JP2000342267A - DNA MARKER LOCATED IN THE VICINITY OF Oryza sativa HYBRID STERILITY LOCUS S-5 LOCUS AND DESCRIMINATION OF GENOTYPE OF S-5 LOCUS USING THE SAME - Google Patents

Abstract

PROBLEM TO BE SOLVED: To cultivate a breed by hybridization between distant relative breeds of Oryza sativa, especially to cultivate a remote crossing hybrid rice by discriminating the genotype of S-5 locus by using specific DNA markers. SOLUTION: The genotype of S-5 locus is discriminated by using DNA markers which are located in the vicinity of S-5 locus which is the hybrid sterility locus of Oriza sativa, and are specified by the base sequences shown by formulas I and II. It is preferable to discriminate the genotype of S-5 locus by carrying out PCR using this set of primers (GTTGGGGTGC AGAAGCGCAT CTTG and ACCGTATGAG GTAGGTTTGA CC) and using DNA extracted from O. sativa to be tested as a template, amplifying a DNA marker located in the vicinity of S-5 locus which is the hybrid sterility locus of O. sativa, followed by determining the genotype of O. sativa to be tested to be S-5n if the amplified DNA marker is cleaved by restriction enzyme XbaI and discriminating the genotype of O. sativa to be S-5i or S-5j if the amplified DNA marker is not cleaved by the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is used for breeding of rice by crossing distantly related varieties, particularly for hybrid rice of distantly related crossing. More specifically, the present invention relates to discrimination of a genotype of a hybrid sterility locus and a DNA marker used for the discrimination.

[0002]

2. Description of the Related Art Hybrid sterility refers to a variety of distantly related varieties such as, for example, Japonica rice (hereinafter sometimes abbreviated as day) and Indica (hereinafter sometimes abbreviated as mark). This indicates a phenomenon in which the fertilization rate of hybrid rice, which is an obtained F1 plant, is reduced when mating is performed.

[0003] Hybrid rice from India and Japan has large heterosis, is vigorous in growth, and has a remarkably large number of paddies. Therefore, if it is put to practical use, it is extremely useful in agriculture. However, as a matter of fact, the above-mentioned hybrid sterility is high, and this strong heterosis is not used for improving rice productivity in rice. If hybrid rice is normally fertile, it can be expected to increase the yield by more than 50% as compared with normal rice, so a method for solving this hybrid sterility through breeding is desired.

[0004] Some knowledge has already been obtained regarding the loci involved in hybrid sterility. Most of the hybrid sterility of the hybrid rice of India and Japan is based on S-5 on chromosome 6.
It has been pointed out that it is due to interactions between alleles at the locus. That is, hybrid sterility is caused by the death of a female gamete having S-5j on a heterozygote of S-5j, a genotype of Japonica species, and S-5i, a genotype of Indica species. to cause. On the other hand, when the Javanica species are used, hybrid sterility does not occur with either the Indica species or the Japonica species. For this reason, the genotype S-5n of the Javanica species is called a hybrid sterility alleviating gene or a broad affinity gene. That is, hybrid sterility does not occur in genotypes of S-5n / S-5j and S-5n / S-5i (H. Ikehashi and H. Araki: Rice Genetics, IRRI, pp. 1).
19-130, (1986)). Therefore, by using the mutual combination of the genes at the S-5 locus, it is possible to grow hybrid rice that does not cause hybrid sterility by crossing Japan and India.

[0005] In order to overcome the hybrid insult of distant crossing between Japan and India, breeding is required in which each allele at the S-5 locus is introduced into the Indica and Japonica varieties.
When using the gene at the -5 locus for breeding hybrid rice, it is necessary to determine which S-5 locus genotype the rice used for crossing has. Conventionally,
The genotype of the S-5 locus in rice can be determined by breeding hybrid rice obtained by crossing the cultivated rice with Indica or Japonica species, examining the fertility of the seed, and clarifying the hybrid sterility. It was done. However, such a method requires a great deal of time and effort. Therefore, a method for easily discriminating the genotype of the S-5 locus at each stage of breeding has been eagerly desired.

[0006] In the case of a trait which is difficult to directly determine a trait such as hybrid sterility, another trait close to the target trait can be used as a marker (trait marker) of the target trait. The presence or absence of a target trait is indirectly determined. The trait of the trait marker must be easier to discriminate than the target trait. In recent years, with rapid progress in DNA analysis technology, a technology for determining the presence or absence of a target gene carrying a target trait at the DNA level has been developed. In this technique, a target gene itself or a base sequence (DNA marker) existing close to the target gene is searched, and the presence or absence of the target gene bearing the target trait is determined by simply detecting the DNA marker. The closer these markers are to the target gene on the chromosome, the higher the contribution to breeding is. For practical breeding, the distance between these markers and the target gene is 5 cM (centimorgan).
It is desirable that:

[0007] For the S-5 locus, trait markers such as the tip color gene (anthocyan chromogen gene C) near S-5n are known. Some RFLP markers linked to the S-5 locus are known as DNA markers at the S-5 locus. Yanagihara et al. Reported that the S-5 locus is located in a 5 cM region between the tip color gene and the RFLP marker RG213 (Yanagihara).
et al., Theor. Appl. Genet., vol. 90, pp. 182-188, (199
Five)). More recently, the RFLP marker R2349 has become S-5
Locus and 1 cM position (K.
D. Liu et al .: Theor.Appl.Genet., Vol. 95, 809-814, (199
7)). However, the method of using these tip color genes and RFLP markers for determining the presence or absence of a target trait is complicated in operation, and has a problem that a great deal of time and labor is required for actual work.

A PCR method has been developed and used as a method for easily amplifying a specific DNA sequence from a very small amount of DNA. It is possible to easily obtain a DNA marker for identifying the target genotype at the S-5 locus using the PCR method, and as such, a DNA marker obtained by converting the RFLP marker RG213 for the PCR method is known. (CEWill
iams et al .: Crop. Sci., vol. 37, pp. 1910-1912). However, when a DNA marker obtained by converting RG213 for PCR is used, the genotype of the target rice is S
Although it was possible to identify -5j or other (S-5n and S-5i), it was not possible to determine whether the genotype was S-5n or S-5i.

[0009] Since the S-5 locus is a locus involved in fertility, the genetic distance varies greatly depending on the test or rice variety, and the precise distance between each marker, the target gene and the DNA marker is not clear. Was. Therefore, the three genotypes S-5n, S-5j, and S-5 at the S-5 locus are further simplified.
i, and those capable of discriminating the genotype in which these are heterozygotes have been required.

