JPH0716421B2 - Synthetic oligonucleotides used to identify watermelon F1 varieties - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a synthetic oligonucleotide used for identifying a watermelon F1 variety, and more specifically, to provide a synthetic oligonucleotide used for identifying a watermelon F1 variety having a specific sequence, The present invention relates to a method for identifying a watermelon variety by DNA analysis and a method for identifying a watermelon variety to determine the purity of watermelon F1 seeds.

[0002]

2. Description of the Related Art In the production of vegetable seeds, most of them are F1 seeds. Therefore, a seed parent (mother) is crossed with a specific pollen parent (father). There is a need. However, in many cases, the self-fertilization of the seed parent is possible, and there is a risk that the self-fertilized seed of the mother is mixed in the F1 seed. Further, this mixing rate changes depending on the degree of crossing error, weather conditions, cultivation conditions, and the like.

Plants derived from mixed self-fertilized seeds are F1
Since the properties are different from those derived from seeds, it becomes a factor that deteriorates the quality of seeds. Therefore, it is necessary to identify the watermelon variety and to test the purity of F1 seeds. Conventionally, the produced seeds are sown in a field or a nursery, and the self-fertilized seeds of the mother and F1 seeds are discriminated from the morphological characteristics of the germinated plants, and the purity is tested (morphological test) or seeds or young seeds. A method (enzymatic assay) in which a plant is ground with an appropriate buffer solution, subjected to electrophoresis, activity staining of a specific enzyme is carried out, and a seed is discriminated from a difference in isozyme (enzymatic assay) is carried out.

However, the former method requires a field, requires a relatively long period of time for plant growth, and in the case of mating between morphologically similar parents, F1 seeds and mother plants are used. Since there is only a slight difference, the determination may not be possible or the determination may be erroneous. In addition, the latter method cannot be used in the case of a combination of parents having the same isozyme, because the enzymes that can be analyzed actually have an upper limit. As described above, in the conventional method, it cannot be said that the identification of the variety and the purity test of the F1 seed are always performed at a satisfactory level.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method capable of identifying watermelon varieties and testing F1 seed purity with high accuracy and efficiency. The present inventor succeeded in distinguishing a watermelon F1 seed from a seed parent by a method of analyzing plant DNA by a polymerase chain reaction (PCR, Polymerase chain reaction) using a synthetic oligonucleotide having an arbitrary sequence. This method can be used not only for identifying the parent-child relationship of seeds but also for identifying varieties, and the present invention has been completed based on such findings.

[0006] That is, the present invention is a screen consisting of the following sequences .
A synthetic oligonucleotide used for identification of squid F1 varieties is provided, and DNA extracted from 5′-ACCACCTGGCTC-3 ′ watermelon is used as a template to perform DN by polymerase chain reaction using the synthetic oligonucleotide.
A method of identifying a watermelon variety characterized by performing amplification analysis of A, and performing electrophoretic analysis of the obtained amplification product, and performing electrophoretic analysis of the amplification product obtained by the reaction, to analyze F1 seed and seed parent (mother). It is intended to provide a method for judging the purity of watermelon F1 seeds by identifying selfed seeds.

The polymorphism of DNA obtained by PCR using a synthetic oligonucleotide having an arbitrary sequence is RAP
Also called D (Random amplified polymorphic DNA),
Many polymorphisms can be easily obtained by changing the type of synthetic oligonucleotide used. The steps required for this method are PC using the extracted DNA as a template.
It is a simple one that only involves the amplification of DNA by R, the electrophoresis of the amplified DNA, and the photographing of the migration image. for that reason,
It has been used for genetic analysis of various organisms and identification of varieties and individuals. The present invention is the first application of this method to the identification of watermelon cultivars and the purity test of F1 seeds.

