TCJP An improved method to quantify Puccinia coronata f. sp. avenae DNA in the host Avena sativa

Authors

M. Acevedo, USDA-ARSFollow
E. W. Jackson, USDA-ARS
A. Sturbaum, USDA-ARS
H. W. Ohm, Purdue University
J. M. Bonman, USDA-ARS

Date of this Version

2010

Comments

Published in Can. J. Plant Pathol., (2010), 32(2): 215–224. DOI: 10.1080/07060661.2010.484182

Abstract

Identification and genetic mapping of loci conferring resistance to polycyclic pathogens such as the rust fungi depends on accurate measurement of disease resistance. We converted an absolute quantification assay of Puccinia coronata DNA to a relative assay by adding a TaqMan^® primers/probe set specific to the oat β-actin gene to simplify and improve quantification of fungal infection. The new multiplex assay estimates the amount of fungal DNA in a sample relative to the amount of host DNA and requires fewer and less labour-intensive steps than previous assays. The relative fungal DNA assay (RFDNA) reliably detected and quantified both host and pathogen DNA over five orders of magnitude and was at least as sensitive as either digital image analysis (DLA) or absolute estimation of fungal DNA (AFDNA) in repeated greenhouse studies using 12 oat cultivars with different resistance responses to P. coronata isolate LGCG. Measuring crown rust resistance to LGCG using DLA, AFDNA and RFDNA in a P8669/P94163 recombinant inbred line population produced segregation ratios that did not differ from the 1:1 Mendelian ratio expected for a single gene. Compared to the AFDNA assessment method, the RFDNA assay is equally sensitive, yet faster and much easier to use than the AFDNA method for precise quantification of the crown rust pathogen in oat leaves. The method will be especially useful for streamlining measurement of partial resistance, since uncovering small differences in resistance requires phenotypic evaluation of large populations.