JP7497035B2 - Method for identifying rice planthoppers, primer set, and identification kit - Google Patents

Description

The present invention relates to a method for identifying rice planthoppers, a primer set, and an identification kit.

Rice planthoppers, which are important pests of rice in Asia, include three species: the brown planthopper (Nilaparvata lugens), the white-backed planthopper (Sogatella furcifera), and the sparse brown planthopper (Laodelphax striatellus). In addition to causing rice wilt by directly sucking sap, rice planthoppers transmit various rice viral diseases, which have a significant negative impact on rice yields. Each species of rice planthopper has developed resistance to different insecticides, so in order to quickly formulate control guidelines for rice planthoppers, it is necessary to grasp the occurrence status of each species of rice planthopper in paddy fields at an early stage.

The three species of rice planthoppers are often found together in paddy fields in Asia, including Japan. Identification of rice planthopper species is mainly based on the morphological characteristics of individuals. However, identifying rice planthopper species based on morphology requires specialized knowledge and experience. It is particularly difficult to accurately identify young rice planthopper larvae from their morphology. This makes it difficult to accurately grasp the occurrence of each species of rice planthopper in paddy fields at an early stage.

One method for identifying rice planthopper species is genetic diagnosis technology. Genetic diagnosis makes it possible to accurately identify rice planthopper species, regardless of specialized knowledge or experience in identifying species based on morphological characteristics, or even the developmental stage of the individual. Non-Patent Document 1 describes identification of three rice planthopper species by a singleplex PCR method using a species-specific primer pair. Non-Patent Document 2 describes identification of three rice planthopper species by a multiplex PCR method using a species-specific primer pair and a fluorescent probe.

Liu et al. 2009 Acta Entomologica Sinica 52(11):1266-1272 Wang et al. 2013 Molecular Ecology Resources 13:811-819

The technique in Non-Patent Document 1 requires multiple rounds of PCR and agarose gel electrophoresis using species-specific primer pairs for three rice planthopper species. The technique in Non-Patent Document 2 requires multiplex PCR using a fluorescent probe, which requires analysis using a sequencer, which is more expensive than agarose gel electrophoresis.

Therefore, there is a demand for technology that can identify rice planthopper species in a lower-cost and simpler way.

One aspect of the present invention aims to realize a technology that can easily identify rice planthopper species.

In order to solve the above problems, one embodiment of the present invention relates to a method for identifying rice planthoppers, which involves subjecting a sample that may contain DNA derived from rice planthoppers to multiplex PCR using a primer set designed to amplify at least a portion of the ITS2 (Internal Transcribed Spacer 2) region in the rDNA of rice planthoppers, and analyzing the electrophoretic pattern of the amplified product.

In addition, the primer set according to one aspect of the present invention comprises:
A primer set for multiplex PCR for identifying rice planthoppers, comprising the following primers (a) to (d):
(a) a first primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:1;
(b) a second primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:2;
(c) a third primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:3; and (d) a fourth primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:4.

Furthermore, the primer set according to one aspect of the present invention comprises:
A PCR primer set for identifying rice planthoppers, comprising the following primer (a) and at least one primer selected from the group consisting of the following primers (b) to (d):
(a) a first primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:1;
(b) a second primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:2;
(c) a third primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:3;
(d) a fourth primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:4.

In addition, a kit for identifying rice planthoppers according to one embodiment of the present invention includes any one of the primer sets described above.

According to one aspect of the present invention, it is possible to easily identify the species of rice planthoppers.

FIG. 1 shows the results of Example 1. FIG. 13 shows the results of Example 2.

The present invention will be described in detail below. All of the documents described in this specification are incorporated herein by reference. In addition, unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more (including A and larger than A) to B or less (including B and smaller than B)."

As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule" and refers to a polymer of nucleotides. Here, the nucleic acid may exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). The DNA or RNA may be double-stranded or single-stranded. The single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). As used herein, the notation of bases uses the one-letter notation defined by IUPAC and IUB as appropriate.