The synthetic oligonucleotide of the present invention can be synthesized by using a commercially available DNA / RNA synthesizer by arbitrarily determining the sequence according to a random number table. Alternatively, commercially available oligonucleotides can be used. To identify the watermelon variety and to test the purity of F1 seeds, the total DNA from the watermelon tissue was analyzed by a conventional method, for example, cetyltrimethylammonium bromide (CTAB) [cloning and sequencing,
p252, 1989, Rural Culture Company]
Use as a DNA standard. The extraction of DNA is a simple method (K. Ed.) In which watermelon tissue is triturated with a buffer solution, and then solid-liquid separation is performed and a solvent such as isopropanol is added to the obtained supernatant.
wards et al., Nucleic Acids Research, vol. 19, p. 1349).

Next, the DNA extracted as described above is used as a template to amplify the DNA by the polymerase chain reaction using the synthetic oligonucleotide. That is,
0.5 ml plastic test tube with template DNA and heat-resistant DN
A polymerase, 4 kinds of deoxynucleotide monomers,
The synthetic primer RA12-12, a 10-fold concentrated reaction solution and distilled water are mixed, the mixture is covered with mineral oil, and the mixture is applied to a PCR machine (for example, manufactured by ASTEC, program temp control, etc.). The reaction is usually a high temperature treatment followed by heat denaturation,
Repeat annealing and extension reaction. For example 94
After pretreatment at 30 ° C. for 30 seconds, heat denaturation is performed at 94 ° C. for 25 seconds, annealing at 40 ° C. for 2 minutes, extension reaction at 72 ° C. for 3 minutes, and 45 cycles.

Thereafter, the obtained amplification product is subjected to electrophoretic analysis. Here, the electrophoretic analysis may be performed by a conventional method, for example, tris-borate-EDTA containing 1.5% agarose.
A voltage of 50 V is applied in a buffer gel and electrophoresis is performed to analyze the separated DNA band pattern. Each amplification product consists of several bands, but only the 850 bp band is different between individuals. That is, when the pollen parent and F1 DNA were used as templates,
A band of 850 bp was amplified, and this band did not appear when the seed parent DNA was used as a template. The amplification of this band is very stable and is seen in pollen parents and all F1 individuals, but not in seed parents at all. Therefore, in the assay of F1 seeds, the seeds to be assayed are germinated, DNA is extracted, the above polymerase chain reaction is carried out using this as a template, and the presence or absence of the 850 bp band in the amplification product is examined. A true F1 seed,
Those that do not exist may be regarded as self-fertilized seeds of seed parents, that is, impurities (contaminated seeds), and the purity of F1 seeds may be calculated.

[0011]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 (1) Extraction of DNA Seed parent Ha of fixed line of Tokyo system × Yamato system, pollen parent Hb belonging to Fuken system and its F1 [Fujimitsu, Hagiwara Farm Co., Ltd.] (Ha
XHb) from the roots on the 4th day after seeding, the total DNA was added to the above-mentioned C
It was extracted by the TAB method. That is, watermelon organization 0.2
~ 1.0 g of the same amount of buffer (2% cetyltrimethylammonium bromide, 20 mM Tris-HCl buffer, pH 8.
The mixture was triturated with 0,20 mM EDTA, 1.4 M NaCl), incubated at 55 ° C. for 10 minutes, and then cooled to room temperature. Then, an equal volume of chloroform was added and mixed, followed by centrifugation to separate into two layers. The upper layer is collected and 1/10 volume of 10
% Cetyltrimethylammonium bromide, 0.7M N
aCl was added and then chloroform was added to obtain an upper layer in the same manner. An equal volume of 1% cetyltrimethylammonium bromide and 50 mM NaCl was added to the upper layer to obtain a nucleic acid precipitate. This precipitate was dissolved in 100 mM Tris-hydrochloric acid buffer and 1M NaCl, and 2.5 volumes of ethanol was added to precipitate again. The precipitate was washed with 70% ethanol, dried, and dissolved in distilled water to obtain a DNA standard.

