A molecular marker primer and a method for detecting potato late blight

Abstract

The present invention discloses a molecular marker primer and a method for identifying Phytophthora infestans of potato late blight. The molecular marker primer is used to identify Phytophthora infestans causing potato late blight, and the molecular markers are as shown in SEQ ID NO: 1-6 in the sequence listing. The identification method comprises the following steps: extracting the tissue DNA of the material to be tested; performing PCR amplification using the shown molecular marker primer; and detecting the PCR amplification product by 1.5% agarose gel electrophoresis. The three pairs of primers provided by the present invention can only obtain positive amplification bands when the DNA of Phytophthora infestans is used as a template. The similarities of the amplification products with the expected sequences are 94.96%, 97.71%, and 96.44% respectively after sequencing analysis, while no cloning bands are obtained when the genomic DNA of related species such as Phytophthora capsici is used as a template. The primers have good specificity and can effectively distinguish related species. The method of the present invention can detect leaves that have been infected with the bacteria but have no lesions, which is quite important for disease prevention and control.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection of potato late blight, and relates to molecular marker primers for detecting potato late blight, and the application of the molecular marker primers in a method for detecting potato late blight. Background Art

[0002] The global planting area and total output of potatoes rank only after rice, wheat and corn, making potatoes the fourth largest crop globally. China is the largest potato producer in the world and lists potatoes as the fourth major staple food after rice, wheat and corn. In potato production practice, tuber propagation is mostly used, but this vegetative propagation method is prone to the generation and continuous accumulation of pathogenic bacteria from generation to generation, especially potato late blight is the most serious. Potato late blight is a devastating disease prevalent in most potato planting areas around the world and is the most devastating disease of potatoes. During the entire growth period of potatoes, as long as the conditions are suitable, its pathogen, Phytophthora infestans, can quickly produce a large number of zoospores and complete several generations of cyclic infections in a short time, resulting in a yield loss of 30-60%, or even a complete crop failure. To date, the economic losses caused by potato late blight still exceed $6 billion annually, and how to effectively prevent and control this disease remains a common problem faced globally.

[0003] Traditional methods for detecting late blight include observing disease symptoms and isolating pathogens for morphological identification. However, direct observation is prone to missing plant materials in the initial stage of disease, and the diseases caused by Phytophthora infestans are easily confused with the disease symptoms caused by Pythium spp., Fusarium spp. and Rhizoctonia spp. Therefore, it is very necessary to isolate Phytophthora infestans. However, the isolation and pure culture of Phytophthora infestans have high technical requirements, and there are few morphological characteristics available for pathogen identification, making it relatively difficult to identify.

[0004] The emergence of molecular detection has provided new ideas and methods for the rapid and accurate diagnosis of plant diseases. In recent years, molecular detection technologies for plant diseases based on PCR technology have played an increasingly important role in plant disease control.

[0005] In the process of implementing the present invention, the inventors found that there is at least one of the following technical problems in the prior art:

[0006] Molecular detection technologies based on targets such as the internal transcribed spacer (ITS), mitochondrial gene Cox1-Cox2, etc. are limited by reasons such as small sequence differences and high GC content, and it is difficult to distinguish the target Phytophthora from its related species or it is not suitable as a molecular target.

[0007] In the prior art, two pairs of primers were designed with the pathogenic Phytophthora infestans Ypt1 sequence as the molecular target to detect 11 different Phytophthora species. The results showed that amplification bands were present in all 11 Phytophthora species, indicating low primer specificity.

[0008] The prior art also disclosed a molecular detection technology system developed with the internal transcribed spacer (ITS) ITS-1 of the pathogenic Phytophthora infestans ribosomal DNA as the detection target. However, this detection target cannot distinguish between closely related species such as Phytophthora colocasiae, Phytophthora cactorum, and Phytophthora palmivora.

