Oligonucleotides and methods for detecting buckwheat downy mildew - Patents.com

Abstract

To provide a method for specifically and efficiently detecting buckwheat downy mildew bacteria.SOLUTION: The present invention provides a method for detecting buckwheat downy mildew bacteria (Peronospora ducommeti), by a pair of oligonucleotides including a specific base sequence, and PCR using the same.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an oligonucleotide, a primer set, a kit and a detection method for detecting buckwheat downy mildew. [Background technology]

[0002] Buckwheat has a short cultivation period of about two months and is suitable for extensive cultivation, so it is cultivated as an important rotation crop in land-use agriculture. Buckwheat is also popular with consumers, so it is used in the sixth industrialization and contributes to the revitalization of the region. Due to this strong demand for buckwheat, continuous cropping is practiced in buckwheat-producing regions to increase buckwheat production. As a result, there are concerns in these producing areas about the occurrence and spread of buckwheat downy mildew.

[0003] Buckwheat downy mildew is caused by infection with the phytopathogenic oomycete Peronospora ducommeti. Infected plants exhibit yellowing of the leaves. In recent years, there has been concern that infection with this disease can cause immature fruit and reduce yields. Because this pathogen is transmitted through seeds, the damage increases when infected seeds are planted. Furthermore, buckwheat residue infected with buckwheat downy mildew is plowed into the soil, becoming a source of infection, and continuous cropping can exacerbate the damage.

[0004] In buckwheat cultivation, the constant use of synthetic chemicals such as pesticides directly leads to increased production costs, so no chemical control system has been established.Kitayuki is known as a buckwheat variety that is resistant to downy mildew, but its resistance is insufficient, and efforts are currently being made to develop new varieties through crossbreeding using genetic resources with excellent resistance.

[0005] A diagnostic method has been reported in which seeds are inspected using an optical microscope to determine whether or not they are infected with Peronospora spp., including the buckwheat downy mildew fungus (Non-Patent Document 1). However, with this method, depending on the condition of the buckwheat seeds, it may not be possible to observe the infecting buckwheat downy mildew fungus, which may result in a false negative.

[0006] There is also a method for distinguishing seeds derived from infected individuals, regardless of whether the seeds are infected, by visually observing the lesions on the leaves (Non-Patent Document 1). However, because the lesions on the leaves may not show any symptoms even if the plant is infected, this method can also result in false negatives.

[0007] It is also known that if the volume density of buckwheat seeds is low, it is suspected that the seeds are infected with buckwheat downy mildew by calculating the weight ratio of the buckwheat seeds to the total weight of the aboveground parts. This method can also be used to distinguish infected seeds. However, because immature seeds can also be caused by physiological disorders, it is nearly impossible to diagnose buckwheat downy mildew infection by visual inspection or seed weight as an indicator.

[0008] Furthermore, Non-Patent Document 2 discloses a method for detecting buckwheat downy mildew using the filamentous fungal universal primers ITS5 / ITS4. However, when the filamentous fungal universal primers ITS5 / ITS4 are used, filamentous fungi other than buckwheat downy mildew are detected as false positives, and the method lacks specificity for buckwheat downy mildew. Furthermore, no PCR primers capable of specifically detecting buckwheat downy mildew have yet been found. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Zimmer et al., 1992, J. Phytopathol, vol. 135, p. 217-223 [Non-patent document 2] Casimiro et al., 2004, J. Appl. Microbiol., vol. 96, p.579-87 [Non-patent document 3] Ikegami et al., 2018, Plant Protection Station Research Report, Vol. 54, pp. 103-105 Summary of the Invention [Problem to be solved by the invention]

[0010] In buckwheat breeding, selecting healthy seeds and using them for cultivation is important for evaluating useful traits. However, because buckwheat downy mildew is seed-transmitted, it is necessary to distinguish healthy seeds from diseased seeds. Traditionally, healthy seeds have been selected by visually determining seed shape and seed weight. However, immature buckwheat fruit can also occur due to physiological disorders, and the symptoms are very similar to those caused by buckwheat downy mildew infection, making it difficult to diagnose buckwheat downy mildew.

