Molecular marker of forest rye powdery mildew resistance related gene PmSESY and application thereof

Abstract

The invention provides a molecular marker of a forest rye powdery mildew resistance related gene PmSESY and application of the molecular marker, and belongs to the technical field of plant molecular biology and genetic breeding. The molecular marker is closely linked with a forest rye powdery mildew resistance gene PmSESY and is marked as PmSESY-M1, and amplification primers of the molecular marker are as shown in SEQ ID No: 1 and SEQ ID No: 2; the molecular marker amplification primer can be subjected to PCR (Polymerase Chain Reaction) amplification in a 1RL chromosome segment material containing the PmSESY gene to obtain a specific product of 383 bp; the molecular marker PmSESY-M1 has the advantages of strong specificity, high stability, good repeatability and the like, can efficiently and accurately detect and track disease-resistant chromosome segments in a wheat-forest rye introgression line, and provides a novel and practical auxiliary selection tool for wheat powdery mildew resistance molecular breeding; the method has an important practical value in wheat powdery mildew resistance molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and genetic breeding technology, specifically relating to a molecular marker of the forest rye powdery mildew resistance gene PmSESY and its application. Background Technology

[0002] Wheat powdery mildew is a foliar disease caused by the grass fungus *Brucea balsamina*, which seriously threatens global wheat production security. Currently, breeding and promoting resistant varieties is the most economical and effective strategy for controlling this disease. However, the existing genetic basis for resistance in major wheat varieties is relatively narrow, and with the continuous variation of the pathogen's physiological races, many identified powdery mildew resistance genes are gradually losing their resistance. Therefore, discovering new powdery mildew resistance genes from closely related wheat species and broadening the genetic basis of wheat resistance is crucial for achieving sustainable development in wheat production.

[0003] Species of the genus *Secale*, especially cultivated rye, are an important gene pool for wheat disease resistance breeding. Several powdery mildew resistance genes, such as Pm7, Pm8, Pm17, Pm20, and Pm56, have been successfully transferred from cultivated rye to common wheat. However, there are few reports of powdery mildew resistance genes discovered from forest rye (*Secale sylvestre*). PmSESY is a novel powdery mildew resistance gene recently identified from the forest rye line SESY-01, located at the end of chromosome 1RL of forest rye (He et al. Characterization of a new gene for resistance to wheat powdery mildew onchromosome 1RL of wild rye Secale sylvestre. Theor Appl Genet. 2021,134:887-896). Introducing this gene into common wheat is expected to provide new genetic resources for wheat disease resistance breeding. However, in actual breeding, after introducing exogenous chromosome fragments into wheat through distant hybridization, relying solely on phenotypic identification to screen for disease-resistant plants suffers from low efficiency, long cycles, and susceptibility to environmental interference. Therefore, developing molecular markers that are closely linked to the target gene and have high specificity in the context of common wheat is crucial for achieving efficient tracking of exogenous genes and accelerating the innovation of disease-resistant germplasm. Currently, no molecular markers for the forest rye PmSESY gene that can be stably applied in the genetic background of common wheat have been reported. Summary of the Invention

[0004] To address some shortcomings in existing technologies, this invention provides a molecular marker for the forest rye powdery mildew resistance gene PmSESY and its applications. The molecular marker described in this invention is closely linked to the forest rye powdery mildew resistance gene PmSESY and is designated PmSESY-M1. The amplification primers for the molecular marker are shown in SEQ ID No:1 and SEQ ID No:2. These primers can be used for PCR amplification of a 383 bp specific product in 1RL chromosome fragments containing the PmSESY gene. The molecular marker PmSESY-M1 possesses advantages such as high specificity, high stability, and good reproducibility, enabling efficient and accurate detection and tracking of disease-resistant chromosome fragments in wheat-forest rye introgression lines. It provides a novel and practical auxiliary selection tool for molecular breeding of wheat powdery mildew resistance and has significant practical value in this field.

[0005] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0006] The present invention first provides a molecular marker PmSESY-M1, which is used to specifically detect the forest rye powdery mildew resistance gene PmSESY. The molecular marker is located on the 1RL chromosome of forest rye containing the forest rye powdery mildew resistance gene PmSESY and is closely linked to the forest rye powdery mildew resistance gene PmSESY.

