JPH05244995A - New primer - Google Patents

Abstract

PURPOSE:To rapidly and simply obtain a DNA fingerprint and detect the polymorphism between rice plant and brassicas which cannot conventionally be detected. CONSTITUTION:A method for detecting the polymorphism of a DNA sequence between rice plant and brassicas according to a polymerase chain reactional(PCR) method is characterized by using a new primer having a base sequence selected from sequence Nos. 1 to 8 and at least one primer thereof.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel primer. The primer of the present invention can quickly and simply obtain a DNA fingerprint, and can detect a DNA polymorphism within the same plant species that has been impossible to detect up to now.

[0002]

2. Description of the Related Art In recent years, with the progress of biotechnology, many new techniques have been introduced for plant breeding. Among them, detecting polymorphisms of DNA fragments on chromosomes,
The method of using a marker in association with a useful trait and the method of identifying individuals and varieties are very useful methods. Restriction Fragment Length Polymorphis
m; hereinafter, abbreviated as RFLP method. The analysis is one of them [Helentjaris et al., Plant Molec. Biol. 5 , 109-118].
(1985); Helentjaris et al., Theor. Appl. Genet. 72 , 761-7.
69 (1986); Neinhuis et al., Crop Science 27, 797-803 (198).
7)]. Also, a method for obtaining a DNA fingerprint was developed [Jeffreys et al., Nature 316 , 76-79 (198
5)], and a method of applying this to plant variety identification and breeding has been studied [Dallas, Proc. Natl. Acad. Sci. 85 , 6831-
6835 (1988), Rogstad et al., Proc. Natl. Acad. Sci. 85 , 917.
6-9178 (1988), Rogstad et al., Theor. Appl. Genet. 79 , 153.
-156 (1990)]. In the method for obtaining a DNA fingerprint, a minisatellite DNA that frequently shows polymorphism
[Jeffreys et al., Nature 314 , 67-73 (1985)], VNTR
[Nakamura et al., Science 235, 1616-1622 (1987)] or microsatellite DNA [Litt et al., Am.J.Hum.Gen].
et .44, 397-401 (1989); Waber et al., Am.J.Hum.Genet .44,
388-396 (1989)] is used.

However, in these methods,
Southern blotting [Southern, EM, J. Molec. Biol. 98, 50
3-517 (1975)], it is a method that requires a large amount of sample DNA, which requires time, labor, and skill. Therefore, only one restriction enzyme fragment length polymorphism clone can be analyzed for each Southern blot. There was a problem such as using expensive radioisotopes that require special facilities that cannot. The PCR (Polymerase Chain Reaction) method was introduced to solve such problems (US Pat. Nos. 4,683,195 and 4,683,202). According to this method, it is possible to analyze a large number of samples simultaneously with a small amount of DNA sample without using radioisotope, using an automatic device, in a short time, at low cost, and at a low cost. ..

In the RFLP method, the difference in the length of the DNA fragment cleaved by the restriction enzyme is compared between individuals or varieties, but in the PCR method, the difference in the length of the DNA fragment is the difference between the two primers hybridized. It can be detected as a difference in distance between soybean sites. That is, when a plurality of individuals or varieties are compared, if primers are prepared for adjacent non-mutational regions on both sides of a specific hypervariable region, a polymorphism based on the variability of the hypervariable region is detected by PCR. Can be detected [Jeffreys et al., Nucleic
Acid Research 16 , 10953-10971 (1988); Nelson et al., Pro.
c.Natl.Acad.Sci. 86 , 6686-6690 (1989); Horn et al., Nucle.
ic Acid Research 17 , 2,140-2140 (1989); D'Ovidio et al., P.
lant Molec. Biol. 15 , 169-171 (1990); Oudet et al., Hum.G.
enet. 84 , 283-285 (1990)]. FIG. 1 shows the site where the primer was prepared between the cultivars A and B. In addition, a PC using one or two of the arbitrarily synthesized 10-base long primers
A method for detecting polymorphism between varieties by the R method has also been reported.

