Development of novel wheat-rye 6RS small fragment translocation lines with powdery mildew resistance and physical mapping of the resistance gene PmW6RS

Novel wheat-rye 6RS small fragment translocation lines with powdery mildew resistance were developed, and the resistance gene PmW6RS was physically mapped onto 6RS-0.58–0.66-bin corresponding to 18.38 Mb in Weining rye. Rye (Secale cereale L., RR) contains valuable genes for wheat improvement. However, most of the rye resistance genes have not been successfully used in wheat cultivars. Identification of new rye resistance genes and transfer of these genes to wheat by developing small fragment translocation lines will make these genes more usable for wheat breeding. In this study, a broad-spectrum powdery mildew resistance gene PmW6RS was localized on rye chromosome arm 6RS using a new set of wheat-rye disomic and telosomic addition lines. To further study and use PmW6RS, 164 wheat-rye 6RS translocation lines were developed by 60Coγ-ray irradiation. Seedling and adult stage powdery mildew resistance analysis showed that 106 of the translocation lines were resistant. A physical map of 6RS was constructed using the 6RS translocation and deletion lines, and PmW6RS was localized in the 6RS-0.58–0.66-bin, flanked by markers X6RS-3 and X6RS-10 corresponding to the physical interval of 50.23–68.61 Mb in Weining rye genome. A total of 23 resistance-related genes were annotated. Nine markers co-segregate with the 6RS-0.58–0.66-bin, which can be used to rapidly trace the 6RS fragment carrying PmW6RS. Small fragment translocation lines with powdery mildew resistance were backcrossed with wheat cultivars, and 39 agronomically acceptable homozygous 6RS small fragment translocation lines were obtained. In conclusion, this study not only provides novel gene source and germplasms for wheat resistance breeding, but also laid a solid foundation for cloning of PmW6RS.


