**3. Results**

## *3.1. Obtaining 5RKu Dissection Lines*

From the self-seeded progeny of MA5RKu, lines 142-30, 142-77, 143-9, 143-61, 237-2, 403-50, 444-12, and 449-4 were obtained (Figures 1–3; Table 1). Probe Oligo-pSc119.2-1 combined with probe Oligo-pTa535-1 can identify individual wheat chromosomes (Figures 1–3). The combination of probes, Oligo-Ku, Oligo-pSc200, Oligo-pSc250, and Oligo-pSc119.2-1, can be used to distinguish rye 5RKu chromosomes from the wheat genome and detect their structural variations (Figures 1–3). For the sake of description, the combination of Oligo-pSc200 and Oligo-pSc250 was denoted as Oligo-pSc200 + 250. Wheat 5B chromosomes disappeared from the five lines 140-30, 142-77, 143-9, 143-61, and 403-50 (Figures 1 and 2A–D). A broken 5RKu chromosome existed in line 142-30, and the breakpoint located between the interstitial Oligo-pSc119.2-1 and the Oligo-pSc200 + 250 signaled sites of the 5RLKu arm (Figure 1A,B). Line 142-77 contained a 5RSKu arm, therefore, it was a 5RSKu(5B) monotelosomic substitution line (Figure 1C,D). Two broken 5RKu chromosomes existed in line 143-9, and the breakpoints were located between the Oligo-pSc200 + 250 signal sites and the telomeres of 5RLKu arms (Figure 1E,F). Lines 143-61, 403-50, and 237-2 were 5RKu(5B) disomic substitution, 5RLKu(5B) ditelosomic substitution, and 5RSKu/5BL translocation lines, respectively (Figure 2). Additionally, a 7B chromosome in line 142-30 had no short arm (Figure 1A) and a wheat 3D chromosome disappeared from line 143-9 (Figure 1E).

Line 444-12 contained two broken 5RKu chromosomes, and the breakpoints were located between the centromeres and the interstitial Oligo-pSc119.2-1 signal sites (Figure 3A,B). In line 449-4, another broken 5RKu chromosome was observed, and the breakpoint occurred on the 5RSKu arm (Figure 3C,D). A wheat 3D chromosome also disappeared from line 449-4 (Figure 3C,D). The chromosome compositions of the eight lines are listed in Table 1. According to the breakpoints, the 5RKu chromosome could be divided into six regions (Figure 4). The broken 5RKu chromosomes in lines 142-30, 143-9, 444-12, and 449-4 were named Del5RKuL3-4, Del5RKuL4, Del5RKuL2-4, and Del5RKuS1, respectively (Figure 4). Therefore, 142-30, 142-77, 143-9, 143-61, 237-2, 403-50, 444-12, and 449-4 comprised a set of 5RKu dissection lines.

**Figure 1.** ND-FISH analysis of root tip metaphase chromosomes of lines 142-30, 142-77, and 143-9 using Oligo-pTa535-1, Oligo-pSc119.2-1, Oligo-Ku, and Oligo-pSc200 + 250 as probes. (**A**) and (**B**) are the same cell, (**C**) and (**D**) are the same cell, and (**E**) and (**F**) are the same cell. Chromosomes were counterstained with 4-,6--diamidino-2-phenylindole (DAPI) (blue). Scale bar is 10 μm.

**Figure 2.** ND-FISH analysis of root tip metaphase chromosomes of lines 143-61, 403-50, and 237-2 using Oligo-pTa535-1, Oligo-pSc119.2-1, Oligo-Ku, and Oligo-pSc200 + 250 as probes. (**A**) and (**B**) are the same cell, (**C**) and (**D**) are the same cell, and (**E**) and (**F**) are the same cell. Chromosomes were counterstained with DAPI (blue). Scale bar is 10 μm.


**Table 1.** Information of the materials used in this study.

**Figure 3.** ND-FISH analysis of root tip metaphase chromosomes of lines 444-12 and 449-4 using Oligo-pTa535-1, Oligo-pSc119.2-1, Oligo-Ku, and Oligo-pSc200 + 250 as probes. (**A**) and (**B**) are the same cell, and (**C**) and (**D**) are the same cell. Chromosomes were counterstained with DAPI (blue). Scale bar is 10 μm.

