A SNP primer combination for identifying blumeria graminis, application and identification method
By developing 10 SNP primer combinations, rapid, accurate, and low-cost identification of wheat powdery mildew was achieved, solving the problems of high cost and limited identification power in existing technologies, and supporting disease-resistant breeding and disease control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2026-06-08
- Publication Date
- 2026-07-14
AI Technical Summary
Existing molecular marker technologies for wheat powdery mildew are costly to develop and have limited distinguishing power, making it difficult to quickly and accurately differentiate strains from different geographical origins and virulence profiles, thus affecting disease resistance breeding and disease control.
A combination of 10 SNP primers was developed to identify the genetic diversity and strain variation of wheat powdery mildew through PCR reaction and sequencing analysis. The 10 SNP primers were used for amplification and detection to determine whether the strains were identical.
It enables rapid, accurate, and low-cost strain identification, distinguishing multiple strains and supporting disease-resistant breeding and disease control.
Smart Images
Figure CN122382249A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to an SNP primer combination, its application, and an identification method for identifying wheat powdery mildew. Background Technology
[0002] Wheat powdery mildew is a significant disease caused by the obligate parasitic fungus *Blumeria graminis* f. sp. *tritici*, posing a persistent threat to global wheat production. Infected wheat suffers from premature leaf blight, reduced photosynthesis, increased respiration, increased transpiration, fewer tillers, and lower ear formation, severely impacting normal growth and development. Thousand-grain weight decreases, typically resulting in yield reductions of 5%–10%, with severely affected fields experiencing yield losses exceeding 20%. Breeding and strategically distributing resistant varieties is the most economical and environmentally friendly strategy for controlling this disease. However, the effectiveness of this strategy highly depends on a precise understanding of the pathogen's genetic structure, virulence variation, and epidemiological patterns. When locating and cloning powdery mildew resistance genes, single strains used for phenotypic identification of genetic populations are prone to contamination during cultivation. Contaminating strains leads to inaccurate phenotypic identification of the population, sometimes even resulting in resistant parents exhibiting susceptibility, thus deviating from the localization of resistance genes. Therefore, achieving rapid, efficient, and stable identification of powdery mildew has become a core research topic in plant pathology and molecular breeding.
[0003] With the development of sequencing technology, single nucleotide polymorphism (SNP) markers based on whole-genome resequencing have shown great potential. SNPs are numerous and widely distributed, theoretically offering extremely high discriminative power. In fungal research, SNP markers provide important clues for revealing species genetic diversity and population history. For example, SNPs have been successfully used for fungal species identification, even simultaneously detecting and identifying 16 Fusarium species. However, the high cost and complex data analysis of whole-genome resequencing-based development protocols greatly limit their widespread application in ordinary laboratories.
[0004] In summary, current molecular marker technology for wheat powdery mildew still faces challenges such as insufficient marker quantity, high development costs, limited discriminative power, and limited ability to detect complex traits (such as herbicide resistance). To address these issues, there is an urgent need for a core molecular marker set with a suitable number of loci, high polymorphism, and stable amplification, capable of rapidly and accurately distinguishing powdery mildew strains from different geographical origins and with varying virulence profiles. This would be crucial for studying disease epidemiology and pathogen transmission pathways, thereby providing strong technical support for sustainable green control of wheat powdery mildew, disease-resistant breeding, and herbicide resistance management. Summary of the Invention
[0005] This invention aims to address the technical problems of existing molecular marker technologies for wheat powdery mildew, such as high development costs and limited identification power. The purpose is to provide a combination of SNP primers, their application, and an identification method for wheat powdery mildew. The 10 developed SNP primers can be used to analyze the genetic diversity, population dynamics, strain variation, and contamination of powdery mildew. They are characterized by simple, rapid, efficient, and accurate identification, and have low development costs.
[0006] This invention is achieved through the following technical solution:
[0007] The first objective of this invention is to provide a combination of SNP primers for identifying wheat powdery mildew, comprising the following SNP primers:
[0008] SNP primer 1: 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;
[0009] SNP primer 2: The upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4;
[0010] SNP primer 3: The upstream primer nucleotide sequence is shown in SEQ ID NO.5, and the downstream primer nucleotide sequence is shown in SEQ ID NO.6;
[0011] SNP primer 4: The upstream primer nucleotide sequence is shown in SEQ ID NO.7, and the downstream primer nucleotide sequence is shown in SEQ ID NO.8;
[0012] SNP primer 6: The upstream primer nucleotide sequence is shown in SEQ ID NO.11, and the downstream primer nucleotide sequence is shown in SEQ ID NO.12;
[0013] SNP primer 7: The upstream primer nucleotide sequence is shown in SEQ ID NO.13, and the downstream primer nucleotide sequence is shown in SEQ ID NO.14;
[0014] SNP primer 8: The upstream primer nucleotide sequence is shown in SEQ ID NO.15, and the downstream primer nucleotide sequence is shown in SEQ ID NO.16;
[0015] SNP primer 9: The upstream primer nucleotide sequence is shown in SEQ ID NO.17, and the downstream primer nucleotide sequence is shown in SEQ ID NO.18;
[0016] SNP primer 10: The upstream primer nucleotide sequence is shown in SEQ ID NO.19, and the downstream primer nucleotide sequence is shown in SEQ ID NO.20;
[0017] SNP primer 11: The upstream primer nucleotide sequence is shown in SEQ ID NO.21, and the downstream primer nucleotide sequence is shown in SEQ ID NO.22.
