JPH0739400A - Method for discriminating plant variety using oligonucleotide - Google Patents

Abstract

PURPOSE:To simply and efficiently carry out the discrimination of a plant variety useful for purity tests, etc., of the variety with a good accuracy by amplifying a mitochondrial DNA of a plant with a specific oligonucleotide used as a primer, separating the resultant amplified mitochondrial DNA according to the electrophoresis and subsequently performing the visual detection CONSTITUTION:This method for discriminating a plant variety comprises amplifying a mitochondrial DNA of a plant with an oligonucleotide with 50-80% contents of GC, composed of 8-15 nucleotides and selected from the sequence group expressed by the formula as a primer according to the polymerase chain reaction, separating the amplified mitochondrial DNA according to the electrophoresis and then carrying out the visual detection of the amplified mitochondrial DNA.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for identifying plant varieties using oligonucleotides.

[0002]

BACKGROUND ART Most of the genes that control the traits of living organisms are present in the nucleus, but genetic information is also present in the cytoplasm.
In plants, subcellular organelles such as chloroplasts and mitochondria have genes, and each has a unique trait. In particular, there are many genes in mitochondria that control important agricultural traits such as male sterility, resistance to pests, resistance to environmental stress, and productivity, and their use is being investigated. Conventionally, as a method for discriminating plant varieties, for example, methods based on differences in pest resistance, environmental stress resistance, etc., and methods based on differences in protein levels such as isozymes are known. On the other hand, recently, the RFLP method (Restrictio
n Fragment Length Polymorphism) is used to identify plant varieties, for example, Kesseli et al., GENOME, Vol. 34, Vol.
Pages 30 to 436 (1991), H. Ichikawa et al., THEORETI
CAL AND APPLIED GENETICS, No. 77, pp. 39 to 43 (1989).

[0003]

However, in the method based on the difference in pest resistance, environmental stress resistance, etc., there was no choice but to cultivate until the above-mentioned traits could be actually investigated and to investigate the characteristics of the variety. Therefore, a large-scale cultivation facility and labor are required, and it takes a time corresponding to the cultivation period. Further, in the method based on the difference in the protein level such as isozyme, it is difficult to obtain a sufficient difference for identifying the variety, and experience and labor are required.
On the other hand, in the identification method by the RFLP method, a great deal of labor is required for restriction enzyme treatment of DNA, Southern blot hybridization (hybridization with a labeled DNA probe) and the like. Genome D, especially in the case of mitochondrial DNA
Since it is present in a very small amount as compared with NA, a large amount of material was required to perform discrimination by visual detection using the RFLP method. Therefore, development of a simpler and more efficient identification method for plant varieties based on the difference in mitochondrial DNA that controls important traits in agriculture has been awaited.

[0004]

Means for Solving the Problems In view of the above situation, the present inventors have conducted extensive studies to find a better method for identifying plant varieties, and as a result, using a certain oligonucleotide as a primer, By amplifying mitochondrial DNA by polymerase chain reaction, a more simple and efficient method for discriminating plant varieties based on the difference in mitochondrial DNA was found, and the present invention was completed. That is, the present invention is to amplify a plant mitochondrial DNA by a polymerase chain reaction using an oligonucleotide having a GC content of 50% or more and 80% or less and consisting of 8 or more and 15 or less nucleotides, After the amplified mitochondrial DNA is separated by electrophoresis, the amplified mitochondrial DNA is visually detected, which is a method for identifying plant varieties (hereinafter referred to as the identification method of the present invention). By performing detection,
The present invention provides a method for selecting plants (hereinafter referred to as the selection method of the present invention), which comprises assaying the presence or absence of a trait derived from mitochondrial DNA and selecting plants having the trait.

