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United States Department of Agriculture

Agricultural Research Service

Research Project: Enhancing Resistance to Diseases and Abiotic Stresses Alfalfa


2009 Annual Report

1a. Objectives (from AD-416)
Objective 1: Develop molecular markers to screen alfalfa for resistance to environmental stresses and identify different physiological races of pathogens responsible for soilborne diseases of alfalfa. Sub-objective 1.1: Identify markers linked to tolerance to lodging in commercial alfalfa populations. Sub-objective 1.2: Develop molecular markers that discriminate unambiguously between Race 1 and Race 2 of Aphanomyces euteiches. Objective 2: Identify and develop sources of disease resistance in alfalfa and common bean. Sub-objective 2.1: Identify bean germplasm with resistance to Clover Yellow Vein Virus.

1b. Approach (from AD-416)
Two alfalfa clones have been identified that differ in resistance to lodging. The lodging susceptible and lodging resistant clones and the F1 resulting from this cross was crossed with each parent to produce two different backcross populations. Replicated clones of each parent, F1 and backcross plants will be produced in the greenhouse. The replicated clones will be transplanted into three different field locations, two in WA and one in WI. DNA from the parents, F1 and BC1s will be screened for the presence of sequence related amplified polymorphisms (SRAPs) using protocols optimized for amplifying SRAP markers from alfalfa. Resistant and susceptible bulk DNA extracts will be subjected to bulk segregant analysis with SRAP primers to identify candidate polymorphic loci associated with resistance to lodging. Candidate markers will be clustered into a linkage group map. Molecular markers will be developed that discriminate unambiguously between Race 1 and Race 2 of Aphanomyces euteiches that cause root rot in alfalfa. Markers specific to each race will be converted into SCAR markers and will then be evaluated for their robustness using a test profile of DNA samples extracted from known race 1 and race 2 isolates of A. euteiches. All candidate markers will be screened across a collection of other isolates A. euteiches collected from other states in the U.S. The assay will be validated on total DNA extracts from alfalfa tissue infected with each race of the pathogen. If a SCAR marker is successfully developed early in the research plan, collaboration will be sought with industry or university breeding programs to identify sources of tolerance and eventually DNA markers linked to tolerance to this pathogen. Sources of resistance will be identified in common bean germplasm with resistance to Clover yellow vein virus (ClYVV). Included in the screening assays will be four sets of bean germplasm: one set representative of each of 12 host groups used in the identification of strains of Bean common mosaic virus and Bean common mosaic necrosis virus; a second set of differential cultivars for identification of Bean yellow mosaic virus strains obtained from the Plant Introduction Station, Pullman, WA.; the third group will consist of reported sources of resistance to ClYVV which include US1140, UI-31, and Jolanda; and lastly, cultivars and breeding lines will be screened to identify sources of resistance already deployed in a snap bean background because this market class is currently at the greatest risk from this disease. A recombinant inbred line (RIL) population segregating for the resistance gene(s) also will be evaluated against strains of ClYVV. Segregation ratios for numbers of resistant and susceptible plants observed within the RIL population(s) and chi-square tests will be used to determine if the genes between sources are allelic, independent, or linked. Formerly 5354-21000-014-00D (3/08).

3. Progress Report
Developing resistance to lodging in alfalfa. An advanced population of replicated alfalfa clones has been developed and evaluated over one year for improved lodging resistance response. Phenotypic data obtained has identified four preliminary groups based on non-lodging response in the field. An additional year of data is being acquired in Washington State and Wisconsin to verify the intitial results. Selected individuals within the four groups consisting of resistance and susceptiblity to lodging will be used to screen for molecular markers that can be used to identify non-lodging traits in alfalfa. Five cultures of Aphanomyces euteiches that cause root rot in alfalfa have been isolated from soil and infected alfalfa plants in Washington State. The isolates are being identified in greenhouse experiments as Aphanomyces Race 1 or Race 2. Additional geographical isolates from other states will be collected to generate a representative population of each race. DNA will be extracted and used for analysis of molecular markers that will differentiate the two races. Once markers have been identified and sequenced, primer/probe sets can then be developed in future work to evaluate germplasm for resistance to each race of the pathogen.

4. Accomplishments
1. Identify bean germplasm with resistance to Clover yellow vein virus. Clover yellow vein virus (ClYVV) continues to be a serious virus in the upper Midwest States where the soybean aphid is the common vector of the virus. Infected plants result in pods with severe necrosis resulting in fresh market and processed snap beans that are unmarketable. A recombinant inbred line (RIL) was developed by the ARS scientists at Prosser, WA from a cross between ClYVV-resistant bean cultivar 'Raven' that posesses the bc-3 gene for resistance to Bean common mosaic virus (BCMV), and susceptible I9365-31, and consisted of 109 lines in total. All lines that showed a resistance response to inoculation with ClYVV were also resistant to BCMV, results of which suggest that resistance to ClYVV may be linked to the bc-3 gene. Hence, lines containing the bc-3 gene may be used in development of resistant breeding lines to reduce future losses in snap bean.

Review Publications
Larsen, R.C., Druffel, K.L., Wyatt, S.D. 2009. The complete nucleotide sequence and genome organization of red clover vein mosaic virus (genus Carlavirus). Archives of Virology. 154:891–894.

Last Modified: 06/24/2017
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