Abstract
The development of wheat-rye-Thinopyrum hybrids is a key strategy for introgressing agronomically valuable traits from multiple genera into common wheat, enriching its genetic diversity. However, the labor-intensive and time-consuming identification of lines carrying chromatin segments from all three donors has limited its widespread application. In this study, a stable hexaploid wheat line, 13-26 (2n=6x=42, AABBDD, T6JvsS·6AL-T5R·2D-T5R·4B), was developed by crossing octoploid Trititrigia with hexaploid triticale, followed by 17 generations of self-pollination. Line 13-26 outperforms its parents agronomically, with well-filled kernels and a TKW exceeding both parental lines, and the higher protein content relative to the male parent. Analyses via smGISH, mcFISH, and short-read sequencing confirmed the integration of wheat, rye, and Thinopyrum chromatin. Approximately 220 Mb was absent from wheat chromosome 6AS and 89 Mb from 2DL, while around 221 Mb of Thinopyrum 6JvsS and 42 Mb of rye 5RL were detected. Line 13-26 carries a Th. intermedium T6JvsS·6AL translocation and terminal segments of rye 5RL translocated to wheat 2DL and 4BL, demonstrating intergeneric translocation and recombination between nonhomologous chromosomes, which represents a valuable germplasm resource with integrated multi-genus traits. This combined cytogenomic approach enhances the sensitivity and specificity of alien chromatin detection, providing a robust framework for characterizing complex chromosomal rearrangements in polyploid wheat.