[0018] The second objective of this invention is to provide an application of an SNP primer combination for identifying wheat powdery mildew in the identification of wheat powdery mildew strain diversity.
[0019] The third objective of this invention is to provide an application of an SNP primer combination for identifying wheat powdery mildew in constructing an identification fingerprint of wheat powdery mildew strains.
[0020] The fourth objective of this invention is to provide a method for identifying the diversity of wheat powdery mildew strains, which uses the aforementioned SNP primer combination for amplification and detection, and determines whether the strains to be identified are the same based on the detection results.
[0021] Furthermore, a method for identifying the diversity of wheat powdery mildew strains includes the following steps:
[0022] Genomic DNA was extracted from the powdery mildew fungus strain to be tested.
[0023] Using the genomic DNA of the powdery mildew strain to be tested as a template, amplification was performed using the aforementioned SNP primer combination to obtain the amplification product;
[0024] The haplotype of the amplified product was determined by sequencing.
[0025] By comparing haplotypes, it can be determined whether the powdery mildew strains being tested belong to the same species.
[0026] Furthermore, the amplification using the aforementioned SNP primer combination specifically involves performing a PCR reaction.
[0027] Furthermore, the PCR reaction system includes a DNA template, DNA polymerase, and SNP primers.
[0028] Furthermore, the PCR procedure is specifically as follows:
[0029] Pre-denaturation at 94 ℃ for 3 minutes; denaturation at 94 ℃ for 30 seconds, annealing at 48–60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, 30 cycles; final extension at 72 ℃ for 5 minutes.
[0030] Furthermore, the criterion for determining whether the powdery mildew strains to be tested belong to the same strain is: if the haplotypes at 10 loci are completely identical, they belong to the same strain.
[0031] Furthermore, the specific method for determining whether the powdery mildew fungal strains being tested belong to the same species by comparing haplotypes is as follows:
[0032] First, compare the haplotypes at any locus to see if they are the same. If they are different, they belong to different strains. If they are the same, further compare the haplotypes at the second locus to see if they are the same, until all loci show the same haplotypes, then they are determined to be the same strain.
[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0034] The 10 SNP primers developed in this invention can be used to analyze the genetic diversity, population dynamics, strain variation and confounding of powdery mildew. Multiple strains can be completely distinguished with only a small number of SNP primers. It has the characteristics of simple, fast, efficient and accurate identification, and low development cost. Attached Figure Description
[0035] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:
[0036] Figure 1 The results are the detection results of the PCR amplification products in Example 1;
[0037] Figure 2 The six haplotypes were obtained by sequencing analysis of the PCR product of site 1 in Example 1. Detailed Implementation
[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.
[0039] Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art.
[0040] The "scope" disclosed in this invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, which define the boundaries of the specific scope. The scope defined in this way can include or exclude end values, and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a scope.
[0041] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.
[0042] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.
[0043] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other substances not listed may also be included, or that only the listed substances may be included.
[0044] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.
[0045] The technical solution of the present invention will be further described in detail below with reference to the embodiments.
[0046] It should be noted that, unless otherwise specified, the experimental methods used in the embodiments are conventional methods. The materials, reagents, methods, and instruments used, unless otherwise specified, are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.