The present invention is extremely useful in, for example, purity test of cultivars, check of mutant strains during storage, arbitration ruling and maternal judgment. That is, omitting the time and effort to test whether the target plant is a desired variety by culturing for a long time until it grows sufficiently, immediately after the seed of the target plant, seedlings or cultured cells, etc. It is possible to test whether or not it is the desired variety, which is rapid / simplified inspection in the purity inspection of the variety, preservation of the original seed species production and genetic seed resource in the check of mutant strains during storage Quick and simple inspection of condition, speedy submission of high-level evidence such as no change in cultivation environment in dispute arbitration when there is infringement of registered variety, and genetic resource search and pure line or F1 variety in maternal assessment The efficiency of breed improvement and cost reduction will be improved by fostering parent strains.

Mitochondrial DNA used in the present invention
For example, the method of LRDeBonte et al. (Plant Mol. Biol.
Rep. 3: 32-36 (1985)).
Specifically, for example, protoplasts derived from cultured cells of the test plant are disrupted in a buffer solution. The disrupted product was centrifuged to collect a mitochondrial fraction, which was
Contaminant genomic DNA is removed by treating with Nase. The purified mitochondrial fraction was treated with proteinase to digest the mitochondrial membrane and then extracted with phenol and chloroform to obtain the resulting mitochondrial DNA.
The RNA mixture is treated with RNase. A method of obtaining mitochondrial DNA by extracting again with phenol and chloroform can be mentioned.

The oligonucleotide used in the present invention is an oligonucleotide having a GC content of 50% or more and 80% or less and 8 or more and 15 or less nucleotides. For example, there may be mentioned an oligonucleotide having a sequence selected from the following sequence group. Sequence Group (1) 5'-TACGGCTAGA-3 '(hereinafter, SEQ ID NO: 1
Is written. ) (2) 5'-CAAACTGGTG-3 '(hereinafter SEQ ID NO: 2
Is written. ) (3) 5'-AGTCCTATGC-3 '(hereinafter, SEQ ID NO: 3
Is written. ) (4) 5'-TACGGCATGA-3 '(hereinafter SEQ ID NO: 4
Is written. ) (5) 5'-AGATCGGGCAT-3 '(hereinafter SEQ ID NO: 5
Is written. ) (6) 5'-GTGGTCAAAC-3 '(hereinafter, SEQ ID NO: 6)
Is written. ) (7) 5'-CGTATCCTGA-3 '(hereinafter, SEQ ID NO: 7)
Is written. ) (8) 5'-AGGCATTACG-3 '(hereinafter, SEQ ID NO: 8)
Is written. ) (9) 5'-AGTACGGCAT-3 '(hereinafter, SEQ ID NO: 9)
Is written. The oligonucleotide used in the present invention can be obtained by a commercially available automatic DNA synthesizer using, for example, the β-cyanoethylphosphoamidite method or the thiophosphite method.

In the present invention, plant mitochondrial DN
Amplification of A is carried out by a polymerase chain reaction using the oligonucleotide used in the present invention as a primer. The polymerase chain reaction is a denaturation step,
This is a reaction in which a DNA replication cycle consisting of a primer annealing step and a DNA polymerase extension step is repeatedly carried out, for example, Saiki et al., Science,
Volume 230, pp. 1350 to 1354 (1985) and Williams
Et al., Nucleic Acids Research, Vol. 18, No. 22, 6531.
From page to page 6535 (1991), etc., ordinary methods are described.

