JP2007054020A - METHOD FOR IDENTIFYING RICE BLAST RESISTANCE WITH Pb1 GENE-LINKED MOLECULAR MARKER AS INDICATOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient method for identifying rice blast resistance. <P>SOLUTION: The method comprises the following procedure: With reference to the physical map of BAC/PAC in the disclosed database, a clone in the vicinity where rice blast resistance-related gene Pb1 is supposed to be present is selected, and a total of 21 new Pb1 markers are prepared. For the homo-type line(F<SB>4</SB>) of "Norin #8"/"St. No.1", whether the homo-type line(F<SB>4</SB>) is "Norin #8" type or "St. No.1" type is determined, along with assaying rice blast resistance in a farm, thereby a Pb1 marker present between the markers "NA-2RG4" and "N2-3RG" is offered as the rice blast resistance marker. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for discriminating rice varieties having resistance to panicle.

  Rice blast occurs throughout the rice cultivation period. In particular, the rice blast that develops after heading is different from the leaf rice bud and directly reduces the yield and quality / taste. For this reason, ear rot control is carried out in many cultivated paddy fields, and the average pesticide spraying is performed about once in all paddy fields. The use of a rice blast resistant variety can be a very effective means for controlling rice blast and reducing the use of pesticides.

  Pb1 is a dominant active gene identified for the first time as a panicle resistance gene (Non-patent document 1, Non-patent document 2). Pb1 was discovered at the Aichi Prefectural Agricultural Experiment Station (hereinafter abbreviated as “Aichi Agricultural Examination”), which is engaged in rice stripe-resistant breeding. Aichi Agricultural Trial has introduced a rice streak-resistant middle mother "St. No.1" derived from the Indian rice cultivar "Modan" and cultivated many rice streak-resistant strains. Through the panicle resistance test of these strains, the presence of the panicle resistance gene: Pb1 linked to rice stripe resistance was discovered.

  Resistance by Pb1 expresses stronger field resistance to panicle than leaf blast. The field resistance to leaf blasts is weak at the seedling stage but becomes remarkable after the 10th stage. The field resistance of Pb1 is the resistance of the main gene and can be easily introduced by normal breeding. Although Pb1 has many excellent features as described above, the most important feature is that the panicle resistance is stable for a long time. 23 years have passed since the practical varieties spread in Gunma, Saitama, and Aichi prefectures, and resistance varieties caused by Pb1 even after years of frequent occurrence of blast in 1991, 1993, and 2003 Then, resistance collapse (breakdown) due to mutation of blast fungus is not observed (Non-patent Document 3).

Despite the fact that resistance to blast caused by Pb1 is a practical and practical important trait, the mechanism of action of Pb1 has not yet been elucidated. In addition, it is necessary to test for rice blast resistance in order to cultivate the rice blast resistant line, but in the past it was necessary to test rice blast resistance after actually cultivating rice in the field and waiting for heading. there were. The Hotomochi test really takes one season. In addition, because there are few places suitable for testing, ear motility test is not performed at an early stage of the breeding process, but after other traits are evaluated to a certain extent and the genetic background of the strain is fixed, the ear motility test is finally performed. There were many cases. For this reason, there were cases in which the lines that had been bred were actually extremely weak in resistance to earing. In order to cultivate the rice blast resistant varieties, it is required to develop an efficient evaluation / testing method that enables the rice blast test at the initial stage of culturing. There have been reports of molecular markers linked to Pb1 so far (Patent Document 1, Non-Patent Documents 5-8), but markers with a higher degree of certainty are required for practical use.
Patent 3153889 Kiyoshi Fujii, Takamichi Toyama, Naoki Sugiura, Noriaki Saka, Masakatsu Inoue, Akio Akimiya (1999) Japanese rice varieties resistant to rice streak virus, moonlight and panicle resistance found in sister lines Properties and pedigree analysis. Breeding studies 1.69-76. Kiyoshi Fujii, Yuriko Hayano, Naoki Sugiura, Nagao Hayashi, Noriaki Saka, Takamichi Toyama, Toshihiko Izawa, Akio Akimiya (1999) Genetic analysis of rice blast resistance varieties resistant to rice stripe blight. Breeding studies 1.203-210. Kiyoshi Fujii, Yuriko Hayano, Naoki Sugiura, Nagao Hayashi, Toshihiko Izawa, Masato Iwasaki (2005) Quantitative evaluation of the rice blast field resistance gene Pb1 for suppressing the rice blast development using rice near-isogenic lines. Breeding studies 7. Printing (scheduled to be issued in June) Fujii, K., Y. Hayano-Saito, K. Saito, N. Sugiura, N. Hayashi, T. Tsuji, T. Izawa and M. Iwasaki (2000) Identification of a RFLP marker tightly linked to the panicle blast resistance gene , Pb1, in rice. Breeding Science 50. 183-188. Allelic diversity among Basmati and non-Basmati long-grain indica rice varieties using microsatellite markers.Journal of Plant Biochemistry and Biotechnology, 2004, vol.13, pp25-32 Yuriko Hayano, Kiyoshi Fujii, Naoki Sugiura, Koji Saito, Nagao Hayashi Analysis of the locus region of rice rice blast field resistance gene Pb1 using inbred lines Takayama Toyama, Yuriko Hayano, Naoki Sugiura, Kiyoshi Fujii, Toshihiko Izawa, Hitoshi Nakamae (1998) Development of a PCR marker linked to the rice blast resistance gene Pb-st (t). Aichi Prefectural Agricultural Experiment Station Research Report 30: 27-34 Chen, X., Temnykh, S., Xu, Y., Cho, YG and McCouch, SR Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.) Theor.Appl. Genet. 95, 553 -567 (1997)

  The present invention has been made in view of such a situation, and the problem to be solved by the present invention is to provide an efficient method for identifying the panicle resistance.

  The present inventors have intensively studied to solve the above problems. Genetic analysis of Pb1 was carried out by Aichi Prefectural Agricultural Research Center and Hokkaido Agricultural Experiment Station (currently Hokkaido Agricultural Research Center). From RFLP analysis, Pb1 gene is present in the long arm of rice chromosome 11 and resistant to rice stripe disease. It is known to be linked to the gene Stvb-i at a genetic distance of 5.2 cM (Non-patent Document 2, Non-patent Document 4). Based on this information, the “Nipponbare” BAC / PAC physical map published by the International Rice Genome Sequence Decoding Project (IRGSP) is located at 90.1 cM and the markers “S723” located on chromosome 11 at 85.7 cM. Ten clones sandwiched between the markers “R2458” were selected. For the 10 clones, an attempt was made to create a Pb1 marker.

  First, the BAC terminal base sequence of Indian rice cultivar “Kasalath” was compared with the base sequence of “Nippon Hare” to find the base difference at the restriction enzyme cleavage site. Primers are designed before and after the nucleotide polymorphism, and PCR is performed using the DNAs of “St. No. 1” and “Noribayashi No. 8” as templates, and the size of the amplified product or the size of the fragment obtained by digesting the product with the restriction enzyme is determined. Differences were detected and markers “K1” and “K3” were made.

