The Physical Location of Stripe Rust Resistance Genes on Chromosome 6 of Rye (Secale cereale L.) AR106BONE

It was reported that the chromosome 6R of rye (Secale cereale L.) carries stripe rust resistance gene Yr83, and the region with the candidate resistance gene(s) still needs to be narrowed down. This study confirmed that the chromosome 6RLAr derived from rye AR106BONE contains stripe rust resistance gene(s). A wheat-rye T6BS.6RLAr translocation chromosome, a wheat-rye small-segment translocation T6RLAr-6AS.6AL, and three kinds of deleted T6BS.6RLAr translocations, T6BS.6RLAr-1, T6BS.6RLAr-2, and T6BS.6RLAr-3, were identified. Translocations T6BS.6RLAr, T6BS.6RLAr-2, and T6RLAr-6AS.6AL were highly resistant to stripe rust and T6BS.6RLAr-1 and T6BS.6RLAr-3 were highly susceptible. The molecular markers specific to 6RL determined that the three regions of the 6RLAr arm from 732,999,830 bp to the telomere, from 735,010,030 to 848,010,414 bp, and from 848,011,262 bp to the telomere were deleted from T6BS.6RLAr-1, T6BS.6RLAr-2, and T6BS.6RLAr-3, respectively. T6BS.6RLAr-2 and T6RLAr-6AS.6AL contained the segment that was deleted in T6BS.6RLAr-3. Therefore, it can be concluded that about 37 Mb segment from 848,011,262 bp to the telomere carried stripe rust resistance gene(s), and it was smaller than that with the Yr83 gene. Gene annotation indicated that about 37 Mb region contains 43 potential resistance genes, and 42 of them are nucleotide-binding site and leucine-rich repeat (NBS-LRR)-like resistance protein genes. The results in this study narrowed down the size of the region with candidate stripe rust resistance gene(s) on the 6RL arm, and the T6RLAr-6AS.6AL is a promising small-segment translocation for improvement of wheat cultivars.


INTRODUCTION
Wheat stripe rust is caused by Puccinia striiformis f. sp. tritici (Pst) and is one of the most serious diseases in wheat. Developing wheat cultivars with resistance to stripe rust is the most practical way to control this disease. More than 80 Yr (yellow rust resistance) genes were officially named; however, most of them lost their resistance because of variations in the prevalence of virulent pathotypes (Ren et al., 2022).
The resistance to stripe rust of 103 wheat lines was tested using pathogenic races CYR32, CYR33, and CYR34, which are currently prevalent in China, and only Yr5, Yr15, and Yr45 exhibited all-stage resistance, and only Yr41, Yr47, and Yr50 were adult plant resistant (Hu et al., 2022). The replacement of historically clonal Pst races by new ones occurred continually (Jamil et al., 2020;Bouvet et al., 2022). Therefore, it is important to explore new resistance genes to stripe rust and enrich the resource pool for wheat resistance breeding.
Wheat-related species contain abundant resistance genes. For example, rye (Secale cereale L.) is an important gene source for wheat disease resistance breeding (Spetsov and Daskalova, 2022). Stripe rust gene Yr 9 located on the 1RS arm was successfully used in commercial wheat cultivars. Additionally, 2R, 4R, 5R, 6R, and 7R chromosomes also carry stripe rust-resistant genes (Lei et al., 2011;Li et al., 2016aLi et al., , 2020aSchneider et al., 2016;An et al., 2019;Xi et al., 2019;Johansson et al., 2020;Ren et al., 2020). Four of these reports indicated that rye chromosome 6R carried stripe rust resistance genes, and they were derived from different rye sources (Schneider et al., 2016;Johansson et al., 2020;Li et al., 2020a,b). A new stripe rust resistance gene on chromosome 6R was named Yr83, and it was located in a bin FL0.73-1.00 of 6RL (Li et al., 2020b). To clone the stripe rust resistance gene(s) on 6RL, it is necessary to narrow the segment with resistant gene(s). In this study, a wheat-rye 6R Ar monosomic addition line (MA6R Ar ) with resistance to stripe rust, a 6RS Ar monotelosomic addition line (MTA6RS Ar ), a 6RL Ar monotelosomic addition line (MTA6RL Ar ), a T6BS.6RL Ar translocation line, and three kinds of deleted T6BS.6RL Ar translocation lines were used to locate the stripe rust resistance gene(s) on a smaller segment of 6RL Ar arm, and some resistance protein genes in this segment were found.

