JPH06113850A - Method for distinguishing variety of rice plant using oligonucleotide and its oligonucleotide - Google Patents

Abstract

PURPOSE:To simply distinguish varieties of rice plant by amplifying genome DNA by using a specific oligonucleotide as a primer and subjecting the oligonucleotide to polymerase chain reaction. CONSTITUTION:An oligonucleotide having a sequence 5'-AGTCCTAAGC-3', 5'-TGGACACTAC-3' or 5'-CTGAACTGAC-3' is used as a primer and subjected to polymerase chain reaction to amplify genome DNA of vegetable of rice plant (Oryza sativa L.). The amplified genome DNA is separated by electrophoresis and the amplified genome DNA is visually detected.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for identifying rice ( Oryza sativa L.) cultivars using oligonucleotides and the oligonucleotides thereof.

[0002]

2. Description of the Related Art Conventionally, rice ( Oryza sativa L.) varieties have been identified by methods such as plant height, shape and size of spikes, shape and size of paddy, shape and size of brown rice, shape of leaves, etc. Method by difference in color, method by difference in color of ears, paddy, brown rice, leaves, etc., heading stage, cold resistance, resistance to pests, method by difference in physiological characteristics such as reaction to photoperiod, isozyme, storage protein, etc. Methods based on differences in protein levels are known. On the other hand, recently, the RFLP method (Restrictio
Rice ( Oryza by n Fragment Length Polymorphism)
sativa L.) Varieties are identified by, for example, ZYWang a
nd S.D.Tanksley, GENOME, Volume 32, pages 1113 to 1118
Page (1989).

[0003]

However, the form,
With the method based on the difference in color and physiological characteristics, there was no choice but to cultivate until the above-mentioned traits could be actually investigated and 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 protein level, 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 genomic DNA, Southern blot hybridization (hybridization with a labeled DNA probe) and the like. Therefore,
The development of a simpler and more efficient identification method for rice ( Oryza sativa L.) varieties has been awaited.

[0004]

[Means for Solving the Problems] In view of the above situation, the present inventors have conducted diligent studies to find a better method for identifying rice ( Oryza sativa L.) cultivars, and as a result, The present invention has been completed by finding a simpler and more efficient method for identifying rice ( Oryza sativa L.) cultivars by utilizing the polymerase chain reaction using the following oligonucleotide as a primer.
That is, the present invention, using an oligonucleotide consisting of a sequence selected from the following sequence group as a primer,
The polymerase chain reaction allows rice ( Oryza sativa)
L.) Amplification of plant genomic DNA to obtain the amplified genomic DN
A method for identifying rice ( Oryza sativa L.) cultivars, characterized in that A is separated by electrophoresis and then the amplified genomic DNA is visually detected (hereinafter referred to as the method of the present invention) Sequence group (1) 5 ' -AGTCCTAAGC-3 '(hereinafter, SEQ ID NO: 1
Is written. ), (2) 5'-TGGACACTAC-3 '(hereinafter, SEQ ID NO: 2)
Is written. ), (3) 5'-CTGAACTGAC-3 '(hereinafter SEQ ID NO: 3
Is written. ), (4) 5'-GTACAGACCT-3 '(hereinafter SEQ ID NO: 4
Is written. ), (5) 5'-ACGACGCTTA-3 '(hereinafter SEQ ID NO: 5
Is written. ), (6) 5'-CAGTCAAGTC-3 '(hereinafter, SEQ ID NO: 6)
Is written. ), (7) 5'-TCCAGACATG-3 '(hereinafter, SEQ ID NO: 7)
Is written. ), (8) 5'-ATTCGCAGCA-3 '(hereinafter, SEQ ID NO: 8)
Is written. ), And its oligonucleotides.

The method of the present invention is extremely useful in, for example, purity test of cultivars, check of mutant strains during storage, dispute arbitrage and paternity test (including F1 plant test). That is, omitting the time and effort to test whether or not the target plant is a desired variety by culturing for a long period of time until it grows sufficiently, the seed of the target plant, the seedlings of the target plant, etc. It is possible to test whether or not it is a variety, which is quick and simple in the purity test of the variety, inspection of the original seed production / preservation state of genetic seed resources in the check of mutant strains during storage Quick and simple,
Accelerate the submission of high-level evidence such as the fact that there is no change in the cultivation environment in the dispute arbitration when there is a violation of the registered variety rights, and improve the efficiency of variety improvement in parentage examination (including F1 plant test), F1 seed production Cost reduction of quality control.

