Molecular detection marker for potato scab farinae and detection method of molecular detection marker

Abstract

The invention discloses a potato scab farinae molecular detection marker and a detection method thereof, the molecular marker is a genome DNA fragment with the length of 2496bp, and the nucleotide sequence of the molecular marker is as shown in SEQ ID NO: 1. Based on the molecular marker, a primer pair, SsF1 / R1, SsF2 / R2 and / or SsF3 / R3, for identifying the molecular detection marker for potato scab farinae is also designed. The detection marker has extremely high species specificity, can accurately identify the potato scab farinae, effectively avoids false positive results, greatly improves the accuracy and reliability of detection, provides powerful technical support for rapid and accurate detection of potato scab farinae, is helpful for taking prevention and control measures in time, and has a wide application prospect. The loss of the potato industry caused by diseases is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and relates to a molecular detection marker for potato powdery scab and a detection method thereof, which are used for quickly and accurately detecting potato powdery scab. Background Art

[0002] Potato (Solanum tuberosum) is the fourth largest food crop in the world. However, potato powdery scab, an important fungal soil-borne disease caused by Spongospora subterranea, seriously threatens the healthy development of the potato industry. This disease mainly harms the tubers and roots of potatoes, resulting in a double decline in tuber quality and yield, a significant reduction in the commodity potato rate, and bringing significant economic losses to growers.

[0003] The transmission routes of Spongospora subterranea are diverse. It can be transmitted through diseased tubers or by means of soil. Moreover, the characteristic that its resting sporangia overwinter in the soil or tubers makes this disease highly persistent and difficult to eradicate. With the increasing frequency of global potato trade and the continuous expansion of the planting area, the transmission risk of powdery scab has also increased, and its quarantine and prevention work has become increasingly important and urgent.

[0004] In terms of symptom manifestation, powdery scab is similar to common scab, which brings great difficulties to direct observational diagnosis. Traditional detection methods for powdery scab mainly rely on microscopy and the plant seedling bait method. However, these methods not only require the detector to have solid taxonomic knowledge but also take a long cultivation time, making it difficult to meet the actual needs of rapid and accurate diagnosis.

[0005] The rise of molecular detection technology has brought new hope for the diagnosis of plant diseases. In particular, detection methods based on the PCR technology play an increasingly crucial role in the field of plant disease control. However, currently commonly used primers designed with target sequences such as the internal transcribed spacer (ITS) and ribosomal genes have problems of matching with genes of other species, resulting in insufficient detection specificity and prone to false positive results. For example, when primers designed with the 18S rRNA gene as a template by Mao Liangan et al. were subjected to sequence alignment in the NCBI database, it was found that there were 100% matching sites with the genomic sequences of various microorganisms such as Plasmodiophora brassicae, Cryptococcus neoformans, and Polymyxa graminis existing in the soil. This may cause serious misjudgments in practical applications. Especially in the case where the cruciferous plant rutabaga is often intercropped with potatoes in the southwestern region of China, primers designed based on the 18S rRNA gene are extremely prone to false positives when detecting potato powdery scab.

[0006] Therefore, exploring new species-specific molecular detection targets is of great practical significance for improving the molecular precision detection system of Spongospora subterranea f. sp. subterranea, enhancing the disease management level of potato powdery scab in China, and ensuring the sustainable development of the potato industry.

[0007] This invention is funded by the Sichuan-Chongqing Science and Technology Innovation Cooperation Project (2022YFC2604501) and the Open Project of the Key Laboratory of Green Germplasm Innovation and Genetic Improvement of Cereal and Oil Crops in Sichuan Province (2024LYKF02). Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a species-specific molecular detection marker, detection primer pair, and detection method for potato powdery scab, so as to solve the problems of insufficient specificity and easy occurrence of false positives in existing detection technologies, and achieve rapid and accurate detection of potato powdery scab.

[0009] Through long-term exploration and attempts, as well as multiple experiments and efforts, and continuous reform and innovation, the technical solution provided by this invention to solve the above technical problems is to provide a molecular detection marker for potato powdery scab, and the molecular marker is a genomic DNA fragment with a length of 2496 bp, and its nucleotide sequence is as shown in SEQ ID NO:1.

[0010] Compared with the existing technology, the beneficial effects of this invention are as follows:

[0011] This detection marker has extremely high species specificity, can accurately identify Spongospora subterranea f. sp. subterranea, effectively avoid the occurrence of false positive results, greatly improve the accuracy and reliability of detection, provide strong technical support for the rapid and accurate detection of potato powdery scab, and help to take prevention and control measures in a timely manner to reduce the losses caused by the disease to the potato industry.