On the other hand, for the purpose of comparison, a DNA is prepared by a simple method.
Was extracted. That is, 100 mg of watermelon tissue 30 mg
μl buffer (200 mM Tris-HCl buffer, pH 7.
5,250 mM NaCl, 25 mM EDTA and
Trituration with 0.5% SDS) and centrifugation followed by addition of an equal volume of isopropanol to the supernatant to recover nucleic acids.

(2) Synthesis of oligonucleotide primer A DNA / RNA synthesizer (manufactured by ABI, type 392) was equipped with four types of protective monomers (manufactured by Wako Pure Chemical Industries) and other necessary synthetic reagents, and the primer cycle (0 Oligonucleotides were automatically synthesized on a CPG column (manufactured by ABI) using a 0.2 μmol scale). After the synthesis, automatic cutting was performed, and the aqueous ammonia solution was collected in a vial tube.

After removing ammonia under reduced pressure, the synthesized oligonucleotide was dissolved in distilled water, washed with n-butanol, ethanol was added to precipitate it, and it was dissolved again in water. Measure and DNA
After calculating the amount, it was used as a primer.

DN extracted by CTAB method or simple method
A5-10ng as template, 200μM dNTPs, 0.2μM
Add 25 units of Tth DNA Polymerase (Toyobo) to the oligonucleotide primer (1 type) and
1 of the reaction solution. The polymerase chain reaction is 94 ° C,
After pretreatment for 30 seconds, heat denaturation 94 ° C., 25 seconds, annealing 40 ° C., 2 minutes, extension reaction 72 ° C., 3 minutes, 45 cycles using a program temp control (ASTEC, PC-700). went.

(3) Agarose gel electrophoresis Agarose gel electrophoresis was performed using an electrophoresis apparatus (Mupid, manufactured by Cosmo Bio). Electrophoresis gel (Tris-borate-EDTA buffer, TBE) on a gel tray,
1.5% agarose and 1 μg / ml ethidium bromide were prepared), and a DNA preparation amplified by the polymerase chain reaction was added thereto, followed by electrophoresis at 50V. In addition, λ phage DNA was used as Ec as a DNA molecular weight marker.
Those digested with oRI and HindIII were electrophoresed at the same time.

In order to discriminate between the parent lines Ha and Hb, a total of 57 types of commercially available primers and synthetic primers (8mer
~ 26mer) was searched. As a result, in the electrophoretic pattern of the product amplified using 9 kinds of primers,
It turned out that Ha and Hb can be distinguished. In this pollen parent Hb
It was an amplification product by the synthetic primer RA12-12 having the following sequence that a clear band was observed that was specific to the enzyme. 5'-ACCACCTGGCTC-3 'That is, the amplification products of pollen parent Hb and F1 (Ha x Hb) showed a band at around 850 bp, but not that of seed parent Ha.

This is because the primer binding site of the synthetic primer RA12-12 was present in the + chain site and the 850 bp − chain site upstream of the synthetic primer RA12-12 in the pollen parent Hb DNA. It is considered that the 850 bp band was not amplified because this set of binding sites was not present in the seed parent Ha DNA. In addition, since this set of amplifiable sites in the pollen parent Hb was inherited by F1 due to inheritance, it is considered that the 850 bp band was also amplified when F1 DNA was used as a template.

Seed parent Ha, pollen parent Hb and F1 (Ha × Hb)
850 when subjected to the polymerase chain reaction in the same manner using DNA extracted separately from 5 individuals respectively as a template
The bp band was not found in all individuals in the seed parent, but in the pollen parent and all individuals in F1. The presence of this amplifiable site was observed in all pollen parent and F1 individuals, while it was presumed that it was not present in all seed parent individuals (see FIG. 1). In addition, it is clear that this seed parent is almost a pure line because it repeats selfing for ten or more generations, and that all selfed individuals of the seed parent do not have an amplifiable site.