[0010]

As described above, the three genotypes S-5n, S-5j and S-5i of the gene related to hybrid asperity located at the S-5 locus of rice and these There is currently no known method for determining the heterozygous genotype. The genotype is D
In order to make a determination using the NA marker, a large number of DNs are required.
A marker, especially D sitting at the position sandwiching the S-5 locus
It is essential to develop an NA marker and dramatically improve the accuracy of the assay, but such a thing has not been known so far.

An object of the present invention is to locate near the S-5 seat,
Three genotypes of the S-5 locus S-5n, S-5j, S-5
i, and DNA markers that can be used to easily determine the genotypes in which they are heterozygous, and three genotypes S-5n, S-5j, and S-5 at the S-5 locus.
It is another object of the present invention to provide a technique for discriminating a genotype in which these are heterozygotes. Specifically, the present invention relates to a DNA that sits near the rice S-5 locus.
An object of the present invention is to provide a marker, a primer for amplifying the marker, and a method for distinguishing three genotypes at the S-5 locus using these DNA markers.

[0012]

Means for Solving the Problems The present inventors RAPD the genomic DNAs of rice with different genotypes at the S-5 locus, that is, rice with genotype S-5n and rice with S-5j.
The comparison was made using the method. As a result, it was possible to identify a DNA specifically amplified in rice having a genotype of S-5n from among a large number of amplified fragments obtained by the RAPD method.

Further, the nucleotide sequence of a DNA fragment specifically amplified in rice having the specified genotype of S-5n is clarified, primers are prepared based on the nucleotide sequence, and the primers are stabilized by PCR. A DNA marker that can be amplified was identified. The DNA marker specified by the present inventors is located near the S-5 locus, and is characterized in that it is amplified in all rice irrespective of the genotype of the S-5 locus. Therefore, when this DNA marker is amplified by the PCR method and then cut with an appropriate restriction enzyme,
The three genotypes of S-5n, S-5i, and S-5j and their heterozygous types can be identified. The present inventors have completed the present invention based on the above findings.

That is, the present invention is as follows. [1] The genotype of the S-5 locus is located near the S-5 locus which is a hybrid sterility locus of rice and defined by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 How to determine. [2] A primer defined by a continuous base sequence having a length of 15 to 40 bases arbitrarily selected from the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 1 or SEQ ID NO: 2 facing the primer And a primer defined by a continuous base sequence having a length of 15 to 40 bases arbitrarily selected from the base sequence described in 1), wherein the DNA marker is located near the S-5 locus which is a rice hybrid sterility locus. Set of primers for detecting C. [3] The set of primers according to [2], wherein the primer sequences are SEQ ID NO: 3 and SEQ ID NO: 4. [4] PCR is performed using the DNA extracted from the test rice as a template using the set of primers according to the above [2] or [3], and S-5, which is a rice hybrid sterility locus,
Amplify a DNA marker located in the vicinity of the locus, determine that the genotype of the tested rice is S-5n when the amplified DNA marker is cleaved by the restriction enzyme XbaI, Is a method for determining the genotype of the S-5 locus, which is determined to be S-5i or S-5j. [5] The genotype of the S-5 locus, which is located near the S-5 locus which is a hybrid sterility locus of rice and defined by the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 How to determine. [6] a primer defined by a nucleotide sequence having a length of 15 to 40 contiguous bases arbitrarily selected from the nucleotide sequence of SEQ ID NO: 5, and a nucleotide sequence of SEQ ID NO: 6 facing the primer S, which is a hybrid sterility locus of rice, comprising a primer defined by a base sequence having a length of 15 to 40 bases which is more arbitrarily selected.
-A set of primers for detecting DNA markers located near the 5 locus. [7] The set of primers according to the above [], wherein the primer sequences are SEQ ID NO: 7 and SEQ ID NO: 8. [8] PCR is performed using the DNA extracted from the test rice as a template using the set of primers according to the above [6] or [7], and S-
The DNA marker located near the 5 loci was amplified, and when the amplified DNA marker was cut with the restriction enzyme BfaI, the genotype of the tested rice was determined to be S-5n, and the restriction enzyme HhaI was detected. In the case of cleavage, the genotype of the tested rice is determined to be S-5i, and when not cut by any of the restriction enzymes, the genotype of the tested rice is determined to be S-5j. A method for determining the genotype of the -5 locus. [9] Using a rice total DNA as a template and amplifying a DNA marker by PCR using primers defined by the nucleotide sequence of SEQ ID NO: 9, the DNA marker was amplified near the S-5 locus, which is a hybrid sterility locus of rice. A method for determining the genotype of the S-5 locus, wherein the genotype of the tested rice is determined to be S-5n when amplification of a DNA marker of about 0.9 kb is performed. [10] The method according to the above [9], wherein a DNA marker of about 0.9 kb located near the S-5 locus which is a hybrid sterility locus of rice is defined by the nucleotide sequence of SEQ ID NO: 10. The described discrimination method. [11] Using a rice total DNA as a template and amplifying a DNA marker by PCR using primers defined by the nucleotide sequence of SEQ ID NO: 11, the DNA marker was amplified near the S-5 locus, which is a hybrid sterility locus of rice. When the positioned DNA marker of about 1.4 kb was amplified, the rice genotype was S-type.
A method for determining the genotype of the S-5 locus, which is determined to be 5n. [12] The base sequences at both ends of the approximately 1.4 kb DNA marker located near the S-5 locus, which is a rice hybrid sterility locus, are the base sequences of SEQ ID NO: 5 and SEQ ID NO: 6 The determination method according to the above [11], characterized by: [13] Located near the S-5 locus which is a rice hybrid sterility locus, the base sequence described in SEQ ID NO: 1 and SEQ ID NO: 2, the base sequence described in SEQ ID NO: 5 and SEQ ID NO: 6, or the SEQ ID NO: 1
A DNA marker defined by the nucleotide sequence described in 0.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA marker at the S-5 locus which is the rice hybrid sterility locus referred to in the present invention is DNA located near the rice hybrid sterility locus S-5 locus, By examining the nucleotide sequence of DNA in rice cells,
-5n, S-5i, and S-5j can be identified.

