Mating Type Locus-Specific Polymerase Chain Reaction Markers for Differentiation of Pyrenophora teres f. teres and P. teres f. maculata, the Causal Agents of Barley Net Blotch

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. Both MAT1-1 and MAT1-2 SNP primers retained respective specificity when used in duplex PCR. No cross-reactions were observed with DNA from P. graminea, P. tritici-repentis, or other ascomycetes, or barley. Single or mixed infections of the two different forms were also differentiated. This study provides the first evidence that the limited SNPs at the MAT locus are sufficient for distinguishing closely related heterothallic ascomycetes at subspecies levels, thus allowing pathogenicity and mating type characteristics of the fungus to be determined simultaneously. Methods presented will facilitate pathogen detection, disease management, and epidemiological studies.