JP2002291488A - Method for detecting contaminated rice seed - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting rice seed of Japonica species having no Indica type genome region contaminated in rice seed of Japonica species having the Indica type genome region and an improved cetyltrimethylammonium bromide(CTAB) method. SOLUTION: This method for detecting contaminated rice seed includes i) to form a primer pair for amplifying the region of the aforesaid Japonica type genome region or a part of it depending on the base arrangement of the Japonica type genome region corresponding to the aforesaid Indica type genome region, ii) carry out a nucleic acid amplification reaction by using a single or a plurality of genome DNAs of the rice seed of the Japonica species as a genetic template, and iii) when a nucleic acid amplification product is detected, it is judged that the rice seed of Japonica species is contaminated. This method also includes to suspend pulverized brown rice in a buffer solution for extracting CTAB, subsequently carry out centrifugation at a low temperature to remove starch.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting Japonica varieties having no Indica genomic region, which are mixed in Japonica varieties having an Indica genomic region.

[0002] The present invention also relates to a nucleic acid amplification primer to be used in the detection method. The present invention further relates to a cetyltrimethylammonium bromide (CTAB) method for preparing DNA from brown rice.

[0003]

2. Description of the Related Art Purity management of seed rice, which is a product at a seed rice production site, is important in terms of guaranteeing the reliability of products. According to the main crop seed method, rice is required to have a seed purity of 99% or more for fixed rice and 98% or more for hybrid rice in Japan. However, in practice, higher seed purity is often required. For example, if a long-culm variety is mixed with a short-culm variety in a general paddy planted with three plants per plant, 300 strains, that is, one strain can be transplanted in a transplant area of only about 14 m ² even if the contamination is about 0.1%. In contrast, different varieties are detected, and some removal work is required. From this, in the estimation of the purity of the seed rice, it is required that contamination of different varieties can be detected with an accuracy of at least 0.1%. Particularly, in recent years, the seed renewal rate has been increasing, and thus its importance has been increasing.

[0004] It is clear that the reliability of the seed rice as a commodity is improved by guaranteeing the seed rice purity. In addition, the purity estimation method, that is, the establishment of a detection method of trace varieties of varieties, cultivation, harvesting, threshing, storage, because it is possible to clarify in which process of contamination has occurred,
It can contribute to the strengthening of the quality control system for seed rice.

The applicant, Japan Tobacco Inc. (J
Many of the rice varieties that T) have so far cultivated are Japonica-type varieties, which are one of the sub-species of the Sativa varieties. It contains genes derived from varieties belonging to the subspecies Indica type. For example, many of them contain a chromosomal region in which the stripe blight resistance gene Stv-bi derived from the Indica cultivar Modan is located.

At present, the estimation of the purity of paddy rice seed is not generally performed, and it is customary as a preventive measure to judge and remove different varieties based on morphological differences during cultivation in a seed field. It was done. However, depending on the combination of planted varieties and mixed different varieties, detection and removal may be extremely difficult. For example, when short culm varieties are mixed in long culm varieties, or when middle cultivars with early heading are slightly mixed in early varieties with early heading, they are characterized by pest and disease resistance. This is the case when a diseased variety is mixed with a variety. Even if such different varieties can be removed, it is not possible to cope with the case where pollen of different varieties crosses in a seeding field or the case where different varieties are mixed in the process of threshing and storage after harvesting.

Therefore, from the viewpoint of quality assurance, it has been pointed out that the estimation of the purity of seed rice is important. Specifically, prior to the present invention, the following method was considered in testing the purity of seed rice.

A) Method of estimating the purity of the seed rice by cultivating the harvested seed rice In this case, it is conceivable that the estimation is performed by extracting the seed rice and conducting a cultivation test of a certain scale. However, for the same reason as when judging and removing different varieties based on morphological differences during cultivation in seed fields,
Finding different strains is not easy. Furthermore, if cultivation is carried out in a paddy field during an appropriate cropping season for the purpose of increasing the reliability of such a test, the shipment of seed rice will be delayed by one year as a result. Due to the low reliability and complexity described above, no such cultivation test has been actually performed.

B) A method for inspecting the purity of plant seeds using a rice microsatellite marker (JP-A-10-5)
7073) JP-A-10-57073 describes a method for inspecting the purity of rice seeds using a microsatellite marker in which AT or TA is repeated 5 times or more. According to this method, DNA is extracted from each rice seed, and the contamination rate is estimated based on the number of seed grains in which a chain length difference is recognized in the amplified DNA fragment by the microsatellite marker.

However, crushing one brown rice at a time requires a large amount of work, and several thousand grains must be handled in order to estimate a statistically significant mixing ratio using a sampling inspection method. Practically difficult.

C) A method for examining the purity of hybrid seeds based on the abundance of male-sterile cytoplasm-specific rice mitochondrial DNA (JP-A-10-286093) JP-A-10-286093 discloses a method for testing male-sterile cytoplasm-specific It describes a method for testing the purity of hybrid seeds based on the abundance of rice mitochondrial DNA. This method makes use of the fact that a specific sequence of mitochondrial DNA is present in the maintenance line and the fertility restoration line but not in the male sterile line, and is represented by the maintenance seeds present in hybrid seeds having sterile cytoplasm. The purpose of this study is to estimate the percentage of mixed seeds of different varieties having normal cytoplasm. DNA is extracted from a mixture (bulk) of 100 brown rice grains, and the contamination rate is estimated by comparing the amount of amplified DNA with the target.

However, since the above method uses a DNA fragment derived from mitochondria, it cannot be applied to estimating the seed purity among general Japonica-type cultivated rice varieties having normal cytoplasm.

As described above, a simple and practical method for detecting Japonica varieties having no Indica-type genomic regions, which are mixed in Japonica varieties having the desired Indica-type genomic regions, is necessary. Not established despite sex.

