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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

2015 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 cellulosic biomass and to amplify alfalfa's environmental services by contributing new knowledge on soil fertility-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 yields, disease resistance, and nutrient cycling efficiency. In experiments to evaluate new breeding strategies, the final year of data have been collected from field trials in Minnesota and Wisconsin to evaluate forage yield potential between semi-hybrid and synthetic populations as well as populations selected for yield per stem. Identification of parental germplasm resulting in highest yield and persistence is underway. Analysis of data is complete and manuscript preparation is underway. The second phase for developing DNA markers for accelerating breeding for resistance to Aphanomyces root rot was accomplished. Plants selected for race-specific resistance were verified and used in crosses to develop mapping populations. Methods for utilizing single nucleotide polymorphism markers and high-resolution melting point analysis were developed for assessing marker-trait relationships. In addition, candidate genes for disease resistance were introduced into alfalfa and methods developed for assessing disease resistance in the transgenic plants. To support this research, a large collection of new field isolates of the pathogen was developed and characterized for race specificity. Field surveys for the pathogen were completed, which identified race 2 as the most prevalent race in Minnesota and also uncovered a high frequency of soils with pathogens causing damping off. A large collection of pathogens was made and characterized from these soils. Three species of Pythium predominated and were found to have resistance to common fungicide seed treatments, partially explaining the high amount of damping off and seed rot observed in these soils. A manuscript has been prepared to report these results. Under Objective 2 progress was made in developing forage germplasm with modified cell wall structure and chemistry to improve livestock and bioenergy productivity. Final field samples from experiments evaluating selection for in vitro neutral detergent fiber digestibly and its impact on livestock nutrition and biofuel conversion efficiency were assayed for forage quality traits and data sets are ready for statistical analyses. The second production year samples were harvested from field trials to investigate whether a near infrared spectroscopy selection methodology used to create alfalfa populations that differ in stem in vitro neutral detergent fiber digestibly can be used to develop alfalfa with increased whole forage in vitro neutral detergent fiber digestibly. Manuscripts are in preparation for both experiments. Under Objective 3 progress was made in developing alfalfa germplasm and crop management systems to improve nitrogen cycling and carbon sequestration. Samples from an intensive four-year project to determine the performance of alfalfa selected for high or low nitrate uptake capacity have been analyzed, data sets were analyzed, and a manuscript is in preparation. The second phase in characterizing the microbial communities associated with alfalfa roots was carried out. Microbial populations associated with alfalfa roots with divergent root morphologies (taprooted or branchrooted) under different soil fertilities were compared using metagenomic analyses. Soil microbial populations from six crop rotations sampled from long-term agricultural research sites is also being analyzed using metagenomic tools. Functional analyses of the microbial populations are underway using multiple enzymatic assays. Finally, high through-put phenotyping methods were developed to identify root morphology at early stages of plant growth and plants were selected for developing mapping populations to identify DNA markers associated with root types.

1. First comprehensive database of alfalfa genes developed. Alfalfa is the fourth most widely grown crop in the U.S., but gains in alfalfa forage yield have lagged behind those of annual grain and seed crops due to the lack of tools for crop improvement. ARS scientists in St. Paul, Minnesota, sequenced the genes expressed in roots, nitrogen-fixing nodules, stems, leaves, and flowers of the two subspecies that comprise cultivated alfalfa to develop the first comprehensive guide to the genes in this important crop. In collaboration with scientists at the Noble Foundation, a website was developed for accessing tissue-specific genes, identifying gene expression differences among tissues, retrieving gene sequences, and developing DNA markers for specific genes. Genes involved in cold tolerance, nutritional quality, antioxidant production, and nitrogen fixation were identified. This database increases the availability of alfalfa gene sequences by ninefold and will be an essential tool used by plant breeders for alfalfa improvement.

2. Fungicide resistant pathogens of alfalfa overcome multiple seed treatments. Establishing a high-yielding and persistent crop of alfalfa is often prevented due to destruction of seeds and young plants by diseases and decay of adult roots in wet soil conditions. ARS scientists in St. Paul, Minnesota, isolated pathogens from soils sampled from commercial alfalfa production fields with high levels of seed rot and seedling damping off disease. Three species of Pythium from the soils were found to cause disease as well as seven Pythium species causing disease on corn or soybean. Most of the pathogens were not controlled by pyraclostrobin and many had resistance to mefanoxam, fungicides used on alfalfa seeds to prevent seedling diseases. These results indicate that neither fungicides nor crop rotation can control seed rot and seedling damping off of alfalfa. The pathogens isolated can be used for selection of disease resistant alfalfa germplasm, which would reduce both seedling and adult root rot to increase stand life and productivity.

