JP4344818B2 - Development of single nucleotide polymorphism discrimination method of rice genome and application to rice variety identification - Google Patents

Description

【図面の簡単な説明】
【図１】図１の上図は、コシヒカリと他品種のゲノム部分配列を示す。コシヒカリと他品種のゲノム部分配列を比較した場合のＳＮＰの位置を、矢印で示す。
図１の中図は、上図に示したゲノム部分配列周辺領域を増幅するＳＮＰ判別用プライマーＡ１〜Ａ６およびＢ１〜Ｂ６の部分配列を示す。ＳＮＰ判別用プライマーの配列中、ゲノムＤＮＡ配列に対応する部分についてのみを図１に示した。「−」は、ゲノムＤＮＡ配列と同一の塩基を示す。ゲノムＤＮＡ配列と異なる塩基を、一文字表記で示した。ＳＮＰ判別用プライマーＡ１〜Ａ６全ての３’末端塩基は「Ｔ」である。一方、ＳＮＰ判別用プライマーＢ１〜Ｂ６全ての３’末端塩基は「Ｃ」である。そのため、図１では、ＳＮＰ判別用プライマーの３’末端の配列を「Ｔ／Ｃ」で示した。
図１の下図：ＳＮＰ判別用プライマーを使用したＰＣＲの結果を示す。
【図２】図２は、配列番号９および１０に記載される配列を有するＳＮＰ判別用プライマーを用いたＰＣＲの結果を示す。ゲルの写真の下に示した品種名は、鋳型ＤＮＡとして使用したゲノムＤＮＡを調製したイネの品種名である。
【図３】図３は、配列番号１〜８に記載される配列を有するＳＮＰ判別用プライマーを用いたＰＣＲの結果を示す。ゲルの写真の下に示した品種名は、鋳型ＤＮＡとして使用したゲノムＤＮＡを調製したイネの品種名である。
【図４】図４は、ＳＮＰ判別プライマーおよびＳＴＳプライマーを使用し、各イネ品種から調製したゲノムＤＮＡを用いてＰＣＲを行った結果を示す図である。使用したプライマーを、配列番号で記載する。ＳＴＳプライマーを使用した場合には判別できないが、ＳＮＰ判別プライマーを使用した場合にのみ識別可能となった品種名に「＊」を付した。[0001]
BACKGROUND OF THE INVENTION
The present invention mainly belongs to a rice variety identification method. In addition, the technique of the present invention can be applied as a DNA marker for selecting useful traits in rice breeding selection. The method of the present invention relates to a method for discriminating rice varieties by simply discriminating a difference of one base level in genomic DNA from a single rice or rice plant.
[0002]
Furthermore, the present invention belongs not only to rice variety identification but also to a method for genotyping a DNA marker site using, for example, the ability to discriminate a difference of one base level as a DNA marker for selection.
[0003]
[Prior art]
In recent years, methods for identifying varieties based on polymorphisms in genomic DNA have been developed.
[0004]
Examples of a method for detecting a DNA polymorphism that does not use the PCR method include AFLP (Amplified Fragment Length Polymorphism). The AFLP method is a genome fingerprinting method that was developed in the 1990s to detect homology of genomic DNA between individuals and varieties (Vos et al. (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407-4414), currently kits for AFLP are also commercially available (eg, AFLP Analysis system, Invitrogen, Carlsbad, CA, USA). In this method, (1) genomic DNA is treated with two types of restriction enzymes and adapters are ligated. (2) PCR is performed using a primer based on the adapter sequence (this primer is the 3 ′ end of the adapter sequence). And (3) a method in which amplification products are separated by electrophoresis, and the presence or absence of specific amplification products is detected. It is. This method requires a large amount of DNA necessary for the reaction, has a complicated process compared with PCR, and cannot detect a change in the level of one base other than the restriction enzyme recognition site. Have.
[0005]
In recent years, polymorphism discrimination methods using the PCR method have been developed. As a method for identifying polymorphisms of genomic DNA using the principle of PCR method, microsatellite method, RAPD method (random amplified polymorphic DNA method), RAPD-derived STS (sequence tagged site) method and the like are used. However, these conventional methods have the following drawbacks.