[0014] In the detection of Japonica varieties having no indica-type genomic region, it is necessary to extract and prepare DNA from brown rice. DNA extraction from plant tissue is usually performed by CTAB or SDS.
The phenol method is used; However, for example, when it is intended to apply to brown rice, it has been pointed out that the SDS-phenol method has a low extraction efficiency of DNA, and that when the DNA is used as a template, the PCR reaction is sometimes inhibited. . On the other hand, in the CTAB method, since a large amount of starch is mixed in the extracted DNA fraction, D
There was a problem that it was difficult to redissolve NA.

[0015]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for detecting a Japonica variety having no Indica genomic region, which is mixed with a Japonica variety having an Indica type genomic region. The method of the present invention comprises: i) forming a primer pair so as to amplify the Japonica-type genomic region or a part thereof based on the nucleotide sequence of the Japonica-type genomic region corresponding to the Indica-type genomic region; Or Genome DN of multiple Japonica varieties seeds
A) a nucleic acid amplification reaction is carried out using A as a template; and iii) when a nucleic acid amplification product is detected, it is judged that Japonica varieties having no indica-type genomic region are mixed. I do.

Another object of the present invention is to provide a nucleic acid amplification primer for use in the detection method. The primer for nucleic acid amplification of the present invention is characterized by being prepared based on the base sequence of a Japonica-type genomic region corresponding to an Indica-type genomic region present in a Japonica variety seed.

The present invention further relates to cetyltrimethylammonium bromide (C) for preparing DNA from brown rice.
In the TAB) method, an object of the present invention is to provide an improved method of the CTAB method, which comprises suspending milled brown rice in a CTAB extraction buffer and centrifuging at a low temperature to remove starch.

[0018]

Means for Solving the Problems According to the present invention, as a result of diligent research for solving the above problems, the indica-type genomic region was obtained by utilizing the base sequence of the Japonica-type genomic region corresponding to the indica-type genomic region. We succeeded in establishing a rapid, simple and accurate method for detecting Japonica varieties having no Indica-type genomic region mixed with Japonica varieties having sap.

That is, the present invention relates to a method for detecting a Japonica variety seed having no Indica genomic region, which is mixed in a Japonica variety having a Indica type genomic region, comprising the steps of: A primer pair is formed so as to amplify the Japonica-type genomic region or a part thereof based on the nucleotide sequence of the Japonica-type genomic region corresponding to the region; ii) Genome DN of single or multiple Japonica-type cultivars
A) a nucleic acid amplification reaction is performed using A as a template; and iii) when a nucleic acid amplification product is detected, it is determined that Japonica varieties having no indica-type genomic region are mixed. provide.

The term "Japonica varieties having indica-type genomic regions" (hereinafter sometimes simply referred to as "Indica-type varieties" in the present specification), which is the object of the present invention, is not particularly limited. One of the subspecies of the Japonica type cultivar belongs to another subspecies of the Indica type for the purpose of improving disease resistance, improving yield, improving quality and taste, etc. Refers to those containing a genomic region containing genes derived from Although not limited, for example, Iwata No. 3, Iwata No. 5, Iwata No. 8, Starlight, Morning Light, Moonlight, Aoi Kaze etc. are originally derived from Japonica type varieties In order to confer resistance to leaf blight, indica type cultivar Modan
An indica-type genomic region containing the derived stripe blight resistance gene Stv-bi has been introduced (Kashiwara et al.
997) Other (1) 174. Ito et al., Aichi Prefectural Agricultural Research Report (1989) 21: 1-17).

On the other hand, there is no particular limitation on japonica variety seeds having no indica-type genomic region (hereinafter sometimes simply referred to as “japonica-type variety” in the present specification).
For example, but not limited to, Koshihikari, Kirara 397, Akihikari, Akitakomachi, Sasanishiki, Kinuhikari, Nipponbare, Hatsuboshi, Koganebaru, Hinohikari, Minneasaki Hikari, Aino Kaori, Hatsimo, Akebono, Fujihikari, Asomi Nori, Mine Snowy, coconoemochi, fukubiki, donkokoi, 500 million stones, hanaechizen, hohohoho, todokiwase, yukinoshimi, haenuki, domanaka,
Yamahikari and the like are known. Both "Indica-type varieties" and "Japonica-type varieties" are well known to those skilled in the art, and those skilled in the art can easily determine which rice varieties can be an object of the present invention.

In the examples described later in this specification, No. 3, No. 11, No. 13, No. 13, and No. 16 have the same indica-type genomic region. Method is available. Furthermore, it can also be applied to varieties grown at the Aichi Prefectural Comprehensive Agricultural Experiment Station, such as starlight, blue sky, moonlight, morning light, aoi breeze, and Akane sky.

When a genomic DNA of a Japonica type cultivar having an indica type genomic region including a contaminated Japonica type cultivar having no indica type genomic region is used as a template, the nucleic acid amplification reaction is carried out in a small amount. Amplification products can be obtained only for certain Japonica varieties,
Amplification product polymorphism (ALP: amplicon length) such that no amplification product is obtained for a Japonica-type variety having an indica-type genomic region, or an amplification product having a length different from that of a Japonica-type variety is obtained
polymorphism). As chain described above, in the detection method of the present invention, to amplify the Japonica type genomic region or a portion thereof based on the nucleotide sequence of the japonica type genomic region corresponding to the Indica type genomic region, a nucleic acid amplification product was detected In this case, it is determined that Japonica variety seeds having no indica-type genomic region are mixed.

The region to be amplified is the nucleotide sequence of the Japonica-type genomic region corresponding to the Indica-type genomic region including the gene region that provides the above-described properties such as improved resistance, improved yield, improved quality and improved taste. Or a part thereof. Not only the Japonica-type genomic region at the locus corresponding to the gene region imparting a specific property of the indica-type genome, but also the region located around the gene region and linked to the gene region and acting together, Can be used for the detection method.