Plant Materials
Octoploid triticale lines Mianyang 11/AR106BONE-4 (MAR4) and Mianyang 11/Kustro (MK) were derived from common wheat (Triticum aestivum L.) Mianyang 11 (MY11) × Secale cereale L. AR106BONE and MY11 × Secale cereale L. Kustro, respectively. Crossing between MAR4 and a wheat line J1025 (T. aestivum L.) and between MK and MY11 was carried out. From the progenies of MAR4 × J1025 and MK × MY11, wheat-rye 6R monosomic addition lines MA6R Ar and MA6R Ku were identified, respectively. These monosomic addition lines were used to investigate the stripe rust resistance of the two kinds of 6R chromosomes. MTA6RS Ar and MTA6RL Ar were identified from the selfed progeny of MA6R Ar . The seeds of MA6R Ar were irradiated with 60 Co-γ rays at a dosage of 200 Gy at Biotechnology and Nuclear Technology Research Institute, Sichuan Academy of Agricultural Sciences, China. From the progeny of irradiated MA6R Ar , a wheat-rye T6BS.6RL Ar translocation line was obtained, and the seeds derived from this translocation line were also irradiated with 60 Co-γ rays and some deleted T6BS.6RL Ar translocation chromosomes were detected. The rye Kustro and AR106BONE and the common wheat MY11 and J1025 were kept in the seed store in our laboratory.

Development of 6RL-Specific Markers
Primers were designed according to the 6RL sequence of rye Lo7 (Rabanus-Wallace et al., 2021) using Primer 3 software (version 4.0), and the optimal melting temperature and size values were set to 60 • C and to 20 bases, respectively. In total, 423 pairs of primers were designed. Additionally, 124 developed 6RL Ku -specific length amplified fragment sequencing (SLAF-seq) markers (Li et al., 2016b) were also used. The PCR amplification and agarose gel electrophoresis were performed following the procedure described by Li et al. (2016b). Chinese Spring (CS), MY11, T6BS.6RL Ar translocation line, and rye AR106BONE were used to test the 6RL Ar specificity of the 423 newly designed primer pairs and the 124 6RL Ku -specific SLAF-seq markers. All the 6RL Ar -specific markers were physically mapped to specific regions of the 6RL Ar arm using deleted T6BS.6RL Ar translocation lines. The sequences of the 124 SLAF-seq primer pairs were used for nucleotide Basic Local Alignment Search Tool (BLAST) searches against the 6R sequence of rye Lo7 (Rabanus-Wallace et al., 2021) using a BLAST tool in the Triticeae Multi-omics Center (http: //202.194.139.32/), and the positions of these markers on 6RL arm were determined according to the BLAST results.

Cloning Partial Sequences of the Candidate Resistance Genes From 6RL Ar
To confirm that the candidate resistance genes on the 6RL arm of Lo7 are similar to that of 6RL Ar , the sequences of two candidate resistance genes SECCE6Rv1G0449960.1 and SECCE6Rv1G0453070.1 of Lo7 were randomly selected for  designing primer pairs. Primer pairs of P60 (5 ′ TGGGG AATAG CTGGC ATTGG3 ′ , 5 ′ TCGGT AGGGT AGACG GTGAG3 ′ ) and P70 (5 ′ AATGG GAGGA CTCTT GCGTG3 ′ , 5 ′ CTGGG AATGA ACCGA CAGCT3 ′ ) were used to amplify the partial sequences of the two genes from 6RL Ar arm. The PCR reactions and product separation were also performed according to the methods described by Li et al. (2016b), and the annealing temperature of the primer pairs was 60 • C. The target products amplified by the two primer pairs, P60 and P70, were cloned and then sequenced by the Tsingke Biotechnology Co., Ltd.

