*4.1. 5R-Specific Markers*

In our previous studies, some PCR-based and rye chromosome-specific markers have been developed using SLAF-seq technology [4,18,24]. Some of these markers have already been used to identify rye chromosomal segments in wheat backgrounds because they have easy application [5,6,28]. It has already been reported that rye 5R chromosomes carry some elite genes for wheat cultivar improvement [7–12]. Rye 5R-specific markers are contributive to identifying 5R chromatin in wheat backgrounds and help in the effective application of 5R elite genes in wheat breeding programs. So far, some PCR-based and 5R-specific markers have been reported. Two *S. cereale* inter-microsatellite (SCIM) markers were found to be 5R-specific [29]. Tomita et al. [30] reported a 5R-specific marker that was derived from the *Revolver* transposon-like sequence. A 5RS-specific and five 5RL-specific markers derived from expressed sequence tags (ESTs) were developed [31]. Sixteen PLUG markers that were developed using the PCR-based landmark unique gene system were proven to be 5R-specific [32]. Twenty-one 5Ra-specific molecular markers were developed [12]. Qiu et al. [24] developed 19 5RSKu-specific and 33 5RLKu-specific markers using SLAF-seq technology. These markers mentioned above are easier to perform because they are PCR-based markers. In addition to distinguishing the 5R chromosome in wheat backgrounds, these markers can be used to construct a map of the 5R chromosome. However, more PCR-based and 5R-specific markers are needed. The 111 markers developed in this study have enriched the 5R-specific markers. In this study, a total of 163 5RKu-specific markers were mapped to six regions of the 5RKu chromosome, and this is beneficial for the identification of introgressed 5R small segments in wheat backgrounds. Additionally, the corresponding *S. cereale* Lo7 scaffolds [26] to 65 5RKu-specific markers were found and they were also mapped to the six regions of the 5RKu chromosome. This might be useful for building the physical map and the high-density genetic map of chromosome 5R. Furthermore, the *S. cereale* Lo7 scaffolds [26] mapped to the region L2 might help to further dissect the stripe resistance gene(s). Twenty-two of the 111 pair-end reads matched some scaffolds that have not ye<sup>t</sup> been assigned to any rye chromosome (0R) and these pair-end reads contributed to mapping a reasonable number of *S. cereale* Lo7 scaffolds to the different regions of the 5R chromosome. The case that some 5RKu-specific pair-end reads hit some Lo7 scaffolds derived from 1R, 3R, or 4R chromosomes might be the result from duplications, members of gene families, etc.

In addition, Silkova et al. reported some 5R deletion lines. However, their breakpoints were unclear, and these deletion lines were not used to map 5R-specific markers [17]. In this study, the signal patterns of probes Oligo-Ku, Oligo-pSc200, Oligo-pSc250, and Oligo-pSc119.2-1 on the rye chromosomes are the same as the ones of the 5RKu chromosomes reported by Li et al. [18]. The rye chromosomes in this study were determined to be chromosome 5R, although the control was not used. Therefore, a new set of 5R dissection lines was developed in this study and the breakpoints on these broken 5R chromosomes were tentatively determined.

### *4.2. Stripe Rust Resistance Gene(s) on the 5RL Arm Might be New One*

It has been reported that 1R chromosomes that were derived from various rye sources carried stripe rust resistance gene(s) [33–37]. Additionally, the 2RL and 5RS arms of *S. africanum* Stapf. [12,38], the 4R chromosome of rye cultivar German White [6], and the 6R chromosome of *S. cereanum* cv. Kriszta [39] also carry stripe rust resistance gene(s). In this study, the 5RLKu arm that was derived from rye Kustro also carried stripe rust resistance gene(s) and the resistance gene(s) was mapped to region L2 of the 5RKu chromosome. Therefore, the stripe rust resistance gene(s) might be new one(s) because of its different source and location from the previously reported ones. The stripe rust resistance gene(s) on the 5RLKu arm reported in this study enriches the resistance source of stripe rust for wheat breeding programs. Additionally, 22 markers and 11 *S. cereale* Lo7 scaffolds were located to the L2 region of the 5RKu chromosome, the same region where the stripe rust resistance gene(s) was mapped, and these markers and scaffolds can facilitate the utilization of the resistance gene(s).

### *4.3. Variations of Wheat Chromosomes*

Compared with the standard signal patterns of probes Oligo-pSc119.2-1 and Oligo-pTa535-1 on the chromosomes of common wheat MY11 [20], it can be noted that the variations of wheat chromosomes occurred in lines 142-30, 143-9, and 449-4. This indicated that wheat-rye 5R addition lines or 5R(5B) substitution lines could cause changes of the wheat genome. However, only alterations of the 7B and 3D chromosomes in the three lines were observed. Therefore, the rules and mechanisms of the alterations of the wheat genome in 5R addition and 5R(5B) substitution lines are not clear, and more attention should be paid to this issue in future research.
