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Research Project: Expanding Resiliency and Utility of Alfalfa in Agroecosystems

2025 Annual Report

Objectives
Objective 1: Develop genomic tools for alfalfa to accelerate breeding and facilitate identification and validation of genes for important agronomic traits. Sub-objective 1.A: Compare alfalfa genomes to improve understanding of genome architecture and complexity. Sub-objective 1B: Identify DNA markers associated with biotic stresses to facilitate germplasm development. Subobjective 1C: Improve transformation and gene editing in elite genotypes to accelerate field testing of novel edited plants. Objective 2: Develop breeding methods and understanding of genetic control of important agronomic traits in alfalfa for plant improvement. Sub-objective 2.A: Improve environmental resiliency and abiotic stress tolerance in alfalfa using genomic selection and machine learning. Sub-objective 2.B: Develop alfalfa germplasm with enhanced forage quality and digestibility. Sub-objective 2.C: Develop germplasm with novel root traits that enhance herbage biomass and utilization of alfalfa in agroecosystems. Sub-objective 2.D: Develop novel germplasm with protein and nutritional profiles desired in human food products. Objective 3: Establish innovative methods and new standards for assessing and evaluating alfalfa quality for multiple uses. Sub-objective 3.A: Quantify and characterize variability of non-structural carbohydrates in alfalfa for improved nutritive value. Sub-objective 3.B: Increase alfalfa fiber digestion by the investigation of cell wall lignification and digestion in alfalfa with reduced lignin concentrations. Objective 4: Increase understanding of the interactions among forage crops, soils, and microbiomes to reduce risk, improve agronomic outcomes, and build resilience. Sub-objective 4.A: Evaluate the impacts of alfalfa cultivar, cutting frequency, and fall dormancy on root production, root turnover, root litter quality, C inputs to soil, and interactions with microbial communities. Sub-objective 4.B: Develop methods to isolate, identify, and characterize emerging plant pathogens. Sub-objective 4.C: Evaluate alfalfa establishment and termination strategies, diverse crop rotations, and winter annual cover crops for improving soil C balances and reducing greenhouse gas emissions. Sub-objective 4.D: Measure total root biomass, root:shoot ratios, root responses to management, and C fractions in forage systems to improve C accounting and model parametrizations of alfalfa and other forage crops.

Approach
Alfalfa is the engine that drives dairy and beef production and is unparalleled for providing environmental services. However, the slow progress in increasing forage yield and re-establishment costs after winter injury have discouraged greater utilization of alfalfa. Modern breeding methods and -omics technologies provide the opportunity to break the yield bottleneck, improve plant persistence and forage nutritive value, and develop novel products and environmental services. In support of these goals, we will assemble, annotate, and carry out an in-depth characterization of the alfalfa genome for structural and repeat number variants using bioinformatic tools. DaRTag SNP markers will be used to identify superior germplasm with improved winter survival, greater forage nutritive value, and root architecture to increase yield potential, and to identify markers associated with resistance to major yield-limiting diseases. Methods will be developed to improve alfalfa gene editing in diverse germplasm. Compositional analyses of alfalfa herbage using biochemical and chromatographic methods will lay the groundwork for utilization of alfalfa in human food products. 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. High throughput sequencing and metagenomic analyses will help fill knowledge gaps in the composition and function of key microbial communities associated with alfalfa in diverse soils and with biotic and abiotic stress. Conventional and novel crop rotations utilizing forages will be evaluated for their effect on greenhouse gas emissions and carbon sequestration using field-scale eddy covariance flux measurements and measurements of total root biomass, root:shoot ratios, root responses to management, and C fractions in forage systems to improve C accounting and model parametrizations of alfalfa and other forage crops.