Stripe Rust Response Test
The response of parental wheat J1025, MY11, lines MA6R Ku , MA6R Ar , MTA6RS Ar , MTA6RL Ar , T6BS.6RL Ar , and the deleted T6BS.6RL Ar to stripe rust was evaluated. The mixed stripe rust prevalent isolates CYR32, CYR33, and CYR34 were used to inoculate seedlings in field according to the method described by Xi et al. (2019). A 0-9 numerical scale of infection types (IT) was scored according to the standard described by Wan et al. (2017) at the adult stage. The disease resistance of J1025, MY11, the lines MA6R Ku , MA6R Ar , MTA6RS Ar , MTA6RL Ar , and T6BS.6RL Ar was evaluated in 2018-2019 in Qionglai, Sichuan Province, China and in 2019-2020, 2020-2021, and 2021-2022

Identification of Wheat-Rye 6R Addition and Translocation Lines
The ND-FISH based on oligo probes was used to analyze the progeny of wheat × rye. Wheat-rye 6R monosomic addition lines MA6R Ar and MA6R Ku were identified (Figures 1A-D). Both the short arms of the 6R Ar and 6R Ku chromosomes (6RS Ar and 6RS Ku ) contained signals of Oligo-pSc119.2-1 and Oligo-pSc250, and no signals of Oligo-pSc250 were observed on their long arms (6RL Ar and 6RL Ku ) ( Figure 1E). The probe Oligo-pSc119.2-1 produced three and four signal bands on 6RL Ar and 6RL Ku , respectively ( Figure 1E). Two and three signal bands of probe Oligo-pSc200 were observed on 6RL Ar and 6RL Ku arms, respectively ( Figure 1E). The results indicated that the structure of the long arms of the 6R Ar and 6R Ku chromosomes is different. Line MA6R Ar was highly resistant to stripe rust (IT = 1), whereas line MA6R Ku was susceptible (IT = 9; Figure 1E).
From the selfed progeny of line MA6R Ar , the 6RS Ar and the 6RL Ar monotelosomic addition lines, MTA6RS Ar and MTA6RL Ar , were identified (Figures 2A,B). Line MTA6RS Ar was susceptible to stripe rust (IT = 9), and line MTA6RL Ar displayed high resistance (IT = 1; Figure 2C). In total, 200 seeds of MA6R Ar were irradiated. A plant 18T231-46 containing a wheat-rye T6BS.6RL Ar translocation chromosome was identified from 1,182 M1 seeds, and a line 19T177-21 containing a pair of T6BS.6RL Ar translocations was identified from 30 seeds of the selfed progeny of 18T231-46 (Figures 3A,B). Additionally, 104 seeds of the selfed progeny of 18T231-46 were also irradiated. Four lines, 21F1, 21F3, 21F7, and 21F11, were identified from the 1,090 seeds (M2) of the progeny of irradiated 18T231-46. In these lines, three kinds of deleted T6BS.6RL Ar translocation chromosomes and a small-segment translocation T6RL Ar -6AS.6AL were found (Figures 3C-I). Line 21F1 contained two deleted translocation chromosomes T6BS.6RL Ar -1, on which the Oligo-pSc200 signals and the distal Oligo-pSc119.2-1 signals on the 6RL Ar arms were disappeared (Figures 3C,I). Line 21F3 contained two deleted translocation chromosomes T6BS.6RL Ar -2, carrying one Oligo-pSc200 signal band and two intercalary Oligo-pSc119.2-1 signal bands (Figures 3D,I). Line 21F7 contained a pair of deleted translocation chromosomes T6BS.6RL Ar -3, and the signals of Oligo-pSc200 on telomeric regions were disappeared (Figures 3E,I). In line 21F11, probe Oligo-pSc200 produced signals on the telomeric regions of the short arms of chromosomes 6A (Figures 3F-I). Therefore, line 21F11 contained a pair of small-segment translocations T6RL Ar -6AS.6AL and a pair of T6BS.6RL Ar -3 (Figures 3F-I). The weak GISH signals on the chromosomes T6RL Ar -6AS.6AL indicated that the segment of 6RL Ar involved in translocation is small (Figures 3H,I).

