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

Location: Plant Science Research

2023 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 1A, the RegenSY27x genome assembly was further improved using genetic mapping data to scaffold assembly pieces into larger fragments. Annotation of the genome is in progress. For annotation of nine additional genome that have been assembled, RNA was extracted from roots, nodules, stems, leaves, and flowers plus seed pods of each genotype and submitted for transcript profiling by Iso-Seq and RNA-seq. The sequenced genotypes were evaluated for resistance to Aphanomyces root rot, Phytophthora root rot, and anthracnose. For Objective 1B, marker-trait analyses for five agronomic traits, stem strength, stem color, stem structure, and winter injury, were completed using genome wide association, random forest, support vector machine, and extreme gradient boost. Heritability was estimated for the five traits, and SNP markers linked to traits were identified. Data collection for stem digestibility traits was completed for samples collected in 2022 and marker-trait association analysis is in progress. In support of Objective 1C, genotyping with 3,000 single nucleotide polymorphism (SNP) markers from Breeding Insight was carried out 1,504 plants from four cycles of selection for root architecture. Phenotyping of root architecture was completed after 28 weeks of growth in the field. From field grown plants, four populations were selected; branched and fibrous roots, branched without fibrous roots, tap and fibrous roots, and tap without fibrous roots; and plants within each population were intermated. The top 10 plants from each population will be used as male parents to transfer the root system architecture genes to elite lines. Previous research indicated that branched and fibrous rooted plant had greater nodulation, potentially increasing nitrogen fixation. A greenhouse experiment was completed to measure nitrogen fixation and biomass yield in plants with diverse root architecture. Samples are currently being prepared for 15N analysis and a follow-up field experiment is underway. In support of Objective 2A, transgenic plants previously selected for expression of fatty acyl-coenzyme A (CoA) diphosphatase (FIT2) and acyl-CoA:diacylglycerol acyltransferase (DGAT2) were imaged using confocal microscopy to quantify the location and number of lipid droplets. The same plants were used to characterize lipid profiles and quantify gene expression. Additionally, plants with both FIT2 and DGAT2 were imaged, and tissue collected for lipid characterization and gene expression. All data for this project have been analyzed and a manuscript is in preparation. In support of Objective 2B, 800 clones of five selected cultivars/lines were established and overwintered in two geographical locations. These plants have been harvested once at vegetative, bud, early flower, and green pod stages at both locations. The second harvest at each of these physiological stages is in progress. Physical stem characteristics were recorded, the seventh internode stored for future microscopic imaging and in vitro digestion analysis, and herbage was collected for forage quality analysis. Additionally, the effect of Genotype x Environment on forage quality of oats was evaluated in collaboration with the South Dakota State University oat forage breeding program. In collaborated with the US Dairy Forage Research Lab, a database of plant traits associated with animal production characteristics was developed. In support of Objective 3B, genotyping with 3,000 SNP markers was done on a second F1 population for mapping Aphanomyces root rot (ARR) resistance, confirming the locations of genes for disease resistance. For Objective 3D, genotyping was completed for F1 plants with edits in the PHOSPHATE2 gene. Three lines were selected, and 720 clones generated and transplanted in the field at two locations to measure phosphate (P) accumulation and winter