Introgression of an adult-plant powdery mildew resistance gene Pm4VL from Dasypyrum villosum chromosome 4V into bread wheat

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) seriously threatens wheat production worldwide. It is imperative to identify novel resistance genes from wheat and its wild relatives to control this disease by host resistance. Dasypyrum villosum (2n = 2x = 14, VV) is a relative of wheat and harbors novel genes for resistance against multi-fungal diseases. In the present study, we developed a complete set of new wheat-D. villosum disomic introgression lines through genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and molecular markers analysis, including four disomic substitution lines (2n=42) containing respectively chromosomes 1V#6, 2V#6, 3V#6, and 6V#6, and four disomic addition lines (2n=44) containing respectively chromosomes 4V#6, 5V#6, 6V#6 and 7V#6. These lines were subsequently evaluated for their responses to a mixture Bgt isolates at both seedling and adult-plant stages. Results showed that introgression lines containing chromosomes 3V#6, 5V#6, and 6V#6 exhibited resistance at both seedling and adult-plant stages, whereas the chromosome 4V#6 disomic addition line NAU4V#6-1 exhibited a high level of adult plant resistance to powdery mildew. Moreover, two translocation lines were further developed from the progenies of NAU4V#6-1 and the Ph1b mutation line NAU0686-ph1b. They were T4DL·4V#6S whole-arm translocation line NAU4V#6-2 and T7DL·7DS-4V#6L small-fragment translocation line NAU4V#6-3. Powdery mildew tests of the two lines confirmed the presence of an adult-plant powdery mildew resistance gene, Pm4VL, located on the terminal segment of chromosome arm 4V#6L (FL 0.6-1.00). In comparison with the recurrent parent NAU0686 plants, the T7DL·7DS-4V#6L translocation line NAU4V#6-3 showed no obvious negative effect on yield-related traits, providing a new germplasm in breeding for resistance.


Introduction
Bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) provides approximately 20% of the calories required by humankind (Liu et al., 2022).However, some diseases seriously threaten wheat production.Powdery mildew (caused by Blumeria graminis f. sp.tritici, Bgt), a wheat major disease in temperate regions, can cause epidemics, resulting in yield losses of up to 70% in susceptible cultivars (Gao et al., 2018).Thus, it is imperative to identify resistance genes from wheat and its wild relatives to control this disease by developing and planting resistant varieties.
Plants have evolved various types of resistance during the longterm coevolution with pathogens.Many host plants have resistance to pathogens at all stages (ASR).Such resistance genes typically encode nucleotide-binding leucine-rich repeat (NLR) receptors, recognizing specific pathogen avirulence (Avr) proteins and providing race-specific resistance (Develey-Rivière and Galiana, 2007).In other cases, the host plant is restricted to adult plant resistance (APR).Some APR genes encode non-NLR receptors and confer race non-specific durable resistance (Whalen, 2005).To date, more than 70 powdery mildew resistance (Pm) genes have been explored in wheat and its wild relatives (McIntosh et al., 2020;Li et al., 2023).However, few of them are APR genes.In addition, 18 Pm genes have been cloned and characterized so far.Most of the isolated Pm genes encode coiled-coil nucleotide-binding leucinerich repeat (CNL) receptors that recognize specific pathogen avirulence (Avr) proteins, whereas the two adult-plant resistance genes Pm38 and Pm46 encode an ATP-binding cassette (ABC) transporter and a hexose transporter involved in sugar uptake, respectively (Moore et al., 2015;Krattinger et al., 2019).Therefore, exploring novel APR genes contribute to not only breeding durable resistance but also dissecting molecular mechanisms of plant immunity.