Progress Report
Significant progress was made on all objectives in the project plan. In support of Objective 1, the assembled alfalfa reference genome for the genotype RegenSY27x was further refined by comparison with published sequences and DNA marker data from genetic maps. The final assembly contains four haplotypes for each chromosome, each approaching chromosome length, with a total length of 3.2 Gb. Annotation identified 226,191 genes in the assembly. The genome sequence has been submitted to GenBank and is available to the public through the Legume Information System. Progress was made in the assembly of genome sequences for eight additional genotypes varying in fall dormancy. DNA from 1,504 plants from four mapping populations was extracted, used for genotyping-by-sequencing, and the markers assembled into genetic maps for each genotype to aid in genome assembly. Each genotype was tested for resistance to five major alfalfa diseases and one mapping population selected for disease phenotyping and genotyping using a panel of 3,000 single nucleotide polymorphic markers from Diversity Array Technologies (DArTag) and Breeding Insight. Tests are complete for Aphanomyces root rot, Phytophthora root rot, and anthracnose using 376 plants in the mapping population. Tests for Fusarium wilt and bacterial wilt will be rated in September from field experiments. QTL mapping for resistance to each disease is in progress. The reference genome and mapped disease resistance traits will benefit alfalfa breeders to accelerate development of improved alfalfa germplasm and cultivars. In support of Objective 2, plant counts were recorded in Fall 2024 and Spring 2025 for the winter survival projects to determine winter survival rates. Additionally, an overall winter survival rate was calculated by comparing the number of surviving plants in 2025 to the total number of plants recorded in Fall 2023. These survival rates were then analyzed using various models to identify DNA markers associated with winter survival. Phenotypic data were collected from global alfalfa germplasm, including canopy architecture traits and resistance to potato leafhopper. In addition, biomass yield and winter survival plant counts were recorded to support ongoing DNA marker association studies. These efforts contribute to the broader goal of identifying traits and genetic resources that can enhance the resilience and productivity of North American alfalfa breeding programs. In 2025, we continued advancing our root system architecture (RSA) research by collecting high-resolution, time-series root growth images using an in-situ scanning system. Live root systems were scanned every two weeks at two field sites. These biweekly scans provide a detailed view of root development over time and will enable comparative analyses of RSA changes. This longitudinal dataset will be instrumental in identifying genotypic and environmental factors influencing root traits, ultimately supporting the development of improved root architectures in breeding programs. In support of Objective 3, stems of five lines that differ in in vitro neutral detergent fiber digestibility (IVNDFD) for the improvement of ruminant nutritionhave been harvested for 2 years. Sectioned stem degradation assays at two physiological time points are complete and most samples imaged. These lines were also analyzed for sequential detergent fiber digestion. The morphological traits and structure of these lines were analyzed , and the data is in the process of being written up for publication. The analysis of nonstructural carbohydrates in fine roots of alfalfa under different management regimes samples have been collected, processed and assays are 50% complete. In support of Objective 4A1, a second research site was established in 2023 in Waseca, Minnesota, and manure was applied prior to establishment of the alfalfa varieties. Yield, stand density, crown health, and root biomass data were collected in 2023 and 2024. The first site at Rosemount, Minnesota, has been rotated to corn and the alfalfa N-credits with varying manure management strategies are being assessed. For Objective 4A2 to identify root-associated microbial communities associated with alfalfa rotation effects, soil and plant samples continue to be collected from long-term rotation experiments, DNA was extracted, and diagnostic genes from microbial communities were sequenced from first and second-year samples following different rotation crops (alfalfa, soy, wheat). Analyses of bacterial, fungal, and arbuscular mycorrhizal marker genes indicate that preceding crops have a significant effect on subsequent plant-associated microbes. This research will help land managers design optimal rotation strategies that leverage microbial-mediated rotation effects. In efforts to determine a core alfalfa microbial community (Goal 4A3), numerous varieties of the alfalfa ‘core’ germplasm collection were selected to represent diverse origins and traits and grown in the greenhouse, root and rhizosphere DNA was extracted and sequencing is planned. Experiments were also performed to characterize the dynamics of microbial communities during plant development and are also awaiting sequencing. This work will benefit researchers and breeders by identifying key microbial taxa of alfalfa and how plant-microbe interactions vary across time and space. Host-mediated microbial engineering experiments (Goal 4A4) have been ongoing to select for microbial communities conferring suppression to Aphanomyces root rot, a major alfalfa disease. This approach involves selection for microbial communities that confer the most effective disease suppression and repeated passaging of effective communities to new generations of plant hosts to generate highly effective, host-adapted consortia. This experiment has required repeated modification to achieve sufficient disease pressure to differentiate between effective and ineffective microbial communities. Selection and characterization of disease-suppressive microbial communities will allow researchers to design effective synthetic consortia for biocontrol applications. In efforts to characterize metagenomes on dryland alfalfa (Goal 4A5), initial sampling of paired dryland and irrigated alfalfa fields has been conducted throughout the year and soil, rhizosphere, and root microbiomes have been sequenced. These analyses will be used to select samples for deep shotgun metagenomic sequencing. Understanding the role of rhizosphere microbial communities in drought response lays the groundwork for biotechnological or breeding applications to harness beneficial microbes. In support of Objective 4C, carbon dioxide flux, soil, and crop yield data collection continued in two production fields, one in a perennial continuous living cover system (intermediate wheatgrass, 2023 - 2025) and the other an annual continuous living cover system (winter barley-sorghum sudangrass-winter camelina in 2024). Data analysis and manuscript preparation are underway, and preliminary findings were presented at the ASA, SSSA, and CSA annual meeting in fall 2024. In support of Objective 4D, the fourth year of data collection of root production, root turnover, root litter quality, and C inputs continued for alfalfa of varying fall dormancy and cutting frequency. The field study is now complete, and data analysis is underway.

