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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Research Project #424185

Research Project: Enhanced Alfalfa Germplasm and Genomic Resources for Yield, Quality, and Environmental Protection

Location: Plant Science Research

2016 Annual Report

Objective 1: Develop strategies and tools for improving alfalfa yields, disease resistance, and nutrient cycling efficiency. Sub-objective 1.1: Identify and compare breeding strategies for alfalfa yield improvement. Sub-objective 1.2: Identify strategies and develop tools for reducing losses from diseases. Sub-objective 1.3: Develop strategies to produce alfalfa germplasm with improved herbage yield and nutrient cycling (phosphorus (P) and potassium (K)) in pure stands and in mixtures with forage grasses. Sub-objective 1.4: Develop a gene expression atlas for two divergent alfalfa gene pools (Medicago sativa subsp. sativa and M. sativa subsp. falcata), identify organ-specific genes, and mine sequences for gene pool diversity (SNPs and SSRs). Objective 2: Develop forage germplasm with modified cell-wall structure and chemistry to improve digestibility, and evaluate impacts on livestock and bioenergy productivity. Sub-objective 2.1: Assess alfalfa germplasm altered for in vitro neutral detergent fiber digestibility (IVNDFD) for forage yield and energy availability for livestock production and biofuel conversion efficiency. Sub-objective 2.2: Identify genetic, metabolic, and developmental processes in alfalfa stems that regulate cell wall composition and energy availability. Objective 3: Develop alfalfa germplasm and crop rotation management systems to improve nitrogen cycling and carbon sequestration. Sub-objective 3.1: Identify and utilize mechanisms to improve nutrient uptake in alfalfa. Sub-objective 3.2: Identify and characterize rhizosphere soil microbes that promote carbon sequestration and improve the agronomic and environmental benefits from crop rotation. Sub-objective 3.3: Measure and predict N credits for second-year corn grown after alfalfa to improve N management and reduce N losses.

To increase yield potential, the contribution of heterosis to yield potential and the effectiveness of selecting for high yield per stem as a yield component in alfalfa will be assessed. Synthetic populations, semi-hybrid populations, parent populations, and two commercial varieties will be established in replicated field trials and total annual forage yield will be evaluated for at least two production years. We will develop DNA markers to increase disease resistance and measure diversity in pathogen populations. A bulked segregant analysis will be done using populations segregating for resistance to Aphanomyces root rot. Histochemical and gene expression studies will be used to gain an understanding of the infection process and mechanisms of resistance in resistant and susceptible plants. Simple sequence repeat (SSR) markers will be identified in the Verticillium albo-atrum genome sequence and tested for polymorphisms on field isolates and standard strains. Plant responses to these strains will be measured with the standard disease severity scale and the amount of pathogen present determined by the qPCR method. Plants with the lowest amount of pathogen present will be retained, intermated, and progeny tested for Verticillium wilt resistance. DNA markers will also be developed from a gene expression atlas for two divergent alfalfa gene pools using transcripts from leaves, roots, nodules, flowers, and elongating and post-elongation stem internodes. To develop alfalfa germplasm with improved herbage yield and nutrient cycling, germplasm differing in root system architecture will be examined in replicated field experiments to determine: P and/or K uptake capacity under low and adequate soil nutrient levels; symbiosis with arbuscular mycorrhizal (AM) fungi; and prevalence of root and foliar diseases. Alfalfa germplasm selected for in vitro neutral detergent fiber digestibility (IVNDFD) and original parents will be evaluated in replicated field trials for forage quality traits, gain from selection, and heritability estimates. Replicated sward plot field trials will be used to determine forage yield and the best crop management methods for germplasm selected for IVNDFD. Energy availability for livestock and biofuel conversion in the harvested forage will be determined by near infrared reflectance spectroscopy. To improve alfalfa stem cellulose content, a comparison of miRNA profiles in elongating stem and post-elongation stem internodes will be used to identify miRNAs that play key roles in the development of secondary xylem. The microbial communities in the rhizosphere that influence plant growth and carbon sequestration will be characterized using culture-dependent and metagenomics approaches. Field tests will determine whether selection for nitrate uptake alters yields of alfalfa-grass mixtures. On-farm field experiments will be established at 10 locations to improve predictions of whether nitrogen contributed by alfalfa to subsequent corn crops will improve farm profit and reduce nitrogen losses.