That is, when an indica-type genomic region containing a desired gene region is introduced into a japonica type, a region linked to the gene region will undergo a gene recombination reaction together with a region corresponding to the gene region, and the japonica type It is highly possible that all or part of the type genome is deleted or mutated. On the other hand, in the Japonica type varieties in which the intended gene region has not been introduced, the indica-type genome has not been introduced not only in the intended gene region but also in the linked region, and the above-described genetic recombination has been performed. No reaction.

As used herein, the term "linkage" refers to the inheritance of a group of genes located on the same chromosome. In meiosis, the genes are linked at a higher frequency than expected by the law of independent inheritance. Means to do. Genes having a high degree of linkage with each other have a high probability of acting together in the event of recombination, meaning that recombination is unlikely to occur between the genes. Therefore, in the present invention, by examining the behavior of the region on the same chromosome that is linked to the indica-type genomic region introduced into the Japonica-type cultivar, it is possible to confirm the presence of a trace amount of the contaminated Japonica-type cultivar Becomes possible.

In general, the closer together on the same chromosome, the closer the linkage. Although not limited, the relative distance to the indica-type genomic region is about 5 cM or less (about 1.5 million bases or less), preferably about 2 cM or less (about 60 cM or less).
The sequence in the region of (within 10,000 bases) has a high degree of linkage and can be used in the detection method of the present invention.

For example, but not limited to, a stripe blight resistance gene (Stv) derived from an Indica type variety
-Bi) sits on the long arm of rice chromosome 11 and contains RFLP
(Restriction fragment len
gth polymorphism) marker G257
And C189 (Kurata et al., Na
cure Genetics (1994) 8:36
5-372). In the course of the study of the present invention, the PCR marker RM21 (Panaude) was used.
t al. , Mol Gen Genet (199
6) 252: 597-607) was also found to be sitting near both RFLP markers. In particular,
G257 (SEQ ID NO: 1) is closely linked to Stv-bi (Hayano-Saito et al.,
Theor Appl Genet (1998)
96: 1044-1049), and Stv-
The relative distance to the bi gene is about 0.9 cM (about 270,000 base pairs).

Whether a certain region in a genomic region is linked to another region is determined by a known method (Allard, Hilgardia (1.
956) 24: 235-278, Immer, Genetics (1930) 15: 81-
98 etc.). For example, whether a known dominant marker is present on the intended indica genomic region that has been introduced into a Japonica cultivar, and whether it is linked to a dominant gene present on that region, as follows: You can find out.

The dominant marker loci X and dominant locus Y has to train F ₂ population of reciprocal crosses combination together separating investigate the phenotype. Note that both loci are X and Y, respectively.
Is dominant and x and y are inferior. For example, the observed values of the phenotypes classified into four types are [XY]: [Xy]: [x
Y]: [xy] = 250 (a): 123 (b): 125
(C): Suppose that it was 2 (d). When the recombination value is r, the estimated value by the maximum likelihood method is (a + b + c + d) (1-
^{r) 4 - (a-2b} -2c-d) (1-r) 2 -2d = 0
, So that this is solved and r =
0.125 (%) is obtained. In addition, Kosambi
(Ann Eugen (1944) 12: 172-
175) z = 0.5 × tanh ⁻¹ (2r / 10
0) to obtain a map distance z = 12.8 (cM).

Genetic mapping of rice genomes is in progress. For example, the aforementioned Kurata et.
al (Nature Genetics (1994)
8: 365-372) shows RF using 1,383 DNA markers for 927 loci in rice.
An LP linkage map is described. Those skilled in the art can determine the intended indica-type genomic region introduced into the Japonica-type variety based on such a known linkage map.

In Examples described later, contaminated Japonica varieties were detected using G257 (SEQ ID NO: 1) linked to the Stv-bi gene as a marker. The present invention is not particularly limited. For example, the black leafhopper resistance gene G
C81 linked to rh-3 (t) (Saka et al.
7) An alternative 155), C600 linked to the bacterial blight resistance gene Xa-1 (Yoshimura et al.,
Theor Appl Genet (1996)
93: 117-122) can also be used in the method of detecting a mixed Japonica variety of the present invention. Further, it is possible to detect and detect other known or new varieties having genomic regions of other varieties which are not present in Japonica rice, based on the technical idea of the present invention.

[0033] In the nucleic acid amplification the present invention, preferably, based on the nucleotide sequence of the japonica type genomic region corresponding to the desired indica type genomic region has been introduced into the Japonica type varieties as shown in Example 2, the Japonica To amplify only the type region,
Create a primer pair.

That is, no amplification product can be obtained for a Japonica cultivar having an indica genomic region.
In this case, if no Japonica-type varieties are mixed, no amplification product is detected, and only for a small amount of Japonica-type varieties, a Japonica-type genomic region corresponding to the Indica-type genomic region according to the mixing ratio And the amplification product of the Japonica-type genomic region or a part thereof based on the nucleotide sequence of One of the advantages of such a dominant marker is that, unlike the case of the co-dominant marker, it is not affected by the positive band of the Indica type cultivar, and enables quantitative detection of contamination of the Japonica type cultivar. .

The nucleic acid amplification reaction is not limited, but may be performed by PCR (Saiki et al., (198)
5), Science 230, p. 1350-135
4).

When preparing primers for nucleic acid amplification, it is essential to design such that DNA is amplified only from the Japonica type region corresponding to the Indica type region. Specifically, it is created as follows.

1) The nucleotide sequence of the indica-type region is compared with the nucleotide sequence of the corresponding Japonica-type region, and a region having a difference between the two is searched. Several bases or more,
More preferably, there is a difference over 100 bases or more. 2) The 3 'end of at least one primer, preferably at least one primer, more preferably both primers are identical to only the base sequence of the japonica type region. Alternatively, a primer is designed so as to be complementary. 3) In the following, a primer can be prepared by a known method.