[0009] Mitochondrial genes Cox1-Cox2 usually have a high GC content, and there is little research on the Lpv gene. It can only be effectively detected for some Phytophthora species and is not applicable to the detection of Phytophthora infestans.

[0010] The key to establishing a molecular detection system for diseases lies in using appropriate molecular targets.

[0011] Therefore, exploring new molecular detection targets is of great significance for improving the molecular precision detection system of Phytophthora infestans and enhancing the management level of potato late blight in China. Summary of the Invention

[0012] In view of this, the purpose of the present invention is to provide a pair of molecular marker primers capable of accurately detecting potato late blight.

[0013] Another purpose of the present invention is to provide a method for detecting potato late blight using the aforementioned molecular marker primers.

[0014] Through long-term exploration and attempts, as well as multiple experiments and efforts, and continuous reform and innovation, the inventors provide the following technical solution to solve the above technical problems: providing a pair of molecular marker primers for detecting potato late blight, and the molecular marker is one or more pairs of the following primer pairs:

[0015] The first primer pair:

[0016] The first upstream primer PiF1: 5'-GCTACCAACATCTTCCACAATCT-3';

[0017] The first downstream primer PiR1: 5'-TCTCTAAAACCTCTGAGCCCCT-3';

[0018] The second primer pair:

[0019] Second upstream primer PiF2: 5'-CTGAAAATGGACGGGGATAGCGA-3';

[0020] Second downstream primer PiR2: 5'-TGCGATGTGAGCGATGGCAAATG-3';

[0021] Third primer pair:

[0022] Third upstream primer PiF3: 5'-ATGGACGGGGATAGCGACTCTTA-3';

[0023] Third downstream primer PiR3: 5'-GATGCCCTTGCTGACTCACCTG-3'.

[0024] The present invention also provides a detection method for potato late blight, using the molecular marker primers described in claim 1, comprising the following steps:

[0025] a) Extract the tissue DNA of the material to be tested;

[0026] b) Using the DNA extracted in step a) as a template, perform PCR amplification with the indicated molecular marker primers. The amplification program is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 59.3°C for 30 s, extension at 72°C for 1 min, for a total of 30 cycles; finally, extension at 72°C for 8 min;

[0027] c) Detect the PCR amplification products by 1.5% agarose gel electrophoresis.

[0028] According to a specific embodiment of the detection method for potato late blight of the present invention, the total system of the PCR amplification is 20 μL: 7 μL of sterile ddH2O, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 10 μL of 2×Easy Taq PCR SuperMix enzyme, and 1 μL of template DNA.

[0029] Compared with the prior art, one of the above technical solutions has the following advantages:

[0030] a) The three pairs of primers provided by the present invention can only obtain positive amplification bands when the DNA of Phytophthora infestans is used as a template. The similarities between the amplified products and the expected sequences are 94.96%, 97.71%, and 96.44% respectively after sequencing analysis. However, no cloning bands are obtained when the genomic DNA of related species such as Phytophthora capsici is used as a template. The primers have good specificity and can effectively distinguish related species.

[0031] b) The 3 pairs of primers of the present invention can all obtain good amplification effects at 59.3°C, and the detection sensitivities reach 26.9, 2.42, and 25.5 copies / μL respectively.

[0032] c) In the method of the present invention, 63 field leaf samples from 3 counties and cities were detected with an optimized detection system, and the detection rates of diseased leaves reached 83.33%, 87.04%, and 85.19% respectively, which were significantly higher than the detection results of the primers Pi-F (TTGTGAAGGCGTCATTCC) and Pi-R (CCGAGAGGATGCTTACGA) by Zhan Fangfang et al.

[0033] d) The method of the present invention can detect 0-level diseased leaves, that is, leaves without any lesions but already infected with bacteria, which is quite important for disease prevention and control. Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. 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.

[0035] Figure 1 It is the electrophoresis diagram of the specific detection of the PCR primers of the present invention. In the figure, A: PiF1 / PiR1; B: PiF2 / PiR2; C: PiF3 / PiR3; CK-: ddH2O.

