Molecular Cytogenetic and Transcriptome Characterization of a Novel Wheat-Thinopyrum intermedium 2J(2D) Disomic Substitution Line with Resistance to Powdery Mildew

Wheat powdery mildew, caused by the biotrophic pathogen Blumeria graminis f. sp. tritici (Bgt), is a destructive disease that causes severe yield losses in susceptible varieties, threatening global food security. Breeding resistant cultivars carrying novel resistance genes through wide hybridization represents a sustainable strategy for powdery mildew management. As a wild relative in the wheat tertiary gene pool enriched in diverse resistance genes, Thinopyrum intermedium serves as a valuable genetic resource for wheat improvement. A wheat–Th. intermedium-derived line SN6307 with powdery mildew resistance and multiple desirable agronomic traits was developed using wheat cultivar YN15 as the recipient crossing to Th. intermedium, and karyotypically characterized as a 2J(2D) disomic substitution line via non-denaturing fluorescence in situ hybridization (ND-FISH), Oligo-FISH painting, genomic in situ hybridization (GISH) and single-nucleotide polymorphism (SNP) analyses. To explore resistance genes in SN6307, we established a novel staining protocol for observing Bgt epiphytic structures and performed integrated second- and third-generation transcriptome sequencing on leaf epidermal tissues of SN6307 and YN15 after Bgt inoculation. Resistance reaction assays revealed that SN6307 exhibits high resistance and near immunity to Bgt isolate E09 at the seedling stage. Significant differences in Bgt-responsive gene expression profiles and defense strategies were observed between SN6307 and YN15. Two Th. intermedium-derived leucine-rich repeat receptor-like kinase (LRR-RLK) family genes and several genes associated with plant–pathogen interactions were comprehensively analyzed and revealed that they coordinately modulate the multi-layered immune interaction network in SN6307. Furthermore, 24 novel Th. intermedium chromosome 2J-specific molecular markers were successfully developed, enabling efficient tracking of 2J chromatin in wheat populations derived from radiation mutagenesis. In conclusion, SN6307 represents novel germplasm for wheat disease-resistance breeding and further characterization of Th. intermedium functional genes.