  Next, SSR (Single sequence repeat) was analyzed, and primers were designed to amplify SSR. PCR was carried out using the DNAs of “St. No. 1” and “Noribayashi No. 8” as a template with the primers, and seven SSR markers: “SSR2”, “SSR4”, “ "SSR6", "SSR11", "SSR16", "SSR34", and "SSR39" were produced.

  In the third method, a primer was designed at an arbitrary position of the base sequence of “Nippon Hare”, and the sizes of the PCR products of “St. No. 1” and “Noribayashi No. 8” were confirmed by agarose gel electrophoresis. When the size of the PCR product was different, it was directly used as a polymorphism. Markers “N3-2”, “N4-1”, and “Pb3810 / 16” were produced by this method. On the other hand, when the size of the PCR product was the same, the base sequence of the PCR product was decoded, analyzed by GENETYX, and the base sequence polymorphism of the restriction enzyme recognition site was used. By this method, the markers “N6-1”, “N6-3”, “NA-2RG4”, “NA-16RG”, “N4-3RG1”, “N2-3RG”, “N2-2RG”, “N2- 1RG "was produced.

Then the F ₂ plants 7,279 individual "Norin 8" /`St.Nanba1 ", DNA and simple extracted by the alkali method from the leaves of seedlings, for the DNA," N6-1 "from the said marker 334 individuals that had undergone recombination between “N6-1” and “Pb3810 / 16” were selected using “Pb3810 / 16”. For the 334 recombinant individuals, the genotypes of 21 molecular markers were determined and the recombination positions were clarified. As a result, it was found that a region that is likely to recombine and a region that is extremely unlikely to occur are adjacent to each other in the assumed region of Pb1.

In addition, 61 homogenized lines (F ₄ ) obtained by immobilizing some of the 334 recombinant heterolines selected from the “Nori No. 8” / “St. The above marker was used to determine whether it was “Noryo No. 8” type or “St. No. 1” type, and at the local field, panicle resistance was tested. As a result, the present inventors succeeded in narrowing down the position of the Pb1 gene, and it became clear that Pb1 exists between the markers “NA-2RG4” and “N2-3RG”.

Furthermore, the present inventors conducted a panicle resistance test using the Pb1 marker of the present invention for rice varieties / lines that had already been confirmed to have panicle resistance. As a result, the panicle resistance test using the Pb1 marker of the present invention was able to produce a correct result and proved to be highly useful. That is, the present invention relates to detection of the panicle resistance gene Pb1, and specifically provides the following inventions.
(1) A molecular marker linked to the rice blast resistance gene Pb1, and using at least one of the molecular markers shown in the linkage map of FIG. 1 as an index, the rice blast resistance of the test rice How to identify,
(2) The method according to (1) above, wherein when the molecular marker exhibits a genotype similar to that of rice having a trait resistance trait, the test rice is determined to be panicle-resistant.
(3) The above (1) or (1), wherein the molecular marker is a molecular marker comprising the base sequence according to any one of SEQ ID NOs: 3, 6, 17, 20, 25, 36, 39, 44, 51 or a partial sequence thereof. The method according to (2),
(4) A method for selecting rice having a panicle-resistant trait including the following steps: (a) mating rice having a panchi-resistant trait and rice having other traits (b) mating Using the method according to any one of (1) to (3) above to select rice having a rice bran resistance trait from rice obtained
(5) SEQ ID NO: 3, 6, 17, 20, 25, 36, 39, 44, having a chain length of at least 15 nucleotides complementary to the DNA consisting of the base sequence according to any one of its complementary strands Oligonucleotides,
(6) At least one primer set shown in any one of (a) to (u) below (a) DNA consisting of the base sequence described in SEQ ID NO: 1 and DNA consisting of the base sequence described in SEQ ID NO: 2
(B) DNA consisting of the base sequence described in SEQ ID NO: 4 and DNA consisting of the base sequence described in SEQ ID NO: 5
(C) DNA consisting of the base sequence set forth in SEQ ID NO: 7 and DNA consisting of the base sequence set forth in SEQ ID NO: 8
(D) DNA consisting of the base sequence set forth in SEQ ID NO: 9 and DNA consisting of the base sequence set forth in SEQ ID NO: 10
(E) DNA consisting of the base sequence set forth in SEQ ID NO: 11 and DNA consisting of the base sequence set forth in SEQ ID NO: 12
(F) DNA consisting of the base sequence set forth in SEQ ID NO: 13 and DNA consisting of the base sequence set forth in SEQ ID NO: 14
(G) DNA consisting of the base sequence set forth in SEQ ID NO: 15 and DNA consisting of the base sequence set forth in SEQ ID NO: 16
(H) DNA consisting of the base sequence set forth in SEQ ID NO: 18 and DNA consisting of the base sequence set forth in SEQ ID NO: 19
(I) DNA consisting of the base sequence set forth in SEQ ID NO: 21 and DNA consisting of the base sequence set forth in SEQ ID NO: 22
(J) DNA consisting of the base sequence set forth in SEQ ID NO: 23 and DNA consisting of the base sequence set forth in SEQ ID NO: 24
(K) DNA consisting of the base sequence set forth in SEQ ID NO: 26 and DNA consisting of the base sequence set forth in SEQ ID NO: 27
(L) DNA consisting of the base sequence set forth in SEQ ID NO: 28 and DNA consisting of the base sequence set forth in SEQ ID NO: 29
(M) DNA consisting of the base sequence set forth in SEQ ID NO: 30 and DNA consisting of the base sequence set forth in SEQ ID NO: 31
(N) DNA consisting of the base sequence set forth in SEQ ID NO: 32 and DNA consisting of the base sequence set forth in SEQ ID NO: 33
(O) DNA consisting of the base sequence set forth in SEQ ID NO: 34 and DNA consisting of the base sequence set forth in SEQ ID NO: 35
(P) DNA consisting of the base sequence set forth in SEQ ID NO: 37 and DNA consisting of the base sequence set forth in SEQ ID NO: 38
(Q) DNA consisting of the base sequence set forth in SEQ ID NO: 40 and DNA consisting of the base sequence set forth in SEQ ID NO: 41
(R) DNA consisting of the base sequence set forth in SEQ ID NO: 42 and DNA consisting of the base sequence set forth in SEQ ID NO: 43
(S) DNA consisting of the base sequence set forth in SEQ ID NO: 45 and DNA consisting of the base sequence set forth in SEQ ID NO: 46
(T) DNA consisting of the base sequence set forth in SEQ ID NO: 47 and DNA consisting of the base sequence set forth in SEQ ID NO: 48
(U) DNA consisting of the base sequence set forth in SEQ ID NO: 49 and DNA consisting of the base sequence set forth in SEQ ID NO: 50;
(7) A panicle resistance identification kit comprising at least one combination shown in any of (a) to (i) below.
(A) DNA consisting of the base sequence set forth in SEQ ID NO: 1, DNA consisting of the base sequence set forth in SEQ ID NO: 2, and restriction enzyme: TaqI
(B) DNA consisting of the base sequence described in SEQ ID NO: 4, DNA consisting of the base sequence described in SEQ ID NO: 5, and restriction enzyme: Sau3AI
(C) DNA consisting of the base sequence set forth in SEQ ID NO: 15 and DNA consisting of the base sequence set forth in SEQ ID NO: 16; and restriction enzyme: RsaI
(D) DNA consisting of the base sequence set forth in SEQ ID NO: 18 and DNA consisting of the base sequence set forth in SEQ ID NO: 19; and restriction enzyme: AluI
(E) DNA consisting of the base sequence set forth in SEQ ID NO: 23, DNA consisting of the base sequence set forth in SEQ ID NO: 24, and restriction enzyme: EcoT22I
(F) DNA consisting of the base sequence set forth in SEQ ID NO: 32, DNA consisting of the base sequence set forth in SEQ ID NO: 33, and restriction enzyme: Sau3AI
(G) DNA consisting of the base sequence set forth in SEQ ID NO: 37, DNA consisting of the base sequence set forth in SEQ ID NO: 38, and restriction enzyme: BclI
(H) DNA consisting of the base sequence set forth in SEQ ID NO: 42, DNA consisting of the base sequence set forth in SEQ ID NO: 43, and restriction enzyme: MspI
(I) DNA consisting of the base sequence set forth in SEQ ID NO: 45, DNA consisting of the base sequence set forth in SEQ ID NO: 46, and restriction enzyme: BanIII.