[0036] Figure 2 It is the optimization of the PCR annealing temperature. In the figure, A: PiF1 / PiR1; B: PiF2 / PiR2; C: PiF3 / PiR3; 1: 55.0 °C; 2: 55.5 °C; 3: 56.4 °C; 4: 57.7 °C; 5: 59.3 °C; 6: 60.6 °C; 7: 61.5 °C; 8: 62.0 °C.

[0037] Figure 3 It is the single-plant diseased leaves of potato late blight. In the figure, A: 0 level; B: 1 level; C: 3 level; D: 5 level; E: 7 level; F: 9 level.

[0038] Figure 4 It is the electrophoresis detection result diagram of the PCR amplification products of the field potato leaf samples in Pengzhou City. In the figure, A: PiF1 / PiR1; B: PiF2 / PiR2; C: PiF3 / PiR3; D: Pi-F / Pi-R; CK-: ddH2O; CK+: Phytophthora infestans DNA.

[0039] Figure 5It is the electrophoresis detection result diagram of the PCR amplification products of the potato leaf samples in Datian, Jintang County. In the figure, A: PiF1 / PiR1; B: PiF2 / PiR2; C: PiF3 / PiR3; D: Pi-F / P-iR; CK-: ddH2O; CK+: Phytophthora infestans DNA.

[0040] Figure 6 It is the electrophoresis detection result diagram of the PCR amplification products of the potato leaf samples in Datian, Chongzhou City. In the figure, A: PiF1 / PiR1; B: PiF2 / PiR2; C: PiF3 / PiR3; D: Pi-F / P-iR; CK-: ddH2O; CK+: Phytophthora infestans DNA. Specific implementation mode

[0041] The following is described in conjunction with the drawings and a specific embodiment.

[0042] To make the purpose, 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 drawings in 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. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art 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 provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention.

[0043] It should be noted that similar reference numerals and letters indicate similar items in the following drawings. Therefore, once an item is defined in one drawing, it may not be further defined and explained in subsequent drawings.

[0044] To illustrate the technical effects of the present invention, the following is described through a specific implementation process.

[0045] In this embodiment, the primer pairs used are:

[0046] The first primer pair is shown in Sequence Listing SEQ ID NO: 1 and SEQ ID NO: 2:

[0047] The first upstream primer PiF1: 5'-GCTACCAACATCTTCCACAATCT-3';

[0048] The first downstream primer PiR1: 5'-TCTCTAAAACCTCTGAGCCCCT-3';

[0049] The second primer pair is shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing:

[0050] The second upstream primer PiF2: 5'-CTGAAAATGGACGGGGATAGCGA-3';

[0051] The second downstream primer PiR2: 5'-TGCGATGTGAGCGATGGCAAATG-3';

[0052] The third primer pair is shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing:

[0053] The third upstream primer PiF3: 5'-ATGGACGGGGATAGCGACTCTTA-3';

[0054] The third downstream primer PiR3: 5'-GATGCCCTTGCTGACTCACCTG-3'.

[0055] Taking the primer Pi-F / Pi-R for late blight detection in the prior art as the control primer pair, the primer Pi-F / Pi-R is shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing:

[0056] Pi-F: 5'-TTGTGAAGGCGTCATTCC-3';

[0057] Pi-R: 5'-CCGAGAGGATGCTTACGA-3'.

[0058] Using the above four primer pairs to perform PCR amplification on the genomic DNA of the collected samples of field potato leaves, and comparing the positive detection rates of the 3 pairs of specific primers and the control primer pair for the field samples.

[0059] The process of this example is specifically as follows.