In the method of the present invention, rice ( Oryza sativa
L.) Genomic DNA of plants is, for example, the basis of the latest agricultural experiments, edited by Tohoku University Faculty of Agriculture, 1990, published by Soft Science Company, Tokyo, Japan
Nucleic Acid Separation / Analysis Method Co-edited by Meiji Izumi et al. 1986 Kyoto
It can be carried out by a usual DNA extraction method described in Kagaku Doujin Co., Ltd. Specifically, for example, tissues such as green leaves of a test plant are ground in liquid nitrogen (when seeds are used, it is desirable to use tissues after removing seed coat, for example). A cetyltrimethylammonium bromide (CTAB) solution is added to the ground product, and after incubation, chloroform-isoamyl alcohol is added and mixed well. The aqueous layer is separated and collected by centrifugation, and isopropyl alcohol is added to this and mixed. After recovering the precipitated portion by centrifugation, for example, a buffer solution containing EDTA or the like is added and dissolved, and then treated with RNase. After the treatment, the solvent is replaced in the order of phenol, phenol and chloroform-isoamyl alcohol, and chloroform-isoamyl alcohol. After the substitution treatment, ethanol can be added and mixed well, and a method of obtaining genomic DNA by centrifugation can be mentioned.

The oligonucleotide used in the method of 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, rice ( Oryza sativa L.
) Amplification of plant genomic DNA is carried out by polymerase chain reaction using the oligonucleotides used in the method of the present invention as primers. The polymerase chain reaction consists of a denaturation step, a primer annealing step, and a DNA polymerase extension step D
A reaction in which the NA replication cycle is repeated, for example, Saiki et al., Science, Vol. 230, p. 1350 to 135.
Page 4 (1985) and Williams et al., Nucleic Acids Researc
h, Vol. 18, No. 22, pp. 6531 to 6535 (1991)
Etc., the usual method is described.

In the present invention, the above-mentioned polymerase chain reaction may be carried out, for example, in advance by the oligonucleotide of the present invention, a DNA polymerase, and four types of bases (dATP, dT).
TP, dCTP, dGTP) and rice ( Oryza sativa L.) plant genomic DNA in a growth buffer containing about 1.0 mM to about 4.0 mM, preferably about 1.5 mM to about 3.0 mM magnesium chloride. Then, the DNA replication cycle can be repeated about 20 to about 50 times, preferably about 25 to about 40 times. 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 for about 1 minute to about 3 minutes, preferably about 1 minute.
It is performed by heating for 1 minute to about 2 minutes. The step of annealing the primer is, for example, usually about 30 ° C to about 60 ° C, preferably about 37 ° C to about 47 ° C, about 1 ° C.
It is carried out by incubating with the primer for about 1 minute to about 3 minutes, preferably for about 1 minute to about 2 minutes. As the primer, the oligonucleotide of the present invention can be used alone or in combination. The extension step with a DNA polymerase is, for example, usually about 70 ° C. to about 73 ° C., preferably about 72 ° C. to about 73 ° C., for about 1 minute to about 4 minutes, preferably for about 2 minutes to about 3 minutes. It is performed by processing. Examples of thermostable DNA polymerases include commercially available thermostable DNA polymerases manufactured by Takara Shuzo Co., Ltd.

Amplified genomic DNA obtained by the above method
Are separated by an electrophoretic method usually used for separating DNA. Generally, short DNA of 1000 base pairs or less
About 3% to about 20% of the polyacrylamide gel was used for fragment separation, and about 3% to about 20% for longer DNA separation.
0.2% to about 2% agarose gel can be mentioned. Preferably, about 1% to about 2% agarose gel is suitable. The buffer used for electrophoresis is Tr
is-phosphoric acid system (pH 7.5-8.0), Tris-acetic acid system (pH 7.5-8.
0), Tris-boric acid system (pH 7.5-8.3), and the like, and preferably 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 5
0V, 80 minutes and the like can be raised. As the size marker, one obtained by completely hydrolyzing lambda DNA with a restriction enzyme Hind III, for example, a commercially available product from Takara Shuzo Co., Ltd. can be used.