[0007] This invention also provides primers for detecting the powdery mildew resistance gene PmSESY or the aforementioned molecular marker PmSESY-M1, the nucleotide sequence of which is as follows:

[0008] PmSESY-M1-F: 5'-CTAGCTTTGATTGAGTCCATTG-3' (SEQ ID No: 1),

[0009] PmSESY-M1-R: 5'-TGGCACGTGACAATGGCACCAT-3' (SEQ ID No: 2).

[0010] The present invention also provides a product for identifying powdery mildew resistance, the product comprising the above-mentioned primers.

[0011] Preferably, the product includes reagents or kits.

[0012] This invention also provides the application of the above-mentioned molecular marker PmSESY-M1, or primers, or products in the detection of wheat, the application including:

[0013] (1) Detect whether wheat carries the powdery mildew resistance gene PmSESY;

[0014] (2) To identify or assist in identifying whether forest rye, wheat, or wheat-forest rye is resistant to powdery mildew;

[0015] (3) Breeding or assisted breeding of wheat resistant to powdery mildew.

[0016] The present invention also provides a method for detecting powdery mildew resistance, the method comprising:

[0017] The genomic DNA of the leaves of the test material was amplified by PCR using primers of the molecular marker PmSESY-M1 or the primers mentioned above, and the amplification products were detected. If a specific target product was obtained, the test material was identified as a powdery mildew resistant material.

[0018] Preferably, the specific target product is a specific amplification fragment with a size of 383 bp.

[0019] Preferably, the 25 μL PCR reaction system used in the PCR amplification procedure comprises: 50 ng of leaf genomic DNA of the test material, 1× PCR buffer, and 1.5 mmol / L. -1 MgCl2, 200 mmol L -1 dNTPs, final concentration 2 mol L -1 Amplification primers, 1 U Taq DNA polymerase, and sterile deionized water were added.

[0020] Preferably, the PCR amplification reaction program includes: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 20 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, 34 cycles; extension at 72°C for 5 minutes; and storage at 4°C.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] Based on the rye genome annotation, this invention locates the powdery mildew resistance gene PmSESY within a physical region of approximately 0.26 Mb on the forest rye 1RL chromosome. Within this region, the gene SECCE1Rv1G0062340 (1RL chromosome: 719,399,788 to 719,404,203) encodes a lectin receptor kinase LecRK that may be involved in the disease resistance response, and this gene was selected as a candidate gene for PmSESY. By comparing and analyzing the genomic sequences of the LecRK gene in cultivated rye and common wheat (Chinese Spring), a specific molecular marker, PmSESY-M1, was developed. This molecular marker can be used to detect new wheat-forest rye germplasm and for precise selection during the breeding of powdery mildew-resistant wheat varieties or lines, providing a novel, practical, and efficient molecular marker-assisted selection technology for breeding new powdery mildew-resistant wheat varieties.

[0023] The molecular marker PmSESY-M1 provided by this invention can accurately identify whether a wheat-forest rye introgression line contains the 1RL chromosome segment of the PmSESY gene. This molecular marker has the advantages of high specificity, high stability, good reproducibility, and simple operation, which can significantly improve the efficiency of disease-resistant germplasm creation and new variety breeding, and provide important technical support for molecular breeding of wheat resistant to powdery mildew. Attached Figure Description

[0024] Figure 1 Phenotypes of the F1 generation of the cross between forest rye and Chinese spring wheat; In the figure, 1-6: F1 generation of the cross between forest rye and Chinese spring wheat; 7: Chinese spring wheat susceptible to powdery mildew; 8: Forest rye line SESY-01, the donor material containing the PmSESY gene.

[0025] Figure 2 The amplification effect of molecular marker PmSESY-M1 in parental materials is shown in the figure. 1-6: F1 of forest rye and Chinese spring wheat; 7: Chinese spring wheat susceptible to powdery mildew; 8: forest rye line SESY-01, the donor material containing the PmSESY gene. The arrow indicates the specific band (383 bp) for tracing the 1RL chromosome segment of forest rye.

[0026] Figure 3 Figure 1-32 shows the results of genotyping of the progeny of the forest rye cultivar SESY-01 and Yangmai 23 through a recurrent cross using the molecular marker PmSESY-M1. In the figure, M represents the DNA molecular weight standard DL2000. Different individual plants from the progeny of the forest rye cultivar SESY-01 and Yangmai 23 are also shown.

