Physical Location of New Stripe Rust Resistance Gene(s) and PCR-Based Markers on Rye (Secale cereale L.) Chromosome 5 Using 5R Dissection Lines

The rye (Secale cereale L.) 5R chromosome contains some elite genes that can be used to improve wheat cultivars. In this study, a set of 5RKu dissection lines was obtained, and 111 new PCR-based and 5RKu-specific markers were developed using the specific length amplified fragment sequencing (SLAF-seq) method. The 111 markers were combined with the 52 5RKu-specific markers previously reported, and 65 S. cereale Lo7 scaffolds were physically mapped to six regions of the 5RKu chromosome using the 5RKu dissection lines. Additionally, the 5RLKu arm carried stripe rust resistance gene(s) and it was located to the region L2, the same region where 22 5RKu-specific markers and 11 S. cereale Lo7 scaffolds were mapped. The stripe rust resistance gene(s) located in the 5RLKu arm might be new one(s) because its source and location are different from the previously reported ones, and it enriches the resistance source of stripe rust for wheat breeding programs. The markers and the S. cereale Lo7 scaffolds that were mapped to the six regions of the 5RKu chromosome can facilitate the utilization of elite genes on the 5R chromosome in the improvement of wheat cultivars.


Introduction
Rye (Secale cereale L.) is a useful gene source supporting disease and insect resistance, stress resistance, and higher yield for wheat (Triticum aestivum L.) breeding programs [1][2][3][4][5][6]. In fact, rye 5R chromosome contains some elite genes that can be used to improve wheat cultivars. The wheat-rye 4BL/5RL translocation chromosome indicates that the 5RL arm carried loci that can increase copper efficiency [7]. The wheat-rye 5R(5A) and 5R(5D) substitution line exhibits some favorite traits, including big spike, multispikelet, high satiation seed, high protein content, and positive effects on somatic embryogenesis [8][9][10]. The rye 5RS arm carries the resistance gene to Russian wheat aphid (RWA) [11]. The 5R a S arm that was derived from S. africanum Stapf. carries genes for stripe rust resistance, increasing spike length and reducing grain hardness [12]. The gene Ddw1 that can reduce plant height in rye is located on the 5RL arm and its close linkage markers have been found [13]. It was also reported that the 5R chromosome harbors genes that determine its growth habit [14]. However, the elite genes on 5R chromosomes have not been successfully used in wheat cultivars because of linkage drag and the non-compensating and low recombination frequency of 5R with its wheat homologues [15,16]. Elite genes can be utilized effectively when segments of 5R chromosomes have been transferred into wheat backgrounds and been accurately identified. Rye chromosome-specific markers are conducive to 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.

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 MA5R Ku 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 5R Ku specific pair-end reads were obtained according to the methods described by Duan et al. [4]. Primers were designed according to 542 randomly selected 5R Ku 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.

PCR Assay, 5R Ku -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 MA5R Ku but not from CS, MY11, MA1R Ku , MA2R Ku , MA3R Ku , MA4R Ku , MA6R Ku , and MA7R Ku were regarded as 5R Ku -specific markers. For each of the primer pairs, PCR reactions were repeated three times. These markers combined with the 52 5R Ku -specific markers developed by Qiu et al. [24] were mapped to specific regions of the 5R Ku chromosome using the 5R Ku dissection lines. Again, PCR reactions of each 5R Ku -specific marker were repeated three times.

Similarity Searches of the Pair-End Reads Against S. cereale L. Lo7 Scaffolds
The original pair-end reads for designing the 5R Ku -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].

Stripe Rust Resistance Testing
The resistance of 5R Ku 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.

Developing 5R Ku -Specific Markers and Physical Mapping
Rye Kustro, CS, MY11, and the seven wheat-rye monosomic addition lines were used to develop 5R Ku -specific markers. In total, 11 of the 542 primer pairs amplified specific bands from rye Kustro

Developing 5R Ku -Specific Markers and Physical Mapping
Rye Kustro, CS, MY11, and the seven wheat-rye monosomic addition lines were used to develop 5R Ku -specific markers. In total, 11 of the 542 primer pairs amplified specific bands from rye Kustro

