JP2964233B2 - Assay method for rice brown planthopper resistance, DNA fragment and PCR marker - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for assaying rice planthopper resistance in rice, a DNA fragment and a PCR marker, and more specifically to the following:

[0002] PCR (Polymna) using genomic DNA of a rice sample as a template and specific primers as PCR markers
Erase Chain Reaction) A method to test rice plant samples for resistance to brown planthopper from polymorphisms obtained by the reaction.

[0003] A DNA fragment that is specifically amplified in a resistant variety having a brown planthopper resistance gene (bph-2) in a PCR reaction using rice genomic DNA as a template, and is useful for setting an assay index and a PCR marker.

A primer for a PCR reaction using rice genomic DNA as a template, which is set in accordance with the nucleotide sequence of the above DNA fragment, and is a PCR marker used for the test of rice planthopper resistance.

[0005]

2. Description of the Related Art Brown planthopper (Nilaparvata lugens)
Stal) is an important pest of rice in warm and temperate regions of Japan, but it is an indica-type resistant variety, "Kanto PL
5 ”(later,“ Middle Mother Hon Nori No. 4 ”), a brown planthopper resistance gene bph-2, which is currently resistant to the biotypes that fly to Japan, is a Japonica-type resistant variety that has this gene. Training is expected.

[0006] At the time of such breeding, a test for resistance to brown planthopper is performed. As a test method, for example, a bioassay (biological test) method or RAPD (Random Amplified Polymo- num) using a DNA marker is used.
rphic DNA) method or RFLP (Restriction Fragm)
ent Length Polymorphism) method is conceivable.

Although the bioassay method can directly test the resistance and is suitable for a multipoint resistance test, the brown planthopper used for the test must be bred / managed.
There are drawbacks, such as being restricted by the growth stage of rice and released insects, and having difficulty in acquiring assay techniques.

[0008] Therefore, when it is necessary to know the presence or absence of a relatively small number of resistance genes in a short period of time in breeding by continuous backcrossing or the like, an indirect resistance test method using a DNA marker is required. Appropriate.

[0009] The RFLP method is a method in which rice genomic DNA is cut with a restriction enzyme and a specific label D designed as a marker is cut.
In this method, the presence or absence of a resistance gene is identified by Southern hybridization using an NA probe.

On the other hand, the RAPD method uses a rice genomic DNA which has not been digested with a restriction enzyme as a template, reacts it with an arbitrary random primer, performs a PCR reaction, and analyzes the polymorphism of the amplified DNA fragment. For the purpose of discriminating the presence or absence of a specific resistance gene, DN that is specifically amplified for a variety having the resistance gene
If the A fragment and the primer for amplifying such a DNA fragment are specified, the assay can be performed extremely conveniently using these.

[0011] Furthermore, the RAPD method can be carried out with the use of a PCR device and electrophoresis equipment, and has the advantage that it does not require any hybridization equipment or operation required for the RFLP method. In addition, the RAPD method also has the advantage that extraction / purification of DNA from a rice specimen can be greatly simplified because a small amount of template DNA is used.

[0012]

However, conventionally, the application of the RAPD method to the rice brown planthopper resistance test has been studied with respect to DNA fragments that are specifically amplified in varieties having the resistance gene bph-2 and PCR. Assay primers (P
CR marker) has not yet been reported.

Accordingly, the present invention provides a method for identifying a DNA fragment as described above, setting a PCR marker capable of amplifying the DNA fragment, and using the same to test for resistance to brown planthopper in rice. And issues to be solved.

[0014]

Means for Solving the Problems (Structure of the First Invention) The structure of the first invention of the present application (the invention as set forth in claim 1) for solving the above problems is to use a genomic DNA of a rice sample as a template,
A PCR reaction was performed using primers that are closely linked to the rice brown planthopper resistance gene bph-2 and amplify a DNA fragment specific to the brown planthopper resistant cultivar as a PCR marker. This is a method for testing rice planthopper resistance in rice, which tests for planthopper resistance.

(Structure of the Second Invention) The structure of the second invention of the present application (the invention according to claim 2) for solving the above problems is as follows.
A DNA fragment specifically amplified in a resistant variety having a brown planthopper resistance gene (bph-2) in a PCR reaction using rice genomic DNA as a template. The DNA fragment has the nucleotide sequence shown in SEQ ID NO: 1 on the side and the nucleotide sequence shown in SEQ ID NO: 2 on the 3 ′ end side.

(Structure of Third Invention) The structure of the third invention (the invention according to claim 3) for solving the above-mentioned problem is as follows.
A primer for a PCR reaction using rice genomic DNA as a template, a sense primer set in accordance with a sequence having an arbitrary number of bases of an arbitrary portion of the nucleotide sequence shown in SEQ ID NO: 1, and a primer for an arbitrary portion of the nucleotide sequence shown in SEQ ID NO: 2 It is a PCR marker consisting of an antisense primer set in accordance with a sequence of an arbitrary number of bases and used for testing rice planthopper resistance.

