which are known to participate in the ABA signal transduction pathway that induces dormancy 10. Candidates showing desirable characteristics will be field-tested and moved into our breeding programs.Oil Quality: It has been shown in other Brassica species that mutations in the FAE1 (FATTY ACID ELONGASE1) and FAD2 (FATTY ACID DESATURASE2) genes result in reductions in erucic acid and linolenic/linoleic acids oil content, respectively 11. Identifying low erucic acid pennycress variants will be of high agronomic importance and is a top priority. The Phippen group has considerable experience analyzing oil composition of pennycress seeds, having quantified seed oil fatty acid compositions associated with dozens of pennycress varieties from their germplasm collection 12.Seed Glucosinolates: After oil extraction using a screw press, approximately 30% of the seed wet weight remains as a press-cake. The unprocessed press-cake potentially can be used as meal (animal feed), owing to its high protein content (27%) and energy content (residual oil). A concern related to pennycress press-cake meal is that it contains the glucosinolate sinigrin (13; Steven Vaughn, personal communication), which decreases palatability. Nour-Eldin et al., 2012 recently identified and characterized two high-affinity glucosinolate-specific transporters in Arabidopsis they named GTR1 and GTR2 14. They found that gtr1 gtr2 double mutants did not accumulate glucosinolates in seeds; the mutations had no adverse affects on plant health or growth. We will identify mutations in the GTR1 and GTR2 genes and then generate pennycress gtr1 gtr2 double mutants for phenotypic assessments. Glucosinolate detection and quantification will be performed using a sephadex/sulfatase protocol originally described by Hogge et al., 1988 and adopted by Kliebenstein et al., 15,16.Flowering Time: Flowering is a primary requirement of seed production, the timing of which must be suitable for a given growth zone. We will target FLC (FLOWERING LOCUS C) and FRI (FRIGIDA) genes, which are well studied as being primary determinants of flowering time in Brassicas 17-19. Variants showing either earlier or later flowering will be further tested to determine the affect on seed yield.Pod Shatter: In Arabidopsis, double mutations in two related genes SHATTERPROOF 1 and 2 (SHP1 and 2) decrease pod shatter by altering the maturation of the separation (dehiscence) zone at the valve margins 20,21. Mutations in these genes will be targeted by TILLING, and shp1 shp2 double mutants will be generated for phenotypic assessments. To quantify reduced pod shatter phenotypes, we will employ a push pull digital force gauge (e.g. Wagner Instruments Mark-10 M5 device with computer interface output). Candidates showing reduced pod shatter over at least one generation will be field-tested and moved into our breeding programs.Seed Size and Oil Content: Several Arabidopsis genes are known to affect seed oil accumulation. For example, loss of function mutations in GLABRA2 (GL2) and MUCILAGE-MODIFIED 4 (MUM4) increase seed oil content 22, whereas overexpression of WRINKLED1 (WRI1), HAIKU2 (IKU2), and KLUH (KLU) increase seed oil content and seed size 23-26. Candidates showing enhanced oil content or seed size will be characterized for fatty acid content.Forward Genetic Screen For forward screening of morphological traits, mutant seeds will be bulk collected in pools of 10 M1 plants. In all, 400 EMS, 400 FN, and 400 gamma ray treated pools, each derived from 10 M1 plants, will be collected for this screen (representing 12,000 M1s). The seeds will be planted in early September 2014 and plants will be allowed to overwinter. In the spring, M2 plants will be screened for flowering time and/or early senescence, seed yield, seed size, plant stature, shatter resistance, flower color, seed coat color, pod shape, and pod fill. Chosen lines will be replanted in the fall of 2015 and rescreened the spring of 2016. The most promising mutants will be rapidly assessed in growth chambers. M2 seeds from individual M1 plants will initially be screened for seed oil content, seed oil composition, and glucosinolate content using nondestructive pulsed NMR and/or Near Infrared Reflectance Spectroscopy (NIRS). NIRS was successfully used by others to rapidly identify rapeseed lines with reduced erucic acid and glucosinolates 27.Literature Cited1 BMC plant biology 9, 147 (2009).2 Genetics 164, 731-740 (2003).3 Plant Physiology 161, 1604-1614 (2013).4 Plant Physiology 156, 1257-1268 (2011).5 Plant Physiology 151, 1077-1086 (2009).6 The Arabidopsis Book/American Society of Plant Biologists 6 (2008).7 Plant Biology 59, 387 (2008).8 Plant J 74, 1003-1015, (2013).9 Plant, cell & environment 35, 1769-1786 (2012).10 The Plant Journal 5, 765-771 (1994).11 New Phytologist 180, 751-765 (2008).12 Phippen, W. B. & Phippen, M. E. Seed oil characteristics of wild field pennycress (Thlaspi arvense L.) populations and USDA accessions. Meeting: Advancement of Industrial Crops Annual Meeting, Washington D.C (2013).13 Journal of chemical ecology 31, 167-177 (2005).14 Nour-Eldin, H. H. et al. NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature (2012).15 Journal of Chromatographic Science 26, 551-556 (1988).16 The Plant Cell Online 13, 681-693 (2001).17 BMC plant biology 12, 21 (2012).18 The Plant Journal 28, 545-553 (2001).19 YJournal of experimental botany 60, 1299-1308 (2009).20 Science 289, 436-438 (2000).21 The Plant Journal 73, 824-835 (2013).22 The Plant Journal 69, 37-46 (2012).23 The Plant Cell Online 24, 5007-5023 (2012).24 Plant Physiology 163, 973-985 (2013).25 Proceedings of the National Academy of Sciences 106, 20115-20120 (2009).26 Plant Physiology and Biochemistry 48, 9-15 (2010).27 Journal of food science and technology 47, 690-692 (2010).