[0012] This invention also provides a primer pair for identifying the molecular detection marker of potato powdery scab, and the primer pair is selected from one or more pairs of the following groups:

[0013] (a) SsF1 / R1, and their sequences are respectively:

[0014] SsF1: 5′-CCTCGTCAATGTAAAGCCGTT-3′,

[0015] SsR1: 5′-TCTAAATCCCCAGAATCAGTCAC-3′;

[0016] (b) SsF2 / R2, and their sequences are respectively:

[0017] SsF2: 5′-GATAGCGGTTTACTCAGCCC-3′,

[0018] SsR2: 5′-TGACCTTAGCGATGATAGCACT-3′;

[0019] (c) SsF3 / R3, the sequences of which are respectively:

[0020] SsF3: 5′-ACCTGTGTCAACCATCTCGCCT-3′,

[0021] SsR3: 5′-GTTATCTCCATTACAATCTGCCA-3′.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The primer pairs provided by the present invention have high specificity and sensitivity, can accurately amplify specific DNA fragments of Spongospora subterranea f. sp. subterranea, effectively distinguish the target pathogen from other microorganisms, significantly improve the accuracy and reliability of detection, provide a powerful tool for the rapid and accurate diagnosis of potato powdery scab, help to take effective prevention and control measures in a timely manner, and reduce the impact of the disease on the potato industry.

[0024] Furthermore, the amplified fragment lengths of the primer pairs a, b, and c in the PCR detection are 274bp, 358bp, and 624bp respectively, the annealing temperature is 60.5°C, and the detection sensitivities all reach the 103 copy number level.

[0025] Furthermore, its use is for detecting potato powdery scab, specifically for PCR amplification reaction, and positive amplification bands can be obtained when using the DNA of Spongospora subterranea f. sp. subterranea as a template.

[0026] The present invention also provides a method for detecting potato powdery scab, comprising the following steps:

[0027] Extract the DNA of the sample to be tested;

[0028] Perform PCR amplification using the aforementioned primer pairs;

[0029] Analyze the PCR amplification product to determine whether the DNA of Spongospora subterranea f. sp. subterranea exists.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] The detection method of the present invention is simple, rapid and efficient. Through PCR amplification with specific primer pairs, it can accurately detect the DNA of the pathogen of potato powdery scab, effectively avoid the false positive problem easily occurring in traditional detection methods, significantly improve the accuracy and reliability of detection, provide powerful technical support for the early diagnosis and prevention and control of potato powdery scab, help to take measures in a timely manner to reduce the damage of the disease to potato crops, and ensure the healthy development of the potato industry.

[0032] Based on the above technical solutions, the present invention can be further improved as follows:

[0033] Further, the sample to be tested includes potato disease-infected tuber samples and soil samples.

[0034] Compared with the prior art, the beneficial effects of adopting the above further technical solutions are as follows:

[0035] By preferably including potato disease-infected tuber samples and soil samples in the sample to be tested, the present invention can more comprehensively cover the detection range of potato powdery scab, effectively improve the accuracy and reliability of detection, and provide more powerful technical support for the early diagnosis and prevention and control of potato powdery scab.

[0036] Further, the conditions for the PCR amplification reaction are: annealing temperature 60.5°C, and the number of cycles is 30 times.

[0037] Further, the analysis method for the PCR amplification product is electrophoresis detection.

[0038] The present invention also discloses a kit for detecting potato powdery scab, including the aforementioned primer pair.

[0039] The kit of the present invention integrates a specific primer pair, making the detection more convenient and efficient, further improving the accuracy and reliability of detection, and strongly promoting the practical application process of potato powdery scab detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0041] Figure 1 It is a detection result diagram of the effectiveness and specificity of the primers in the PCR system. Figure 1 In it, Figure A: PCR amplification result of SsF1 / R1; Figure B: PCR amplification result of SsF2 / R2; Figure C: PCR amplification result of SsF3 / R3; Figure 1 In each lane are respectively: CK-: ddH 2 O; 1: Spongospora subterranea f. sp. subterranea; 2: Streptomyces scabies; 3: Phytophthora infestans; 4: Phytophthora capsici; 5: Ralstonia solanacearum; 6: Globodera pallida; 7: Potato tissue culture seedlings.