[0006]
The microsatellite method is a method of amplifying a several base repetitive sequence (microsatellite) present in genomic DNA and discriminating a polymorphism such as a difference in the number of repeats of the repetitive sequence. Since this method detects a slight PCR amplification fragment length difference, that is, a difference in the number of repeats of a repetitive sequence of several bases, a high-concentration gel is prepared and carefully analyzed when analyzing the amplification product. It is necessary to perform electrophoresis for a long time. Moreover, it has the fault that single nucleotide polymorphism cannot be discriminated.
[0007]
The RAPD method is a method for discriminating polymorphisms by amplifying genomic DNA using a random primer of generally about 12 bases and detecting the amplified band pattern by electrophoresis. In the RAPD method, although primer preparation is simple, it is necessary to set PCR conditions precisely, and different results occur depending on slight differences in conditions (for example, temperature). Therefore, in the RAPD method, it is difficult to set PCR conditions, and even if the conditions are set, reproducibility is poor and stable discrimination is difficult. In addition, the RAPD method is affected by the purity of the template DNA, and further has a drawback that multiple PCRs and electrophoresis are required.
[0008]
The RAPD-derived STS method is a method in which a specific band identified by the RAPD method is sequenced, primers are designed based on the sequence information, and PCR is performed. Since this method is based on a band identified by the RAPD method, it can be used only in a genomic portion having a polymorphism with a considerably different base sequence, and it is difficult to discriminate single base substitution.
[0009]
The SNPs method is a method capable of discriminating genotypes of single nucleotide polymorphisms, but for stable discrimination, an expensive apparatus such as a multi-label counter of PerkinElmer (Boston, MA, USA) is used. Need to use.
[0010]
The present invention provides a PCR primer for overcoming the drawbacks of these conventional methods and distinguishing polymorphisms at a single base level that is simple and highly reproducible.
[0011]
As an application example of the DNA polymorphism discrimination method, breed identification based on polymorphism of genomic DNA sequences has also become important in recent years. For example, in the case of rice, classic cultivar identification has been carried out by comparing fruit variability when mated, rice plant type, rice grain type, enzyme polymorphism, and the like. However, these classic techniques are effective for discriminating between indica and japonica, discriminating between indica and japonica, and distant rice. It was. As in recent years, there is a problem that it cannot be used for distinguishing closely related good-tasting rices developed mainly with “Koshihikari”.
[0012]
With the enforcement of the Food Law, it was obliged to display the varieties, origins, and year of milled rice on the milled rice packaging. These indications can be one of varietal identification indicators, but it is extremely difficult to identify closely related good-tasting rice varieties if there is no information on plants, rice bran, and brown rice. .
[0013]
Recently, it is necessary to specify the varieties of rice that is supplied as cooked rice at convenience stores, commercial rice cooking services, restaurants, and the like. However, since gelatinization of starch, protein denaturation, and the like occur by cooking rice, it is impossible to extract DNA and identify varieties by classical techniques.
[0014]
However, conventional methods for identifying polymorphisms in genomic DNA have the disadvantages described above. Therefore, there is a need for a simple and highly reproducible polymorphism identification method.
[0015]
The technology for distinguishing polymorphisms on the genome in selective breeding of DNA markers, which has the same principle as that of breed identification by genome analysis, has the same disadvantages as the current technology, and requires a simple and reliable polymorphism discrimination technology. .
[0016]
[Patent Document 1]
Japanese Patent Laid-Open No. 6-113850
[Patent Document 2]
Japanese Patent Laid-Open No. 6-113851
[Patent Document 3]
Japanese Patent Laid-Open No. 6-113852
[Patent Document 4]
Japanese Patent Laid-Open No. 7-39400
[Patent Document 5]
Japanese Patent Laid-Open No. 2001-95589
[0017]
[Problems to be solved by the invention]
An object of the present invention is to provide a simple and highly reproducible method for identifying genomic DNA polymorphisms. The method of the present invention can be used, for example, as a DNA marker in rice variety identification and further in rice breeding selection.
[0018]
[Means for Solving the Problems]
The above-mentioned problems have been achieved by investigating primers used in the SNPs discrimination method and finding for the first time a primer that is simple and highly reproducible and can be used to identify polymorphisms in genomic DNA.
[0019]
Specifically, the third base from the 3 ′ end of the primer was replaced with another base to create an artificial mismatch and enhance the PCR selectivity. When making this mismatch, for example, if the original base is G, it will be T, if it is A, it will be C, if it is T, if it is C, it will be A. You can make a marker.