In the present invention, "near" means a distance at which the DNA marker according to the present invention is practically used for breeding rice. It is known that the distance by genetic analysis varies depending on the type of individual population and the number of individuals used in the analysis. In this sense, the neighborhood in the present invention specifically means that the distance between the DNA marker and the target gene is 1
It is within 0 cM (centiMorgan), preferably within 5 cM. To measure the distance between the DNA marker and the target gene, for example, in the case of the present invention, the genotype S
-5n, Athen 2 and S-5j, Japonica species M
Indica species IR of genotype S-5i in F1 of HB25
Calculated by analyzing about 250 individuals crossed with 36. D located near the S-5 locus of the rice of the present invention
Examples of the NA marker include DNAs having the nucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6.

Next, a method for identifying a DNA marker at the S-5 locus of rice will be described. First, a DNA marker in the vicinity of the S-5 locus is identified by comparing the differences in the nucleotide sequences of the entire genomes of a plurality of rice plants having different genotypes at the S-5 locus. Rice having a different genotype at the S-5 locus includes:
Examples include F2 obtained by crossing two varieties having different genotypes at the S-5 locus, a near-isogenic line related to the S-5 locus, an individual in the middle of backcrossing for raising a near-isogenic line, and the like. As a method for comparing the difference in the nucleotide sequence of the whole genome, the RAPD method, the AFLP method, or the known RFLP method is used.
There is a method using a marker, a microsatellite marker, or the like. By using these methods and comparing the genomic DNAs of rice having different genotypes at the S-5 locus,
A DNA marker located near the locus can be identified.

For example, a plurality of rice having a genotype S-5n and a plurality of rice genomes DN having a genotype S-5j
By comparing A using the RAPD method, the RAPD
By the method, a plurality of DNA markers that specifically amplify only the rice having S-5n can be identified.

Furthermore, the identified DNA marker at the S-5 locus can be easily and stably amplified using the PCR method. For that purpose, the base sequence of the DNA marker is determined. The nucleotide sequence can be determined by directly cloning the DNA marker or by cloning into a vector.
Based on the determined base sequence, a primer consisting of 15 to 40 bases is designed, and the DNA marker near the S-5 locus is
Amplify using CR method. Furthermore, by using the TAIL-PCR method or the inverse-PCR method, S-5
It is possible to successively obtain DNA markers adjacent to the locus and DNA markers adjacent to the locus.

For example, one S-5n obtained by the RAPD method
The DNA fragment located in the vicinity of the gene was cloned into a vector by TA-cloning method,
The nucleotide sequences at both ends of the A marker excluding the central portion are the nucleotide sequences described in SEQ ID NOS: 5 and 6. Based on the nucleotide sequence of another DNA marker located near S-5n represented by SEQ ID NO: 10 obtained in the same manner, TAIL-PC
R is performed to obtain a base sequence adjacent to the DNA marker obtained by the RAPD method. Thereby, a DNA marker having a longer nucleotide sequence than the DNA marker of the RAPD method can be specified. Sequences of the DNA marker thus obtained include, for example, SEQ ID NO: 1 and SEQ ID NO: 2.

Next, a method for discriminating the genotype of the S-5 locus using a DNA marker which can be easily and stably amplified by the PCR method will be described. First, DNA as a template is extracted from the rice to be tested. As a tissue for extracting the DNA, any part of a plant such as a root, a leaf, or a seed can be used. As a method for extracting DNA, any method can be used, and a roughly purified DNA can also be used. Using the extracted DNA as a template, the DNA marker
Amplify by CR method.

As a method of discriminating a genotype using the PCR method, (1) a method of amplifying a specific fragment only in a certain genotype and discriminating the genotype based on the presence or absence of the amplification;
(2) a method of determining the genotype by comparing the lengths of the amplified fragments based on the fact that the lengths of the amplified fragments differ between genotypes; (3) a method of amplifying all genotypes having the same length When a fragment is treated with a certain restriction enzyme, there is a method of determining the genotype by comparing the cleavage patterns based on the fact that the cleavage pattern differs depending on the genotype. PC to suit each of (1), (2) and (3)
Design the R primer. In the discrimination methods (2) and (3), it is possible to simultaneously discriminate whether a genotype is homozygous or heterozygous.

For example, a fragment amplified by PCR using the oligonucleotides represented by SEQ ID NO: 7 and SEQ ID NO: 8 as primers is treated with a restriction enzyme BfaI to give S-
In 5n rice, the fragment is cut, but the genotype is S-5.
It is not cut in rice that is j or S-5i. On the other hand, when the same amplified fragment is treated with the restriction enzyme HhaI, the fragment is cleaved in rice having the genotype S-5i,
It is not cut in rice of n or S-5j. When all of the PCR amplified fragments were cut with the restriction enzyme BfaI, S-
5n / S-5n, S-5n / S-5i or S-5n / S-5j when partially cut with the restriction enzyme BfaI,
If it is not cut at all, S-5i / S-5i or S-5i
5j / S-5j or S-5i / S-5j.
When all of the PCR amplified fragments were cut with the restriction enzyme HhaI, S-5i / S-5i, and a part thereof was restricted with the restriction enzyme HhaI.
S-5i / S-5n or S-5i
/ S-5j, S-5n / S- if not cut at all
5n or S-5j / S-5j or S-5n / S-5j.

In the present invention, the term "sandwich" or "sandwich" refers to the 3'-side DN with respect to a certain base sequence.
This indicates that the arbitrary base sequences of the A region and the 5′-side DNA region exist in pairs.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments. Example 1 Ketan Nangka of Javanica
Roken No. 2 with the hybrid sterility alleviation gene S-5n and Japonica MHB25 with S-5j
gers and Bendich's method (Rogers, SO. and Bendich, AJ., P
Total DNA was extracted by the CTAB method according to lant Mol. Biol., VOL. 5, PP. 69-76). Using the obtained total DNA as a template, a reaction of the RAPD method was carried out using 1000 kinds of commercially available 10-mer oligonucleotides (Operon) as primers. The composition of the reaction solution and the reaction conditions for the RAPD method are as follows. The reaction solution was 10 mM Tris-HCl.
(PH8.3), 50 mM KCl, 2 mM MgCl
₂ , 0.001% gelatin, 100 each
μM dATP, dCTP, dGTP and dTTP,
3 pmole primer, 15 ng template DNA, 0.
3 units Ampli Taq DNA polym
erase (Perkin-Elmer) and adjusted with water to a total of 15 μl. The reaction cycle was held at 94 ° C. for 2 minutes, and then repeated 45 times at 94 ° C. for 10 seconds, 40 ° C. for 1 minute and 72 ° C. for 1 minute.
The reaction was carried out at 10 ° C for 10 minutes, and then maintained at 4 ° C. GeneAmp PCR System 9600 type (Perkin-Elmer) was used for the reaction. After the reaction,
10 μl of the reaction solution was electrophoresed with 2.0% agarose containing 0.5 μg / ml ethidium bromide and TAE buffer (pH 7.6) to detect amplified polymorphism under UV irradiation. As a result, 147 DNA fragments specifically amplified by KEN No. 2 and 182 DNA fragments specifically amplified by MHB25 were detected.