**Figure 4.** Cut-pasted 5RKu dissections and the schematic diagram of 5RKu chromosome. (**A**) Cut-pasted 5RKu, 5RSKu, 5RLKu, and the four kinds of broken 5RKu. 5RKu chromosome was divided into six regions. (**B**) The schematic diagram of 5RKu chromosome. Red arrows indicate broken points. White arrow and "Cen" indicate centromere. Scale bar is 10 μm.

### *3.2. Developing 5RKu-Specific Markers and Physical Mapping*

Rye Kustro, CS, MY11, and the seven wheat-rye monosomic addition lines were used to develop 5RKu-specific markers. In total, 11 of the 542 primer pairs amplified specific bands from rye Kustro and MA5RKu, but not from CS, MY11, MA1RKu, MA2RKu, MA3RKu, MA4RKu, MA6RKu, and MA7RKu (Figure 5).

**Figure 5.** Developing 5RKu-specific markers. ( **A**) Products amplified by marker Ku5R-58. (**B**) Products amplified by marker Ku5R-516. M: DNA marker; CS: Chinese Spring; MY11: Mianyang 11; Kustro: rye kustro; 1R-7R: MA1RKu-MA7RKu. Arrows indicate the target bands.

Therefore, the 111 primer pairs were regarded as 5RKu-specific markers and the information of these markers is listed in Table S1. Subsequently, the 111 markers and the 52 5RKu-specific markers developed by Qiu et al. [24] were physically mapped to six regions of the 5RKu chromosome using lines 142-30, 142-77, 143-9, 143-61, 237-2, 403-50, 444-12, and 449-4 (Figures 6 and 7; Table S2).

**Figure 6.** Physically localized 5RKu-specific markers using 5RKu dissection lines. ( **A**) Products amplified by primer pair Ku5R-290 representing the markers mapped to region S1. (**B**) Products amplified by primer pair Ku5R-271 representing the markers mapped to region S2. ( **C**) Products amplified by primer pair Ku5R-120 representing the markers mapped to region L1. ( **D**) Products amplified by primer pair Ku5R-342 representing the markers mapped to region L2. (**E**) Products amplified by primer pair Ku5R-48 representing the markers mapped to region L3. (**F**) Products amplified by primer pair Ku5R-9 representing the markers mapped to region L4. ( **G**) Cut-pasted 5RKu chromosome, 5RSKu arm, 5RLKu arm, 5RSKu/5BL translocation chromosome, and the four kinds of broken 5RKu chromosomes corresponding to their own amplified products in each electrophoresis lane. Arrows indicate the target bands amplified by each marker.

**Figure 7.** The schematic diagram for the physical map of 5RKu-specific markers. These markers were mapped to six regions on 5RKu chromosome according to their amplicons in 5RKu dissection lines. The six regions are divided by five dark lines and named S1, S2, L1, L2, L3, and L4, respectively. The red arrows corresponding to each dark line indicate the breakpoints on the 5RKu chromosome. The blue arrow and "Cen" indicate the centromere. In each region, the names of the markers are listed on the right. In the schematic diagram, green bands represent FISH signals of Oligo-pSc119.2-1 and the red bands represent the FISH signals of Oligo-pSc200 + 250. The markers named "KU + number" were developed by Qiu et al. [24], and those named "Ku5R + number" were developed in this study.

Thirty markers amplified their target bands from lines 143-16 (5RKu), 142-77 (5RSKu), 237-2 (5RSKu/5BL), 444-12 (Del5RKuL2-4), 142-30 (Del5RKuL3-4), and 143-9 (Del5RKuL4) but not from lines 403-50 (5RLKu) and 449-4 (Del5RKuS1) (Figure 6A). So, the 30 markers were mapped to region S1 of the 5RKu chromosome (Figure 7). The target products of 15 markers only disappeared from line 403-50 (5RLKu); therefore, they were mapped to region S2 of the 5RKu chromosome (Figures 6B and 7). The target products of 34 markers only disappeared from lines 142-77 (5RSKu) and 237-2 (5RSKu/5BL), and they were mapped to region L1 of the 5RKu chromosome (Figures 6C and 7). The target products of 22 markers existed in lines 143-16 (5RKu), 403-50 (5RLKu), 142-30 (Del5RKuL3-4), 143-9 (Del5RKuL4), and 449-4 (Del5RKuS1) but not in lines 142-77 (5RSKu), 237-2 (5RSKu/5BL), and 444-12 (Del5RKuL2-4) (Figure 6D). Therefore, the 22 markers were mapped to region L2 of 5RKu chromosome (Figure 7). Thirty-nine markers did not amplify their target products from lines 142-77 (5RSKu), 237-2 (5RSKu/5BL), 444-12 (Del5RKuL2-4), and 142-30 (Del5RKuL3-4), and 23 markers did not amplify their target products from lines 142-77 (5RSKu), 237-2 (5RSKu/5BL), 444-12 (Del5RKuL2-4), 142-30 (Del5RKuL3-4), and 143-9 (Del5RKuL4) (Figure 6E,F). Therefore, the 39 and 23 markers were mapped to regions L3 and L4 of 5RKu chromosome, respectively (Figure 7).