In the method of the present invention, for the visual detection of amplified genomic DNA, for example, a method of detecting DNA by a staining method using a phenanthridine dye such as ethidium bromide and a substance capable of interacting with nucleic acid is used. Can be raised. The staining method is carried out by using a buffer solution used for electrophoresis in advance, for example, as a final concentration.
By adding 0.5 μg / ml ethidium bromide and other substances, the red band of the DNA-ethidium bromide conjugate can be detected during electrophoresis by irradiating the gel with ultraviolet rays of 254 nm or 366 nm in the dark. But,
Usually, after the electrophoresis is completed, the gel is immersed in a solution of a substance such as ethidium bromide for about 15 to about 60 minutes, and then the gel is irradiated with ultraviolet rays of 254 nm or 366 nm in the dark to bind the DNA to ethidium bromide. Detect the red band on the body. As a result, rice ( Oryza sativa L.) was detected depending on the presence or absence of detected amplified genomic DNA.
It becomes possible to distinguish the variety. Rice ( Oryza sativa L.) cultivars can also be identified by combining presence or absence of amplified genomic DNA of various sizes (molecular weights) separated by electrophoresis as necessary.
Furthermore, if the oligonucleotides used in the present invention are used as a primer in various combinations, more detailed and accurate discrimination between varieties can be achieved. In order to further improve the accuracy of identification or the ability to discriminate between varieties, whether the same behavior can be obtained by changing the conditions of the polymerase chain reaction such as the temperature of the primer annealing step and the magnesium concentration in the reaction buffer. It is possible by investigating. It is expected that the oligonucleotide used in the method of the present invention can be applied to plants other than rice ( Oryza sativa L.).

[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 (Extraction of Genomic DNA) As rice ( Oryza sativa L.) varieties, Golden Hare (Japonica, hereinafter referred to as variety number 1) and Akenohoshi (Japonica, hereinafter referred to as variety number 2). , Koshihikari (Japonica, hereafter referred to as breed number 3), Nipponbare (Japonica, hereafter referred to as breed number 4), Kanto 13
No. 8 (Japonica, hereafter, breed number 5), Ochikara (Japonica, hereafter, breed number 6), Toyohatamochi (Japonica, hereafter, breed number 7), S201 (Japonica, hereafter, breed) Number 8), Calrose 76 (Japonica, hereafter referred to as breed number 9), Tenma (Japonica, hereafter referred to as breed number 10), Kaharu No. 3 (Japonica, hereafter referred to as breed number 11) , No. 21 (Japonica, hereafter, breed number 1)
Write 2. ), Taichung No. 65 (Japonica, hereinafter referred to as variety number 13), Bahia (Japonica, hereinafter referred to as category number 14), Lido (Japonica, hereinafter referred to as variety number 1)
Write as 5. ), IRAT 13 (Javonica, hereafter breed number 16), Dubovsky 129 (Indica, hereafter breed number 17), Toyonishiki (Japonica, hereafter breed number 18), Lokt-jan ( Javanica, hereinafter referred to as variety number 19,) IR36 (Indica, hereinafter referred to as variety number 20), Aoni dwarf (Indica, hereinafter referred to as variety number 21), Koyo dwarf (Indica, hereinafter, Variety number 22), Miryang No. 23 (Indica, hereinafter referred to as variety number 23), Krishna (Indica, hereinafter referred to as variety number 24), Mahsuri (Indica, hereinafter referred to as variety number 25) ), RD3 (Indica, hereinafter referred to as breed number 26), Taichung conventional 1
No. (Indica, hereinafter referred to as breed number 27), Chin
surah Boro II (Indica, hereafter, breed number 28), BG367-4 (Indica, hereafter, breed number 29), L201 (Javanica, hereafter, breed number 30)
Is written. ), Kartuna (Javanica, hereafter, breed number 3)
Write 1. ), Mudgo (Indica, below, breed number 32
Is written. ), Saji (Javanica, hereafter, breed number 33)
Is written. ) And Musashi kogane (Japonica, hereinafter referred to as variety number 34). Rice above ( Oryza sati
va L.) Green leaf tissues of various varieties of plants (3 g fresh weight) were finely chopped, and the tissue pieces were immersed in about 100 ml of liquid nitrogen for 1 minute and frozen. The frozen product was powdered in liquid nitrogen using a mortar and pestle, and then liquid nitrogen was vaporized. The obtained powder was added to 10 ml of a 2% (w / v) CTAB solution which had been kept at 60 ° C. in advance and mixed well. After incubating at 60 ° C. for 30 minutes, an equal amount of chloroform-isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) was further added and shake-mixed for 15 minutes. The mixture was centrifuged (1940 g,
15 minutes) and the water layer obtained is recovered and added to 0.7
Double the amount of isopropyl alcohol was added and mixed by shaking. The precipitate obtained by centrifuging the mixture (1940 g, 15 minutes) was collected. After air-drying for 5 minutes, 1 ml of 20 mM 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 45 minutes. Further, 1 ml of phenol was added and shaken for 10 minutes. And centrifugation (1940g, 10 minutes)
After collecting the aqueous layer obtained by, 0.5 ml of phenol and 1 ml of chloroform-isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) were collected.
Was added and shaken for 10 minutes. This is centrifuged (194
The water layer obtained by
l of chloroform-isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) was added, and the mixture was shaken for 10 minutes. Centrifuge this (1940g, 5 minutes)
The aqueous layer thus obtained was recovered, and then 0.1 ml of 3M sodium acetate and 2.5 ml of ethanol were added, and the mixture was shaken and mixed several times. After recovering the precipitate obtained by centrifugation (1940 g, 10 minutes), 1 ml of 70% was added to the precipitate.
Ethanol of was added and washed. Ethanol was removed by air-drying for 60 minutes to obtain about 100 μg of genomic DNA of various rice ( Oryza sativa L.) plant varieties.