Specifically, although not limited,
For example, the following conditions 1) -5): 1) The length of each primer is 15-30 bases; 2) The ratio of G + C in the base sequence of each primer is 30-
3) The distribution of A, T, G, and C in the base sequence of each primer is not partially largely biased; 4) The length of the nucleic acid amplification product amplified by the primer pair is 50- 3000 bases, preferably 100-2000
And 5) identical or complementary to the base sequence of the japonica type region so as to satisfy the absence of a complementary sequence in the base sequence of each primer or between base sequences of the primers. Producing a single-stranded DNA having a unique base sequence,
Alternatively, if necessary, a primer pair can be prepared by a method including producing the single-stranded DNA modified so as not to lose the binding specificity to the linked region.

It should be noted that a PC was prepared using the prepared primer pair.
It is preferable to perform an R reaction and confirm that the PCR product is amplified only when the japonica type region is used as a template. In the examples described below, primers having the base sequences of SEQ ID NOS: 2 and 8 described below were used to amplify G257 (SEQ ID NO: 1) linked to the Stv-bi gene.

The thus prepared primer for nucleic acid amplification for use in the detection method of the present invention is also included in the scope of the present invention. The procedure and conditions for the nucleic acid amplification reaction are not particularly limited, and are well known to those skilled in the art. Those skilled in the art can appropriately adopt conditions depending on various factors such as the nucleotide sequence of the linkage region, the nucleotide sequence of the primer pair, and the length. In general, the longer the length of the primer pair, the higher the ratio of G + C, and the smaller the bias in the distribution of A, T, G, and C, the more stringent conditions (annealing reaction and nucleic acid extension at higher temperatures). Reaction, a smaller number of cycles). By employing more stringent conditions, an amplification reaction with high specificity becomes possible.

Although the amplification reaction is not limited, preferably, the denaturation reaction is performed at 90 ° C. to 95 ° C. for 20 seconds to 2 minutes, the annealing reaction is performed at 55 ° C. to 65 ° C. for 20 seconds to 2 minutes, and the extension reaction is performed. 70 ° C to 75 ° C 3
One cycle is from 0 seconds to 2 minutes, and this is performed for 25 cycles to 40 cycles. More preferably, the denaturation is 94
The cycle is 30 cycles at 30 ° C., 30 cycles of annealing at 57 ° C., and 2 cycles of extension at 72 ° C. for 29 cycles.

Without limitation, a stable PC
To obtain the R amplification result, the volume of the PCR reaction solution is about 1
It is 0 μl to about 100 μl, preferably about 50 μl. The minimum amount of template DNA required to detect a PCR product derived from a contaminated seed is about 3 ng. Therefore, for example, when the mixing ratio is 1/100, the required amount of template genomic DNA is about 300 ng.

After completion of the nucleic acid amplification reaction, the presence or absence of an amplification product subjected to electrophoresis on an agarose gel is detected. In order to more clearly identify the presence or absence of nucleic acid amplification products, using an electrophoresis tank capable of loading a relatively large volume of sample,
For example, 10 μl to 20 μl of a nucleic acid amplification product-containing sample is loaded. After electrophoresis, electrophoretic bands are visualized by a known method, for example, ethidium bromide (EtBr) staining.

In the method of the present invention, the ratio of the Japonica variety seeds mixed in the Japonica variety seeds having the indica type genomic region is about 1/300 or more, preferably about 1/300 or more. In the case of / 100 or more, detection is possible (Example 5). That is, for example, seed rice is divided into batches each having 100 grains, and the detection method of the present invention is performed for each batch. Even if one batch of Japonica-type genomic paddy is mixed in each batch (mixing rate 1%)
Because it is detectable, it can be a practical seed purity assay.

Further, the amount of the amplified nucleic acid product increases in proportion to the mixing ratio of Japonica variety seeds. Therefore, it is possible to determine the mixing ratio of Japonica varieties having no indica-type genomic region according to the amount of the nucleic acid amplification product.

For example, as described in Example 6, 10
0 batches were prepared as 30 batches, and 2 batches were prepared.
If nucleic acid amplification products are detected in one batch, the contamination rate can be determined to be at least 21/3000. In addition, the mixing ratio is known (for example, 100%,
(1%, 0%) can be used as a control to conduct a nucleic acid amplification reaction at the same time to perform the assay, whereby the contamination rate can be determined more accurately. If necessary, the amount of the nucleic acid amplification product can be analyzed more accurately by a known method, for example, a method of labeling a nucleic acid with fluorescence, radiation, or the like, and analyzing the image. In Example 6, which will be described later, the mixing ratio actually determined by the method of the present invention coincided very well with the estimation based on the field survey.

Therefore, according to the present invention, it is possible to perform not only the detection of the mixed Japonica type but also the estimation of the mixing ratio. Differences between the present invention and the prior art a) Compared with the method of estimating the purity of the seed rice by cultivating the harvested seed rice, the present invention is an assay method using DNA markers. It is possible to compare them collectively by bulk in units of individuals. Therefore, there are no drawbacks such as a delay in shipment and a complication.

B) Compared with the method using microsatellite markers, the present invention makes it possible to compare not only individual individuals but also bulks in units of 100 individuals. Also, unlike the microsatellite marker, which is a co-dominant marker, the ALP marker, which is a dominant marker, can always be selected.

C) In comparison with the method for examining the purity of hybrid seeds based on the abundance of male-sterile cytoplasm-specific rice mitochondrial DNA, the present invention tests fixed variety seeds rather than hybrid seeds. Therefore,
It can be used for detection of general Japonica type cultivated rice varieties having normal cytoplasm. The present invention converts mutations in nuclear DNA, but not mitochondrial DNA, into PCR markers.

Improved CTAB Method The present invention further provides an improved method of cetyltrimethylammonium bromide (CTAB) for preparing DNA from brown rice. The improved CTAB method of the present invention is characterized in that ground brown rice is suspended in a CTAB extraction buffer, and then the starch is removed by centrifugation at a low temperature.

