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United States Department of Agriculture

Agricultural Research Service

Research Project: Enhancing Resistance to Diseases and Abiotic Stresses Alfalfa

Location: Vegetable and Forage Crops Production Research

2010 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. Objective 3. Identify markers linked to the adaptation of alfalfa to abiotic stresses, particularly limited water availability, salinity, and other stresses that may be intensified by climate change.

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). FY10 Program Increase $180,000.

3.Progress Report
A backcross population (BC1) of alfalfa composed of 180 progeny was developed by crossing a parent alfalfa line susceptible to lodging with a line tolerant to lodging and then back crossing the resulting F1 to the susceptible parent. A linkage map was developed based on the segregation of 336 markers among the backcross progeny. A total of 72 sequence related amplified polymorphism (SRAP) and 134 amplified fragment length polymorphism (AFLP) markers were distributed among 24 linkage groups. Replicated clones of both parents and all BC1 progeny have been evaluated for general appearance, tolerance to lodging, and spring vigor in the field over two experimental years in Washington State and Wisconsin. QTL analysis was performed using the combined data of both locations in both years. One unlinked AFLP marker demonstrated significant associations with general appearance over both years. One SRAP marker and one AFLP marker showed significant associations for lodging resistance over both years. For spring vigor the same unlinked AFLP marker associated with general appearance was significantly associated with the trait in one experimental year only. The SRAP marker is the most likely candidate marker for further consideration of use in marker assisted selection for lodging resistance. We will continue to identify more SRAP markers associated with general appearance, spring vigor and lodging tolerance and to integrate these results into a consensus map of tetraploid alfalfa.

Isolates of Aphanomyces euteiches Race 1 (R1) and Race 2 (R2) were obtained from alfalfa fields in Illinois, North Carolina, Washington, and Wisconsin. 395 DNA molecular markers have been screened to independently identify R1 and R2 of the pathogen. A single sequence related amplified polymorphism (SRAP) marker was identified across five R2 isolates that did not occur in DNAs of four R1 isolates. No polymorphisms were detected in R1 or R2 using random amplified polymorphic DNA (RAPD) primers so far. Additional SRAP and RAPD markers are being screened to identify markers specific to R1 and to obtain additional markers specific to R2. More isolates of both races are being collected to increase the screening panel size in order to better evaluate the robustness of the markers to specifically detect each race.

Genetic crosses have been made between four common bean parental lines resistant to Clover yellow vein virus, the causal virus of the 'chocolate pod' disease in snap bean. One cultivar contains the bc-3 gene demonstrated earlier in the project to be linked to complete resistance to the virus. The gene(s) conferring resistance in the other three lines is currently unknown. F1 progeny obtained from the crosses have been generated and the F2 progeny population is now complete. The population will be screened in allelism tests to determine the nature of the other sources of resistance, with the potential of discovering new genes for resistance to the virus that can be introgressed into new germplasm and variety development.

1. Identification of molecular markers linked to general appearance, lodging resistance and spring vigor traits in alfalfa. Good general appearance, lodging resistance and spring vigor are important agronomic traits for alfalfa. ARS scientists at Prosser, WA, developed a genetic map of these traits based on the segregation of DNA molecular markers from a uniquely developed population of alfalfa. Two of the DNA markers showed significant associations for lodging resistance while other markers were associated with spring vigor and good general appearance. Most recently, a unique marker was identified as the most likely candidate for further consideration of use in marker assisted selection of alfalfa lines with these desired traits. Upon conversion of the marker to a more highly specific marker type, it will be useful in the rapid examination of many alfalfa samples for these traits in a short period of time.

Review Publications
Riga, E., Larsen, R.C., Guerra, N., Eastwell, K., Crosslin, J. 2009. Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus allius from Greenhouse and Field Specimens. Journal of Nematology. 41:60-63.

Naderpour, M., Lund, O., Larsen, R.C., Johansen, E. 2010. Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc-3 is associated with the homozygotic presence of a mutated eIF4E allele. Molecular Plant Pathology. 255-263.

Last Modified: 4/18/2014
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