[0060] 1 PCR system establishment

[0061] 1.1 Collection of field potato leaf samples

[0062] From March 15, 2020 to April 18, 2021, healthy potato leaf samples were collected from disease-free fields in three cities and counties of Pengzhou City, Jintang County, and Chongzhou City (the occurrence of late blight in these fields was continuously monitored until after harvest to confirm that no late blight occurred), marked as CK, as the negative control; potato leaf samples with different degrees of disease were collected from diseased fields. According to the grading standard of potato late blight, the disease susceptibility was divided into the following 6 grades (Table 1). Three replicates were taken for each disease grade, photographed and placed in disposable self-sealing bags. One diseased leaf was placed in each collection bag, and the bag mouth was sealed and then placed in an ice box or ice bottle and taken back to the laboratory.

[0063] Table 1 Grading standard of potato late blight

[0064]

[0065] 1.2 Test strains and vectors

[0066] The detection strains involved in this example include fungi and bacteria. The test fungi include: Phytophthora infestans, Phytophthora sojae, Phytophthora capsici, Phytophthora colocasiae, Phytophthora nicotianae; the test bacteria include: Ralstonia solanacearum, Streptomyces scabies. Among them, Phytophthora infestans was isolated from diseased potato leaves, and Phytophthora sojae, Phytophthora nicotianae, Phytophthora colocasiae, and Phytophthora capsici were kindly provided by the Plant Protection Institute of Sichuan Academy of Agricultural Sciences. The rest of the strains were stored in our laboratory. The cloning vector pEasy-T1 vector was purchased from TransGen Biotech Co., Ltd.

[0067] 1.3 Preparation of culture media

[0068] Rye medium: Weigh 100 g of rye, wash it and add 1 L of distilled water, sterilize it at 121 °C for 40 min in a high-pressure steam sterilizer. After cooling, squeeze out the juice with three layers of gauze, filter and collect the filtrate. Add 20 g of sucrose and 15 g of agar powder, and make up to 1 L with distilled water, then sterilize it at 121 °C for 20 min in a high-pressure steam sterilizer.

[0069] Rye selective medium: Take 0.2 g of ampicillin, 0.02 g of rifampicin, and 0.1 g of nystatin, dissolve them in 2 mL of dimethyl sulfoxide to prepare "red mercury".

[0070] Cool the sterilized rye medium to about 50 °C, add 1 mL of "Mercurochrome", gently shake and mix well, and then pour into plates.

[0071] 1.4 Isolation, purification and identification of Phytophthora infestans

[0072] The method of "potato chip with leaf" was used to isolate and purify the pathogen of potato late blight: Wash fresh and healthy potatoes, gently wipe the surface with 75% alcohol, and pass through the flame until the alcohol volatilizes completely; Cut the cooled potatoes into potato chips 1 cm thick with a knife, sandwich the diseased leaf tissue collected in the field between two potato chips, place in the dark at 18 °C for cultivation. When Phytophthora mycelium grows on the potato chips, pick the mycelium and inoculate it on the rye selective medium for isolation, and culture it in the dark at 18 °C in an inverted position; After the mycelium grows on the rye selective medium, pick the mycelium again and inoculate it on the ordinary rye medium for purification.

[0073] 1.5 Genomic DNA extraction

[0074] Genomic DNA of field potato leaf samples was extracted using the EasyPure Plant Genomic DNA Kit (product number: EE111-01); Genomic DNA of Phytophthora infestans, Phytophthora sojae, Phytophthora nicotianae, Phytophthora colocasiae, Phytophthora capsici was extracted using PlantZol (product number: P10927); Genomic DNA of bacteria was extracted using the EasyPure Bacteria Genomic DNA Kit (product number: EE161-11). All the above kits were purchased from Beijing TransGen Biotech Co., Ltd. The concentration and purity of genomic DNA were measured using NanoDrop400.

[0075] 1.6 Primer design and synthesis

[0076] Taking the DNA fragment specific to the Phytophthora infestans genome discovered by the inventors as the molecular target, 3 pairs of specific primers (PiF1 / R1, PiF2 / R2, and PiF3 / R3, see SEQ ID NO: 1-6 in the sequence listing in turn) were designed using the Premier5.0 primer design software, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. According to the primer synthesis order instructions, add sterile ddH2O to dilute the primers to the working concentration of 10 μM and store them at -20 °C for standby.

