A molecular marker closely linked to wheat powdery mildew resistance gene PmCW116926 and application thereof

Abstract

This invention discloses a molecular marker, YTU2BS-P12, tightly linked to the wheat powdery mildew resistance gene PmCWI16926. The upstream primer nucleotide sequence is shown in SEQ ID NO:1, and the downstream primer nucleotide sequence is shown in SEQ ID NO:2. Using this molecular marker, PCR amplification of the wheat genomic DNA to be tested yielded a corresponding amplification product with a molecular weight of 105 bp. The YTU2BS-P12 molecular marker, tightly linked to the wheat powdery mildew resistance gene PmCWI16926, provided by this invention, can more efficiently detect the genetic mapping population of PmCWI16926, which is beneficial for map-based cloning of PmCWI16926. Using this marker for marker-assisted selection of PmCWI16926 can shorten the breeding cycle, improve breeding efficiency, and be better applied to wheat disease resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI16926 and its application. Background Technology

[0002] Powdery mildew in wheat, caused by the grass bacterium *Blumeria graminis* f. sp. *tritici* (Bgt), seriously threatens wheat yield and quality. This epidemic disease occurs in wheat-growing regions worldwide (Li, H., Zhou, Y., Xin, W., Wei, Y., Zhang, J., Guo, L. Wheat breeding in northern China: Achievements and technical advances. Crop J. 2019, 7, 718-729.). Identifying and cloning powdery mildew resistance genes carried in different wheat varieties, and then breeding superior resistant varieties, is the most economical, effective, and environmentally friendly approach to controlling wheat powdery mildew. To date, over 100 powdery mildew resistance genes and their alleles have been reported in different wheat types (Zhang, J., Yang, H., Han, G., Xu, H., Liu, R., Yu, N., Han, R., Li, Y., Li, J., Dai, Y., Liu, C., Jin, Y., Ma, P. Fine mapping of Pm71, a novel powdery mildew resistance gene from emmer wheat. Crop J. 2025,13, ​​62-68.). However, with the continuous evolution of pathogen races, many resistance genes have lost their resistance in production. Therefore, in order to reduce the occurrence of powdery mildew and improve wheat yield and quality, it is essential to continuously explore and deploy new resistance resources for breeding programs. Wheat's close relatives, such as rye (Secale cereale L, 2n = 2x = 14, RR), icegrass (Agropyron cristatum, 2n = 2x = 14, PP), and tufted wheat (Dasypyrum villosum, 2n = 2x = 14, VV), as well as common wheat's ancestors, such as goatgrass (Aegilops tauschii, 2n = 2x = 14, DD), wild wheat (Triticum urartu, 2n = 2x = 14, AA), emmer wheat (T. monococcum L, 2n = 2x = 14, AA), wild emmer wheat (T. dicccum, 2n = 4x = 28, AABB), and timopheevii wheat (T. timopheevii, 2n = 4x = 28, AAGG), are all valuable resources containing disease-resistant genes.It is worth mentioning that more than half of the recorded powdery mildew resistance genes are derived from the resources of the aforementioned species (Han, G., Wang, J., Yan, H., Cao, L., Liu, S., Li, X., Zhou, Y., Liu, W., Gu, T., Shi, Z., Liu, H., Li, L., An, D. Development and molecular cytogenetic identification of anew wheat-rye 6RL ditelosomic addition and 1R (1B) substitution line with powdery mildew resistance, J. Integr. Agric. 2025, 24, 72-84.).