[0051] The CTAB method is a widely used method for extracting DNA from rice seeds.
Murray and Thompson Nucle
icAcids Res (1980) 8: 4321
-4325 and the like. Generally, the CTAB method can be performed in the following steps.

The cell wall is broken by crushing the tissue, and a CTAB extraction buffer, which is a surfactant, is added to the cell wall to remove DAB in the tissue.
Dissolve the NA. After the permeation, an organic solvent such as a mixed solution of chloroform / isoamyl alcohol = 24: 1 is added to denature proteins existing in the cells. After removing the organic phase by centrifugation, a high salt concentration aqueous solution such as an ammonium acetate solution and an alcohol solution such as isopropanol and ethanol are added to concentrate and purify the DNA. Finally TE
Dissolve in buffer.

The above-described CTAB method has an advantage that a substantially equal amount of DNA can be extracted from individual rice grains, but has a drawback that a large amount of starch is mixed in a DNA preparation. The improved CTAB method of the present invention is characterized in that, in the above step, the ground brown rice is sufficiently removed by suspending the milled brown rice in a CTAB extraction buffer and performing a low-temperature and centrifugal treatment. Without limitation, at 0 ° C. to room temperature,
1,000 rpm to 20,000 rpm, 5 minutes to 20 minutes, preferably at about 4 ° C., about 3000 rpm
Centrifuge for about 10 minutes. These conditions may change depending on the size of the container.

When DNA extraction from brown rice is not possible,
The seeds are germinated, a certain amount of leaf pieces are collected from each individual, and DN
A extraction must be performed. This involves a very complicated task. Therefore, the improved CATB method described above can contribute to the rice genomic DNA extraction step in the detection method of the present invention.

REFERENCES The following publications are incorporated herein by reference. 1. Kashiwabara et al. (1997) Sep. (1) 174. 2. Ito et al. Aichi Prefectural Agricultural Research Report (1989) 21:
p. 1-17. 3. Kurata et al. , Nature
Genetics (1994) 8: p. 365-3
72. 4. Hayano-Saito et al. , T
heor ApplGenet (1998) 96:
p. 1044-1049. 5. Panaud et al. , Mol Gen
Genet (1996) 252: p. 597-6
07 6. Allard, Hilgardia (195
6) 24: p. 235-278 7. Immer, Genetics (1930)
15: p. 81-98 8. Saka et al. (1997) 1 55 9 Yoshimura et al. , Theo
r Appl Genet (1996) 93: p.
117-122 10. Saiki et al. , (1985),
Science 230, p. 1350-1354 11. Murray and Thompson N
ucleic Acids Res (1980)
8: p. 4321-4325 Hereinafter, the present invention will be described specifically with reference to Examples.
They are not intended to limit the technical scope of the present invention. Those skilled in the art can easily make modifications and changes to the present invention based on the description in this specification, and they are included in the technical scope of the present invention.

[0056]

EXAMPLES Example 1 PCR markers G257ALP and
Creation of CLP and C189ALP RFLP markers G257 and C189 were obtained from Kurata
(Nature Genetics (1994)
8: 365-372) and rice 11th
Located on the long arm of the chromosome. Both markers, especially G257,
It is known to be linked to the stripe blight resistance gene (Stv-bi) (Hayano-Saito et al.).
al. , Theor Appl Genet (19
98) 96: 1044-1049).

The PCR markers G257ALP and C189 were prepared based on the RFLP markers G257 and C189. Specifically, nucleic acid amplification primers were prepared based on the nucleotide sequences of G257 (SEQ ID NO: 1) and C189.

Plasmid clones G257 and C189, which are RFLP marker probes, were obtained from the Agricultural and Biological Resources Institute of the Ministry of Agriculture, Forestry and Fisheries and transformed into Escherichia coli. After culturing the transformed Escherichia coli, the plasmid is purified, and the base sequence of the inserted portion is compared with Applied Biosystem.
It was revealed using MS Sequencer Model 373A. Nucleic acid amplification primers were prepared based on the terminal portions of each sequence. 1) Primer sequence

[0059]

Embedded image G257F1; 5′-CTGCAGGGTACGTGAACAA-3 ′ (SEQ ID NO: 2) G257R1; 5′-CTGCAGGCTGCACGCCATT-3 ′ (SEQ ID NO: 3) C189-1; 5′-AAGATGCTGGAGGGGATCATT-3 ′ (SEQ ID NO: 4 ) C189-2; 5'-ACGCTCTGCTACTACTAACATCA-3 '(SEQ ID NO: 5) Using the above primers, a PCR reaction was performed using the following PCR reaction solution composition and reaction conditions. 2) PCR reaction solution composition

[0060]

Table 1 3 ng / μl DNA solution 2.0 μl 10 × TaKaRa Taq attached buffer (Mg2 + plus) 1.0 μl 2.5 mM dNTP 0.4 μl 5 pmole / μl Primer 0.4 μl TaKaRa Taq (manufactured by TaKaRa) 0.08 μl H ₂ O 5.72 μl total 10.00 μl 3) PCR reaction conditions Denaturation -94 ° C, 30 seconds; Extension -57 ° C, 30 seconds; Annealing -72 ° C, 1 minute cycle, 30 cycles, 72
° C, 4 ° C after 10 minutes. PCR marker G257 created
For ALP and C189ALP, the sitting position was investigated using a recombinant inbred line group (available from Kyushu University) created by crossing Asinominori with IR24. These converted PCR markers were confirmed to be identical to the RFLP marker G257 locus and C189 locus on chromosome 11.