[0011] To diagnose buckwheat downy mildew seed infection, it is necessary to examine the presence or absence of conidia using an optical microscope. However, diagnosis using an optical microscope is not efficient, and there is a need to develop a faster and simpler method for selecting healthy seeds. Furthermore, if it were possible to diagnose buckwheat downy mildew during the early stages of growth, when symptoms are not yet apparent, it would be possible to collect only symptomless cultivated individuals, crossbreed them, and accumulate resistance genes.

[0012] Therefore, an object of the present invention is to provide a method for specifically and efficiently detecting buckwheat downy mildew. [Means for solving the problem]

[0013] To solve the above problems, the present inventors designed primer sets A and B using the nucleotide sequence information of P. parasitica and performed PCR using DNA extracted from buckwheat leaves as a template. As a result, nonspecific bands were detected when primer set A was used, but amplification products specific to P. parasitica were obtained when primer set B was used. Primer set B was also able to specifically detect P. parasitica when DNA extracted from germinated buckwheat seedlings was used as a template. Furthermore, primer set B was also able to specifically detect P. parasitica when DNA extracted from symptomless buckwheat leaves was used as a template. Based on these results, the present inventors have discovered oligonucleotides capable of specifically and quantitatively detecting P. parasitica in buckwheat seeds and symptomless buckwheat.

[0014] The present invention provides an oligonucleotide for detecting buckwheat downy mildew (Peronospora ducommeti), which comprises a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3 or 4, or a complementary sequence thereof.

[0015] The present invention also provides a primer set for detecting buckwheat downy mildew (Peronospora ducommeti), comprising a first primer and a second primer, wherein the first primer is an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, and the second primer is an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 4.

[0016] The present invention also provides a kit for detecting buckwheat downy mildew (Peronospora ducommeti), comprising a primer set and an instruction manual, wherein the primer set consists of a first primer and a second primer, the first primer being an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, and the second primer being an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 4.

[0017] The present invention also provides a method for detecting buckwheat downy mildew (Peronospora ducommeti), which comprises the steps of performing PCR using a primer set and a nucleic acid in a sample as a template, and detecting the PCR amplification product, wherein the primer set consists of a first primer and a second primer, the first primer is an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, and the second primer is an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 4.

[0018] The present invention also provides the above-described detection method, wherein the sample is buckwheat seeds, seedlings or leaves.

[0019] The present invention also provides a method for diagnosing buckwheat downy mildew, comprising the steps of: performing PCR using a primer set with DNA extracted from buckwheat seeds, seedlings, or leaves as a template; and detecting an amplified product obtained by PCR, wherein the primer set consists of a first primer and a second primer, the first primer being an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3; and the second primer being an oligonucleotide comprising a sequence of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 4; and determining that the buckwheat is infected with downy mildew if an amplified product specific to the buckwheat downy mildew fungus (Peronospora ducommeti) is detected in the detecting step. [Effects of the Invention]

[0020] The present invention makes it possible to diagnose whether seeds with immature fruits and symptomless buckwheat plants are infected with buckwheat downy mildew. By removing buckwheat downy mildew-infected plants and seeds, healthy seeds that are not infected with buckwheat downy mildew can be efficiently selected. [Brief explanation of the drawings]

[0021] [Figure 1] A diagram illustrating how to germinate buckwheat seeds. [Figure 2] FIG. 1 shows the results of PCR using the filamentous fungal universal primers ITS5 / ITS4 of a comparative example. [Figure 3] FIG. 1 shows the results of PCR using primer set B according to one embodiment of the present invention. [Figure 4] FIG. 1 shows the results of PCR using primer set B according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides oligonucleotides for detecting buckwheat downy mildew (Peronospora ducommeti).

[0023] As used herein, "oligonucleotide" includes 10-35 base deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogs thereof. Analogs include, for example, peptide nucleic acids. The oligonucleotides of the present invention encompass single-stranded and double-stranded nucleic acids. The oligonucleotides of the present invention can be, for example, primers and probes.

[0024] As used herein, a "primer" refers to a single-stranded polynucleotide having a free 3'OH group, which hybridizes with a target sequence to promote the polymerization of a polynucleotide complementary to the target by DNA polymerase. The oligonucleotides of the present invention can be used as primers for PCR. The oligonucleotides of the present invention can be used, for example, in general PCR (conventional PCR) and real-time PCR, in which amplified fragments are detected by agarose electrophoresis or the like after the PCR reaction.