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.
An oligonucleotide used in the method of the present invention consisting of the sequence shown by (commercial compound manufactured by Takara Shuzo Co., Ltd.), 2.5 units of thermostable DNA polymerase (Takara Shuzo Co., Ltd.) 1.0 nmole of each of four bases (dATP, dTTP, dCT
P, dGTP) and 0.1 μg of the various rice ( Oryza sativa L.) varieties obtained in Example 1 plus 0.01% keratin, 50 mM potassium chloride, 10 mM containing 1.5 mM magnesium chloride. Tris-HCl buffer (p
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 37 ° 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 37 ° C.
Incubate with the primer for 2 minutes, extend with DNA polymerase at 72 ° C for 3 minutes, heat-treat with DNA polymerase for 3 minutes, and perform the second cycle 40 times. Further, heat at 94 ° C for 1 minute to anneal the primer. Was incubated with the primers for 2 minutes at 37 ° C and the extension step with DNA polymerase was 72
Third, heat-resistant DNA polymerase treatment for 10 minutes at ℃
Do one cycle. The amplified genomic DNA obtained is
Separation was performed by electrophoresis on a 1.5% agarose gel in 40 mM Tris-20 mM acetate buffer (pH 8.0) containing 1 mM EDTA at 50 V for 80 minutes. At that time, as a size marker, pHY D manufactured by Takara Shuzo Co., Ltd.
NA marker was used. After separation, gel at 0.5 μg / ml
By immersing it in an aqueous solution of ethidium bromide for 30 minutes and then irradiating the gel with 254 nm ultraviolet light in the dark.
The red band of the conjugate of DNA and ethidium bromide was detected. The results are shown in Tables 1 to 3. Amplified genomic DNA of about 2070 and about 780 bp was detected, and 33 varieties of rice ( Oryza
sativa L.) plants could be distinguished into 4 types.

[0015]

[Table 1]

[0016]

[Table 2]

[0017]

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

Example 3 (Method of identifying a variety using an oligonucleotide consisting of the sequence shown in SEQ ID NO: 2) Instead of the oligonucleotide used in the method of the present invention consisting of the sequence shown in SEQ ID NO: 1 The same test as in Example 2 was carried out except that an oligonucleotide having the sequence shown was used. The results are shown in Table 4 ~
6 shows. Amplified genomic DNA of about 1030 and about 1250 bp was detected, and 33 rice varieties ( Oryza sativa L.)
Could be identified by type.

[0019]

[Table 4]

[0020]

[Table 5]

[0021]

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

Example 4 (Method of identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 3) Instead of the oligonucleotide used in the method of the present invention having the sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 is used. The same test as in Example 2 was carried out except that an oligonucleotide having the sequence shown was used and that the primer annealing step in the polymerase chain reaction was carried out at 42 ° C instead of 37 ° C. The results are shown in Tables 7 to 9. Amplified genomic DNA of about 1030 bp was detected, and 33 types of rice ( Oryza sativa L.) plants could be distinguished into two types.

