Primer of a molecular marker closely linked to wheat powdery mildew resistance gene PmXP544 and application thereof

Abstract

This invention relates to the field of bioengineering technology, specifically to a gene for wheat resistance to powdery mildew. PmXP544 Primers for a tightly linked molecular marker and their application. The molecular marker is YTUXP35; the nucleotide sequence of the upstream primer is shown in SEQ ID NO:1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2; PCR amplification of the genomic DNA of the wheat sample to be tested was performed using the marker primers for YTUXP35, and the corresponding amplification product with a molecular weight of 348 bp was obtained, which is associated with the wheat powdery mildew resistance gene. PmXP544 Tightly linked molecular markers. This invention provides a marker associated with wheat powdery mildew resistance genes. PmXP544 The tightly linked molecular marker YTUXP35 can be used for... PmXP544 Molecular marker-assisted selection can significantly improve breeding efficiency.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a gene for wheat resistance to powdery mildew. PmXP544 Primers for tightly linked molecular markers and their applications. Background Technology

[0002] Wheat powdery mildew is a fungal disease caused by *Erysiphe graminearum*, a wheat-specific fungus. Blumeria graminis. sp. tritici, Bgt (Dean R, Van Kan J, Pretorius Z, Hammond-Kosack, K, Di Pietro A, Spanu P, Rudd J, Dickman M, Kahmann R, Ellis J, et al. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant. Pathol. 2012, 13:417-30). In the early stages of wheat infection with powdery mildew, chlorotic necrotic spots form on the leaf surface. As the disease progresses, the spots expand into elliptical or nearly circular shapes, covered with a white mycelial layer composed of conidiophores and chains of conidia. In the middle and late stages, the mycelial layer gradually changes from pure white to grayish-white, and in the later stages, a large number of grayish-brown cleistothecia containing asci and ascospores are formed. In mild infections, the mycelium is scattered; in severe infections, the mycelial layer merges into patches that can completely cover the leaf surface. The mesophyll tissue below the infected area rapidly turns chlorotic and yellow, accompanied by programmed early wilting. When the pathogen spreads to the stems and leaf sheaths, the mechanical strength of the stem base decreases significantly, making it prone to lodging. Ultimately, the plant exhibits weakened growth, reduced plant height, and fewer effective tillers; the ear length is shortened, the seed setting rate decreases, and the thousand-grain weight is significantly reduced, leading to severe yield loss (Liu Ruishan. Wild Emmer Wheat Powdery Mildew Resistance Gene). PmLF540Precise positioning. Master's thesis, Yantai University, 2025. In recent years, due to the widespread planting of susceptible varieties and the continuous mutation of the pathogen, wheat powdery mildew has spread to almost all wheat-producing areas in China, affecting approximately 6 million hectares of wheat fields annually, resulting in a yield loss of 10%-15%, and in severe cases, even as high as 62% (Wang, B, Meng T, Xiao, B, Yu, T, Yue, T, Jin, Y, Ma, P. Fighting wheat powdery mildew: from genes to fields. Theor. Appl. Genet. 2023, 136:196). In order to effectively control wheat powdery mildew and reduce the damage caused by it, various powdery mildew control measures have been adopted in production, such as agricultural control measures, chemical control, and the use of disease-resistant varieties. In comparison, breeding disease-resistant varieties is the preferred measure to reduce and control wheat powdery mildew (He Z, Zhang P, Jia H, Zhang S, Nishawy E, SunX, Dai M. Regulatory mechanisms and breeding strategies for crop drought resistance. New. Crops 2024, 1:100029).

