The most widespread and harmful disease of barley is net form of net blotch caused by the ascomycete Pyrenophora teres f. teres Drechsler (Ptt). A cost effective and environmentally sustainable strategy for barley protection against Ptt is to develop barley cultivars possessing genetic resistance. In previous GWA analysis, we identified SNP-markers associated with a resistance locus on chromosome 3H in the interval of 45.82–54.53 cM. These SNPs have been described previously in the literature to be located within the same region of chromosome 3H. The aim of the study was to validate QTL markers controlling resistance to Ptt on chromosome 3H in this region by KASP genotyping in four F₂ populations of crosses between the resistant cultivars, Morex, Fox, and Zolo, and the accession, Local k-21578, with the susceptible barley cv. Gesine and in a doubled haploid (DH) population of Canadian Lake Shore (CLS)/Harrington. Eleven of fifteen studied markers showed high efficacy (97.5–100%) for co-segregation with resistance to Ptt in the DH population, CLS/Harrington. Three of these markers located at 54.53 cM and one at 51.27 cM were effective in two F₂ populations of crosses of Morex and Fox with susceptible cv. Gesine. These markers are also located close to each other on the physical map (442,203,921–443,119,491 bp). Apparently, in cultivars, CLS, Morex, and Fox, resistance to Ptt is determined by the same locus. Markers JHI-Hv50k-2016-166392 (47.1 cM, 112,536,071 bp), Clone ID 3255462_1 (51.63 cM, 363,531,898 bp), and Clone ID 3255462_2 (51.63 cM, 363,531,871 bp) showed high efficacy in the DH population and in the F₂ population, Local k-21578/Gesine. Apparently, at least two loci controlling Ptt resistance exist in the chromosome region of 47.0–54.3 cM: one at 46.0–48.44 cM and another at 51.27–54.8 cM. These regions were found to harbor several genes involved in important plant functions, including disease response and signaling pathways. Allele-specific PCR markers were developed based on the KASP assay data and tested on six resistant, two moderately resistant, and two susceptible barley genotypes. Four markers were found to be effective to differentiate susceptible and resistant barley genotypes. The KASP and allele-specific PCR markers associated with Ptt resistance on chromosome 3H will be useful for pyramiding resistance QTLs in barley marker-assisted selection.

Two forms of the pathogenic fungus Pyrenophora teres, P. teres f. teres (a net form) and P. teres f. maculata (a spot form), cause different disease signs, net or spot blotch, on barley leaves. The net form of P. teres is widespread wherever barley is cultivated, while the spot form was first identified in Krasnodar krai of Russia in 2011 and Brest oblast of Belarus in 2016. The two forms of the pathogen easily mate each other in laboratory conditions, but their hybrids either do not form or are difficult to detect in nature. The question as to whether hybrids between the net and spot forms are produced and maintained in natural populations is pressing and bears applied significance because different genes determine resistance to the different P. teres forms in barley. Hybrid forms may be virulent to resistance donors used in breeding. The objective of this work was to search Russian and Belarussian natural populations for hybrids between P. teres f. teres and P. teres f. maculata with the use of new form-specific markers, Ptt and Ptm. The study included 138 single-conidium isolates from four P. teres f. maculatа and four P. teres f. teres populations. The isolates were collected from commercial barley plantations of Leningrad oblast, Krasnodar krai (Russia), and Brest oblast (Belarus) from 2013 to 2016. A genotyping with 10 form-specific markers was performed in all isolates. Several isolates were found to combine markers of both of the P. teres forms and were conventionally identified as hybrids between the forms. Hypotheses were advanced to explain the occurrence of hybrids in natural populations. The most plausible hypothesis suggests that sexual or somatic hybridization between the two forms coexisting in barley plantations accounts for the origin of the P. teres isolates that combine markers specific to P. teres f. teres and markers specific to P. teres f. maculata in their genomes. It is also possible that a third, possibly ancestral, intermediate form was preserved during divergence in the species P. teres.