One aspect of the present invention is a technology for identifying rice planthoppers. One aspect of the present invention identifies rice planthoppers by amplifying at least a portion of the ITS2 (Internal Transcribed Spacer 2) region present in the rDNA of rice planthoppers and analyzing the electrophoretic pattern of the amplified product.

Rice planthoppers are important pests of rice in Asia, sucking rice sap and causing rice wilt and various diseases. Rice planthoppers include the brown planthopper (Nilaparvata lugens), the white-backed planthopper (Sogatella furcifera), and the sparse brown planthopper (Laodelphax striatellus). One embodiment of the present invention is a technique for distinguishing between three rice planthopper species, the brown planthopper, the white-backed planthopper, and the sparse brown planthopper. Insects closely related to rice planthoppers in paddy fields are leafhoppers. Leafhoppers are found in paddy fields at the same time as rice planthoppers, and similarly suck rice sap and transmit viral diseases. Leafhoppers include the green rice leafhopper (Nephotettix cincticeps) and the lightning leafhopper (Maiestas dorsalis).

In order to quickly formulate control guidelines for rice planthoppers, it is necessary to grasp the occurrence status of each species of rice planthopper in paddy fields at an early stage, and according to the present invention, it is possible to identify multiple species of rice planthoppers at once. In other words, according to the present invention, species of rice planthoppers can be easily identified.

(Method of identifying rice planthoppers)
A method for identifying rice planthoppers according to one embodiment of the present invention involves subjecting a sample that may contain DNA derived from rice planthoppers to multiplex PCR using a primer set designed to amplify at least a portion of the ITS2 region in the rDNA of the rice planthoppers, and analyzing the electrophoretic pattern of the amplified product.

In one embodiment of the method for identifying rice planthoppers according to the present invention, DNA derived from rice planthoppers in a sample is amplified by multiplex PCR. The DNA derived from rice planthoppers to be amplified is DNA derived from individual rice planthoppers, and is extracted from any tissue of rice planthoppers according to a conventionally known method, purified as necessary, and subjected to an amplification reaction.

The tissues of rice planthoppers from which DNA is extracted include the whole body, legs, head, thorax, abdomen, etc. In addition, rice planthoppers may be at any developmental stage from young larvae to adults, but larvae (especially young larvae) may be preferable from the viewpoint of early identification and considering the difficulty of identification due to morphology. In the identification method according to one embodiment of the present invention, the rice planthoppers to be identified are not limited to rice planthoppers (including larvae and adults) captured by the sticky board brushing method, but may also be individuals captured using an insect net, aspirator, etc.

The DNA extraction method is not particularly limited, and examples include the phenol extraction method and the alkaline SDS method, but simple extraction may also be performed using the Chelex method. DNA may also be extracted from rice planthoppers using a commercially available DNA extraction kit.

The sample that may contain DNA derived from rice planthoppers and that is subjected to multiplex PCR may be a sample that contains DNA derived from insects (adults, larvae, etc.) captured from rice. The method for capturing insects from rice is not particularly limited, and may be any of the capture methods described above. In addition, the insects captured from rice may be rice planthoppers and leafhoppers.

A primer set designed to amplify at least a portion of the ITS2 region (between the 5.8S rRNA gene and the 28S rRNA gene) in the rDNA of rice planthoppers means a primer set created to include a partial sequence within the 5.8S rRNA gene or the ITS2 region, or a complementary sequence thereof, so as to specifically anneal to a predetermined position within the ITS2 region in the rDNA of rice planthoppers during the amplification reaction.

The primer set used in the identification method according to one embodiment of the present invention was designed based on the base sequence information obtained by DNA sequence analysis of the 5.8S rRNA gene and ITS2 region of 10 rice planthopper species collected in Japan from different collection locations and collection years, and the base sequence information of three rice planthopper species registered in GenBank (GenBank accession numbers: KC660120-KC660122 and AB625596-AB625609). In this way, the primer set used in the identification method according to one embodiment of the present invention is designed based on the gene sequences of multiple individuals collected from different collection locations and collection times, making it possible to identify rice planthopper species corresponding to base sequence polymorphisms.