[0023]

[Table 7]

[0024]

[Table 8]

[0025]

[Table 9] * +: Amplified genomic DNA was detected. -: Amplified genomic 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 used in the method of the present invention having the sequence represented by SEQ ID NO: 1, SEQ ID NO: 4 is used. The same test as in Example 2 was carried out except that an oligonucleotide having the sequence shown was used and that the primer annealing step in the polymerase chain reaction was carried out at 42 ° C instead of 37 ° C. The results are shown in Tables 10-12. Amplified genomic DNAs of about 1120 and about 710 bp were detected, and 33 types of rice ( Oryza sativa L.) plants could be distinguished into 4 types.

[0027]

[Table 10]

[0028]

[Table 11]

[0029]

[Table 12] * +: Amplified genomic DNA was detected. -: Amplified genomic DNA could not be detected.

Example 6 (Method for identifying a variety using an oligonucleotide having the sequence shown in SEQ ID NO: 5) Instead of the oligonucleotide used in the method of the present invention having the sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 Using an oligonucleotide consisting of the sequence shown, and the polymerase chain reaction contains 0.01% gelatin, 50 mM potassium chloride, 1.5 mM magnesium chloride.
Instead of 0 mM Tris-HCl buffer (pH 8.3), 10 mM Tris-HCl buffer (pH 8.3) containing 0.01% gelatin, 50 mM potassium chloride and 2.5 mM magnesium chloride was used. The same test as in Example 2 was performed except that the primer annealing step in the chain reaction was performed at 47 ° C instead of 37 ° C. The results are shown in Tables 13 to 15. About 1000, about 750, about 620bp
Amplified genomic DNA of 33 rice varieties ( Oryz
a sativa L.) plants could be distinguished into 5 types.

[0031]

[Table 13]

[0032]

[Table 14]

[0033]

[Table 15] * +: Amplified genomic DNA was detected. -: Amplified genomic DNA could not be detected.

Example 7 (Method of identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 6) Instead of the oligonucleotide used in the method of the present invention having the sequence represented by SEQ ID NO: 1, SEQ ID NO: 6 is used. Using an oligonucleotide consisting of the sequence shown, and the polymerase chain reaction contains 0.01% gelatin, 50 mM potassium chloride, 1.5 mM magnesium chloride.
Carried out in 10 mM Tris-HCl buffer (pH 8.3) containing 0.01% gelatin, 50 mM potassium chloride, 2.5 mM magnesium chloride instead of 0 mM Tris-HCl buffer (pH 8.3). The same test as in Example 2 was conducted. The results are shown in Tables 16-18. About 700, about 510, about 480, about
Amplified genomic DNA of 400 bp was detected, and 33 types of rice ( Oryza sativa L.) plants could be distinguished into 6 types.

[0035]

[Table 16]

[0036]

[Table 17]

[0037]

[Table 18] * +: Amplified genomic DNA was detected. -: Amplified genomic 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 used in the method of the present invention, SEQ ID NO: 7 is used. Using an oligonucleotide consisting of the sequence shown, and the polymerase chain reaction contains 0.01% gelatin, 50 mM potassium chloride, 1.5 mM magnesium chloride.
Instead of 0 mM Tris-hydrochloric acid buffer (pH 8.3), it was carried out in 10 mM Tris-hydrochloric acid buffer (pH 8.3) containing 0.01% gelatin, 50 mM potassium chloride and 3.0 mM magnesium chloride. The results are shown in Tables 19 to 21. About 1180, about 5
Amplified genomic DNA of 80 bp was detected, and 33 types of rice ( Oryza sativa L.) plants could be distinguished into 3 types.

[0039]

[Table 19]

[0040]

[Table 20]

[0041]

[Table 21] * +: Amplified genomic DNA was detected. -: Amplified genomic DNA could not be detected.