[0042] Figure 2 It is the optimization of the annealing temperature of the PCR primer (at an interval of 0.5°C).Figure 2 Among them, CK-: negative control (ddH 2 O); M: Maker (band size is expressed in bp).

[0043] Figure 3 It is the optimization of the concentration of PCR primers SsF2 / SsR2. Figure 3 Among them, each lane is respectively 1: 0.5 μM; 2: 0.4 μM; 3: 0.3 μM; 4: 0.2 μM; 5: 0.1 μM; CK-: negative control (ddH 2 O). M: Maker (band size is expressed in bp).

[0044] Figure 4 It is the result of the sensitivity test of the PCR amplification system. Figure 4 Among them, A: PCR amplification result of SsF1 / R1. B: PCR amplification result of SsF2 / R2. C: PCR amplification result of SsF3 / R3. The template concentrations (orders of magnitude) are respectively in lane 1: 10 8 copies / μL; 2: 10 7 copies / μL; 3: 10 6 copies / μL; 4: 10 5 copies / μL; 5: 10 4 copies / μL; 6: 10 3 copies / μL; 7: 10 2 copies / μL; 8: 10 1 copies / μL. The PCR detection is carried out under a reaction volume of 20 μL, the template dosage is 1 μL, the annealing temperature is 60.5 °C, and the number of cycles is 30 times.

[0045] Figure 5 It is the electrophoresis detection result of the PCR amplification products of field potato diseased tubers and soil samples. Figure 5 Among them, A: PCR amplification result of SsF1 / R1; B: PCR amplification result of SsF2 / R2; C: PCR amplification result of SsF3 / R3. Lanes 1 - 41: DNA of potato diseased tubers; 42 - 61: DNA of diseased soil samples. M: DNA marker (band size is expressed in bp). CK-: negative control (ddH 2 O). -: negative control (DNA of healthy soil sample). +: positive control (DNA of Spongospora subterranea f. sp. subterranea) Specific embodiments

[0046] The following is described in conjunction with specific embodiments.

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed present invention, but merely represents the selected embodiments of the present invention.

[0048] In the present invention, unless otherwise specified, all devices and raw materials can be purchased from the market or are commonly used in this industry. The methods in the following embodiments are conventional methods in this field unless otherwise specified.

[0049] 1. Mining of genomic DNA fragments specific to Spongospora subterranea

[0050] Total DNA was extracted from Spongospora subterranea samples, and the genome of Spongospora subterranea was sequenced using high-throughput sequencing technology to obtain complete genome sequence data. The NT nucleic acid database of NCBI was selected as the comparison database. This database contains a large amount of nucleic acid sequence information and is one of the most comprehensive nucleic acid sequence databases globally.

[0051] The genomic sequence of Spongospora subterranea and the sequences in the NT database were subjected to multiple sequence alignment using professional bioinformatics software (such as BLAST). The alignment parameters were set to default parameters to ensure the accuracy and reliability of the alignment results.

[0052] 2. Design of specific primers and detection application for diseases

[0053] Based on the 2496bp Spongospora subterranea-specific DNA fragment, 3 pairs of PCR primers were designed: SsF1 / R1, SsF2 / R2, and SsF3 / R3.

[0054] The 3 pairs of primers and their amplified fragment lengths are as follows:

[0055] SsF1: 5'-CCTCGTCAATGTAAAGCCGTT-3';

[0056] SsR1: 5'-TCTAAATCCCCAGAATCAGTCAC-3';

[0057] The amplified fragment length is 274bp (SEQ ID NO:4).

[0058] SsF2: 5'-GATAGCGGTTTACTCAGCCC-3';

[0059] SsR2: 5'-TGACCTTAGCGATGATAGCACT-3';

[0060] The amplified fragment length is 358 bp (SEQ ID NO: 7).

[0061] SsF3: 5'-ACCTGTGTCAACCATCTCGCCT-3';

[0062] SsR3: 5'-GTTATCTCCATTACAATCTGCCA-3';

[0063] The amplified fragment length is 624 bp (SEQ ID NO: 10).

[0064] Positive amplification bands could only be obtained when using the DNA of Spongospora subterranea as the template for the three pairs of primers. The similarities between the amplified products and the expected sequences were 99.64%, 100%, and 98.40% respectively after sequencing analysis. However, no cloning bands were obtained when using the genomic DNA of common potato diseases such as Streptomyces scabies as the template ( Figure 1 ). When comparing the three pairs of primers with the genomes of related species in Plasmodiophoromycetes, the matching rates of 5 primers with the "potential" binding sites on the genomic DNA of related species were all below 70%, and at least two bases at the 3'-end of the primers did not match any potential binding sites; although the highest matching rate of SsR2 with the potential binding site reached 81.82%, there were two base mismatches at the 3'-end of the primer. The three pairs of primers had good specificity and could effectively distinguish related species.