(Structure of the Fourth Invention) The structure of the fourth invention according to the present invention for solving the above-mentioned problems is a primer for a PCR reaction using rice genomic DNA as a template.
A sense primer selected from the nucleotide sequences of SEQ ID NO: 5 or SEQ ID NOs: 9 to 11, and SEQ ID NO: 6
And a antisense primer selected from the nucleotide sequence shown in SEQ ID NO: 11, and is a PCR marker used for testing rice planthopper resistance.

(Structure of the Fifth Invention) In the structure of the fifth invention of the present application for solving the above-mentioned problems, the sense primer in the fourth invention is a primer having the base sequence shown in SEQ ID NO: 4, and the antisense primer is It is a PCR marker that is a primer having the nucleotide sequence shown in SEQ ID NO: 7.

(Structure of the Sixth Invention) The structure of the sixth invention (the invention according to claim 4) for solving the above-mentioned problems is as follows.
A PCR marker wherein the sense primer is a primer having the nucleotide sequence shown in SEQ ID NO: 5 and the antisense primer is a primer having the nucleotide sequence shown in SEQ ID NO: 7.

[0020]

Operation and Effect of the Invention (Operation and Effect of the First Invention) In the first invention, the resistance of brown rice planthopper, which is a sample, can be assayed from the polymorphism obtained as a result of the PCR reaction. That is, whether or not a specific DNA fragment is amplified in the PCR reaction product is determined by the amplification D in the electrophoresis pattern.
The determination can be made by judging the presence or absence of a specific band representing the NA fragment.

This test method is not intended to directly amplify the DNA fragment containing the brown planthopper resistance gene bph-2, and therefore, the DNA fragment specifically amplified in the resistant varieties contains Gene bph-2
It does not matter whether or not is included.

However, the primer (in other words, the specific DNA fragment to be amplified), which is a PCR marker, is closely linked to the resistance gene bph-2.
From the fact that the fragment was amplified, the presence of the resistance gene bph-2 in the genomic DNA can be indirectly confirmed.

According to this assay method, there are no difficulties such as the need for breeding / managing of brown planthoppers, such as the bioassay method, and the restriction of the growth stage of rice or released insects, and the acquisition of assay techniques is relatively easy. Out.

Further, according to this assay method, it can be performed if a PCR device and an appropriate polymorphism analysis means (for example, electrophoresis equipment) are used, and the equipment and operation for hybridization as in the case of the RFLP method are performed. Is not required, and the amount of template DNA used is small.
Extraction / purification can be greatly simplified.

(Function / Effect of the Second Invention) Since the DNA fragment according to the second invention has been specified, a PCR reaction is carried out using many random primers using rice genomic DNA as a template to specifically amplify the resistant variety. A difficult and laborious preliminary process of determining a DNA fragment to be performed can be omitted, and the test for rice planthopper resistance can be performed very conveniently using the DNA fragment as a test index.

Further, the DNA fragment is useful for setting a PCR marker, and the 5 'end of the DNA fragment and the 3'
According to the base sequence on the terminal side, a variety of PCR markers consisting of an arbitrary sense primer and an antisense primer can be set.

That is, the 5 'end of the DNA fragment and the 3'
The sense primer and the antisense primer set according to the sequence with the terminal side should specifically amplify the DNA fragment as a PCR marker of the PCR reaction using the rice genomic DNA as a template in the resistant variety,
Then, from these various PCR markers, those having excellent specificity and ease of assay (for example, a PCR marker that produces a specific and distinct single band in the electrophoresis pattern of a PCR reaction product) are selected. be able to.

(Functions and Effects of Third to Sixth Inventions) Third
Using any of the PCR markers of the invention to the sixth invention,
The operation and effect of the assay method of the first invention can be obtained.

[0029] In particular, the PCR marker according to the sixth aspect of the present invention differs from the other markers in the so-called STS (Sequence).
Tagged Site) and improved to generate a specific and distinct single band in the electrophoresis pattern of the PCR reaction product, which is particularly useful.

Although not confirmed experimentally, among the arbitrary and various types of PCR markers that can be set according to the third invention, in addition to the PCR markers according to the sixth invention, the PCR markers are at least equivalent to these. One or more PCR markers that produce a specific and distinct single band in the electrophoresis pattern of the PCR reaction product may be included.

The PCR markers according to the third to sixth aspects of the present invention are specific to the PCR cultivar from a resistant variety having only the brown planthopper resistance gene Bph-1 different from the brown planthopper resistance gene bph-2 by PCR. Since the DNA fragment is not amplified, it can be used for testing whether or not the rice sample has the resistance gene bph-2 regardless of whether or not the rice sample has the resistance gene Bph-1.

The PCR markers according to the third to sixth aspects of the present invention can be used as varieties resistant to other pests currently used as mother plants in rice breeding in Japan (for example,
Also, the specific DNA fragment is not amplified by the PCR reaction. Therefore, PCR markers capable of detecting resistance genes against these other pests (for example, Hayano et al., "Molecular markers closely linked to rice stripe disease resistance gene Stv-bi". (19
97) P for detecting rice stripe blight resistance gene described in 97)
It is also possible to perform both tests at the same time by using it simultaneously with the CR marker “ST10”).

