An integrated molecular cytogenetic platform enables precise characterization of Thinopyrum ponticum chromatin and its structural modifications in wheat background.

By integrating ND‑FISH, Oligo‑FISH painting, and chromosome microdissection, we established a comprehensive platform for decaploid Thinopyrum ponticum, enabling karyotyping, sequencing, and marker development, which provides precise tools for the targeted introgression and utilization of alien chromatin in wheat breeding. Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey, a decaploid wild relative of wheat, represents a valuable genetic reservoir for improving abiotic stress tolerance and disease resistance in wheat breeding. However, its complex genome structure and polyploid nature have long impeded both precise genome assembly and comprehensive characterization of individual chromosomes for wheat-Thinopyrum introgressions. In the present study, we established a reference karyotype of individual Th. ponticum chromosomes by integrated cytogenetic methods combining non‑denaturing fluorescence in situ hybridization (ND‑FISH) and bulked oligonucleotide pool‑based FISH (Oligo‑FISH) painting. This platform was subsequently applied to cytogenetically analyze the wheat-Th. ponticum partial amphiploid 'Agrotana'. Immunostaining assays revealed that Thinopyrum chromosomes maintain normal centromeric histone localization and exhibit distinct methylation patterns in the wheat background. The group-6 chromosomes of the Thinopyrum species harbor several superior resistance genes, we performed microdissection and sequencing of Th. ponticum 6JSS chromosome, generating a genomic resource that enables both comparative genome analysis and targeted functional gene discovery. The specificity of Thinopyrum introgressions in various wheat-Thinopyrum derivatives was further validated by seed storage protein profiles. Collectively, this study establishes an integrated molecular and cytogenetic platform for the accurate identification and assembly of Thinopyrum chromatin, thereby accelerating the targeted introgression and effective deployment of its agronomically valuable traits in wheat improvement programs.