[0065] The above primers were synthesized by a professional primer synthesis company and purified to ensure the quality and purity of the primers. The synthesized primers were used to perform PCR amplification on the genomic DNA of Spongospora subterranea to verify the specificity and amplification effect of the primers.

[0066] Further optimize the template dosage, annealing temperature, primer dosage, extension time, and cycle number of the three pairs of primers, analyze the specificity and sensitivity, and determine the reaction system. The specific process is as follows:

[0067] 1), Setting of the initial PCR reaction program and detection of primer effectiveness

[0068] Use 2×Easy Taq PCR Super Mix enzyme (TRANSGEN, AS111-11) to construct the initial PCR reaction system: 2×Easy Taq PCR Super Mix 10 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, DNA template 1 μL, sterile ddH 27 μL. The initial reaction program was set as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 20 s, for a total of 30 cycles, and finally extension at 72°C for 6 min. The DNA of Spongospora subterranea was identified by PCR amplification using the SsF1 / SsR1, SsF2 / SsR2, and SsF3 / SsR3 primers respectively to detect the effectiveness of the primers. The results showed that the amplified products of the three pairs of primers all had a single bright DNA band of the expected size after agarose gel electrophoresis detection.

[0069] 2), Analysis of the template dosage in the PCR system

[0070] The PCR amplification products were used to construct the recombinant plasmids pEASY-Ss1, pEASY-Ss2, and pEASY-Ss3 by TA cloning using the pEASY-T1 Simple Cloning Kit (TRANSGEN, CT111). After sequencing, it was found that the similarities between the DNA sequences amplified by the three primer pairs and the expected sequences were 99.64%, 100%, and 98.40% respectively. The recombinant plasmids were extracted, their concentrations were measured and converted to copy numbers (Table 1). The pEASY-Ss1, pEASY-Ss2, and pEASY-Ss3 plasmids were all diluted to 1.0×10 10 copies / μL and then serially diluted 10-fold. Samples with concentrations ranging from 1.0×10 8 copies / μL to 1.0×10 1 copies / μL, a total of 8 different concentrations of plasmids, were used as templates for PCR respectively to determine that in a 20 μL PCR reaction system, when the template dosage was 1.0×10 8 to 1.0×10 5 copies, the brightness of the electrophoresis bands did not change significantly, but the band brightness at 1.0×10 4 copies and 1.0×10 3 copies became significantly weaker. Therefore, plasmids with a concentration of 1.0×10 3 copies / μL (the PCR amplification for 30 cycles did not reach the plateau phase) were selected for the subsequent optimization of the PCR detection system.

[0071] Table 1 Calculation of plasmid concentration and copy number

[0072]

[0073] 3), Optimization of the annealing temperature in the PCR system

[0074] Eight different annealing temperatures were set at 55 - 65 °C (the gradient PCR instrument automatically generated 65.0 °C, 64.3 °C, 63.0 °C, 61.1 °C, 58.8 °C, 56.9 °C, 55.7 °C, 55.0 °C), and the annealing temperatures of SsF1 / SsR1, SsF2 / SsR2, and SsF3 / SsR3 were optimized respectively. When the annealing temperatures of SsF1 / SsR1 and SsF3 / SsR3 were 55.0, 55.7, 56.9, 58.8, and 61.1 °C, the electrophoretic band brightness of the amplification products was relatively high and there was no obvious difference. When the annealing temperature was 65 °C, the electrophoretic band brightness of the reaction products of SsF2 / SsR2 was slightly darker than that of the other seven temperatures. Further, gradient annealing with an interval of 0.5 °C was set to detect the amplification effects of the three pairs of primers ( Figure 2 ).

[0075] Figure 2 Among them,

[0076] Figure A: Optimization of the annealing temperature of SsF1 / SsR1 (interval 0.5 °C). The annealing temperatures were set as 1: 63.5 °C; 2: 63.0 °C; 3: 62.5 °C; 4: 62.0 °C; 5: 61.5 °C; 6: 61.0 °C; 7: 60.5 °C; 8: 60.0 °C; 9: 59.5 °C.