[0033]

Next, embodiments of the first invention to the sixth invention will be described. In the following, simply "the present invention"
When this is the case, the first to sixth inventions are collectively referred to.

(Rice) The kind of rice to which the present invention is applied is not limited as long as the test method of the first invention is effectively established, and includes rice and upland rice, and is not limited to Japonica type or Indica type. Because genomic DNA
Due to the basic commonality of rice varieties, there can be a brown planthopper resistant variety and a susceptible variety in that type of rice line,
Moreover, it is highly likely that a DNA fragment specific to the resistant variety is amplified by the PCR reaction using the PCR marker of the present invention.

(Genomic DNA) Genomic DNA refers to the entire DNA constituting a chromosome or a gene contained in a gamete, according to a general definition.
A fragment that has not been subjected to cleavage processing into the A fragment. The genomic DNA to be tested is not restricted by the growth stage of rice,
In addition, the method of extracting and purifying genomic DNA from a rice specimen is not limited at all. However, a CTAB method, an alkali extraction method, a method using ISOPLANT (trade name, manufactured by Nippon Gene) and the like are particularly preferable.

(Linking) The primer used as the PCR marker used in the first invention is the third primer if it is closely linked to the rice brown planthopper resistance gene bph-2.
Even those other than the PCR markers of the sixth to sixth aspects of the present invention can be used. The reason is that the DNA fragment specifically amplified by such a PCR marker is also the same as the DNA fragment of the second invention amplified by the PCR marker of the third to sixth inventions, and the genomic DNA which is the basis of these DNA fragments. The nucleotide sequence of is transmitted to the progeny along with the resistance gene bph-2 with extremely high probability at the time of chromosomal recombination during meiosis,
Therefore, it can be an effective test index of the first invention.

The degree of closeness of this chain can be calculated experimentally, and can be stochastically expressed as a “genetic distance” in centiMorgans (cM). However, the genetic distance required in the first invention depends on the accuracy required for the test,
It cannot be defined uniformly in relation to the number of generations that are crossed repeatedly for breeding, and it is sufficient that they are linked closely enough to meet the purpose of the test. To give just one specific example,
The genetic distance is within 5 cM, more preferably 0 to 1 cM.

(PCR Marker) As a PCR marker, in the process of specifying a DNA fragment that is specifically amplified in a brown planthopper resistant variety by a PCR reaction using random primers, a sense primer selected from a variety of random primers is used. / Antisense primer may be used, and at least the base sequence at the 5 ′ end and 3 ′ end of the above DNA fragment is specified, and a sense / antisense primer arbitrarily set therefrom may be used. good.

At present, the present inventors have considered that the PCR marker of the sixth invention is the most effective PCR marker because it produces a specific and distinct single band in the electrophoresis pattern of the PCR reaction product. I have. However, in the future, a more effective PCR marker will be
The possibility of being set based on the DNA fragment of the invention is not denied.

Among the PCR markers according to the present invention, one or more sense primers selected from the nucleotide sequences shown in SEQ ID NOs: 3 to 5 and the nucleotide sequences shown in SEQ ID NOs: 6 to 8 A PCR marker consisting of one or more selected antisense primers is
The DNA fragment according to the second invention is amplified. On the other hand, PCR comprising a sense primer and an antisense primer selected from the nucleotide sequences shown in SEQ ID NOS: 9 to 11
The marker amplifies a DNA fragment corresponding to a band on an electrophoresis image different from the DNA fragment according to the second invention, as described later in Examples.

In the case where at least the DNA fragment of the second invention is amplified to be used as an indicator of the assay, the sense / antisense primer constituting the PCR marker must have its D
As long as the base sequence corresponds to the 5 'end / 3' end portion of the NA fragment, it can be set according to the sequence of any number of bases in any portion of the DNA fragment.

In general, if the number of bases of the primer is too small, a large number of bands are generated, so that it is difficult to observe a target polymorphism, and a problem that the amplification of the band is unstable tends to occur. On the other hand, as the number of bases of the primer increases, the specificity as a PCR marker increases. However, if the number of bases is excessive, the Tm value increases and becomes higher than the annealing temperature. Is liable to occur. From these points, it is generally considered that a base having about 10 to 30 bases is appropriate.

The STS conversion for the purpose of setting the PCR marker according to the sixth invention is described in, for example, Monna L. et.
al., "Determination of RAPD markers in rice and t
heirconversion into sequence tagged sites (STSs) a
nd STS-specific primers "DNA Res. 1,139-148 (1994) and other known methods.