The Physical Location of 6RL Ar -Specific Markers
Among the 423 newly designed primer pairs, 204 amplified specific bands from T6BS.6RL Ar translocation line and rye AR106BONE, but not from CS and MY11, indicated that these 204 primer pairs are 6RL Ar -specific markers (Figure 4 FIGURE 5 | Ideograms of wheat-rye 6RL translocation chromosomes and stripe rust response test. (A) Ideograms of T6BS.6RL Ar , deleted T6BS.6RL Ar, and the small-segment translocation T6RL Ar -6AS.6AL. Brown, blue, and orange indicate segments of 6B, 6R, and 6A chromosomes, respectively. Green and red bands indicate the signals of Oligo-pSc119.2-1 and Oligo-pSc200, respectively. "BR" indicates a break point. The right brace indicates the 6RL Ar segment that was transferred on the 6A chromosome. (B) Parental wheat J1025 and MY11 and lines 21F1 and 21F7 were highly susceptible to stripe rust and lines 19T177-21, 21F3, and 21F11 were highly resistant.
and Supplementary Table S1). Additionally, 95 of the 124 SLAF-seq markers amplified target bands from both T6BS.6RL Ar and AR106BONE. A total of 299 (204 + 95) 6RL Ar -specific markers were obtained, and their target amplification regions on the 6RL arm are listed in Supplementary Table S1. The breakpoints on the 6RL Ar arms of T6BS.6RL Ar -1, T6BS.6RL Ar -2, and T6BS.6RL Ar -3 were determined using the 299 markers. In total, 103 of the 299 markers amplified 6RL Ar -specific bands from translocation T6BS.6RL Ar -1 (Figure 4A), and these markers covered the region of 6RL from 333,302,151 to 732,900,689 bp (Supplementary Table S1). The first distal marker (Lo7.6RL-71) that did not amplify products from T6BS.6RL Ar -1 occupied the region from 732,999,830 to 733,000,692 bp (Supplementary Table S1). Therefore, the breakpoint in T6BS.6RL Ar -1 was located in the region of the 6RL Ar arm between 732,900,689 and 732,999,830 bp ( Figure 5A and Supplementary Table S1). Totally, 118 and 52 markers that were distributed into two regions of 6RL from 333,302,151 to 733,480,409 bp and from 848,011,262 to 885,003,466 bp, respectively amplified target bands from T6BS.6RL Ar -2, and 129 markers that distributed the region of 6RL from 735,010,030 to 848,010,414 bp did not produce amplicons from this translocation chromosome (Figures 4B-D and Supplementary Table S1). It can be determined that the two breakpoints in T6BS.6RL Ar -2 occurred in the two regions of 6RL Ar arm between 733,480,409 and 735,010,030 bp and between 848,010,414 and 848,011,262 bp ( Figure 5A and Supplementary Table S1). For T6BS.6RL Ar -3 translocation, 247 markers that were distributed on the 6RL region from 333,302,151 to 848,010,414 bp amplified target bands, and the rest 52 markers, which were the same as those amplified products from T6BS.6RL Ar -2, did not amplify amplicons ( Figure 4D and Supplementary Table S1). According to the amplification of these markers, the breakpoint regions on 6RL Ar in the three deleted T6BS.6RL Ar translocations can be determined (Figure 5A and Supplementary Table S1). For the T6BS.6RL Ar -3 chromosome, the breakpoint between 848,010,414 and 848,011,262 bp was the same as the second breakpoint in the T6BS.6RL Ar -2 chromosome ( Figure 5A and Supplementary Table S1). Therefore, the segments from 732,999,830 bp to telomere, from 735,010,030 to 848,010,414 bp, and from 848,011,262 bp to telomere of 6RL Ar were deleted from T6BS.6RL Ar -1, T6BS.6RL Ar -2, and T6BS.6RL Ar -3, respectively ( Figure 5A). The 6RL Ar segment deleted from T6BS.6RL Ar -3 was transferred to T6RL Ar -6AS.6AL (Figures 4, 5A).

Location of Stripe Rust Resistance Genes on Segment of 6RL Ar
Stripe rust response test indicated that translocation lines T6BS.6RL Ar , 21F3 (T6BS.6RL Ar -2), and 21F11 (T6BS.6RL Ar -3 and T6RL Ar -6AS.6AL) were highly resistant to stripe rust (IT = 1), and translocation lines 21F1 (T6BS.6RL Ar -1) and 21F7 (T6BS.6RL Ar -3) were highly susceptible (IT = 9; Figure 5B). According to the resistance of 21F3, 21F7, and 21F11, and the 6RL Ar segments existed in T6BS.6RL Ar -2, T6BS.6RL Ar -3, and T6RL Ar -6AS.6AL, it was determined that the 6RL Ar segment in T6RL Ar -6AS.6AL carried the stripe rust resistance gene. Therefore, it was determined that the 6RL Ar region from 848,371,946 bp to the telomere (885,153,844 bp) carried the stripe rust resistance gene. Gene annotation indicated that there are 43 potential resistance genes located in the region from 848,011,885 to 885,153,844 bp of chromosome 6R (Supplementary Table S2). Only one of the 43 genes belongs to the kinase family protein gene and the other 42 genes are nucleotide-binding site and leucine-rich repeat (NBS-LRR)-like resistance protein genes (Supplementary Table S2).