Introduction
Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the major food crops in the world, but its production is threatened by various diseases (Singh et al. 2016). Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease of wheat occurring throughout the wheat-growing areas worldwide. Growing resistant cultivars is the most economical, effective, and environmentally friendly way to control this disease. However, the limited disease resistance gene diversity of cultivars and the prevalence of new virulent races capable of overcoming deployed resistance genes make it urgent to continuously explore new gene sources of broad-spectrum resistance and apply these genes to wheat breeding.
Rye (Secale cereale L., 2n = 2x = 14, RR), a close relative of common wheat, possesses valuable genes for biotic and abiotic stress resistance, high yield, and high-protein content for wheat improvement . The 1RS chromosome arm of rye carries multiple disease resistance genes, such as Pm8, Pm17,PmCn17,Sr31,Sr50,Yr9,YrCn17,YrR212,and Lr26, as well as genes associated with high yield and wide adaptability, which were transferred to wheat in the form of T1RS . 1BL and T1RS . 1AL translocations (Hurni et al. 2013;Mago et al. 2005Mago et al. , 2015 Communicated by Thomas Miedaner. Ren et al. 2009;Singh et al. 2018;Wang et al. 2017;Yang et al. 2016a). In addition to 1RS, other rye chromosomes also contain powdery mildew resistance genes and were introduced into wheat in the form of wheat-rye addition lines, substitution lines, or translocation lines. Pm7 derived from Rosen rye 2RL was transferred into wheat in the form of T4BS . 4BL-2RL translocation (Driscoll et al. 1965), Pm56 from Qinling rye 6RS was brought into wheat in the form of T6RS . 6AL translocation (Hao et al. 2018;Ren et al. 2022b), and Pm20 from Prolific rye was introduced into wheat in the form of T6BS . 6RL translocation (Friebe et al. 1994). Recent studies also reported some other rye chromosomes carried novel powdery mildew resistance genes, including 1RS from Weining and Baili rye (Ren et al. , 2018, 1R from Austrian rye (Yang et al. 2016b), 2RL from Jingzhou and German White rye (An et al. 2006;Zhuang et al. 2011), 4RL from Kustro and German White rye Fu et al. 2014;Ma et al. 2020), 6RL from Kustro, Qinling, Jingzhou, and German White rye Du et al. 2018;Ren et al. 2022a;Wang et al. 2010), and 7RL from Baili rye (Ren et al. 2020). However, with the prevalence of new virulent races, the widely used rye powdery mildew resistance genes Pm7, Pm8, and Pm17 are no longer effective, and the ratio of rye powdery mildew resistance genes being applied in wheat breeding is very low because of agronomic disadvantages, possibly caused by linkage drag of the rye chromosomes (Zeng et al. 2014). Small fragment translocation lines may eliminate the deleterious effects carried by the alien chromosomes and make the alien genes more available for wheat breeding (Dilbirligi et al. 2004). Many efforts have been made to produce small fragment translocation lines of wheat relatives, such as wheat-rye 2RL small fragment translocation lines with stripe rust (Puccinia striiformis f.sp. tritici) resistance , wheat-rye 6RL small fragment translocation line with powdery mildew resistance (Du et al. 2018), wheat-Thinopyrum ponticum ) and wheat-Th. elongatum (Guo et al. 2023;Zhang et al. 2022) 7EL small fragment translocation lines with fusarium head blight resistance, wheat-Th. ponticum small fragment translocation lines with powdery mildew and stem rust (Puccinia graminis f. sp. tritici) race Ug99 resistance Niu et al. 2014), wheat-Agropyron cristatum 6PS small fragment translocation lines with stripe rust resistance (Zhang et al. 2017b), and wheat-Haynaldia villosa 4VS small fragment translocation lines with wheat yellow mosaic virus resistance (Zhao et al. 2013). The small fragment translocation lines transferred alien resistance genes into wheat without affecting yield traits can be used as appropriate germplasm for wheat resistance breeding.
The development of translocation lines is not only crucial for the successful use of alien genes for wheat improvement, but also useful for constructing physical and cytological maps, mapping genes, and allocating molecular markers to specific physical regions together with the deletion lines. For instance, in rye (Du et al. 2018;Duan et al. 2017;Li et al. 2020aLi et al. , 2020bXi et al. 2019), Ag. cristatum (Lin et al. 2022;Song et al. 2016;Zhang et al. 2017b), and H. villosa (Dai et al. 2020;Zhao et al. 2013), translocation lines and deletion lines were used to divide chromosome regions and localize resistance genes.
In a previous study, a whole set of wheat-rye disomic and telosomic addition lines were developed by distant hybridization between wheat 843 and rye PI613196 ). In the present study, a broad-spectrum powdery mildew resistance gene, tentatively designated as PmW6RS, was localized on rye chromosome arm 6RS using this set of wheat-rye addition lines. Wheat-rye 6RS small fragment translocation lines with powdery mildew resistance were developed and transferred to wheat cultivars. Physical mapping of the locus of PmW6RS was conducted. Gene annotation based on the Weining rye reference genome ) was carried out, and molecular markers were developed which can be used to trace the resistance gene effectively in wheat breeding. The newly developed wheat-rye 6RS small fragment translocation lines carrying PmW6RS will be valuable germplasms for wheat resistance breeding.

Plant materials
The wheat cultivar parent 843, the diploid rye parent PI 613196, and the set of wheat-rye disomic and telosomic addition lines developed by distant hybridization between 843 and PI 613196 were kindly provided by Prof. Fangpu Han, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing . Susceptible wheat cultivars Gao 8901, Shixin 828, and Shiluan 02-1 were used as recurrent parents.