(PCR reaction and analysis of reaction product) The PCR reaction in the assay method of the present invention uses the genomic DNA of a rice sample as a template and the PCR marker of any of the third to sixth inventions as a primer. There are no particular restrictions except for the following. Therefore, template DNA, primer, Mg
Ions, four types of deoxyribonucleotide triphosphates (hereinafter referred to as "dNTP"), and DNA polymerase (heat-resistant, such as Taq polymerase is more preferable).
Amount, temperature and time of each process of thermal denaturation / annealing / extension reaction, number of cycles of these processes,
Can be arbitrarily set as necessary.

In order to confirm whether or not a specific DNA fragment has been amplified in the PCR reaction product, analysis is performed by an appropriate means. As a typical example of the analysis means, there is a method of checking the expression of a specific band by performing gel electrophoresis.

As described above, one of the advantages of the present invention is that "there is no need to perform a hybridization reaction".
However, if it is desired to reconfirm the reliability of the band expressed in the electrophoresis as a test index, as described in Examples below, the resistant varieties that have already been obtained separately The insert fragment of the clone of the DNA fragment specific to DNA may be labeled by a suitable means (for example, using a radioisotope or a chemiluminescence method) to form a probe, and Southern hybridization may be carried out to confirm a signal by hybridization. It is possible. However, this operation is not a component of the assay method of the first invention.

(DNA Fragment) The DNA fragment of the second invention is
It has been found that, in a PCR reaction using rice genomic DNA as a template, when a specific appropriate primer is used, it is specifically amplified in a resistant variety having the brown planthopper resistance gene bph-2. Therefore, PCR to be used as a test index for brown planthopper resistance and in the test method thereof
It is a DNA fragment useful for setting a marker.

This DNA fragment was composed entirely of about 1.3 Kbp, and the 5 '
It has been confirmed that the 3 ′ terminal side has the nucleotide sequence shown in SEQ ID NO: 2. The nucleotide sequence of the intermediate part has not been determined at present.

In addition to the DNA fragment of the second invention, DN which is closely linked to the brown planthopper resistance gene (bph-2) and which is specifically amplified by the brown planthopper resistant variety
A fragment can be used in the assay method of the first invention together with a PCR primer that amplifies the fragment. For example, in Example 1 to be described later, when a primer 9 is used, a DNA which produces a specific band shown in FIG.
Fragments and DNA fragments that produce the specific bands shown in FIG. 2 when primer 12 is used.

[0050]

Embodiments of the present invention will be described below.

Example 1 In this example, the genome DN of rice plants of the brown planthopper resistant variety and the susceptible variety was
A PCR reaction was performed with various RAPD primers against A. From the analysis of the obtained polymorphism, a DNA fragment closely linked to the rice brown planthopper resistance gene bph-2 and specifically amplified in the brown planthopper resistant cultivar And a PCR marker capable of detecting the brown planthopper resistance gene bph-2 was selected from the above RAPD primers.

[0052] F ₄ 30 system as a (EUT species) test system, insect-resistant seedlings test has been carried out in the Aichi Prefecture Nogyosogoshikenjo-Crops Research Institute for-breeding laboratory, "Aoi wind / education Tobi 1137" As reference varieties, “Kanto PL5”, from which the brown planthopper resistance gene bph-2 of “Ikutobi 1137” was introduced, “Aichino Kaori”, a susceptible variety,
"Starlight" was used.

(Extraction of Genomic DNA from Test Varieties) Seeds of each of the test varieties described above were sown in vermiculite, and 3 g of each of the seedlings germinated in the dark was crushed in liquid nitrogen using a mortar. MG and Thompson WF ”Rapid
isolation high molecular weight plant DNA ”Nucle
CTAB described in ic acid Res8, 4321-4325 (1980)
Genomic DNA was extracted according to the method.

The extracted genomic DNA was dissolved in a 1/10 TE buffer and treated with RNase to degrade RNA.
The amount of DNA was estimated by 0.8% agarose gel electrophoresis. Based on the results of the insect resistance seedling test, 25 out of the 30 F ₄ lines, excluding the 5 lines with a small amount of DNA, were subjected to 4 separations of S separation, S fixation, R separation, and R fixation in Table 1. Classified into groups.

[0055]

[Table 1] In Table 1, the "test number" is the specimen number of consecutive granted to arbitrarily Sample Varieties of the F ₄ 30 lines, "insect resistance assay R (%)" and resistance to test individuals body number Indicates the percentage of the number of individuals, and indicates “S” in the item “Group”.
"Separation" means that the strain is susceptible but did not lead to a decision. "S fixed" means that the strain was determined to be susceptible. "R segregation" means that the strain was resistant. Group did not come to a conclusion, "R
“Fixed” means each group in which the system is determined to be resistant. In Table 1, "-" in the "Group" section means five lines excluded because of a small amount of DNA.

(RAPD Analysis Using Bulk DNA) Genomic DNAs of strains classified into the same group in Table 1 were mixed in equal amounts, and four types of bulk DNA corresponding to each group were mixed.
Samples were prepared, and these were used as template DNA for the PCR reaction.