  The present invention has provided a novel method for identifying rice blast resistance. According to the method of the present invention, it is possible to determine the presence or absence of panicle resistance at an early stage of the rice growth stage. By selecting rice having resistance to panicles by the method of the present invention, it is expected that the breeding period of panicle-resistant rice will be greatly shortened.

  The present invention is a molecular marker linked to the rice blast resistance gene Pb1, and among the molecular markers shown in the linkage map of FIG. 1, using at least one molecular marker as an index, the rice blast resistance of the test rice Provide a way to identify gender. In the identification method of the present invention, it is possible to specifically and efficiently identify the rice blast resistance of the test rice by examining whether or not the test rice has the rice blast resistance gene Pb1.

  In the present invention, “ear glutinous resistance gene Pb1” (hereinafter sometimes referred to simply as “Pb1”) is present in the long arm of rice chromosome 11 and in rice varieties having resistance to rice stripe disease. It is linked to the rice stripe blight resistance gene Stvb-i at a genetic distance of 5.2 cM.

  In the identification method of the present invention, in a desired rice (hereinafter referred to as “test plant” in some cases) for identifying the presence or absence of panicle resistance, when the Pb1 gene is contained, the test plant is panicle. When it is determined that the rice has resistance, and it does not have the Pb1 gene, the test plant is determined to be rice that does not have panicle resistance.

  In a preferred embodiment of the identification method of the present invention, a molecular marker linked to Pb1 is used. “Molecular marker” in the present invention refers to a DNA region genetically linked to Pb1, which can be distinguished from other DNA regions. As a preferred molecular marker in the present invention, the molecular marker shown in FIG. 1 (marker number: 1 to 21) can be exemplified.

  In general, a molecular marker is more useful because the shorter the map distance expressed in units cM, the closer to the gene and the inheritance of the gene is. Although the exact position of Pb1 has not yet been determined, the present inventors narrowed down the position of Pb1, and Pb1 is changed from marker “NA-2RG4” (marker number: 7) to marker “N2-3RG” (marker number: 16) (FIG. 1). Therefore, in the method of the present invention, among the 21 molecular markers shown in FIG. 1, markers existing between the two markers (“NA-2RG4”, “NA-16RG”, “SSR4”, “N4-3RG1”) ”,“ SSR34 ”,“ N4-1 ”,“ SSR39 ”,“ K3 ”,“ N3-2 ”,“ N2-3RG ”) are likely to be particularly useful markers.

  One embodiment of the method of the present invention is a method in which a test rice is determined to be ear resistant when it exhibits a genotype similar to that of a rice having a trait resistant trait. The above-mentioned “rice having a rice blast resistance trait” refers to rice that has been confirmed to have the rice blast resistance gene Pb1. For example, when the molecular marker in the test plant shows the same genotype as “rice having a rice blast resistance trait”, the test plant is determined to have the rice blast resistance. Comparison of the molecular marker in the test plant with the molecular marker in “rice having a trait resistance” is performed not only by comparing the DNA sequence of the molecular marker but also by comparing the information characterized by the DNA sequence. can do. Information characterized by the DNA sequence of the molecular marker includes, but is not limited to, information about the presence or absence of the molecular marker and information about the presence or absence of the mutation site or polymorphic site contained in the molecular marker. It is not something. For example, as in the examples of the present invention, the molecular weight of the amplification product when PCR is performed using the DNA of the test plant as a template and primers for amplifying the molecular marker region is “rice having rice bran resistance trait “The molecular weights of the amplified products are compared by electrophoresis. If the molecular weights are the same, it can be said to“ show similar genotype ”. Thus, “showing the same genotype” includes not only the case where the DNA sequences of the molecular markers are completely identical, but also the case where the information characterized by the DNA sequence is the same.

  In one embodiment of the present invention, two or more molecular markers shown in the linkage map of FIG. 1 are selected as appropriate, and the identification method of the present invention is performed, so that identification with higher accuracy is possible.

  In the present invention, “use a molecular marker” means that the molecular marker is used as an index for identifying rice ear resistance. That is, in a preferred embodiment of the present invention, when the molecular marker of the test plant shows a genotype similar to that of rice having a panicle-resistant trait, the test plant is judged to have a panicle-resistant trait. Is done.

  In the present invention, the “test rice” is not particularly limited as long as it is rice, and may be an Indica species or a Japonica species, or a hybrid variety / line of an Indica species and a Japonica species or a wild species. It may be rice, or a hybrid / crossbred variety of cultivar and wild rice. In the present invention, the `` rice having a rice blast resistance trait '' may be any rice having Pb1, and examples thereof include `` Modan '' and `` St.No.1 ''. It is not limited. The identification method of the present invention can be suitably implemented by using as a control the molecular marker type in rice varieties / lines already known to have Pb1.

  Moreover, in the preferable aspect of this invention, a "test plant" refers to the system | strain etc. in the middle of the growth which the parent knows reliably. That is, in the test plant, a plant having the same genotype as that of the “ear blast resistance” parent is determined to have a pan blast resistance trait (having a Pb1 gene) with a high probability. The probability in this case can be represented by 1-0.01xP when the recombination value is P (%).

  Examples of the molecular marker of the present invention include an STS (Sequence Tagged Site) marker, a CAPS (Cleaved Amplified Polymorphic Sequence) marker, and an SSR (Single Sequence Repeat) marker. An STS marker refers to a DNA region that can be used to determine the presence or absence of a polymorphism in a sequence tag site (STS) on DNA, and STS refers to a sequence site specific to a specific position on DNA. A polymorphism of STS is obtained by amplifying a DNA region containing the specific sequence site by a nucleic acid amplification method such as PCR method, and subjecting the amplified product to agarose or polyacrylamide gel electrophoresis. Can be detected. The CAPS method is a method in which a region containing a specific sequence site on DNA is further treated with a restriction enzyme to detect a polymorphism in the specific sequence site. For example, when no polymorphism is found in the amplification product by STS, the amplification product is treated with various restriction enzymes, and the polymorphism is detected by electrophoresis or the like. The SSR marker is a marker also called a microsatellite marker. There are sequence repeats consisting of several bases on DNA, and the number of repeats varies depending on the system. The SSR marker is a marker for detecting this polymorphism difference in the number of repetitions.

