Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 10, 2010
Publication Date: November 10, 2010
Citation: Lu, S., Platz, G., Edwards, M.C., Friesen, T.L. 2010. Mating Type (MAT) Locus -Specific PCR Markers for Differentiation of Pyrenophora teres f. teres and P. teres f. maculata, the Causal Agents of Barley Net Blotch. Phytopathology. 100(12):1298-1306. Interpretive Summary: Pyrenophora teres causes barley net blotch, one of the most important barley diseases in North America and worldwide. The fungus occurs in the field as two forms: P. teres f. teres, which causes “net-form” (NF) net blotch and P. teres f. maculata, which causes “spot form” (SF) net blotch. Differentiation of these two forms largely relies on pathogenicity assays using differential barley lines that are time-consuming and often have limitations. The objective of this study was to develop conventional polymerase chain reaction (PCR) markers for accurate differentiation of NF and SF isolates. The results indicated clearly that the NF and SF isolates with opposite mating types can be precisely differentiated using PCR primers designed to the mating type (MAT) locus that contains single nucleotide polymorphisms (SNPs). The method was sensitive, highly specific and applicable to all NF and SF isolates collected from different geographic origins worldwide. In comparison with other procedures such as RAPD or AFLP markers, the major advantage of this method was that both subspecies identity and mating types can be determined simultaneously in the same PCR reactions, thus facilitating disease management and epidemiological studies. To the best of our knowledge, this is the first example that the limited genetic variation at the MAT locus is sufficient for PCR-based identification of heterothallic ascomycetes at subspecies levels.
Technical Abstract: Fourteen single nucleotide polymorphisms (SNPs) were identified at the mating-type (MAT) loci of Pyrenophora teres f. teres (Ptt), which causes net form (NF) net blotch, and P. teres f. maculata (Ptm), which causes spot form (SF) net blotch of barley. MAT-specific SNP primers were developed for polymerase chain reaction (PCR) and the two forms were differentiated by distinct PCR products: PttMAT1-1 (1,143 bp) and PttMAT1-2 (1,421 bp) for NF MAT1-1 and MAT1-2 isolates; PtmMAT1-1 (194 bp) and PtmMAT1-2 (939 bp) for SF MAT1-1 and MAT1-2 isolates, respectively. Specificity was validated using 37 NF and 17 SF isolates collected from different geographic regions worldwide. Duplex PCR including both MAT1-1 and MAT1-2 SNP primers gave the same specificity. No cross-reactions were observed with DNA from P. graminea, P. tritici-repentis and other ascomycetes or barley. Plants infected with a single or mixed isolates of the two different forms were also differentiated. This study provides the first example that the limited SNPs at the MAT locus are sufficient for distinguishing closely related heterothallic ascomycetes at subspecies levels so that the pathogenic and mating type characteristics of the fungus can be determined simultaneously. Methods presented will facilitate pathogen detection, disease management and epidemiological studies.