[0025] As used herein, a "probe" refers to an oligonucleotide used to detect complementary DNA or RNA. The oligonucleotide of the present invention can be used, for example, in DNA microarrays, Southern blotting, Northern blotting, and fluorescent in situ hybridization.

[0026] The oligonucleotide of the present invention comprises a sequence consisting of 10 or more, preferably 15 or more, and more preferably 20 or more consecutive bases in the base sequence represented by SEQ ID NO: 3 or 4, or a complementary sequence thereof. The oligonucleotide of the present invention may consist of the base sequence represented by SEQ ID NO: 3 or 4.

[0027] As used herein, the term "complementary sequence" refers to a sequence that can hybridize to a target sequence under stringent conditions. "Stringent conditions" can be, for example, conditions such as "hybridization in a hybridization solution having a salt concentration of 6×SSC or an equivalent thereto at a temperature of 50 to 70°C for 16 hours, pre-washing with 6×SSC or a solution having a salt concentration equivalent thereto as needed, followed by washing with 1×SSC or a solution having a salt concentration equivalent thereto."

[0028] The oligonucleotides of the present invention can be prepared by, for example, known chemical synthesis methods, including, but not limited to, synthesizing oligonucleotides using a DNA synthesizer by the phosphoramidite method and purifying them using anion exchange column chromatography.

[0029] The present invention also provides a primer set for detecting buckwheat downy mildew (Peronospora ducommeti).

[0030] The primer set of the present invention consists of a first primer and a second primer. The first primer is an oligonucleotide comprising a sequence consisting of 10 or more, preferably 15 or more, and more preferably 20 or more consecutive bases in the nucleotide sequence represented by SEQ ID NO: 3. The second primer is an oligonucleotide comprising a sequence consisting of 10 or more, preferably 15 or more, and more preferably 20 or more consecutive bases in the nucleotide sequence represented by SEQ ID NO: 4. The first primer may be an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3. The second primer may be an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4.

[0031] The oligonucleotide of the present invention may have one or several bases, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases, added, inserted, deleted, and / or substituted. When the oligonucleotide is a primer or a probe, the addition, insertion, deletion, and / or substitution may be made within a range that does not impair the function of the primer or probe. For example, the oligonucleotide may have a sequence in which one or several bases are added to the 5' end.

[0032] The oligonucleotide of the present invention may have any of the nucleotides constituting the oligonucleotide modified. For example, the 5'-terminal nucleotide, the 3'-terminal nucleotide, or both may be modified. Alternatively, the oligonucleotide may have a nucleotide at an internal position other than the terminal nucleotide modified. Modifications include radioisotopes, enzymes, fluorescent substances, luminescent substances, organic compounds, and the like. Radioisotopes include 32P, 33P, and 35S, and the like. Enzymes include alkaline phosphatase and peroxidase, and the like. Fluorescent materials include Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, EvaGreen®, Fluorescein (FAM)™, Tetrachlorofluorescein (TET)™, Hexachlorofluorescein (HEX)™, Tetramethylrhodamine (TAMRA)™, JOE™, VIC®, NED™, PET®, Alexa® fluorescent dyes, Texas Red®, Rhodamine Red X (ROX)™, and Exciton dyes. Luminescent materials include luminescent compounds such as acridinium esters. Organic compounds include digoxigenin and biotin.

[0033] The present invention also provides a kit for detecting P. parasitica. The kit of the present invention includes the primer set described above. The kit of the present invention may further include an instruction manual, a PCR enzyme, a buffer, a dNTP mixture, a fluorescent dye, and a DNA solution containing 18S rDNA of P. parasitica.

[0034] The present invention also provides a method for detecting buckwheat downy mildew (Peronospora ducommeti). The detection method of the present invention includes the steps of performing PCR using the above-mentioned primer set and nucleic acid in a sample as a template, and detecting the PCR amplification product. The sample may be any sample suspected of containing buckwheat downy mildew, and is not particularly limited, and may include, for example, buckwheat seeds, seedlings, leaves, and soil suspensions.

[0035] The present invention also provides a method for diagnosing buckwheat downy mildew. The diagnostic method of the present invention includes the steps of performing PCR using DNA extracted from buckwheat seeds, seedlings, or leaves as a template and the above-mentioned primer set, and detecting the PCR amplification product. In the diagnostic method of the present invention, if an amplification product specific to the buckwheat downy mildew fungus (Peronospora ducommeti) is detected in the detection step, the buckwheat is determined to be infected with buckwheat downy mildew.