Progress 09/01/14 to 08/31/18

Outputs
Target Audience:Students of plant biology at all levels. Scientists engaged in plant research in both academic and private industrial settings. Farming interest groups. State and Federal legislators. Investors including start-up company CoverCress, Inc. (formerly Arvegenix, Inc.). Ecological groups. Biofuels researchers at all levels. Agriculture undergraduates. Local and regional producers. Changes/Problems:Switched from TILLING to forward screens of mutagenized populations and CRISPR-Cas. Problems- some loss of time due to fungal outbreaks in growth chambers. What opportunities for training and professional development has the project provided?Marks (UMN): Five technicians directly after completing their undergraduate degree have worked on this project. Two of these have left for other positions (one at the Salk Institute and one at U Colorado-Boulder). One left to enter graduate school a Washington University (St. Louis, MO) where he is pursuing a Ph.D. in plant sciences. We have lost track of a fourth, while the fifth is still working in the lab. One graduate student finished their PhD early in the project (Kevin Dorn). Kevin was recently hired as a USDA scientist in Fort Collins, CO. In June 2017, a postdoc focused on the genomics work joined our group (Ratan Chopra). Already Ratan has been the lead author on two recent publications. In August 2017, a graduate student studying the early flowering and reduced lodging mutants joined our group. More 15 undergraduates have participated in this project including six who worked full time over summers. Sedbrook (ISU): This project provided training for three PhD students (one graduated and is working at the pennycress start-up company CoverCress, Inc., while the other two are still students in the lab), three Master's students (one graduated and entered our PhD program, while the other two are still students in the lab), and 34 undergraduate students (25 have graduated and moved on to professional schools, molecular graduate programs, and biotech-related jobs, while nine are still students in the lab). All six graduate students did/are doing internships at CoverCress, Inc. (formerly Arvegenix, Inc). Pennycress-related molecular experiments and content were integrated into introductory and upper-level courses taken by over 300 undergraduate and graduate students. Courses included BSC203 Cell Biology, BSC353 Biotechnology Laboratory, BSC365 Plant/Microbe Bioenergy and the Environment, and BSC450 Issues in Biotechnology. Phippen (WIU): Through this 4-year project, a total of 8 undergraduate students worked part time in the greenhouse, field, and laboratory supporting the efforts of growing pennycress lines. Six students completed independent research projects focused on pennycress breeding, seed storage, and evaluating herbicide tolerance in pennycress. The use of pennycress was also integrated into a traditional plant breeding course (AGRN-472 Principles of Plant Breeding) as a model plant for studying plant domestication. This course reached 28 students in 2015 and 11 students 2017. How have the results been disseminated to communities of interest?Marks - Oral Presentations: January 19, 2017 Minnesota Department of Health meeting focus on nutrient contamination of rural well heads. February 22, 2017 Botany and Plant Pathology, Purdue University May 5, 2017 PepsiCo Senior Vice President and others May 17, 2017 Minnesota Crop Improvement Association August 15, 2017 Deep Dive presentation to potential investors February 2018 Department of Plant Science, University Arizona July 2018 Danforth Research Center, St. Louis, MO September 2018 Department of Biochemistry, University of Nebraska-Lincoln Sedbrook - Oral Presentations: May 9, 2018: "Employing synthetic biology approaches to facilitate value-added oil production in the oilseed cover crop pennycress.," AOCS (American Oil Chemists' Society) Annual Meeting, American Oil Chemists' Society, Minneapolis, MN. May 8, 2018: "Improving agronomic traits of the oilseed-producing winter cover crop pennycress using CRISPR-Cas9 genome editing and EMS mutagenesis.," University of Minnesota Plant and Microbial Biology Colloquium Series, University of Minnesota, St. Paul, MN. March 3, 2018: Maliheh Esfahanian (Graduate student presenter), "Improving agronomic traits of the oilseed-producing winter cover crop pennycress (Thlaspi arvense) using CRISPR-Cas9 genome editing and EMS mutagenesis.," American Society of Plant Biologists (ASPB) Midwest Meeting, Ames, IA. February 27, 2018: "Advancing Field Pennycress as a New Oilseed Biofuels Feedstock that does not Require New Land Commitments.," USDA NIFA/DOE Project Director/Principal Investigator Meeting, Washington D.C. November 4, 2017: Katy Haag, Ashley King (Undergraduate presenters), "Reducing seed pod shatter in the new oilseed winter cover crop pennycress (Thlaspi arvense).," Integrative Plant Biology and Bioenergy (IPBB) Symposium, Normal, IL. June 28, 2017: "Genetic improvements to pennycress for commercialization.," Arvegenix Annual Board Meeting, St. Louis, MO. June 21, 2017: Michaela McGinn (Graduate student presenter), "Rapidly domesticating the new oilseed crop pennycress (Thlaspi arvense) by translating findings from Arabidopsis research.," International Conference on Arabidopsis Research (ICAR), St. Louis, MO. May 31, 2017: Maliheh Esfahanian (Graduate student presenter), "Characterization of the pennycress (Thlaspi arvense) reduced pod shatter1 (rps1) mutant.," Plant Cell Dynamics Meeting, Madison, WI. May 31, 2017: Brice Jarvis (Graduate student presenter), "Generating high erucic acid seed oil content in the oilseed plant pennycress.," Plant Cell Dynamics Meeting, Madison, WI. March 13, 2017: "Improving crop plants for their use in generating biofuels from biomass.," Ain Shams University, Department of Botany Seminar, Cairo, Egypt. February 7, 2017: "Advancing Field Pennycress as a New Oilseed Biofuels Feedstock that does not Require New Land Commitments.," USDA NIFA/DOE Project Director/Principal Investigator Meeting, Washington D.C. Phippen - Oral Presentations: May 24, 2018 9th Annual Pennycress Field Day. Macomb, IL October 18, 2017 Illinois Extension Agriculture Association (IEAA). Plant breeding efforts on pennycress. May 10, 2017 LaMoine River Ecosystem Partnership. Plant breeding efforts on pennycress. May 12, 2017 8th Annual Pennycress Field Day. Macomb, IL April 27, 2017 Farmweek Magazine. Advances in Pennycress Production February 10, 2017 Illinois AgriNews. Pennycress research February 8, 2017 Intellifarm Conference. The Field Pennycress Future: Production, Applications and Market Opportunities. Kansas City, MO. Marks- Poster Presentations: June 2017 Annual American Society for Plant Biology Meeting Advancing Field Pennycress as a New Oilseed Biodiesel Feedstock-Focus on New Mutants Ratan Chopra, Nicole Folstad, Ryan Emenecker, Kevin Dorn1, M. David Marks Engineering a New Oil-Seed Cover Crop: Domesticating Pennycress Nicole Folstad, David Marks Sedbrook - Poster Presentations: March 3-4, 2018: Suo, T. (Presenter), Esfahanian, M., Chopra, R., Woodworth, J., Haag, K., King, A., Janowiak, K., Marks, D., Sedbrook, J. "Reducing seed coat fiber content to improve seed meal nutritional value of the oilseed crop pennycress (Thlaspi arvense).", American Society of Plant Biologists (ASPB) Midwest Meeting, Ames, IA. January 13-17, 2018: Esfahanian, M. (Presenter), McGinn, M., Jarvis, B., Suo, T., Sedbrook, J. "Utilizing CRISPR genome editing to rapidly domesticate the winter annual oilseed crop pennycress (Thlaspi arvense).", Plant and Animal Genome (PAG) Conference, San Diego, CA. January 29-February 3, 2017: Esfahanian, M. (Presenter), Durrett, T., Cahoon, E., Sedbrook, J. "Engineering medium chain fatty acids into seed triacylglycerols of the oilseed crop pennycress (Thlaspi arvense).", Gordon Research Conference on Plant Lipids, Galveston, TX. January 29-February 3, 2017: McGinn, M. (Presenter), Jarvis, B., Cahoon, E., Sedbrook, J. "Utilizing CRISPR-Cas9 to improve seed oil quality in the winter annual oilseed crop pennycress (Thlaspi arvense).", Gordon Research Conference on Plant Lipids, Galveston, TX. Phippen - Poster Presentations: September 24-28, 2016 Advancement of Industrial Crops Annual Meeting. Evaluation of the germination rates of Pennycress (Thlaspi arvense L.) in different conditions of storage and temperature. Rochester, NY What do you plan to do during the next reporting period to accomplish the goals?We have one additional publication under review and we will be continuing this work using funds from two additional awards from the DOE/USDA feedstock program (Sedbrook PI and Anderson PI).