  The marker of the above method that can be used in the present invention is not particularly limited as long as it is a molecular marker shown in FIG. 1, and any marker can be used.

  When an STS marker or SSR marker is used as the molecular marker of the present invention, the identification method of the present invention can be performed, for example, as follows. First, a DNA sample is prepared by a well-known method from a test rice and rice having a rice blast resistance trait. Next, a nucleic acid amplification reaction (for example, PCR method) is performed using the prepared DNA as a template and a primer DNA. When the size of the amplified DNA fragment is compared between the test rice and the panicle resistance by electrophoresis or the like, and shows the same genotype, the test plant is characterized by the panicle resistance trait. It is determined that

  When a CAPS marker is used as the molecular marker of the present invention, the identification method of the present invention can be performed, for example, as follows. First, a DNA sample is prepared by a well-known method from a test rice and rice having a rice blast resistance trait. Next, a nucleic acid amplification reaction (for example, PCR method) is performed using the prepared DNA as a template and a primer DNA. When the amplified DNA fragment is cleaved with a restriction enzyme, the size of the cleaved DNA fragment is compared between the test rice and panicle resistance by electrophoresis or the like, and when showing the same genotype, The test plant is determined to have a panicle-resistant trait.

As a primer DNA used in the identification method of the present invention, those skilled in the art can appropriately design an optimal primer in consideration of sequence information on various molecular markers. Usually, the above-mentioned primer is present specifically in rice having Pb1 and is specific to the nucleotide sequence linked to Pb1, or is specifically present in rice having Pb1 and sandwiches the nucleotide sequence linked to Pb1 It is a pair of primer sets designed to amplify the base sequence. The primer set for the CAPS marker is preferably used in combination with a specific restriction enzyme. Specifically, the following primer sets and restriction enzymes can be exemplified.
As a primer set of STS marker,
Primer set N4-1 consisting of primer N4-1F (SEQ ID NO: 28) and primer N4-1R (SEQ ID NO: 29),
Primer set N3-2 consisting of primer N3-2F (SEQ ID NO: 34) and primer N3-2R (SEQ ID NO: 35),
Primer set K1 consisting of primer K1F (SEQ ID NO: 47) and primer K1R (SEQ ID NO: 48),
Primer set Pb3810 / 16 consisting of primer Pb3810 / 16F (SEQ ID NO: 49) and primer Pb3810 / 16R (SEQ ID NO: 50),
These four primer sets can be exemplified.
In addition, as a primer set and restriction enzyme of CAPS marker,
Primer set N6-1 consisting of primer N6-1F (SEQ ID NO: 1) and primer N6-1R (SEQ ID NO: 2), and restriction enzyme: TaqI,
Primer set N6-3 consisting of primer N6-3F (SEQ ID NO: 4) and primer N6-3R (SEQ ID NO: 5), and restriction enzyme: Sau3AI,
Primer set NA-2RG-4 consisting of primer NA-2RG-4F (SEQ ID NO: 15) and primer NA-2RG-4R (SEQ ID NO: 16), and restriction enzyme: RsaI,
Primer set NA-16RG consisting of primer NA-16RGF (SEQ ID NO: 18) and primer NA-16RGR (SEQ ID NO: 19), and restriction enzyme: AluI,
Primer set N4-3RG1 consisting of primer N4-3RG1F (SEQ ID NO: 23) and primer N4-3RG1R (SEQ ID NO: 24), and restriction enzyme: EcoT22I
Primer set K3 consisting of primer K3F (SEQ ID NO: 32) and primer K3R (SEQ ID NO: 33), and restriction enzyme: Sau3AI
Primer set N2-3RG consisting of primer N2-3RGF (SEQ ID NO: 37) and primer N2-3RGR (SEQ ID NO: 38), and restriction enzyme: BclI
Primer set N2-2RG consisting of primer N2-2RGF (SEQ ID NO: 42) and primer N2-2RGR (SEQ ID NO: 43), and restriction enzyme: MspI
Primer set N2-1RG consisting of primer N2-1RGF (SEQ ID NO: 45) and primer N2-1RGR (SEQ ID NO: 46), and restriction enzyme: BanIII
9 primer sets can be exemplified.
Furthermore, as a primer set of SSR marker,
Primer set SSR-16 consisting of primer SSR-16F (SEQ ID NO: 7) and primer SSR-16R (SEQ ID NO: 8),
Primer set SSR-2 consisting of primer SSR-2F (SEQ ID NO: 9) and primer SSR-2R (SEQ ID NO: 10),
Primer set SSR-24 consisting of primer SSR-24F (SEQ ID NO: 11) and primer SSR-24R (SEQ ID NO: 12),
Primer set SSR-11 consisting of primer SSR-11F (SEQ ID NO: 13) and primer SSR-11R (SEQ ID NO: 14),
Primer set SSR-4 consisting of primer SSR-4F (SEQ ID NO: 21) and primer SSR-4R (SEQ ID NO: 22),
Primer set SSR-34 consisting of primer SSR-34F (SEQ ID NO: 26) and primer SSR-34R (SEQ ID NO: 27),
Primer set SSR-39 consisting of primer SSR-39F (SEQ ID NO: 30) and primer SSR-39R (SEQ ID NO: 31),
Primer set SSR-6 consisting of primer SSR-6F (SEQ ID NO: 40) and primer SSR-6R (SEQ ID NO: 41),
The eight primer sets can be exemplified.

  The PCR primer of the present invention can be prepared by those skilled in the art using, for example, an automatic oligonucleotide synthesizer. Those skilled in the art can also carry out the method of the present invention by a known polymorphism detection method, for example, the PCR-SSCP method using the above PCR primers.

  When the molecular marker of the present invention is present in an exon of genomic DNA, RT-PCR using mRNA as a template can be used. Further, if a Taqman (quantitative PCR detection) system (Roche) is used, the presence or absence of an amplification product can be detected by fluorescence. According to this system, it is possible to perform the identification method of the present invention in a short time since the labor of electrophoresis can be saved.

  The DNA sample used in the identification method of the present invention is not particularly limited, but usually genomic DNA extracted from rice as a test plant is used. Further, the collection source of genomic DNA is not particularly limited, and it can be extracted from any tissue of rice. For example, it can be extracted from ear, leaf, root, stem, seed, endosperm part, bran, embryo and the like.

  A person skilled in the art can prepare (extract) the above-described DNA sample of the present invention by a known method. As a preferable preparation method, for example, a method of extracting DNA using the CTAB method can be mentioned.

  Furthermore, the electrophoretic analysis of the present invention may be performed according to a conventional method. For example, electrophoresis is performed by applying voltage in an agarose or polyacrylamide gel, and the separated DNA pattern is analyzed.