survival. Additional lines were tested for P uptake and cold tolerance in a low temperature chamber. In support of Objective 4A, spring wheat was planted in 2022, followed by fall seeded winter camelina in September 2022. Due to limited fall precipitation, winter camelina establishment was poor. The camelina was terminated in spring 2023 and a full-season soybean was planted. In a separate field, alfalfa was terminated in August 2022 and intermediate wheatgrass ‘MN-Clearwater’ was seeded in September. Data collection continued in 2023 with spring soil sampling to 90-cm depth in the two research fields with ongoing eddy covariance, crop leaf area index, height, and yield data collection. In support of Objective 4B, measuring the effects of dairy manure application on alfalfa productivity and soil health, the fourth year of fall soil sampling was conducted at three research locations across Minnesota. Soils have been dried, ground, and analyzed for soil fertility, soil organic carbon (SOC), and mineralizable carbon (C). Data analysis and manuscript preparation are underway. In support of process-based modelling to examine tradeoffs in C, water, and nutrient flows associated with dairy forage production, baseline and alternative alfalfa management soil C modeling is complete, and data analysis and manuscript preparation are underway. Preliminary analysis of the EPIC model output shows a wide range of responses to crop rotation across six sites within Minnesota and Wisconsin. Relative to a baseline management scenario of continuous silage corn, including alfalfa in rotation with silage corn at 33% frequency (2 of 6 years) improved SOC storage by 0.14 Mg C/ha/year, though net C losses are still observed at this alfalfa frequency. Model simulations show that a 50% alfalfa frequency (3 of 6 years) is required to achieve field SOC equilibrium, and that SOC gains are not realized until 67% alfalfa frequency in rotation (4 of 6 years). The climate mitigation potential of changes in SOC is not commonly or consistently included in C footprints of agricultural products. A manuscript outlining a novel method of including short-term changes in biogenic C (such as SOC) into C footprints and Global Warming Potential frameworks is currently under review. This newly developed method is being used to integrate modelled soil C change into the life-cycle assessment of alfalfa in dairy supply chains being conducted by a collaborator at the University of Minnesota. Analysis of a microcosm experiment testing the roles of biotic and abiotic factors in determining how soil fungal communities respond to dairy manure amendments was completed. Similar to previous observations for bacteria, results suggest that indigenous soil fungi primarily respond to abiotic inputs associated with manure, and that individual taxa that responded to manure inputs varied among soils from different sites. However, manure tended to reduce fungal diversity consistently among soils. Few fungal taxa were associated with manure, and these did not tend to persist in soil. Additional metagenomic sequencing and re-analysis of microbial communities from soils that received a single high-rate application of manure were conducted. Preliminary results confirmed that manure had minimal impacts on the composition and diversity of soil communities, and that ‘priming’ effects were not apparent in the following growing season. Rather, within- and among-site spatial variation and crop rotational phase remained primary drivers of soil microbiomes at each time point. Soil, rhizosphere, and root samples were collected to evaluate the impacts of different manure application strategies on soil and alfalfa-associated microbiomes. Preliminary analyses suggest that surface broadcast and banding applications had greater impacts on soil communities than did shallow injection and were enriched with putative manure-borne bacteria. These differences were also reflected in microbial communities in alfalfa rhizospheres.