In the present invention, as the polymerase chain reaction, for example, an oligonucleotide, a DNA polymerase, four kinds of bases (dATP, dTTP, dCTP, dGTP) previously used in the present invention and plant mitochondrial DNA of about 1.0 mM are added. To about 4.0 mM, preferably about 1.5 mM to about 3.0 mM magnesium chloride, etc., in the amplification buffer, about 20 to about 50 times, preferably about 25 to about 40 times. You can name what you do. Furthermore, each step in the polymerase chain reaction can be performed, for example, under the following conditions. The denaturation step is, for example, usually about 90 ° C to about 95 ° C, preferably about 94 ° C to about 95 ° C.
It is carried out by heating at 0 ° C. for about 1 minute to about 3 minutes, preferably about 1 minute to about 2 minutes. The primer annealing step is typically performed at, for example, about 30 ° C to about 50 ° C.
At about 37 ° C. to about 47 ° C. for about 1 minute to about 3 minutes, preferably about 1 minute to about 2 minutes. As the primer, the oligonucleotides used in the present invention can be used alone or in combination. The extension step using a DNA polymerase is usually carried out, for example, from about 70 ° C. to about 73
Heat resistant DN at ℃, preferably about 72 ℃ to about 73 ℃, about 1 minute to about 4 minutes, preferably about 2 minutes to about 3 minutes
A polymerase treatment and the like. Heat resistance D
Examples of NA polymerase include commercially available products such as heat-resistant DNA polymerase manufactured by Takara Shuzo Co., Ltd.

The amplified mitochondrial DNA obtained by the above method is separated by the electrophoresis method usually used for separating DNA. Generally, about 3% to about 20% polyacrylamide gel is used for the separation of DNA fragments having a length of 1000 base pairs or less, and about 0.2% to about 2% agarose gel is used for the separation of DNA longer than that. I can give you. Preferably, about 1% to about 2% agarose gel is suitable. Buffers used for electrophoresis include Tris-phosphate system (pH 7.5-8.0), Tris-acetate system (pH
7.5-8.0), Tris-boric acid system (pH 7.5-8.3), etc.
Preferred is the Tris-acetic acid system. Moreover, EDTA etc. can also be added as needed. The conditions for electrophoresis include, for example, 100 V, 40 minutes and 50 V, 80 minutes. As the size marker, one obtained by completely hydrolyzing lambda DNA with a restriction enzyme Hind III, for example, a commercially available product manufactured by Takara Shuzo Co., Ltd. can be used.

In the present invention, amplified mitochondrial DNA
Examples of the visual detection of DNA include a method of detecting DNA by a staining method using a phenanthridine-based dye such as ethidium bromide and a substance that interacts with a nucleic acid. In the staining method, when a substance such as ethidium bromide at a final concentration of about 0.5 μg / ml is added to a buffer solution used for electrophoresis in advance, the gel is irradiated with ultraviolet rays at 254 nm or 366 nm in a dark place. By doing so, the red band of the conjugate of DNA and ethidium bromide can be detected even during electrophoresis. Usually, after completion of electrophoresis, soak the gel in a solution of a substance such as ethidium bromide for about 15 minutes to about 60 minutes. The red band of the conjugate of DNA and ethidium bromide is detected by irradiating the gel with ultraviolet light of 254 nm or 366 nm in the dark. As a result, it becomes possible to distinguish plant varieties based on the presence or absence of detected amplified mitochondrial DNA. Plant varieties can also be distinguished by combining presence or absence of amplified mitochondrial DNA of various sizes (molecular weights) separated by electrophoresis as necessary. Furthermore, various combinations of the oligonucleotides used in the present invention, which are primers, enable more detailed and accurate discrimination between varieties. In order to further improve the accuracy of identification or the ability to discriminate between varieties, change the conditions of the polymerase chain reaction such as the temperature of the primer annealing step and the magnesium concentration in the reaction buffer to determine whether the same behavior is exhibited. It is possible by investigating.