Impacts
What was accomplished under these goals? University of Minnesota (UMN) It was found that pennycress shares extensive whole genome similarity to the model plant Arabidopsis (see Chopra et al. (2018) Plant Journal- https://doi.org/10.1111/tpj.14147). We have identified the most likely causative mutations for one early maturing line and six reduced seedpod shatter lines. We have identified more than 15 seed coat mutants and have characterized the causative mutations. We found that we could use preexisting canola NIRS calibration equations to estimate the chemical contents of pennycress seeds, including glucosinolates, oil%, protein%, as well the % of various fatty acids. We have analyzed our NIRS scan and have identified lines that qualify as 0 glucosinolates and 0 for erucic acid. Illinois State University (ISU) We generated and performed forward genetic screens of a large pennycress EMS mutant population (Spring-type background instead of the Winter-type background mutagenized by UMN), and also generated/grew out a large EMS mutant population (low seed dormancy winter-type background) in partnership with the Phippen group at WIU. We also developed the inbred line Spring 32-10 (10 generations of single seed descent; the Marks group sequenced the Spring 32-10 genome), to be used for day-to-day laboratory experimentation akin to the Columbia ecotype of Arabidopsis (Spring 32-10 seeds donated to the Arabidopsis Biological Resource Center (ABRC). We developed an efficient pennycress Agrobacterium-mediated floral dip vacuum infiltration transformation protocol and effective synthetic biology tools including CRISPR-Cas gene editing. We identified and have characterized mutants improving virtually every agronomic trait for which we searched, including reduced seed dormancy, improved seed oil fatty acid composition, higher seed oil content, reduced glucosinolate content, reduced pod shatter, reduced seed coat fiber, and larger seed size. We have had great success employing CRISPR gene editing to mutagenize nearly 20 genes orthologous to known domestication trait genes. We have also demonstrated success in targeting multiple genes with a single construct, which is allowing us to rapidly introduce domestication trait mutations into our top breeding lines. Western Illinois University (WIU) 51 wild populations collected from across the Midwest were evaluated in replicated plots for seed yield, early flowering time, seed size, stand establishment, and days to maturity. Studies were completed in establishing the optimum seeding rate for fall planting. Several lines were identified having the non-dormancy trait of the spring type, along with short stature dwarf lines and a very early maturing line. 24 mutant lines identified by UMN and ISU were grown in single rows for field evaluation and seed increase. Traits identified include: large pods, fast germination, thick stems, tillerless, dwarf, and early flowering. Seed increases of several hundred mutant lines created at ISU for seed shatter reduction and yellow seed coat were grown in the greenhouse. Large 150' strip trials were conducted on 12 advanced breeding lines to evaluate fall establishment, time to flowering, and seed yield. Eight lines with yellow seed coat and improved seed fiber were increased under field conditions. Other experiments completed during the 2018 growing season included a fungicide trial on pennycress to improve seed yield.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Thomas, JB, Hampton, ME, Dorn, KM, Marks, MD, and Carter, CJ (2017) The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization. BMC plant biology 17, 201. https://doi.org/10.1186/s12870-017-1146-8
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Anderson, JV, Horvath, DP, Do?ramaci, M, Dorn, KM, Chao, WS, Watkin, EE, Hernandez, AG, Marks, MD, and Gesch, R (2018) Expression of FLOWERING LOCUS C and a frameshift mutation of this gene on chromosome 20 differentiate a summer and winter annual biotype of Camelina sativa. Plant Direct 2 (7), e00060 https://doi.org/10.1002/pld3.60
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: McGinn, M, Phippen, WB, Chopra, R, Bansal, S, Jarvis, BA, Phippen, ME, Dorn, KM, Esfahanian, M, Nazarenus, TJ, Cahoon, EB, Durrett, TP, Marks, MD, and Sedbrook, JC (2018) Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop. Plant Biotechnology Journal https://doi.org/10.1111/pbi.13014
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Chopra, R., E.B. Johnson, E. Daniels, M. McGinn, K.M. Dorn, Maliheh Esfahanian, Nicole Folstad, Kirk Amundson, Kayla Altendorf, Kevin Betts, Katherine Frels, James A. Anderson, Donald L. Wyse, John C. Sedbrook, M. David Marks (2018) Translational genomics using Arabidopsis as a model enables the characterization of pennycress genes through forward and reverse genetics. The Plant Journal https://doi.org/10.1111/tpj.14172 (selected for the December cover and research highlights: https://onlinelibrary.wiley.com/doi/10.1111/tpj.14172)
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dorn, KM, Johnson, EB, Daniels, E, Wyse, DL, and Marks, DM (2018) Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times. Plant Direct, https://doi.org/10.1002/pld3.97
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Chopra, R, Folstad, N, Lyons, J, Ulmasov, T, Gallaher, C, Sullivan, L, McGovern, A, Mitacek, R, Frels, K, Altendorf, K, Killiam, A, Ismail, B, Anderson, JV, Wyse, DL, and Marks, MD (2019) The adaptable use of Brassica NIRS calibration equations to identify pennycress variants to facilitate the rapid domestication of a new winter oilseed crop. Industrial Crops and Products 128: 5561. https://doi.org/10.1016/j.indcrop.2018.10.079


Progress 09/01/16 to 08/31/17

Outputs
Target Audience:Students of plant biology at all levels. Scientists engaged in plant research in both academic and private industrial settings. Farming interest groups. State and Federal legislators. Investors. Ecological groups. Biofuels researchers at all levels. Agriculture undergraduates. Local and regional producers. Changes/Problems:Switched from TILLING to forward screens of mutagenized populations and to the use of CRISPR-Cas to create desirable lines. Problems- some loss of time due to fungal outbreaks in growth chambers. What opportunities for training and professional development has the project provided?Marks: At the start of the New Year, we hired two new technicians. One of these technicians left to enter graduate school a Washington University (St. Louis, MO) where he is pursuing a Ph.D. in plant sciences. In June, a postdoc focused on the genomics work joined our group. In August, a graduate student studying the early flowering and reduced lodging mutants joined our group. In addition, 15 undergraduates have participated in this project including two who worked full time over the summer. Phippen: In the fall of 2016, 4 undergraduate students worked part time in the greenhouse and laboratory supporting the efforts of growing all the pennycress lines. Three students completed an independent research project focused on pennycress seed storage and an additional honors student is evaluating herbicide tolerance in pennycress and conducting controlled crosses for the introgression of ALS resistance into the current production line. How have the results been disseminated to communities of interest?Marks - Oral Presentations entitled "Domestication of Pennycress" given to: January 19, 2017 Minnesota Department of Health meeting focus on nutrient contamination of rural well heads. February 22, 2017 Botany and Plant Pathology, Purdue University May 5, 2017 PepsiCo Senior Vice President and others May 17, 2017 Minnesota Crop Improvement Association August 15, 2017 Deep Dive presentation to potential investors Phippen - Oral Presentations February 8, 2017 Intellifarm Conference. The Field Pennycress Future: Production, Applications and Market Opportunities. Kansas City, MO. February 10, 2017 Illinois AgriNews. Pennycress research April 27, 2017 Farmweek Magazine. Advances in Pennycress Production May 12, 2017 8th Annual Pennycress Field Day. Macomb, IL Sedbrook - Oral Presentations February, 2017 USDA/DOE Plant Feedstocks Genomics for Biofuels PD meeting June, 2017 Arvegenix, Inc. Annual Board Meeting Poster Presentations: Phippen: September 2016 Annual Meeting of the Association for the Advancement of Industrial Crops Evaluation of the germination rates of Pennycress (Thlaspi arvense L.) in different conditions of storage and temperature. C. S. Nascimento, G. M. Regalado de Oliveira, T. R. Baran, and W. Phippen. Marks: June 2017 Annual American Society for Plant Biology Meeting Advancing Field Pennycress as a New Oilseed Biodiesel Feedstock-Focus on New Mutants