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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

2017 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
Under Objective 1 progress was made on developing strategies and tools for improving alfalfa yield, disease resistance, and nutrient acquisition. The fourth phase for developing DNA markers for accelerating breeding for resistance to Aphanomyces root rot was accomplished. Genotyping by sequencing results were obtained and identified loci for resistance to race 1 of Aphanomyces euteiches on chromosome 1 and resistance to race 2 on chromosome 2. Primers for markers to continue to evaluate these loci were developed and are being tested on parental and progeny lines. When confirmed, these markers will be useful in marker assisted breeding for disease resistance in this important soil-borne disease. A third cycle of selection was completed for enhancing resistance to brown root rot of alfalfa. Seed was produced from selected plants and is currently being tested for disease resistance. Surveys for seed-borne diseases of alfalfa identified Pythium and Fusarium seed rot and damping off as an emerging disease problem. Three populations of alfalfa were selected for resistance to Pythium and seed produced from selected plants. The epidemiology of bacterial stem blight was investigated in Fall 2016 and Spring 2017 in six locations within California and Utah. Significant damage from the disease was identified in all locations. Methods were developed for assessing population density of the pathogen. A collection of the pathogen was made and genetics and pathogenic diversity investigated. Antimicrobial peptides from tomato, spinach, alfalfa, and Medicago truncatula were evaluated for activity against crown rot pathogens of alfalfa. Peptides showed diverse activity, with M. truncatula peptides active against both bacterial and fungal pathogens. Transgenic alfalfa plants producing M. truncatula peptides were generated and will be tested for disease resistance. Bacterial mutants were generated to investigate the mechanism of peptide activity. Genome editing constructs were developed for mutating genes involved in phosphate accumulation, salt and cold tolerance, and disease resistance. Transgenic alfalfa plants have been produced with each construct and are currently being evaluated for alterations in these traits. 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. Rigorous statistical 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. A manuscript describing the material is in preparation and will result in a germplasm release. Under Objective 3 progress was made in developing alfalfa germplasm and crop management systems to improve nutrient cycling and carbon sequestration. Methods were developed for rapid phenotyping of root system architecture of alfalfa plants and a fourth cycle of selected plants was evaluated in field conditions. Greater progress was obtained by growth chamber selection for both branch roots and tap roots than for field selection and decreased the time for selection from 22 weeks to 2 weeks. All data have been analyzed and a manuscript is in preparation. A biomass type of alfalfa was used to produce a protein extract for use in aquaculture. A yellow perch feeding study was completed and found that the alfalfa protein could substitute effectively for fishmeal in the diet. Various extraction methods were evaluated for yield and extract composition. Current experiments are evaluating diverse alfalfa germplasm for their effect on extract composition.

1. DNA markers for disease resistance genes in alfalfa. Aphanomyces root rot is currently the most important disease of alfalfa nationwide. Breeding for resistance is complicated by the high amount of diversity in the pathogen population and the lack of molecular markers associated with resistance genes in alfalfa. ARS scientists in St. Paul, Minnesota and Prosser, Washington determined the mechanism for disease resistance involves rapid responses of infected root cells that limits the penetration of the pathogen followed by production of chemical barriers in the root that prevent further infection. The chromosomal location of disease resistance genes, DNA sequences associated with resistance, and candidate resistance genes were determined and the results support genetic evidence for a large cluster of resistance genes. The DNA markers can be used to accelerate selection of resistant plants and make selection of plants with broad resistance to the pathogen more accurate. Higher levels of disease resistance will increase seedling establishment and lead to higher yields and longer stand life.

2. Plant antimicrobial proteins for control of bacterial and fungal pathogens. Many plant diseases are caused by a complex of multiple pathogens that include several bacteria and fungi. Crown rot of alfalfa is caused by such a complex of pathogens and is found in all alfalfa stands more than 2 years old, resulting in yield losses and decreased stand life. ARS scientists in St. Paul, Minnesota and collaborators at the University of Minnesota and the Donald Danforth Plant Science Center, Saint Louis, Missouri tested synthetic core regions of antimicrobial peptides to identify those with activity against crown rot pathogens and found that the core regions predicted activity of full-length native peptides, which facilitates selection of effective peptides. The genes for these peptides can be used to engineer plants for enhanced resistance to multiple pathogens. Plant antimicrobial peptides represent an untapped resource for combatting bacteria with resistance to conventional antibiotics and for protection of plants against diseases.

3. Sustainable protein for aquaculture from alfalfa. The growing demand for fish and seafood products is accelerating development of aquaculture nationwide and alternative feed ingredients are needed to meet these demands. Some alternative proteins extracted from crop plants lack essential nutrients or have anti-nutritional components. ARS scientists in St. Paul, Minnesota and collaborators at the University of Minnesota tested a protein concentrate made from alfalfa foliage as a replacement for fishmeal in the diet for yellow perch and found that growth of fish was equivalent, indicating that alfalfa protein concentrate can substitute for this feed ingredient. Several methods were tested for producing the protein concentrate from leaves of a biomass type of alfalfa and a simple heat treatment after juicing was found to result in the highest yield of protein concentrate. Alfalfa stems, the press cake resulting from leaf juicing, and the de-proteinized juice have potential as additional value-added products in biorefining of alfalfa. High value products derived from alfalfa will increase the value of the crop and farm gate revenue.

Review Publications
Samac, D.A., Ophel-Keller, K., Caffier, D. 2017. Detection of Clavibacter michiganensis subsp. insidiosus in alfalfa. In: Fatmi, M., Walcott, R.R. Schaad, N.W., editors. Detection of Plant Pathogenic Bacteria in Seed and Other Planting Material. St. Paul, MN: APS Press. p. 103-107.
Hu, W., Samac, D.A., Liu, X., Chen, S. 2017. Microbial communities in the cysts of soybean cyst nematode affected by tillage and biocide in a suppressive soil. Applied Soil Ecology. 119(2017):396-406.