Progress Report
This project aims to increase alfalfa yields and utilization for livestock and to promote the environmental services provided by alfalfa by contributing new knowledge on nitrogen contribution to soil fertility by alfalfa rotations and plant root-microbial interactions. Progress was made on all project objectives. Under Objective 1 progress was made on developing strategies and tools for improving alfalfa yield, disease resistance, and nutrient acquisition. The third phase for developing DNA markers for accelerating breeding for resistance to Aphanomyces root rot was accomplished. From the 120 populations developed, 30 mapping populations (F1 populations derived from crossing resistant and susceptible parents) were phenotyped for disease resistance and two populations were selected for marker development. From the two populations, 400 plants were phenotyped for resistance to two races of the pathogen and DNA extracted from each plant. The DNA was submitted for genotyping-by-sequencing. The mapping populations are currently being used to screen for novel races of the pathogen. In addition, transgenic alfalfa expressing candidate genes for Aphanomyces root rot resistance were evaluated for response to a broad host range strain. Expression of the candidate genes resulted in a moderate increase in resistance of adventitious roots. Seed from plants was generated and will be tested for disease resistance. Additionally, a third cycle of selection was completed for enhancing resistance to brown root rot of alfalfa. Surveys for foliar diseases of alfalfa identified bacterial stem blight as an emerging disease problem in several areas of the U.S. The pathogen genome was sequenced, the pathogen host range characterized, a standard test developed for identifying resistant germplasm and standard check cultivars developed. These tools will be useful in developing resistant cultivars to reduce spring and fall forage losses due to this disease. Under Objective 2 progress was made in developing forage germplasm with modified cell wall structure and chemistry to improve livestock and bioenergy productivity. Alfalfa lines bred over three cycles of selection for digestibility of stem cell walls were evaluated from field experiments in two locations over two years with material harvested at a range of maturities. Preliminary analysis shows that lines selected for increased stem digestibility have consistently greater digestibility at later maturities than current high quality alfalfa cultivars and total lignin concentration in stems is significantly reduced. Earlier studies found that small gains in digestibility have very large impacts on improving animal performance and reducing manure waste. This material would be a non-GMO alternative to the recently deregulated reduced lignin cultivars developed through genetic engineering. Germplasm releases of the improved lines will make this material available for commercial development. Under Objective 3 progress was made in developing alfalfa germplasm and crop management systems to improve nitrogen cycling and carbon sequestration. The second phase in characterizing the microbial communities associated with alfalfa roots was completed. Microbial communities were identified using metagenomic approaches from alfalfa roots with divergent root morphologies (taprooted or branchrooted) under different soil fertilities. Bioinformatic analyses found that location was the primary driver of bacterial and fungal communities but that specific core members were present in all locations. Specific taxa associated with the nitrogen fixing microsymbiont were identified for additional characterization. Analysis of sampling methodologies was completed to determine the optimal strategy for identifying soil microbial communities. Manuscripts describing these studies are in preparation. Using a culture-based approach, a collection of the microsymbiont and nodule-associated bacteria was made from two locations. Diversity analysis of the microsymbiont found that populations in the two locations are distinct and are not derived from the bacteria originally inoculated onto seeds. A majority of the nodule-associated bacteria were found to have plant growth promoting activities in vitro, including activity against alfalfa fungal pathogens. Further characterization of the microsymbiont and nodule associated bacteria is in progress. Methods were developed for rapid phenotyping of branchrooted and taprooted alfalfa plants and a fourth cycle of selection was completed. In addition, research was initiated to utilize a biomass type alfalfa for production of leaf protein concentrate as a sustainable protein source for aquaculture. Methods for protein extraction were optimized and used for large-scale extraction to produce protein for feeding studies.

1. Bacterial stem blight disease of alfalfa is an increasing threat to forage production. Economic damage of alfalfa fields by late spring and early fall frosts have increased in the past several years and in some locations increased sensitivity to frost was associated with herbicide application. ARS researchers in Saint Paul, Minnesota found damaged alfalfa was infected by the bacterial stem blight pathogen, which as the ability to increase temperatures at which frost damage occurs. The genome of the pathogen was sequenced and comparisons with other bacterial genomes found that it is closely related to bacteria infecting pears and beets. Methods were developed for identifying resistant alfalfa plants and cultivars with moderate numbers of resistant plants were identified. These methods, bacterial isolates, and plant materials will be valuable for developing cultivars with high levels of resistance to the disease for integrated disease management and sustainable forage production.

2. Rapid selection of root system architecture that promotes enhanced alfalfa forage yields. Selection for yield in alfalfa has focused on aboveground plant traits, largely ignoring the potential contribution of the root system to improve yield due to enhanced water and nutrient acquisition. 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 20 weeks to identify plants with the branching root phenotype. ARS researchers in Saint Paul, Minnesota 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. 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.

Review Publications
Harrison, J., Dornbusch, M.R., Samac, D.A., Studholme, D.J. 2016. Draft genome sequences of pseudomonas syringae pv. syringae ALF3 isolated from alfalfa. Genome Announcements. 4(1):e01722-15.
Castell-Miller, C.V., Gutierrez-Gonzlez, J.J., Tu, Z.J., Bushley, K., Hainaut, M., Henrissat, B., Samac, D.A. 2016. Genome assembly of the fungus Cochliobolus miyabeanus, and transcriptome analysis during early stages of infection on American wild rice (Zizania palustris L.). PLoS One. 11(6):e0154122. doi:10.1371/journal.pone.0154122.
Samac, D.A., Bucciarelli, B., Miller, S.S., Yang, S.H., O'Rourke, J.A., Shin, S., Vance, C.P. 2015. Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis. Biomed Central (BMC) Plant Biology. 15:283. doi:10.1186/s12870-015-0649-4.