  The present invention also provides a chain length of at least 15 nucleotides complementary to DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 3, 6, 17, 20, 25, 36, 39, 44, 51 or a complementary strand thereof. An oligonucleotide is provided.

  Here, “complementary strand” refers to the other strand with respect to one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs. Further, the term “complementary” is not limited to a case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the homology on the above base sequences. An algorithm for determining homology may be one known to those skilled in the art.

  The oligonucleotide of the present invention specifically hybridizes to DNA consisting of the base sequence described in any of SEQ ID NOs: 3, 6, 17, 20, 25, 36, 39, 44, 51 or a complementary strand thereof. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the second edition 1989) means that no significant cross-hybridization with other DNA occurs.

  The oligonucleotide of the present invention is used as a probe or primer used for detection or amplification of DNA consisting of the base sequence described in any of SEQ ID NOs: 3, 6, 17, 20, 25, 36, 39, 44, 51 be able to. Moreover, the oligonucleotide of the present invention can be used in the form of a substrate of a DNA array.

  When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least part of the DNA of the present invention or its complementary strand. When used as a primer, the 3 ′ region can be complementary, and a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 ′ side. In addition, the primer set used for detection and amplification of DNA consisting of the base sequence described in SEQ ID NOs: 3, 6, 17, 20, 25, 36, 39, 44, 51 corresponds to the primer set described above. Primer set (primer set N6-1, primer set N6-3, primer set NA-2RG-4, primer set NA-16RG, primer set N4-3RG1, primer set N3-2, primer set N2-3RG, primer set N2 -2RG, primer set Pb3810 / 16).

  Further, when the oligonucleotide is used as a probe, the probe is a DNA comprising the base sequence set forth in any of SEQ ID NOs: 3, 6, 17, 20, 25, 36, 39, 44, 51 or its complementary There is no particular limitation as long as it specifically hybridizes to at least a part of the chain. The probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp.

When the oligonucleotide of the present invention is used as a probe, it is preferably used after appropriately labeling. As a labeling method, a method of labeling by phosphorylating the 5 ′ end of the oligonucleotide with ³² P using T4 polynucleotide kinase, and a random hexamer oligonucleotide or the like using a DNA polymerase such as Klenow enzyme are used. Examples of the primer include a method of incorporating a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin (random prime method or the like).

  The oligonucleotide of the present invention can be prepared, for example, by a commercially available oligonucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.

  In the reagent for identifying rice bran resistance of the present invention, in addition to the active ingredient oligonucleotide, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffering agent In addition, a preservative or the like may be mixed as necessary.

  If the identification method of this invention is utilized, it will become possible to select the rice identified as panicle resistance early. The present invention also provides a method for early selection of rice identified as such panicle resistance. Here, “early” refers to the state before the heading of rice, preferably the state immediately after germination. By using the selection method of the present invention, it becomes possible to cultivate varieties having a panicle-resistant trait in a shorter period of time than before.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

[Example 1] Preparation of marker The information on the nucleotide sequence of a publicly disclosed rice (variety: Nipponbare) was used. With reference to the molecular genetic map of Pb1, from the BAC / PAC physical map published by the International Rice Genome Sequence Decoding Project (IRGSP), the marker “S723” located on chromosome 11 at 85.7cM and the marker located at 90.1cM About 10 clones sandwiched between “R2458”, a total of 21 Pb1 peripheral markers were prepared by the following three methods.

  In the first method, the BAC terminal base sequence of the Indian rice cultivar “Kasalath” was compared with the base sequence of “Nippon Hare” to find out whether there was a base difference in the restriction enzyme cleavage site. Primers were designed before and after the nucleotide polymorphism, and PCR was performed using the DNAs of “St. No. 1” and “Noribayashi No. 8” as templates. “St.No.1” is a variety cultivated by backcrossing Japanese-style rice “Noryo No. 8” five times to “Modan”. "St.No.1" has Pb1, but "Nori No. 8" has no Pb1. A difference in the size of the product amplified by the PCR or the size of the fragment obtained by cleaving the product was detected and used as a marker. Markers “K1” and “K3” were prepared by the above method. “K1” is an STS (Sequence Tagged Site) marker, and “K3” is a CAPS (Cleaved Amplified Polymorphic Sequence) marker.

  Next, SSR (Single sequence repeat) was analyzed, and primers were designed to amplify SSR. Using the primers, PCR was performed using “St. No. 1” and “Noribayashi No. 8” genomic DNA as templates, and the size of the band was confirmed by acrylamide gel electrophoresis for each PCR amplification product. Select the ones with different bands of amplification products of “St.No.1” and “Noribayashi No.8”, and select 7 SSR markers: “SSR2,” “SSR4,” “SSR6,” “SSR11,” “SSR16” "SSR34" and "SSR39" were produced.

The third method was performed as follows. Primers were designed at arbitrary positions in the base sequence of “Nipponbare”, and the sizes of the PCR products of “St. No. 1” and “Noribayashi No. 8” were confirmed by agarose gel electrophoresis. When the size of the PCR product was different, it was directly used as a polymorphism. Markers “N3-2”, “N4-1”, and “Pb3810 / 16” were produced by this method. On the other hand, when the size of the PCR product was the same, the base sequence of the PCR product was decoded, analyzed by GENETYX, and the base sequence polymorphism of the restriction enzyme recognition site was used. By this method, the markers “N6-1”, “N6-3”, “NA-2RG4”, “NA-16RG”, “N4-3RG1”, “N2-3RG”, “N2-2RG”, “N2- 1RG "was produced. All of these 8 are CAPS markers.
A brief description of each of the 21 markers produced above is given below.

a) Molecular marker number 1: “N6-1” CAPS marker primer N6-1F: 5′-GCCCAGTAGTACCACCTCCA-3 ′ (SEQ ID NO: 1)
Primer N6-1R: 5'-CAGTCTTCTACCGCGAAAGG-3 '(SEQ ID NO: 2)
Restriction enzyme type: TaqI
SEQ ID NO: 3 shows the partial sequence: 774 bp of the base sequence: 942 bp of the PCR product using the rice cultivar “St. No. 1” as a template using the above primers. There is a TaqI restriction enzyme cleavage site from position 529 to position 532 of the 774 bp partial sequence (SEQ ID NO: 3). Therefore, not only the PCR product consisting of 942 bp but also a 774 bp partial sequence can be used as a panicle resistance identification marker.

b) Molecular marker number 2: “N6-3” CAPS marker primer N6-3F: 5′-TCATGTCTCCTCTCCCATCC-3 ′ (SEQ ID NO: 4)
Primer N6-3R: 5'-GACCAAGAAGGCGTCAAGAG-3 '(SEQ ID NO: 5)
Restriction enzyme type: Sau3AI
SEQ ID NO: 6 shows the base sequence of the PCR product: 953 bp partial sequence: 784 bp when the rice cultivar “St. No. 1” was used as a template using the above primers. A restriction enzyme cleavage site of Sau3AI exists from the 631 to the 634th position of the 784 bp partial sequence (SEQ ID NO: 6). Therefore, not only the PCR product consisting of 953 bp but also the 784 partial sequence can be used as a panicle resistance identification marker.