[0012]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (Plant mitochondrial DNA
As a carrot ( Daucus carota L. ) variety, trade name: Kokubun Benin Daicho (hereinafter referred to as variety number 1), trade name: Long fat gold jelly (hereinafter referred to as variety number 2), product Name: Impe
rater (hereinafter referred to as variety number 3), trade name: high agricultural fresh red three dimensions (hereafter referred to as variety number 4), trade name: Nantes
A scarlet (hereinafter referred to as variety number 5) and a trade name: Kikuyo Gosho (hereinafter referred to as variety number 6) were used. Sowing of each cultivar of the above carrot ( Daucus carota L. ) plant 7-1
Each day 0 seedling was sterilized with a 3% sodium hypochlorite aqueous solution and thoroughly washed with sterile water, and the hypocotyl portion was cut into a length of about 1 cm. This hypocotyl is 0.5mg / L 2,4
The MS medium containing -D was placed on a solid medium solidified with 0.8% (wt / v) agarose and cultured at a temperature of 25 ° C. Callus formed after about 1 month. This callus about 2
g was subdivided and suspended in 30 ml of MS medium containing 0.5 mg / L of 2,4-D, and cultured at 25 ° C. with shaking. When the cell density reached about 1 × 10 ⁷ cells / ml, the cells were subcultured into the fresh medium and further cultured. The suspension cells of each variety were obtained by centrifuging the cultured cells on the fifth day of passage. The resulting suspension cell (10 fresh weight)
g) 50 ml of 1.0% (wt / v) dolicerase,
0.5% (wt / v) cellulase, 0.01% (wt /
v) Pectolyase and 0.1% (wt / v) MES
[2- (N-Morpholino) ethane-sulfonic acid, monohy
float on 0.5M mannitol solution containing drate
Enzyme treatment was performed at 0 ° C. for 4 hours. Collect the released protoplasts by centrifugation (690Xg, 3 minutes) and collect 0.5
It was washed twice with M mannitol. The isolated protoplasts were treated with 40 ml of 100 mM Tris-hydrochloric acid, 1 mM E.
DTA, 1% (wt / v) BSA and 0.3% (wt
/ V) Suspended in 0.4M sorbitol solution containing 2-mercaptoethanol and incubated on ice for 20 minutes. The protoplast contained in the suspension was adjusted to 18 ga
The protoplast membrane was gently broken by poking 1-3 times with a uge needle. The disrupted protoplast membrane was centrifuged at 3000 × g for 10 minutes to precipitate nuclei and plastids.
The supernatant was taken and centrifuged at 6000 × g for 10 minutes to precipitate a mixed fraction of plastid and mitochondria. After collecting the supernatant and repeating the above treatment once, the supernatant was collected again and centrifuged at 15000 × g for 15 minutes to collect the precipitate (mitochondrial fraction). The resulting precipitate was gently washed with DNase, 10 mM MgCl ₂ and 0.3M.
50 mM Tris-hydrochloric acid buffer containing sucrose (pH
7.5) for 30 minutes at 4 ° C. To remove DNase, add 5 ml 0.02M mitochondrial solution.
10 mM containing EDTA and 0.6 M sucrose
Tris-hydrochloric acid buffer solution (pH 7.2) was overlayered, 15
Centrifuged at 000 × g for 10 minutes. The precipitate obtained is 1
00 μg Proteinase K, 1% (wt / v) N-lauryl
50m containing sarcosine and 20mM EDTA
M Tris-hydrochloric acid buffer solution (pH 8.0) at 37 ° C., 1
It was treated for a time and the mitochondrial membrane was digested. To this, an equal amount of phenol and chloroform-isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) were added and shake-mixed for 15 minutes. The aqueous layer obtained by centrifuging the mixture (1940 × g, 15 minutes) was collected, and equal amounts of phenol and chloroform-isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) were added thereto again, and the mixture was added for 15 minutes. Mixed by shaking. The aqueous layer obtained by centrifuging the mixture (1940 × g, 15 minutes) was collected, 2.5 volumes of ethanol was added,
Mix several times with shaking. After recovering the precipitate obtained by centrifugation (1940Xg, 15 minutes), 70% ethanol was added to the precipitate for washing and centrifugation (1940Xg, 15 minutes).
The precipitate obtained after 10 minutes) was dried under reduced pressure. 0.
4 ml of Tris-hydrochloric acid buffer solution (pH 7.5) containing 1 mM EDTA was added and suspended. After the precipitate was completely dissolved, 2.5 units of RNase (Boehringer Mannheim) was added, and the mixture was incubated at 37 ° C for 1 hour. Further, equal amounts of phenol and chloroform-isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) were added, and the mixture was shaken and mixed for 15 minutes. The aqueous layer obtained by centrifuging the mixture (12000 × g, 15 minutes) was recovered, and equal amounts of phenol and chloroform-isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) were added thereto again for 15 minutes. Mixed by shaking. The mixture was centrifuged (12000Xg, 1
The aqueous layer obtained after 5 minutes) was collected, 2.5 times the amount of ethanol was added, and the mixture was shaken and mixed several times. Centrifuge (194
After recovering the precipitate obtained by (0Xg, 15 minutes), 70% ethanol was added to the precipitate to wash the precipitate, which was then centrifuged (19
40 Xg, 15 minutes) the precipitate obtained by drying under reduced pressure to remove ethanol to remove carrot ( Daucus carota
L. ) About 5 to 1 mitochondrial DNA of various plant varieties
0 μg was obtained.