Accomplishments
1. Alfalfa breeding benefits from universal DNA markers. Use of DNA markers has revolutionized modern molecular plant breeding by accelerating the ability of breeders to select parents, identify chromosomal regions encoding traits of agronomic interest, and harness hybrid vigor for increasing crop yields and nutritional value. Development of markers for improving alfalfa has lagged behind other major crops because many marker types do not capture sufficient information to accurately identify gene locations due to the high level of genetic heterogeneity in alfalfa and due to the high cost of marker development. ARS scientists at St. Paul, Minnesota; Madison, Wisconsin; Logan, Utah; and Prosser, Washington, collaborated with Breeding Insight at Cornell University to develop a set of DNA markers that can be used in any alfalfa population, are equally distributed across the genome, and are inexpensive to use with large numbers of individual plants in a breeding program. The 3,000 markers were validated in alfalfa populations segregating for resistance to the disease Aphanomyces root rot and identified the same chromosomal regions for disease resistance genes as more expensive and time-consuming methods. Candidate genes for resistance to the two races of the pathogen were identified. The markers also successfully grouped plants by stem strength, stem color, stem structure, and winter injury. The set of markers are available to the alfalfa research community as a powerful new tool for crop improvement.

2. Characterization of bacterial stem blight disease in alfalfa. Bacterial stem blight of alfalfa recently emerged as a serious disease of alfalfa in the western United States causing up to 50% yield loss from the first forage harvest. Although the disease was recognized in 1904, little is known about the pathogen, the mechanism of infecting plants, or host plant resistance. To fill this knowledge gap, ARS scientists in St. Paul, Minnesota, and University of Minnesota collaborators isolated the pathogen from alfalfa samples obtained from California, Utah, Oregon, Minnesota, and Ohio, extending the known range of the disease. In addition to the known bacterial stem blight pathogen, Pseudomonas syringae pv. syringae, a second pathogen, P. viridiflava, was discovered to be associated with diseased samples at a high frequency and shown to cause bacterial stem blight symptoms. A comprehensive genomic study of 94 strains of P. syringae and 29 strains of P. viridiflava showed that both species are genetically diverse, evidence that the populations are widespread and have been established on alfalfa plants across the U. S. for a long period of time. The high frequency of ice nucleation activity in P. syringae isolates indicates that the pathogen moves in precipitation and can cause frost damage on alfalfa as a means of infecting plants. The complete genome sequence was obtained for 20 strains, which identified unique genes for toxins causing disease symptoms. Plants resistant to BSB were identified in the cultivars Maverick and ZG9830, some that inhibit pathogen growth and some with novel tolerance to high levels of bacteria without showing disease symptoms. Several candidate genes involved in resistance and tolerance were identified. Collectively, the studies found that the emergence of bacterial stem blight is not due to a recent pathogen introduction or evolution of more aggressive strains, and that breeding for resistance is a viable means of reducing damage from the disease.

3. Single, high-rate manure applications on soil microbial communities have no imact on soil communities. Organic amendments, such as livestock manure, are commonly used as fertilizers and can impact the composition and diversity of soil microbial communities. Single applications of high rates of manure are suggested to induce lasting shifts in soil microbiomes, prime nutrient cycling, and improve soil health. To test this assertion, ARS scientists in St. Paul, Minnesota, evaluated the impacts of single, high-rates of liquid dairy manure on soil microbial communities in different crop phases of an alfalfa-corn rotation in multiple locations over a two-year period. Contrary to expectations, manure had little impact on the composition and diversity of soil communities at crop harvest or in the following growing season. Rather, location, year, season, and cropping phase all had significant effects on soil microbiomes. This work indicates that single, high-rate applications of liquid dairy manure do not appear to prime persistent shifts in soil microbial communities, but that microbial dynamics track crop rotational phases.

4. RUBY reporter gene expression in alfalfa results in high levels of betalain production. Introducing new genes into alfalfa for genome editing or for novel traits currently requires genetic transformation, which can be low in many alfalfa varieties, creating a bottleneck for crop improvement. A new reporter called RUBY was developed for noninvasively monitoring plant transformation and gene expression. Expression of the RUBY construct results in conversion of the amino acid tyrosine to betalain, the red pigment found in beet roots. Betalains are water-soluble, nitrogen-rich pigments known for their bright colors that are used in food applications, such as color additives and nutraceuticals, and have antimicrobial and anti-inflammatory properties. ARS scientists at St. Paul, Minnesota, found that expression of RUBY in alfalfa was useful to identify DNA elements that increase the frequency of transformation of alfalfa cells by 340%, demonstrating that the marker is an effective new tool for alfalfa improvement. Additionally, alfalfa plants with RUBY are vividly red purple throughout the plant. Betalain accumulation in alfalfa was 20- to 300-fold greater than in beet root and significantly higher than in other plant species expressing RUBY. Use of alfalfa for production of betalain would be a highly efficient and sustainable means of producing this important natural product.