Next, 20 ng of template DNA and 200 μm of dNTP were used.
M, 0.5 U of Tth polymerase manufactured by Boehringer Manheim Biochemica, and 0.2 μM of a known primer (296 types) consisting of 10 bases selected optionally, and a PCR reaction system was constituted. A PCR reaction was performed using various types of primers. Each of these primers has the same base sequence as a sense primer and an antisense primer.

The reaction conditions for the above PCR reaction are as follows:
The temperature was set at 4 ° C./30 seconds, the annealing at 40 ° C./2 minutes, and the extension reaction at 72 ° C./3 minutes, and these were repeated 45 cycles.

Next, each PCR reaction product was used for 1.5 times.
% Of the primers, and the band patterns of the above four groups were compared for each primer. As a result, a bulk-specific polymorphism was observed for 13 of the 296 primers. The breakdown is as follows: 7 types of primers produced a band specific to the resistant bulk (R-fixed group), 5 types of primers produced a band specific to the sensitive bulk (S-fixed group), One of the primers produced a band specific to S-separation and R-separation groups).

FIG. 1 shows an electrophoresis image of the case where a primer having a band specific to the resistant bulk and having the sequence shown in SEQ ID NO: 9 (named “primer 9”) was used. In the figure, lane 1 is resistant bulk, lane 2 and lane 3 are separated bulk, lane 4 is susceptible bulk, lane 5 is genomic DNA of "Hoshi no Hikari" which is a susceptible variety, and lane M is a size marker. The specific band generated in lane 1 is indicated by an arrow. The DNA fragment appearing in this band is about 1.2 Kbp.

FIG. 2 shows an electrophoresis image obtained when a primer having a band specific to the resistant bulk and having the sequence shown in SEQ ID NO: 10 (named “Primer 12”) was used. In the figure, lane 1 is resistant bulk, lane 2 is susceptible bulk, lane 3 is "Kanto PL5" of a comparative variety, and lane M is a size marker. Specific bands generated in lanes 1 and 3 are indicated by arrows. The DNA fragment that appeared in this band was approximately 0.3
Kbp.

FIG. 12 shows an electrophoretogram obtained when another primer (UBC148 described later) that produced a band specific to the resistant bulk was used. In the figure, lane 1 is an R fixed system, lane 2 is an R separation system, and lane 3 is S
Lane 4 shows an S-fixed line, and Lane M shows a size marker. Specific bands generated in lane 1 and lane 2 are indicated by arrows. The DNA fragment appearing in this band is approximately 1.3 Kbp.

(Narrowing down of primers) The band pattern of the above seven types of primers that produced a band specific to the resistant bulk was examined again using existing resistant and sensitive varieties and lines. Four types of primers are used for the rice brown planthopper resistance gene bph
It was narrowed down to show a band specific to only the line having -2.

These four types of primers consist of the primer 9, the primer 12, the primer named “primer 181” shown in SEQ ID NO: 11, the sense primer of SEQ ID NO: 3 and the antisense primer of SEQ ID NO: 6. A primer called "UBC148". Therefore, any of these primers can be used as an effective PCR marker in the present invention.

In the case of the primer 181 alone, there is a band that does not show polymorphism in the vicinity of the specific band of interest, so that it is difficult to cut out the band after electrophoresis, for example. It does not hinder the implementation itself.

(Cloning of Polymorphic Fragment) The above three types of primers (primer 9, primer 12, and UBC
148), specific target bands obtained as a result of electrophoresis were cut out, and their DNA fragments were
Stratagene plasmid pBleusscript
It was cloned by inserting it into the cleavage site of IISK + by the restriction enzyme SmaI.

Next, these inserts were M13 primers (a primer set which hybridizes to both outer sides of the multicloning site of pBluescriptIISK + and amplifies the insert and the multicloning site).
After the amplification, the size was confirmed by electrophoresis as shown in FIG. 3, and it was confirmed that the DNA fragment relating to the target specific band could be cloned.

In the figure, lane M is a size marker, lane 148K is a polymorphic fragment of Kanto PL5 by primer UBC148, lane 9 is a polymorphic fragment of a resistant bulk by primer 9, and lane 12 is a polymorphic fragment of primer Bu by The polymorphic fragment, lane 148R, shows the polymorphic fragment of the resistant bulk by the primer UBC148, and the lane 9, lane 12, and lane 148R show the band size and polycloning site size where the polymorphism was recognized in the bulk. Since it is almost equal to the sum of the above, the success of the cloning can be confirmed.

Example 2 In this example, of the cloned DNA fragment (a DNA fragment specifically amplified in a rice strain having the brown planthopper resistance gene bph-2), the primer UBC148 was used. The nucleotide sequence at both 5 'and 3' ends of the insert fragment of the clone relating to the DNA fragment to be amplified was analyzed, and specific primers particularly effective as PCR markers were set.

Further, the homology between UPCR148R5C and the PCR product amplified from the rice genomic DNA of the brown planthopper resistant cultivar using the specific primers was confirmed.

(Clone Analysis) Of the DNA fragments cloned in the first embodiment, the primer UBC148 was used.
A clone (hereinafter, referred to as “UBC148R5C”) relating to a DNA fragment amplified when was used for analysis.