Assessment of powdery mildew resistance
Powdery mildew reactions of wheat 843, rye PI 613196, and the wheat-rye disomic and telosomic addition lines at the seedling stage were assessed three times using the widely prevalent single-spore Bgt isolate E09 in the greenhouse as previously described (Qie et al. 2019). For each line, 20 seedlings identified by genomic in suit hybridization (GISH) were planted in rectangular trays (54 × 28 × 4.2 cm) with 128 wells (3 × 3 × 4.2 cm), which were placed in the greenhouse with daily light of 14 h at 22 °C and darkness of 10 h at 18 °C and a relative humidity > 80%. The seedlings were inoculated with fresh conidia at the one-leaf stage. Infection types (ITs) were recorded 15 days after inoculation when the pustules were fully developed on the first leaf of the susceptible control Mingxian 169, with IT 0-2 being considered resistant and IT 3-4 susceptible (Si et al. 1992).
The wheat-rye 6RS telosomic addition line along with Mingxian 169 and 48 wheat genotypes carrying known Pm gene(s) as controls, including LM47-6 with Pm56 derived from rye 6RS chromosome arm, TAM104R/Thatcher with Pm20 derived from 6RL chromosome arm, Kavkaz with Pm8 and Amigo with Pm17 both from 1RS chromosome arm, and CI14189 with Pm7 from 2RL chromosome arm, were tested for powdery mildew resistance at the seedling stage to 24 single-pustule-derived Bgt isolates by separate inoculations with three replicates in the greenhouse as above described.
Powdery mildew resistance assessment at the adult stage of wheat-rye 6RS and 6RL addition lines, wheat 843, and rye PI 613196 was carried out using a mixture of Bgt isolates E09, E18, and E20 collected from northern China to infect field nurseries in early spring from 2017 to 2022 (five growing seasons with three replicates each year) at Luancheng Agro-Ecological Experimental Station, Chinese Academy of Sciences, Shijiazhuang, China (37°53′15′′N, 114°40′47′′E). Each line was planted in two 1.5-m rows with 20 seeds per row and 25-cm apart, and all lines were surrounded by Mingxian 169 as inoculum spreader. After heading stage, when Mingxian 169 showed severe disease symptoms, powdery mildew reactions were scored using a 0-9 scale, with 0-4 being considered resistant and 5-9 being considered susceptible (Sheng and Duan 1991).

Development and improvement of wheat-rye 6RS translocation lines
Wheat-rye 6RS translocation lines were developed by pollen irradiation. The spikes of wheat-rye 6RS and 6R addition lines at flowering stage were radiated by 60 Coγ-ray with a dose of 18 Gy and dosage rate of 1 Gy/min (Guo et al. 2023). The pollens were then used to pollinate Gao 8901, Shixin 828, and Shiluan 02-1 with the stamens removed in advance. The F 1 seeds were screened by GISH to identify wheat-rye 6RS translocation lines.
The breakpoints of 6RS translocation and deletion chromosomes were estimated by comparing the physical length of the 6RS translocation and deletion chromosomes with the complete 6RS chromosome by 6RS-specific markers. For each 6RS translocation and deletion line, 5-10 metaphase root-tip cells were photographed, and the length of the 6RS chromosome segments was measured using Photoshop CS 6.0. The centromere of the 6RS chromosome is considered as 0, while the telomere is considered as 1.
Powdery mildew reactions of the 6RS translocation and deletion lines were assessed three times at both seedling and adult stages. Wheat-rye 6RS telosomic addition line, wheat recurrent parents Gao 8901, Shixin 828, and Shiluan 02-1, and susceptible control Mingxian 169 were also included in the test. The seedlings were inoculated with Bgt isolate E09. The plants were then transplanted to the field nursery to test their powdery mildew reactions at adult stage using a mixture of Bgt isolates comprised of E09, E18, and E20.
We focused on 6RS small fragment translocation lines with the translocation fragments significant shorter than the entire 6RS chromosome arm and still immune to powdery mildew to develop 6RS small fragment translocation germplasms. The 6RS small fragment translocation lines were backcrossed three times with wheat recurrent parents Gao 8901, Shixin 828, and Shiluan 02-1 to replace chromosome mutations induced by irradiation and improve their agronomic performance. Homozygous 6RS translocation germplasms were obtained by selfing of the plants with desirable performance, and their genetic constitutions were detected by GISH and non-denaturing fluorescence in situ hybridization (ND-FISH).