[0003] In the process of discovering disease-resistant genes in wheat, ancestral wheat species resources are an important gene pool, among which wild emmer wheat ( Triticum turgidum ssp. Dicoccoides The *A. AABB* strain (2n = 4X = 28, AABB) is one of the key ancestral resources of hexaploid common wheat, carrying a large number of excellent disease resistance allelic variations and serving as an important gene donor for improving wheat resistance to powdery mildew (Avni R, Nave M, Barad O, Baruch K, Twardziok S, Gundlach H, HaleI, Mascher M, Spannagl M, Wiebe K, et al. Wild emmer genome architecture and diversity elucidate wheat evolution and domestication. Science. 2017, 357:93–97.). Currently, officially named powdery mildew resistance genes that have been successfully transferred from wild emmer wheat into common wheat include... Pm16 , Pm26 , Pm30 , Pm36 ,Pm41 , pm42 , Pm64 and Pm69 These studies significantly improved the resistance of common wheat to powdery mildew (Liu R, Xu H, Yu N, Zhang J, Li Y, Li J, Dai Y, Xiao B, Pan G, Li D, et al. Fine mapping of a powdery mildew resistance gene). PmLF540 from wild emmerwheat. Theor. Appl. Genet. 2025, 138:178).

[0004] In the history of wheat genetic breeding, traditional breeding has been primarily based on empirical methods, relying mainly on field selection of target phenotypic traits based on breeding experience. However, field phenotypes are easily affected by environmental interactions, resulting in low breeding accuracy, long breeding cycles, and limited efficiency. In contrast, marker-assisted selection (MAS) utilizes molecular markers that are highly linked to or co-segregate with the target gene to achieve direct genotyping and targeted selection, significantly improving selection accuracy and genetic gain. Currently, this strategy is widely used in the breeding of disease-resistant wheat varieties.

[0005] To discover more powdery mildew resistance genes for application in wheat powdery mildew resistance breeding, the wild emmer wheat line XP544, an ancestral species of wheat, was previously identified as exhibiting high levels of powdery mildew resistance in the field, making it an excellent powdery mildew-resistant germplasm resource. Further genetic analysis of seedling resistance showed that XP544 carries a single dominant gene. PmXP544 They used molecular markers to locate it on chromosome 4AL. Therefore, development... PmXP544 Closely linked molecular markers, for PmXP544 Precise localization and cloning are crucial and can promote PmXP544 The conversion and utilization of these technologies greatly improves breeding efficiency and mitigates wheat powdery mildew. Summary of the Invention

[0006] The purpose of this invention is to provide a gene for resistance to powdery mildew in wheat. PmXP544 Primers for tightly linked molecular markers and their applications to utilize these markers to target wheat powdery mildew resistance genes. PmXP544 Gene mapping was performed, and the marker was used for... PmXP544 Molecular marker-assisted selection breeding can shorten the breeding cycle, improve breeding efficiency, and be better applied to wheat disease resistance breeding.

[0007] This invention is achieved through the following method: a gene associated with wheat powdery mildew resistance. PmXP544 Primers for a tightly linked molecular marker, the marker being the co-dominant INDEL marker YTUXP35; The upstream primer for the molecular marker YTUXP35 is YTUXP35-F, and its nucleotide sequence is as follows: 5'-CTACTCCATCGCTTCCCCTG-3', as shown in SEQ ID NO: 1; The downstream primer for the molecular marker YTUXP35 is YTUXP35-R, and its nucleotide sequence is as follows: 5'-GGTTTGTGGTATTCTGGTTCACC-3', as shown in SEQ ID NO: 2; PCR amplification of the wheat genomic DNA to be tested was performed using primers labeled with YTUXP35, yielding an amplification product with a molecular weight of 348 bp, which corresponds to the wheat powdery mildew resistance gene. PmXP544 Tightly linked molecular markers.

[0008] The appropriate PCR amplification system for this molecular marker is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

[0009] The applicable PCR amplification program for this molecular marker is as follows: 95℃ pre-denaturation for 2 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℃.

[0010] 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 2.0 μL of 10× Loading Buffer, and 0.8 μL of the mixture is loaded onto the gel. Electrophoresis is performed at a constant voltage of 170 V for 80-120 mins. The gel is then stained with silver nitrate and photographed.

[0011] The wheat powdery mildew resistance gene provided by this invention PmXP544 Tightly linked molecular markers in wheat powdery mildew resistance genes PmXP544 Applications of gene mapping, map-based cloning, and molecular marker-assisted selection breeding.

[0012] The application described in this invention is to detect whether a wheat variety carries a gene for resistance to powdery mildew. PmXP544 The main steps include: (1) Extract genomic DNA from fresh leaves of the wheat sample to be tested; (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTUXP35 to obtain the amplification product; (3) If a specific band of 348 bp can be amplified, it indicates that the wheat being tested contains a gene for resistance to powdery mildew. PmXP544 Otherwise, the wheat sample tested does not contain the wheat powdery mildew resistance gene. PmXP544 .

