1a.Objectives (from AD-416)
1) Develop rapid detection/diagnostic procedures for identifying new Pgt races in plant tissues and urediniospores;. 2)Organize and conduct training courses for National Plant Diagnostic Network (NDPN);. 3)Use new detection/diagnostic procedures to monitor for wheat rust urediniospores, including new Pgt races in rain samples across central and southern U.S.; and. 4)Improve resistance in wheat germplasm to new races of Pgt through marker selected selection.
1b.Approach (from AD-416)
There is an urgent need to develop a rapid PCR procedure for the diagnosis of Ug99. The present diagnostic method is based on infecting a set of wheat differential lines and evaluating avirulence/virulence phenotypes. A TaqMan PCR will be developed based on the current genome sequence of P. graminis f. sp. tritici and the resequencing of several isolates (including Ug99 and two variants). Comparison of genomic sequences will identify potential DNA targets for assay development. A suite of real-time assays will be combined to identify individual variants within the Ug99 genetic lineage. Plant Diagnostic Center technicians will be trained to implement the new combination assay for detecting Ug99 in wheat and barley. A second more sensitive assay will be developed to detect low concentrations of Ug99 spores in rainwater. The increased sensitivity will be achieved by integrating TaqMan assays in a nested design. This sensitive nested assay design will be used to detect Ug99 spores in rain samples collected from the nationwide NADP/NTN network. This detection will help monitor long distance transport of Ug99 spores and validate stem rust aerobiological model predictions. In addition, rain samples will be monitored for P. graminis and P. triticina to better understand the movement of rust spores through the central and southern U.S. Finally, marker-selected-selection will be used to facilitate movement of known stem rust resistance genes into advanced lines of wheat.
Genetic variation between isolates of the wheat stem rust fungus was identified by analysis of DNA sequence data generated in a related project. Fifty primer/probe sets were developed and tested with a representative set of Puccinia graminis f. sp. tritici (Pgt) isolates. Seven primer/probes that passed the initial screen were analyzed and larger set of isolates (150) of which, four primers/probe sets passed. This final set of four primers/probes was able to distinguish Ug99 and related races from all other Pgt isolates tested. The target regions for these four sets were cloned from a number of wheat stem rust isolates in order to refine the specificity of the assay. Work continues on expanding the core set of primer/probes and developing assays that will be able to distinguish individual members of this group.
Regular e-mails, telephone calls, and meetings were used to plan and coordinate the project.