In the method for detecting polymorphism between individual plants and varieties, the polymorphism can be actually detected only when the relative relations between individuals and varieties to be compared are apart to some extent. On the other hand, many of the agriculturally important plants are artificially cultivated cultivars and self-fertilizing plants, and since they have been crossed with varieties with high similarity for many years, the above PCR method is used. However, the polymorphism is rarely detected. For example, among Japanese-type rice varieties such as Koshihikari, Sasanishiki, and Nihonbare, although the phenotypic traits vary in taste, disease resistance, etc., the frequency of obtaining DNA polymorphisms is Japanese. It is less than one tenth of the frequency obtained between Indian type rice. Therefore, it is considered that a study that requires 2-3 years even for a normal plant, such as making a genetic map, requires 20 years or more when using Japanese type rice as a material. Further, a method of synthesizing primers at random and experimentally selecting useful primers one by one is not an efficient method among varieties in which polymorphism is difficult to detect.

[0006]

SUMMARY OF THE INVENTION The present invention provides a novel primer for detecting polymorphism between highly related rice and brassica.

[0007]

[Means for Solving the Problems] In order to detect a polymorphism between highly related rice plants and a polymorphism between brassicas by the PCR method, the present inventors have used as primers primers adjacent to non-variable sites of DNA. However, a DNA fragment that is amplified by using a specific sequence among a plurality of sequences (hereinafter abbreviated as multiple sequences) existing on a chromosome containing repetitive sequences is electrophoretically analyzed. It is possible to obtain a plurality of electrophoretically separable DNA fragments (DNA fingerprints) without forming a series of inseparable and continuous lengths, and to obtain the DN thereof.
It was found that the A fingerprint shows a high frequency of polymorphism. As a result, not only can individuals and varieties with high affinity be identified, but the identification and selection of F1 individuals, which are extremely important in testing the mutation rate of seedlings produced by tissue culture and in breeding, can be time-consuming and time-consuming. Can also be applied, and it becomes possible to create a genetic map, which was impossible with the conventional method in time, cost and labor.

The present invention will be described in detail below. The present invention relates to a novel primer having one of the nucleotide sequences selected from SEQ ID NOs: 1 to 8. Furthermore, the present invention relates to a method for detecting polymorphism between rice and brassica by PCR using the primer. Examples of varieties belonging to rice include Japanese type rice, Indian type rice, Java type rice, and the like, and varieties belonging to Brassica include rape,
Chinese cabbage, cabbage, broccoli, etc. can be mentioned. The present invention is useful for detecting polymorphisms among these varieties or between the same varieties.

The sequence of the primer of the present invention is a plant of each plant to be distinguished, preferably leaves (dry weight 0.1 to 5).
g) from the standard method [Saghai-Maroof et al., Proc. Natl. Acad. Sc.
i. 81 , 8014-8018 (1984)], DNA is extracted, one or more existing sequences of the DNA sequence are focused on, and at least one or more regions are specified to determine. The sequence of this primer includes a plurality of existing sequences. Synthesis of the primer of the present invention is performed according to a standard method, and D
Performed by NA synthesizer cyclone.

In the method of detecting a polymorphism by the PCR method using the primer of the present invention, at least one of the primers of the present invention may be present in the reaction solution. Moreover, you may mix and use with the primer which has another sequence. The reaction conditions for the PCR method are conventional [Saiki et al., Science 239, 487-491 (19
88)] The annealing temperature is determined for each primer, but it is easier to detect polymorphism by setting the temperature lower than usual, for example, 32 ° C. Also, usually at 72 ℃ 1
In many cases, an extension reaction of about a minute is incorporated, but in the PCR method using the primer of the present invention, this extension reaction is omitted because the length of the reaction product DNA fragment is 1 kilobase or less.