Ratan Chopra, Nicole Folstad, Ryan Emenecker, Kevin Dorn1, M. David Marks Engineering a New Oil-Seed Cover Crop: Domesticating Pennycress Nicole Folstad, David Marks Sedbrook: January 2017, Gordon Research Conference on plant lipids. Utilizing CRISPR-Cas9 to improve seed oil quality in the winter annual oilseed crop pennycress (Thlaspi arvense). Michaela G. McGinn, Brice A. Jarvis, Edgar B. Cahoon, John C. Sedbrook Engineering medium chain fatty acids into seed triacylglycerols of the oilseed crop pennycress (Thlaspi arvense). Malihe Esfahanian, Timothy P. Durrett, Edgar B. Cahoon, and John C. Sedbrook February 2017, USDA/DOE Plant Feedstocks Genomics for Biofuels PD meeting Advancing Field Pennycress as a New Oilseed Biofuels Feedstock that does not Require New Land Commitments. John Sedbrook (jcsedbr@ilstu.edu), Winthrop Phippen, and David Marks. June 2017, Midwest Plant Cell Biology Meeting Generating High Erucic Acid Seed Oil Content in the Oilseed Plant Pennycress. Brice A. Jarvis, Michaela G. McGinn, Li-Hua Zhu, John C. Sedbrook. Chosen for oral presentation. Characterization of the pennycress (Thlaspi arvense) reduced pod shatter1 (rps1) mutant. Malihe Esfahanian, Michaela McGinn, M. David Marks, and John C. Sedbrook. Chosen for oral presentation. June 2017, International Conference on Arabidopsis Research (ICAR) Rapidly domesticating the new oilseed crop pennycress (Thlaspi arvense) by translating findings from Arabidopsis research. Michaela G. McGinn, Evan Johnson, Malihe Esfahanian, Erin Daniels, M. David Marks, John C. Sedbrook. Chosen for oral presentation. What do you plan to do during the next reporting period to accomplish the goals?Marks: Continue the analysis and stacking of mutant traits. Characterize zero erucic acid oil. Conduct field trials to test performance of new traits and multiple locations in Minnesota. Sedbrook: Complete the generation of 10,000 new M3-generation EMS mutant lines and begin screening for agronomically-relevant traits. Continue analysis of mutations conferring improved oil composition, reduced glucosinolate, reduced seed coat fiber, and reduced pod shatter as well as generation/identification of new mutant alleles; Introgress the best mutations into breeding lines; Deposit pennycress germplasm for curation/distribution by the Arabidopsis Biological Resource Center (ABRC). Phippen: Continue with seed increases, crosses, and mutant line evaluations in the greenhouse. Conduct field evaluations of 1500 mutant lines, 54 wild populations, and advanced breeding lines for improved seed germination and yellow seed coat color.

Impacts
What was accomplished under these goals? The goals for year 3 were to : Characterize mutants identified in the various screens and outcross those with traits of interest to wild lines with superior characteristics. IL and MN Publish characterizations of interesting mutants IL and MN University of Minnesota (UMN) We were able to identify key mutants in our mutagenized populations needed for domestication of pennycress. The traits exhibited by these mutants have been followed for multiple generations, which supports a genetic vs. an environmental basis for these traits. We have identified six reduced shatter mutants. Whole genome sequencing (WGS) has shown that two of these have mutations in candidate orthologs of Arabidopsis genes known to involved in seedpod development, and further, that mutations in these genes in Arabidopsis result in reduced shatter phenotypes. We have identified an early flowering mutant that matures a week ahead of wild type. Again, WGS has revealed that this mutant has a mutation in a candidate ortholog of a well-characterized early flowering gene in Arabidopsis. This assignment has been confirmed by co-segregation analyses and by the finding of a second mutant allele. We have identified two allelic mutants exhibiting an absence of erucic acid in the seed oil. This loss is compensated by increases in oleic, linoleic, and linolenic acids. Erucic acid is not allowed in oils that are used for human consumption. Its removal makes the mutant oil edible for humans and other monogastric species. We have identified a mutant that exhibits reduced levels of polyunsaturated fatty acids. This should result in more stable oil with a longer shelf life. For all of these traits we have identified the causative mutations. We have developed KASP markers for each of these. We are currently using these markers to follow the traits in F1s and should have the first version of a domesticated pennycress that matures early, exhibits reduced seedpod shatter, and that produces a stable edible oil. We anticipate having lines ready for field-testing in the fall of 2018. For the next generation of domestication we will be adding increased yield, increased oil content, increased seed size, reduced glucosinolates, reduced lodging, and reduced fiber. We are currently working with genetically stable mutants exhibiting all of these traits and analyses are underway to identify the causative genes in these mutants. At the same time we have been exhaustively backcrossing all of the mutants to an elite wild type line to remove extraneous mutations. Recombining the cleaned-up traits will create another generation of domesticated pennycress. Additional advances will come from traditional breeding. Illinois State University (ISU) In coordination with efforts at UMN and WIU, we have employed both forward and reverse genetic strategies (EMS and CRISPR-Cas9 mutagenesis along with phenotypic analyses and Next Generation Sequencing), to generate and identify a number of pennycress mutants and underlying mutations affecting key domestication traits including oil quality (low erucic 22:1 as well as reduced 18:2 and 18:3), reduced seed coat fiber (improves meal nutritional value), and reduced pod shatter (reduces pre-harvest seed loss). We filed provisional patent applications covering the pennycress oil quality, pod shatter, and seed coat fiber traits, and have signed related licensing agreements with the startup company Arvegenix, Inc. We are assisting Arvegenix to develop commercial pennycress varieties by 2020. Highlights of mutant generation/analysis led by ISU: 1. Oil quality: fae1 knockout mutants were generated by CRISPR-Cas9, and the CRISPR-Cas9 transgenes have been segregated away. These lines, which have low or undetectable seed oil erucic acid content and germinate/grow comparable to wild type, have been crossed with elite breeding lines and are being used for stacking with other traits including reduced sinigrin, reduced seed coat fiber, and reduced pod shatter. We also generated loss of function mutations that reduced seed oil polyunsaturated fatty acid content thereby improving seed oil oxidative stability. These plants are being analyzed for growth characteristics including abiotic stress responses. 2. Seed coat fiber: Generated/identified light seed coat mutants with mutations in 8 of the 19 known TRANSPARENT TESTA (TT) genes and may have identified a mutation in a TT-like gene this is uncharacterized in any plant species. Phenotypic analyses have shown at least some of these lines contain reduced seed coat fiber and higher oil content. While some lines do not grow as well as wild type, others grow comparably well and have been crossed into elite breeding lines for further analyses. 3. Pod shatter: Performed bulk segregant analysis and Next Generation Sequencing to confirm that the reduced pod shatter phenotype of our E42 EMS mutant line is due to a mutation in a key pod shatter-controlling transcription factor. We have also employed CRISPR-Cas9 to generate over 10 mutations in genes known to control pod shatter in Arabidopsis and other Brassicaceae. These mutants all grow like wild type. We are currently evaluating the pod shatter phenotypes in these lines and are performing genetic crosses for trait stacking and introduction into elite breeding lines. 4. Sinigrin (glucosinolate): We have generated a number of CRISPR-Cas9 lines to target mutations in glucosinolate-related genes including those encoding transcription factors, biosynthetic enzymes, and transporters. Mutations have been confirmed in some of these genes and phenotypic analyses are underway. We also have employed NIR spectroscopy to screen our EMS mutant populations to identify low glucosinolate candidates. Western Illinois University (WIU) We continue to conduct field and greenhouse evaluations of mutant lines and advanced breeding lines in small and large-scale experiments. Seed increases of several hundred mutant lines created at ISU for seed shatter reduction and yellow seed coat were grown in the greenhouse during the winter of 2016. Promising traits were crossed into the current advanced breeding lines and evaluated under field conditions during the 2017 summer growing season. An additional 51 populations were evaluated in replicated plots for improved seed yield, early flowering time, seed size, and stand establishment. Three new varieties have been identified with significantly larger pod and seed sizes. Nine lines have improved shatter reduction. Other experiments completed during the 2017 growing season include: fungicide trails on pennycress to improve seed yield, planting density trials, and large seed increases on M1 mutant seed for further gene identification. Large 150' strip trials were conducted on 12 advanced breeding lines to evaluate fall establishment, time to flowering, and seed yield.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization (2017) Jason B. Thomas, Marshall E. Hampton, Kevin M. Dorn, M. David Marks and Clay J. CarterEmail author BMC Plant 17:201 https://doi.org/10.1186/s12870-017-1146-8