[Example 2] MHB25 and F1 of Atsushi No. 2
MHB25 was backcrossed to an individual (MHB25 /
From the Institute of Thermal Engineering No. 2 // MHB25), the tip color and RF
From the analysis of the LP marker RG213, 3 individuals estimated to have S-5n and 1 individual estimated to not have S-5n
Individuals were used. From these 4 individuals, all D
The NA was extracted and used as a template.
Analysis by the APD method was performed. As a result, of the 147 DNA fragments specifically amplified in Athen 2
Three DNA fragments that were amplified in common by three individuals having 5n and were not amplified by one individual without S-5n were found.

[Example 3] MHB25 and F1 of Atsushi No. 2
MHB25 was backcrossed to an individual (MHB25 /
Atsushi No. 2 // MHB25), and MHB25 and Atsushi 2
MHB25 was backcrossed twice to F1 of No. 2 (MHB25 / Nken 2 // MHB25 /// MHB2
DNA was obtained from a total of 19 individuals of 5) by the method of Edwards et al. (Nucleic Acids Research, Vol. 19, No. 6 p. 1349, (199)
The crude extraction was performed by 1)). The 19 individuals include the four individuals of Example 2. Using this as a template and three primers to amplify the three fragments selected in Example 2,
The reaction of the RAPD method was carried out by the method described above. In addition, the tip color and the genotype of the RFLP marker RG213 were examined for 19 individuals. The genotype of the RFLP marker RG213 is S
All individuals having -5n / S-5j exhibited a tip color.
The genotype of RFLP marker RG213 is S-5n / S-
5j, the fragment of about 0.9 kb was amplified when the primer represented by SEQ ID NO: 9 was used, and when the primer represented by SEQ ID NO: 11 was used, about 1.
A 4 kb fragment was amplified. On the other hand, RFLP marker R
Individuals whose G213 genotype was S-5j / S-5j did not show amplification of these fragments. From this result, it became clear that two of the three DNA fragments selected in Example 2 were linked to RG213 and the tip color.

[Example 4] Total DNA was extracted from the leaves of Atsushi No. 2, MHB25, Akihikari, Japanese Masali, and Ketan Nangka by the method of Example 1. Using this as a template, RAPD analysis was performed by the method of Example 1 using the primers of SEQ ID NO: 9 and SEQ ID NO: 11 that amplify the two fragments obtained in Example 3. As a result, a fragment of about 0.9 kb in SEQ ID NO: 9
In No. 1, a fragment of about 1.4 kb was specifically amplified only when a cultivar having S-5n, namely, Kenken No. 2 or Ketan Nangka was used as a template.

Example 5 Approximately 0.9 kb and about 1.4 kb fragments amplified in Example 4 using Asahi No. 2 as a template were recovered from an agarose gel, and were subjected to a TA cloning kit (In Vitrogen). Plasmid p
Subcloned into CRTMII. The nucleotide sequence of the obtained clone was compared with a 373S DNA sequencer (AB
(Company I). The thus obtained nucleotide sequence of about 0.9 kb amplified by the primer of SEQ ID NO: 9 is shown in SEQ ID NO: 10. For the fragment of about 1.4 kb amplified by the primer of SEQ ID NO: 11, the nucleotide sequences at both ends excluding the center are shown in SEQ ID NOs: 5 and 6.

Example 6 Atsushi No. 2, MHB25, IR
PCR was performed using all DNAs extracted by the method of Example 1 from No. 36, IR24 and Asominori as a template and primers synthesized based on the nucleotide sequence obtained in Example 5. That is, with respect to the DNA marker represented by SEQ ID NO: 10, the combination of the primers represented by SEQ ID NO: 12 and SEQ ID NO: 13
For markers, PCR was performed with the combination of the primers of SEQ ID NO: 7 and SEQ ID NO: 8. The composition of the reaction solution and the reaction conditions for this PCR are as follows. 10 mM
Tris-HCl (PH8.3), 50 mM KCl,
1.5 mM MgCl ₂ , 0.001% gelati
n, 200 μM each of dATP, dCTP, dGT
P and dTTP, each 12 pmole primer, 1
ng template DNA, 0.3 units Ampli Ta
q DNA polymerase (Perkin-E
1mer) and adjusted with water to a total of 15 μl. The reaction cycle was held at 94 ° C. for 2 minutes,
After repeating repetition of 10 seconds at 60 ° C., 30 seconds at 60 ° C. and 30 seconds at 72 ° C. for 40 times, the reaction was carried out at 72 ° C. for 10 minutes, and then maintained at 4 ° C. PCR is GeneAmp PCR
System 9600 type (Perkin-Elme
r company). After the reaction, 10 μl of the reaction solution was
0.8 containing 0.5 μg / ml ethidium bromide
% Agarose and TAE buffer (pH 7.6), and the reaction product was detected under UV irradiation. Regardless of which primer combination was used, the DNA marker was amplified in all varieties used.

[Example 7] Using as a template all the DNAs of Nken No. 2, IR36 and MHB25 extracted by the method of Example 1,
Using the primer combination of SEQ ID NO: 7 and SEQ ID NO: 8, a DNA marker was amplified by PCR according to the method of Example 6. The obtained reaction product was treated with the restriction enzyme HhaI or the restriction enzyme Bfa1, and fractionated by agarose gel electrophoresis. When treated with the restriction enzyme HhaI, IR3
When only the DNA fragment amplified from No. 6 was cleaved and treated with the restriction enzyme Bfa1,
Only the NA fragment was cut. That is, the DNA marker near the S-5 locus amplified by the PCR method is a restriction enzyme H.
Genotype S-5i when digested with haI, genotype S-5n when digested with restriction enzyme Bfa1, and genotype S-5j when digested with none of the restriction enzymes. Was.