In the case of F1 seed production, since the seed parent is fertile, there is a possibility that the self-fertilized seed of the seed parent may be mixed into the F1 seed due to a mistake in the mating work, which is a major factor that reduces the purity of the F1 seed. Cause. The contamination rate of this self-fertilized seed was determined by germinating the seed, extracting the DNA, and performing the polymerase chain reaction using the synthetic primer RA12-12 by the above-mentioned method to determine the contamination rate of the self-fertilized seed from the presence or absence of the band of 850 bp. It can be calculated and the purity of the F1 seed can be determined.

FIG. 2 shows the results of an assay by polymerase chain reaction using the synthetic primers RA12-12 described above after the F1 seeds and the self-fertilized seeds of the seeds were actually mixed and germinated. A band of 850 bp was not observed in 4 individuals among 14 individuals examined from the electrophoretic pattern, and it was possible to determine that these were self-fertilized seeds.

On the other hand, FIG. 3 shows DN extracted by a simple method.
The results obtained by using A are shown. As is clear from the figure, also in this case, the band of 850 bp was F1 as in the case of using the DNA extracted by the CTAB method.
It was found in individuals and not in seed parents.

FIG. 4 shows another combination of seed parent Hc ×.
For F1 seed of pollen parent Hd, the synthetic primer R
The result of the polymerase chain reaction performed using A12-12 is shown. As is clear from the figure, it is 850 in F1 seeds.
A band of bp was seen, but not the seed parent.

[0024]

EFFECTS OF THE INVENTION According to the present invention, a synthetic oligonucleotide for use in identifying watermelon F1 varieties is provided, and by using it, identification of watermelon varieties and F1 seed purity test can be performed with high accuracy and high efficiency. You can The combination of the parent line and the Metropolitan line × Fuken line used in the examples of the present invention is a general combination used for the production of the F1 variety of watermelon in Japan, and other than the F1 variety'Fujimitsu 'seed production. Hc × Hd
In many other combinations such as the production of F1 seeds, the purity test of F1 seeds can be performed with this primer. Therefore, with the development of this method, the watermelon variety identification and F
The purity test of one seed can be performed quickly, accurately and easily.

[Brief description of drawings]

FIG. 1 Synthetic oligonucleotide primer RA12
Electrophoresis photograph showing polymorphisms of watermelon parent lines Ha, Hb and its F1 DNA obtained using -12. 1 to 5 in the figure
Are seed parents Ha, 6-10 are F1 (Ha × Hb), Fujimitsu, 11
-15 shows pollen parent Hb. Arrow is used for identification 850
It is a band of bp.

FIG. 2 Synthetic oligonucleotide primer RA12
Electrophoresis photograph showing the assay of seeds using −12. The arrow indicates the 850 bp band used for identification. Both sides in the photograph are molecular weight markers.

FIG. 3 is an electrophoretic photograph showing the assay of seeds performed on DNA extracted by a simple method. The left 5 individuals are seed parent Ha and the right 5 individuals are pollen parent Hb. Arrows are used for identification 85
It is a band of 0 bp. The right end in the photograph is a molecular weight marker.

FIG. 4 Synthetic oligonucleotide primer RA12
-12 Watermelon Seed Parent Hc and its F1
Electrophoresis photograph showing polymorphism of (Hc × Hd) DNA. The arrow indicates the 850 bp band used for identification. In addition, the left end in the photograph is a molecular weight marker, and the following 7 individuals are seed parents.
Hc and the remaining 7 individuals show F1 (Hc × Hd).

Claims (3)

[Claims] 1. Identification of a watermelon F1 variety consisting of the following sequences:
Synthetic oligonucleotide used separately . 5'-ACCACCTGGCTC-3 ' 2. A DNA extracted from watermelon is used as a template,
A method for identifying watermelon varieties, characterized in that DNA is amplified by polymerase chain reaction using the synthetic oligonucleotide according to claim 1, and the obtained amplification product is subjected to electrophoretic analysis. 3. A method for determining the purity of watermelon F1 seeds by subjecting the amplification product obtained by the method of claim 2 to electrophoretic analysis to distinguish F1 seeds from selfed seeds of seed parents (mothers).