[0077] 1.7 TA cloning of the target fragment and construction of recombinant plasmids

[0078] Based on primers PiF1 / R1, PiF2 / R2, and PiF3 / R3, the genomic DNA of Phytophthora infestans was amplified using 2×Easy Taq PCR Super Mix enzyme (TransGen Biotech Co., Ltd., Beijing, catalog number: O10708) (the 3' end of the amplification product has an "A" base). The amplification product was purified and recovered for the target fragment using the EasyPure Quick Gel Extraction Kit (TransGen Biotech Co., Ltd., Beijing, catalog number: EG101-02). The recovered target fragment was subjected to TA cloning and positive clone detection according to the pEASY-T1 Simple Cloning Kit (TransGen Biotech Co., Ltd., Beijing, catalog number: CT111) to construct a recombinant plasmid containing the target fragment.

[0079] 1.8 Preliminary establishment of the PCR reaction system

[0080] According to the instructions of 2×Easy Taq PCR Super Mix and the annealing temperature provided by the primer design software Premier5.0, the initial PCR reaction conditions were designed, and the reaction program was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 1 min, for a total of 30 cycles, and finally extension at 72°C for 8 min, and stored at 4°C. The PCR amplification product was detected by 1.5% agarose gel electrophoresis to observe the size and band brightness of the amplified fragment. The total PCR reaction system was 20 μL: 7 μL of sterile ddH2O, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 10 μL of 2×Easy Taq PCR Super Mix enzyme,

[0081] 1 μL of template DNA.

[0082] 1.9 Screening of specific primers

[0083] The genomic DNAs of Phytophthora infestans, Ralstonia solanacearum, Streptomyces scabies, Phytophthora capsici, Phytophthora sojae, Phytophthora nicotianae, Phytophthora colocasiae, and virus-free potato seedlings were used as templates for PCR reactions, and amplified with primer pairs PiF1 / R1, PiF2 / R2, and PiF3 / R3 according to the above PCR reaction system.

[0084] 1.10 Optimization of the annealing temperature of the PCR reaction system

[0085] To optimize the establishment of suitable PCR reaction amplification conditions and achieve rapid and effective detection of specific DNA segments in the genome of Phytophthora infestans, the annealing temperature of the PCR system was optimized. The reaction system was placed on a gradient PCR instrument, and a gradient annealing temperature of 55 - 62 °C was set, with 8 temperature gradients of 55.0 °C, 55.5 °C, 56.4 °C, 57.7 °C, 59.3 °C, 60.6 °C, 61.5 °C, and 62.0 °C. The annealing temperatures of the three primer pairs PiF1 / PiR1, PiF2 / PiR2, and PiF3 / PiR3 were optimized respectively. All other conditions were the same as in 1.8 except for the annealing temperature.

[0086] 1.11 Sensitivity test

[0087] The concentrations of recombinant plasmids constructed with primer pairs PiF1 / R1, PiF2 / R2, and PiF3 / R3 were measured using NanoDrop400, and a gradient dilution solution of recombinant plasmids was constructed by sequential 10-fold gradient dilution. Using this as a template for PCR amplification, the lowest template concentration at which nucleic acid amplification bands could be detected was determined, and the copy number of target DNA template molecules was calculated. The plasmid copy number conversion formula is as follows:

[0088]

[0089] The PCR amplification conditions were the optimized amplification conditions, and the amplification system was as described in 1.8. The sensitivity amplification results were analyzed by 1.5% agarose gel electrophoresis.