[0020]
The Tm of the primer is 50 ° C. or higher, preferably 55 ° C. or higher, more preferably 60 ° C. or higher, and a primer having a Tm of 65 ° C. or higher and 70 ° C. or higher can also be used.
[0021]
Accordingly, the present invention provides the following.
1. A PCR primer for discriminating the genotype of a single nucleotide polymorphism, wherein the first base from the 3 ′ end of the primer corresponds to the position of the single nucleotide polymorphism to be discriminated, and the 3 ′ end of the primer The third base from is replaced by a base complementary to the template sequence that the primer anneals, the substitution being a G to T, A to C, T to G, or C to A substitution .
2. The primer of item 1 which has Tm of 50 degreeC or more.
3. The primer of item 1 which has Tm of 60 degreeC or more.
4). The primer of item 1 which has Tm of 65 degreeC or more.
5. Primer pairs selected from the group consisting of the following primer pairs:
(1) SEQ ID NOs: 1 and 2;
(2) SEQ ID NOs: 3 and 4;
(3) SEQ ID NOs: 5 and 6;
(4) SEQ ID NOs: 7 and 8; and
(5) SEQ ID NOs: 9 and 10.
6). A set of primers comprising at least two primer pairs according to item 5.
7). Item 7. The primer set according to Item 6, comprising two or more primers selected from the group consisting of SEQ ID NOs: 1-12.
8). A method for determining a genotype of a single nucleotide polymorphism using the primer according to item 1.
9. A kit for determining a genotype of a single nucleotide polymorphism, comprising the primer according to item 1.
10. A method for discriminating a genotype of a single nucleotide polymorphism, which comprises the following steps:
a) selecting a single nucleotide polymorphism genotype to be discriminated;
b) a step of preparing a polymorphism discrimination primer, wherein the first base from the 3 ′ end of the polymorphism discrimination primer corresponds to the position of the single nucleotide polymorphism to be discriminated; The third base from the 3 ′ end of the type discrimination primer is substituted with a base complementary to the template sequence that the polymorphism discrimination primer anneals. The substitution is from G to T, from A to C, from T A step of preparing a polymorphism discrimination primer which is a substitution of G or C to A;
c) performing PCR using the polymorphism discrimination primer and at least one other primer; and
d) a step of discriminating the genotype of the single nucleotide polymorphism from the presence or absence of amplification of the PCR product;
Including the method.
11. Item 11. The method according to Item 10, wherein at least two or more primer pairs are used.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.
[0023]
Listed below are definitions of terms particularly used in the present specification.
[0024]
As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. A “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides and is used interchangeably. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, derivative oligonucleotides in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. Is an N3′-P5 ′ phosphoramidate bond derivative oligonucleotide, a ribose and phosphodiester bond in the oligonucleotide converted to a peptide nucleic acid bond, and uracil in the oligonucleotide is C− A derivative oligonucleotide substituted with 5-propynyluracil, a derivative oligonucleotide wherein uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide is substituted with C-5 propynylcytosine Derivative oligonucleotides, derivative oligonucleotides in which cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in DNA is substituted with 2′-O-propylribose, and Examples thereof include derivative oligonucleotides in which ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Optionally, the primer includes a base substituted with a deoxyinosine residue.
[0025]
The term “nucleic acid” is also used herein interchangeably with gene, DNA, cDNA, mRNA, oligonucleotide, and polynucleotide. Particular nucleic acid sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.
[0026]
In the present specification, the “nucleotide” may be natural or non-natural. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioate, phosphoramidate, methyl phosphonate, chiral methyl phosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA). .
[0027]
In the present specification, when the nucleotide is represented by one letter, G represents guanine, T represents thymine, A represents adenine, and C represents cytosine.
[0028]
In the present specification, a “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit.