As the reaction liquid, for example, template D
NA 10ng, primer 20pmol each, 10X buffer [50
0 mM potassium chloride, 100 mM Tris-HCl buffer (pH 8.
4), 25 mM magnesium chloride] 10 μl, 2.5 mM dNTP 8 μl
2 units of Taq polymerase (manufactured by Perkin-Elmer Cetus) were added with water to make 100 μl. This reaction solution is put into a 0.5 ml reaction tube and the reaction is carried out using a thermocycler. The reaction is
For example, after reacting at 95 ℃ for 5 minutes, at 32 ℃ for 30 seconds, 93
Repeat the reaction for 30 seconds at 40 ° C for 40 times, or at 95 ° C for 5 times.
After reacting for 30 minutes, the reaction at 55 ° C. for 30 seconds and at 93 ° C. for 30 seconds is repeated 40 times to amplify the DNA.

The reaction products are separated by gel electrophoresis, high performance liquid chromatography (HPLC), capillary electrophoresis, etc., with gel electrophoresis being preferred. As the gel, an agarose gel, a polyacrylamide gel, etc. are used, but a polyacrylamide gel is preferable for detecting a minute difference. A polyacrylamide gel having a concentration of about 5% is usually used. Electrophoresis is usually
Perform at 100-120V for 2-3 hours. 1 to 10 after electrophoresis
stain with ethidium bromide at μg / ml for 10-30 minutes,
Detects separation patterns under UV light.

Examples of the present invention will be shown below.

[0014]

Example 1 DNA was extracted from each of Japanese rice cultivars, Koshihikari, Sasanishiki, Nihonbare and yakan leaves (0.5 g dry weight) by a standard method. Next, the primer of SEQ ID NO: 1 (primer 1) and the primer of SEQ ID NO: 2 (primer 2) were each synthesized using a DNA synthesizer Cyclone (manufactured by Milligen). DNA of each rice
10 ng, 20 pm each of primer 1 and primer 2
ol, 10X buffer 10 μl, 2.5 mM dNTP 8 μl and 2 units of Taq polymerase (Perkin Elmer Cetus) were added water to make 100 μl reaction solution, and put it in a 0.5 ml volume reaction tube. Use a thermocycler (Ericomp) at 95 ° C for 5
After reacting for 30 minutes, the reaction at 32 ° C. for 30 seconds and at 93 ° C. for 30 seconds was repeated 40 times to amplify the DNA. The reaction products were separated by electrophoresis at 120V for 2.5 hours using a 5% polyacrylamide gel, stained with ethidium bromide, and the separation pattern was detected under ultraviolet light to examine the pattern difference between cultivars. The result is shown in FIG. As shown in FIG. 2, polymorphism was detected among the four cultivars.

Example 2 The primer of SEQ ID NO: 3 (primer 3) and the primer of SEQ ID NO: 4 (primer 4) were each synthesized using a DNA synthesizer Cyclone (manufactured by Milligen).
Primer 3 instead of primers 1 and 2 respectively
The reaction was performed in the same manner as in Example 1 except that and 4 were used. The reaction products were separated by 120%, 2.5 hours, 5% polyacrylamide gel electrophoresis, stained with ethidium bromide, and then the separation pattern was detected under ultraviolet light to examine the difference in pattern between varieties. The result is shown in FIG. As shown in FIG. 3, polymorphism was detected among the four cultivars.

Example 3 The primer of SEQ ID NO: 5 (primer 5) and the primer of SEQ ID NO: 6 (primer 6) were each synthesized using a DNA synthesizer Cyclone (manufactured by Milligen).
Primer 5 instead of Primer 1 and 2 respectively
The reaction was carried out in the same manner as in Example 1 except that and 6 were used. The reaction product was separated by 5% polyacrylamide gel electrophoresis at 120 V for 2.5 hours, stained with ethidium bromide, and then the separation pattern was detected under ultraviolet light to examine the difference in pattern between varieties. The result is shown in FIG.
As shown in FIG. 4, polymorphism was detected among the four cultivars.