Plant materials
A durum-D. villosum amphiploid line AABBVV-3 (2n = 6x = 42, AABBVV) was developed from the crossing between D. villosum accession 01I139 (#6) and durum wheat ZY1286 (2n = 4x = 28, AABB, introduced from CIMMYT) (Hou et al., 2024).Spring bread wheat cv NAU0686 (highly susceptible to powdery mildew) was used to cross with AABBVV-3.To accelerate the alien introgression process, the progenies of NAU0686/AABBVV-3 were planted in a custom-made growth chamber (3 m × 3 m × 3 m) illuminated with LED lights set to an 18-h photoperiod (6 h of darkness) and temperatures of 22°C and 18°C.From the progenies of BC 2 F 2 and BC 2 F 3 of AABBVV-3/2*NAU0686, eight disomic lines were identified.A locally adapted ph1b-carrying line NAU0686-ph1b, deriving from a BC 4 plant obtained by backcrossing the Chinese Spring Ph1b deletion line CS-ph1b to NAU0686, was used to cross with the DA4V#6 line to induce the alien chromosome translocation.Their F 2 individuals were genotyped by PCR markers, 4VL-24 and 4VS-99, to ensure that they had retained the chromosome 4V#6 in a ph1bph1b background.The PCR primers are listed in Supplementary Table S1, following the methods described by Zhang et al. (2022).All the genetic resources used and developed in the present study are listed in Table 1 and are maintained at the Cytogenetics Institute, Nanjing Agricultural University (CINAU).

Cytogenetic analysis
A platform described by Zhang et al. (2022) was used to accelerate alien gene introgression into bread wheat by combining chromosome engineering and speed breeding.D. villosum chromosomes were detected by the combination of genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) techniques.The mitotic metaphase chromosome spreads were obtained from the seedling root-tip cells soaked in cetyltrimethylammonium bromide (0.2 mmol/L) solution for 2 h at 24°C (King et al., 2017).D. villosum 01I139 genome DNA, labeled with fluorescein-12-dUTP and showing green signal, was used as a probe for GISH.The oligonucleotide probes Oligo-pAs1-2,5 and (GAA) 10 , labeled with 6-carboxytetrmethylrhodamine (TAMRA, red signal) and 6-carboxyfluorescein (6-FAM, green signal), respectively, and synthesized by Shanghai Sangon Biotech Co., Ltd.(Shanghai, China), were used to respectively identify the wheat D and B genome chromosomes by nondenaturing FISH, following the procedures described by Du et al. (2017).To complete the process, the chromosomes on the slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen Life Science, Carlsbad, CA, USA).All cytological images were observed under an Olympus BX60 microscope (Olympus Co., Tokyo) and captured with a SPOT Cooled Color Digital Camera (Diagnostic Instruments, Sterling Heights, MI, USA).

Molecular marker analysis
DNA was extracted from young leaves of materials using the cetyl trimethyl ammonium bromide (CTAB) method.Sixteen previously reported IT molecular markers specific to 1V-7V chromosome arms listed in Supplementary Table S1 were used to screen for the introgression lines from D. villosum 01I139 (Zhang et al., 2017a).PCR amplification was carried out in an iCycler thermalcycler (Bio-Rad Laboratories, Emeryville, Calif., USA) in a 10-mL reaction, containing 40 ng of genomic DNA, 2.0 mmol/L of each primer, 2.5 mmol/L of each dNTP, and 0.2 U Taq DNA polymerase.The amplification was conducted at 95°C for 4 min followed by 32 cycles at 94°C, 55-60°C for 45 s, and 60 s of elongation at 72°C, with a final extension at 72°C for 8 min.The amplification samples were separated on 8% non-denaturing polyacrylamide gels and the band patterns were visualized by silver staining.

Powdery mildew evaluation
The alien introgression lines were evaluated for their responses to powdery mildew at the three-leaf stage in a greenhouse at 18/22°C (night/day) with a photoperiod of 14 h of light per day.Ten seeds per line were planted in a single pot (10 cm ×10 cm).NAU0686 seeds were planted as a susceptible control.The local mixture Bgt isolates (E09 and E26) were used for inoculation on the seedling leaves.The response of each plant was recorded on a 0-4 IT scale at 10 days post-inoculation when susceptible NAU0686 control plants were heavily diseased.Plants with IT 0-2 were considered resistant and those with IT 3-4 were susceptible (Jin et al., 2021).
In addition, all the V#6 disomic introgression lines and translocation lines were planted in the field nurseries at the