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: New Pathways to Sustainability in Agroecological Systems (2016) Anne Cristina de la Vega-Leinert, Ludger Brenner, Susanne Stoll-Kleemann, Katlyn S Morris, Gabriela Bucini, Adrienne C Shelton, William F Tracy, Christian J Peters, Jamie Picardy, Amelia F Darrouzet-Nardi, Jennifer L Wilkins, Timothy S Griffin, Gary W Fick, Maywa Montenegro de Wit, Alastair Iles, Yolanda H Chen, Selena Ahmed, John Richard Stepp, NR Jordan, K Dorn, B Runck, P Ewing, A Williams, KA Anderson, L Felice, K Haralson, J Goplen, K Altendorf, A Fernandez, W Phippen, J Sedbrook, M Marks, K Wolf, D Wyse, G Johnson, Deborah K Letourneau, Sara G Bothwell Allen, Robert R Kula, Michael J Sharkey, John O Stireman III, Genevi�ve S Metson, Elena M Bennett, Liz Carlisle, Matt Liebman, Lisa A Schulte. Policy4, 000139


Progress 09/01/15 to 08/31/16

Outputs
Target Audience:Students of plant biology at all levels. Scientists engaged in plant research in both academic and private industrial settings. Farming interest groups. State and Federal legislators. Investors. Ecological groups. Biofuels researchers at all levels. Agriculture undergraduates. Local and regional producers. Changes/Problems: The key goals for this project were to identify new lines of pennycress with key domestication traits, to begin field testing these new lines, and to advance promising lines into our breeding programs. At the time the proposal was written it seemed that the best and most efficient mechanism for achieving these goals was through the use of such reverse genetic strategies as TILLING and DeTILLING. We predicted that we could use information from Arabidopsis research to identify key gene targets, where it is known that recessive mutations in many genes result in agronomically desirable traits. For example, mutations in the DOG1, DA1, and PHYB genes result in reduced dormancy, larger seeds, and early flowering. These are all important traits needed for the domestication of pennycress. During the early course of these studies we published the first draft for the pennycress genome. From comparisons between the Arabidopsis and pennycress genomes it became clear that pennycress and Arabidopsis share similar whole genome duplication histories and that for most genes it is possible to identify a single good candidate ortholog in pennycress for each gene in Arabidopsis. This led to the prediction that we should be able to directly use forward genetic screens to identify the desirable recessive mutants in our mutagenized populations. Further, we predicted that for many of the mutants that there should be good single good mutant candidate gene based on the comparisons between the pennycress and Arabidopsis mutant phenotypes. Indeed, we have identified pennycress mutants corresponding to the Arabidopsis mutants phytochrome B, leafy, agamous, flowering locus C, assymetric1, and pistillata. Furthermore, a wild isolate with a reduced dormancy phenotype has been shown to carry a natural mutation in DOG1. The one-one gene correspondence between pennycress and Arabidopsis also has facilitated the use of CRISPR-Cas9 technology to make mutations in candidate target genes. This technology has largely surpassed such techniques as TILLING and DeTILLING. What opportunities for training and professional development has the project provided?Under Marks, two lower level technicians moved onto higher paying positions with more responsibility. In addition, nine undergraduates received extensive research experience. Six of these students are continuing with the project this year. Under Phippen at WIU, 3 undergraduate students were employed Sedbrook: Training for 2 PhD students, 1 Master's student, and 17 undergraduate students. The grad students and 8 undergrads are still in the lab; 3 undergrads have gone on to graduate programs; 4 undergrads have gotten jobs with biology/medical/agriculture-related companies. The Sedbrook lab has initiated weekly lab meetings using Skype with researchers at the St. Louis-based pennycress startup company Arvegenix. Besides synergistically advancing the science, this is a wonderful opportunity for students to learn how research is performed at a company. In year 3, students will be traveling to St. Louis and working closely with Arvegenix scientists, some of whom have extensive experience working at Monsanto. We envision internship opportunities as well. How have the results been disseminated to communities of interest?UMN - Marks UMN pennycress presentations: Minnesota state legislators and environmental groups including Governor Dayton. April 19, 2016 National Association of Conservation Districts. July 18,2016 Environmentalist, producers, Minnesota Department of Agriculture employees. August 24, 2016. Minnesota corn and soy growers. November 24, 2015 Invited presentation. 4th Plant Genomics Congress. Pennycress, a fortunate choice for a new oilseed feedstock. September 12 and 13, 2016. Philadelphia, PA WIU- Phippen W. B. Phippen, J. Sedbrook, and D. M. Marks. 2015. Advances in Pennycress Breeding. Advancement of Industrial Crops Annual Meeting, Lubbock, Texas. October 18-22, 2015 Field Day Conducted 7th annual Pennycress Field Day. Attended by 50 people. Guest speakers included; Terry Isbell - USDA NCAUR; Dennis Plumber- Arvegenix, LLC, May 23, 2016. Sedbrook: Presentations Michaela McGinn, Malihe Esfahanian, and Brice Jarvis (Hands-on learning activities). "Building with Biology". Children's Discovery Museum, Normal, IL. (September 24th, 2016). Brice Jarvis (Presenter), Sedbrook, J. "Manipulating the lipid biosynthetic pathway in the new oilseed crop pennycress (Thlaspi arvense)," Integrative Plant Biology and Bioenergy (IPBB) Symposium, Normal, IL. (November 5, 2016). Malihe Esfahanian (Presenter), Sedbrook, J. "The identification of agronomically-relevant mutants of the new oilseed crop pennycress (Thlaspi arvense)," Integrative Plant Biology and Bioenergy (IPBB) Symposium, Normal, IL. (November 14, 2015). John Sedbrook (Presenter). "Prospects for developing pennycress, an Arabidopsis relative, as a new winter annual oilseed energy crop." University of California, Berkeley, Energy Biosciences Institute. (June 25, 2015). What do you plan to do during the next reporting period to accomplish the goals?Continue to screen for new mutants and characterize existing mutants with the following desirable traits. a. early flower/maturation. b. improved yield. c. better oil content. d. reduced seed coat fiber and lignin. e. reduced dormancy. f. reduced seed pod shatter. g. reduced lodging. h. larger seeds. i. reduced glucosinolates. j. larger flowers. k. higher harvest index. We have identified mutants with all of these traits. We are beginning to test individual lines in the field at multiple locations. The reduced shatter mutants will likely be the first to be used in commercial production as losses during harvest of wild type pennycress can reach over 70% of the theoretical yield. These stand-alone mutants should greatly boost yield. The mutagenesis-induced mutants will be subjected to backcrossing to reduce extraneous mutation loads. Representative mutants that pass field trial tests will be whole genome sequenced. The sequenced mutant genomes will be scanned for mutations in genes candidate genes gleaned from surveying the Arabidopsis literature. In Arabidopsis there are known mutations that result in similar phenotypes for each of the traits listed above. These analyses should result in the identification of the mutations that can be used to facilitate the stacking of multiple traits into single lines. For the CRISPR-Cas9 work, plants with confirmed mutations will be tested for the presence of the predicted phenotypes. In Illinois, seeds will be collected from the large M1 mutant population for subsequent screening.