First, the size of the inserted fragment (inserted DNA fragment) of UBC148R5C was determined using pBluescriptI.
After cutting with HindIII and BamHI near the SmaI site of the multiple cloning site of ISK +, running on a 1.5% agarose gel, and judging by comparison with a size marker, the SmaI site and HindIII and BamHI were determined to be approximately 1.3 Kbp. 34 additional bases
It was found to be about 1.3 Kbp on the basis that it was considered to be an error range for the base.

Next, the base sequences at both ends of the 5 ′ and 3 ′ ends of the inserted fragment of UBC148R5C were determined using Perkin-Elmer J
It was determined by a terminal sequencing reaction using a DNA die terminator cycle sequencing ready reaction kit manufactured by apan Applied Biosystems using a sequencer model 373A. As a result, the base sequence at the 5 ′ end was as shown in SEQ ID NO: 1, and the base sequence at the 3 ′ end was as shown in SEQ ID NO: 2.

(Setting of specific primer) Then, a specific sequence recognition site was formed from the above nucleotide sequence, and the sense primer was a primer having the nucleotide sequence shown in SEQ ID NO: 5 extended inside primer UBC148. An STS-specific primer (hereinafter, referred to as "A3 primer"), which is a primer having the nucleotide sequence shown in SEQ ID NO: 7 and extending inward including the primer UBC148, was set. The A3 primer is currently the best mode of the PCR marker of the present invention.

(Confirmation of Homology of DNA Fragment) In the same manner as in Example 1, PCR was carried out using genomic DNA extracted from Kanto PL5 as a template and A3 primer. Then, the amplified PCR product was cloned using the pGEM-TEasy vector system manufactured by Promega, and the plasmid was extracted and purified using an Easy prep kit manufactured by Pharmacia. After that, 5 ′ of the DNA fragment amplified by the terminal sequencing reaction was extracted. The nucleotide sequence of both 3 'ends was analyzed.
As a result, the nucleotide sequence at both 5 '/ 3' ends of this DNA fragment was the same as that of the inserted fragment of UBC148R5C.
The homology with the base sequence at both 5 '/ 3' ends shown in FIG.
9.8%.

[Example 3] In this example, the resistance to brown planthopper using the STS-specific primers was evaluated for a large number of brown planthopper-resistant varieties / susceptible varieties that grew more than the test variety used in Example 1. A sex test was performed, and a clear single band was produced only in the resistant strain, confirming its practicality.

(Test Varieties) Table 2 shows the varieties of the resistant strains that were tested during growth. These are breeding materials from Aichi Prefectural Agricultural Research Institute, Crop Research Institute and Breeding Laboratory.
Among the lines that are growing in 1996, two generations before the previous generation, the previous generation and the current generation, based on the test for insect resistance seedlings, the resistance gene to brown planthopper
We selected 22 strains that had bph-2 and had a relatively large number of backcrosses to Kanto PL5.

[0078]

[Table 2] The breakdown of these lines, Test No. 1-6 "F ₆ Koshihikari / (F ₆ Koshihikari / (F ₄ months of light // Aichi 56 Issue /
Kanto PL5)) ", test numbers 7 to 11 are the above-mentioned test numbers 1
More than 6 numbers of backcrossing "F ₄ Aoi wind // Aoi wind / education Tobi 1284", "F ₇ Aoi wind / education Tobi Test No. 12 to 15 hits the progeny varieties used in Example 1 113
7 ", test number 16 to 22 is" F ₄ education 1274 // Aichi of fragrance / Kanto PL5 ".

In Table 2, "system number" indicates the progress of selection. “R ₂ ” indicates that the individual was selected in the F ₂ generation, “P ₃ ” indicates that the individual was selected in the F ₃ generation, and “−” indicates the progress of the generation, and the number indicates the individual number. Is shown. With respect to the column of "Powdered brown planthopper resistance" in Table 2,
"R" indicates resistance, "S" indicates sensitivity, and the resistance test of the selected individual generation is based on the progeny test.

Test No. 23 in Table 2 is "Koshihikari" used as a comparative cultivar of sensitivity, Test Nos. 24 and 25
"Kanto PL" used as comparative varieties of resistance
5 "and" Nankai 133 ".

On the other hand, Table 3 shows the susceptible varieties tested.
These are donated by the Aichi Prefectural Agricultural Research Institute, Crop Research Institute, Breeding Laboratory and the Aichi Prefectural Mountain Agriculture Research Institute, Rice Cultivation Laboratory, and are used as breeding bases for paddy rice in Japan. And 32 main varieties and lines used as the backcross susceptible parents of the resistant lines shown in Table 2.

[0082]

[Table 3] The breakdown of these susceptible varieties includes the stripe blight resistance gene (Stv-b ⁱ ), which is derived from very early varieties (maturity category in Aichi prefecture), middle-aged varieties, glutinous rice, sake rice, and Modan. "Moonlight" with ear blast resistance gene, "Aichi 77" and "Aichi96" with black leafhopper resistance gene (Grh-3 (t))
"Sally Queen", a fragrant rice derived from Basmati370, and "Akenohoshi", a high-yielding rice.