Line
Chromosome structure High-yield wheat cultivar highly susceptible to powdery mildew, used as the recurrent parent for all alien introgression lines NAU0686-ph1b AABBDD (2n=42) The Ph1 gene mutation line, pedigree: NAU0686 adding a pair of chromosome 7V#6 of 01I139 Approximately 15 plants were grown in each 1.0-m rows with a 25cm spacing between rows.Each line was grown in five rows with three replications.The recurrent parent NAU0686 was planted on both sides of each experimental row and served as a susceptible control.A local mixture of Bgt isolates (E09 and E26) was used to infect all materials at the jointing stage.Reactions evaluated at the heading stage were recorded on a 0-9 scale with 0-4 considered as resistant and 5-9 considered as susceptible (An et al., 2021).

Evaluation of agronomic traits
The wheat-D.villosum translocation lines NAU4V#6-1, NAU4V#6-2, and NAU4V#6-3 and their recurrent parent NAU0686 were planted in the natural field without powdery mildew disease.Each plot consisted of six rows with each row being 1.5 m long and 0.25 m wide.Twenty seeds per row were hand seeded.Yield trials of each line were performed in a randomized plot design with three replications.The agronomic traits were evaluated in 2022 and 2023 planting seasons.Field management followed local practices.At the physiological maturity stage, 20 plants located in the middle of the internal rows of each material were randomly selected for the analysis of yield-related traits, including plant height, spike length, spike number per plant, seeds per spike, thousand-kernel weight, and grain yield per plant.The mean values and standard errors of the treatments were determined by Microsoft Excel.Differences in agronomic traits between the lines were evaluated by means of Tukey's posthoc test (SPSS 26.0, USA) at the p = 0.05 significance level.

Results
Development of a complete set of wheat-D.villosum (#6) disomic lines D. villosum accession 01I139 was crossed as a male parent with spring durum cv ZY1286, and their F 1 plants were subjected to chromosome doubling using colchicine to generate the durum-D.villosum amphiploid AABBVV-3 (Hou et al., 2024).Subsequently, AABBVV-3 was crossed with bread wheat cv NAU0686, and backcrossed with NAU0686 three to four times through speed breeding technology.Plants with V chromosomes were firstly identified in the BC 1 F 1 and BC 2 F 1 generations using molecular markers that were specific to 14 chromosome arms of D. villosum (Supplementary Table S1).Furthermore, the alien chromosomes were confirmed by using GISH with a D. villosum 01I139 probe and FISH with D genome-specific Oligo-pAs1-2,5 probes.A total of eight fully fertile disomic introgressions were selected in the BC 3 F 2 or BC 4 F 2 generations by using molecular cytogenetic technologies (Figure 1).
To confirm whether the resistance was linked to T7DL•7DS-4V#6L, a cross was made between NAU0686 and NAU4V#6-3.All F 1 seedlings were susceptible to a mixture of Bgt isolates (E09 and E26) (IT 4) but showed adult-plant resistance (IT 1-2), similar to NAU4V#6-3.The F 2 seedlings were firstly examined using a combination of GISH/FISH and the 4VL-specific marker 4VL-48.Of the 180 seeds identified, 46 individuals lacked alien chromatin, 95 were heterozygous and 39 were disomic for a T7DL•7DS-4V#6L recombinant chromosome pair, suggesting normal gametic transmission of the translocated chromosome relative to its intact chromosome 7D (c 21:2:1 = 1.1, p > 0.05).These plants were further tested for their reaction to a mixture of Bgt isolates at both seedling and adult-plant stages.All of the F 2 plants were susceptible at the seedling stage.However, the plants with T7DL•7DS-4V#6L translocated chromosome showed high resistance (IT 1-2) at the adult-plant stage, whereas the remaining 46 plants lacking 4V#6L chromatin were still susceptible (IT 7-8) (Figure 4C; Table 2).As a result, we concluded that a dominant powdery mildew APR gene is located on the alien segment in T7DL•7DS-4V#6L, henceforth designated as Pm4VL.

