Research Project: Genetic Improvement and Cropping Systems of Alfalfa for Livestock Utilization, Environmental Protection and Soil Health

Location: Plant Science Research

2021 Annual Report

Objectives
1. Develop genomic tools to enhance genetic selection of alfalfa for beneficial traits. Subobjective 1.A: Develop a reference genome sequence for cultivated alfalfa to facilitate breeding and genome editing for agronomic traits. Subobjective 1.B: Utilize universal DNA markers for accelerating breeding in alfalfa for improved forage digestibility. Subobjective 1.C: Utilize universal DNA markers for accelerating breeding in alfalfa for root system architecture. 2. Establish innovative, science-based methods and standards for assessing and evaluating alfalfa quality for multiple end uses. Subobjective 2.A: Develop new tools for enhancing nutritive value of alfalfa. Subobjective 2.B: Investigate cell wall lignification and digestion in alfalfa with reduced lignin concentrations. 3. Develop germplasm and crop management strategies to enhance productivity and environmental resiliency of forages. Subobjective 3.A: Evaluate expression of antimicrobial proteins for enhancing disease resistance in alfalfa. Subobjective 3.B: Map genes for resistance to Aphanomyces root rot. Subobjective 3.C: Evaluate seed treatments for enhancing germination and establishment of alfalfa plants. Subobjective 3.D: Evaluate modified alfalfa plants for phosphate uptake to remediate soil and reclaim phosphate. Subobjective 3.E: Evaluate alfalfa plants with an edited disease susceptibility gene for altered responses to pathogens. Subobjective 3F: Develop genomic and management strategies to reduce winterkill and increase persistence of alfalfa. 4. Develop knowledge and tools to increase understanding of the interactions among forage crops, soil nutrients, soil and plant health, and animal productivity. Sub-objective 4.A: Compare conventional and novel crop rotations utilizing forages for their effect on greenhouse gas emissions, C sequestration, nutrient cycling, and beneficial and pathogenic microbial populations. Sub-objective 4B: Measure effects of dairy manure application on productivity of alfalfa and rotational crops, forage quality, and soil health. Subobjective 4.C: Develop and utilize novel methods to identify and characterize pathogens of alfalfa.

Approach
Alfalfa is the most widely grown perennial forage crop in the U.S. and plays key roles in livestock nutrition, protecting water and soil resources, enhancing soil fertility, and sequestering soil carbon. However, there has been slow progress in alfalfa improvement and the contributions of alfalfa to agroecosystem sustainability are undervalued. To meet these needs, we aim to develop tools to accelerate breeding; test strategies to improve stand establishment and persistence; and measure carbon dioxide emissions, carbon sequestration, and nutrient cycling benefits of alfalfa in crop rotations and in the broader dairy production system. We will sequence and assemble the genome of alfalfa and provide web-accessible platforms to retrieve data. New breeding strategies including genome editing will be used for improving forage quality and stress tolerance. New seed treatments will be tested for improving stand establishment and soil indexing methods will be developed to determine risk of soilborne diseases. In-depth analyses of stem cell wall development and ruminal degradation will be done to gain a better understanding of developmental and structural changes that improve forage quality. Conventional and novel crop rotations utilizing forages will be evaluated for their effect on greenhouse gas emissions, carbon sequestration, nutrient cycling, and beneficial and pathogenic microbial populations. The effect of dairy manure application on productivity of alfalfa and rotational crops, forage quality, and soil health will be measured. The research will be of use to public and private plant breeders who will utilize the alfalfa genome sequence, markers, and breeding strategies in developing new cultivars; alfalfa farmers who will use soil indexing methods and seed fungicides to reduce damage from plant diseases; and the dairy food industry which has increased emphasis on reducing the environmental impacts of dairy, especially greenhouse gas emissions.