[0013] In the application described above, the primers for the molecular marker YTUXP35 in step (2) include an upstream primer YTUXP35-F and a downstream primer YTUXP35-R. The nucleotide sequence of the upstream primer YTUXP35-F is shown in SEQ ID NO:1, namely: YTUXP35-F: 5'-CTACTCCATCGCTTCCCCTG-3'; the nucleotide sequence of the downstream primer YTUXP35-R is shown in SEQ ID NO:2, namely: YTUXP35-R: 5'-GGTTTGTGGTATTCTGGTTCACC-3'.

[0014] The application of this molecular marker is based on a 10 μL PCR amplification system, which includes: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

[0015] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 2 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℃.

[0016] Detection of PCR amplification products: Electrophoresis was performed using an 8% non-denaturing polyacrylamide gel. The amplification product was mixed with 2.0 μL of 10× Loading Buffer, and 0.8 μL of this mixture was loaded onto the gel. Electrophoresis was performed at a constant voltage of 170 V for 80-102 mins. The gel was then stained with silver nitrate and photographed. The electrophoresis results were interpreted as follows: if a specific band of 348 bp was amplified, it indicated that the tested wheat germplasm carried a gene for resistance to powdery mildew. PmXP544 Otherwise, the wheat germplasm being tested does not carry the wheat powdery mildew resistance gene. PmXP544 .

[0017] This invention, through genetic analysis and molecular marker detection of seedling powdery mildew resistance, demonstrates that the resistance of wild emmer wheat XP544 seedlings to the prevalent powdery mildew strain E09 is controlled by a single dominant gene, which is named...PmXP544 Polymorphism was detected in 156 pairs of molecular markers evenly distributed throughout the genome in wild emmer wheat XP544, susceptible wheat Langdon (LDN), and F2 populations using XP544×LDN, comprising 10 homozygous resistant and 10 homozygous susceptible families. Sixteen marker pairs showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible families. These markers were then used to detect polymorphism in 152 F2 populations using XP544×LDN. 2:3 Genotyping of family pedigrees PmXP544 The sequence was located within the region of 732.87-736.29 Mb on wheat chromosome 4AL. Further, based on the sequence within this region of the Chinese spring wheat reference genome, Primer 5.0 software was used to design and screen for sequences related to the gene. PmXP544 The tightly linked INDEL marker YTUXP35 was used to detect well-segregating genetic populations through phenotypic analysis. Linkage calculations revealed that YTUXP35 is associated with the target gene. PmXP544 The genetic distance is only 1.7 cM, indicating tight linkage, which can significantly promote... PmXP544 Precise positioning and map cloning, while assisting PmXP544 Molecular marker-assisted selection breeding was used to precisely transfer common wheat varieties, thereby improving the powdery mildew resistance level of common wheat.

[0018] This invention provides a gene for resistance to powdery mildew in wheat. PmXP544 Tightly linked molecular markers can be used for fine mapping and map-based cloning of disease resistance genes, and also for marker-assisted selection breeding of wheat to resist powdery mildew. This can effectively promote the breeding process of wheat to resist powdery mildew and has important practical significance. Attached Figure Description

[0019] Figure 1 To label YTUXP35 in XP544 ×LDN derived F 2:3 The results of partial pedigree expansion.

[0020] M in the image: pUC19 Msp 1: XP544; 2: LDN; 3-17: F2 population of XP544×LDN, where 3-7: homozygous resistant families, 8-12: homozygous susceptible families, and 13-17: segregating resistant and susceptible families; arrows indicate... PmXP544 Specific bands. Detailed Implementation

[0021] 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.

[0022] Example 1: Wheat powdery mildew resistance gene PmXP544 Development of materials for the molecular marker YTUXP35 The resistant parent was wild emmer wheat XP544, and the susceptible parent was durum wheat LDN. XP544 and LDN were crossed, and the resulting F1 generation was self-crossed to obtain the F2 population and its F3 generation. 2:3 Family lineage.