GISH and ND-FISH analyses
GISH was used to detect wheat-rye 6RS translocation and deletion lines. Chromosome spread preparation was performed as previously described . Genomic DNA of rye was labeled with Alexa Fluor-488-dUTP (Invitrogen). The centromeric retrotransposon of wheat (CRW) (Zhang et al. 2004) was labeled with Texas-red-5-dUTP (PerkinElmer, USA). Chromosomes were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Images were taken using an epifluorescence Olympus BX53 microscope fitted with a cooled CCD digital camera and processed with Photoshop CS 6.0.

Molecular marker development for physical mapping of 6RS chromosome arm
6RS-specific markers were used to detect wheat-rye 6RS translocation and deletion lines, which were designed based on sequence differences between the annotated disease resistance-related genes of Weining rye ) and Chinese Spring RefSeq v2.1 (Zhu et al. 2021), and based on specific length amplified fragment sequencing (SLAFseq) ). These 6RS-specific markers were mapped onto the physical map of 6RS chromosome arm.
All the primers used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR amplification system contained 5 μl of 2 × Taq Master Mix (Vazyme Biological, Nanjing, China), 1 μl of template DNA (100 ng/μl), 1 μl of a mixture of forward and reverse primers (10 μmol), and up to 10 μl of ddH 2 O. The PCR procedure was as follows: initial denaturation at 94 °C for 5 min, followed by 36 cycles of denaturing at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 40 s, and a final extension at 72 °C for 10 min. The PCR products were separated in 8% non-denaturing polyacrylamide gels with 29:1 ratio of acrylamide and bis-acrylamide, and silver-stained prior to visualizing the banding patterns.

Results
The powdery mildew resistance gene in rye PI 613196 was preliminarily localized on 6RS chromosome arm Rye PI 613196 was immune to Bgt isolate E09 at the seedling stage and a mixture of Bgt isolates E09, E18, and E20 at the adult stage (Fig. 1). To explore which chromosome was involved in powdery mildew resistance, seedling responses of a set of wheat-rye addition lines of wheat 843 and rye PI 613196 to Bgt isolate E09 were examined. The results showed that wheat-rye 6R addition line and rye parent PI 613196 were immune to E09 with IT 0, while wheat-rye 1R, 2R, 3R, 4R, 5R, and 7R addition lines and the wheat parent 843 were highly susceptible with high IT (4) (Fig. 1a). To further determine which chromosome arm carried the powdery mildew resistance gene, the wheat-rye 6RS and 6RL telosomic addition lines were tested with Bgt isolate E09. The results showed that the 6RS telosomic addition line was immune with an IT 0, while 6RL addition line was highly susceptible with a high IT (4) (Fig. 1a). These results suggested that a powdery mildew resistance gene was localized on rye chromosome arm 6RS, which was tentatively named PmW6RS.