Example 2 (Method of identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 1) 80 pmol of SEQ ID NO: 1 was used as a primer in advance.
Oligonucleotide consisting of the sequence shown in (Takara Shuzo Co., Ltd. consignment compound), 2.5 units of thermostable DNA polymerase (Takara Shuzo Co., Ltd.) 1.0 nmol of each of 4 kinds of bases (dATP, dTTP, dCTP, dGTP) and 0.02 μ
g of various carrots obtained in Example 1 ( Daucus carot
a L. ) 0.01% (w containing mitochondrial DNA)
t / v) 10 mM Tris-hydrochloric acid buffer (p) containing gelatin, 50 mM potassium chloride, 2.5 mM magnesium chloride
Polymerase chain reaction was performed in H8.3). The reaction volume should be 20 μl and approximately 2 to prevent evaporation of the reaction volume.
0 μl mineral oil was added. Each step in the above polymerase chain reaction was performed under the following conditions. The denaturation step is heated at 94 ° C. for 5 minutes, the primer annealing step is incubated at 40 ° C. for 2 minutes with the primer, and the DNA polymerase extension step is performed for 7 minutes.
First, heat-resistant DNA polymerase treatment for 3 minutes at 2 ℃
After one cycle, the denaturing step was heated at 94 ° C for 1 minute and the primer annealing step was at 40 ° C.
Incubation with primers for 2 minutes, extension step with DNA polymerase at 72 ° C for 3 minutes, heat-resistant DNA polymerase treatment for 2nd cycle 40 times, and denaturation step at 94 ° C for 1 minute, primer annealing step Was incubated with the primers for 2 minutes at 41 ° C and the extension step with DNA polymerase was 72
Third, heat-resistant DNA polymerase treatment for 10 minutes at ℃
Do one cycle. Obtained amplified mitochondrial D
NA was 1 mM EDTA using 1.5% agarose gel.
40 mM Tris-20 mM acetate buffer (pH 8.
0) in 50 V for 80 minutes for separation. At that time, as a size marker, pHY manufactured by Takara Shuzo Co., Ltd.
A DNA marker was used. After separation, gel 0.5 μg /
The red band of the conjugate of DNA and ethidium bromide was detected by immersing the gel in ml of ethidium bromide aqueous solution for 30 minutes and then irradiating the gel with 254 nm ultraviolet light in the dark. The results are shown in Table 1. About 3100, about 2860, about
Amplified mitochondrial DNA of 2490, about 2260, about 2200, about 1660 bp was detected, and 6 varieties of carrot ( Daucus carota
L. ) plants could be distinguished into 3 types.

[0015]

[Table 1] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 3 (Method of identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 2) Instead of the oligonucleotide having the sequence represented by SEQ ID NO: 1, the oligo having the sequence represented by SEQ ID NO: 2 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 2. About 1080, about 860, about 57
0, about 330 bp amplified mitochondrial DNA was detected,
Six types of carrot ( Daucus carota L. ) plants could be distinguished into four types.