[0027] Figure 4 Figure shows the results of powdery mildew resistance phenotype identification in wheat-forest rye introgression progeny; in the figure, 1-6: single plants carrying the PmSESY gene; 7-8: single plants not carrying the PmSESY gene; 9: Yangmai 23, the recurrent parent susceptible to powdery mildew. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the embodiments of the present invention, unless otherwise described, conventional experimental methods are used. The processes involved in the embodiments, unless otherwise described, are those that can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0029] Example 1:

[0030] The inventors' previous research (He et al. Characterization of a new gene for resistance to wheat powdery mildew on chromosome 1RL of wild rye Secalesylvestre. Theor Appl Genet. 2021,134:887-896) found that the powdery mildew resistance gene PmSESY is located in a physical region of about 0.26 Mb on the forest rye 1RL chromosome. Based on the reference genome annotation of the rye variety Lo7 (accession number: PRJEB35461), the SECCE1Rv1G0062340 gene (chromosome 1RL: 719,399,788 to 719,404,203) at the genetic locus where the PmSESY gene is located was found to encode a lectin receptor kinase, which is an immune receptor with both sugar recognition and signal transduction functions and plays a key role in the process of plant defense against pathogen invasion. The rye SECCE1Rv1G0062340 gene can be considered as a potential candidate gene for forest rye PmSESY.

[0031] In light of the above research findings, and in order to further develop molecular markers for identifying or assisting in the identification of wheat resistance to powdery mildew, this embodiment designs specific primers PmSESY-M1-F and PmSESY-M1-R targeting the sequence of the potential candidate gene rye SECCE1Rv1G0062340, PmSESY, from forest rye.

[0032] PmSESY-M1-F: 5'-CTAGCTTTGATTGAGTCCATTG-3' (SEQ ID No: 1),

[0033] PmSESY-M1-R: 5'-TGGCACGTGACAATGGCACCAT-3' (SEQ ID No: 2).

[0034] The reliability of the above-mentioned specific primers was verified by PCR using F1 cells of the disease-resistant forest rye strain SESY-01 (Polish Genebank: National Centre for Plant Genetic Resources, website: https: / / bankgenow.edu.pl / en) and common wheat Chinese Spring (susceptible, publicly known material, provided by Jiangsu Lixiahe Agricultural Science Research Institute), as well as genomic DNA from the susceptible Chinese Spring and disease-resistant forest rye strain SESY-01.

[0035] The amplification conditions are as follows:

[0036] PCR reaction system: 25 μL reaction system contains approximately 50 ng template DNA (genomic DNA), 1×PCR buffer, 1.5 mmol L⁻¹ MgCl₂, and 200 mmol L⁻¹ PCR buffer. -1 dNTPs, with a final concentration of 2 μmol / L for each primer. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water.

[0037] PCR reaction procedure: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 34 cycles; 72℃ final extension for 5 minutes; store at 4℃.

[0038] PCR products were detected by 8% polyacrylamide gel electrophoresis: the PCR products were mixed with 5 μL of loading buffer and separated by electrophoresis on an 8% non-denaturing polyacrylamide gel. Electrophoresis was performed at a constant voltage of 120 V for 2 hours and then stained with silver.

[0039] Staining results showed that a clear 383 bp band, namely the molecular marker PmSESY-M1 described in this invention, was stably amplified only in the forest rye line SESY-01 and hybrid F1, while this band was not found in Chinese spring rye. This demonstrates that the molecular marker PmSESY-M1 has high specificity and can be used to track chromosome 1R carrying PmSESY in a wheat background.

[0040] Example 2:

[0041] In this embodiment, the powdery mildew resistance gene PmSESY from forest rye was introduced into common wheat through systematic distant hybridization and backcrossing. The molecular marker PmSESY-M1 obtained in Example 1 was used to assist in the creation of new disease-resistant wheat-forest rye germplasm. The specific steps are as follows:

[0042] (1) Hybrid parents and design:

[0043] Intergeneric hybridization was carried out using the common wheat variety China Spring, which is highly susceptible to powdery mildew, as the female parent (recipient) and the disease-resistant forest rye line SESY-01 carrying the PmSESY gene as the male parent (donor).

[0044] (2) Embryo rescue and F1 generation acquisition:

[0045] Due to reproductive barriers in intergeneric hybridization, embryos are taken for in vitro culture approximately 14-16 days after hybridization (embryo rescue technique) to obtain hybrid F1 plants. The authenticity of the hybrid F1 plants is confirmed by powdery mildew identification and detection of the molecular marker PmSESY-M1.

