United States Department of Agriculture Final Report. Alfalfa and Forage Program. Departments Monteros Lab {NO DATA ENTERED}

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1 Title: Root Traits to Enhance Nutrient and Water Use in Alfalfa Sponsoring Agency Funding Source Accession No. Project Start Date Reporting Period Start Date Submitted By NIFA Non Formula /01/ /01/2014 Deborah Samac Project Status COMPLETE Reporting Frequency Final Grants.gov No. Award No Proposal No Project End Date 08/31/2016 Reporting Period End Date 08/31/2016 Date Submitted to NIFA 11/14/2016 Program Code: AFRP Program Name: Alfalfa and Forage Program Project Director Deborah Samac Research Plant Pathologist Recipient Organization AGRICULTURAL RESEARCH SERVICE 1815 N UNIVERSITY ST Peoria, IL DUNS No Co-Project Directors Monteros, Maria Topp, Christopher Performing Department Departments Monteros Lab Non-Technical Summary The root system is the fundamental means for plants to explore the soil environment and acquire the water and mineral nutrients needed for growth. New crop cultivars that can more efficiently capture resources from the soil are needed for future low-input, high-yield agricultural production systems. In alfalfa, root system architecture (RSA) is believed to affect stand persistence and crop yield. However, selection for persistence and yield has focused on aboveground plant traits, largely ignoring the potential contribution of the root system for crop improvement. The goal of this project is to gain an understanding of root system development in alfalfa for developing germplasm with improved nutrient acquisition and water use to increase persistence and productivity of the crop. Rapid methods for characterizing root traits in alfalfa will be developed. Plants with specific root traits will be identified and used for associating DNA markers with the root traits. These methods and markers can be used for accelerating breeding improved cultivars of alfalfa that require fewer inputs and have improved reliency to variable environmental conditions. Accomplishments Major goals of the project The goal of this project is to develop phenotyping methods to accelerate breeding of alfalfa for increased yield, nutrient uptake capacity, and adaptation to abiotic stress. The objectives are: (1) utilize two- and three-dimensional imaging systems to characterize root system architecture traits (RSA) in alfalfa; (2) identify genetic loci associated with root system architecture traits in alfalfa; and (3) develop and implement education and outreach programs to transfer information to alfalfa producers, the alfalfa seed industry, crop consultants and state forage educators. What was accomplished under these goals? The root system is the fundamental means for plants to explore the soil environment and acquire the water and mineral nutrients needed for growth. New crop cultivars that can more efficiently capture resources from the soil are needed for future low-input, high-yield agricultural production systems. In alfalfa, root system architecture (RSA) is believed to affect stand persistence and crop yield. However, selection for persistence and yield has focused on aboveground plant traits, largely Page 1 of 5