3. Emerging pathogens a threat to alfalfa production. Developing disease resistant alfalfa is the most sustainable means of controlling diseases and obtaining highly productive alfalfa stands but failure of cultivars with resistance to race 1 of Aphanomyces root rot has been observed frequently. ARS scientists in St. Paul, Minnesota, conducted surveys of 45 fields in Minnesota and 40 fields in New York to determine the distribution of root rot and seed rot pathogens. In both states there was a high risk for Aphanomyces root rot and race 2 of the pathogen was the most common race in those soils. Additionally, new pathotypes were obtained that could overcome race 1 and race 2 resistance. A fungicide seed treatment was found to provide protection of young plants from all races but did not provide protection to adult plants. Fungicide treatments may be used as a stopgap measure until nonrace-specific disease resistance to Aphanomyces root rot can be developed.

4. Complete genome sequence of the alfalfa bacterial wilt pathogen Clavibacter michiganensis subsp. insidiosus. Bacterial wilt of alfalfa occurs throughout most alfalfa growing regions worldwide and for this reason the pathogen is considered a phytosanitary risk for international seed movement. The lack of a complete genome sequence for the pathogen causing this disease has hampered progress in understanding pathogen biology and developing tests to detect the pathogen. ARS scientists and University of Minnesota colleagues in St. Paul, Minnesota, obtained the complete genome sequence from three strains of the bacterium using two sequencing methods, resulting in very high quality sequence information. Availability of the complete genome sequence will be valuable for developing molecular diagnostic tools and understanding the mechanisms the pathogen uses for causing disease.

Review Publications
Lamb, J.F., Jung, H.G., Russelle, M.P., Sheaffer, C.V., Samac, D.A., Vance, C.P. 2014. Alfalfa non-feed uses. Legume Perspectives. 4:38.
O'Rourke, J.A., Fu, F., Bucciarelli, B., Yang, S.S., Samac, D.A., Lamb, J.F., Monteros, M., Graham, M.A., Gronwald, J.W., Krom, N., Li, J., Dai, X., Zhao, P.X., Vance, C.P. 2015. The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies. BioMed Central (BMC) Genetics. 16:502. DOI: 10.1186/s12864-015-1718-7.
Samac, D.A., Schraber, S., Barclay, S. 2015. A mineral seed coating for control of seedling diseases of alfalfa suitable for organic production systems. Plant Disease. 99(5):614-620.
Vail, A.W., Wang, P., Uefuji, H., Samac, D.A., Vance, C.P., Wackett, L.P., Sadowsky, M.J. 2015. Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene. Transgenic Research. 24:475-488.
Yost, M.A., Coulter, J.A., Russelle, M.P., Davenport, M.A. 2014. Opportunities exist to improve alfalfa and manure nitrogen crediting in corn following alfalfa. Agronomy Journal. 106(6):2098-2106.
Yost, M.A., Russelle, M.P., Coulter, J.A., Bolstad, P.V. 2014. Alfalfa stand length and subsequent crop patterns in the upper Midwestern United States. Agronomy Journal. 106(5):1697-1708.
Yost, M.A., Russelle, M.P., Coulter, J.A., Schmitt, M.A., Sheaffer, C.C., Randall, G.W. 2015. Stand age affects fertilizer nitrogen response in first-year corn following alfalfa. Agronomy Journal. 107:486-494. DOI: 10.2134/agronj14.0493.
Lu, Y., Samac, D.A., Glazebrook, J., Ishimaru, C.A. 2015. Complete genome sequence of Clavibacter michiganensis subsp. insidiosus R1-1 using PacBio single-molecule real-time technology. Genome Announcements. 3:e00396-15.
Zhang, T., Yu, L., Mccord, P.H., Miller, D., Bhamidimarri, S., Johnson, D., Monteros, M., Ho, J., Reisen, P., Samac, D.A. 2014. Identification of molecular markers associated with verticillium wilt resistance in alfalfa (Medicago sativa l.) using high-resolution melting. PLoS One. 1-18.