[0023] 2. Extraction of wheat genomic DNA Wheat genomic DNA was extracted using the CTAB method, and the procedure is as follows: 1) Take tender fresh leaves of the wheat to be tested, freeze them quickly with liquid nitrogen, grind them into powder, and put them into 2 mL EP tubes; 2) Add 600-800 μL of CTAB extraction solution and incubate in a 65°C water bath for 1 h, inverting and mixing every ten minutes during the process; 3) Add an equal volume of chloroform and mix on a shaker for 30 minutes; 4) Centrifuge at 8000 rpm at room temperature for 10 min, aspirate 400 μL of supernatant into a 1.5 mL EP tube, add 3 times the volume of pre-cooled 95% ethanol, mix well, and allow to settle at -20°C for 0.5 h. 5) Centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant, and wash three times with 800 μL of 75% ethanol; 6) Air dry the precipitate and dissolve it in 50 μL of 1×TE or ddH2O.

[0024] 7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.

[0025] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of resistance Wheat seedling powdery mildew resistance identification was completed in a greenhouse. The resistant parent XP544, the susceptible parent LDN, F1 hybrids, and F2 hybrids were used. 2:3 The families were planted in 128-cell seed trays (3.2×3.2×4.2 cm). Both parents and F1 strains were identified as having at least 20 seeds per cell. 2:3Each family was identified with at least 25 seeds. 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 analysis was conducted 10-17 days later, when the susceptible control TN18 showed full disease development. Infection type (IT) was recorded according to a 0-4 grade standard. Disease resistance grades were classified as follows: 0-2 for resistant types, and 3-4 for susceptible types (Si Quanmin et al., 1987).

[0026] The results showed that XP544 exhibited high resistance to powdery mildew strain E09, high susceptibility to LDN, and all F1 plants showed high resistance, indicating that XP544 carries a dominant resistance gene. Resistance identification of the F2 population of this combination showed a resistance-susceptibility segregation ratio of 147:51, which, according to the chi-square test, conformed to a 3:1 segregation ratio for a single dominant gene (χ²). 2 =0.061, P =0.806), further analysis of F 2:3 Family pedigree analysis revealed a segregation ratio of 47:100:51 (χ²) between homozygous resistant families, segregating families, and homozygous susceptible families. 2 =0.192, P =0.909), which conforms to a segregation ratio of 3:1 for a single dominant gene (1:2:1). In conclusion, the resistance of XP544 to the powdery mildew strain E09 is controlled by a single dominant gene, and this powdery mildew resistance gene is named... PmXP544 .

[0027] 4. PmXP544 fine localization of molecular markers Based on 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 wild emmer wheat XP544, durum wheat LDN, and the resistant and susceptible pools using 156 pairs of molecular markers evenly distributed throughout the genome. Sixteen pairs of markers showed consistent polymorphism in the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to analyze the F1 generation of 152 XP544×LDN lines. 2:3 Genotyping of family pedigrees PmXP544 It was initially located within the region of 732.87-736.29 Mb on wheat chromosome 4AL.

[0028] 5. with PmXP544 Development of tightly linked molecular markers Based on the sequence information of the Chinese spring wheat reference genome within the candidate region of 732.87-736.29 Mb, INDEL markers were designed using Primer 5.0 software, and F of XP544×LDN was analyzed. 2:3Genotyping of the family pedigree yielded results related to the genes. PmXP544 The closely linked INDEL marker YTUXP35 has a genetic distance of only 1.7 cM.

[0029] The primers for the molecular marker YTUXP35 consist of one upstream primer and one downstream primer: The nucleotide sequence of the upstream primer YTUXP35-F is: 5'-CTACTCCATCGCTTCCCCTG-3'; The nucleotide sequence of the downstream primer YTUXP35-R is: 5'-GGTTTGTGGTATTCTGGTTCACC-3'.

[0030] The applicable PCR amplification system for this marker is 10 μL, including: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

[0031] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.