[0003] Cultivated emmer wheat (T. dicoccum, 2n = 4x = 28, AABB), domesticated from wild emmer wheat, is a tetraploid wheat that shares the A and B genomes with common wheat and is considered the ancestor of common wheat (Zaharieva, M., Ayana, N., Hakimi, A., Misra, S., Monneveux, P. Cultivated emmer wheat (Triticum dicoccon Schrank), an old crop with promising future: a review. Genet. Resour. Crop Evol. 2010, 57, 937-962.). This species possesses a wealth of desirable traits, especially resistance to biotic and abiotic stresses, making it an important genetic resource for the genetic improvement of common wheat (Feng, K., Cui, L., Lv, S., Bian, J., Wang, M., Song, W., Nie, X. Comprehensiveevaluating of wild and cultivated emmer wheat (Triticum turgidum L.)genotypes response to salt stress, Plant Growth Regul. 2018, 84, 261-273.).Taking powdery mildew resistance genes as an example, important genes such as Pm4a, Pm49, Pm50, and Pm71 are all derived from cultivated emmer wheat (Zhu, K.,Li, M., Wu, H., Zhang, D., Dong, L., Wu, Q., Chen, Y., Xie, J., Lu, P., Guo, G., Zhang, H., Zhang, P., Li, B., Li, W., Dong, L., Wang, Q., Zhu, J., Hu, W., Guo, L., Wang, R., Yuan, C., Li, H., Liu, Z., Hua, W. Fine mapping of powdery mildew resistance gene MlWE74 derived from wild emmer wheat (Triticum turgidum ssp. dicoccoides) in an NBS-LRR gene cluster. Theor. Appl. Genet. 2022, 135, 1235-1245.).

[0004] After identifying superior resistance genes, the key lies in how to efficiently apply them to wheat disease resistance breeding. In recent years, wheat breeding technology has continued to innovate, and molecular marker-assisted selection breeding has gradually become the core method for wheat powdery mildew resistance breeding. Having efficient, accurate, and easy-to-use molecular markers is the foundation for conducting molecular marker-assisted selection of target genes. Tracking and detecting target genes through molecular markers can significantly shorten the breeding cycle and greatly improve breeding efficiency.

[0005] The cultivated emmer wheat CWI16926-4Y exhibits excellent agronomic traits and has demonstrated good resistance to powdery mildew in multi-year, multi-location field trials, making it an extremely superior wheat germplasm resource resistant to powdery mildew. Genetic analysis and molecular marker detection of powdery mildew resistance in seedlings revealed that, at the seedling stage, the resistance of CWI16926-4Y to the prevalent powdery mildew strain E09 is controlled by a pair of dominant genes, PmCWI16926, located on the wheat 2BS chromosome. This gene is a newly discovered wheat powdery mildew resistance gene / allele. Therefore, developing molecular markers closely linked to the PmCWI16926 gene and applying them to marker-assisted selection breeding for the PmCWI16926 powdery mildew resistance gene is crucial for breeding powdery mildew-resistant wheat varieties and achieving effective control of powdery mildew. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI16926 and its application, so as to locate and detect the wheat powdery mildew resistance gene PmCWI16926 using this molecular marker. Using this marker for marker-assisted selection of PmCWI16926 can shorten the breeding cycle, improve breeding efficiency, and be better applied to wheat disease resistance breeding.

[0007] The present invention is achieved by the following method: a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI16926, the molecular marker being the dominant INDEL marker YTU2BS-P12;

[0008] The upstream primer for the molecular marker YTU2BS-P12 is YTU2BS-P12-F, and its nucleotide sequence is as follows:

[0009] 5'-AGGTAATGTTGAGTGCCGC-3', as shown in SEQ ID NO: 1;

[0010] The downstream primer for the molecular marker YTU2BS-P12 is YTU2BS-P12-R, and its nucleotide sequence is as follows:

[0011] 5'-ATCAACTTGTCTTCTCAGTTGTACC -3', as shown in SEQ ID NO: 2;

[0012] The marker primers for the molecular marker YTU2BS-P12 were used to amplify the wheat genomic DNA to be tested by PCR. The corresponding amplification product had a molecular weight of 105 bp, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI16926.

[0013] The appropriate PCR amplification system for this molecular marker is 10 μL, comprising: 1.2 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4.3 μL of sterile deionized water.

[0014] The applicable PCR amplification program for this molecular marker is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0015] The electrophoretic separation procedure for the amplification products applicable to this molecular marker is as follows: electrophoresis is performed using an 8% non-denaturing polyacrylamide gel. The amplification product is mixed with 2.5 μL of 10× Loading Buffer, and 1.3 μL of the mixture is loaded onto the gel. Electrophoresis is performed at a constant voltage of 220 V for 1.5 h. The gel is then stained with silver nitrate and photographed.

[0016] The present invention relates to the application of the molecular markers closely linked to the wheat powdery mildew resistance gene PmCWI16926 in gene localization, map-based cloning, and marker-assisted selection breeding of the wheat powdery mildew resistance gene PmCWI16926.