Powdery mildew responses of the wheat-rye 6RS telosomic addition line to different Bgt isolates
The 6RS telosomic addition line and 48 wheat genotypes carrying known Pm genes were tested for their responses to 24 Bgt virulent isolates at the seedling stage. The results showed that 6RS telosomic addition line was immune to all the 24 isolates, which was different from the genotypes with documented Pm genes from rye, including LM47-6 with Pm56 derived from 6RS of Qinling rye resistant to seven while susceptible to 15 Bgt isolates, TAM104R/ Thatcher with Pm20 derived from 6RL of Prolific rye resistant to 17 and susceptible to seven Bgt isolates, Kavkaz with Pm8 derived from 1RS of Petkus rye resistant to Bgt isolate E60 but susceptible to the other 18 Bgt isolates, Amigo with Pm17 derived from 1RS of Insave rye resistant to 10 while susceptible to 11 Bgt isolates, and CI14189 with Pm7 derived from 2RL of Rosen rye susceptible to all the 24 Bgt isolates (Table S1). Thus, the broad-spectrum powdery mildew resistance gene PmW6RS on 6RS appears to be a new resistance gene.
Powdery mildew reactions of 6R telosomic addition lines to a mixture of Bgt isolates E09, E18, and E20 were also scored at adult stage during five wheat-growing seasons (2017)(2018)(2019)(2020)(2021)(2022). The 6RS telosomic addition line and rye PI 613196 were immune with IT 0, whereas the 6RL telosomic addition line and wheat 843 showed susceptible with ITs 8-9 (Fig. 1b). These results were consistent with the powdery mildew reactions at the seedling stage. Therefore, PmW6RS conferred all-stage powdery mildew resistance.

Development of wheat-rye 6RS translocation lines
Wheat-rye 6RS translocation lines were developed by 60 Coγray irradiation treatment on the spikes of the 6RS and 6R addition lines at flowering stage. The fresh pollens were used Fig. 1 Identification of powdery mildew resistance of wheat-rye addition lines. a Identification of resistance of wheat-rye addition lines 1R, 2R, 3R, 4R, 5R, 6R, 6RS, 6RL, and 7R, their wheat parent 843, and rye parent PI613196 to Bgt isolate E09 at seedling stage; b iden-tification of resistance of wheat-rye 6RS and 6RL telosomic addition lines and their parents to a mixture of Bgt isolates E09, E18, and E20 at adult stage to pollinate emasculated spikes of the recurrent parents Gao 8901, Shixin 828, and Shiluan 02-1. A total of 2472 F 1 seeds were obtained and cytologically analyzed by GISH, and 164 6RS translocation lines were identified at a frequency of 6.63% (Fig. S1). The translocation lines were classified into terminal, intercalary, and telocentric types by the position of the translocation fragments, and the terminal translocation lines were further classified into short segmental translocation if the length of the translocation fragment is less than half of the 6RS chromosome arm, long segmental translocation if the length of the translocation fragment is over half of the 6RS chromosome arm, whole-arm translocation, and terminal heterochromatin translocation by the size of the translocation fragments. We obtained 31 short segmental translocation lines, 41 long segmental translocation lines, 34 whole-arm translocation lines, 11 terminal heterochromatin translocation lines, four intercalary translocation lines, 27 telocentric translocation lines, and 16 deletion lines. These plants and the recurrent parents Gao 8901, Shixin 828, and Shiluan 02-1 were tested for powdery mildew resistance to Bgt isolate E09 at the seedling stage and to a mixture of Bgt isolates E09, E18, and E20 at the adult stage. We obtained 106 translocation lines immune to powdery mildew at both stages, including 19 short segmental translocation lines, 33 long segmental translocation lines, 34 whole-arm translocation lines, three intercalary translocation lines, 10 telocentric translocation lines, and seven deletion lines. The other translocation lines and the recurrent parents Gao 8901, Shixin 828, and Shiluan 02-1 were susceptible at both seedling and adult stages.