2 ignoring the potential contribution of the root system for crop improvement. The goal of this project was to gain an understanding of root system development in alfalfa for developing germplasm with improved nutrient acquisition and water use to increase persistence and productivity of the crop. Rapid methods for characterizing root traits in alfalfa were developed. Previous research found that alfalfa plants with a highly branched root system supported greater forage yields than plants with a typical root system; however, selection required a minimum of 22 weeks to identify plants with the branching root phenotype. In this project we developed a method to identify plants with a strong taproot or strong branch roots after only 2 weeks of growth with the number and length of tertiary roots the key measurement for distinguishing root types. Plants could be identified consistently even with mild drought stress, nutrient stress and with nodulation by symbiotic bacteria. Several candidate genes were identified that were associated with the branching root phenotype. We developed improved germplasm with a greater proportion of plants with taproots or branch roots and developed populations that are being used to identify the genes involved in root traits. The outcomes of this project will facilitate 'root breeding' approaches aimed at modifying root system architecture to increase the absorptive capacity of roots for water and nutrients to increase alfalfa productivity, persistence, and resilience to environmental stresses. Objective 1: Utilize two- and three-dimensional imaging systems to characterize root system architecture traits (RSA) in alfalfa. Two-dimensional (2D) systems for evaluating root architecture were developed that included optimizing the plant growth medium, growth conditions, and time course of development. Methods were optimized for visualizing roots and measuring root length, number of secondary roots and number of tertiary roots in a more streamlined manner by integrating software. This resulted in the ability to identify plants with a fibrous root system after only 2 to 3 weeks of growth; a tremendous improvement over the 22 weeks previously required to determine root architecture traits in alfalfa. Using a multivariate ANOVA method, we could identify the effects of watering regime on the expression of fibrous or taprooted architectures (i.e. root plasticity). The 2D system was used to screen over 200 alfalfa individuals from three populations (base population of germplasm and two divergently-selected populations based on root traits). Based on the new rapid phenotyping method, we identified parental plants with either a branch or taproot phenotype and developed new populations by intermating (cycle 4). Seeds of these populations were compared by the rapid phenotyping method and traditional field-based method and found to have similar results. A manuscript describing this work is in preparation and a germplasm release of the cycle 4 materials is planned. Three-dimensional (3D) systems were developed to visualize root systems over time without destruction of the roots. In general, the information from the 3D systems tracked with those from the 2D, although future investigations into root growth dynamics would be enabled by non-destructive 3D systems. Objective 2: Identify genetic loci associated with root system architecture traits in alfalfa. Leaf tissue was collected from plants with divergent root growth phenotypes and DNA was extracted from those lines. The DNA samples were used for genotyping single nucleotide polymorphism (SNP) via high resolution melting (HRM) of root candidate genes and at the whole genome level using Genotyping by Sequencing (GBS). The scientific literature was mined to identify transcription factors and target genes associated with root growth and development in plants. Primers targeting 22 root-related genes were developed and used to genotype three alfalfa individuals with tapped and three individuals with branched roots. SNPs between tapped vs. branched alfalfa were identified in transcription factors MYB77 and MYB93 in alfalfa. MYB77 affects lateral root density (Shin et al., 2007; Plant Cell. 19: ) and MYB93 is a negative regulator of lateral root development (Gibbs et al., New Phytol. 203: ) in Arabidopsis. Based on the phenotyping results, we identified three genotypes with branched and three genotypes with tapped roots and these were used as parents to generate bi-parental mapping populations. Over 200 individuals from a cross between (tap rooted) and (fibrous root) were germinated and grown using treepots in the greenhouse. Root phenotypes were documented with images and analyzed using various software programs developed for root analysis. Both univariate and multivariate statistics were used to classify branch or taproot phenotypes and genetic variation for these classes were observed in the progenies generated from the population. Leaf tissue was collected and DNA extraction is ongoing to prepare samples for genotyping with the polymorphic SNP identified between the parental genotypes targeting the root candidate genes and genome-wide SNP discovery efforts. The combined phenotypic and genotypic datasets can be used to identify genetic determinants and quantitative trait loci (QTL) for root system architecture in alfalfa. Objective 3: Develop and implement education and outreach programs to transfer information to alfalfa producers, the alfalfa seed industry, crop consultants and state forage educators. During the grant period we reached alfalfa seed industry representatives, extension educators, producers and students (middle school, high school and undergraduates). A presentation on the project was given in March 2015 to the Industry- Extension Forage Advisory Council in LaCrosse, WI. Posters describing the research were presented at the National Association for Plant Breeders meeting in Pullman, WA; Molecular Breeding of Forage and Turf 2016 in Lanzhou, China; and Page 2 of 5