Developing 5R Ku -Specific Markers and Physical Mapping
Rye Kustro, CS, MY11, and the seven wheat-rye monosomic addition lines were used to develop 5R Ku -specific markers. In total, 11 of the 542 primer pairs amplified specific bands from rye Kustro and MA5R Ku , but not from CS, MY11, MA1R Ku , MA2R Ku , MA3R Ku , MA4R Ku , MA6R Ku , and MA7R Ku ( Figure 5).  Therefore, the 111 primer pairs were regarded as 5R Ku -specific markers and the information of these markers is listed in Table S1. Subsequently, the 111 markers and the 52 5R Ku -specific markers developed by Qiu et al. [24] were physically mapped to six regions of the 5R Ku 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).  Therefore, the 111 primer pairs were regarded as 5R Ku -specific markers and the information of these markers is listed in Table S1. Subsequently, the 111 markers and the 52 5R Ku -specific markers developed by Qiu et al. [24] were physically mapped to six regions of the 5R Ku Figure 5). Therefore, the 111 primer pairs were regarded as 5R Ku -specific markers and the information of these markers is listed in Table S1. Subsequently, the 111 markers and the 52 5R Ku -specific markers developed by Qiu et al. [24] were physically mapped to six regions of the 5R Ku 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).  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 5R Ku chromosome, 5RS Ku arm, 5RL Ku arm, 5RS Ku /5BL translocation chromosome, and the four kinds of broken 5R Ku 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 5R Ku -specific markers. These markers were mapped to six regions on 5R Ku chromosome according to their amplicons in 5R Ku 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 5R Ku 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.

Similarity between S. cereale Lo7 Scaffolds and Pair-End Reads Used for 5R Ku -Specific Primers Design
The original pair-end reads that were used to design 5R Ku -specific primer pairs were deposited in the GenBank Database (GenBank accession numbers: MN325158-MN325268) ( Table S1). The corresponding pair-end reads to 5R Ku -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 Agronomy 2019, 9, 498 9 of 14 served to validate the chromosomal localization of the majority of the 5R Ku -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 5R Ku chromosome because they hit single scaffolds (Figure 8).

Similarity between S. cereale Lo7 Scaffolds and Pair-End Reads Used for 5R Ku -Specific Primers Design
The original pair-end reads that were used to design 5R Ku -specific primer pairs were deposited in the GenBank Database (GenBank accession numbers: MN325158-MN325268) ( Table S1). The corresponding pair-end reads to 5R Ku -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 5R Ku -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 pairend 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 5R Ku chromosome because they hit single scaffolds (Figure 8).

R-Specific Markers
In our previous studies, some PCR-based and rye chromosome-specific markers have been developed using SLAF-seq technology [4,18,20]. 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][8][9][10][11][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 intermicrosatellite (SCIM) markers were found to be 5R-specific [29]. Tomita et al. [30] reported a 5Rspecific 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 5R a -specific molecular markers were developed [12]. Qiu et al. [24] developed 19 5RS Ku -specific and 33 5RL Ku -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 5R Ku -specific markers were mapped to six regions of the 5R Ku 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 5R Ku -specific markers were found and they were also mapped to the six regions of the 5R Ku 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 yet 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 5R Ku -specific pairend reads hit some Lo7 scaffolds derived from 1R, 3R, or 4R chromosomes might be the result from duplications, members of gene families, etc.

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][8][9][10][11][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 5R a -specific molecular markers were developed [12]. Qiu et al. [24] developed 19 5RS Ku -specific and 33 5RL Ku -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 5R Ku -specific markers were mapped to six regions of the 5R Ku 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 5R Ku -specific markers were found and they were also mapped to the six regions of the 5R Ku 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 yet 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 5R Ku -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 5R Ku 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.

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][34][35][36][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 5RL Ku 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 5R Ku 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 5RL Ku 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 5R Ku 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).

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.

Conclusions
In the present study, a new set of 5R Ku dissection lines was identified, and 111 new PCR-based and 5R Ku -specific markers were developed. The 111 markers were combined with the 52 5R Ku -specific markers reported previously, and 65 S. cereale Lo7 scaffolds were physically mapped to six regions of the 5R Ku chromosome. Additionally, the 5RL Ku arm carried stripe rust resistance gene(s) and it was mapped to region L2 of the 5R Ku chromosome, the same region where 22 5R Ku -specific markers and 11 S. cereale Lo7 scaffolds were mapped. The stripe rust resistance gene(s) located in the 5RL Ku arm enriches the resistance source of stripe rust for wheat breeding programs, and the markers and the 11 S. cereale Lo7 scaffolds that were mapped to the L2 region of the 5R Ku chromosome facilitates the utilization of the stripe rust resistance gene(s) in the improvement of wheat cultivars.