Impacts
What was accomplished under these goals? Year 2 report Marks UMN Our efforts in forward screening for desirable mutants have been successful. As reported last year we identified a large number of lines with desirable traits. For example, we identified 59 individuals that flowered early, 27 semi-dwarves, 9 seed coat mutants, and 6 with reduced shatter. This past spring we observed that progeny from 18 of the early flower, 10 semi-dwarf, 6 seed coat, and 4 reduced shatter mutants inherited the desirable parental traits. Seeds from these lines are now entering field trials at 3 different locations in Minnesota. Given these successes, we screened additional M2s derived from approximately 7000 EMS treated M1 plants. This screen identified extra early flowering/maturing, reduced shatter, altered seed coat, and semi-dwarf mutants. Progeny from these plants have sowed in the field and will be phenotyped during spring 2017. During the fall of 2015 M3 seeds from 8000 individual M2 plants were scanned using a Perten 7250 diode NIR instrument. Data from Canola seeds was used as a preliminary proxy to analyze the spectral information from the pennycress scans. More recently, chemical wet analyses of pennycress seed have indicated that pennycress and Canola seeds behave identically when subjected to NIR with the Perten 7250 NIR. Importantly, we have found wide variation in our populations for such traits as percent oil content, erucic acid content, oleic acid content, as well as glucosinolate content. Seeds from lines exhibiting either high or low levels of these constituents have been replanted into the field during this past summer and will be reanalyzed next spring. Within these populations are lines exhibiting either lower erucic acid or glucosinolates. Given the success of this screen, M3 seeds from an additional 6000 M2 individual plants were collected this past summer. These are currently being screened via NIR. We anticipate that lines with either increased oil, reduced erucic acid or reduced glucosinolate will be ready for field testing next year. In addition to testing via NIR, the seeds from the 8000 M3 populations were tested for seed size. This screen was aided by the fact that the seeds were just the right size to be collectively sieved using cheap food strainers. A team of seven undergraduates tested for larger seeds that did not readily pass through the strainers. This analysis identified 15 lines with larger seeds (wild type seeds were ~1.3 mg/seed, whereas selected lines were over 1.7 mg/seed). The selected lines were replanted last March (we have found that winter pennycress is sufficiently vernalized to flower when planted in the early spring in Minnesota). Seeds from individuals from 5 of the lines showed the same large seed phenotype. These are now being retested in the field. In conclusion, we have identified lines showing either reduced dormancy, early flowering, reduced shatter, larger seeds, increased oil content, reduced erucic acid, reduced glucosinolate, reduced tannins (the seed coat mutants), potentially higher yield (the semi-dwarfs), or reduced lodging (the semi-dwarfs). These are currently being field tested at multiple locations. As described below, our key goal for next year will be to identify causative mutations in these mutant lines. These causative mutations will then be used as molecular markers to facilitate the stacking of these traits into elite production ready pennycress lines. Sedbrook, Phippen ISU and WIU A) We have generated and identified pennycress plants homozygous for heritable CRISPR-Cas9 induced loss-of-function frameshift mutations in the FATTY ACID ELONGASE1 (FAE1) gene and the REDUCED DORMANCY5 (RDO5) gene. fae1 loss of function specifically is responsible for the low erucic acid trait in canola seed oil, whereas rdo5 loss of function specifically reduces seed dormancy in Arabidopsis. These pennycress mutants are being phenotypically analyzed as well as crossed with breeding lines. More than 10 other pennycress genes are being targeted for knockout by CRISPR-Cas9 in an effort to improve pennycress traits including reduced glucosinolate, reduced seed coat fiber, improved oil quality (high oleic acid content), and reduced pod shatter. B) As noted in the Year 1 progress report, we have identified nearly 100 EMS mutant lines exhibiting agronomically beneficial phenotypes including reduced pod shatter, early flowering/senescing, and larger flowers/pods/seeds. While we are in the process of performing next-generation sequencing to identify causative mutations in candidate genes for a large number of these mutants, there are more mutants than we have hands to specifically analyze, so only the most promising mutants are being phenotypically and genotypically characterized in detail as well as crossed with breeding lines and field tested. C) We identified a natural single base deletion in the DELAY OF GERMINATION1 (DOG1) gene of variety Spring32. DOG1 mutations confer reduced seed dormancy in canola and Arabidopsis without causing adverse phenotypes. We are assessing how widespread loss of function DOG1 mutations are in pennycress varieties and are introgressing this natural variant mutation into breeding lines. D) Both our in-field and in-lab experiments have shown that the Elizabeth variety has superior seed germination, stand establishment, and yield compared to most other pennycress varieties we have tested in Central Illinois. Elizabeth was isolated by Terry Isbell at the USDA (Peoria, IL) as a natural variant within the Beecher strain. We have generated a segregating population of Elizabeth x Beecher crossed plants and will be scoring in Year 3 the reduced seed dormancy phenotype; bulk segregant analysis and next gen sequencing will be performed in order to identify the genetic basis for these agronomically superior traits. E) We have generated transgenic pennycress plants overexpressing the WRI1 and DGAT1 genes. WRI1 is a master regulator of fatty acid biosynthesis whereas DGAT1 is the rate limiting step in triacylglycerol biosynthesis. Work in rapeseed and Arabidopsis has shown that co-overexpressing these two genes synergistically increases seed oil content. WRI1 OX and DGAT1 OX pennycress plants are currently being phenotypically and genotypically analyzed in collaboration with Ana Alonzo (Ohio State U.) as well as genetically crossed to generate double OX lines. F) This fall (2016), we EMS mutagenized 40,000 Elizabeth variety seeds and planted the seeds in the field at WIU. While we have already generated/are screening large mutant populations in the MN106 and Spring32 backgrounds, given how well Elizabeth has performed in the field in central Illinois, we decided it may be quicker to directly introduce beneficial mutations into the Elizabeth background as opposed to introgressing in from the MN106 and Spring32 backgrounds. That being said, we are still pursuing the introgression route for our most promising mutations, in part because we are seeing higher yields/plant fitness improvements in our segregating breeding populations.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Michael B Kantar, Catrin E Tyl, Kevin M Dorn, Xiaofei Zhang, Jacob M Jungers, Joe M Kaser, Rachel R Schendel, James O Eckberg, Bryan C Runck, Mirko Bunzel, Nick R Jordan, Robert M Stupar, M David Marks, James A Anderson, Gregg A Johnson, Craig C Sheaffer, Tonya C Schoenfuss, Baraem Ismail, George E Heimpel, Donald L Wyse (2016) Perennial Grain and Oilseed Crops. Annu. Rev. Plant Biol. 67:703-729