[0090] 1.12 Detection of potato diseased leaves in the field

[0091] Using the optimized PCR reaction conditions and reaction system, the genomic DNA of potato leaf samples in the field was subjected to PCR amplification using the aforementioned 3 pairs of specific primers and the primer Pi-F / Pi-R (see Sequence Listing SEQ ID NO: 7 - 8) used by Zhan Fangfang et al. for late blight detection as a control primer, and the positive detection rates of the 3 pairs of specific primers and the control primer for field samples were compared.

[0092] 1.13 Specificity detection of PCR primers

[0093] The genomic DNAs of Phytophthora infestans, Ralstonia solanacearum, Streptomyces scabies, Phytophthora capsici, Phytophthora nicotianae, Phytophthora colocasiae, Phytophthora sojae, and virus-free potato seedlings were used as templates for PCR reactions, and ddH2O was used as a negative control. PCR amplifications were performed using primer pairs PiF1 / PiR1, PiF2 / PiR2, and PiF3 / PiR3 respectively. The results are as Figure 1Shown as follows: For the 3 pairs of primers, no target bands could be amplified from the genomic DNA of the tested strains Ralstonia solanacearum, Streptomyces scabies, Phytophthora capsici, Phytophthora sojae, Phytophthora nicotianae, Phytophthora colocasiae, and virus-free potato seedlings, while a single target DNA fragment could be amplified using the genomic DNA of Phytophthora infestans as the template. The amplification products were used to construct recombinant plasmids by TA cloning, and the recombinant plasmids were sequenced and identified. The sequencing results showed that the sequence similarities of the target fragments amplified by the 3 pairs of primers to the expected sequences were 94.96%, 97.71%, and 96.44% respectively, indicating that the 3 pairs of primers all had good specificity and could be used for subsequent experiments.

[0094] 1.14 Optimization of PCR annealing temperature

[0095] Using the software DNAMAN to evaluate the annealing temperatures of the 3 pairs of primers, the annealing temperatures of PiF1 and PiR1 were determined to be 59.5°C and 60.7°C; the annealing temperatures of PiF2 and PiR2 were 68.2°C and 72.3°C; the annealing temperatures of PiF3 and PiR3 were 65.1°C and 66.6°C. To optimize and obtain the optimal annealing temperatures of the 3 pairs of primers, 8 annealing temperatures were set between 55 and 62°C: 55.0°C, 55.5°C, 56.4°C, 57.7°C, 59.3°C, 60.6°C, 61.5°C, 62.0°C, and PCR amplifications were carried out respectively. The results are as Figure 2 shown: At the 8 annealing temperatures, the 3 pairs of primers could all amplify target bands. Among them, the target bands amplified by the PiF1 / PiR1 primers at the annealing temperatures of 59.3°C, 60.6°C, 61.5°C, and 62.0°C were brighter than those amplified at 55.0°C, 55.5°C, 56.4°C, and 57.7°C, and the amplification effect was better. Finally, 59.3°C was selected as the optimal annealing temperature for PiF1 / PiR1; the target bands amplified by the PiF2 / PiR2 primers at the 8 annealing temperatures had uniform brightness and no significant difference. Finally, 59.3°C was determined as the optimized optimal annealing temperature; the bands of the PiF3 / PiR3 primers at the annealing temperatures of 55.0°C, 55.5°C, 56.4°C, 57.7°C, 59.3°C, and 60.6°C were brighter than those at 61.5°C and 62.0°C. Finally, 59.3°C was determined as the optimized optimal annealing temperature. In summary, the optimized PCR reaction procedures for the 3 pairs of primers are shown in Table 2.