3 at the North American Alfalfa Improvment Conference, July The outreach and education activities to students from middle school, undergraduate and graduate on root, agriculture and molecular based approaches will generate the next generation of scientists and policymakers with knowledge on these topics. The outcomes from the alfalfa root phenotyping and genomics work were presented during a workshop at the Noble Foundation (Nov. 5-7, 2015) dedicated to roots entitled 'Laying the Roots for Year Round Grazing in the Southern Great Plains'. Experts in the fields of root phenotyping, roots and abiotic stress, root-microbe interactions and roots and soils were invited to present and the group discussed strategies for root system architecture improvement in forages. Additionally, Co-PI Monteros visited operations of cooperators with various production systems in Enid, Oklahoma on May 7, 2015 and on May 12, 2016, went to Middle Creek ranch in Dustin, OK and the Chocktaw Nation ranch in Krebs, OK. These two ranches differ in their management practices and strategies to enhance water and nutrient utilization through modified roots was discussed. What opportunities for training and professional development has the project provided? The project has provided training for a visiting scientist in use of phenotyping methodologies, data analysis, and alfalfa genetics/breeding and plant-microbe interactions. Technical staff have received professional development and training in use of new software and equipment for phenotyping and strategies for alfalfa breeding and genetics. Students from the Southern Oklahoma Technology Center and Oklahoma State University have received training on various strategies for root growth evaluation and discussions on experimental design. Outreach and education efforts included hands-on activities for 50 middle school students from Dickson Middle School in Oklahoma focused on DNA extraction and learning about the role of roots for plant growth and development. Science in Ag Day was held on May 5-6, 2015 and May 4-5, 2016 at the Noble Foundation campus in Ardmore, OK and had the participation from almost 300 8th grade students per year from surrounding area schools in Oklahoma. The focus was to teach students about how science and biotechnology is being used to advance agriculture. These activities were pursued jointly with the Noble Academy (more information available at Ongoing activities include engaging students with a 'root viewing station' and developing a lesson plan focused on the development and role of plant roots. A facility tour, oral presentation and demonstration of root phenotyping approaches, sensors and molecular techniques for genotyping forage species was given to 64 undergrad and graduate students from the Universidad Autonoma de Chapingo, Mexico (June 22-23, 2015). How have the results been disseminated to communities of interest? A presentation on the project was given in March 2015 to the Industry-Extension Forage Advisory Council in LaCrosse, WI. Posters describing the research were presented at the National Association for Plant Breeders meeting in Pullman, WA, Molecular Breeding of Forage and Turf 2016 in Lanzhou, China; and the North American Alfalfa Improvmeent Conference, July The outreach and education activities to students from middle school, undergraduate and graduate on root, agriculture and molecular based approaches will generate the next generation of scientists and policymakers with knowledge on these topics. The outcomes from the alfalfa root phenotyping and genomics work were presented during a workshop at the Noble Foundation (Nov. 5 to 7, 2015) dedicated to roots entitled 'Laying the Roots for Year Round Grazing in the Southern Great Plains'. Experts in the fields of root phenotyping, roots and abiotic stress, root-microbe interactions and roots and soils were invited to present and the group discussed strategies for root system architecture improvement in forages. Additionally, Co-PI Monteros visited operations from Noble Foundation cooperators with various production systems in Enid, Oklahoma on May 7, 2015 and on May 12, 2016, went to Middle Creek ranch in Dustin, OK and the Chocktaw Nation ranch in Krebs, OK. These two ranches differ in their management practices and strategies to enhance water and nutrient utilization through modified roots was discussed. What do you plan to do during the next reporting period to accomplish the goals? {Nothing to report} Participants Actual FTE s for this Reporting Period Role Non-Students or faculty Students with Staffing Roles Undergraduate Graduate Post-Doctorate Computed Total by Role Scientist Professional Technical Page 3 of 5

4 Actual FTE s for this Reporting Period Role Non-Students or faculty Students with Staffing Roles Undergraduate Graduate Post-Doctorate Computed Total by Role Administrative Other Computed Total Student Count by Classification of Instructional Programs (CIP) Code Target Audience During the reporting period we reached alfalfa seed industry representatives, extension educators, producers and students (middle school, high school and undergraduates). Products Conference Papers and Published 2016 Hernández, T., D. Samac, C. Topp, M.J. Monteros Characterization of alfalfa populations contrasting for root system architecture. Molecular Breeding of Forage and Turf. August Lanzhou, China. Hernández, T., D. Samac, C. Topp, M.J. Monteros Evaluation of alfalfa populations contrasting for root system architecture. Proceedings of the North American Alfalfa Improvement Conference. July Nedley, A., C.M. Motes, T. Hernández, M.J. Monteros Phenotyping strategies to evaluate root growth of alfalfa cultivars ranging in fall dormancy. National Association of Plant Breeders. July Pullman, WA. Bucciarelli, B., Cao, Y., Samac, D.A Rapid phenotyping of alfalfa root system architecture. Proceedings of the North American Alfalfa Improvement Conference, July Other Products Page 4 of 5

5 Data and Research Material Data generated from this project provided foundational work for additional funding opportunities realized through the Noble Foundation s Forage 365 initiative and a new grant An Integrated Imaging and Modeling Toolbox for Accelerated Development of Root-focused Crops at Field Scales recently submitted for funding consideration to the Department of Energy (DOE), ARPA-E program. New Germplasm Cycle 4 of breeding for root traits completed. A germplasm release is in progress. New Germplasm Mapping population for root traits completed. These will be used for identifying genes controlling traits. Other A visiting scientist from China has been mentored under this project and has gained skills in plant phenotyping, microbiology and molecular biology. Changes/Problems {Nothing to report} Page 5 of 5