[0017]

[Table 2] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 4 (Method for identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 3) Instead of the oligonucleotide having the sequence represented by SEQ ID NO: 1, the oligo having the sequence represented by SEQ ID NO: 3 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 3. About 1540, About 1230, About 98
0, about 250 bp amplified mitochondrial DNA was detected,
Six types of carrot ( Daucus carota L. ) plants could be distinguished into three types.

[0019]

[Table 3] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 5 (Method of identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 4) Instead of the oligonucleotide having the sequence represented by SEQ ID NO: 1, the oligo having the sequence represented by SEQ ID NO: 4 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 4. About 2620, About 2160, About 197
0, about 1300, about 1050, about 580 bp amplified mitochondrial D
NA was detected and six varieties of carrot ( Daucus carota
L. ) plants could be distinguished into 6 types.

[0021]

[Table 4] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 6 (Method for identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 5) Instead of the oligonucleotide having the sequence represented by SEQ ID NO: 1, an oligo having the sequence represented by SEQ ID NO: 5 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 5. 4210, 3630, 310
0, about 2440, about 2160, about 1740, about 1290, about 1090, about 930b
Amplified mitochondrial DNA of p was detected, and 6 kinds of carrot ( Daucus carotaL. ) plants could be distinguished into 4 types.

[0023]

[Table 5] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 7 (Method for identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 6) Instead of the oligonucleotide having the sequence represented by SEQ ID NO: 1, an oligo having the sequence represented by SEQ ID NO: 6 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 6. About 550, about 450, about 37
0, about 290, about 210, about 200 bp amplified mitochondrial D
NA was detected and six varieties of carrot ( Daucus carota
L. ) plants could be distinguished into 6 types.

[0025]

[Table 6] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 8 (Method for identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 7) Instead of the oligonucleotide having the sequence represented by SEQ ID NO: 1, an oligo having the sequence represented by SEQ ID NO: 7 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 7. About 4320, About 3880, About 380
Amplified mitochondrial DNA of 0, about 3430, and about 350 bp was detected, and 6 kinds of carrot ( Daucus carota L. ) plants could be distinguished into 3 types.

[0027]

[Table 7] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 9 (Method for identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 8) Instead of the oligonucleotide having the sequence represented by SEQ ID NO: 1, an oligo having the sequence represented by SEQ ID NO: 8 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 8. About 6250, About 5060, About 480
Amplified mitochondrial DNA of 0, about 4440, about 2840, about 2100, about 230 bp was detected, and 6 varieties of carrot ( Daucus c
arota L. ) plants could be distinguished into 3 types.

[0029]

[Table 8] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 10 (Method for identifying a variety using an oligonucleotide consisting of the sequence represented by SEQ ID NO: 9) Instead of the oligonucleotide consisting of the sequence represented by SEQ ID NO: 1, the oligo consisting of the sequence represented by SEQ ID NO: 9 The same test as in Example 2 was performed except that nucleotides were used. The results are shown in Table 9. About 2840, about 2160, about 1380
The amplified mitochondrial DNA of bp was detected, and 6 carrot ( Daucus carota L. ) plants could be discriminated into 2 types.

[0031]

[Table 9] * +: Amplified mitochondrial DNA was detected. -: Amplified mitochondrial DNA could not be detected.

Example 11 (Method of identifying varieties using various oligonucleotides) Various carrots of variety numbers 1 to 5 ( Daucus carota)
L. ) Mitochondrial DNA was extracted from seeds of varieties according to Example 1, and amplified mitochondrial DNA according to the above Examples using oligonucleotides having the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 5 separately. To detect. Based on the detected amplified mitochondrial DNA, the variety type in various oligonucleotides is distinguished, and further the variety is identified from the variety type. Results, Table 1
As shown in 0, all five types can be identified.