[0017] The application described in this invention, which detects whether a wheat variety carries the powdery mildew resistance gene PmCWI16926, mainly includes the following steps:

[0018] (1) Extract genomic DNA from fresh leaves of the wheat sample to be tested;

[0019] (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTU2BS-P12 to obtain the amplification product;

[0020] (3) If a specific band of 105 bp can be amplified, it indicates that the powdery mildew resistance gene PmCWI16926 exists in the wheat to be tested; otherwise, the powdery mildew resistance gene PmCWI16926 does not exist in the wheat to be tested.

[0021] In the application described above, the primers for the molecular marker YTU2BS-P12 in step (2) include an upstream primer YTU2BS-P12-F and a downstream primer YTU2BS-P12-R. The nucleotide sequence of the upstream primer YTU2BS-P12-F is shown in SEQ ID NO:1, namely: YTU2BS-P12-F: 5'-AGGTAATGTTGAGTGCCGC-3'; the nucleotide sequence of the downstream primer YTU2BS-P12-R is shown in SEQ ID NO:2, namely: YTU2BS-P12-R: 5'-ATCAACTTGTCTTCTCAGTTGTACC-3'.

[0022] The application of this molecular marker is based on a 10 μL PCR amplification system, which includes: 1.2 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4.3 μL of sterile deionized water.

[0023] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0024] Detection of PCR amplification products: Electrophoresis was performed using an 8% non-denaturing polyacrylamide gel. The amplification product was mixed with 2.5 μL of 10× Loading Buffer, and 1.3 μL of this mixture was loaded onto the gel. Electrophoresis was carried out at a constant voltage of 220 V for 1.5 h. The gel was then stained with silver nitrate and photographed. The electrophoresis results were used to determine the presence of the powdery mildew resistance gene PmCWI16926 in the tested wheat germplasm. Otherwise, the powdery mildew resistance gene PmCWI16926 was not present in the tested wheat germplasm.

[0025] This invention demonstrates, through genetic analysis and molecular marker detection of seedling powdery mildew resistance, that the resistance of cultivated emmer wheat CWI16926-4Y seedlings to the prevalent powdery mildew strain E09 is controlled by a single dominant gene, named PmCWI16926. Polymorphism detection was performed on 198 pairs of molecular markers evenly distributed throughout the genome in cultivated emmer wheat CWI16926-4Y, susceptible wheat Langdon (LDN), and F2 populations consisting of 10 homozygous resistant and 10 homozygous susceptible families. Twelve pairs of markers showed consistent polymorphism in the resistant and susceptible parents and the resistant and susceptible pools. Subsequently, these markers were used to genotype 498 F2 populations of CWI16926-4Y×Langdon (LDN). Referring to the durum wheat cv. Svevo genome, PmCWI16926 was located in the 920kb physical region (21.70-22.62Mb) on chromosome 2BS. Based on the sequence of the reference genome within this region, the insertion-deletion (INDEL) marker YTU2BS-P12, which is closely linked to the gene PmCWI16926, was designed and screened using Primer 5.0 software. The molecular marker YTU2BS-P12 for the wheat powdery mildew resistance gene PmCWI16926 provided by this invention, after genetic segregation population testing, showed a genetic distance of only 0.2 cM from the gene PmCWI16926, indicating close linkage. This allows for more accurate and efficient detection of the genetic mapping population of PmCWI16926, which is beneficial for map-based cloning and fine localization of PmCWI16926.

[0026] This invention provides a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI16926. When applied to powdery mildew resistant wheat breeding, it can not only greatly save costs and shorten the breeding cycle, but also be more precise and efficient, and can be better applied to powdery mildew resistant wheat breeding work. Attached Figure Description

[0027] Figure 1 The results of primer detection of the molecular marker YTU2BS-P12 on disease-resistant emmer wheat CWI16926-4Y and disease-susceptible durum wheat Langdon (LDN) and their hybrid progeny segregating populations.