Major agronomic traits investigation of chromosome 4V#6 introgression lines
To evaluate the differences in agronomic traits of 4V#6S and 4V#6L translation lines, field traits, including plant height, spike length, spike number per plant, seeds per spike, thousand-kernel weight, and grain yield per plant of NAU4V#6-1, NAU4V#6-2, and NAU4V#6-3, were investigated.All the introgression lines have similar development stages to their background parent NAU0686 under the field conditions in two grown seasons.Statistical analysis revealed no significant differences between the recurrent parent NAU0686 and the T7DL•7DS-4V#6L translation line NAU4V#6-3 in terms of the major traits (Figures 4C-E, 5A-F).However, the DA4V#6 line NAU4V#6-1 exhibited lower spike length, seeds per spike, and grain yield per plant in comparison to NAU0686 plants.In contrast, the T4DL•4V#6S translocation line NAU4V#6-2 showed no significant differences in spike length, spike number per plant, seeds per spike, thousand-kernel weight, and grain yield per plant, but displayed a reduction in plant height of approximately 4.0 cm compared to NAU0686 (Figure 5A).These results suggested that T4DL•4V#6S and T7DL•7DS-4V#6L translocations could have no negative impact on wheat yieldrelated traits.
wheat diseases, such as powdery mildew, rust, and sharp eyespot (De Pace et al., 2011).In this study, we developed a complete set of new wheat-D.villosum disomic introgression lines from D. villosum accession 01I139.All the individual chromosomes of 1V#6 to 7V#6 were identified by GISH/FISH and molecular markers and transferred into a modern wheat cv NAU0686.These disomic addition/substitution lines along with the molecular markers provide the resource bridge to introgress useful variations from the wild species D. villosum into the wheat genome using markerassisted chromosome engineering.
Previously, inoculation of two panels of wheat-D.villosum introgression lines with Bgt isolate E09 revealed that four chromosome arms, 1VS (Pm67), 2VL (Pm62), 5VS (Pm55/Pm5V), and 6VS (Pm21), carried genes conferring four types of powdery mildew resistance (Xing et al., 2018;Zhang et al., 2018Zhang et al., , 2021Zhang et al., , 2022)).Among them, the T5DL•5V#4S translocation line with Pm55 exhibited adult-plant resistance but with susceptible lower leaf sheaths, which is opposite to the response of the T1DL•1V#5S line with Pm67, which confers seedling resistance but powdery mildew develops on the leaves of adult plants.Pm5V, in a T5DL•5V#5S translocation chromosome, and Pm21, in a T6AL•6V#4S translocation chromosome, confer broad-spectrum resistance to powdery mildew at all stages, whereas Pm62, in a T2BS•2V#5L translocation chromosome, confers a high resistance to powdery mildew at the adult-plant stage (Zhang et al., 2018).In this study, we evaluated the powdery mildew responses of the third panel of wheat-  D. villosum introgression lines and showed that chromosomes, 3V#6, 5V#6, and 6V#6, conferred all-stage resistance to powdery mildew, whereas the chromosome 4V#6 introgression line exhibited resistance to powdery mildew at the adult-plant stage.Through the evaluation of powdery mildew responses of T4DL•4V#6S and T7DL•7DS-4V#6L translocation lines, we further confirmed the presence of an APR gene Pm4VL located on the terminal segment (FL 0.6-1.0) of 4V#6L from D. villosum accession 01I139.However, the introgression lines of chromosomes 4V#2, 4V#4, and 4V#5 did not confer the resistance to wheat powdery mildew (Zhang et al., 2022).Thus, chromosome 4V-related introgression lines with and without resistance can be used to map-based clone the alien genes through normal recombination on chromosome 4V in wheat background.Previously, Pm gene isolation has shown that introgressed alien genes may be orthologous to resistance genes in wheat, for example, rye-derived Pm8 homologous to wheat Pm3 and wheat-derived Pm12 homologous to D. villosum Pm21 (Hurni et al., 2013;Zhu et al., 2022).To date, three of the cataloged powdery mildew resistance genes in wheat and its relatives are located on homoeologous group 4 chromosomes, of which