Example 9 (Method of identifying a variety using an oligonucleotide having the sequence represented by SEQ ID NO: 8) Instead of the oligonucleotide used in the method of the present invention having the sequence represented by SEQ ID NO: 1, SEQ ID NO: 8 is used. Using an oligonucleotide consisting of the sequence shown, and the polymerase chain reaction contains 0.01% gelatin, 50 mM potassium chloride, 1.5 mM magnesium chloride.
Carried out in 10 mM Tris-HCl buffer (pH 8.3) containing 0.01% gelatin, 50 mM potassium chloride, 3.0 mM magnesium chloride instead of 0 mM Tris-HCl buffer (pH 8.3). The same test as in Example 2 was conducted. The results are shown in Tables 22-24. Approximately 1640 and approximately 390 bp of amplified genomic DNA were detected, and 33 varieties of rice ( Oryza sativa
L.) plants could be distinguished into 3 types.

[0043]

[Table 22]

[0044]

[Table 23]

[0045]

[Table 24] * +: Amplified genomic DNA was detected. -: Amplified genomic DNA could not be detected.

Example 10 (Method of Identifying Varieties by Changing Conditions of Polymerase Chain Reaction) Rices of variety No. 20 and variety No. 34 ( Oryza sati)
genomic DNA was extracted from the green leaf tissue of the va L.) variety according to Example 1, and the amplified genomic DNA was detected according to Example 9 using the oligonucleotide of the present invention having the sequence shown in SEQ ID NO: 8. . At this time, the temperature conditions of the primer annealing step in the polymerase chain reaction were 37 ° C and 47 ° C. The results are shown in Table 25.

[0047]

[Table 25]

Under the temperature condition of 37 ° C., about 500 bp of amplified genomic DNA was not detected in both test cultivars. on the other hand,
The temperature condition of 47 ° C., but the amplified genomic DNA is detected in rice (Oryza sativa L.) cultivar varieties number 20 was not detected in rice (Oryza sativa L.) cultivar varieties number 34. The varieties could be identified by this difference.

Example 11 (Method of identifying varieties using various oligonucleotides) Variety numbers 1, 2 (or 3, 4, 13), 5, 6, 7
(Or 33), 8, 9 (or 14), 10 (or 11, 18), 12, 15 (or 30), 16, 1
7, 19, 20, 21, 22, 23, 24, 25, 2
Genomic DNA was extracted from the seeds of various rice ( Oryza sativa L.) varieties of 6, 27, 28, 29, 31 and 32 in accordance with Example 1 to obtain SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 4, sequence. Amplified genomic DN was prepared according to the above-mentioned Examples by separately using the oligonucleotides consisting of the sequences of SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8.
Detect A. Based on the detected amplified genomic DNA, distinguish the variety type in various oligonucleotides,
Further, various kinds are identified from the kind type. As a result, all 25 types of products can be identified as shown in Tables 26 to 28.

[0050]

[Table 26]

[0051]

[Table 27]

[0052]

[Table 28]

Example 12 (Method of identifying varieties using various oligonucleotides) Varieties Nos. 1 and 2 (or 3, 4 and 13), 5, 6 and 7
(Or 33), 8, 9 (or 14), 10 (or 11, 18), 12, 15 (or 19, 30), 1
6, 17, 20, 21 (or 28), 22, 23, 2
4, 25 (or 26, 32), 27, 29 and 31
Genomic DNA was extracted from the seeds of various rice ( Oryza sativa L.) varieties according to Example 1 to obtain oligonucleotides consisting of the sequences shown in SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 4 and SEQ ID NO: 2. Separately used, amplified genomic DNA is detected according to the previous examples. Variety types in various oligonucleotides are distinguished based on the detected amplified genomic DNA, and various varieties are further distinguished from the variety types. As a result, all 20 types of products can be identified as shown in Tables 29 to 31.

[0054]

[Table 29]

[0055]

[Table 30]

[0056]

[Table 31]

Example 13 (Purity test of variety) Rice of variety number 24 ( Ory cultivated for seed production
za sativa 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 genomic DNA is extracted from 20 healthy growing young plants according to Example 1, and the amplified genomic DNA is detected according to Example 7 using the oligonucleotide having the sequence shown in SEQ ID NO: 6. . Amplified genomic DNA detected and rice of variety number 24 ( Oryza sativa
L.) Amplified genomic DNA originally owned by the cultivar (about 700b
p, about 510bp, about 480bp). As a result, the amplified genomic DNA originally possessed by the rice ( Oryza sativa L.) variety of variety number 6 (about 700 bp, about 510 bp, about 480 b
The test seed in which amplified genomic DNA different from p) 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 in combination.