[0028] In the figure, M: pUC19; 1: CWI16926-4Y (resistant parent); 2: Langdon (LDN) (susceptible parent); 3-17: F2 population of CWI16926-4Y × Langdon (LDN), where 3-7: homozygous resistant lineage, 8-12: segregating resistant and susceptible lineage, 13-17: homozygous susceptible lineage; the white arrows are specific bands of PmCWI16926. Detailed Implementation

[0029] The following examples are provided to better understand and use the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials and reagents used in the examples are commercially available.

[0030] Example 1: Development of the molecular marker YTU2BS-P12 for the wheat powdery mildew resistance gene PmCWI16926

[0031] Material

[0032] The resistant parent was cultivated emmer wheat CWI16926-4Y, and the susceptible parent was durum wheat Langdon (LDN). CWI16926-4Y and Langdon (LDN) were crossed, and the resulting F1 generation was self-crossed to obtain the F2 population.

[0033] 2. Extraction of wheat genomic DNA

[0034] Wheat genomic DNA was extracted using the CTAB method, and the procedure is as follows:

[0035] (1) Take tender fresh leaves of wheat to be tested, freeze them quickly with liquid nitrogen, grind them into powder, and put them into 2 mL EP tubes;

[0036] (2) Add 600-800 μL of CTAB extract and incubate in a 65°C water bath for 1 h, inverting and mixing every ten minutes during the incubation period;

[0037] (3) Add an equal volume of chloroform:isoamyl alcohol (24:1) and mix on a shaker for 30 min;

[0038] (4) Centrifuge at 8000 rpm / min at room temperature for 10 min, take 400 μL of supernatant into a 1.5 mL EP tube, add 3 times the volume of pre-cooled 95% ethanol, mix well, and precipitate at -20°C for 0.5 h;

[0039] (5) Centrifuge at 12000 rpm / min at room temperature for 10 min, discard the supernatant, and wash 3 times with 800 μL of 75% ethanol;

[0040] (6) Air dry the precipitate and dissolve it in 50 μL of ddH2O.

[0041] (7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.

[0042] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of resistance

[0043] Wheat seedling powdery mildew resistance identification was conducted in a greenhouse. The resistant parent CWI16926-4Y, the susceptible parent Langdon (LDN), F1 hybrids, and F2 populations were planted in 128-cell trays (3.2 × 3.2 × 4.2 cm). At least 20 seeds from each parent and F1 were identified, and at least 25 seeds from each F2 population were identified. The susceptible control TN18 was randomly sown and tagged for identification. Greenhouse conditions were controlled at 18-20°C, 80% relative humidity, and a photoperiod of 14 h light / 10 h dark. Powdery mildew strain E09 was inoculated using the sweeping method at the one-leaf stage. Phenotypic assessment was conducted 10-14 days later, when the susceptible control TN18 showed full disease development. Infection type (IT) was recorded according to a 0-4 scale. Disease resistance classification: 0-2 are disease-resistant types, and 3-4 are disease-susceptible types (Si Quanmin, Zhang Xinxin, Duan Xiayu, Sheng Baoqin. Identification of physiological races of wheat powdery mildew fungus. Chinese Agricultural Science. 1987, 20: 64-70).

[0044] The results showed that CWI16926-4Y exhibited high resistance to powdery mildew strain E09 (IT=0) and high susceptibility to Langdon (LDN) (IT=4). All F1 plants showed resistance (IT = 0-1), indicating that CWI16926-4Y carries a dominant resistance gene. Resistance identification of the F2 population of this combination showed a resistance-susceptibility segregation ratio of 154:53, which, according to the chi-square test, conformed to a segregation ratio of 3:1 for a single dominant gene (χ²=0). 2=0.02, P=0.84). In summary, the resistance of CWI16926-4Y to powdery mildew strain E09 is controlled by a single dominant gene, and this powdery mildew resistance gene is named PmCWI16926.

[0045] 4. Fine localization of molecular markers for PmCWI16926

[0046] Based on the phenotypic identification results, 10 homozygous resistant families and 10 homozygous susceptible families were selected to construct resistant and susceptible pools, respectively. Polymorphism detection was performed on cultivated emmer wheat CWI16926-4Y, durum wheat Langdon (LDN), and the resistant and susceptible pools using 198 pairs of molecular markers evenly distributed throughout the genome. Twelve pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to genotype the F2 population of 498 CWI16926-4Y×Langdon (LDN), locating PmCWI16926 within the 21.70–22.62 Mb region of wheat 2BS chromosome.