[0036] DNA can be extracted from buckwheat seeds, seedlings, or leaves using known DNA extraction methods. For example, the seeds, seedlings, or leaves can be crushed in a mortar and pestle, and then extracted using a commercially available DNA extraction kit. Sterile water or a DNA extraction buffer can be used to crush the seeds, seedlings, or leaves.

[0037] Buckwheat seedlings can be obtained by germinating buckwheat seeds. For example, buckwheat seeds can be sown on a paper sheet such as Kimwipes moistened with water and left to germinate for about two weeks to one month. The number of buckwheat seeds sown is not particularly limited, but 1 to 10 seeds or more may be sown. The paper sheet on which the seeds are sown may be folded in half, for example, with the seeds at the center, and placed in a plastic or other container. The conditions for leaving the sheet to germinate may be, for example, a dark room at 10 to 20°C. The sweat-bag seedling method described in Non-Patent Document 3 may also be used to germinate buckwheat seeds.

[0038] In the method of the present invention, PCR can be performed using a general PCR method (conventional PCR method) or a real-time PCR method. When real-time PCR is used, an intercalator method or a hybridization method can be used. General reaction conditions can be used for PCR, and for example, three steps of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds can be performed for 25 to 45 cycles.

[0039] In the step of detecting the PCR amplification product, the amplification product can be detected using any method, including, but not limited to, electrophoresis, intercalator method, probe method (5'-nuclease method), cycling probe detection, and SYBR Green method. Electrophoresis methods include agarose gel electrophoresis and capillary electrophoresis. The amplification product may also be detected by melting curve analysis. If an amplification product specific to P. parasitica is detected, the sample is determined to be infected with P. parasitica.

[0040] An amplification product specific to P. parasitica is a DNA fragment containing a DNA sequence derived from P. parasitica. In the detection step, whether or not the amplification product is the target amplification product can be determined based on the size of the amplification product to be amplified by the primer set used. For example, the length of the amplification product may be determined by electrophoresis or the like. Alternatively, the electrophoretic fraction may be hybridized with a labeled probe obtained by labeling the oligonucleotide of the present invention with a labeling substance, and the label may be detected to determine whether or not the amplification product is the target amplification product. Furthermore, the base sequence of the amplification product may be identified to determine whether or not the amplification product is the target amplification product. [Example]

[0041] Example 1 Based on the nucleotide sequence information (KP271924) of the 18S ribosomal RNA gene and ITS region of P. parasitica, two sets of primers, primer set A (SEQ ID NOs: 1 and 2) and primer set B (SEQ ID NOs: 3 and 4), were designed to detect P. parasitica. The designed primers are shown in Table 1 below.

[0042] [Table 1]

[0043] (PCR test) PCR tests were carried out using the prepared primers.

[0044] Fifty milligrams of buckwheat leaves were ground, and DNA was extracted using the DNeasy Plant Mini Kit (QIAGEN). PCR was performed using 50-150 ng / μl of DNA extract and a KOD FX Neo (Toyobo) under the following conditions: 2 minutes at 94°C, followed by 35 cycles of 10 seconds at 98.3°C and 30 seconds at 68°C. The amplified products were detected by electrophoresis on a 2% agarose gel containing Gel Red.

[0045] As a result, when primer set A (Pd-3F / Pd-3R) was used, nonspecific bands (false positives) were detected. In addition, there was a problem that PCR amplification did not occur in diseased leaves in some areas (not shown).

[0046] On the other hand, when primer set B (Pd-4F / Pd-4R) was used, an amplification product (196 bp) specific to P. parasitica was obtained when DNA from diseased leaves was used as a template. Figure 3 shows the results of PCR performed with primer set B using DNA from healthy and diseased leaves as templates. This suggests that primer set B can be used to specifically detect P. parasitica.

[0047] Example 2 Primer set B was used to diagnose whether buckwheat seeds were infected with buckwheat downy mildew.