[0077] Figure B: Optimization of the annealing temperature of SsF2 / SsR2 (interval 0.5 °C). The annealing temperatures were set as 1: 62.0 °C; 2: 62.5 °C; 3: 63.0 °C; 4: 63.5 °C; 5: 64.0 °C; 6: 64.5 °C; 7: 65.0 °C; 8: 65.5 °C; 9: 66.0 °C.

[0078] Figure C: Optimization of the annealing temperature of SsF3 / SsR3 (interval 0.5 °C). The annealing temperatures were set as 1: 64.0 °C; 2: 63.5 °C; 3: 63.0 °C; 4: 62.5 °C; 5: 62.0 °C; 6: 61.5 °C; 7: 61.0 °C; 8: 60.5 °C; 9: 60.0 °C.

[0079] All reactions were repeated three times.

[0080] The results showed that there was no obvious difference in the brightness of the electrophoresis bands of the SsF1 / SsR1 amplification products at temperatures from 59.5°C to 60.5°C. When the annealing temperature was from 61.0°C to 63.5°C, the brightness of the electrophoresis bands gradually decreased with the increase of the annealing temperature. There was no obvious difference in the brightness of the electrophoresis bands of the SsF2 / SsR2 amplification products at 62.0°C to 65.0°C, and the brightness of the electrophoresis bands was slightly weaker at 65.5°C and 66.0°C. From 63.0°C to 64.0°C, the brightness of the electrophoresis bands of the SsF3 / SsR3 amplification products gradually decreased with the increase of the annealing temperature, and there was no obvious difference in the brightness of the electrophoresis bands when the annealing temperature was between 60.0°C and 62.5°C. Combining with the Tm values evaluated by DNAMAN 5.0, 60.5°C was finally selected as the annealing temperature for the 3 pairs of primers for the subsequent PCR system optimization experiment.

[0081] 4), Optimization of the primer dosage in the PCR system

[0082] Five different final primer concentrations of 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, and 0.5 μM were set. Using the recombinant plasmids pEASY-Ss1, pEASY-Ss2, and pEASY-Ss3 (concentration 1.0×103 copies / μL) as templates, 60.5°C was selected as the annealing temperature for PCR. The results showed that the brightness of the electrophoresis bands was high and the difference was small when the primer concentrations were 0.5, 0.4, 0.3, and 0.2 μM respectively. Therefore, a final primer concentration of 0.2 μM was selected for the subsequent experiment ( Figure 3 ).

[0083] Using plasmid pEASY-Ss2 (concentration 1.0×10 3 copies / μL) as a template for PCR detection. The final substrate concentrations were set as 1: 0.5 μM; 2: 0.4 μM; 3: 0.3 μM; 4: 0.2 μM; 5: 0.1 μM. All reactions were repeated three times. CK-: negative control (ddH 2 O). M: Maker (the band size is represented in bp).

[0084] 5), Optimization of the extension time in the PCR system

[0085] Based on a 20 μL PCR system, with an annealing temperature of 60.5°C, four different extension times of 15 s, 20 s, 25 s, and 30 s were set to analyze the three pairs of primers for plasmids pEASY-Ss1, pEASY-Ss2, and pEASY-Ss3 (concentrations 1.0×103 copies / μL, 1.0×103 copies / μL, 1.0×10 3The PCR effect at (copies / μL). The results showed that the primers SsF1 / SsR1 and SsF2 / SsR2 had good amplification effects at 4 different extension times, and the primer SsF3 / SsR3 had a better amplification effect at an extension time of 20 s. Therefore, 20 s was selected as the extension time for the 3 pairs of primers for subsequent experiments.

[0086] 6), Optimization of the number of PCR cycles

[0087] Based on a 20 μL PCR system, the annealing temperature was set at 60.5 °C and the extension time was set at 20 s. The plasmid pEASY-Ss1, pEASY-Ss2, pEASY-Ss3 (concentrations were 1.0×10 3 copies / μL, 1.0×10 3 copies / μL, 1.0×10 3 copies / μL) were used for PCR amplification, and the number of cycles was set to 25, 30, 35. The results showed that the amplification effect was the best at 35 PCR cycles, but there was also a good amplification result when the number of cycles was set to 30. Therefore, 30 PCR cycles were selected for subsequent experiments.

[0088] In summary, the optimized PCR system for SsF1 / SsR1, SsF2 / SsR2, SsF3 / SsR3 was: 2×Easy Taq PCR Super Mix 10 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, DNA template 1 μL, sterile ddH 2 O 8.2 μL.