Review Publications
Heuschele, D.J., Gamble, J.D., Vetsch, J., Shaeffer, C., Coulter, J., Kaiser, K., Lamb, J., Lamb, J., Samac, D.A. 2023. Influence of potassium fertilization on alfalfa leaf and stem yield, forage quality, nutrient removal, and plant health. Agrosystems, Geosciences & Environment. 6(1). Article e20346. https://doi.org/10.1002/agg2.20346.
Rosier, A., Pomerleau, M., Beauregard, P.B., Samac, D.A., Bais, H.P. 2023. Surfactin and Spo0A-dependent antagonism by bacillus subtilis strain UD1022 against medicago sativa phytopathogens. Plants. 12(5). Article 1007. https://doi.org/10.3390/plants12051007.
Xu, Z., Heuschele, D.J., Lamb, J., Jung, H.G., Samac, D.A. 2023. Improved forage quality in alfalfa (Medicago sativa L.) via selection for increased stem fiber digestibility. Agronomy. 13(3). Article 770. https://doi.org/10.3390/agronomy13030770.
Larsen, L., Schlatter, D.C., Nimpoeno, J., Hines-Snider, C., Samac, D.A. 2023. Rhizosphere and root community analysis of oomycetes associated with poor alfalfa (Medicago sativa) seedling establishment. Phytobiomes Journal. 7(4):526-537. https://doi.org/10.1094/PBIOMES-02-23-0005-R.
Schlatter, D.C., Gamble, J.D., Castle, S., Rogers, J., Wilson, M. 2022. Abiotic and biotic filters determine the response of soil bacterial communities to manure amendment. Applied Soil Ecology. 180. Article 104618. https://doi.org/10.1016/j.apsoil.2022.104618.
Welikhe, P., Williams, M.R., King, K.W., Bos, J.H., Akland, M., Baffaut, C., Beck, G., Bierer, A.M., Bosch, D.D., Brooks, E., Buda, A.R., Cavigelli, M.A., Faulkner, J., Feyereisen, G.W., Fortuna, A., Gamble, J.D., Hanrahan, B.R., Hussain, M., Kovar, J.L., Lee, B., Leytem, A.B., Liebig, M.A., Line, D., Macrae, M., Moorman, T.B., Moriasi, D.N., Mumbi, R., Nelson, N., Ortega-Pieck, A., Osmond, D., Penn, C.J., Pisani, O., Reba, M.L., Smith, D.R., Unrine, J., Webb, P., White, K.E., Wilson, H., Witthaus, L.M. 2023. Uncertainty in phosphorus fluxes and budgets across the U.S. long-term agroecosystem research network. Journal of Environmental Quality. 52(4):837-885. https://doi.org/10.1002/jeq2.20485.
Yin, C., Schlatter, D.C., Hagerty, C., Hulbert, S.H., Paulitz, T.C. 2023. Disease-induced assemblage of the rhizosphere fungal community in successive plantings of wheat. Phytobiomes Journal. 7 (1):100-112. https://doi.org/10.1094/PBIOMES-12-22-0101-R.
Alexander, J.R., Baker, J.M., Gamble, J.D., Venterea, R.T., Spokas, K.A. 2023. Spatiotemporal distribution of roots in a maize-kura clover living mulch system: Impact of tillage and fertilizer N source. Soil & Tillage Research. 227(3). Article 105590. https://doi.org/10.1016/j.still.2022.105590.
Lipps, S., Samac, D.A., Ishii, S. 2022. Genome sequence resource for strains of Pseudomonas syringae phylogroup 2b and Pseudomonas viridiflava phylogroup 7a causing bacterial stem blight of alfalfa. Plant Disease. 112(9):2028-2031. https://doi.org/10.1094/PHYTO-12-21-0511-A.
Loveland, C., Orloff, S., Yost, M., Bohle, M., Galdi, G., Getts, T., Putnam, D., Ranson, C., Samac, D.A., Wilson, R., Creech, E. 2023. Glyphosate-resistant alfalfa can exhibit injury after glyphosate application in the Intermountain West. Agronomy Journal. 115(4):1827-1841. https://doi.org/10.1002/agj2.21352.
Schlatter, D.C., Gamble, J.D., Castle, S.C., Rogers, J., Wilson, M. 2023. Abiotic and biotic drivers of soil fungal communities in response to dairy manure amendment. Applied and Environmental Microbiology. 89(6). Article e01931-22. https://doi.org/10.1128/aem.01931-22.