Location:2011 Annual Report
1a. Objectives (from AD-416)
Objective 1: Identify molecular markers to screen alfalfa for resistance to environmental and biotic stresses including problems caused by nematodes and diseases. 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.
3. Progress Report
During FY2011 new objectives were added due to funding enhancement to this project. An agronomist and a plant pathologist were re-directed to this project. A postdoc (Molecular Biology) was hired to develop markers for resistance to abiotic and biotic stresses in alfalfa. The additional funding was terminated in FY2011 CR, which resulted in discontinuation of new objectives and reduction in SY pool. As a part of our studies toward developing markers for Verticillium wilt resistance, we extracted DNA from each of the Cal/West Company entries and bulked according to respective resistant and susceptible phenotyping. The bulks have been screened with over 90 simple sequence repeat (SSR) markers developed by the Noble Foundation, Ardmore, OK. Markers linked to resistance or susceptibility have been located on chromosomes 7 and 8 by screening the individual genotypes comprising the bulks. Toward developing markers for stem nematode resistance, we bulked DNA samples from Cal/West and Forage Genetics company entries and screened with the pool of SSR markers mentioned above. To date, not many polymorphic markers have yet been identified, and any putative markers do not seem to work well across different varieties. Toward developing drought-adapted alfalfa germplasm, we collected alfalfa germplasm from three locations in eastern Washington which were being grown without irrigation with only < 150 mm annual precipitation. Seed increase of these populations is currently underway. The seed from these cages will be used to plant a replicated experiment to compare the yield of these populations with elite varieties under normal and reduced irrigation. Significant progress has also been made towards developing biological controls to manage white mold in alfalfa seed production. Field soil was collected from 68 locations within an alfalfa field and survival structures (sclerotia) of the white mold pathogen were removed from the soil using sieves. Sclerotia were plated onto a selective agar medium to determine if they were viable. Eleven fungi were collected from non-viable sclerotia that will be assessed as potential biological control agents to manage this pathogen. Burning alfalfa crop residue reduced the number of sclerotia found in crop soil by 41 to 71%. Burning also helped promote natural biological control of sclerotia in the soil by the Fusarium acuminatum fungus.