[0047] Example 1
[0048] Development and validation of SNP molecular markers
[0049] Whole-genome resequencing data (fastq files) of 100 *B. tumefaciens* (Powdery mildew) strains were downloaded from the NCBI (National Center for Biotechnology Information) database (https: / / www.ncbi.nlm.nih.gov / sra / ?term=SRP062198). These data were aligned to the *B. tumefaciens* reference genome Bgt_genome_v3.16 (Wicker et al., 2013) using the Burrows-Wheeler-Alignment (BWA) tool, and genetic variations were identified using the Genome Analysis Toolkit (GATK) (McKenna et al., 2010). Based on the genotype data, a 1 Kb sliding window analysis was performed. The window with the highest π value on each chromosome was selected as the candidate region with the highest SNP diversity. Within the candidate regions on 11 chromosomes, an average of 20.2 single nucleotide polymorphism sites were found per chromosome, with an average π value of 0.0074. Among them, chromosome LR026987.1 (0.980 Mb – 0.981 Mb region) had the most SNPs, with a total of 43 and a π value of 0.0149; while chromosome LR026991.1 (1.377–1.378 Mb region) had the fewest SNPs, with only 6 and a π value of 0.0052. Based on the sequencing data of 245 powdery mildew (Bgt) strains in our laboratory, we analyzed the haplotypes of the target loci. The results are shown in Table 1. The loci on chromosome 4 had the highest haplotype diversity (17 haplotypes), while the loci on chromosomes 3 and 8 had the lowest haplotype diversity, with only 5 haplotypes each.
[0050] Table 1. Polymorphic sites on each chromosome of wheat powdery mildew.
[0051]
[0052] Conidia of powdery mildew were collected, and DNA was extracted according to the steps and requirements of the Fungal DNA Extraction Kit (D3195-02, OMEGA). Primers were designed based on the sequences of each candidate region in Table 1, as shown in Table 2. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).
[0053] Table 2. Primer sequences for molecular markers of wheat powdery mildew.
[0054]
[0055] Using the primer sequences in Table 2, four purified single strains of powdery mildew were randomly selected from the laboratory, and their genomic DNA was extracted for PCR. The total volume of the PCR reaction system was 20 μL, including 1 µL of DNA template (100 ng / µL), 17 µL of DNA polymerase Green Mix (Beijing Qingke Biotechnology Co., Ltd.), and 1 µL each of forward and reverse primers (10 µM). The PCR program was as follows: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, followed by annealing at an appropriate temperature (48–60 ℃) for 30 seconds according to the primer sequences, and extension at 72℃ for 30 seconds, for 30 cycles; and a final extension at 72℃ for 5 minutes.
[0056] The PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that, except for locus 5, the products at other loci were all around 1000 bp, and the size was as expected (e.g., ...). Figure 1 ).
[0057] First-generation sequencing analysis of the PCR products revealed that these loci all exhibited high polymorphism; for example, locus 1 had six haplotypes (e.g., ...). Figure 2 The vertical lines of different colors on the sequence represent SNPs generated compared to the reference genome. Theoretically, these 10 molecular markers can distinguish up to 1,889,244,000 different powdery mildew strains.
[0058] Example 2
[0059] A method for identifying the diversity of wheat powdery mildew strains, comprising the following steps:
[0060] (1) Extract genomic DNA from the powdery mildew strain to be tested;
[0061] (2) Using the genomic DNA of the powdery mildew strain to be tested as a template, PCR reaction was performed using the SNP primer combination to obtain the amplification product; the total volume of the PCR reaction system was 20 μL, including 1 µL DNA template (100 ng / µL), 17 µL DNA polymerase Green Mix (Beijing Qingke Biotechnology Co., Ltd.), and 1 µL (10 µM) each of the upstream and downstream primers; the PCR program was as follows: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30 seconds, 48–60°C annealing for 30 seconds, 72°C extension for 30 seconds, 30 cycles; and finally 72°C extension for 5 minutes.
[0062] (3) Sequencing analysis of PCR products to obtain haplotypes at different sites. By comparing the haplotypes of PCR products, it can be determined whether two wheat powdery mildew strains are the same. If the haplotypes at site 1 are the same, the haplotypes at site 2 can be compared, and so on, until the haplotypes at all sites are the same, then they are judged to be the same strain.
[0063] Example 3
[0064] The diversity of 40 powdery mildew strains was evaluated using the 10 SNP primers of the present invention, as described in Example 2. The results are shown in Table 3.
[0065] Analysis shows that only 4-5 markers are needed to completely distinguish all 40 strains. Specifically, haplotypes at locus 1 can divide the 40 strains into 7 types; for example, B30-A2, B20-A3, and B47-A3 all belong to haplotype 1. Haplotypes at locus 2 can further divide the 40 strains into 5 types; for example, B30-A2 belongs to haplotype 1, B20-A3 belongs to haplotype 2, and B47-A3 belongs to haplotype 3. Since B30-A2 and B20-A3 are different from the other 39 strains in both haplotypes at loci 1 and loci 2, we can conclude that B30-A2 and B20-A3 are distinct from the other 39 strains. Meanwhile, B47-A3 and the other 6 strains share the same haplotype at loci 1 and loci 2. Therefore, we need to analyze whether the haplotype at loci 3 is the same as that of these 6 strains. If they are different, then strain B47-A3 is also different from the other 39 strains. If they are the same, then we need to analyze the haplotype at loci 4, and so on. Finally, we can determine whether the strain is unique by using 10 molecular markers.