[0029]
As used herein, the term “primer” means under appropriate conditions (eg, in the presence of four different nucleoside triphosphates and a polymerization agent (eg, DNA polymerase, RNA polymerase, or reverse transcriptase)). ) Refers to a single-stranded oligonucleotide that acts as a point to initiate template-directed DNA synthesis in an appropriate buffer and at an appropriate temperature. The appropriate length of the primer depends on the intended use of the primer but is typically in the range of 15-30 nucleotides. Short primer molecules generally require lower temperatures in order to form a sufficiently stable hybrid complex with the template. A primer need not exactly match the exact sequence of the template, but should be sufficiently complementary to hybridize with the template. The term “primer site” refers to the sequence of the template nucleic acid to which the primer hybridizes. The term “primer pair” refers to a 5 ′ (upstream) primer that hybridizes to the 5 ′ end of the nucleic acid sequence to be amplified and a 3 ′ (downstream) primer that hybridizes to the complement of the 3 ′ end of that sequence to be amplified. A set of primers including
[0030]
As used herein, “single nucleotide polymorphism” refers to a state in which the nucleotide sequence of a gene differs only by one place and its site. In the present specification, “single nucleotide polymorphism” is used interchangeably with “SNP” and “SNPs”.
[0031]
As used herein, a “polymorphism discrimination primer” is used to discriminate the genotype of a polymorphic site from the presence or absence of the amplification product by a nucleic acid amplification reaction such as PCR using the primer. Means a primer. In the present specification, the term “polymorphism discrimination primer” is used interchangeably with “SNP discrimination primer” and “SNPs discrimination primer”.
[0032]
In this specification, the term “PCR (polymerase chain reaction)” is used as an example of a nucleic acid amplification reaction, but a nucleic acid amplification reaction other than PCR can also be used for carrying out the present invention. Therefore, the primer of the present invention is not limited to PCR.
[0033]
In this specification, when counting bases from the 3 ′ end of the “primer”, “the first base from the 3 ′ end of the primer” refers to the base at the 3 ′ end. Thus, for example, in the primer having the sequence of SEQ ID NO: 1 (5′-CCAACTGCTGGCATAATAGCCC-3 ′), “the first base from the 3 ′ end of the primer” is “C” and “3” from the 3 ′ end of the primer The “th base” is “G”.
[0034]
In this specification, the “base corresponding to the first base from the 3 ′ end of the primer” means that when the primer hybridizes with the template nucleic acid, the first base from the 3 ′ end of the primer hybridizes. The base present at the position.
[0035]
Preparation of template DNA in PCR using the primer of the present invention can be performed by preparing genomic DNA from cells, tissues, etc. by various well-known methods (for example, CTBA method, boil method, etc.).
[0036]
When using a DNA fragment as the template nucleic acid, the template nucleic acid can be prepared, for example, by digesting genomic DNA or plasmid DNA, or by using PCR. Oligonucleotides for use as primers or probes are described by methods known in the art of chemical synthesis of polynucleotides (by way of non-limiting example, Beaucage and Carruthers (Tetrahedron Lett 22: 1859-1862 (1981)). Chemically synthesized by the phosphoramidite method and the triester method provided by Matteucci et al. (J. Am. Chem. Soc., 103: 3185 (1981), both of which are incorporated herein by reference). These syntheses may use an automated synthesizer as described in Needham-Van Devanta, DR, et al. (Nucleic Acids Res. 12: 6159-6168 (1984)). Oligonucleotide purification can be performed using native acrylamide gel electrophoresis or anion exchange HPLC as described in Pearson, JD and Regnier, FE (J. Chrom, 255: 137-149 (1983)). Next, if desired, the double-stranded fragment can be annealed under appropriate conditions or with appropriate primer sequences, if desired. It can be obtained by synthesizing the complementary strand using DNA polymerase, given the specific sequence for the nucleic acid probe, it is understood that the complementary strand is also identified and included. Target strands work equally well when the target is a double-stranded nucleic acid.
[0037]
The sequence of a synthetic oligonucleotide or any nucleic acid fragment can be obtained using either the dideoxy chain termination method or the Maxam-Gilbert method (Sambrook et al., Molecular Cloning-a Laboratory Manual (2nd edition), 2nd edition). 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989), incorporated herein by reference). This manual is referred to herein as “Sambrook et al.”; “Zyskind et al. (1988)”. Recombinant DNA Laboratory Manual, (Acad. Press, New York).
[0038]
In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). .
[0039]
General molecular biology techniques that can be utilized in the present invention include Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, etc. can be easily implemented by those skilled in the art.