The length of each primer included in the primer set used in the identification method according to one embodiment of the present invention may be, for example, 15 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, or 19 bp or more, or 50 bp or less, 40 bp or less, or 30 bp or less. Each primer may be an oligonucleotide further modified with a labeling substance or the like.

The primer set used in the identification method according to one embodiment of the present invention comprises the following primers (a) to (d):
(a) a first primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:1;
(b) a second primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:2;
(c) a third primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 3; and (d) a fourth primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 4.

The first primer is a primer that contains 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 1, and an example of this example is primer Unka01F3. The first primer is a forward primer common to the three species of rice planthoppers.

The second primer is a primer that contains 15 or more consecutive bases in the base sequence shown in SEQ ID NO:2, and an example of this example is primer UnkaT01R. The second primer is a reverse primer that amplifies DNA derived from the brown planthopper.

The third primer is a primer that contains 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 3, and an example of this example is primer UnkaS01R. The third primer is a reverse primer that amplifies DNA derived from the white-backed planthopper.

The fourth primer is a primer that contains 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 4, and an example of this example is primer UnkaH01R. The fourth primer is a reverse primer that amplifies DNA derived from Laodelphax striatellus.

Amplification of DNA derived from rice planthoppers using the above primer set is carried out by multiplex PCR, which uses multiple primer pairs simultaneously in one PCR reaction system and can amplify multiple DNAs simultaneously. The above primer set can also be applied to a method for identifying rice planthoppers by using one pair of primers in one PCR reaction system and performing PCR reactions multiple times.

The PCR reaction conditions can be appropriately set according to the type of DNA polymerase and PCR device used, the length of the amplified fragment, etc. As cycle conditions, a three-step PCR method in which one cycle is a denaturation step, an annealing step, and an extension step, and a two-step PCR method in which one cycle is a denaturation step, an annealing step, and an extension step, can be applied. An example of the PCR reaction conditions is 90-100°C for 40-60 seconds (e.g., 95°C for 60 seconds), followed by 90-100°C for 20-40 seconds (e.g., 94°C for 30 seconds), 50-60°C for 20-40 seconds (e.g., 56°C for 30 seconds), and 65-75°C for 20-40 seconds (e.g., 72°C for 30 seconds) for 25-35 cycles (e.g., 30 cycles), and finally 65-75°C for 4-6 minutes (e.g., 72°C for 5 minutes). The PCR reaction conditions may be adjusted according to the state of the template DNA.

The amount of each primer contained in the primer set used in the PCR reaction may be 100 nM to 500 nM, preferably 120 nM to 450 nM, for example, 150 nM. The amount of extracted DNA to be amplified in the PCR reaction may be 0.5 μL to 3 μL, preferably 0.75 μL to 2 μL, for example, 1 μL.

In one embodiment of the identification method of the present invention, the amplified products obtained by multiplex PCR are subjected to electrophoresis, and the obtained electrophoretic pattern is analyzed to identify rice planthoppers. Electrophoresis of the amplified products is performed by known methods such as agarose gel electrophoresis and acrylamide gel electrophoresis. Bands separated by electrophoresis may be detected by ethidium staining, or, if fluorescently labeled, may be detected using fluorescent coloring as an indicator.

The electrophoretic pattern refers to the pattern of bands (nucleic acid fragments) obtained by electrophoresis of the amplification products, and includes the presence or absence of bands and their positions (base lengths of the nucleic acid fragments). The electrophoretic pattern may also include the shading of the bands (abundance of the nucleic acid fragments).

In the analysis of the electrophoretic pattern, when the size of the amplified product is 480 bp to 560 bp, preferably 490 bp to 540 bp, more preferably 500 bp to 520 bp, for example, 501 bp, it can be determined that the DNA is derived from the brown planthopper.

In the analysis of the electrophoretic pattern, when the size of the amplified product is 165 bp to 245 bp, preferably 170 bp to 220 bp, more preferably 180 bp to 200 bp, for example, 184 bp, it can be determined that the DNA is derived from the white-backed planthopper.