c) Molecular marker number 3: “SSR16” SSR marker primer SSR-16F: 5′-TGATGACCATACAAGTGGGG-3 ′ (SEQ ID NO: 7)
Primer SSR-16R: 5'-GACTGTTCATCGAATGCCCT-3 '(SEQ ID NO: 8)
d) Molecular marker number 4: “SSR2” SSR marker primer SSR-2F: 5′-CACATGCAACACAACACAGG-3 ′ (SEQ ID NO: 9)
Primer SSR-2R: 5'-ATGGAGGTGGAGAAGGGG-3 '(SEQ ID NO: 10)
e) Molecular marker number 5: “SSR24” SSR marker primer SSR-24F: 5′-GGAGAGGGGGAGGATTAGAA-3 ′ (SEQ ID NO: 11)
Primer SSR-24R: 5'-TAGGACGAGCCAAGCTCAAG-3 '(SEQ ID NO: 12)
f) Molecular marker number 6: “SSR11” SSR marker primer SSR-11F: 5′-CCGATATCCAATTAACAATGC-3 ′ (SEQ ID NO: 13)
Primer SSR-11R: 5'-CAACCCTCCACCACTCACTT-3 '(SEQ ID NO: 14)

g) Molecular marker number 7 “NA-2RG4” CAPS marker primer NA-2RG-4F: 5′-CAGGATGGACAGGATATGTGG-3 ′ (SEQ ID NO: 15)
Primer NA-2RG-4R: 5'-CATGTGCCTTGCATTTCTTC-3 '(SEQ ID NO: 16)
Restriction enzyme type: RsaI or ClaI
When the rice cultivar “St. No. 1” is used as a template using the above primers, a PCR product having a base sequence of 323 bp is obtained. SEQ ID NO: 17 shows the sequence 906 bp including before and after the PCR product 323 bp. The PCR product 323 bp is located at positions 535 to 857 of the 906 bp (SEQ ID NO: 17). The sequence (ATAC) from position 763 to position 766 in the 906 bp (SEQ ID NO: 17) (ATAC position from position 228 to position 231 of the PCR product 323 bp) is not cleaved by restriction enzymes, but Nipponbare and Norin 8 (both varieties have no Pb1) The corresponding sequence in is the restriction enzyme cleavage position of RsaI. Therefore, the PCR product 323 bp can be used as a panicle resistance identification marker by combining with RsaI.
Similarly, the sequence ATCTAT of positions 619 to 624 (PCR product 323 bp, positions 84 to 89) of 906 bp (SEQ ID NO: 17) is not cleaved by restriction enzymes, but the corresponding sequence in Nipponbare and Norin No. 8 is ClaI This is the restriction enzyme cleavage position. Therefore, the PCR product 323 bp can be used as a panicle resistance identification marker by combining with ClaI.
Furthermore, the 906 bp sequence ACTAGT from positions 172 to 177 bp is cleaved by the restriction enzyme SpeI, but the corresponding sequence in Nipponbare and Norin No. 8 is not cleaved. Therefore, the above 906 bp (SEQ ID NO: 17) can be used as a marker that can be distinguished from Nipponbare or Norin No. 8 by combining with SpeI.

h) Molecular marker number 8 “NA-16RG” CAPS marker primer NA-16RGF: 5′-GACAGCCTTATCGTTCAGCA-3 ′ (SEQ ID NO: 18)
Primer NA-16RGR: 5'-TTGAAGCAATCTAATAATGGAGGA-3 '(SEQ ID NO: 19)
Restriction enzyme type: AluI
When the rice cultivar “St. No. 1” is used as a template using the above primers, a PCR product having a base sequence of 336 bp is obtained. SEQ ID NO: 20 shows a sequence of 1118 bp including before and after the above PCR product of 336 bp. The PCR product 336 bp is located at positions 552 to 887 of the above 1118 bp (SEQ ID NO: 20). The sequence (AGCC) from position 624 to position 627 (position 72 to position 75 of the PCR product 336 bp) in the above 1118 bp (SEQ ID NO: 20) is not cleaved by the restriction enzyme, but the corresponding sequence in Nihonbare, Norin 8 is AluI This is the restriction enzyme cleavage position. Therefore, the PCR product 336 bp can be used as a panicle resistance identification marker by combining with AluI.

i) Molecular marker number 9 “SSR4” SSR marker primer SSR-4F: 5′-GTTGATGAGCTCCATCGCTC-3 ′ (SEQ ID NO: 21)
Primer SSR-4R: 5'-TCCTCTTAAGCAGCCACCAT-3 '(SEQ ID NO: 22)

j) Molecular marker number 10 “N4-3RG1” CAPS marker primer N4-3RG1F: 5′-CGCCAAGCTACCCAAGTTAC-3 ′ (SEQ ID NO: 23)
Primer N4-3RG1R: 5'-TTGCAGCATTGTCTTCATCC-3 '(SEQ ID NO: 24)
Restriction enzyme type: EcoT22I
When the rice cultivar “St. No. 1” is used as a template using the above primers, a PCR product having a base sequence of 378 bp is obtained. SEQ ID NO: 25 shows a sequence of 1150 bp including around 378 bp of the PCR product. The PCR product 378 bp is located at positions 270 to 647 of 1150 bp (SEQ ID NO: 25). The sequence (ATGCAT) from position 315 to position 320 (position 45 to position 50 of the PCR product 378 bp) in the above 1150 bp (SEQ ID NO: 25) is cleaved with the restriction enzyme EcoT22I. The PCR product 378 bp can be used as a panicle resistance identification marker in combination with EcoT221.

k) Molecular marker number 11: “SSR34” SSR marker primer SSR-34F: 5′-GCGGGGAGTAGAACTCCAG-3 ′ (SEQ ID NO: 26)
Primer SSR-34R: 5'-CTGGCTCTTCAAGTCCATCC-3 '(SEQ ID NO: 27)
l) Molecular marker number 12: “N4-1” STS marker (dominant marker)
Primer N4-1F: 5'-GAGAGGCTGAACCGTTGAAG-3 '(SEQ ID NO: 28)
Primer N4-1R: 5'-CCAATGCTACCACCACTCCT-3 '(SEQ ID NO: 29)
m) Molecular marker number 13: “SSR39” SSR marker primer SSR-39F: 5′-CGCTGAAGTGAGGGGAAGT-3 ′ (SEQ ID NO: 30)
Primer SSR-39R: 5'-GACAACACGCAAACATGACC-3 '(SEQ ID NO: 31)
n) Molecular marker number 14 “K3” CAPS marker primer K3F: 5′-AAAACGATTTCCACGGAGAC-3 ′ (SEQ ID NO: 32)
Primer K3R: 5'-AATCTCGGCAGCTGATAGGA-3 '(SEQ ID NO: 33)
Restriction enzyme type: Sau3AI

o) Molecular marker number 15: “N3-2” STS marker primer N3-2F: 5′-CGCAGCGTTTTTATGGATTT-3 ′ (SEQ ID NO: 34)
Primer N3-2R: 5'-ACGTCCATCCAGCGTTTATC-3 '(SEQ ID NO: 35)
SEQ ID NO: 36 shows the partial sequence: 775 bp of the base sequence: 1131 bp of the PCR product using the above-mentioned primers and the rice cultivar “St. No. 1” as a template. When the partial sequence 775 bp is compared with the corresponding sequence of Nipponbare and Norin No. 8, a deletion of 349 to 3 bp and deletion of 368 to 27 bp are observed in 775 bp (SEQ ID NO: 36). If a primer is designed across the deletion, the partial sequence of 775 bp can be used as a panicle resistance identification marker.