[0089] The optimized reaction program was: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 60.5 °C for 30 s, extension at 72 °C for 20 s, for 30 cycles; additional extension at 72 °C for 6 min.

[0090] The optimized reaction system was applied to the detection of diseased potato and soil samples, and the results are shown in Table 2, Figure 4 and Figure 5 .

[0091] Table 2 Primers for amplifying the specific DNA segment of Spongospora subterranea

[0092]

[0093] All three pairs of primers could achieve good amplification effects at 60.5°C. The detection sensitivities of the 20 μL detection system reached 1,079, 1,030, and 1,060 copies respectively, and the corresponding template concentrations were 53.95 copies / μL, 51.50 copies / μL, and 53.00 copies / μL respectively. The primers had good specificity and could effectively distinguish Spongospora subterranea from other related species.

[0094] Using the optimized detection system (pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 60.5°C for 30 s, extension at 72°C for 20 s, 30 cycles; final extension at 72°C for 6 min) to detect 41 diseased potato samples, the detection rate of Spongospora subterranea reached 100%. When detecting 20 soil samples, the detection rates were 90%, 90%, and 75% respectively, indicating that this method had high accuracy and reliability in the detection of potato powdery scab.

[0095] In the description of the present invention, it should be understood that "-" and "~" represent the range between two numerical values, and this range includes the endpoints. For example: "A - B" represents a range greater than or equal to A and less than or equal to B. "A ~ B" represents a range greater than or equal to A and less than or equal to B.

[0096] In the description of the present invention, the term "and / or" herein is merely a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone.

[0097] In the description of the invention, numerical values such as time, temperature, ratio, and mass involved can be based on actual measurements, equipment standard parameters, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0098] In the description of the present invention, the term "about" or "approximately" is used to express an approximate value of a numerical value or interval, allowing for a certain error to ensure the flexibility and practicability of the description, while remaining within an acceptable error range, and the maximum error range does not exceed 10% of the corresponding numerical value or numerical range.

[0099] The above is only the preferred embodiment of the present invention. It should be noted that the above preferred embodiment should not be regarded as a limitation of the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art in this technical field, without departing from the spirit and scope of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A molecular detection marker for potato powdery scab, characterized in that: The molecular marker is a genomic DNA fragment with a length of 2496 bp, and its nucleotide sequence is shown in SEQ ID NO:

1.

2. A primer pair for identifying the potato powdery scab molecular detection marker according to claim 1, characterized in that: The primer pairs are selected from one or more pairs in the following group: (a) SsF1 / R1, the sequences of which are: SsF1: 5′-CCTCGTCAATGTAAAGCCGTT-3′, SsR1: 5′-TCTAAATCCCCAGAATCAGTCAC-3′; (b) SsF2 / R2, the sequences of which are: SsF2: 5′-GATAGCGGTTTACTCAGCCC-3′, SsR2: 5′-TGACCTTAGCGATGATAGCACT-3′; (c) SsF3 / R3, the sequences of which are: SsF3: 5′-ACCTGTGTCAACCATTCCGCT-3′, SsR3: 5′-GTTATCTCCATTACAATCTGCCA-3′.

3. The primer pair according to claim 2, characterized in that: The lengths of the amplified fragments of the primer pairs a, b, and c in PCR detection were 274 bp, 358 bp, and 624 bp, respectively, the annealing temperature was 60.5°C, and the detection sensitivity was 10 3 Copy order.

4. The primer pair according to claim 2, characterized in that The method is used for detecting potato powdery scab, and is specifically used for PCR amplification reaction. When powdery scab fungus DNA is used as a template, a positive amplification band can be obtained.

5. A method for detecting potato powdery scab, characterized in that: The following steps are involved: Extracting DNA from the sample to be tested; Perform PCR amplification using the primer pair described in claim 2; Analyze the PCR amplification products to determine whether P. sphaerocephala DNA is present.

6. The method according to claim 5, characterized in that The samples to be tested include diseased potato samples and soil samples.

7. The method according to claim 5, characterized in that The conditions of the PCR amplification reaction are: annealing temperature 60.5° C., and cycle number 30 times.

8. The method according to claim 5, characterized in that The analysis method of the PCR amplification product is electrophoresis detection.

9. A kit for detecting potato powdery scab, characterized in that: Comprising the primer pair described in claim 2.

Images