In the analysis of the electrophoretic pattern, when the size of the amplified product is 300 bp to 380 bp, preferably 310 bp to 360 bp, more preferably 315 bp to 350 bp, for example, 319 bp, it can be determined that the DNA is derived from Laodelphax striatellus.

In addition, according to one embodiment of the identification method of the present invention, multiplex PCR does not produce amplification products that amplify DNA derived from leafhoppers. Therefore, rice planthoppers can be clearly identified without being confused with the closely related leafhoppers.

According to one embodiment of the identification method of the present invention, a sample that may contain DNA derived from rice planthoppers is subjected to multiplex PCR, and the electrophoretic pattern of the amplified product is analyzed, thereby making it possible to identify multiple types of rice planthoppers in a single PCR reaction.

(Primer set)
A primer set according to one embodiment of the present invention is a primer set for multiplex PCR for identifying rice planthoppers, comprising the following primers (a) to (d):
(a) a first primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:1;
(b) a second primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:2;
(c) a third primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:3; and (d) a fourth primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:4.

The primer set used in the method for identifying rice planthoppers according to one embodiment of the present invention described above is one embodiment of the primer set according to the present invention. Therefore, details of the primer set according to the present invention are as described above for the method for identifying rice planthoppers according to one embodiment of the present invention.

A primer set according to one embodiment of the present invention is a primer set for PCR for identifying rice planthoppers, comprising the following primer (a) and at least one primer selected from the group consisting of the following primers (b) to (d):
(a) a first primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:1;
(b) a second primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:2;
(c) a third primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:3;
(d) a fourth primer comprising 15 or more consecutive bases in the base sequence shown in SEQ ID NO:4.

In other words, by using the primer set of the present invention, rice planthoppers can be identified by using one pair of primers in one PCR reaction system and performing PCR reactions multiple times.

(Rice planthopper identification kit)
A rice planthopper identification kit according to one embodiment of the present invention is a kit for identifying rice planthoppers, comprising a primer set according to one embodiment of the present invention. Therefore, details of the rice planthopper identification kit according to the present invention are in accordance with the description of the rice planthopper identification method and primer set according to one embodiment of the present invention described above.

The rice planthopper identification kit according to one embodiment of the present invention may include, in addition to the above-mentioned primer set, reagents necessary for multiplex PCR, DNA extraction reagents, instructions for use, etc.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments.

An embodiment of the present invention will be described below. Three species of rice planthoppers were genetically diagnosed using a primer set according to one embodiment of the present invention.

(Primer design)
We performed DNA sequence analysis of the 5.8S rRNA gene and ITS2 region of 10 rice planthopper species collected in Japan from different locations and years. Based on the sequence information obtained from the above analysis and the sequence information of three rice planthopper species collected in several countries overseas registered in GenBank (GenBank accession numbers KC660120-KC660122 and AB625596-AB625609), we designed a new primer set for the purpose of identifying the three rice planthopper species (Table 1).

Example 1
Legs were collected from adults and mature larvae of three rice planthoppers species that were reared indoors and obtained from Koshi City, Kumamoto Prefecture and Minamisatsuma City, Kagoshima Prefecture. In addition, whole bodies of young larvae of rice planthoppers that were reared indoors and obtained from Koshi City, Kumamoto Prefecture and Minamisatsuma City, Kagoshima Prefecture were used. DNA was extracted from the collected legs and whole bodies of each individual by the Chelex method.

Using the primer set shown in Table 1 and the extracted DNA as a template, PCR was performed (95°C for 1 minute, followed by 30 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, and finally 72°C for 5 minutes). The amount of primers used in the PCR reaction was 150 nM each, and the amount of extracted DNA was 1 μL.