[Example 8] DNA of SEQ ID NO: 10 amplified by the primer represented by SEQ ID NO: 9 in Example 3
The region adjacent to the marker was amplified and cloned by the TAIL-PCR method. TAIL-PCR was performed according to Example 1.
The method was performed according to the method described below using the DNA of Atsushi No. 2 and MHB25 extracted as described above as a template. All reactions were performed with GeneAmp PCR System 9600 (P
erkinElmer). 10 ng of template DN was contained in 20 μl of the reaction solution of the first PCR.
A, 0.5 units TAKARA Ex Taq
(TAKARA), 4nmole dNTP, 10pm
ole primer, 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 mM MgCl ₂
Is included. PCR conditions were 95 ° C. for 10 seconds denature, 55 ° C. for 30 seconds annealing, and 72 ° C. for 30 seconds.
The elongation reaction for one second was defined as one cycle, and the reaction was performed for 40 cycles. The combination of SEQ ID NO: 19 and SEQ ID NO: 14 and the combination of SEQ ID NO: 19 and SEQ ID NO: 16 were used as PCR primers.

0.5 units of TAKARAEx Taq (TAK) was contained in 20 μl of the reaction solution of the second PCR.
ARA), 4 nmole dNTP, 10 pmole
Primer, 10 mM Tris-HCl (pH 8.
3), 50 mM KCl, 1.5 mM MgCl ₂ . To this, 1 μl of the first reaction solution diluted with 100 μl of sterile water was added as a template. P
CR conditions were 95 ° C. for 10 seconds denature, 55
Annealing at 30 ° C. for 30 seconds and elongation at 72 ° C. for 30 seconds were defined as one cycle, and 40 cycles were performed. The combination of SEQ ID NO: 19 and SEQ ID NO: 3 and the combination of SEQ ID NO: 19 and SEQ ID NO: 17 were used as PCR primers.

Further, 0.5 units of TAKARAEx Taq (TAK) was contained in 20 μl of the third reaction solution.
ARA), 4 nmole dNTP, 10 pmole
Primer, 10 mM Tris-HCl (pH 8.
3), 50 mM KCl, 1.5 mM MgCl ₂ . To this, 1 μl of the second reaction solution diluted with 100 μl of sterilized water was added as a template. P
CR conditions were 95 ° C. for 10 seconds denature, 55
Annealing at 30 ° C. for 30 seconds and elongation at 72 ° C. for 30 seconds were defined as one cycle, and 40 cycles were performed. The combination of SEQ ID NO: 19 and SEQ ID NO: 15 and the combination of SEQ ID NO: 19 and SEQ ID NO: 18 were used as PCR primers.

The third reaction solution was fractionated with 0.8% agarose, and amplified about 0.8 kbp and about 0.4 kb.
The amplified DNA fragment of p was recovered from the gel and subcloned into the plasmid pCRTMII using a TA cloning kit (In Vitrogen). The nucleotide sequence of the obtained clone was determined using a 373S DNA sequencer (ABI). The clone derived from Atsushi No. 2 contained DNA markers represented by SEQ ID NOs: 1 to 819 and 1589 to 2012. On the other hand, the clone derived from MHB25 contained the DNA markers represented by SEQ ID NOs: 1 to 820 and 1590 to 2013.

Example 9 In the sequence obtained in Example 8, in SEQ ID NO: 1 derived from Atsushi No. 2, bases from SEQ ID NOs: 667 to 672 were the recognition sequence of the restriction enzyme XbaI. On the other hand, in SEQ ID NO: 2 derived from MHB25, the base at position 669 corresponding to base C at position 668 in SEQ ID NO: 1 was T, and was not a recognition sequence for restriction enzyme XbaI. P is inserted between the restriction enzyme XbaI recognition sequence.
CR primers were designed and PCR was performed. As a template for the PCR, the thermolab No. 2, MHB25, I used in Example 7 was used.
R36 genomic DNA was used. The oligonucleotides shown in SEQ ID NO: 3 and SEQ ID NO: 4 were used as primers. The DNA marker amplified by PCR according to Example 6 was treated with a restriction enzyme and fractionated on an agarose gel. As a result, the fragments of the desired length were amplified in all the varieties used, and only the DNA fragments amplified from Atsushi No. 2 were cut by the restriction enzyme XbaI treatment. That is, PCR
Genotype S-5n when the DNA fragment in the vicinity of the S-5 locus amplified by the method was cleaved with the restriction enzyme XbaI, and genotype S when the DNA fragment was not cleaved with the restriction enzyme XbaI.
-5j or S-5i could be determined.

[Example 10] SEQ ID NO: 1 and SEQ ID NO: 2,
A DNA marker represented by SEQ ID NO: 5 or SEQ ID NO: 6,
The genetic distance and positional relationship with known markers near the S-5 locus were determined. MHB25 (S-5j) and F1 of Atsushi No. 2 (S-5n) and IR36 (S-5i) were crossed to obtain 263 individuals of "MHB25 / Atsushi No. 2 // IR36". The genotype of these individuals at the S-5 locus is S-5.
It is a hetero type of j / S-5i or S-5n / S-5i. A DNA was roughly extracted from the 263 plants by the method of Example 3 as a template, and PC was obtained by the method described in Example 6.
The DNA marker amplified by the R method was treated with a restriction enzyme and fractionated on an agarose gel. For the tip color gene, the tip color was visually observed after heading. Eight of the 263 individuals examined had recombination between the RFLP marker RG213 and the tip color gene.
The distance between 213 and the tip color gene is Kosambi ((1944)
Ann Eugen 12: 172-175), 3.0 cM ±
It was estimated to be 1.1 cM. In addition, 263 RG21
3. As a result of analyzing the genotypes of the tip color gene and the DNA marker, the DNA markers represented by SEQ ID NO: 1 and SEQ ID NO: 2, and SEQ ID NO: 5 and SEQ ID NO: 6 were RFLP marker R
It was found that the gene was located between G213 and the tip color gene. That is, the DNA markers represented by SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 5 and SEQ ID NO: 6 are located between known markers sandwiching the S-5 locus at a distance of 5 cM. Was determined to be 5 cM or less.

[0038]

According to the present invention, a plurality of DNA markers located near the S-5 locus of rice are provided. By using the DNA marker of the present invention to identify the genotype of the S-5 locus by a method combining PCR and restriction enzyme treatment, it is easy to discriminate hybrid sterility which is a phenotype of the S-5 locus in rice. Can be performed.