Development of new powdery mildew-resistant wheat-rye 6RS small fragment translocation germplasms
We focused on 39 of the wheat-rye 6RS small fragment translocation lines in which the translocated 6RS chromosome segments were significantly shorter than the entire 6RS chromosome arm and immune to powdery mildew for backcrossing with main wheat cultivars Gao 8901, Shixin 828, and Shiluan 02-1. After three times backcrossing, the performance of the newly developed 6RS small fragment translocation lines was improved. Homozygous 6RS translocation lines were obtained from selfing plants with desirable performance, i.e., the homozygous 6RS small fragment translocation lines In14 and T15 which were cytologically stable (Fig. 4). GISH analysis indicated that In14 and T15 both had 42 chromosomes, including one pair of wheat-rye 6RS translocation chromosomes. ND-FISH analysis showed that in In14, the 6RS fragment was inserted into the long arm of the wheat chromosome 1B, and in T15, the 6RS fragment was translocated to the long arm of the wheat chromosome 4B. No obvious abnormalities of the wheat chromosomes were observed in both lines (Fig. 4). Molecular marker analysis suggested that 19 6RS chromosome-specific markers could trace the 6RS FL0.45-0.79 fragment in In14, and 22 6RS-specific markers could trace the 6RS FL0.34-1.00 fragment in T15 (Fig. 2). Powdery mildew assessment using Bgt isolate E09 at seedling stage and a mixture of Bgt isolates E09, E18, and E20 at adult stage showed that In14 and T15 were both immune to powdery mildew (Fig. S3).

PmW6RS was a novel gene resource for wheat powdery mildew resistance breeding
With the rapid progress in modern wheat breeding, the genetic diversity of powdery mildew resistance genes in wheat cultivars becomes increasingly narrow. It is urgent and important to expand the genetic basis of powdery c and d GISH and ND-FISH detection of wheat-rye T4BS . 4BL-6RS translocation line T15, respectively. In a and c, the rye genome was labeled in green, and CRW was labeled in red. In b and d, Oligo-pSc119.2 was labeled in green, and Oligo-pTa535 was labeled in red. Arrows indicate the translocated chromosomes. Bar = 10 μm. (color figure online) mildew resistance genes in wheat breeding. Rye, as a naturally cross-pollinating relative of common wheat, carries rich genetic diversity among and within rye varieties, and is a valuable resource for wheat improvement. The powdery mildew resistance genes on rye chromosome 6R also appear to be distinct. The 6RL chromosome of Prolific rye carries powdery mildew resistance gene Pm20 and was brought into wheat in the form of T6BS . 6RL translocation (Friebe et al. 1994). The 6RL chromosomes of rye cultivars German White, Jingzhou, Kustro, Qinling, and Secale africanum also carry powdery mildew resistance genes, and the powdery mildew resistance gene of German White rye was shown to be different from Pm20 Du et al. 2018;Li et al. 2020a;Ren et al. 2022a;Wang et al. 2010). The powdery mildew resistance gene PmW6RS of the newly developed wheat-rye 6RS telosomic addition line was derived from rye PI 613196 and was immune to all 24 Bgt isolates tested, including E01, E06, E07, E09, E11, E15, E16, E23-(1), E23-(2), E26, E30-(1), E50, E60, and E69, which were virulent to Pm56 from 6RS of Qinling rye. Thus, the broadspectrum powdery mildew resistance gene PmW6RS might be a new gene and enrich the resistance gene resources for wheat breeding. Further fine mapping and cloning of these genes are needed to explain their relationships. Broad-spectrum powdery mildew resistance genes are highly desirable for developing elite wheat cultivars; however, only a few of such genes have been identified, such as Pm12 , Pm16 (Reader and Miller 1991), Pm21 (He et al. 2018;Xing et al. 2018), Pm24 (Lu et al. 2020), and Pm36 (Blanco et al. 2008). Pm21 has been widely transferred into wheat varieties in wheat breeding. PmW6RS confers broad and high level of resistance, which could contribute to durable resistance breeding.

