Development of diagnostic markers for the disease susceptibility gene Tsn1 in wheat reveals novel resistance alleles and a new locus required for ToxA sensitivity

The wheat Tsn1 gene recognizes the necrotrophic effector ToxA, which is produced by three different necrotrophic fungal pathogens. A compatible Tsn1 -ToxA interaction leads to host-induced responses that result in the development of disease. Therefore, marker-assisted elimination of functional Tsn1 alleles is an effective strategy for the development of disease resistant varieties. To develop such markers, available wheat genome assemblies were used to compare gene and transposable element content in lines with and without Tsn1 ( Tsn1 - and Tsn1 +), revealing two conserved haplotypes. Because Tsn1 is almost always absent in insensitive lines, Kompetitive Allele Specific PCR (KASP) markers were designed in flanking syntenic regions of Tsn1 - and Tsn1  + assemblies. The KASP markers were validated in more than 1,500 diverse lines. The markers correctly predicted a ToxA-insensitive phenotype in 99.33–100% of the lines, but they were less effective at predicting a ToxA-sensitive phenotype (89.50-94.55%) due to 60 insensitive lines with sensitive marker alleles. Sequence analysis of Tsn1 from these lines revealed that some were not transcribed and others contained point mutations. However, some carried and expressed the dominant Tsn1 allele, and subsequent analysis of two such lines revealed a second locus controlling ToxA sensitivity on chromosome 2B, termed Tsn1-B2 . Genetic mapping of Tsn1-B2 in a biparental durum population define the locus to a 4.8 cM region corresponding to 8.6 Mb in Svevo Rel 2.0. The markers presented here could be used for reliable and robust marker-assisted elimination of Tsn1 in a high-throughput manner, furthering the development of wheat genetically resistant to multiple pathogens.