[0096] Table 2 PCR reaction procedures

[0097]

[0098] 1.15 PCR sensitivity test

[0099] The amplification products of primers PiF1 / PiR1, PiF2 / PiR2, and PiF3 / PiR3 were used to construct recombinant plasmids containing the corresponding specific DNA sequences through TA cloning. The concentrations of the recombinant plasmids were measured and serially diluted 10-fold. Meanwhile, their copy numbers were calculated to construct recombinant plasmid dilution series with copy numbers of 10 9 、10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 copies / μL. In a 20-μL reaction system, amplification was performed under the optimized PCR conditions and system (Table 2) to detect the sensitivity of the reaction system. The amplification results showed that as the copy number of the template DNA molecules decreased, the brightness of the gel electrophoresis bands of the PCR amplification products using PiF1 / PiR1, PiF2 / PiR2, and PiF3 / PiR3 as primers decreased in a gradient manner. Among them, the lowest template concentration detectable by primer PiF1 / PiR1 was 1.31 pg / μL, and the template copy number was 2.69×10 5 copies / μL; the lowest template concentration detectable by primer PiF2 / PiR2 was 1.18 pg / μL, and the lowest template copy number was 2.42×10 5 copies / μL; the lowest template concentration detectable by primer PiF3 / PiR3 was 1.24 pg / μL, and the lowest template copy number was 2.55×10 5 copies / μL.

[0100] To explore the effect of the number of cycles on sensitivity and considering that in general, 35 PCR cycles have reached the amplification plateau, the inventors increased the number of cycles of the optimized PCR reaction system (Table 2) to 35 cycles and gradient-diluted the plasmid solution corresponding to the lowest detected concentration above to construct recombinant plasmid solutions with copy numbers of 10 4 、10 3 、10 2 、10 1 、10 0 、10 -1 in the order of magnitude. The lowest template concentration detectable by primer PiF1 / PiR1 was 1.31×10 -1 fg / μL, corresponding to a template copy number of 26.9 copies / μL; the lowest template concentration detectable by primer PiF2 / PiR2 was 1.18×10 -2 fg / μL, and the template copy number was 2.42 copies / μL; the lowest template concentration detectable by primer PiF3 / PiR3 was 1.24×10 -1 fg / μL, and the lowest template copy number was 25.5 copies / μL.

[0101] 2. Application of PCR Detection System in Detecting Field Potato Leaf Samples

[0102] 2.1 Collection of Field Potato Leaf Samples

[0103] In this example, a total of 63 field potato leaf samples from 3 counties and cities were collected (the specific sampling information is shown in Table 3), with 21 field potato leaf samples each from Pengzhou City, Jintang County, and Chongzhou City. The disease grading of each potato leaf sample is as Figure 3 .

[0104] Table 3 Information of Field Potato Leaf Samples and Genomic DNA Concentration

[0105]

[0106] Note 1: The unit of nucleic acid concentration is g / mL;

[0107] Note 2: 1-3 are healthy leaves, and according to subsequent follow-up investigations, no diseases occurred until harvest. 4-21 are diseased leaves at levels 0, 1, 3, 5, 7, and 9 respectively, with 3 replicates for each disease level.

[0108] 2.2 DNA Extraction and Detection of Field Potato Leaf Samples

[0109] Extract the genomic DNA of field potato leaf samples, measure the concentration and purity of the extracted DNA with Nano Drop400 (Table 3), and finally detect the results by 1.5% agarose gel electrophoresis. The genomic DNA extracted from a total of 63 samples in 3 counties and cities all had relatively bright electrophoresis bands, which could meet the requirements of subsequent test detections.

[0110] 2.3 Detection of Field Potato Leaf Samples by PCR Detection System

[0111] Use 3 pairs of specific primers (PiF1 / PiR1, PiF2 / PiR2, PiF3 / PiR3) of the present invention and 1 pair of control primers (Pi-F / Pi-R) to perform PCR molecular detection on a total of 63 field potato leaf samples from 3 counties and cities. The electrophoresis detection results are respectively as Figure 4 , Figure 5 , Figure 6 shown.

[0112] The detection results of the field potato leaf samples collected from Pengzhou City show( Figure 4):Among them, primer pair PiF1 / PiR1 could detect 14 positive samples out of 18 samples collected from diseased fields, with a positive detection rate of 77.78%; primer pair PiF2 / PiR2 could detect 16 positive samples, with a positive detection rate of 88.89%; primer pair PiF3 / PiR3 could detect 15 samples, with a detection rate of 83.33%; primer pair Pi-F / Pi-R could detect 13 samples, with a positive detection rate of 72.22%.