New wheat-rye 6RS small fragment translocation germplasm lines for wheat powdery mildew-resistant breeding
Developing translocation lines is an effective way to utilize alien genes for wheat breeding. Linkage drag is a key factor affecting the successful transfer of alien genes (Zhang et al. 2017a). Although lots of wheat-alien translocation lines have been developed, only a few of them are successfully applied in wheat breeding practice. Wheat-rye T1RS . 1BL translocation lines and wheat-H. villosa T6VS . 6AL translocation lines are two successful examples of application of alien chromosomes for wheat breeding Xing et al. 2021). However, the 1RS chromosome arm also carries the Sec-1 locus, which is associated with poor bread-making quality (Barak et al. 2013). Therefore, breeders made efforts to develop 1RS small fragment translocation lines to break the linkage between the Sec-1 locus and the disease resistance loci (Fu et al. 2010;Li et al. 2015). Also, wheat-H. villosa 6VS small fragment translocation lines were developed to create high yield breeding lines with disease resistance ). In our study, a total of 164 6RS translocation lines were systemically developed, and 106 of them showed immune to powdery mildew. The large amount of translocation lines contained various translocation breakpoints and translocation types, which may eliminate deleterious effects of the entire 6RS chromosome arm and contribute to abundant genetic and morphological variations and enable selection for desirable agronomic traits. Moreover, the 6RS small fragment translocation lines with high level of powdery mildew resistance were backcrossed to three wheat cultivars and have been transferred to many elite wheat cultivars to improve the comprehensive agronomic traits. PmW6RS resistance could stably expressed in all these wheat backgrounds, thus highlighting the potential utility of the 6RS small fragment translocation lines for wheat powdery mildew resistance breeding.

Physical mapping of PmW6RS promotes the cloning of its candidate gene
It is an efficient way to use various translocation and deletion lines to divide alien chromosomes into different regions for gene localization studies. Using five wheat-rye 4R Ku translocation lines, the powdery mildew resistance gene was localized on the 4RL Ku segment between L.4 and L.8 (Duan et al. 2017). A powdery mildew resistance gene was localized on a 6RL Ku small translocation segment between L2.5 and L2.8 (Du et al. 2018). Yr83 was localized on a 6RL chromosomal bin with FL0.73-1.00 using wheat-rye translocation and deletion lines (Li et al. 2020b). By constructing a physical map of 6VS chromosome using wheat-H. villosa introgression lines, the Pm21 locus was narrowed to a small physical interval which was of great help for the successful cloning of its candidate gene NLR1-V (Xing et al. 2018). In this study, we developed 164 6RS translocation and deletion lines containing different fragment sizes of 6RS chromosome arm to construct a physical map of 6RS and narrowed the location of PmW6RS to an 18.38 Mb physical interval in the Weining rye reference genome. The physical mapping result of PmW6RS appears to be credible as all the translocation and deletion lines showed consistent results. Moreover, the relatively small chromosome region laid a solid ground work for further cloning of PmW6RS. The rapid progress of sequencing technology has greatly accelerated the cloning of alien genes in wheat. The release of high-quality rye genome sequences will greatly promote gene cloning and functional studies in rye and related cereal crops Rabanus-Wallace et al. 2021). As for our study, in the 18.38 Mb physical interval, 23 resistance-related genes were annotated, including two CC-NBS-LRR genes, 13 receptorlike kinase genes, seven receptor-like protein genes, and one transmembrane and coiled-coil protein gene. As most of the cloned disease resistance genes were NBS-LRR type genes, further functional verifications of these resistance genes especially for the two CC-NBS-LRR genes such as VIGS and gene transformation are needed to identify the candidate gene of PmW6RS. Nine markers co-segregated with PmW6RS in this interval, which can be used for tracking the 6RS translocation chromosomes in wheat breeding.
In conclusion, we identified a novel broad-spectrum powdery mildew resistance gene PmW6RS and developed a series of the wheat-rye 6RS small fragment translocation lines immune to powdery mildew. These 6RS small fragment translocation lines have been introduced into various wheat cultivars. Meanwhile, the location of PmW6RS was narrowed to FL 0.58-0.66 on 6RS, corresponding to 50.23-68.61 Mb physical interval in Weining rye reference genome. This study not only provided valuable gene source and germplasms for wheat resistance breeding, but also laid solid foundation for cloning of PmW6RS.