*2.1. Plant Materials*

The octoploid triticale line MK was derived from common wheat (*T. aestivum* L.) Mianyang 11(MY11) × *S. cereale* L. Kustro. Some progeny was obtained by controlled backcrossing of MK with MY11 followed by strict self-seeding [18]. Seven wheat-rye monosomic addition lines, including MA1RKu, MA2RKu, MA3RKu, MA4RKu, MA5RKu, MA6RKu, and MA7RKu, were developed according to the methods described by Li et al. [18]. From the self-seeded progeny of the monosomic addition line MA5RKu, a 5RKu(5B) disomic substitution line, 5RSKu(5B) monotelosomic substitution line, 5RLKu(5B) ditelosomic substitution line, 5RSKu/5BL translocation line, and four 5RKu deletion lines were obtained. The progeny of MA5RKu comprised a set of 5RKu dissection lines. In addition, rye Kustro (*S. cereale* L.), common wheat (*T. aestivum* L.) Chinese Spring (CS), and MY11 were also used in this study. The materials used in this study are available upon request to interested researchers.

### *2.2. Non-Denaturing Fluorescence in situ Hybridization (ND-FISH) Analysis*

Oligonucleotide (oligo) probes, including Oligo-Ku [19], Oligo-pSc119.2-1, Oligo-pTa535-1 [20], Oligo-pSc200, and Oligo-pSc250 [21], were used for ND-FISH analysis of the materials used in this study. Oligo-pSc119.2-1 and Oligo-pTa535-1 can be used to identify individual wheat chromosomes [20]. Oligo-Ku, Oligo-pSc200, and Oligo-pSc250 can together be used to distinguish rye chromosomes from the wheat genome [19]. In this study, the combination of Oligo-pSc200 and Oligo-pSc250 was denoted as Oligo-pSc200 + 250. Oligo probes were synthesized by Tsingke Biological Technology Co. Ltd. (Beijing, China), and they were 5'-end-labeled with 6-carboxytetramethylrhodamine (TAMRA), 6-carboxyfluorescein (6-FAM), or Cyanine Dye 5 (Cy5). The chromosome spreads of the root tips were prepared following the methods described by Han et al. [22]. Images were made using an epifluorescence Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) equipped with a cooled charge-coupled device camera and with the HCIMAGE Live software (Hamamatsu Corporation, Sewickley, PA, USA). Images were processed using Adobe Photoshop CS 3.0.
