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.
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 United States. 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:
This is a new project that replaced project 5354-21000-015-00D, Enhancing Resistance to Diseases and Abiotic Stresses in Alfalfa". USDA, ARS Prosser, the Samuel Roberts Noble Foundation, Forage Genetics International (FGI), Pioneer Hi-bred, and Cal-West seeds agreed on a joint effort to identify publically available markers for selection for resistance to Verticillium wilt and alfalfa stem nematode. Pioneer and Cal-West generated and phenotyped biparental populations 188 and 164 individuals, respectively, using parents either resistant or susceptible to Verticillium wilt. We screened the parents and a subset of individuals from both Pioneer and Cal-West populations with 156 single nucleotide polymorphisms (SNP) markers, using the high-resolution melting technique. Approximately 50% of the SNPs were polymorphic in the Cal-West population, whereas approximately 40% of the SNPs were polymorphic in the Pioneer population. Associations between marker genotypes and Verticillium wilt phenotype were performed using the PROC MIXED procedure of SAS. Based on the results from PROC MIXED, several markers were significantly associated with the trait at p<0.05, but at most 8 percent of the explained phenotypic variation. To increase the power and resolution of marker-trait association, we are using genotyping by sequencing which will increase thousands of SNP markers throughout whole genome, and by increasing the size of mapping population by combining several populations. Improved methods would increase the probability for identifying molecular markers associated with VW resistance.