1a.Objectives (from AD-416):
Identify molecular markers specific to Aphanomyces eiteiches Race 1 and 2.
1b.Approach (from AD-416):
Collect fungal isolates from various geographical regions. Extract DNA from each and combine from each respective race (bulked DNA). Use RAPD, SRAP, or other primers to screen for polymorphisms specific to each race. Develop SCAR markers from sequence derived from the latter markers.
Aphanomyces euteiches Drechs. is an Oomycete that causes seedling root rot and poor stand establishments in alfalfa and results in stand decline of established fields with a heavy soil structure. Within the alfalfa pathotype, there have been two races identified, namely race 1 (R1) and race 2 (R2), based on the disease response of standard resistant and susceptible check cultivars. Abundant alfalfa cultivars are available with resistance to R1 but fewer are currently resistant to R2. At present, 57.7% of the available certified alfalfa cultivars are rated as resistant or highly resistant to R1 of A. euteiches, with only 7.5% of these varieties rated as resistant or highly resistant to R2 of A. euteiches. At present, the only method to identify the two races is through virulence testing which requires greenhouse evaluation over time after applying R1 and R2 inoculum to test plants. As such, DNA markers that can rapidly and unambiguously distinguish the two races would be highly useful.
A single SRAP marker ca 190 bp in length was identified across all five R2 isolates that did not occur in DNAs of R1. No polymorphisms were detected in R1 or R2 using RAPD primers. The SRAP marker is being developed into a sequence-characterized amplified region (SCAR) that will be useful for rapid identification of race 2. Additional SRAP and RAPD markers are being screened to identify markers specific to R1 and to obtain additional markers specific to R2, which addresses objective 1.2. of the related in-house project, "Develop molecular markers that discriminate unambiguously between Race 1 and Race 2 of Aphanomyces euteiches".