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: NR Jordan, K Dorn, B Runck, P Ewing, A Williams, KA Anderson, L Felice, K Haralson, J Goplen, K Altendorf, A Fernandez, W Phippen, J Sedbrook, M Marks, K Wolf, D Wyse, G Johnson (2016) Sustainable commercialization of Field Pennycress: a strategy for sustainable intensification of agriculture in central North America. Elementa: Science of the Anthropocene 4:000081
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: DeHaan, LR; Van Tassel DL; Anderson, JA; Asselin, SR; Barnes, R; Baute, GJ; Cattani, DJ; Culman, S; Dorn, KM ; Hulke, BS; Kantar, M; Larson, S; Marks, MD; Miller, AJ; Poland, J; Ravetta, DA; Rude, E; Ryan, MR; Wyse, DL; Zhang, X. (2016) A Pipeline Strategy for Crop Domestication. Crop Science 56:917-930
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Invited presentation. 4th Plant Genomics Congress. Pennycress, a fortunate choice for a new oilseed feedstock. September 12 and 13, 2016. Philadelphia, PA


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:Students of plant biology at all levels. Scientists engaged in plant research in both academic and private industrial settings. Farming interest groups. State and Federal legislators. Investors. Ecological groups. Biofuels researchers at all levels. Agriculture undergraduates. Local and regional producers. Changes/Problems:Seeds from the fast neutron mutagenized population germinated at a much lower rate than expected. Thus, an insufficient number of plants matured to make the planned DeTILLING DNA pools. Given that the EMS mutagenized populations have shielded many more desirable mutants than expected, DeTILLING will not be pursued and more effort will be made toward working with the EMS populations. What opportunities for training and professional development has the project provided?Under Marks, one PhD student, Kevin Dorn, has completed training. Two technicians who were hired directly after completing their undergraduate degrees, Evan Johnson and Erin Daniels, have moved on to more advanced positions elsewhere. Three undergraduates in the Marks lab are currently working on related projects. Under Anderson one student, Kayla Altendorf, will be completing an M.S. degree early in 2016. Under Sedbrook, two PhD students and 10 undergraduate students are being trained on this project and an additional three undergrads have graduated with BS degrees. Under Phippen, three undergraduate students were trained with students completing projects that were presented at the WIU Undergraduate Research Day. How have the results been disseminated to communities of interest?Oral Presentations: 10/2014 Marks presented on the Domestication of Pennycress at the workshop entitled: New Roots for Ecological Intensification in Estes Park, CO. 1/2015 Sedbrook presented a videoconference seminar to researchers in Wisconsin, Michigan, Texas, and Vancouver, Canada as part of the DOE Great Lakes Bioenergy Research Center Plants Area seminar series. Seminar entitled: Pennycress, a new oilseed cover crop. 2/2015 Marks represented the pennycress research team at the Genomic Science Contractors-Grantees Meeting XIII USDA-DOE Plant Feedstock Genomics for Bioenergy Meeting in Washington, DC. 4/2015 Phippen, Sedbrook, and Marks provided individual presentations on their pennycress progress at the Inaugural Pennycress stakeholders meeting hosted by Commercial Aviation Alternative Fuels Initiative in Peoria, IL. 6/2015 M. McGinn (PhD student) gave an oral presentation on her work, entitled: Demonstrating Agrobacterium-mediated transformation of Pennycress (Thlaspi arvense) by expressing EaDAcT to alter seed oil composition. Midwest Plant Cell Dynamics Meeting in Madision, WI. Poster Presentations: 1/2015 K.M. Dorn, E.B. Johnson, D.L.Wyse, and M.D. Marks. Toward an anchored reference genome for field pennycress (Thlaspi arvense L.). Plant and Animal Genome XXIII meeting in San Diego, CA. 2/2015 M. McGinn, S. Bansal, C. Cass, T. Durrett and J. Sedbrook. Demonstrating Agrobacterium-mediated transformation of Pennycress (Thlaspi arvense) by expressing EaDAcT to alter seed oil composition. Gordon Research Conference on Plant Lipids: Structure, Metabolism & Function in Galveston, TX. 4/2015 T. Lima Marques and W. Phippen. 2015. Effects of Planting Density on Field Pennycress (Thlaspi arvense L.) Seed Production. WIU Undergraduate Research Day. April 16, 2015 4/2015 T. Carvalho Carli and W. Phippen. 2015. Effects of Nitrogen on Field Pennycress (Thlaspi arvense L.) Seed Production. WIU Undergraduate Research Day. April 16, 2015 4/2015 J. N. Rudy and W. Phippen. 2015. Determination of Vernalization Requirements of Field Pennycress (Thlaspi arvense L.) Seed Production. WIU Undergraduate Research Day. April 16, 2015 4/2015 J. N. Rudy and W. Phippen. 2015. Field pennycress (Thlaspi arvense L.) cross pollination in a greenhouse. WIU Undergraduate Research Day. April 16, 2015 6/2015 M. Esfahanian, M. McGinn, E. Johnson, E. Daniels, M.D. Marks and J. Sedbrook. Mutant and TILLING populations of Pennycress (Thlaspi arvense), a new model plant and oilseed winter annual cover crop. Midwest Plant Cell Dynamics Meeting in Madision, WI. 7/2015 K.M. Dorn, E.B. Johnson, E. Daniels, and M.D. Marks. Mutation breeding approaches to domesticate field pennycress (Thlaspi Arvense) in a new winter oilseed crop at Plant Biology 2015 meeting in Minneapolis, MN. What do you plan to do during the next reporting period to accomplish the goals? M3-generation mutants derived from M2s with phenotypes of interest described above will be assessed for the heritability of these phenotypes. Seeds from plants that show good heritability will be sowed at multiple locations in MN and IL along side parental plants to identify both lines that perform better at some locations than others and also to identify lines that perform better at all locations. F2 seeds from crosses described above will be planted into the field in the early spring as well as in growth chambers. These populations will be screened for single and double mutants. We will screen over 100,000 M2 plants in 700 two plots derived from the second round of EMS mutagenized seed for additional mutants with desirable phenotypes as described above. In addition, seeds will be collected from another 10,000 individual for NIR analysis. DNA will be isolated from EMS lines to begin making pools for TILLING. We will screen through progeny of plants transgenic for the various Crisper/Cas9 constructs noted above to identify loss-of-function mutations followed by phenotypic and genotypic characterization. We will continue to work toward the generation of overexpressing lines that produce higher seed oil content and/or specialty oils such as high erucic acid content.