Progress Report
This project made significant progress on all objectives. In support of Objective 1, long read Hi-Fi DNA sequences were obtained from the reference alfalfa genotype RegenSY27x and assembled into a draft genome sequence of four distinct haplotypes. Genetic mapping of RegenSY27x was initiated to aid in assembly of the sequence. For mapping, DNA from 300 plants from a biparental cross was purified and submitted for genotyping by sequencing. To aid in gene annotation, RNA sequencing was completed from roots, nodules, stems, leaves, and seed pods of RegenSY27x. Hi-Fi DNA sequence was obtained and assembled from five additional alfalfa plants varying in fall dormancy to aid in identifying the core alfalfa genome. In support of identifying DNA markers associated with stem digestibility, over 1,300 plants were established in the greenhouse and leaf material harvested for genotyping. Replicated field plots were established for plant phenotyping. In support of developing alfalfa plants with distinct root system architecture, an analysis of data on rapid phenotyping of alfalfa root systems was completed and a manuscript submitted. Studies are in progress to evaluate nodulation, nitrogen fixation, and biomass accumulation of plants with increased root branching. In support of Objective 2, transgenic plants expressing genes for increased lipid production were evaluated by fluorescence microscopy and the plants with highest expression identified. New transgenic plants have been developed to repeat the experiment. In order to assay the plants for in vitro methane production, the prototype of an autosampler has been built by modifying a previously published instrumentation design. For evaluating stem composition and digestibility, plants from diverse genetic backgrounds were established in a spaced plant nursery and methods established for evaluation of traits. In support of Objective 3, transgenic alfalfa plants expressing an antimicrobial peptide were tested for resistance to Fusarium wilt and bacterial wilt using a standard protocol. Evaluated at three months after inoculation, transgenic plants were similar to control plants. New transgenic plants have been developed to repeat the experiment with less stringent conditions that would be found under field conditions. To aid in further mapping of genes for resistance to Aphanomyces root rot, over 300 plants from a backcross population were phenotyped for resistance to race 1 and race 2 strains. Leaf material was collected from all plants and submitted for genotyping using DaRT markers. For testing new fungicidal seed treatments, five on-farm trials were completed testing six treatments. Seedling establishment and forage yields were measured. A second year of trials were established at three locations. To identify mutations in phosphate uptake regulatory genes, genome edited transgenic plants were screened using next generation sequencing technologies. This analysis identified a suite of single, double, triple and quadruple mutant plants. A phosphate analytical assay was developed to quantify Pi concentration in the mutant and wild-type plants and a 5-10x fold increase Pi concentration was confirmed in the mutant plants. Introgression of the hyper-accumulation trait is being made into a range of alfalfa genotypes. Transgenic alfalfa plants with mutations in DMR6, a putative disease susceptibility gene, were evaluated for resistance to Phytophthora root rot using a standard protocol. At the end of the trial, mutant plants had similar symptoms as control wildtype plants. Wildtype plants showed a 50% increase in DMR6 expression with infection. Roots from mutated plants were collected for gene expression analysis. In support of Objective 4, two suitable fields were identified and planted to alfalfa and silage corn in 2020 and the first year of soil sampling, eddy covariance, and plant harvest data were collected. Data collection continued in 2021 with spring soil sampling to 90-cm depth in the two research fields and ongoing eddy covariance data collection. Winter camelina was harvested and short-season soybean will be planted. In support of measuring the effects of dairy manure application on alfalfa productivity and soil health, the second year of spring soil sampling was conducted at three research locations across Minnesota. Soils have been dried, ground, and analyzed for soil fertility, soil organic carbon, and mineralizable carbon, and have been sent to a commercial lab for genomic sequencing. In support of process-based modelling to examine tradeoffs in carbon, water, and nutrient flows associated with dairy forage production, multiple process models have been assessed for their suitability to meet project objectives and one model has been chosen as the best candidate model. Baseline alfalfa management and crop rotation scenarios have been determined for the Midwest U.S. region, and alternative scenario development is ongoing. Simulation of baseline scenarios is complete and long-term (100 year) impacts on soil carbon have been quantified. To investigate the pathogens limiting alfalfa establishment, DNA was isolated from bulk soil, rhizosphere soil, and alfalfa roots from five sites with poor establishment. Taxonomic marker genes for bacteria, fungi, and oomycetes were amplified from DNA extracts and submitted for sequencing. Oomycete amplicons were denoised and community profiles were constructed for alfalfa root and rhizosphere samples. Over 350 distinct oomycete amplicon sequence variants, including known and potential pathogens belonging to the genera Aphanomyces, Phytophthora, and Pythium were identified from alfalfa rhizosphere and endospheres.