[0040]
(Description of Preferred Embodiment)
In one aspect, the present invention provides a primer in which the first, second, third, or fourth base is substituted from the 3 ′ end. This substitution may be only one or a combination of two or more. Preferably, only the third base from the 3 ′ end is substituted. The primer substitution may be any substitution, but is preferably a G to T, A to C, T to G, or C to A substitution.
[0041]
In one aspect, the Tm of the primer of the present invention is preferably 50 ° C. or higher, more preferably 55 ° C. or higher, more preferably 60 ° C. or higher, and has a Tm of 65 ° C. or higher and 70 ° C. or higher. Primers can also be used.
[0042]
3 ′ of the primer of the present invention corresponds to the position of the single nucleotide mutation to be discriminated. Accordingly, the base at the position of the single base mutation and the first base from the 3 ′ end of the primer of the present invention are in a position where they can be annealed. When using the primer of the present invention, the first base from the 3 ′ end may be complementary to the base of one genotype of the SNP or may be complementary to the other genotype of the SNP. . When using a plurality of the primers of the present invention, it is preferable that the first base from the 3 ′ end is complementary to the base of one genotype of SNP and the 3 ′ end base is another gene of SNP. Used in combination with a primer that is complementary to the base of the type.
[0043]
Preferably, the primer of the present invention can amplify only a template having a base complementary to the base at the 3 ′ end of the primer. Accordingly, when an amplification product is detected in the PCR reaction using the primer of the present invention, the base at the position corresponding to the primer 3 ′ end in the template is a base complementary to the primer 3 ′ end. .
[0044]
Examples of the template used in the PCR reaction in the present invention include, but are not limited to, genomic DNA, cDNA, RNA, and mRNA. Genomic DNA used in the present invention includes, but is not limited to, bacterial chromosomal DNA, eukaryotic nuclear DNA, mitochondrial DNA, chloroplast DNA, and viral DNA. The template used in the present invention may be in a state isolated from nature or may have been isolated and then treated with an enzyme or the like. Examples of the enzyme that processes the template include, but are not limited to, reverse transcriptase, restriction enzyme, and polymerase.
[0045]
The primer used in the present invention may be labeled with, for example, a radioactive substance or a fluorescent substance. In the SNP discrimination method using the primer of the present invention, an STS primer may be used at the same time.
[0046]
Examples of the method for detecting a PCR product include, but are not limited to, electrophoresis and a real-time PCR detection apparatus.
[0047]
Various well-known or commercially available enzymes can be used for the PCR reaction. The conditions for the PCR reaction are well known.
[0048]
The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.
[0049]
【Example】
(Example 1: Preparation of genomic DNA)
Methods for preparing genomic DNA from various organisms are well known. For example, when preparing genomic DNA from polished rice or brown rice, the following method was used.
[0050]
Take 1 grain of polished rice or brown rice (for rice with rice bran, use Fujiwara Seisakusho (Tokyo) 's rice grinder to brown rice, and to use brown rice for milled rice, use a test parquet made in Tokyo (Tokyo) ), Wrapped in medicine wrapping paper, crushed with a hammer, and placed in a 1.5 mL Eppendorf tube (a small test tube made of polypropylene for testing). 400 μL of DNA extract (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 1M KCl) was added and stirred. After standing at room temperature for 10 minutes, the mixture was centrifuged at 15000 rpm for 10 minutes in a Tommy centrifuge MX-100, the supernatant was taken, 400 μL of 2-propanol was added, and the mixture was stirred and centrifuged at 15000 rpm for 10 minutes to precipitate DNA. The supernatant was discarded, the precipitate was dried, dissolved in 20 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0), and used as template DNA (genomic DNA).
[0051]
(Example 2: Optimization of SNP discrimination primer)
Primers that specifically amplify different genotypes of SNPs in the PCR reaction were prepared, the specificity was examined, and the primers were optimized.
[0052]
As a candidate primer when optimizing a primer, a nucleic acid sequence having “T” base as “base corresponding to the first base from the 3 ′ end of the primer” is specifically used as in SEQ ID NO: 13 (A1 primer) A2-A6 primers were synthesized as primers to be amplified (Fig. 1). A2-A6 primers (SEQ ID NOs: 14-18) are primers in which bases corresponding to the 3rd to 5th bases from the 3 ′ end of the primer are substituted with various bases. The B1 primer (SEQ ID NO: 19) is a primer in which “T” at the 3 ′ end of the A1 primer is replaced with “C”. Similarly, the B2 to B6 primers (SEQ ID NOs: 20 to 24) are primers in which “T” at the 3 ′ end of the A2 to A6 primers is replaced with “C”.
