Development and validation of a quantitative PCR assay method of assessing relative resistance of oat (Avena sativa) to crown rust (Puccinia coronata f. sp. avenae)

Authors: YIMER, BELAYNEH - Orise Fellow; Gordon, Tyler; Bonman, John; Esvelt Klos, Kathy

Submitted to: Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2019
Publication Date: 3/3/2019
Citation: Yimer, B.A., Gordon, T.C., Bonman, J.M., Esvelt Klos, K.L. 2019. Development and validation of a quantitative PCR assay method of assessing relative resistance of oat (Avena sativa) to crown rust (Puccinia coronata f. sp. avenae). Plant Pathology. 68:669-677. https://doi.org/10.1111/ppa.12988.
DOI: https://doi.org/10.1111/ppa.12988

Interpretive Summary: Crown rust resistance is usually measured based on visual assessment, which is subjective, prone to rater bias and require expert knowledge. Another method, counting, has greater accuracy, but it is labor intensive and time consuming. Quantitative assessments combine the speed of visual assessment with the accuracy, objectivity and reproducibility of counting. In this study we developed a PCR based quantitative method that estimates the fungal DNA content in crown rust infected oat tissue. The fungal DNA content in oat tissues is extrapolated into resistance level of oat plants to crown rust resistance. This method provides a measure of oat resistance to crown rust that is less subjective than visual assessment without the high skill requirement, and with the accuracy and reproducibility of direct-counting method.

Technical Abstract: Evaluation of oat crown rust resistance is usually based on visual assessment of disease severity or infection types. Visual assessment is subjective, prone to rater bias and requires expert knowledge. PCR-based quantitative assays can overcome challenges associated with visual assessment. We designed TaqMan primers and probes from Puccinia coronata f. sp. avenae (Pca) sequences. The new primer-probe sets were specific to Pca, amplified fungal DNA (FDNA) as low as 0.5 pg and allowed for scaling to variation in sample total DNA quantity. The qPCR assay was validated using recombinant inbred lines (RILs) from the Provena x 94197A1-9-2-2-2-5 cross evaluated under a controlled environment. For comparison with fungal DNA load (FDNA), inoculation with the Pca race ‘LCBB’ provided segregation data on the hypersensitive response, while Pca race ‘LSLG’ provided data on segregation for reduced pustule number. FDNA content was positively correlated with both pustule number and infection type (IT). Composite interval mapping identified two QTL on oat linkage group Mrg12 and Mrg20 using visual and qPCR assessments (pustule number, IT and FDNA). In this study we refined and validated a quantitative PCR assay method that can be used to assess the relative resistance of oat to crown rust and identified SNPs closely linked with two QTL derived from the crown rust resistant line ‘94197A1-9-2-2-2-5’.