[0033]

[Table 10]

Example 12 (Purity test of variety) Carrot of variety number 4 cultivated for seed production ( Da
ucus carota L. ) Obtain seeds from plants. 50 seeds are sown one by one in a pot filled with seedling cultivation soil and cultivated in a greenhouse. Each mitochondrial DNA is extracted from 20 healthy seedlings according to Example 1, and the amplified mitochondrial DNA is detected according to Example 2 using the oligonucleotide having the sequence represented by SEQ ID NO: 3. . The difference between the detected amplified mitochondrial DNA and the amplified mitochondrial DNA (about 1230 bp, about 250 bp) originally possessed by the carrot ( Daucus carota L. ) variety of variety number 4 is examined. As a result, carrot ( Daucus c
arota L. ) Amplified mitochondrial DN originally owned by the variety
The test seed in which amplified mitochondrial DNA different from A (about 1230 bp, about 250 bp) was detected is an impurity, and the purity can be calculated from this number. The accuracy of the purity test can be improved by using a plurality of oligonucleotides used in the present invention together.

Example 13 (Check of mutant strain during storage) Each mitochondrial DNA was obtained from 50 seeds of carrot ( Daucus carota L. ) variety of variety No. 5 which was preserved as a genetic seed resource, according to Example 1. The amplified mitochondrial DNA is detected according to Example 2 by extracting and using the oligonucleotide having the sequence represented by SEQ ID NO: 6. The amplified mitochondrial DNA detected and the amplified mitochondrial DNA originally possessed by the carrot ( Daucus carota L. ) variety of variety number 5 (about 450 bp, about 370 bp, about 2
90bp, about 210bp). As a result, product number 5
Amplified mitochondrial DNA (about 450bp, about 370bp, about 290) originally owned by the carrot ( Daucus carota L. ) variety of
The tested seeds in which amplified mitochondrial DNA different from bp, about 210 bp) were detected are mutant strains, and the mutation rate can be calculated from this number. The accuracy of the mutation rate can be improved by using a plurality of oligonucleotides used in the present invention together.

Example 14 (Use in Dispute Arbitration) 20 seeds of a variety are sown one by one in a pot filled with seedling cultivation soil and cultivated in a greenhouse. 10 healthy seedlings
Mitochondrial DNA was extracted from an individual according to Example 1, and the oligonucleotides having the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9 were separately used, and Examples 2 to 10 were used.
Amplified mitochondrial DNA is detected according to. next,
Amplified mitochondrial DNA detected based on the result of determination of breed type or according to Example 11.
Based on the above, the product type of each oligonucleotide is distinguished, and the identification type of the product is determined from the product type. 10 seeds of No. 1 variety are sown one by one in a pot filled with seedling cultivation soil and cultivated in a greenhouse. Example 1 Mitochondrial DNA from 5 healthy growing seedlings
The amplified mitochondrial DNA is detected according to Examples 2 to 9 by separately using the oligonucleotides having the sequences shown in SEQ ID NOs: 1 to 9 separately. Then decide the breed type based on this result,
Alternatively, the breed type of each oligonucleotide is discriminated based on the amplified mitochondrial DNA detected according to Example 11, and the discrimination type of the breed is examined from the breed type. As a result, by comparing the variety type or identification type of the variety and the variety type or identification type of the No. variety, quick and accurate evidence that the species is almost the same can be obtained accurately and without change due to the environment, etc. You can
It is possible to further improve the identification accuracy by changing the conditions of the polymerase chain reaction and examining whether or not the same behavior is exhibited.

Example 15 (Use in Mother Identification ) Seed of a plant obtained by using a carrot ( Daucus carota L. ) variety of variety number 1 as a mother and a carrot ( Daucus carota L. ) variety of variety number 4 as a father. Each mitochondrial DNA is extracted from 50 grains according to Example 1, and the amplified mitochondrial DNA is detected according to Example 2 using the oligonucleotide having the sequence represented by SEQ ID NO: 3. The amplified mitochondrial DNA to be detected and the carrot ( Daucus carota L. ) variety of variety number 1 are originally owned, but the carrot ( Daucuscarota L. ) variety of variety number 4 is not originally owned. Amplified mitochondrial DNA (about 1540 bp)
Check the difference with. As a result, it was investigated that the test plant possessing an amplified mitochondrial DNA of approximately 1540 bp had a mother of carrot ( Daucus carota L. ) of breed number 1 and had a mitochondrial DNA-derived trait similar to that of the mother. It can be cultivated until it is possible and can be quickly determined without investigating the characteristics of the variety.