Further, as shown in FIG. 4, a close linkage with the brown planthopper resistance gene bph-2 possessed by Kanto PL5 has been reported (Ryoichi Ikeda, “The brown planthopper resistance gene in rice and the brown planthopper resistance and virus disease resistance in rice”). Breeding research on the complexation of rice. Agricultural Research Center Research Report. 3,1-54 (1985)) Seven species in the breeding line of the brown planthopper resistance gene Bph1 (underlined in FIG. 4. Lines are susceptible varieties, and double underlines are resistant lines.).

(Extraction of Genomic DNA from Test Varieties and PCR Analysis) Each of the test varieties described above was raised, and genomic DNA was extracted from the leaf blade and leaf sheath of the three-leaf stage in the same manner as in the first example.
The DNA was extracted by the TAB method and used as a template DNA for the PCR reaction.

Then, 10 ng of template DNA, 96 μM of A3 primer / 110 mM of Tris-HCL, 150 mM of KCl, 150 mg of MgC
l ₂ 1.5 mM, dNTPs 150 μM, Taq polymerase 1U manufactured by Takara, total volume adjusted to 25 μl, and PC
An R reaction system was constructed. The reaction conditions of the PCR reaction were as follows: heat denaturation at 94 ° C / 1 minute, annealing at 55 ° C / 1 minute,
The extension reaction was set at 72 ° C / 2 minutes, and these were repeated 30 cycles, and the extension reaction was performed at 72 ° C for 7 minutes.

Next, 5 μl of each PCR reaction product was electrophoresed on a 2% agarose gel and stained with ethidium bromide. The results are shown in FIGS. In each figure, lane M is a size marker, lanes 1 to 25 in FIGS. 5 and 6 show varieties corresponding to test numbers 1 to 25 in Table 2, and lanes 1 to 32 in FIGS. The varieties corresponding to test numbers 1 to 32 in Table 3 are shown,
7 to 9, lane K indicates Kanto PL5, and lane N indicates Nankai 133.

As indicated by the arrows in FIGS. 5 to 9, the UBC148R5 was used for all the resistant varieties tested.
Approximately 1,300 bp, corresponding to the insert size of C
A clear single band was observed. On the other hand, in the susceptible varieties, such band amplification was not observed.

FIG. 10 shows the results of PCR analysis using the A3 primer with respect to the above seven genera of the brown planthopper resistance gene Bph1 shown in FIG. In this figure, lanes 1 to 7 are in the order of “Mudgo, No. 53
, Saikai 165, Saikai 190, Aichi 97, Hoyoku, Nipponbare ", lane K indicates" Kanto PL5 ", and lane M indicates a size marker.

In the Kanto PL5 used for comparison, a clear single band of about 1.3 Kbp was observed as indicated by the arrow, but “Mudgo to Aichi 97” in Lanes 1 to 5 having the brown planthopper resistance gene Bph1 were observed. And the sensitive "Hoyoku, Nipponbare" did not show amplification of the band.

(PCR-Southern Hybridization) The above PCR using the genomic DNAs of the varieties of the resistant breeding lines according to Test Nos. 1 to 22 in Table 2 and the comparative varieties according to Test Nos. 23 to 25 in Table 2 as templates For the reaction product, insert fragment of clone UBC148R5C was
Hybridization was performed using a probe labeled with an ECL gene detection kit manufactured by the company. The result is shown in FIG.
Shown in The lane numbers in the figure correspond to the test numbers.

As is clear from the figure, a signal due to hybridization was observed in all resistant lines including the comparative resistance variety, and no signal was observed only in the sensitive lane 23 (Koshihikari).

[0092]

[Sequence list]