### *3.3. Similarity between S. cereale Lo7 Sca*ff*olds and Pair-End Reads Used for 5RKu-Specific Primers Design*

The original pair-end reads that were used to design 5RKu-specific primer pairs were deposited in the GenBank Database (GenBank accession numbers: MN325158-MN325268) (Table S1). The corresponding pair-end reads to 5RKu-specific markers were used for nucleotide BLAST search against the *S. cereale* Lo7 scaffolds database using the blastn tool in GrainGenes [25] The *S. cereale* Lo7 scaffolds

served to validate the chromosomal localization of the majority of the 5RKu-specific markers. The results were listed in Table S1. The 111 pair-end reads had 83% to 100% similarity with some of the *S. cereale* Lo7 scaffolds (Table S1). The left and the right 1 to 60 bp nucleotide sequences of 13 pair-end reads, respectively, hit two Lo7 scaffolds that were derived from different rye chromosomes (Table S1). Twenty-two pair-end reads only hit the Lo7 scaffolds that derived from the 0R chromosome (Table S1). Four pair-end reads respectively hit the five Lo7 scaffolds that were derived from 1R, 3R, or 4R chromosomes (Table S1). The left and the right 1 to 60 bp nucleotide sequences of seven pair-end reads hit two different Lo7 scaffolds that were derived from 5R chromosome (Table S1). Sixty-five pair-end reads hit 65 single Lo7 scaffolds that were derived from the 5R chromosome, respectively (Table S1). Although there were 72 5R-derived Lo7 scaffolds matching the pair-end reads, only 65 Lo7 scaffolds were mapped to six regions of the 5RKu chromosome because they hit single scaffolds (Figure 8).

**Figure 8.** The schematic diagram for the physical map of *S. cereale* Lo7 scaffolds that were derived from the 5R chromosome. The names of the scaffolds are listed on the right and their corresponding markers are listed in brackets. The means of the other symbols are the same as the ones in Figure 7.

### *3.4. Location of Stripe Rust Gene(s) on 5RKu Chromosome*

The resistance of lines 142-30, 142-77, 143-9, 143-61, 237-2, 403-50, 444-12, and 449-4 and parental wheat MY11 to stripe rust was tested. According to the standard scale of infection types that was described by Wan et al. [27], the infection types of lines 143-16 (5RKu), 403-50 (5RLKu), 142-30 (Del5RKuL3-4), 143-9 (Del5RKuL4), and 449-4 (Del5RKuS1) were 2 to 3 (Figure 9). The infection types of MY11 and the lines 142-77 (5RSKu), 237-2 (5RSKu/5BL), and 444-12 (Del5RKuL2-4) were 8 to 9 (Figure 9). These results indicated that the 5RKu chromosome carried stripe rust resistance gene(s) and it was located on the 5RLKu arm. Further, the resistance gene(s) was mapped to regions L2-L4 because line 444-12 (Del5RKuL2-4) was highly susceptible to stripe rust (Figure 9). At last, the stripe rust resistance gene(s) could be mapped to region L2 of the 5RKu chromosome because lines 142-30 (Del5RKuL3-4) and 143-9 (Del5RKuL4) exhibited resistance to stripe rust (Figure 9).

**Figure 9.** Stripe rust resistance testing. MY11, and lines 142-77, 237-2, and 444-12 are highly susceptible to stripe rust. Lines 143-9, 142-30, 449-4, 403-50, and 143-16 display resistance to stripe rust.