p) Molecular marker number 16: “N2-3RG” CAPS marker primer N2-3RGF: 5′-TGACGCAGCAACTAAGATGG-3 ′ (SEQ ID NO: 37)
Primer N2-3RGR: 5'-TCTTCTAGTTCATTCTGGATTCTAAA-3 '(SEQ ID NO: 38)
Restriction enzyme type: BclI or Sau3AI
When rice cultivar “St. No. 1” is used as a template using the above primers, a PCR product having a base sequence of 224 bp is obtained. SEQ ID NO: 39 shows a sequence 812 bp including before and after the PCR product 224 bp. The PCR product 224 bp is located at positions 46 to 270 of the 812 bp (SEQ ID NO: 39). The sequence (TGATCA) from position 166 to position 171 (position 120 to position 125 of the PCR product 224) in the 812 bp is cleaved with the restriction enzyme BclI. Similarly, the sequence GATC from position 167 to 170 in 812 bp (position 121 to position 124 of the PCR product 224 bp) is cleaved by the restriction enzyme Sau3AI, and the corresponding sequence of Nihonbare and Norin 8 is not cleaved. 224 bp can be used as a panicle resistance identification marker in combination with BclI or Sau3AI.

q) Molecular marker number 17: “SSR6” SSR marker primer SSR-6F: 5′-TTTTTATGGGATTGAGGGAGT-3 ′ (SEQ ID NO: 40)
Primer SSR-6R: 5'-GTCAAGCTAGGCGGATTGTC-3 '(SEQ ID NO: 41)

r) Molecular marker number 18: “N2-2RG” CAPS marker primer N2-2RGF: 5′-TCTTGGGTGTCAGGTTTGAA-3 ′ (SEQ ID NO: 42)
Primer N2-2RGR: 5'-ATAGCTGTGATGGAGGTGGG-3 '(SEQ ID NO: 43)
Restriction enzyme type: MspI
When the rice cultivar “St. No. 1” is used as a template using the above primers, a PCR product having a base sequence of 202 bp is obtained. The sequence 762 bp including before and after the PCR product 202 bp is shown in SEQ ID NO: 44. The PCR product 202 bp is located from position 386 to position 670 of the above 762 bp (SEQ ID NO: 44). The sequence (CCGG) from position 425 to position 428 (position 39 to position 42 of the PCR product 202 bp) in the 762 bp is cleaved with the restriction enzyme MspI.

s) Molecular marker number 19: “N2-1RG” CAPS marker primer N2-1RGF: 5′-CCTTTAGGTCCACCGAATCA-3 ′ (SEQ ID NO: 45)
Primer N2-1RGR: 5'-GACACTATGCAGGTGCAGGA-3 '(SEQ ID NO: 46)
Restriction enzyme type: BanIII
t) Molecular marker number 20: “K1” STS marker primer K1F: 5′-TCTGGATAAAAATGGCGGAG-3 ′ (SEQ ID NO: 47)
Primer K1R: 5'-CCAGGCCGAACCCTAATATC-3 '(SEQ ID NO: 48)

u) Molecular marker number 21: “Pb3810 / 16” STS marker primer Pb3810 / 16F: 5′-TCTACGAGAGTTACAGCTTCTCC-3 ′ (SEQ ID NO: 49)
Primer Pb3810 / 16R: 5'-GAGTTGCATGGTACACCTAGCC-3 '(SEQ ID NO: 50)
PCR product base sequence 240bp (PCR product using rice cultivar "St. No. 1" as template) (SEQ ID NO: 51)

For F ₂ plants 7,279 individuals [Example 2] narrowed down Pb1 region "Norin 8" /`St.Nanba1 ", DNA was simplified extracted by the alkali method from the leaves of seedlings. Among the 21 molecular markers, PCR was performed using “N6-1” (marker number 1) and “Pb3810 / 16” (marker number 21) primers (see FIG. 1) located on the outermost side. 334 individuals having recombination between “-1” and “Pb3810 / 16” were selected.

  For the 334 recombinant individuals, the genotypes of 21 molecular markers were determined and the recombination positions were clarified. First, DNA of each individual was extracted from seedlings. Using the DNA as a template, PCR was performed using the 21 marker primers prepared in Example 1, and the pattern was analyzed by agarose gel electrophoresis. The electrophoretic pattern of 21 markers was compared with St. No. 1 and Norin 8 to determine whether the pattern of each marker was St. No. 1 or No. 8 type. As a result, it was found that a region that is likely to recombine and a region that is extremely unlikely to occur are adjacent to each other in the assumed region of Pb1.

Next, among the 334 recombination lines selected from the “Noririn No. 8” / “St. No. 1” crossing population, a part of the 334 recombination lines were fixed in the next generation, and 61 homogenized lines (F ₄ ) obtained were Panicle resistance was tested in the field. The above-mentioned panicle resistance test was conducted for two years, 2003 and 2004, at the Aichi Prefectural Agricultural Experiment Station, a mountain farming research institute field, which is a place where rice blasts usually occur. At the beginning of June, 15 strains of one line were transplanted to the test field by two plantings, and normal cultivation management was performed. Rice blast started in early July. From 10 days after heading, the survey criteria of Asaga are expressed in 11 stages from 0 to 10 (Saga 1981 Study on field resistance test for rice varieties Agricultural Experiment Station Research Report No. 35 51- 138.), the disease was investigated. The survey value 21-23 days after heading of each line was regarded as the disease severity. The disease incidence was determined by determining the rabies morbidity rate. The morbidity rate was 0%, the morbidity rate was 0, and the morbidity rate was 100%. The onset values of “St. No. 1” (with Pb1) and “Nori No. 8” (without Pb1) were 2.7 and 9.1, respectively. With reference to this value, the panicle resistance of the recombinant fixed line was determined. On the other hand, for 61 homogenized lines, each of the 21 markers of the present invention was examined to indicate whether it represents St. No. 1 type or Norin No. 8 type. Table 1 shows the types of the 61 homogenized lines. In Table 1, I represents St. No. 1 type, and J: represents No. 8 type. In addition, since [J / H] in the table is a dominant marker, the band is the same size for both “J” Japonica and “H” heterotypes, and distinguish between “J” and “H”. Indicates that you cannot. As a result of the series of studies described above, Pb1 was identified between the markers “NA-2RG4” (marker number 7)-“N2-3RG” (marker number 16) (Table 1, FIG. 1).

[Example 3] Ear motility test using Pb1-linked marker For 96 varieties / lines mainly including cultivars grown in Aichi Prefecture, the ear motility test was performed using the Pb1-linked marker of the present invention. The varieties and lines used were confirmed to be resistant to panicles, and it was examined whether the test results using the marker of the present invention were correct.