SEQ ID NO: 1 Sequence length: 300 Sequence type: Nucleic acid Sequence characteristics Other information: The following sequence shows a sequence of 300 bases from the 5 'end in the DNA fragment according to the second invention. SEQ: TGTCCACCAG GCAGCCTGAT AAAACCTAGG TCCAAACCCG TCCGGGCCCG GTGCACTGTT 60 CCTGTCCATT TGCCGGATTG CCAACAGTAT TTCAGTCTGG GTGAATGGAG CGATCAGACT 120 GTCGCAATTC CAAACACGAT GACGATACAA TGCTGCAAGA TCAAAATGCC ACTCAGAGTT 180 TGAGGTAGAC CCTAGAATTG CACTGTAATA ATCATGCATG ATCGACGCCT TAGCAGCATG 240 ATCGGAGAAT TCGACACCCT GAACTTTAAT CGAGCGCACA TAAGTTTTGC GATGATTGTT 300 SEQ ID NO: length of the two sequences: 290 Type of sequence : Nucleic acid Antisense: YES Sequence characteristics Other information: The following sequence shows the sequence from the 3 'end to the 290 bases in the DNA fragment according to the second invention in order from the 5' side. SEQ: TGTCCACCAG ATCTTAAGGG ACGGGGGGGG GGGGGGGTTG GTCTCTGTCT GGGATAGCAC 60 TTCGCTCTCA AAAACTTCGT ATATCTCTAG TCACCATACG CTTTTGATTA CTTTCGAATC 120 TCTAATTGAN TCGATCTCCC CTCACTGTCA CCAATGTTTA TGCTCCATCC GACCACGCCT 180 ACACTGATAT TTCCTGCTGA GCTGATTGAT CTTGATAGAA CCATCTCGGG ACATTGGCTT 240 CTTATCGGAG ACTTTAACCT AATCCGTTCC CCAGCAGATA AGAACAATGA 290 SEQ ID NO: 3 Length of sequence: 10 SEQ type: Nucleic acid sequence: TGTCCACCAG 10 SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid sequence: TGTCCACCAG GCAGCCTGAT 20 SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid sequence: GCAGCCTGAT AAAACCTAGG T 21 SEQ ID NO: 6 Sequence length: 10 Sequence type: nucleic acid Sequence: TGTCCACCAG 10 SEQ ID NO: 7 Sequence length: 20 Sequence type: nucleic acid Sequence: TGTCCACCAG ATCTTAAGGG 20 SEQ ID NO: 8 Sequence length: 21 Sequence type: nucleic acid Sequence: CACCAGATCT TAAGGGACGG G 21 SEQ ID NO: 9 Sequence length: 10 Sequence type: Nuclear SEQ: CTCTGGAGAC 10 SEQ ID NO: 10 SEQ Length: 10 sequence types: nucleic acid sequence: TTATCGCCCC 10 SEQ ID NO: 11 SEQ Length: 10 SEQ Type: nucleic acid sequence: ATGACGACGG 10

[Brief description of the drawings]

FIG. 1 is an electrophoretic image negative of a PCR reaction product.

FIG. 2 is a negative image of an electrophoresis image of a PCR reaction product.

FIG. 3 is an electrophoretic image negative of a cloned DNA fragment.

FIG. 4 is a diagram showing the genealogy of the brown planthopper resistance gene.

FIG. 5 is an electrophoretic image negative of a PCR reaction product.

FIG. 6 is an electrophoretic image negative of a PCR reaction product.

FIG. 7 is a negative electrophoresis image of a PCR reaction product.

FIG. 8 is an electrophoretic image negative of a PCR reaction product.

FIG. 9 is a negative image of an electrophoresis image of a PCR reaction product.

FIG. 10 is a negative drawing showing the results of PCR analysis using the A3 primer.

FIG. 11 is a negative drawing showing the results of Southern hybridization.

FIG. 12 is a negative image of an electrophoresis image of a PCR reaction product.

──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Norikuni Saka Mikamine, Nagakute-cho, Aichi-gun, Aichi Prefecture, 1 Aichi Prefectural Agricultural Research Station, No. 1 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/09 C12Q 1/68 G01N 33/50 REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A genomic DNA of rice sample as a template, the following
The PCR fragment of (2) below, which amplifies the DNA fragment of (1)
A method for detecting rice planthopper resistance in rice, comprising performing a PCR reaction in a rice plant and testing the rice sample for resistance to brown planthopper from the resulting polymorphism. (1) PCR reaction using rice genomic DNA as a template
Have the brown planthopper resistance gene (bph-2)
DNA fragments that are specifically amplified for resistant varieties
And the whole is about 1.3 Kbp and the 5 'end
The base sequence shown in SEQ ID NO: 1 is on the end side, and the sequence is
DNA fragments each having the nucleotide sequence shown in No. 2 . (2) a sense primer having the nucleotide sequence shown in SEQ ID NO: 3
Is an antisense primer having the nucleotide sequence of SEQ ID NO: 6
Or a base sequence shown in SEQ ID NO: 5.
Of the base sequence shown in SEQ ID NO: 7
PCR marker consisting of chisense primer . 2. In a PCR reaction using rice genomic DNA as a template, a brown planthopper resistance gene (bph-
A DNA fragment specifically amplified by the resistant cultivar having 2), which is composed entirely of about 1.3 Kbp, and has a nucleotide sequence represented by SEQ ID NO: 1 at the 5 'end and a sequence at the 3' end A DNA fragment having the nucleotide sequence shown in No. 2. 3. A primer for a PCR reaction using rice genomic DNA as a template, the primer comprising any one of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.
A primer having any of the above nucleotide sequences or a sense primer having the nucleotide sequence shown in SEQ ID NO: 3
And the antisense primer having the nucleotide sequence of SEQ ID NO: 6
Or a sense primer having the nucleotide sequence shown in SEQ ID NO: 5.
Of the antisense primer having the nucleotide sequence of SEQ ID NO: 7
Consisting of either sense / antisense primer ,
A PCR marker, which is used for detecting rice planthopper resistance in rice.