[0039]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2012 Sequence form: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism: Oryza sativa L. Strain name: cultivar "heat Labs No. 2" sequence: CATCGGCGGA GAGGAAATCT GTAAAAGAAG ATCAGCTAAC AGTTGGTGTT GCAGAGCTGA 60 AAAAAACGAT GTATGTCAAT GAACAACATG CTTGATGTAT ATGAAGATAC ATATTTATGT 120 ATGCAAGATA TGAAAAAGTA CGAAATGAGA GTGAATAAAG TGCGATATAT GTAAATGGTA 180 ACATCTAAAT TTGGCACCTA AGACGGAAAA ACAGGAAAAC GCGGTCAAAT TTGGAAAAGA 240 CCCGGTTTAC CGTATGAGGT AGGTTTGACC GCCGCATGGA GTCAGTCAGA CCGACTTCAA 300 GCCCAATTTG TCGGTTCTTG GGTTTTCTTC ATTGACCCTG GGTTTTTTTT TAAGTTCTTA 360 TTGATCTCTA AACTTAGTAG GACATAAAAA AGATCCTGTG GAGACAAGAC AAACTTGTTT 420 ATAAGAGCTA TGGCTAGTTC AATTTTTAAC ACCCTAACCA TAATCCCTAC AATCTATCGA 480 ATCATTCTTT TGTCTTTACT TTTTAGGCAT GGTTTCTACT TTACCCATGT TTTGTCTATC 540 TTTATCGATT TGTGTCGTAA ATTGTCCTCC TTCGCTGCTC GAGTGCAATA TCCTTTGTAG 600 GTTTGTCCCA TAAATTTTCT AATTCACCCA CGAGATGATA GTTATATCCA TATCGATCTA 660 AATCGGTCTA GAGGCTAGTT TTGATATTTA AATCGACTCT GCTAGTCGGT TTAATCTTCA 720 AAGCCCATCT TGGTTTAGAG TTTTCTAAAT CAAGGTGATT CGTGACTCCA TTAACACCGA 780 AAATCATTAG GTAAGTCCGT GTTGGCTAGA AAAGGCATCA ACA ATAAATA TCCATAAGAA 840 AAAAATGGTT AAAACAAGAT GAGCTTCTGC ACCCCAACAC AACAAGCAAC AATCGAGTAG 900 CTAAATGCAT TCACAATAGA AGCAACTTAT ATGAGTGTCC TTAAGATGCC ATACAGGACT 960 AAGACCACAC CTCTAAAAAG TTATTTTACC ATAGTGCAAG CGGCTTCAAG TAATAACCTC 1020 CACTTTTCAT GTCCGTCTTG ATATTTTTCT ATTTCAGTCC TCATATTTTA TTAATTCATA 1080 TGGAAGTCCA AAATTTTATA CTATACATAC CAAAATACAT AAACTAATAT AAAATCTCTC 1140 CTCACGTCTT CCCCTCTACC ATTCTCTACC ACCCCCTCTC CACTATTCTT TGTCGTGTTT 1200 CTTCTCCCGA GTCCTTCTCT TGTCACCCCT AAACTTTTTA ATCTAGTCAG CCATTGCCAA 1260 ACATCATTGC ATCGTCGAGC GCATGACACC GTCCTTCTCG TCTCGATGCC AAAGTAACCC 1320 AAGCTGACAC TAGGTCAATT TTCTGCCACG GACAATCAAT CATGCCAAAG CCTCGTCGGA 1380 GCTTGTTATG AGCTCACCAT GCTGGCCACG AGCTCGTAGG TGGTGGGTAG ACGGGCTCGG 1440 ACTCGTCGTC ATCATCTTCC TGGTCAACGG TGACGAGGTC CCCCAGCTGG TATGTCCCTC 1500 CCAACCACCG CTCCTCCACT CTCCATATTT GGCCACCGTC GTCCGCTATC TCCATCCACC 1560 AACTTCATTT ATTTTATCGG ACGTTGTTCT CCAGTAACCG CTCTAGCTCA ACCCTCTAGA 1620 TCTAGCGGCA GAAAAGTACC ACACACCGTG TGCAGCGTGA CGTGTTCGCC C AACTTAGAT 1680 GTCTACTTAC TGAAGAAACC ATGTCTGCTG CAAGGCACGA ACACCTTATT ACCGAGGCCA 1740 CTCACCCACA CCCCCGCTCT TCGCAGCAGC ATGGCTCCTA TGTGGCAACC AGCAAGCTAA 1800 AGTCACTCTA CCACTCATCA TGTAGGAGCG CTGTGAATGG CCTAATTCAA CCATCACTAC 1860 ATGCATGCAG CGCAGAGATG CAGTTGATAA CAGTACGGAA CAACGTAACC TCCTTGCTCG 1920 ATCGATCGAC CTCGCTACAC CATCCCTACT ACAAAGCCTA CAGCAATCCT ACTCCATCAG 1980 AGACACTGAC CCATCAACAT CTGACTCGCA CA 2012