Example 16 (Cluster analysis of mitochondrial DNA) Various carrots of varieties number 1 to 6 ( Daucus) were subjected to polymerase chain reaction using various oligonucleotides having the sequences shown in SEQ ID NOS: 1 to 9 as primers. The mitochondrial DNA of the carota L. ) variety was amplified, the amplified mitochondrial DNA was separated by electrophoresis, and the amplified mitochondrial DNA was visually detected to classify the mitochondrial DNA. Mitochondrial DNA is classified by the WPGMA method (weighted pair-
group method using arithmetic averages, Peter HAS
Cluster analysis was performed based on neath et al. NUMERICAL TAXONOMY (1973). As a result, as shown in Table 11, 6 types of mitochondrial DNA could be classified. In this way, it is possible to test the presence or absence of a trait derived from mitochondrial DNA and select plants having this trait.

[0039]

[Table 11]

[0040]

INDUSTRIAL APPLICABILITY The present invention makes it possible to more easily and efficiently identify plant varieties based on differences in mitochondrial DNA by amplifying mitochondrial DNA by polymerase chain reaction using certain oligonucleotides as primers. Has the effect that can be done well.

[Sequence list]

SEQ ID NO: 1 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TACGGCTAGA 10

SEQ ID NO: 2 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CAAACTGGTG 10

SEQ ID NO: 3 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AGTCCTATGC 10

SEQ ID NO: 4 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TACGGCATGA 10

SEQ ID NO: 5 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AGATCGGCAT 10

SEQ ID NO: 6 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GTGGTCAAAC 10

SEQ ID NO: 7 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CGTATCCTGA 10

SEQ ID NO: 8 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AGGCATTACG 10

SEQ ID NO: 9 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AGTACGGCAT 10

Claims (5)

[Claims] 1. A plant mitochondrial DNA is amplified by a polymerase chain reaction using an oligonucleotide having a GC content of 50% or more and 80% or less and consisting of 8 or more and 15 or less nucleotides, and the amplification is performed. A method for identifying plant varieties, which comprises performing visual detection of amplified mitochondrial DNA after separating mitochondrial DNA by electrophoresis. 2. The method for identifying plant varieties according to claim 1, wherein the oligonucleotide according to claim 1 is an oligonucleotide having a sequence selected from the following sequence group. Sequence group (1) 5'-TACGGCTAGA-3 ', (2) 5'-CAAA
CTGGTG-3 ', (3) 5'-AGTCCTATGC-
3 ', (4) 5'-TACGGCATGA-3', (5) 5'-AG
ATCGGGCAT-3 ', (6) 5'-GTGGTCAAAC
-3 ', (7) 5'-CGTATCCTGA-3', (8) 5'-A
GGCATTACG-3 ', (9) 5'-AGTACGGCA
T-3 ' 3. A plant chain mitochondrial DNA is amplified by a polymerase chain reaction using an oligonucleotide having a GC content of 50% or more and 80% or less and consisting of 8 or more and 15 or less nucleotides, and the amplification is performed. After the mitochondrial DNA is separated by electrophoresis, the presence or absence of a trait derived from mitochondrial DNA is assayed by visually detecting the amplified mitochondrial DNA, and a plant having the trait is selected. 4. The mitochondrial DN of the plant according to claim 3.
A is mitochondrial DNA derived from seedlings or cultured cells
The method for selecting plants according to claim 3, wherein 5. An oligonucleotide comprising a sequence selected from the following sequence group. Sequence group (1) 5'-AGTCCTAGTC-3 ', (2) 5'-TACG
GCATGA-3 ', (3) 5'-CGTATCCTGA-
3 ', (4) 5'-AGGCATTACG-3'