pm61 is a recessive gene located on 4AL-0.8-1.00 (Sun et al., 2018), Pm66 is an ASR gene on the short chromosome arm of 4S l S•4BL from Aegilops longissima (Li et al., 2020), and Pm46/Lr67/Yr46/Sr55 on chromosome arm 4DL confers pleiotropic resistance (Moore et al., 2015).In addition, a wheat-rye line WR41-1 carrying a T4BL•4RL translocated chromosome was resistant to powdery mildew at all stages, indicating that an ASR gene is contained on chromosome arm 4RL of rye (An et al., 2019).The T7DL•7DS-4V#6L translocation line with Pm4VL showed powdery mildew resistance at the adult-plant stage that is distinct from the resistance conferred by pm61, Pm66, Pm46, and the ASR gene locus on 4RL.We thus conclude that the APR gene Pm4VL could be a novel resistance locus of homoeologous group 4 chromosomes in Triticeae species.Since adult-plant resistance is regarded to be more durable than all-stage resistance, further work to transfer Pm4VL to different bread wheat backgrounds would be valuable.
Generally, the translocations induced by ph1b mutation are compensating, as the recombination can occur among homologous/ homoeologous chromosome segments without the control of Ph1b (Türkösi et al., 2022).The T4DL•4V#6S translocation induced by the ph1b mutation is expected to produce compensating translocations, but T7DL•7DS-4V#6L appears to be a noncompensating translocation.However, marker analysis using wheat-D.villosum deletion lines showed that several markers located on the short arms of CS 7A/7B/7D produced amplicons in DA4V and were physically mapped into the bins of chromosome arm 4VL, suggesting that D. villosum chromosome 4V showed some affinity to both wheat homoeologous groups 4 and 7 (Zhang et al., 2017b).Subsequently, the size of the segment translocated from 7VS to 4VL was confirmed by the genome sequencing of D. villosum accession 91C43 DH , which was collinearly larger than that of the fragment translocated from 7BS to 4AL (Zhang et al., 2023).Consequently, the genetic recombination between 4VL and 7DS could be due to the 4VL chromosome segment compensating for the missing fragment of 7DS wheat chromosome.Powdery mildew is a serious disease in the main wheat production area of China.The race structure of Bgt in China is complex, and only a few known Pm genes, such as Pm21, Pm24, Pm48, and Pm55, remain effective (Lu et al., 2020;Jin et al., 2022).Accordingly, further novel powdery mildew resistance genes are required to broaden the genetic variability available for use by breeders.The compensatory translocation T7DL•7DS-4V#6L line with APR gene Pm4VL may be useful in wheat improvement.Although the effect of this translocated chromosome on yield-and quality-related traits is unknown at present, there were no significant differences in morphological characters between NAU0686 and NAU4V#6-3 when tested in two grown seasons.The plants with T7DL•7DS-4V#6L showed good viability, together with high genetic stability.The similar values obtained for agronomic parameters between NAU0686 and NAU4V#6-3 could be due to the 4VL chromosome segment being homoeologous to the missing fragment of chromosome arm7DS.It is imperative to further evaluate the yield impact of the T7DL•7DS-4V#6L translocated chromosome in various genetic backgrounds in future studies.

Conclusion
In conclusion, we developed a complete set of new wheat-D.villosum disomic introgression lines by molecular cytogenetic methods and found chromosome 4V#6 with a novel adult-plant powdery mildew resistance gene Pm4VL.Pm4VL was physically located on the terminal segment of 4VL (FL 0.6-1.00).The T7DL•7DS-4V#6L translocation line NAU4V#6-3 with Pm4VL showed no obvious negative effect in yield-related traits, providing a new germplasm in breeding for resistance.

TABLE 1
Summary of the genetic stocks developed and used in this study.
Baima Experiment Station of Nanjing Agricultural University for evaluation of powdery mildew responses at the adult-plant stage.

TABLE 2
Responses of F 1 and F 2 populations derived from cross NAU0686/NAU4V#6-3 when infected with a mixture of Bgt isolates at the adult-plant stage.