[0048] (Germination of seeds infected with buckwheat downy mildew) Buckwheat seeds were germinated using the method shown in Figure 1. First, buckwheat seeds were sown on a Kimwipe moistened with sterilized water, folded in half around the seed, and placed in a plastic bag with a Ziploc®. Next, the plastic bag was filled with air and left to stand at 15°C in a dark room for two weeks to one month. As air escaped from the plastic bag, it was refilled with air once a week.

[0049] Seedlings that germinated in the plastic were collected and crushed in a mortar and pestle. DNA was extracted from the crushed sample according to the protocol of the commercially available DNeasy plant mini kit (Qiagen) and used as template DNA.

[0050] (PCR test) Conventional PCR (94°C for 1 minute, followed by 35 cycles of 96°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds) was performed using primer set B prepared in Example 1. As a comparative example, the filamentous fungal universal primers ITS5 / ITS4 (Non-Patent Document 2; Table 1) were used.

[0051] The results of PCR using the primers of the comparative example are shown in Figure 2, and the results of PCR using primer set B are shown in Figure 3. When the primers of the comparative example were used, nonspecific amplification was observed regardless of whether healthy or diseased buckwheat seeds were used as templates, and no amplification specific to P. parasitica was observed (Figure 2). On the other hand, when primer set B was used and DNA derived from diseased seeds was used as a template, an amplification product (196 bp) specific to P. parasitica was obtained (Figure 3). Therefore, it was confirmed that primer set B can be used to specifically detect P. parasitica.

[0052] Example 3 We investigated whether diagnosis was possible for buckwheat plants infected with the buckwheat downy mildew fungus but showing no symptoms (asymptomatic buckwheat). Leaves were collected from buckwheat plants under cultivation, and DNA was extracted from the leaves. Conventional PCR was performed using the extracted DNA as a template and primer set B under the same conditions as in Example 2.

[0053] The results of PCR using DNA obtained from symptom-free buckwheat leaves as a template are shown in Figure 4. As shown in Figure 4, PCR results showed that amplification products specific to P. parasitica were detected even when symptom-free buckwheat leaves were used.

[0054] These results showed that by using primer set B, it is possible to evaluate the presence or absence of buckwheat downy mildew infection even at a stage when no symptoms of buckwheat downy mildew are observed.

[0055] As shown in this example, the use of the primers and method of the present invention makes it possible to specifically and efficiently detect whether buckwheat seeds and buckwheat plants are infected with buckwheat downy mildew. The use of the primers and method of the present invention also makes it possible to detect the presence or absence of infection in symptomless plants. Therefore, the present invention can contribute to improving the efficiency of selection for useful traits in cultivar development. [Industrial Applicability]

[0056] The present invention can be used to diagnose buckwheat downy mildew, particularly seed infection, not only in areas where buckwheat downy mildew is present, but also in areas where it is not present. For example, the present invention can contribute to the surveillance and diagnosis of buckwheat downy mildew in major buckwheat-producing areas. The present invention can also contribute to the formulation of countermeasures after buckwheat downy mildew outbreaks. The present invention can also minimize the spread of damage in areas where buckwheat downy mildew is present by quickly and reliably distinguishing healthy seeds from infected seeds and selecting healthy seeds.

Claims

1. A method for producing buckwheat seedlings, comprising: performing PCR using a primer set and nucleic acid in the seedling as a template; and detecting an amplified product by PCR, the primer set consists of a first primer and a second primer, the first primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, A detection method, wherein the second primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO:

4.

2. A method of producing buckwheat seedlings by germinating seeds of buckwheat; performing PCR using the DNA extracted from the seedling as a template and a primer set; and detecting the PCR amplification product, the primer set consists of a first primer and a second primer, the first primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, the second primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 4, A method for diagnosing buckwheat downy mildew, wherein if an amplification product specific to buckwheat downy mildew (Peronospora ducommeti) is detected in the detection step, the plant is determined to be infected with buckwheat downy mildew.

3. A primer set for use in the detection method of claim 1 or the diagnostic method of claim 2, comprising: a first primer and a second primer; the first primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, A primer set in which the second primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO:

4.

4. A kit for use in the detection method of claim 1 or the diagnostic method of claim 2, comprising: A primer set and an instruction manual are included. the primer set consists of a first primer and a second primer, the first primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, A kit, wherein the second primer is an oligonucleotide containing a sequence consisting of 10 or more consecutive bases in the base sequence represented by SEQ ID NO:4.

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