The microsatellite marker RM2
When the seating position of No. 1 was also examined, it was seated between both markers. Therefore, in the indica-type genome region of morning light, the stripe blight resistance gene (Stv-bi), PC
It is considered that the R markers G257 and C189 and the microsatellite marker RM21 are located. Example 2 257 ALP, C189 ALP and RM2
Detection of nucleic acid amplification product polymorphism by three kinds of PCR markers
討 Shimaha blight resistant varieties and G257ALP showing the DNA polymorphisms between the susceptible variety, 3 C189ALP and RM21
Preliminary tests were performed on the three PCR markers. Specifically, Koshihikari DN was added to DNA No. 3 at various ratios.
A was mixed, and a PCR reaction was performed using the mixture as a template.

1) G257ALP 1-1) Primer sequence

[0063]

G257F1; 5'-CTGCAGGGTACGTGAACAA-3 '(SEQ ID NO: 2) G257R1; 5'-CTGCAGCTGCACGCCATTT-3' (SEQ ID NO: 3) 1-2) Composition of PCR reaction solution

[0064]

TABLE 2 100 ng / [mu] l DNA solution 10 [mu] l 10 × PCR buffer (TaKaRa Co.) 5μl 2.5mM dNTP 2μl 5 pmole/μl G257 -F1 2μl 5 pmole/μl G257-R1 2μl rTaq polymerase (manufactured by TaKaRa Co.) 0.4 [mu] l H ₂ O _28.6μl Total 50.0μl 1-3) PCR reaction conditions denaturation -94 ° C., 30 sec; extension -57 ° C., 30 sec; annealing -72 ° C., 30 cycles of 1 minute, 72
° C, 4 ° C after 10 minutes.

2) C189ALP 2-1) Primer sequence

[0066]

Embedded image C189-1; 5′-AAGATGCTGGAGGGGATCATT-3 ′ (SEQ ID NO: 4) C189-2; 5′-ACGCTCTCGCTAACTTAACATCA-3 ′ (SEQ ID NO: 5) 2-2) Composition of PCR reaction solution

[0067]

TABLE 3 100 ng / [mu] l DNA solution 10 [mu] l (manufactured by TaKaRa Co.) 10 × PCR buffer 5μl 2.5mM dNTP 2μl 5 pmole/μl C189-1 2μl 5 pmole/μl C189-2 2μl rTaq polymerase (manufactured by TaKaRa Co.) 0.4 [mu] l H ₂ O _28.6μl Total 50.0μl 2-3) PCR reaction conditions denaturation -94 ° C., 20 sec; extension -57 ° C., 30 sec; annealing -72 ° C., 30 cycles of 1 minute, 72
° C, 4 ° C after 10 minutes.

3) RM21 3-1) Primer sequence

[0069]

RM21-F; 5'-ACAGATTTCCGTAGGCACGG-3 '(SEQ ID NO: 6) RM21-R; 5'-GCTCCATGAGGGTGGTAGAG-3' (SEQ ID NO: 7) 3-2) Composition of PCR reaction solution

[0070]

TABLE 4 100 ng / [mu] l DNA solution 10 [mu] l 10 × PCR buffer (TaKaRa Co.) 5μl 2.5mM dNTP 2μl 5 pmole/μl RM21 -F 5μl 5 pmole/μl RM21-R 5μl rTaq polymerase (manufactured by TaKaRa Co.) 0.4 [mu] l H ₂ O _22.6μl Total 50.0μl 3-3) PCR reaction conditions denaturation -94 ° C., 30 sec; extension -59 ° C., 30 sec; annealing -72 ° C., 35 cycles of 1 minute, 72
° C, 4 ° C after 10 minutes. For each DNA solution, total DN
The ratio of Koshihikari DNA in A is 1, 1/30, 1
/ 10, 1/33, 1/100, 1/333, 1/10
Dilution systems of 00, 1/3333, 1/10000, and 0 were prepared and used.

As a result, C189ALP and RM21 were 1
Contaminated DNA up to / 33 was reliably detected. G257A
LP could be reliably detected up to 1/100. This is G2
It is believed that 57ALP is a dominant marker. It was judged that the higher the detection sensitivity was, the more suitable it was for estimating the purity. In the following examples, the examination was continued using G257ALP.

Example 3 Based on G257ALP
Japonica varieties and japoni with a deca-type genomic region
Examination of discrimination with mosquito rice varieties DNA polymorphism was investigated in the same manner as in Example 2 using G257ALP for Japonica rice varieties and several indica rice varieties mainly planted in Japan. As a result, it was classified as having an amplified DNA product or not as shown in Table 5 below.

[0073]

[Table 5] Table 5 Genotype Japonica type at G257 locus Indica type cultivar Koshihikari Iwata 3 Kirara 397 Iwata 5 Akihikari Iwata 8 Akitakomachi Starlight Sasanishiki Morning light Kinuhikari Moonlight Nihonharu Aoi wind Hatsusei St. No. 1 Koganeharu IR24 Hinohikari Dual Minaasahi Calotoc Aichi no Kaori Shinsei Dwarf 1 Hatsimo Shinko Akebono IR64 Fujihikari Kasalath -Queen M302 M401 The classification results shown in Table 5 almost matched the classifications of the Japonica varieties and the Indica varieties determined based on the conventional phenotypes and the like. Of the 20 major rice varieties planted in Japan (Rice Map '99, Rice Data Bank), G2
The varieties designated as Indica by 57ALP are only morning light. This is because Morning Light introduced a region containing the stripe blight resistance gene Stv-bi from Modan as in Iwata-3. Therefore, the rice cultivar whose G257 locus is of the Japonica type is different from the stripe blight resistance gene Stv.
It can be said that this is a general Japonica-type variety having no region including -bi.

Based on the above, the main varieties grown by JT (No. 3, No. 5, No. 11, No. 1,
No. 3 and No. 16) were mixed with seeds of general Japonica type varieties except for some varieties having a region containing the stripe blight resistance gene Stv-bi such as morning light,
By using 7ALP, the presence or absence of a general Japonica-type variety can be accurately detected.