[0032] 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 2.0 μL of 10× Loading Buffer, and 0.8 μL of this mixture is loaded onto the gel. Electrophoresis is carried out at a constant voltage of 170 V for 80-120 mins. The gel is then stained with silver nitrate and photographed. If a specific band of 348 bp is amplified, it indicates the presence of a powdery mildew resistance gene in the tested wheat germplasm. PmXP544 Otherwise, the wheat germplasm tested does not contain the wheat powdery mildew resistance gene. PmXP544 .

[0033] Molecular marker detection results are shown below Figure 1 .in Figure 1 To label YTUXP35 in XP544×LDN derived F 2:3 Partial amplification results from the family. M in the figure: pUC19 Msp I; 1: XP544; 2: LDN; 3-17: F of XP544×LDN 2:3 Family pedigrees, where 3-7: homozygous resistant pedigrees, 8-12: homozygous susceptible pedigrees, and 13-17: segregating resistant and susceptible pedigrees; red arrows indicate... PmXP544The specific band was observed. Amplification results showed that the marker YTUXP35 amplified a specific band of 348 bp in the resistant parent XP544 and resistant families, but did not amplify the target band in the susceptible parent LDN and susceptible families.

[0034] wheat powdery mildew resistance gene PmXP544 Derived from the wild emmer wheat XP544 in my country, this is a novel gene exhibiting excellent resistance. Currently, there are no reports on the localization, map-based cloning, or molecular breeding of this gene, and primers for the molecular marker YTUXP35 have also not been reported, thus demonstrating significant innovation. Using the primers for the molecular marker YTUXP35 provided in this invention for large-scale genetic mapping detection is beneficial for achieving gene [specific goals / achievements]. PmXP544 Fine-grained localization and map cloning. (The sentence is incomplete and lacks context.) PmXP544 After being introduced into major wheat varieties susceptible to powdery mildew, the molecular marker YTUXP35 developed in this invention can be used to efficiently and accurately detect large breeding populations, greatly improving the transfer of disease-resistant genes. PmXP544 The efficiency and accuracy of this are crucial for gene sequencing. PmXP544 It is of great significance to conduct in-depth analysis of the efficient transformation and disease resistance mechanism.

[0035] 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. A gene associated with wheat powdery mildew resistance PmXP544 Primers for the tightly linked molecular marker YTUXP35 were used. 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. PCR amplification of the genomic DNA of the wheat sample was performed using primers for the molecular marker YTUXP35. The corresponding amplification product with a molecular weight of 348 bp was obtained, which corresponds to the wheat powdery mildew resistance gene. PmXP544 Tightly linked molecular markers.

2. The wheat powdery mildew resistance gene as described in claim 1 PmXP544 A tightly linked molecular marker, applicable to a 10 μL PCR amplification system, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCRMasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

3. The wheat powdery mildew resistance gene as described in claim 1 PmXP544 The tightly linked molecular markers were subjected to the following PCR amplification program: 95°C pre-denaturation for 2 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 40 s, for 35 cycles; 72°C extension for 10 min; and storage at 4°C.

4. A gene for wheat powdery mildew resistance as described in claim 1 PmXP544 Tightly linked molecular markers in wheat powdery mildew resistance genes PmXP544 The application of gene localization and molecular marker-assisted breeding.

5. The application according to claim 4, detecting whether the test variety carries the wheat powdery mildew resistance gene. PmXP544 The main steps include: (1) Extract genomic DNA from the wheat sample to be tested; (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTUXP35 to obtain the amplification product; (3) Perform electrophoresis on the amplification products and judge the results. If the wheat DNA can amplify a specific band of 348 bp, it indicates that the wheat being tested carries the powdery mildew resistance gene. PmXP544 Otherwise, the wheat being tested does not carry the wheat powdery mildew resistance gene. PmXP544 .

6. In the application according to claim 5, the primers for the molecular marker YTUXP35 in step (2) include an upstream primer YTUXP35-F and a downstream primer YTUXP35-R, wherein the nucleotide sequence of the upstream primer YTUXP35-F is shown in SEQ ID NO:1 and the nucleotide sequence of the downstream primer YTUXP35-R is shown in SEQ ID NO:

2.

7. According to claim 5, the PCR amplification system suitable for the labeling in step (2) is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

8. According to the application of claim 5, the PCR amplification program applicable to the label in step (2) is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.

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