[0046] The steps for identifying powdery mildew include:

[0047] Forest rye cultivar SESY-01 was crossed with common wheat (Chinese Spring) as the female parent to obtain F1 generation plants. At the one-leaf stage, the F1 generation and its parents (Forest rye cultivar SESY-01 and common wheat (Chinese Spring)) were inoculated with powdery mildew strain BgtYZ011 (collected by the inventor from Yangzhou, Jiangsu Province). Leaf phenotypes of the F1 generation and parental plants were observed on the eighth day after inoculation to assess powdery mildew resistance. All plants used in this study were cultured in a greenhouse under a diurnal cycle of 16 hours light and 8 hours dark, with the temperature maintained at approximately 22℃. Results are as follows: Figure 1 As shown: Numbers 1-6 represent leaf phenotypes in the F1 generation plants exhibiting resistance to powdery mildew; number 7 represents the susceptible phenotype of the maternal parent, common wheat (Chinese Spring); and number 8 represents the resistant phenotype of the paternal parent, forest rye cultivar SESY-01. Since the disease resistance trait controlled by the PmSESY gene is dominantly inherited, and the leaf phenotype of the F1 generation plants is consistent with that of the paternal parent, forest rye cultivar SESY-01, this indicates that the hybridization combination was successful, and the resulting F1 plants are true hybrids.

[0048] The steps for detecting the molecular marker PmSESY-M1 include:

[0049] Genomic DNA from F1 leaves was amplified by PCR using amplification primers PmSESY-M1-F and PmSESY-M1-R. The amplification products were then detected. If a specific target product was obtained, the tested material carried the powdery mildew resistance gene PmSESY and was therefore resistant to powdery mildew. Otherwise, the material did not carry the powdery mildew resistance gene PmSESY.

[0050] The PCR reaction system used in the PCR amplification procedure includes: a 25 μL reaction system containing: 50 ng of leaf genomic DNA from the test material, 1×PCR buffer, 1.5 mmol L⁻¹ MgCl₂, and 200 mmol L⁻¹ MgCl₂. -1 dNTPs, final concentration 2 mol L -1 Amplification primers, 1 U Taq DNA polymerase, and sterile deionized water were added.

[0051] The PCR amplification reaction procedure includes: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 34 cycles; 72℃ extension for 5 minutes; storage at 4℃.

[0052] The results are as follows Figure 2As shown, lanes 1-6 represent the bands corresponding to the F1 generation plants identified using the molecular marker PmSESY-M1; lane 7 represents the band of the maternal parent, common wheat (Chinese Spring); and lane 8 represents the band of the paternal parent, forest rye line SESY-01. Since this molecular marker can amplify a specific 383 bp band in materials containing the PmSESY gene, and both the F1 plants in lanes 1-6 and the paternal parent forest rye line SESY-01 in lane 8 showed this band, it further confirms that the F1 generation is a true hybrid.

[0053] (3) Backcrossing and population building:

[0054] To restore the agronomic background of common wheat and retain the target disease-resistant genes, Yangmai 23 (publicly known and provided by the Jiangsu Lixiahe Agricultural Science Research Institute), a common wheat variety with excellent comprehensive traits and high susceptibility to powdery mildew, was used as the recurrent parent and multiple crosses were performed with the aforementioned F1 plants. After each cross, powdery mildew resistance was identified using the method described in step (2), and genomic DNA was extracted from the plants at the seedling stage. PCR detection was performed using PmSESY-M1 to assist selection, and the results are as follows: Figure 3 As shown: Lanes 1-32 represent the detection results of individual plants in the recurrent hybridization progeny of the forest rye line SESY-01 and Yangmai 23 using this molecular marker. Lanes 2, 6, 9, 10, 11, 14, 21, 22, 25, 26, 28, and 32 all showed specific bands of 383 bp, indicating that these individual plants are positive for carrying the PmSESY gene.

[0055] Single plants that are disease-resistant and whose agronomic traits tend to be similar to those of the recurrent parent were selected and backcrossed with Yangmai 23 to obtain BC3F1 plants.

[0056] (4) Self-pollination purification and creation of high-generation germplasm:

[0057] The BC3F1 plants obtained in step (3) were self-pollinated to form the BC3F2 segregating population. In this population, the molecular marker PmSESY-M1 was used to screen for single plants containing the target gene. After two self-pollinations and confirmation by the molecular marker PmSESY-M1, a high-generation wheat-forest rye introgression line carrying the PmSESY gene was created.