SAFELOOK® Road Green (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the PCR product, and 3% agarose gel electrophoresis was performed. The separated bands were observed using a blue LED. The results are shown in Figure 1. As shown in Figure 1, bands of nucleic acid fragments of the brown planthopper (501 bp), white-backed planthopper (184 bp), and striate brown planthopper (319 bp) were detected. In this way, specific PCR amplification products were obtained for each of the three rice planthopper species, and the species of rice planthopper individuals (adults, final instar larvae, and young larvae) could be clearly identified.

Example 2
Three species of rice planthoppers were collected from outdoor paddy fields using the sticky board brushing method used in infestation forecasting surveys. Leafhoppers closely related to planthoppers, the green rice leafhopper and the lightning leafhopper, which are found in paddy fields together with the rice planthoppers, were also collected.

DNA was extracted from the legs of adult rice planthoppers, the legs of final stage larvae, and the entire bodies of young larvae, as well as the legs of adult leafhoppers, for each individual using the Chelex method.

Using the primer set shown in Table 1 and the extracted DNA as a template, PCR was performed (95°C for 1 minute, followed by 30 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, and finally 72°C for 5 minutes). The amount of primers used in the PCR reaction was 150 nM each, and the amount of extracted DNA was 1 μL.

SAFELOOK® Road Green (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the PCR product, and 3% agarose gel electrophoresis was performed. The separated bands were observed using a blue LED. The results are shown in Figure 2. As shown in Figure 2, bands of nucleic acid fragments of the brown planthopper (501 bp), white-backed planthopper (184 bp), and striate brown planthopper (319 bp) were detected. It was also confirmed that no PCR amplification products were obtained for the green rice leafhopper and lightning leafhopper.

In this way, we were able to clearly identify the species of rice planthoppers without confusing them with leafhoppers.

The present invention can be used in the fields of agriculture, pesticides, etc.

Claims (6)

A method for identifying rice planthoppers, comprising subjecting a sample that may contain DNA derived from rice planthoppers to multiplex PCR using a primer set designed to amplify at least a portion of an ITS2 (Internal Transcribed Spacer 2) region in rDNA of the rice planthoppers, and analyzing an electrophoretic pattern of the amplified product ,
The primer set includes the following primers (a) to (d):
(a) a first primer comprising 20 consecutive bases in the base sequence shown in SEQ ID NO:1;
(b) a second primer comprising 21 consecutive bases in the base sequence shown in SEQ ID NO:2;
(c) a third primer comprising 20 consecutive bases in the base sequence shown in SEQ ID NO:3; and
(d) a fourth primer comprising 21 consecutive bases in the base sequence shown in SEQ ID NO:4;
The rice planthopper is selected from the group consisting of the brown planthopper, the white-backed planthopper, and the striate brown planthopper . The method according to claim 1 , wherein the DNA derived from rice planthoppers is DNA derived from individual rice planthoppers including larvae and adults captured by a sticky-plate brushing method, an insect net, or an insect aspirator. In analyzing electrophoretic patterns,
When the size of the amplification product is 480 bp to 560 bp, it is determined to be DNA derived from the brown planthopper;
When the size of the amplification product is 165 bp to 245 bp, it is determined to be DNA derived from the white-backed planthopper;
When the size of the amplification product is 300 bp to 380 bp, it is determined that the DNA is derived from Laodelphax striatellus.
The method according to claim 1 or 2 . The method according to claim 1 , wherein the multiplex PCR does not produce an amplification product that amplifies DNA derived from leafhoppers. A primer set for multiplex PCR for identifying rice planthoppers, comprising the following primers (a) to (d):
(a) a first primer comprising 20 consecutive bases in the base sequence shown in SEQ ID NO:1;
(b) a second primer comprising 21 consecutive bases in the base sequence shown in SEQ ID NO:2;
(c) a third primer comprising 20 consecutive bases in the base sequence shown in SEQ ID NO: 3; and (d) a fourth primer comprising 21 consecutive bases in the base sequence shown in SEQ ID NO: 4 ;
The rice planthoppers are the brown planthopper, the white-backed planthopper, and the striate brown planthopper . A kit for identifying rice planthoppers, comprising the primer set according to claim 5 .