Impacts
What was accomplished under these goals? Note: This is a collaborative award, with half of the funds going to the University of Minnesota to support the activities of PI Marks and Co-PIs Anderson and Wyse, and half of the funds going to support the activities of Co-PIs Sedbrook at Illinois State University and Phippen at Western Illinois University. Marks, Sedbrook and Phippen are the key PIs to which most of the funding has been allocated. Excellent progress was made in year 1. A) The screening of EMS-mutagenized spring- and winter-type mutant populations, carried out during 2015, has exceeded our most optimistic predictions. Seed has been collected from many different individual M2 plants exhibiting the following important agronomic traits, in both MN and IL. 59 early flower - Needed for successful double cropping 39 early maturing - Needed for successful double cropping 28 semi dwarf - green revolution type yield increase with reduced lodging? 8 enlarged stems - lodging resistance? 1 enlarged stems and bigger flowers - lodging resistance? 4 tillerless - improved plant spacing in the field, higher harvest index? 9 reduced pod shatter - reduced harvest losses? 3 more nectar * - better for honeybees? 2 bigger flowers and more nectar *- better for honeybees? 8 bigger pods - more seeds or larger seeds? 5 smaller pods - less shading to enhance overall photosynthesis rates? 3 larger seeds ~2x larger - better harvest - better field establishment? 12 yellow seeds - easier chemical processing, more oil, reduced dormancy? 10 reduced dormancy - faster field establishment and reduced seed bank production? 6 waxy bright green - altered wax composition or chlorophyll content may impart greater resistance to insects and diseases or reduced water loss or higher photosynthesis capacity. During the summer of 2015, M3 seeds from individual plants from the winter-type population were sowed into small field plots. Plants will be evaluated in the spring of 2016 for the inheritance of these traits. Evaluation of M3 seeds from individual plants from the spring-type population, planted and grown in growth chambers, is on-going. In many cases sufficient M3 seed was collected from individuals to allow field testing in both MN and IL. In addition, crosses are being conducted on select mutants as follows: 1) backcrosses to parental lines to reduce mutation loads, study heritability, and identify the affected genes; 2) double mutant crosses to study the effects of combining desirable mutations; and 3) crosses to introgress mutations into elite breeding lines. B) M3 seeds were collected from 10,000 M2 winter-type individual plants and 2,000 M2 spring-type individual plants. The winter-type seeds have been subjected to NIR scans with the goal of identifying lines with improved oil composition and reduced glucosinolates. During the winter of 2015/2016 chemical analyses will be preformed on over 100 of the scanned lines. These data will allow a predictive program to be generated that can be used to predict the oil and glucosinolate composition of the 10,000 scanned lines. Candidate lines will be sowed into the field in early April and then seeds from these lines will be retested in the fall of 2016. C) Tissue samples and seeds have been isolated from more than 3,000 M2 individuals from the winter- and spring-type EMS populations. Plans are being made to isolate and pool DNA from this tissue for conducting the reverse genetic TILLING screens. To avoid redundancy and extra work, we first will be analyzing individuals collected in the forward screens for the presence of mutations in genes contained in our TILLING target gene list. D) We anticipated that we might find interesting pod mutations that might convert the penny shaped pod to a silique shape that we predict to be ancestral in the pennycress lineage. However, only mutants affected in pod size and shatter resistance have been identified to date. Another round of mutant screening will be conducted in 2016 to search for additional pod mutants. E) 700 pools of M2 seeds were collected from another 7,000 EMS-treated M1 winter-type plants during the spring of 2015. These 700 pools have been sowed into the field and will be screened for additional mutants harboring agronomic traits of interest during the spring of 2016. F) Seeds from 5 high-yielding semi-dwarf plants have been sowed into larger plots in anticipation of conducting larger evaluation trials in 2016. Of note, one of these plants produced over 45 grams of seeds with a harvest index of 37% (seed weight to total dry matter). G) Crispr/Cas9 genome editing constructs have been generated and transformed into spring-type pennycress, targeting knockout of the following agronomically-relevant genes: 1) FAE1: Erucic acid production in seed oil (Screening T3-generation plants for mutations); 2) HAG1: Glucosinolate production (Screening T3-generation plants for mutations); 3) DOG1: Seed dormancy (Screening T2-generation plants for mutations); 4) GTR1/2: Glucosinolate production (Screening T1-generation plants for construct); 5) RDO5: Seed dormancy (Screening T1-generation plants for construct); 6) SHP1/2: Pod shatter (Screening T1-generation plants for construct; this target may be dropped due to success in finding pod shatter mutants in the forward mutant screens). H) In collaboration with Terry Isbell at the USDA in Peoria, IL, we are genotypically and phenotypically characterizing two pennycress natural variants that he isolated and named Elizabeth and Kaitlyn, which exhibit reduced seed dormancy. We are particularly excited about Elizabeth. Laboratory tests show that Elizabeth seeds germinate much more rapidly and those of the Beecher population of seeds from which Elizabeth was isolated. Moreover, field tests in Illinois show that Elizabeth was one of the best, if not the best, variety at germinating and establishing this fall, 2015. Elizabeth will be a platform line into which mutations we are identifying conferring improved agronomic traits will be introgressed. I) We have generated and biochemically characterized T2- and T3-generation pennycress plants transformed with the EaDAcT gene, finding that those plants produce significant amounts of low viscosity Acetyl-TAG seed oil. This oil has application as a drop-in fuel to be used in number 4 diesel engines. This work is being written up in a manuscript that will also describe an Agrobacterium-mediated floral dip method we developed as well as the use of the Spring 32 pennycress inbred line as a model akin to the Columbia ecotype of Arabidopsis. J) We are collaborating with a group in Sweden (Li-Hua Zhu group), which is working to develop Lepidium campestre as an oilseed crop to be grown at high latitudes. The joint goal is to generate Pennycress and Lepidium varieties that produce oil having high erucic acid content. Erucic acid is a value-added chemical feedstock. The Zhu group is excited about pennycress serving as a model for Lepidium, not to mention a crop in its own right.K) Completed in field trials in Illinois of wild collected pennycress populations and seed increases of advanced agronomically sound parental lines for subsequent breeding and evaluations.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: JC Sedbrook, WB Phippen, MD Marks (2014) New approaches to facilitate rapid domestication of a wild plant to an oilseed crop: Example pennycress (Thlaspi arvense L.) Plant Science 227, 122-132.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: KM Dorn, JD Fankhauser, DL Wyse, MD Marks (2015) A draft genome of field pennycress (Thlaspi arvense) provides tools for the domestication of a new winter biofuel crop DNA Research doi: 10.1093/dnares/dsu045.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Michael Kantar, Catrin Tyl, Kevin Dorn, Xiaofei Zhang, Jacob Jungers, Joe Kaser, Rachel Schendel, James Eckberg, Bryan Runck, Mirko Bunzel, Nick Jordan, Robert Stupar, M. David Marks, James Anderson, Gregg Johnson, Craig Sheaffer, Tonya Schoenfuss, Baraem Ismail, George Heimpel, and Donald Wyse (2015) Perennial Grain and Oilseed Crops. Annu. Rev. Plant Biol. doi: 10.1146/annurev-arplant-043015-112311
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Jordan, NR; Dorn, KM ; Runck, B; Ewing, P; Williams, A; Anderson, KA; Felice, L; Haralson, K; Goplen, J; Altendorf, K; Fernandez, A; Phippen, W; Sedbrook, J; Marks, MD; Wolf, KE; Wyse, DL; Johnson, G. (2015) Sustainable commercialization of Field Pennycress: a strategy for sustainable intensification of agriculture in central North America. Elementa accepted for publication
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: DeHaan, LR; Van Tassel DL; Anderson, JA; Asselin, SR; Barnes, R; Baute, GJ; Cattani, DJ; Culman, S; Dorn, KM ; Hulke, BS; Kantar, M; Larson, S; Marks, MD; Miller, AJ; Poland, J; Ravetta, DA; Rude, E; Ryan, MR; Wyse, DL; Zhang, X. (2015) A Pipeline Strategy for Crop Domestication. Under Review at Crop Science