  DNA was extracted from the seedlings, PCR was performed using the primer of marker number 15: “N3-2” from the molecular markers prepared this time, and the pattern was analyzed by 1.5% agarose gel electrophoresis. The PCR product of “St. No. 1” was detected as a 1129 bp band that was 99 bp shorter than “Noribayashi No. 8” and “Nihonbare”, and could be distinguished from other varieties. The varieties in which a band was formed at this position were limited to varieties and lines introduced with panicle resistance from “St. No. 1” (FIG. 2).

It is a figure which shows the presumed area | region of the panicle resistance gene Pb1, and the position of a molecular marker. Numbers without an underline (1-21) indicate the marker number, and numbers with an underline indicate the number of recombinant individuals between each marker. The panicle resistance gene Pb1 is located in the center of the long arm of chromosome 11. Pb1 is limited to 278 kb, and 21 molecular markers were set before and after the Pb1 predicted region and inside. It is a photograph which shows the discrimination | determination result of panicle resistance by this invention marker. When PCR is performed with the marker “N3-2” primer, an amplified band of about 1.1 kb is detected. The presence or absence of a band at this position was consistent with the strength of the panicle resistance.

Claims (7)

A molecular marker linked to the rice blast resistance gene Pb1, which identifies the rice blast resistance of the test rice using at least one of the molecular markers shown in the linkage map of FIG. Method. The method according to claim 1, wherein when the molecular marker exhibits a genotype similar to that of rice having a panicle-resistant trait, the test rice is determined to be panicle-resistant. The molecular marker according to claim 1 or 2, wherein the molecular marker is a molecular marker comprising the base sequence according to any one of SEQ ID NOs: 3, 6, 17, 20, 25, 36, 39, 44, 51 or a partial sequence thereof. Method. A method for selecting rice having a panicle-resistant trait comprising the following steps.
(A) a step of mating rice having a trait resistance trait and rice having other traits (b) from a rice obtained by mating, using the method according to any one of claims 1 to 3 The process of selecting rice having a trait resistance SEQ ID NO: 3, 6, 17, 20, 25, 36, 39, 44, 51. An oligonucleotide having a chain length of at least 15 nucleotides complementary to DNA consisting of the base sequence described in any one of the above or a complementary strand thereof. At least one primer set shown in any one of (a) to (u) below.
(A) DNA consisting of the base sequence described in SEQ ID NO: 1 and DNA consisting of the base sequence described in SEQ ID NO: 2
(B) DNA consisting of the base sequence described in SEQ ID NO: 4 and DNA consisting of the base sequence described in SEQ ID NO: 5
(C) DNA consisting of the base sequence described in SEQ ID NO: 7 and DNA consisting of the base sequence described in SEQ ID NO: 8
(D) DNA consisting of the base sequence set forth in SEQ ID NO: 9 and DNA consisting of the base sequence set forth in SEQ ID NO: 10
(E) DNA consisting of the base sequence set forth in SEQ ID NO: 11 and DNA consisting of the base sequence set forth in SEQ ID NO: 12
(F) DNA consisting of the base sequence set forth in SEQ ID NO: 13 and DNA consisting of the base sequence set forth in SEQ ID NO: 14
(G) DNA consisting of the base sequence set forth in SEQ ID NO: 15 and DNA consisting of the base sequence set forth in SEQ ID NO: 16
(H) DNA consisting of the base sequence set forth in SEQ ID NO: 18 and DNA consisting of the base sequence set forth in SEQ ID NO: 19
(I) DNA consisting of the base sequence set forth in SEQ ID NO: 21 and DNA consisting of the base sequence set forth in SEQ ID NO: 22
(J) DNA consisting of the base sequence described in SEQ ID NO: 23 and DNA consisting of the base sequence described in SEQ ID NO: 24
(K) DNA consisting of the base sequence set forth in SEQ ID NO: 26 and DNA consisting of the base sequence set forth in SEQ ID NO: 27
(L) DNA consisting of the base sequence set forth in SEQ ID NO: 28 and DNA consisting of the base sequence set forth in SEQ ID NO: 29
(M) DNA consisting of the base sequence set forth in SEQ ID NO: 30 and DNA consisting of the base sequence set forth in SEQ ID NO: 31
(N) DNA consisting of the base sequence set forth in SEQ ID NO: 32 and DNA consisting of the base sequence set forth in SEQ ID NO: 33
(O) DNA consisting of the base sequence set forth in SEQ ID NO: 34 and DNA consisting of the base sequence set forth in SEQ ID NO: 35
(P) DNA consisting of the base sequence set forth in SEQ ID NO: 37 and DNA consisting of the base sequence set forth in SEQ ID NO: 38
(Q) DNA consisting of the base sequence set forth in SEQ ID NO: 40 and DNA consisting of the base sequence set forth in SEQ ID NO: 41
(R) DNA consisting of the base sequence set forth in SEQ ID NO: 42 and DNA consisting of the base sequence set forth in SEQ ID NO: 43
(S) DNA consisting of the base sequence set forth in SEQ ID NO: 45 and DNA consisting of the base sequence set forth in SEQ ID NO: 46
(T) DNA consisting of the base sequence set forth in SEQ ID NO: 47 and DNA consisting of the base sequence set forth in SEQ ID NO: 48
(U) DNA consisting of the base sequence listed in SEQ ID NO: 49 and DNA consisting of the base sequence described in SEQ ID NO: 50 A panicle resistance identification kit comprising at least one combination shown in any of (a) to (i) below.
(A) DNA consisting of the base sequence set forth in SEQ ID NO: 1, DNA consisting of the base sequence set forth in SEQ ID NO: 2, and restriction enzyme: TaqI
(B) DNA consisting of the base sequence described in SEQ ID NO: 4, DNA consisting of the base sequence described in SEQ ID NO: 5, and restriction enzyme: Sau3AI
(C) DNA consisting of the base sequence set forth in SEQ ID NO: 15 and DNA consisting of the base sequence set forth in SEQ ID NO: 16; and restriction enzyme: RsaI
(D) DNA consisting of the base sequence set forth in SEQ ID NO: 18 and DNA consisting of the base sequence set forth in SEQ ID NO: 19; and restriction enzyme: AluI
(E) DNA consisting of the base sequence set forth in SEQ ID NO: 23, DNA consisting of the base sequence set forth in SEQ ID NO: 24, and restriction enzyme: EcoT22I
(F) DNA consisting of the base sequence set forth in SEQ ID NO: 32, DNA consisting of the base sequence set forth in SEQ ID NO: 33, and restriction enzyme: Sau3AI
(G) DNA consisting of the base sequence set forth in SEQ ID NO: 37, DNA consisting of the base sequence set forth in SEQ ID NO: 38, and restriction enzyme: BclI
(H) DNA consisting of the base sequence set forth in SEQ ID NO: 42, DNA consisting of the base sequence set forth in SEQ ID NO: 43, and restriction enzyme: MspI
(I) DNA consisting of the base sequence set forth in SEQ ID NO: 45, DNA consisting of the base sequence set forth in SEQ ID NO: 46, and restriction enzyme: BanIII