[0053]
Each of these primers was subjected to PCR under the following conditions, using a primer prepared from Koshihikari as a template DNA together with a primer having the sequence described in SEQ ID NO: 25 (5′-gctgggtgctatagctgttatcctc-3 ′).
(PCR solution)
Template DNA 1.0 μL
10 x Taq polymerase reaction buffer 2.0 μL
dNTPs (each 2.5 mM) 1.6 μL
Sterile water 14.5μL
Taq polymerase (5 U / μL) 0.1 μL
Primer (forward direction) (50 μM) 0.4 μL
Primer (reverse direction) (50 μM) 0.4 μL
Total 20.0μL
In this experiment, a primer having a Tm (melting point) of 60 ° C. or higher was designed. As a polymerase for PCR, Takara's EX Taq polymerase, which is Taq polymerase for HotStart, was used. The PCR amplifier uses Takara's Thermal Cycler SP, but is not limited to these.
[0054]
PCR conditions are as follows:
94 ° C 2 minutes
After 30 cycles
94 ° C for 10 seconds
60 ° C for 10 seconds
72 ° C 20 seconds
Finally, the PCR product was stored by incubating at 4 ° C.
[0055]
The results of confirmation of PCR amplification and non-amplification by gel electrophoresis are shown in FIG. The primers used in the experiments shown in each lane of FIG. 1 are as follows:
Lane A1 (SEQ ID NO: 13 and SEQ ID NO: 25),
Lane B1 (SEQ ID NO: 19 and SEQ ID NO: 25),
Lane A2 (SEQ ID NO: 14 and SEQ ID NO: 25),
Lane B2 (SEQ ID NO: 20 and SEQ ID NO: 25),
Lane A3 (SEQ ID NO: 15 and SEQ ID NO: 25),
Lane B3 (SEQ ID NO: 21 and SEQ ID NO: 25),
Lane A4 (SEQ ID NO: 16 and SEQ ID NO: 25),
Lane B4 (SEQ ID NO: 22 and SEQ ID NO: 25),
Lane A5 (SEQ ID NO: 17 and SEQ ID NO: 25),
Lane B5 (SEQ ID NO: 23 and SEQ ID NO: 25),
Lane A6 (SEQ ID NO: 18 and SEQ ID NO: 25),
Lane B6 (SEQ ID NO: 24 and SEQ ID NO: 25).
[0056]
The primer sequences used are as follows:
A1 primer: SEQ ID NO: 13: gcggctctggggccggccgtggtat
A2 primer: SEQ ID NO: 14: gcggctcttagggccggcctggaat
A3 primer: SEQ ID NO: 15: gcggctcttagggccggcctgctat
A4 primer: SEQ ID NO: 16: gcggctcttagggccggcctcgaat
A5 primer: SEQ ID NO: 17: gcggctctagggccggccgtggat
A6 primer: SEQ ID NO: 18: gcggctcttagggccggccgtgttat
B1 primer: SEQ ID NO: 19: gcggctctggggccggccgtggtac
B2 primer: SEQ ID NO: 20: gcggctctagggccggcctggaac
B3 primer: SEQ ID NO: 21: gcggctctagggccggccctgctac
B4 primer: SEQ ID NO: 22: gcggctctagggccggcctcgaac
B5 primer: SEQ ID NO: 23: gcggctctggggccggccgtggac
B6 primer: SEQ ID NO: 24: gcggctcttagggccggcctgttac
AB-reverse primer: SEQ ID NO: 25: gctgggtgctatatagctgttatcctc.
[0057]
As shown in the electrophoresis results of FIG. 1, primers different only at the 3 ′ end could not specifically discriminate SNPs (lanes A1 and B1). Specifically, since the base corresponding to the 3 ′ end of the used A1 primer and B1 primer is “T” in Koshihikari, amplification using the B1 primer is a false positive amplification. Therefore, SNPs could not be specifically identified even when primers differing only at the 3 ′ end were used.