In an actual paddy field, when the mixing ratio of a variety having a different morphological characteristic, for example, a long culm variety of seed rice exceeds 0.1%, that is, 1/1000, it becomes conspicuous. Risk factor 5
In order to guarantee 0/1000 in%, it is necessary to investigate 3000 seeds and to show that no different varieties of seeds are mixed therein. However, investigating each of the 3,000 seeds with a PCR marker is labor intensive and inefficient. Therefore, it is desirable that the particles are grouped as much as possible into a bulk, and that the estimation can be performed for each bulk. As described above, since the mixing rate of about 1/100 can be detected with G257ALP, it was considered that 30 bulks should be investigated.

Example 4 DNA extraction from 100 brown rice grains
Establishment of Method For extraction of DNA from plant tissue, the CTAB method or the SDS-phenol method is usually used. Examination of both methods revealed that the SDS-phenol method had low DNA extraction efficiency, and that the DNA was used as a template for PCR.
In some cases, the reaction was inhibited. So, CTA
The B method was adopted. However, in the usual CTAB method, a large amount of starch is mixed in the extracted DNA fraction.
Has been found to be difficult to redissolve.

Therefore, after the ground brown rice is suspended in the CTAB extraction buffer, it is centrifuged (3000 ° C.) at a low temperature (4 ° C.).
rpm, 10 minutes), a large amount of starch precipitated. Then, the DNA is obtained by the usual operation of the CTAB method.
When purified, the amount of starch which was clearly mixed was reduced. Furthermore, when PCR was performed using this DNA as a template, the PCR product was clearly confirmed. Example 5 Examination of Optimal PCR Conditions for G257ALP As confirmed in Example 3, PCR marker G257
ALP is a dominant marker and is characterized in that a PCR product is amplified from a Japonica-type genome but not from an Indica-type genome. However, as a result of the preliminary test, it was clarified that even in rice cultivar Iwata No. 3 having an indica-type genome, a slight amplification of the PCR product occurred. Therefore, the primer was redesigned.

Specifically, similarly to G257R1 used in Examples 2 and 3, the primer chain length is set to 21 mer, and the base sequence is shifted one base at a time toward the 3 ′ end to newly add four primers in both forward and reverse directions. Was synthesized.

As a result of the PCR experiment, F1 / R3 was used in place of the conventionally used primer G257 F1 / R1.
When the DNA of No. 3 was used as a template, the intensity of the G257 band amplified was relatively weak, and the amplification of non-specific DNA was small. Therefore, the subsequent experiments were performed with the combination of F1 / R3.

[0080]

G257F1: 5′-CTGCAGGGTACGTGAACAA-3 ′ (SEQ ID NO: 2) G257R1: 5′-CTGCAGCTGCACGCCATTT-3 ′ (SEQ ID NO: 3) G257R3: 5′-GCAGCTGCACGCCATAA-3 ′ (SEQ ID NO: 8) The conditions were examined. To obtain stable PCR amplification results, the PCR reaction volume was set to 50 ul. Next, when the minimum amount of template DNA required for detecting the PCR product derived from the contaminated seeds was examined, it was found to be 3 ng. This result and 30, 100, 30
From the comparison experiment of the amount of the template DNA of 0 ng, the mixing ratio was 1/10.
At 0, the required amount of template DNA was about 300 ng. Further, when the number of cycles of the PCR reaction was examined from 27, 29, and 31 cycles, 29 cycles were optimal.

Specifically, the conditions shown in Table 2 below were adopted as preferred PCR reaction compositions and PCR reaction conditions.

[0082]

Table 6 PCR reaction composition DNA (approx. 100 ng / μl) 3 μl 10 × TaKaRa Taq Buffer 5 μl 2.5 mM each dNTP 2 μl G257F1 primer (5 μM) 2 μl G257R3 μlαμl 2 μl 2 μl ₂ μl Further, electrophoresis conditions were examined. P amplified under the above conditions
In order to clearly distinguish the presence or absence of the CR product, it was appropriate to run 20 μl of the PCR reaction solution on an agarose gel prepared using relatively wide teeth.

[0083]

Table 7: Electrophoresis conditions Gels are made using combs with wide teeth.

Load about 20 ul of sample. Following electrophoresis, ethidium bromide (EtBr) staining is performed. Except for the above examination items, the conventional PCR method and its modification were followed. Under the above-mentioned PCR reaction composition, PCR reaction conditions, and electrophoresis conditions, the relationship between the mixing ratio of amplified rice and Japonica varieties without the indica-type genomic region mixed with the Japonica varieties having the indica-type genomic region under the conditions of the indica-type genomic region Was confirmed (FIG. 1).

99, 199 or 2 Iwata No. 3 brown rice
One 99 grains of brown rice of Kinuhikari, a common Japonica rice variety, were mixed with 99 grains, and DNA was extracted by an improved CTAB method. A PCR reaction was performed under the above PCR conditions, and electrophoresis was performed. As shown in FIG. 1, as shown in FIG. 1, only trace DNA bands were observed from No. 3 DNA, but clear from the DNA of the test plot in which 99 No. 3 No. 3 particles were mixed with one Kinuhikari. Na D
The NA band was detected.

Example 6 Examination of Contamination Rate Ordinary Japonica rice seed rice was randomly mixed with Iwata No. 3 seed rice. According to the present invention, the contamination rate was estimated, and confirmation by field cultivation was also performed.

Specifically, after the seed rice was turned into brown rice by hulling, 30 batches were prepared with 100 grains as one batch.
DNA was prepared from each batch by a modified CTAB method. A PCR reaction was performed under the above PCR conditions, and electrophoresis was performed.

As a result, clear PCR products were detected in 21 of the 30 batches. That is, it became clear that at least 21 Japonica rice seeds were mixed in 3000 grains. Further, it was considered that there was a possibility that at most about 30 particles were mixed as estimated from the density of the band. Some of the results are shown in FIG.

On the other hand, according to the field survey, 21/3000
Or about 27/3000.
Therefore, the estimated value according to the present invention agreed well with the confirmation by field cultivation.

