*2.3. Developing PCR-Based Markers*

Genomic DNA of *S. cereale* L. Kustro has already been sequenced using specific length amplified fragment sequencing (SLAF-seq) technology (Biomarker, Beijing, China) [4]. In this study, the genomic DNA of wheat-rye monosomic addition line MA5RKu was also sequenced using the SLAF-seq technique. Sixty base sequences of both ends of the sequences with sizes between 450 to 500 bp were obtained. The sequencing procedure was carried out according to the methods described by Duan et al. [4]. The primary 5RKu specific pair-end reads were obtained according to the methods described by Duan et al. [4]. Primers were designed according to 542 randomly selected 5RKu specific pair-end reads using the software Primer 3 (version 4.1.0) [23], and the optimal melting temperature and size values were set to 60 ◦C and 20 bases, respectively. The 542 pair-end reads were deposited in the GenBank Database (GenBank accession numbers: MN325158-MN325699). A total of 542 primer pairs were designed.

### *2.4. PCR Assay, 5RKu-Specific Markers Testing and Physical Location*

The PCR amplifications and electrophoresis were carried out according to the procedure described by Duan et al. [4]. The markers that could produce bands from both rye Kustro and MA5RKu but not from CS, MY11, MA1RKu, MA2RKu, MA3RKu, MA4RKu, MA6RKu, and MA7RKu were regarded as 5RKu-specific markers. For each of the primer pairs, PCR reactions were repeated three times. These markers combined with the 52 5RKu-specific markers developed by Qiu et al. [24] were mapped to specific regions of the 5RKu chromosome using the 5RKu dissection lines. Again, PCR reactions of each 5RKu-specific marker were repeated three times.

### *2.5. Similarity Searches of the Pair-End Reads Against S. cereale L. Lo7 Sca*ff*olds*

The original pair-end reads for designing the 5RKu-specific markers were used for Nucleotide BLAST searching against the sequences in the *S. cereale* L. Lo7 scaffolds database in GrainGenes [25]. The *S. cereale* L. Lo7 scaffolds were reported by Bauer et al. [26].

### *2.6. Stripe Rust Resistance Testing*

The resistance of 5RKu dissection lines and the parental wheat MY11 to stripe rust was evaluated under field conditions. Plants were grown in Qionglai, Sichuan, China. The materials were inoculated twice with the mixed stripe rust prevalent isolates CYR32, CYR33, and CYR34 in China at approximately 6 weeks and 9 weeks after sowing. Infection types (IT) were scored according to a 0–9 numerical scale as described by Wan et al. [27] in the adult stage.