Accomplishments
1. Genome editing of alfalfa to develop plants for reclamation of phosphorus from soil. Phosphorus (P) is one of the most essential plant macronutrients supporting global food systems and ensuring high crop yields. While global sources of P are declining, soils with a history of manure applications often have very high P concentrations, which can lead to eutrophication of aquatic ecosystems. To aid in reclamation of P from soils, ARS scientists in Saint Paul, Minnesota, developed alfalfa plants through genome editing of a P regulatory gene that accumulate five- to tenfold more P than unedited plants. Mutations were inherited in self-pollinated and cross-pollinated plants. This is the first demonstration of genome editing of all copies of a gene in tetraploid alfalfa, which has four copies of each gene. Alfalfa lines with these genome edits can be deployed to remediate high P soils and subsequently recover P for fertilizer use or can be used in buffer strips to protect waterways from P runoff.

Review Publications
Castle, S.C., Samac, D.A., Gutknecht, J.L., Sadowsky, M.J., Rosen, C.J., Schlatter, D.C., Kinkel, L.L. 2021. Impacts of cover crops and nitrogen fertilization on agricultural soil fungal and bacterial communities. Plant and Soil. https://doi.org/10.1007/s11104-021-04976-z.
Zivanov, D., Zivanov, S., Samac, D.A. 2021. First report of Mycoleptodiscus terrestris causing crown and root rot of alfalfa (Medicago sativa) in Minnesota. Plant Disease. 105(1). https://doi.org/10.1094/PDIS-12-19-2742-PDN.
Gamble, J.D., Feyereisen, G.W., Griffis, T.J., Wente, C.D., Baker, J.M. 2021. Long-term ecosystem carbon losses from silage maize-based forage cropping systems. Agricultural and Forest Meteorology. 306. Article 108438. https://doi.org/10.1016/j.agrformet.2021.108438.
Gordon, B., Lenhart, C., Peterson, H., Nieber, J., Gamble, J.D., Current, D. 2021. Reduction of nutrient loads from agricultural subsurface drainage water in a small, edge-of-field constructed treatment wetland. Ecological Engineering. 160. Article 106128. https://doi.org/10.1016/j.ecoleng.2020.106128.
Xu, Z., Kurek, A., Cannon, S.B., Beavis, W.D. 2021. Predictions from algorithmic modeling result in better decisions than from data modeling for soybean iron deficiency chlorosis. PLoS ONE. 16(7). Article e0240948. https://doi.org/10.1371/journal.pone.0240948.
Gamble, J.D., Johnson, G., Current, D., Wyse, D., Zamora, D., Sheaffer, C. 2020. Alley cropping affects perennial bioenergy crop root distribution, carbon, and nutrient stocks. Agronomy Journal. 112(5):3718-3732. https://doi.org/10.1002/agj2.20350.
Venterea, R.T., Petersen, S., de Klein, C., Pederson, A.R., Noble, A., Rees, R., Gamble, J.D., Parkin, T.B. 2020. Global research alliance N2O chamber methodology guidelines: Flux calculations. Journal of Environmental Quality. 49(5):1141-1155. https://doi.org/10.1002/jeq2.20118.
Samac, D.A., Temple, S.J. 2021. Biotechnology advances in alfalfa. In: Yu, X. and Kole, C., editors. The Alfalfa Genome, Compendium of Plant Genomics. Cham, Switzerland: Springer. p. 65-86.
Samac, D.A., Yu, L., Missaoui, A.M. 2021. Identification and characterization of disease resistance genes in alfalfa and Medicago truncatula for breeding improved cultivars. In: Yu,X. and Kole, C., editors. The Alfalfa Genome, Compendium of Plant Genomics. Springer, Cham:Switzerland. p. 211-233. https://doi.org/10.1007/978-3-030-74466-3_13.