[0058]
Next, PCR and gel electrophoresis were performed using primers A2 to A6 and B2 to B6 in which the bases corresponding to the 3rd to 5th bases from the 3 ′ end of the A1 primer and the B1 primer were substituted with various bases. . As a result, only when the A5 primer and the B5 primer were used, neither false positive nor negative was observed. Therefore, it can be understood that in order to design an optimal SNP discrimination primer, it is preferable to replace the third base from the 3 ′ end of the primer with a base complementary to the template sequence that the primer anneals.
[0059]
The results shown in FIG. 1 (lanes A2, B2, A5 and B5) are consistent with the expectation that a purine-purine base pair mismatch would destabilize the nucleic acid duplex. Surprisingly, however, the single nucleotide polymorphism is greater when primers that form G / A mismatches (lanes A5 and B5) than with primers that form A / A mismatches (lanes A2 and B2). The specificity of the primer in the discrimination was remarkably improved, and when a primer that actually forms a G / A mismatch was used, no false positive was detected.
[0060]
This result shows that (1) the formation of an A / A mismatch causes steric hindrance between the 6-position amino groups of adenine of different chains, and (2) the formation of a G / A mismatch, When hydrogen bonds can be formed between the guanine ring and the adenine ring, the case of (2) has a greater influence on the base pairing of the primer, and as a result, a single base It is predicted that the specificity of the primer in type discrimination is improved. Therefore, it is preferable for the improvement of the primer characteristics in single nucleotide polymorphism discrimination to form not only a purine-purine mismatch base pair but also a G / A mismatch base pair capable of forming a hydrogen bond.
[0061]
Similarly, in the case of pyrimidine, not only the formation of a pyrimidine-pyrimidine mismatch base pair but also the formation of a T / C mismatch base pair capable of forming a hydrogen bond is a primer in single nucleotide polymorphism discrimination. It is considered preferable for improving the characteristics.
[0062]
From the above results, in designing a PCR primer for discriminating the genotype of a single nucleotide polymorphism, the third base from the 3 ′ end of the primer is replaced with a base complementary to the template sequence that the primer anneals. And the substitution is either (1) a C to A substitution, or a T to G substitution, so as to form a G / A base pair, or (2) a T / C base pair is formed. In addition, the substitution from G to T or the substitution from A to C is preferable.
[0063]
(Example 3: PCR amplification reaction using a primer corresponding to the position of the SNP in which the first base from the 3 ′ end of the primer is to be distinguished)
In order to identify rice varieties, the primer pairs of SEQ ID NOs: 9 and 10 were used. These primer pairs were designed as follows based on the base sequence (region 31P2) containing SNPs on the first chromosome of rice.
[0064]
In the 31P2 region, an SNP that was an “A” base in Koshihikari but a “T” base in Nipponbare was identified. In order to determine the genotype of this SNP, the first base from the 3 ′ end of the primer is made to correspond to the position of this SNP site, and as a primer for amplifying Koshihikari genomic DNA, the sequence 5′-ctatagaataacttgtctttggcaagaca-3 A primer having '(SEQ ID NO: 10) was designed. In PCR, a primer having the sequence 5′-gagcctgttgatgggtgaagc-3 ′ (SEQ ID NO: 9) was designed as a primer to be used with this primer, and a PCR amplification reaction was performed.
[0065]
The composition of the reaction solution is as follows:
Template DNA 1.0 μL
10 x Taq polymerase reaction buffer 2.0 μL
dNTPs (each 2.5 mM) 1.6 μL
Sterile water 14.5μL
Taq polymerase (5 U / μL) 0.1 μL
Primer 1; (5′-ctatacaaaaacttgtctttggacaagcaa-3 ′ SEQ ID NO: 10) (50 μM) 0.4 μL
Primer 2; (5′-gagcctgttgatgggtgaagc-3 ′ SEQ ID NO: 9) (50 μM) 0.4 μL
Total 20.0μL
In this experiment, Taq polymerase uses Takara's Ex Taq polymerase, and PCR amplifier uses Takara's Thermal Cycler SP. However, the present invention is not limited to these.
PCR conditions are as follows:
94 ° C 2 minutes
After 30 cycles
94 ° C for 10 seconds
60 ° C for 10 seconds
72 ° C 20 seconds
Finally, the PCR product was stored by incubating at 4 ° C.
[0066]
The use of PCR reactions other than those described above is well known to those skilled in the art, so that PCR amplification for increasing normal DNA is not limited to the above reaction cycle, and other cycles can be used as well. is there.