[0090]

The detection method of the present invention has the following advantages as compared with the prior art. a) Advantages compared to the method of estimating the purity of the seed rice by cultivating the harvested seed rice The result is obtained in about 3 to 4 days after the seed rice is harvested and sampling is performed. In addition, estimation can be made even in the case of the following mixed forms.・ When varieties with the same plant type and heading time are mixed ・ When short culm varieties are mixed with long culm varieties ・ Various varieties with early heading are slightly late When there is a mixture ・ When a diseased variety is mixed with a variety that is characterized by pest and disease resistance b) Advantages compared to the method using microsatellite markers DNA is extracted from each grain because it is compared in units of 100 grains Labor is reduced, and time and reagents are saved. Since a dominant marker is used instead of a co-dominant marker, highly accurate estimation can be performed.

C) Advantages compared to the method of examining the purity of hybrid seeds based on the abundance of male-sterile cytoplasm-specific rice mitochondrial DNA Estimation of seed purity among common Japonica-type cultivated rice varieties having normal cytoplasm Applicable to

[0092]

[Sequence List] SEQUENCE LISTING <110> JAPAN TABACCO INC.Zeneca LIMITED <120> Method for detecting immixed seed rice <130> 012025 <160> 8 <210> 1 <211> 572 <212> DNA <213> Rice <400 > ctgcaggtac gtgaacaata gcacggtacg tacagcgcta tcgtccaagc tgcgaatggt 60 ggaaagcaga gaaatggaac ccggcaaagg cagagaaatg gatctgacag tgaagggttg 120 ggtgcacgtg tttaaagcag ccagacctcc tcaatgatcg agatgcgctt aattaattaa 180 ttaattgtgt tccgggtgat ttcagctttg gatcgagcaa ctgttgttgt ttgttgtgtc 240 gtacgcactc cttccatcaa aagaaaaaaa aaacaaatat aaaattgaat ttgatacaat 300 ttaatacaac taatctgaat ataaatatat cctgattcgt tgtaataaat tgtgttaaat 360 ttaattctag attggtttta tttaagatgg agctgagtat aaactcgtcc agtgagtgcg 420 <? > 2 ctgcaggtac gtgaacaa 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 ctgcagctgc acgccatt 18 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: primer <400> 4 aagatgctgg aggggatcat t 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400 > 5 acgctctgct aacttaacat ca 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 acagtattcc gtaggcacgg 20 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 gctccatgag ggtggtagag 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: primer <400> 8 gcagctgcac gccattaa 18

[Brief description of the drawings]

FIG. 1 is an electrophoretic diagram of PCR products of Iwata No. 3 (Japonica cultivar having an indica genomic region) and / or Kinuhikari (Japonica cultivar having no indica genomic region) under various PCR reaction conditions. Is shown.

FIG. 2 shows an electropherogram of a PCR product for determining the contamination rate of Kinuhikari (a Japonica cultivar having no indica-type genomic region).

Continued on the front page (72) Inventor, Naoto Nitta 700, Higashihara, Toyotamachi, Iwata-gun, Shizuoka Prefecture Inside Orinoba Co., Ltd. (Reference) 4B024 AA08 CA01 HA19 4B063 QA13 QQ42 QR32 QR62 QS25

Claims (12)

[Claims] 1. A method for detecting a Japonica variety seed having no Indica-type genomic region mixed in a Japonica variety seed having an Indica-type genomic region, i) corresponding to the Indica-type genomic region A primer pair is formed based on the nucleotide sequence of the Japonica-type genomic region to amplify the Japonica-type genomic region or a part thereof; ii) Genome DN of single or plural Japonica-type cultivars
A) a nucleic acid amplification reaction is performed using A as a template; and iii) when a nucleic acid amplification product is detected, it is determined that Japonica varieties having no indica-type genomic region are mixed. 2. The detection method according to claim 1, further comprising, in step iii), determining the contamination rate of Japonica varieties having no indica genomic region according to the amount of the nucleic acid amplification product. 3. In the step (i) of preparing a primer pair,
The following conditions 1) -5): 1) The length of each primer is 15-30 bases; 2) The ratio of G + C in the base sequence of each primer is 30-
3) The distribution of A, T, G, and C in the base sequence of each primer is not partially largely biased; 4) The length of the nucleic acid amplification product amplified by the primer pair is 50- The detection method according to claim 1 or 2, wherein the number of bases is 3000 bases; and 5) the absence of a complementary sequence in the base sequence of each primer itself or between base sequences of the primers is satisfied. 4. The method according to claim 1, wherein the nucleic acid amplification in step ii) is performed by PCR. 5. The indica-type genomic region present in the Japonica variety rice paddy is characterized by a stripe blight resistance gene (Stv.
The detection method according to any one of claims 1 to 4, wherein the detection method is a region including -bi). 6. The detection method according to claim 5, wherein the Japonica-type genomic region corresponding to the indica-type genomic region to be amplified contains G257 having the nucleotide sequence of SEQ ID NO: 1. 7. The detection method according to claim 6, wherein the primer pair has the sequence of SEQ ID NOs: 2 and 8. 8. A method according to claim 1, wherein the method is based on the nucleotide sequence of a Japonica genomic region corresponding to an Indica genomic region, which is present in a Japonica variety seed. Primer for nucleic acid amplification. 9. The primer for nucleic acid amplification according to claim 8, for amplifying the base sequence of SEQ ID NO: 1 or a part thereof. 10. The primer for nucleic acid amplification according to claim 9, which has the sequence of SEQ ID NO: 2 or 8. 11. In a cetyltrimethylammonium bromide (CTAB) method for preparing DNA from brown rice, starch is removed by suspending ground brown rice in a CTAB extraction buffer and centrifuging at low temperature. A method that includes: 12. The method according to claim 11, for preparing genomic DNA of a Japonica variety seed rice for use in the detection method according to any one of claims 1 to 7.