Example 4 The primer of SEQ ID NO: 7 (primer 7) and the primer of SEQ ID NO: 8 (primer 8) were each synthesized using a DNA synthesizer Cyclone (manufactured by Milligen).
Instead of primers 1 and 2, primer 7 respectively
After reacting at 95 ° C for 5 minutes with 30 and 55 ° C,
The same procedure as in Example 1 was repeated except that the reaction was repeated 40 times at 93 ° C. for 30 seconds for 40 seconds. The result is shown in FIG. As shown in FIG. 5, polymorphism was detected among the four cultivars.

Example 5 DNA was extracted from leaves (dry weight: 0.5 g) of four cultivars of broccoli by a standard method. A reaction product was obtained by carrying out the reaction in the same manner as in Example 4 except that this DNA was used instead of the rice rice DNA. This reaction product was separated by 5% polyacrylamide gel electrophoresis at 120 V for 2.5 hours, stained with ethidium bromide, and the separation pattern was detected under ultraviolet light to examine the difference in pattern between varieties. The result is shown in FIG. As shown in FIG. 6, polymorphism was detected among the four cultivars.

[0019]

INDUSTRIAL APPLICABILITY The present invention provides a novel primer for detecting polymorphism between highly related rice and brassica. The primer of the present invention can quickly and simply obtain a DNA fingerprint, and can detect a DNA polymorphism within the same plant species that has been impossible to detect up to now.

[0020]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin: Oryza sativa Sequence features Part of S repeat unit Sequence CAATATGGTG CGCGACAAT 19

SEQ ID NO: 2 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Origin: Oryza sativa Sequence Features S repeat unit Partial sequence TGCCATTCCT ATACTAACCA 20

SEQ ID NO: 3 Sequence Length: 9 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Characteristics S 5'UTR Sequence AGCCATGGC 9

SEQ ID NO: 4 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin: Oryza sativa Sequence features S repeat unit Partial sequence GCCGTGCTGC CCCTGGTA 18

SEQ ID NO: 5 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics S repeat unit sequence TAGATAGATA GATAGA 16

SEQ ID NO: 6 Sequence Length: 16 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Characteristics S repeat unit Sequence GACAGACAGA CAGACA 16

SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics S repeat unit Sequence TGTGTGTGTG TGTGTGTGCC 20

SEQ ID NO: 8 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Origin: Zea maize Sequence Features S repeat unit Partial sequence CCAGAAATCC AAGAATGTGA 20

[Brief description of drawings]

FIG. 1 is a diagram showing a site for preparing a primer between varieties A and B in the PCR method. → represents a primer.

FIG. 2 is a diagram showing the result of electrophoresis of rice DNA amplified by using primers 1 and 2. From the left lane, from the molecular weight marker, Koshihikari, Sasanishiki,
Shows the leaves of Nihonbare and Yakan respectively.

FIG. 3 is a diagram showing the result of electrophoresis of rice DNA amplified by using primers 3 and 4. From the leftmost lane, Koshihikari, Sasanishiki, Nipponbare, and yakan leaves are shown.

FIG. 4 is a diagram showing the result of electrophoresis of rice DNA amplified using primers 5 and 6. From the leftmost lane, Koshihikari, Sasanishiki, Nipponbare, and yakan leaves are shown.

FIG. 5 is a diagram showing the results of electrophoresis of rice DNA amplified using primers 7 and 8. From the left lane, from the molecular weight marker, Koshihikari, Sasanishiki,
Shows the leaves of Nihonbare and Yakan respectively.

FIG. 6: Amplification was performed using primers 7 and 8 4
FIG. 6 is a diagram showing the results of electrophoresis of DNA of seed broccoli.

Claims (5)

[Claims] 1. A novel primer having one of the nucleotide sequences selected from SEQ ID NOs: 1 to 8. 2. A method for detecting a polymorphism in a DNA sequence between rice plants by a PCR method, which comprises using at least one of the primers according to claim 1. 3. A method for detecting a polymorphism in a DNA sequence between brassicas by the PCR method, which comprises using at least one of the primers according to claim 1. 4. The method according to claim 2, wherein the rice is Japanese type rice. 5. The method according to claim 3, wherein the brassica is broccoli.