[0066] Table 3. Results of diversity analysis of 40 powdery mildew strains
[0067]
[0068]
[0069] Note: In the strain column, B+ numbers represent the sample number, and A+ numbers represent different isolated and purified strains; in the locus column, different numbers represent different haplotypes, and "NA" indicates that no valid typing data was obtained, possibly due to PCR failure or poor sequencing quality.
[0070] In summary, the 10 SNP primers developed in this invention can be used to analyze the genetic diversity, population dynamics, strain variation and contamination of powdery mildew. Multiple strains can be completely distinguished with only a small number of SNP primers, and the identification is simple, rapid, efficient and accurate.
[0071] Finally, it should be noted that the above specific embodiments are only used to describe the purpose, technical solution, and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation of the present invention and is not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the foregoing specific embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions or improvements can be made to some or all of the technical features. These modifications, equivalent substitutions, and improvements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.
Claims
1. A combination of SNP primers for identifying wheat powdery mildew, characterized in that, Including the following SNP primers: SNP primer 1: 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; SNP primer 2: The upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4; SNP primer 3: The upstream primer nucleotide sequence is shown in SEQ ID NO.5, and the downstream primer nucleotide sequence is shown in SEQ ID NO.6; SNP primer 4: The upstream primer nucleotide sequence is shown in SEQ ID NO.7, and the downstream primer nucleotide sequence is shown in SEQ ID NO.8; SNP primer 6: The upstream primer nucleotide sequence is shown in SEQ ID NO.11, and the downstream primer nucleotide sequence is shown in SEQ ID NO.12; SNP primer 7: The upstream primer nucleotide sequence is shown in SEQ ID NO.13, and the downstream primer nucleotide sequence is shown in SEQ ID NO.14; SNP primer 8: The upstream primer nucleotide sequence is shown in SEQ ID NO.15, and the downstream primer nucleotide sequence is shown in SEQ ID NO.16; SNP primer 9: The upstream primer nucleotide sequence is shown in SEQ ID NO.17, and the downstream primer nucleotide sequence is shown in SEQ ID NO.18; SNP primer 10: The upstream primer nucleotide sequence is shown in SEQ ID NO.19, and the downstream primer nucleotide sequence is shown in SEQ ID NO.20; SNP primer 11: The upstream primer nucleotide sequence is shown in SEQ ID NO.21, and the downstream primer nucleotide sequence is shown in SEQ ID NO.
22.
2. The application of the SNP primer combination for identifying wheat powdery mildew as described in claim 1 in the identification of wheat powdery mildew strain diversity.
3. The application of the SNP primer combination for identifying wheat powdery mildew as described in claim 1 in constructing an identification fingerprint of wheat powdery mildew strains.
4. A method for identifying the diversity of wheat powdery mildew strains, characterized in that, After amplification using the SNP primer combination described in claim 1, detection is performed, and the identification results are used to determine whether the strains to be identified are the same.
5. The method for identifying the diversity of wheat powdery mildew strains according to claim 4, characterized in that, Includes the following steps: Genomic DNA was extracted from the powdery mildew fungus strain to be tested. Using the genomic DNA of the powdery mildew strain to be tested as a template, amplification was performed using the SNP primer combination described in claim 1 to obtain the amplification product; The haplotype of the amplified product was determined by sequencing. By comparing haplotypes, it can be determined whether the powdery mildew strains being tested belong to the same species.
6. The method for identifying the diversity of wheat powdery mildew strains according to claim 5, characterized in that, Amplification using the SNP primer combination described in claim 1 specifically involves performing a PCR reaction.
7. The method for identifying the diversity of wheat powdery mildew strains according to claim 6, characterized in that, The PCR reaction system includes a DNA template, DNA polymerase, and SNP primers.
8. The method for identifying the diversity of wheat powdery mildew strains according to claim 6, characterized in that, The PCR procedure is as follows: Pre-denaturation at 94 ℃ for 3 minutes; denaturation at 94 ℃ for 30 seconds, annealing at 48–60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, 30 cycles; final extension at 72 ℃ for 5 minutes.
9. The method for identifying the diversity of wheat powdery mildew strains according to claim 5, characterized in that, The standard for determining whether powdery mildew fungal strains belong to the same strain is: if the haplotypes at 10 loci are completely identical, they belong to the same strain.
10. The method for identifying the diversity of wheat powdery mildew strains according to claim 5, characterized in that, The specific method for determining whether powdery mildew fungal strains belong to the same species by comparing haplotypes is as follows: First, compare the haplotypes at any locus to see if they are the same. If they are different, they belong to different strains. If they are the same, further compare the haplotypes at the second locus to see if they are the same, until all loci show the same haplotypes, then they are determined to be the same strain.