[0058] (5) Identification of powdery mildew resistance phenotype:

[0059] Artificial inoculation of the obtained high-generation wheat-forest rye infiltration lines carrying the PmSESY gene was performed to investigate powdery mildew resistance. Powdery mildew was identified using the method described in step (2). The powdery mildew strain BgtYZ01 was inoculated into the seedlings of the recurrent parent common wheat Yangmai 23 and the high-generation wheat-forest rye infiltration lines at the one-leaf stage. Powdery mildew resistance was assessed on the eighth day after inoculation. The results are as follows: Figure 4As shown in the figure, lines 1-6 are advanced wheat-forest rye introgression lines carrying the PmSESY gene, exhibiting a leaf phenotype resistant to powdery mildew; lines 7-8 are advanced wheat-forest rye introgression lines not carrying the PmSESY gene, also exhibiting a susceptible phenotype; and line 9 is the recurrent parent Yangmai 23, exhibiting a susceptible phenotype.

[0060] The results showed that all materials carrying the PmSESY-M1 specific band exhibited high or moderate resistance to powdery mildew, while materials without this band and the recurrent parent Yangmai 23 showed high susceptibility. This confirms that the PmSESY resistance in forest rye SESY-01 was successfully transferred to common wheat via this technical pathway. This further confirms the accuracy and practicality of the molecular marker PmSESY-M1 in assisted selection. In summary, this invention provides a molecular marker for identifying the forest rye powdery mildew resistance gene PmSESY and its applications. The molecular marker is closely linked to the forest rye powdery mildew resistance gene PmSESY and is designated PmSESY-M1. The amplification primers for the molecular marker are shown in SEQ ID No:1 and SEQ ID No:2. These primers can be used for PCR amplification of a 383 bp specific product in 1RL chromosome fragments containing the PmSESY gene. The molecular marker PmSESY-M1 possesses advantages such as high specificity, high stability, and good reproducibility, enabling efficient and accurate detection and tracking of disease-resistant chromosome fragments in wheat-forest rye introgression lines. It provides a novel and practical auxiliary selection tool for molecular breeding of wheat powdery mildew resistance and has significant practical value in this field.

[0061] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Molecular marker PmSESY-M1, characterized in that, The molecular marker is used to specifically detect the forest rye powdery mildew resistance gene PmSESY. The molecular marker is located on the 1RL chromosome of forest rye containing the powdery mildew resistance gene PmSESY and is closely linked to the forest rye powdery mildew resistance gene PmSESY.

2. Primers for detecting the powdery mildew resistance gene PmSESY or the molecular marker PmSESY-M1 as described in claim 1, characterized in that, The nucleotide sequence of the primer is as follows: PmSESY-M1-F: 5'-CTAGCTTTGATTGAGTCCATTG-3' (SEQ ID No: 1), PmSESY-M1-R: 5'-TGGCACGTGACAATGGCACCAT-3' (SEQ ID No: 2).

3. A product for identifying powdery mildew resistance, characterized in that, The product includes reagents or kits, and the product contains the primers as described in claim 2.

4. The application of the molecular marker of claim 1, or the primer of claim 2, or the product of claim 3, wherein the application includes: (1) Detect whether wheat carries the powdery mildew resistance gene PmSESY; (2) To identify or assist in identifying whether forest rye, wheat, or wheat-forest rye is resistant to powdery mildew; (3) Breeding or assisted breeding of wheat resistant to powdery mildew.

5. A method for detecting powdery mildew resistance, characterized in that, The method includes: The genomic DNA of the leaf of the test material is amplified by PCR using the primers of the molecular marker described in claim 1 or the primers described in claim 2, and the amplification products are detected. If a specific target product is obtained, the test material is a powdery mildew resistant material.

6. The method according to claim 5, characterized in that, The specific target product is a specific amplification fragment with a size of 383 bp.

7. The method according to claim 5, characterized in that, The 25 μL PCR reaction system used in the PCR amplification procedure included: 50 ng of leaf genomic DNA from the test material, 1× PCR buffer, and 1.5 mmol / L. -1 MgCl2, 200 mmol L -1 dNTPs, final concentration 2 mol L -1 Amplification primers, 1 U Taq DNA polymerase.

8. The method according to claim 5, characterized in that, The PCR amplification reaction procedure includes: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 34 cycles; 72℃ extension for 5 minutes; storage at 4℃.

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