Example 14 (Checking Mutant Strain during Storage) Rice (variety: O of cultivar No. 12 stored as a genetic seed resource)
ryza sativa L.) varieties of seeds were each extracted with genomic DNA in accordance with Example 1 and the oligonucleotide of SEQ ID NO: 6 was used in Example 7.
The amplified genomic DNA is detected according to Amplified genomic DNA detected and rice of variety number 12 ( Oryza sativa
L.) Amplified genomic DNA originally owned by the cultivar (about 700b
p, about 480 bp). As a result, product number 12
Rice ( Oryza sativa L.) cultivar, which has amplified genomic DNA (700 bp, 480 bp) different from that originally possessed, is a mutant seed, and the mutation rate should be calculated from this number. You can The accuracy of mutation rate can be improved by using a plurality of oligonucleotides in combination.

Example 15 (Use in Dispute Arbitration) 20 seeds of the registered variety are sown one by one in a pot filled with seedling cultivation soil and cultivated in a greenhouse. Genomic DNA was extracted from 10 healthy grown young plants according to Example 1,
The amplified genomic DNAs are detected according to Examples 2 to 9 by separately using the oligonucleotides having the sequences shown in SEQ ID NOs: 1 to 8. Next, the breed type of each oligonucleotide of the present invention is distinguished based on the amplified genomic DNA detected according to Example 11, and the identification type of the registered breed is determined from the breed 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. Healthy growing seedlings 5
Genomic DNA was extracted from an individual according to Example 1, and amplified genomic DNA was detected according to Examples 2 to 9 using the oligonucleotides of the present invention consisting of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8 separately. To do. Next, the breed type of each oligonucleotide is distinguished based on the amplified genomic 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 identification type of the registered variety and the identification type of the No.
It is possible to quickly and accurately obtain evidence that it is almost the same varieties, without any change due to the environment. 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.

[0060] (available in paternity testing: F1 plant test) Example 16 Rice (Oryza sativa L.) varieties model number 16 as a mother to obtain a rice (Oryza sativa L.) varieties model number 12 as the father Each genomic DNA is extracted from 50 seeds of the plant according to Example 1 and the amplified genomic DNA is detected according to Example 7 using the oligonucleotide having the sequence represented by SEQ ID NO: 6. Amplified genomic DNA detected and rice of variety number 16 ( Oryza sativa
L.) Amplified genomic DNA (about 400b
p) and rice of variety number 12 ( Oryza sativa L.)
Amplified genomic DNA originally owned by the variety (about 700 bp, about 48
0 bp) and the difference. As a result, the test plants possessing the amplified genomic DNAs of about 700 bp, about 480 b, and about 400 bp derived from the mother and the father were identified, and it was possible to quickly determine that they were F1 plants.

[0061]

INDUSTRIAL APPLICABILITY According to the method of the present invention, rice ( Oryza sativa L.) is used by utilizing the polymerase chain reaction using the oligonucleotide of the present invention as a primer.
It has the effect of identifying varieties more easily and efficiently.

[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 AGTCCTAAGC 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 TGGACACTAC 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 CTGAACTGAC 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 GTACAGACCT 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 ACGACGCTTA 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 CAGTCAAGTC 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 TCCAGACATG 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 ATTCGCAGCA 10

Claims (2)

[Claims] 1. A genomic DNA of a rice ( Oryza sativa L.) plant is amplified by a polymerase chain reaction using an oligonucleotide consisting of a sequence selected from the following sequence group as a primer, and the amplified genomic DNA is subjected to electrophoresis. A method for identifying rice ( Oryza sativa L.) cultivars, which comprises performing visual detection of amplified genomic DNA after isolation. Sequence group (1) 5'-AGTCCTA AGC-3 ', (2) 5'-TGGA
CATAC-3 ', (3) 5'-CTGAACTGAC-3', (4) 5'-GTAC
AGACCT-3 ', (5) 5'-ACGACGCTTA-3', (6) 5'-CAGT
CAAGTC-3 ', (7) 5'-TCCAGACATG-3', (8) 5'-ATTC
GCAGCA-3 ' 2. An oligonucleotide consisting of the following sequence. Sequence 5'-TGGACACTAC-3 '