Stripe rust caused by Puccinia striiformis f. sp. tritici and powdery mildew caused by Blumeria graminis f. sp. tritici are devastating diseases of wheat worldwide. Exploration of new disease-resistant genes from cultivated wheat and wild relatives are the most effective means of reducing the amounts of fungicides applied to combat these diseases. Thinopyrum scirpeum (2n = 4x = 28, EEEE) is an important promising reservoir of useful genes, including stripe rust and powdery mildew resistance, and may be useful for enhancing wheat disease resistance. Here, we characterize a novel wheat–Th. scirpeum disomic substitution line, K16-730-3, and chromosome-specific markers were developed that can be used to trace the Th. scirpeum chromosome or chromosome segments transferred into wheat. Genomic in situ hybridization and fluorescence in situ hybridization analyses indicated that K16-730-3 is a new 4E (4D) chromosomal substitution line. Evaluation of seedling and adult disease responses revealed that K16-730-3 is resistant to stripe rust and powdery mildew. In addition, no obvious difference in grain yield was observed between K16-730-3 and its wheat parents. Genotyping-by-sequencing analyses indicated that 74 polymerase chain reaction -based markers can accurately trace chromosome 4E which were linked to the disease resistance genes in the wheat background. Further marker validation analyses revealed that 13 specific markers can distinguish between the E-genome chromosomes of Th. scirpeum and the chromosomes of other wheat-related species. The new substitution line K16-730-3 carrying the stripe rust and powdery mildew resistance genes will be useful as novel germplasm in breeding for disease resistance. The markers developed in this study can be used in marker-assisted selection for improvement of disease resistance in wheat.

Understanding the genetic diversity of wheat is important for wheat breeding and improvement. However, there have been limited attempts to evaluate wheat diversity using fluorescence in situ hybridization (FISH). In this study, the chromosomal structures of 149 wheat accessions from 13 countries located between the latitudes of 30° and 45°N, the principal growing region for wheat, were characterized using FISH with pTa535 and pSc119.2 probes. The ranges of the numbers of FISH types in the A-, B-, and D-genomes were 2–8, 3–7, and 2–4, respectively, and the average numbers in the A- and B-genomes were greater than in the D-genome. Chromosomal translocations were detected by these probes, and previously undescribed translocations were also observed. Using the FISH, the genetic relationships among the 149 common wheat lines were divided into three groups (G1, G2, and G3). G1 mainly consisted of Southern European lines, G2 consisted of most lines from Japan and some lines from Western Asia, China, and Korea, and G3 consisted of the other lines from Southern Europe and most of the lines from Western Asia, China, and Korea. FISH karyotypes of wheat chromosomes distinguished chromosomal structural variations, revealed the genetic diversity among wheat varieties. Furthermore, these results provide valuable information for the further genetic improvement of wheat in China.