Accomplishments
1. First U.S. alfalfa reference genome assembly released. Reference genome sequences, composed of the digital DNA sequences of organisms assembled into chromosome sized lengths, are a requirement for modern plant and animal breeding methods. Alfalfa, which is a cornerstone for dairy and beef livestock feed, has lacked a reference genome sequence for U.S. cultivars due to the complex nature of its genome, which has four copies of each of the eight chromosomes. ARS scientists in St. Paul, Minnesota, with colleagues at the University of Minnesota and National Center for Genome Resources used long-read next-generation DNA sequencing, genetic mapping, and long-range chromosome mapping to assemble a high-quality reference genome of RegenSY27x, which is widely used in molecular biology research. The assembled genome consists of 3.2 billion base pairs of DNA with highly contiguous sequences approximately the length of each chromosome, separating the four copies of each chromosome. A total of 226,191 genes were identified with help of sequencing expressed genes from roots, nitrogen-fixing root nodules, stems, leaves, flowers, and seeds, of which 178,985 have an assigned function. The RegenSY27x genome assembly sets a new benchmark in gene content and annotation quality with a total gene count that exceeds all previously published alfalfa genome assemblies and achieves the highest assembly completeness to date, establishing it as the most comprehensive and high-quality alfalfa genome currently available. The RegenSY27x reference genome will provide a stable foundation for analysis of DNA variation in alfalfa cultivars, benefit alfalfa breeders and growers by accelerating development of improved alfalfa cultivars and will serve as a model for assembly of other complex genomes. The gene annotated sequence is available on the National Institutes of Health-GenBank and ARS Legume Information System.

2. Breeding program develops high yielding reduced lignin alfalfa for livestock feed. Alfalfa is highly valued as a source of protein, fiber, and essential nutrients in ruminant livestock production but as the plants mature the stems accumulate lignin and become less digestible, so the crop is harvested at an immature stage, sacrificing crop yield. A long-term breeding program performed by ARS scientists in St. Paul, Minnesota, identified unique alfalfa plants with more digestible stems and used them in several cycles of selection and breeding. The resulting population has reduced amounts of lignin and cellulose in stems, making them digestible at mature stages. Tests in multiple locations over multiple years and harvests showed that the selected population can be harvested 14 days later than conventional alfalfa and retain high digestibility, producing greater biomass, and reducing the number of harvests each year. DNA markers associated with high digestibility were identified that will benefit alfalfa breeders and farmers by simplifying selection of plants from any alfalfa population and accelerate development of improved alfalfa cultivars. This approach can be used by plant breeders in other forage crops in which stem maturity reduces fiber digestibility.

3. Invention of a novel adaptor for crop sample processing. In crop breeding and improvement, thousands of samples must be processed by each breeding program before testing can occur for various traits such as nutritional content. After harvesting and drying the materials, the next step is to grind the materials into small particles. This process is time consuming and can result in sample loss from spilling. ARS scientists in St. Paul, Minnesota, and a collaborating engineering student designed a simple 3D printed cone-shaped adaptor that attaches to the grinder used by most breeding programs. This adapter allows for samples to be collected directly into sample bags, replacing the jar provided by the manufacturer that had to be removed, the sample transferred, and the jar cleaned between samples. The use of the adapter accelerates grinding and sample transfer by 27% and reduces sample loss by 5%. The design and printing instructions were published on an open-source digital commons (DOI:10.17632/35z724djjv.2) and shared with collaborators who also found it to result in significant savings in time and resources.

4. Improved accounting of forage crop impacts on soil carbon loss and dairy farm emissions. When assessing the net emissions impact of management practices, soil organic carbon (SOC) storage is often aggregated with nitrous oxide and methane after converting all components to carbon dioxide equivalents (CO2e) and assuming a fixed duration of storage. However, such analyses do not consider potential re-emission of SOC or apply consistent assumptions about storage duration. Moreover, these efforts have been exclusively focused on how to quantify the emissions offsets of potential increases in SOC storage and have yet to consider the implications of agricultural systems with prevailing SOC loss. Soils in the Midwest U.S. managed for corn silage production are a substantial source of carbon dioxide (CO2) emissions to the atmosphere, with SOC losses ranging from 13.5 to 25.6 Mg CO2 per hectare each year. ARS scientists in St. Paul, Minnesota, demonstrated that including emissions associated with SOC losses under dairy forage production can increase the carbon footprint of milk nearly two-fold. This represents a more accurate estimate of the emissions impact of milk production and suggests that gains in the emissions efficiency of milk have come, in part, at the expense of SOC where forage rotations are predominated by silage corn. The U.S. dairy industry has pledged to achieve net zero emissions by 2050, but reliance on corn silage as a primary forage source undermines progress toward this goal. Findings from this work help forage growers and dairy managers improve the health and quality of their soils and improve the overall efficiency of their production systems.

U.S. DEPARTMENT OF AGRICULTURE

Plant Science Research: St. Paul, MN