[0047] 5. Development of molecular markers closely linked to PmCWI16926

[0048] Based on the sequence information of the durum wheat reference genome (Durum wheat cv. Svevo) within the candidate region of 21.70-22.62 Mb, an insertion-deletion (INDEL) marker was designed using Primer 5.0 software. Genotyping was performed on the F2 population of CWI16926-4Y×Langdon (LDN), and the INDEL marker YTU2BS-P12, which is closely linked to the gene PmCWI16926, was obtained, with a genetic distance of only 0.2 cM.

[0049] The primers for the molecular marker YTU2BS-P12 include one upstream primer and one downstream primer:

[0050] The nucleotide sequence of the upstream primer YTU2BS-P12-F is: 5'-AGGTAATGTTGAGTGCCGC-3';

[0051] The nucleotide sequence of the downstream primer YTU2BS-P12-R is: 5'-ATCAACTTGTCTTCTCAGTTGTACC-3'.

[0052] The applicable PCR amplification system for this marker is 10 μL, including: 1.2 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4.3 μL of sterile deionized water.

[0053] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0054] The electrophoretic separation procedure for the amplified products is as follows: Electrophoresis is performed using an 8% non-denaturing polyacrylamide gel. The amplified product is mixed with 2.5 μL of 10× Loading Buffer, and 1.3 μL of this mixture is loaded onto the gel. Electrophoresis is carried out at a constant voltage of 220 V for 1.5 h. After silver nitrate staining, the sample is photographed. If a specific band of 105 bp is amplified, it indicates the presence of the powdery mildew resistance gene PmCWI16926 in the tested wheat germplasm; otherwise, the powdery mildew resistance gene PmCWI16926 is not present in the tested wheat germplasm.

[0055] Molecular marker detection results are shown below Figure 1 .in Figure 1 The results of primer detection for the molecular marker YTU2BS-P12 were obtained from the segregating populations of disease-resistant emmer wheat CWI16926-4Y and susceptible durum wheat Langdon (LDN) and their hybrid offspring. In the figure, M: pUC19; 1: CWI16926-4Y (disease-resistant parent); 2: Langdon (LDN) (susceptible parent); 3-17: F2 populations of CWI16926-4Y × Langdon (LDN), where 3-7: homozygous disease-resistant families, 8-12: segregating families of resistance and susceptibility, and 13-17: homozygous susceptible families; the white arrows represent specific bands of PmCWI16926. The amplification results showed that the marker YTU2BS-P12 amplified a specific band of 105 bp in the resistant parent CWI16926-4Y and the resistant family, but no such band was amplified in the susceptible parent Langdon (LDN) and the susceptible family.

[0056] The wheat powdery mildew resistance gene PmCWI16926, derived from cultivated emmer wheat CWI16926-4Y, is a novel gene exhibiting excellent resistance. Currently, there are no reports on its localization, map-based cloning, or molecular breeding applications. Using the molecular marker YTU2BS-P12 provided in this invention to detect large-scale genetic mapping populations facilitates the precise localization and map-based cloning of the PmCWI16926 gene. Introducing PmCWI16926 into major wheat varieties susceptible to powdery mildew, and utilizing the molecular marker YTU2BS-P12 developed in this invention, allows for efficient and accurate detection of large breeding populations, significantly improving the efficiency and accuracy of transferring the disease-resistant gene PmCWI16926. This is of paramount importance for the efficient transfer of the PmCWI16926 gene and for in-depth analysis of its resistance mechanisms.

[0057] The above embodiments are optimized implementations of the present invention and are used only to illustrate the present invention, not to limit it. Modifications or equivalent substitutions made by those skilled in the art without departing from the spirit and principles of the embodiments of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. Application of a primer containing a molecular marker closely linked to a wheat powdery mildew resistance gene in the breeding of wheat powdery mildew resistant varieties; The nucleotide sequence of the upstream primer of the molecular marker is as follows: 5'-AGGTAATGTTGAGTGCCGC-3', as shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer of the molecular marker is as follows: 5'-ATCAACTTGTCTTCTCAGTTGTACC -3', as shown in SEQ ID NO: 2.

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