Polymorphism of 6R Chromosomes
The complex cytological structure of the 6R chromosome has been reported. It was known that the long arm of the rye 6R chromosome contains homologous groups 3 and 7 segments in the distal region (Devos et al., 1993;Li et al., 2013). FISH signal patterns of tandem repeats pSc119.2 on S. cereale 6R and S. africanum 6R afr were different, and this indicated the structural alteration between the two kinds of 6R chromosomes (Li et al., 2020a). In this study, the cytological structure of 6R chromosomes derived from rye AR106BONE and Kustro is different. Some 6RL Ku -specific SLAF-seq markers that did not amplify products from 6R Ar also exhibited the diversity between chromosomes 6R Ku and 6R Ar . The 6RL arm with stripe rust resistance gene Yr83 carried weak pSc119.2 signals in the intercalary and telomeric regions (Li et al., 2020b), and it is different from that of the 6RL Ar arm. However, the pSc119.2 signal pattern of 6RL Ar is similar to that on the 6RL arm of rye cv. Qinling (Hao et al., 2018). These results exhibited the abundant genetic diversity of chromosome 6R in the genus Secale. Therefore, it is necessary to continue to study the allelic variations of 6R chromosomes as they may enrich the genetic diversity of the resistance genes on them. Especially, the difference among the stripe rust resistance genes on different 6R chromosomes needs to be confirmed. Additionally, the results in this study make us think about whether the stripe rust resistance of the 6RL segment is controlled by a single gene or multiple genes.

Using 6R Deletions to Locate Stripe Rust Resistance Genes
Rye chromosome deletion lines are useful for the location of resistance genes. The powdery mildew resistance gene Pm56 was located in the subtelomeric region of the 6RS arm using 6R deletion lines (Hao et al., 2018). The stripe rust resistance gene Yr83 was mapped to the bin of FL0.73-1.00 of 6RL using deletion mapping (Li et al., 2020b). Similarly, 6R chromosome deletion and translocation lines were used to locate the powdery mildew resistance gene on the 6RL arm at FL0.85-1.00 and the stripe rust resistance gene on the 6RS arm at FL0.95-1.00 (Li et al., 2020a). However, the detailed information about the physical location of these reported resistance genes on 6R chromosomes is still unclear. In this study, 6RL-specific markers have been anchored to the exact physical positions on the 6R chromosome using reference genomic sequences of rye, and these markers combined with deleted T6BS.6RL translocation chromosomes were used to reveal that the 6RL segment with stripe rust resistance gene(s) is about 37 Mb. The primer sequences of the 33 markers that were located in the bin with the Yr83 gene were used for nucleotide BLAST searches against the 6R sequence of rye Lo7 (Rabanus-Wallace et al., 2021) using a BLAST tool in the Triticeae Multi-omics Center (http: //202.194.139.32/), and these markers covered the 6R segment between 743,894,430 and 880,064,740 bp. Therefore, in this study, the 6RL segment with stripe rust resistance is smaller than that reported by Li et al. (2020b), and this narrows down the candidate genes with stripe rust resistance on 6R chromosome. The high similarity of the candidate resistance gene sequences between rye Lo7 and AR106BONE indicated that the physical mapping based on the reference genome of rye Lo7 can be applied to 6RL Ar arm. Additionally, the new markers developed in this study enrich the 6R-specific molecular markers. It is a pity that the effect of the T6RL Ar -6AS.6AL chromosome on agronomic traits is unclear because it exists in a plant together with the T6BS.6RL Ar -3 chromosome. To get rid of the T6BS.6RL Ar -3, the cross between line 21F11 and some wheat lines that are susceptible to stripe rust has already been carried out. The T6RL Ar -6AS.6AL chromosome is a promising small-segment translocation for the improvement of wheat cultivars.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
ZT and SF conceived and designed the study, created the materials, analyzed the data, and wrote the manuscript. YD and JL performed the experiments and analyzed the data. ZY and GL created the materials. All authors read and approved the final manuscript.