Leymus mollis Trin. (2n = 4x = 28, NsNsXmXm), an important wild relative of common wheat (Triticum aestivum L.), harbors abundant genes for disease resistance and excellent agronomic traits genes, representing a valuable genetic resource for wheat improvement. Cytological observation and genomic in situ hybridization (GISH) analyses confirmed that both lines contain 40 wheat chromosomes and a pair of L. mollis chromosomes. Fluorescence in situ hybridization (FISH) revealed that DM160 lacked chromosome 6B and exhibits structural variations in chromosome 5A and 7B, while DM252 lacks chromosome 6D and carries a mutated chromosome 7B. Combined molecular markers and liquid array analyses confirmed DM160 was a wheat–L. mollis 6Ns (6B) disomic substitution line, and DM252 was a wheat–L. mollis 6Ns (6D) disomic substitution line. Agronomic and disease resistance evaluations demonstrated that DM160 produces more tillers and exhibits stripe rust resistance, whereas DM252 displays reduced plant height, and confers resistance to both powdery mildew and stripe rust. Transcriptome sequencing revealed distinct gene expression profiles among DM160, DM252, and 7182, leading to the identification of six key candidate disease resistance genes. Furthermore, 15 DNA markers specific to Lm#6Ns were developed to track this chromatin in wheat backgrounds. These novel substitution lines provide valuable germplasm for enhancing disease resistance and agronomic traits in wheat, and serve as essential materials for further characterization and utilization of Lm#6Ns chromosomes.

Thinopyrum ponticum (2n=10×=70), a wild relative of common wheat (Triticum aestivum L.), is considered an invaluable genetic resource for wheat improvement due to its abundance of genes conferring resistance to biotic and abiotic stresses. This study focused on the CH97 line, derived from the BC₁F₇ progeny of a cross between wheat cv. 7182 and Th. ponticum. Cytological evidence showed that CH97 has 42 chromosomes, forming 21 bivalents at meiotic metaphase I, with the bivalents subsequently separating and moving to opposite poles during meiotic anaphase I. Through a combination of fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH), multicolor GISH (mc-GISH), and liquid array analysis, it was determined that CH97 comprises 40 wheat chromosomes and two alien chromosomes from the E^e genome of Th. ponticum, featuring the absence of a pair of 5D chromosomes and variations in 1B, 6B, and 7B chromosomes. These findings confirm that CH97 is a stable wheat-Th. ponticum 5E (5D) alien disomic substitution line. Inoculation experiments revealed that CH97 exhibits high resistance to wheat powdery mildew and stripe rust throughout the growth period, in contrast to the highly susceptible common wheat parent 7182. Compared to 7182, CH97 displayed improvements in thousand-kernel weight and kernel length. Additionally, utilizing specific-locus amplified fragment sequencing (SLAF-seq) technology, chromosome 5E-specific molecular markers were developed and validated, achieving a 33.3% success rate, facilitating marker-assisted selection for disease resistance in wheat. Overall, the CH97 substitution line, with its resistance to diseases and improved agronomic traits, represents valuable new germplasm for wheat chromosome engineering and breeding.