[0113] The detection results of potato leaf samples from fields in Jintang County showed ( Figure 5 ):Among them, primer pairs PiF1 / PiR1, PiF2 / PiR2, and PiF3 / PiR3 could all detect 16 positive samples out of 18 samples collected from diseased fields, with a positive detection rate of 88.89%; while primer pair Pi-F / Pi-R could detect 15 samples, with a positive detection rate of 83.33%.

[0114] The detection results of potato leaf samples from fields in Chongzhou City showed ( Figure 6 ):Among them, primer pairs PiF1 / PiR1, PiF2 / PiR2, and PiF3 / PiR3 could all detect 15 positive samples out of 18 samples collected from diseased fields, with a positive detection rate of 83.33%; while primer pair Pi-F / Pi-R could detect 14 positive samples, with a positive detection rate of 77.78%.

[0115] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and 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 of this technology, 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. Sequence Listing <110> Sichuan Ruyuan Biotechnology Co., Ltd. <120> A Molecular Marker Primer and a Detection Method for Potato Late Blight <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 23 <212> DNA <213> Phytophthora infestans <400> 1 gctaccaaca tcttccacaa tct 23 <210> 2 <211> 22 <212> DNA <213> Phytophthora infestans <400> 2 tctctaaaac ctctgagccc ct 22 <210> 3 <211> 23 <212> DNA <213> Phytophthora infestans <400> 3 ctgaaaatgg acggggatag cga 23 <210> 4 <211> 23 <212> DNA <213> Phytophthora infestans <400> 4 tgcgatgtga gcgatggcaa atg 23 <210> 5 <211> 23 <212> DNA <213> Phytophthora infestans <400> 5 atggacgggg atagcgactc tta 23 <210> 6 <211> 22 <212> DNA <213> Phytophthora infestans <400> 6 gatgcccttg ctgactcacc tg 22 <210> 7 <211> 18 <212> DNA <213> Phytophthora infestans <400> 7 ttgtgaaggc gtcattcc 18 <210> 8 <211> 18 <212> DNA <213> Phytophthora infestans <400> 8 ccgagaggat gcttacga 18

Claims

1. A molecular marker primer for detecting potato late blight, characterized in that, The molecular marker primer is one or more pairs of the following primer pairs: The first primer pair: The first upstream primer PiF1: 5'-GCTACCAACATCTTCCACAATCT-3'; The first downstream primer PiR1: 5'-TCTCTAAAACCTCTGAGCCCCT-3'; The second primer pair: The second upstream primer PiF2: 5'-CTGAAAATGGACGGGGATAGCGA-3'; The second downstream primer PiR2: 5'-TGCGATGTGAGCGATGGCAAATG-3'; The third primer pair: The third upstream primer PiF3: 5'-ATGGACGGGGATAGCGACTCTTA-3'; The third downstream primer PiR3: 5'-GATGCCCTTGCTGACTCACCTG-3'.

2. A detection method for potato late blight, characterized in that, Using the molecular marker primer described in claim 1, the following steps are included: a) Extract the tissue DNA of the material to be tested; b) Using the DNA extracted in step a) as a template, perform PCR amplification with the indicated molecular marker primer. The amplification program is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 59.3°C for 30 s, extension at 72°C for 1 min, for a total of 30 cycles; finally, extension at 72°C for 8 min; c) Detect the PCR amplification product by 1.5% agarose gel electrophoresis.

3. The method according to claim 2, characterized in that, The total volume of the PCR amplification system is 20 μL: 7 μL of sterile ddH2O, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 10 μL of 2× Easy Taq PCR Super Mix enzyme, and 1 μL of template DNA.

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