SEQ ID NO: 2 Sequence length: 2013 Sequence shape: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism name: Oryza sativa L. Strain name: cultivar "MHB25" sequence: CATCGGCTGA GAGGAAATCT GTAAAAGAAG ATCAGCTAAC AGTTGGTGTT GCAGAGCTGA 60 AAAAAACGAT GTATGCCAAT GAACAACATG CTTGATGTAT ATGAAGATAC ATATTTATGT 120 ATGCAAGATA CGAAAAAGTA CGAAATGAGA GTGAATAAAG TGTGATATAT GTAAATGGTA 180 ACATCTAAAT TTGGCACCTA AGATGGAAAA ACAGGAAAAC ACGGTCAAAT TTGGAAAAGA 240 CCCGGTTTAC CGTATGAGGT AGGTTTGACC GCCGCATGGA GTCAGTCATA CCGACTTCAA 300 GCCCAATTTG TCGGTTCTTG GGTTTTTTTC ATTGACCCTG GGTTTTTTTT TTAAGTTCTT 360 ATTGATCTCT AAACTTAGTA GGACATAGAA AAGATCCTGT GGAGACAAGA CAAACTTGTT 420 TATAAGAGCT ATGGCTAGTT CAATTTTTAA CACCCTGACC ATAATCCCTA CAATCTATCG 480 AATCATTCTC TTGTCTTTAC TTTTTAGGCA TGGTTTCTAC TTTACCCATG TCTTGTCTAT 540 CTTTATCGAT TTGTGTCGTA AATTGTCCTC CTTCGCTGCT CGAGTGCAAT ATCCTTTGTA 600 GGTTTGTCCC ATAAATCTTC TGATTCACCC ACGAGATGAT AGTTATATCC ATATCGATCT 660 AAGTCGGTTT AGAGGCTAGT TTTGATATTT AAATCGACTC TGCTAGTCGG TTTAATCTTC 720 AAAGCCCATC TTGGTTTAGA GTTTTCTAAA TCAAGGTGAT TCGTGACTCC ATTAACACCG 780 AAAATCATTA GGTAAGTCCG TGTTGGCTAG AAAAGGCATC AACAATAAAT ATCCATAAGA 840 AAAAAATGGT TAAAACAAGA TGAGCTTCTG CACCCCAACA CAACAAGCAA CAATCGAGTA 900 GCTAAATGCA TTCACAATAG AAGCAACTTA TATGAGTGTC CTTAAGATGC CATACAGGAC 960 TAAGACCACA CCTCTAAAAA GTTATTTTAC CATAGTGCAA GCGGCTTCAA GTAATAACCT 1020 CCACTTTTCA TGTCCGTCTT GATATTTTTC TATTTCAGTC CTCATATTTT ATTAATTCAT 1080 ATGGAAGTCC AAAATTTTAT ACTATACATA CCAAAATACA TAAACTAATA TAAAATCTCT 1140 CCTCACGTCT TCCCCTCTAC CATTCTCTAC CACCCCCTCT CCACTATTCT TTGTCGTGTT 1200 TCTTCTCCCG AGTCCTTCTC TTGTCACCCC TAAACTTTTT AATCTAGTCA GCCATTGCCA 1260 AACATCATTG CATCGTCGAG CGCATGACAC CGTCCTTCTC GTCTCGATGC CAAAGTAACC 1320 CAAGCTGACA CTAGGTCAAT TTTCTGCCAC GGACAATCAA TCATGCCAAA GCCTCGTCGG 1380 AGCTTGTTAT GAGCTCACCA TGCTGGCCAC GAGCTCGTAG GTGGTGGGTA GACGGGCTCG 1440 GACTCGTCGT CATCATCTTC CTGGTCAACG GTGACGAGGT CCCCCAGCTG GTATGTCCCT 1500 CCCAACCACC GCTCCTCCAC TCTCCATATT TGGCCACCGT CGTCCGCTAT CTCCATCCAC 1560 CAACTTCATT TATTTTATCG GACGTTGTTC TCCAGTAACC GCTCTAGCTC AACCCTCTAA 1620 ATCTAGCGGC AGAAAAGTAC CACACACCGT GTGCAGCGTG ACGTGTTCG C CCAACTTAGA 1680 TGTCTACTTA CTGAAGAAAC CATGTCCGCT GCAAAGCACG AACACCTTAT TACCGAGGCC 1740 ACTCACCCAC ACCCCCGCTC TTCGCAGCAG CATGGCTCCT ATGTGGCAAC CAGCAAGCTA 1800 AAGTCACTCT ACCACTCATC ATGTAGGAGC GCTGTGAATG GTCTAATTCA ACCATCACTA 1860 CATGCATGCA GCGCAGAGAT GCAGTTGATA ACAGTACGGA ACAACGTAAC CTCCTTGCTC 1920 GATCGATCGA CCTCGCTACA CCATCCCTAC TACAAAGCCT ACAGCAATCC TACTCCATCA 1980 GAGACACTGA CCCATCAACA TCTGACTCGT ACA 2013

SEQ ID NO: 3 Sequence length: 24 Sequence form: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GTTGGGGTGC AGAAGCGCAT CTTG 24

SEQ ID NO: 4 Sequence length: 22 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: ACCGTATGAG GTAGGTTTGA CC 22

SEQ ID NO: 5 Sequence length: 480 Sequence form: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism name: Oryza sativa L. Strain name: cultivar "heat Labs No. 2" sequence: GAGGGACCTC GGGGTTGAAA ACTACACATC CCTTAAAACT GCACTATTCG GTGCATCTCA 60 CTACCTTGCA GAGGTCACAA CCAACACCAT CAGTGATGAA TCCATGATGT AAATTGAGGG 120 GGTGCTAGTG TAAAAATACA ATATGATTAA AACAAATAAA GATATATCAA CTAAAGTATT 180 TTCCTATGAA AACCAAATGT GCATATGATC AAAAGAACTT TCTCTATTTT GATTTGTGGC 240 TATATTACTA ATATATGTGA ATATATTGTT ATGTATACTC GGAGTAAGTT TTGTTTTATC 300 AGTCTGGGTA GTTTGGGTAG TAGAAGTAGT GATGCAACCT GTGTCCTTGT CATCTAGGAA 360 ATATCTTAGG AATAGAATTT CGGGATACTC TTGGTGGATG CTATTACTGG GATAGGCGAG 420 AGATAGGAGG AGAGAGGCGC CGTGATTTCG GGTGGGCGTT CTGCGTGGGA GGGGAGGCTG 480

SEQ ID NO: 6 Sequence length: 360 Sequence form: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism name: Oryza sativa L. Strain name: cultivar "heat Labs No. 2" sequence: ATAGTCCCGG CCGTTTAATT CGTCTCATTT TTGTATCACC GGTACGTTCA GCTACTACTA 60 TATATACATA CCACCGCTGC GTTGTGGCTT GGAAATGACC AGACACTAAA CCTGCATGAC 120 TGCATCCGTA GGAGTGCAGA GGAAACTTCA ATTCAGCCAA CGAAAGGGTC TCGTTAATCA 180 ACTTTTCCAC GTATGTATAT ATGTTCATGT GACCATTTGG TTGAGAGTTA TGACGAGCAA 240 AGTTGACTAG CTAGCTGTTT GTCATGATTA GACATATGTA GTAACATATG TAGTAACATA 300 TGTCTAATCA TGACAAAACA GGGTGTGATT TACTTAATTT GCAGACGGAT GAGGTCCCTC 360

SEQ ID NO: 7 Sequence length: 23 Sequence form: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: CTACACATCC CTTAAAACTG CAC 23

SEQ ID NO: 8 Sequence length: 24 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: AGTAGCTGAA CGTACCGGTG ATAC 24

SEQ ID NO: 9 Sequence length: 10 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GGTCTAGAGG 10

