Powdery mildew is a fungal disease that threatens wheat production throughout the world. Breeding resistant cultivars is an effective way to reduce harm caused by powdery mildew. In this study, 35 wheat-Psathyrostachys huashanica-derived lines were developed by crossing common wheat and P. huashanica Keng (2n = 2x = 14, NsNs) using embryo culture. Resistance to powdery mildew in the derived lines was identified at the seedling and adult stages. Line H5-5-4-2 was selected with immunity to powdery mildew at both growth stages. The chromosome structure of this line was characterized by cytology, genomic in situ hybridization (GISH), and expressed sequence tag-sequence-tagged site (EST-STS) analysis. The chromosome configuration was 2n = 44 = 22II. Two P. huashanica chromosomes with strong hybridization signals were detected by GISH analysis. Among 83 EST-STS markers that covered all seven homologous groups in wheat, three pairs of STS markers, BE497584, BF202643, and BG262410, located in wheat homologous group 1 amplified clear specific bands related to P. huashanica. The results indicated that resistant line H5-5-4-2 was a wheat-P. huashanica 1Ns disomic addition line.

Psathyrostachys huashanica (2n = 2x = 14, NsNs) and Leymus mollis (2n = 4x = 28, NsNsXmXm) are important wild relatives of common wheat. The Ns chromosomes from two species have been successfully introgressed into wheat through distant hybridization. To compare the genetic effects and evolutionary relationship of Ns chromosomes from different genera in a wheat background, wheat-P. huashanica derivative WH15 and wheat-L. mollis derivative WM14-2 were selected. Sequential FISH-GISH showed that both WH15 and WM14-2 contained 40 wheat chromosomes (with 2D deletion) and two Ns chromosomes with different FISH karyotypes. Molecular markers and SNP array analysis revealed that the two lines both introduced 2Ns chromosomes. However, the P. huashanica 2Ns and L. mollis 2Ns had distinct sequence compositions, and the different SNPs between the two species 2Ns chromosomes were primarily clustered on the short arm. WH15 and WM14-2 exhibited significant differences in spike-related morphologies but shared leaf rust resistance and susceptibility to powdery mildew and Fusarium head blight. Cytogenetic analysis confirmed stable meiotic inheritance of the introduced 2Ns chromosomes. We further developed universal diagnostic markers for 2Ns chromosomes based on SLAF-seq. Therefore, substantial divergence likely exists between the Ns genomes of P. huashanica and L. mollis, and P. huashanica is probably not the direct Ns genome donor for Leymus. Our research-developed derivatives provide unique resources for comparative studies of the structural and functional evolution of homoeologous Ns chromosomes across genera, while offering valuable alleles for wheat improvement.