SEQ ID NO: 10 Sequence length: 958 Sequence form: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism name: Oryza sativa L. Strain name: cultivar "heat Labs No. 2" sequence: GGTCTAGAGG CTAGTTTTGA TATTTAAATC GACTCTGCTA GTCGGTTTAA TCTTCAAAGC 60 CCATCTTGGT TTAGAGTTTT CTAAATCAAG GTGATTCGTG ACTCCATTAA CACCGAAAAT 120 CATTAGGTAA GTCCGTGTTG GCTAGAAAAG GCATCAACAA TAAATATCCA TAAGAAAAAA 180 ATGGTTAAAA CAAGATGAGC TTCTGCACCC CAACACAACA AGCAACAATC GAGTAGCTAA 240 ATGCATTCAC AATAGAAGCA ACTTATATGA GTGTCCTTAA GATGCCATAC AGGACTAAGA 300 CCACACCTCT AAAAAGTTAT TTTACCATAG TGCAAGCGGC TTCAAGTAAT AACCTCCACT 360 TTTCATGTCC GTCTTGATAT TTTTCTATTT CAGTCCTCAT ATTTTATTAA TTCATATGGA 420 AGTCCAAAAT TTTATACTAT ACATACCAAA ATACATAAAC TAATATAAAA TCTCTCCTCA 480 CGTCTTCCCC TCTACCATTC TCTACCACCC CCTCTCCACT ATTCTTTGTC GTGTTTCTTC 540 TCCCGAGTCC TTCTCTTGTC ACCCCTAAAC TTTTTAATCT AGTCAGCCAT TGCCAAACAT 600 CATTGCATCG TCGAGCGCAT GACACCGTCC TTCTCGTCTC GATGCCAAAG TAACCCAAGC 660 TGACACTAGG TCAATTTTCT GCCACGGACA ATCAATCATG CCAAAGCCTC GTCGGAGCTT 720 GTTATGAGCT CACCATGCTG GCCACGAGCT CGTAGGTGGT GGGTAGACGG GCTCGGACTC 780 GTCGTCATCA TCTTCCTGGT CAACGGTGAC GAGGTCCCCC AGC TGGTATG TCCCTCCCAA 840 CCACCGCTCC TCCACTCTCC ATATTTGGCC ACCGTCGTCC GCTATCTCTCCA TCCACCAACT 900 TCATTTATTT TATCGGACGT TGTTCTCCAG TAACCGCTCT AGCTCAACCC TCTAGACC 958

SEQ ID NO: 11 Sequence length: 10 Sequence form: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: GAGGGACCTC 10

SEQ ID NO: 12 Sequence length: 20 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TCTTCAAAGC CCATCTTGG 20

SEQ ID NO: 13 Sequence length: 21 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TTGAGCTAGA GCGGTTACTG G 21

SEQ ID NO: 14 Sequence length: 25 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GAGGTGTGGT CTTAGTCCTG TATGG 25

SEQ ID NO: 15 Sequence length: 25 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GATGCCTTTT CTAGCCAACA CGGAC 25

SEQ ID NO: 16 Sequence length: 25 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GTCGTCATCA TCTTCCTGGT CAACG 25

SEQ ID NO: 17 Sequence length: 24 Sequence form: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: CGTCCGCTAT CTCCATCCAC CAAC 24

SEQ ID NO: 18 Sequence length: 24 Sequence form: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: CTCCAGTAAC CGCTCTAGCT CAAC 24

SEQ ID NO: 19 Sequence length: 16 Sequence form: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: AGWGNAGWAN CAWAGG 16

Claims (13)

[Claims] The present invention is characterized in that the S-5 locus is located near the S-5 locus which is a hybrid sterility locus of rice, and is determined by using a DNA marker defined by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2. A method for determining genotype. 2. A primer defined by a continuous nucleotide sequence having a length of 15 to 40 nucleotides arbitrarily selected from the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 1 or a sequence facing the primer. And a primer defined by a continuous base sequence having a length of 15 to 40 bases arbitrarily selected from the base sequence described in No. 2 and located near the S-5 locus which is a rice hybrid sterility locus. One set of primers for detecting DNA markers. 3. The set of primers according to claim 2, wherein the sequences of the primers are SEQ ID NO: 3 and SEQ ID NO: 4. 4. A PCR using the DNA extracted from a test rice as a template using the set of primers according to claim 2 or 3, and S-
Amplify a DNA marker located in the vicinity of the 5 loci. If the amplified DNA marker is cleaved by restriction enzyme XbaI, judge that the genotype of the tested rice is S-5n. A method for determining the genotype of the S-5 locus, which is determined to be S-5i or S-5j. 5. The S-5 locus located near the S-5 locus, which is a hybrid sterility locus of rice, and defined using the DNA marker defined by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6. A method for determining genotype. 6. A primer defined by a base sequence having a length of 15 to 40 contiguous bases arbitrarily selected from the base sequence of SEQ ID NO: 5, and a primer of SEQ ID NO: 6 facing the primer. Detecting a DNA marker located in the vicinity of the S-5 locus, which is a rice hybrid sterility locus, comprising a primer defined by a nucleotide sequence having a length of 15 to 40 consecutive nucleotides arbitrarily selected from the nucleotide sequence. A set of primers for 7. The set of primers according to claim 6, wherein the sequences of the primers are SEQ ID NO: 7 and SEQ ID NO: 8. 8. A DNA extracted from a rice to be tested using the set of primers according to claim 6 or 7.
Was used as a template to perform PCR, and
The DNA marker located near the −5 locus is amplified, and when the amplified DNA marker is cut with the restriction enzyme BfaI, the genotype of the tested rice is determined to be S-5n, and the restriction enzyme is determined. If cut with HhaI, the genotype of the tested rice is determined to be S-5i, and if not cut by any of the restriction enzymes, the genotype of the tested rice is determined to be S-5j. A method for determining the genotype of the S-5 locus. 9. PCR using rice total DNA as a template and primers defined by the nucleotide sequence of SEQ ID NO: 9
To amplify a DNA marker, and a 0.9 kb DNA located near the S-5 locus which is a rice hybrid sterility locus
A method for determining the genotype of the S-5 locus, wherein the genotype of the tested rice is determined to be S-5n when the marker is amplified. 10. The DNA marker of about 0.9 kb located near the S-5 locus which is a rice hybrid sterility locus is defined by the nucleotide sequence of SEQ ID NO: 10. Determination method described in. 11. The method using SEQ ID NO: 1 with rice whole DNA as a template
Using the primer defined by the nucleotide sequence described in 1,
The DNA marker was amplified by CR, and an approximately 1.4 kb D fragment located near the S-5 locus, which is a rice hybrid sterility locus.
A method for determining the genotype of the S-5 locus, wherein the genotype of rice is determined to be S-5n when the NA marker is amplified. 12. The nucleotide sequence at both ends of the approximately 1.4 kb DNA marker located near the S-5 locus, which is a hybrid sterility locus of rice, excluding the central portion is the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6. The method according to claim 11, wherein: 13. The nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, or the nucleotide sequence or sequence of SEQ ID NO: 5 and SEQ ID NO: 6, which is located near the S-5 locus which is a rice hybrid sterility locus. A DNA marker defined by the nucleotide sequence of No. 10.