[0067]
(Confirmation of PCR amplification and non-amplification by gel electrophoresis)
In the cultivar identification in this example, rice varieties are identified based on the polymorphism of the genome, using as an index whether or not nucleic acids are amplified by PCR amplification reaction using arbitrarily selected rice genomic DNA. It was.
[0068]
The method for confirming amplified nucleic acid by gel electrophoresis was as follows.
Using 0.8 to 2.0% agarose gel, take a part of PCR product (2 μL or more) and perform electrophoresis. As an electrophoresis apparatus, a mini gel (trade name: Mupid) of Cosmo Bio (Tokyo) was used. After electrophoresis for about 15 minutes, the gel was stained with ethidium bromide and the DNA was confirmed with a UV lamp (FIG. 2).
[0069]
As a result, when Koshihikari genomic DNA was used as a template, a nucleic acid fragment was amplified by the PCR method. In the same way, nucleic acids in “Hitomebore”, “Hinohikari”, “Akitakomachi”, “Kirara 397”, “Haenuki”, “Sasanishiki”, “Dontokoi”, and “Akihikari” having the same genotype as Koshihikari The fragment was amplified.
[0070]
On the other hand, when genomic DNA of Nipponbare was used as a template, the nucleic acid fragment was not amplified by PCR. Similarly, the nucleic acid fragments were not amplified in “Kinuhikari”, “Yuki no Sei”, “Koi Shibuki”, “Hanaetizen”, “Hohoho no Hoho” and “500 000 stones” having Nippon Sunny type SNP. .
[0071]
Therefore, the base corresponding to the first nucleic acid from the 3 ′ end of the primer of SEQ ID NO: 10 is not “T” but the amplification of the nucleic acid fragment by PCR using the primer pair of SEQ ID NO: 9 and 10 as an index. A ”can be discriminated.
[0072]
In addition, when the amplified nucleic acid is confirmed when the above primers are used, the varieties obtained by adjusting the template DNA used for PCR are, for example, “Koshihikari”, “Hitomebore”, “Hinohikari”, “Akitakomachi” ”,“ Kirara 397 ”,“ Haenuki ”,“ Sasanishiki ”,“ Donto Koi ”, or“ Akihikari ”,“ Kinuhikari ”,“ Yuki no Sei ”,“ Koi Buki ”,“ Hanaetizen ”, It is also possible to identify whether it is neither “hohohonoho” nor “500 million stones”.
[0073]
(Example 4: Multiplex PCR)
In order to identify rice varieties, a plurality of primer pairs are combined, and the presence or absence of an amplified band in each primer pair is examined. For this reason, a PCR reaction in a plurality of tubes is usually required.
[0074]
Therefore, in order to simplify this work, it is also possible to mix several primer pairs and perform the reaction in one PCR (one tube). This method is called a multiplex method. The example of the reaction liquid composition in this case is shown.
[0075]
For four types of PCR markers
Template DNA 1.0 μL
10X PCR reaction buffer 2.0 μL
dNTPs (each 2.5 mM) 1.6 μL
Sterile water 13.86 μL
Taq polymerase (5 U / μL) 0.1 μL
Primers (SEQ ID NOs: 1 to 8) 50 μM each
Total 20μL
The primers used in the above reaction are as follows:
6P1N-522 primer: SEQ ID NO: 1: CCAACTGCTGCAATAATGCC
6P1N-322 primer: SEQ ID NO: 2: CGTAGCAACTCAATATAACATCAGCTAAA
26P1K-5 primer: SEQ ID NO: 3: TGTTTTAGAACAAAAGTAAGGAGCAATGCT
26P1-3-1 primer: SEQ ID NO: 4: GATACTTAGGGGCCCGTTTGG
17P1K-51 primer: SEQ ID NO: 5: TGACAACGTAGATATAGGTTTCTATTCAATAATAC
17P1K-32 primer: SEQ ID NO: 6: ACAATCCCTGTTTGCCAGG
4P1K-5-2 primer: SEQ ID NO: 7: TTGTATCCAAAATAAATCAGGACCACTCTG
4P1-3-11 primer: SEQ ID NO: 8: GGTCTTGTAGGCTCTCAC.
[0076]
In the case of multiplex PCR, 3% agarose gel was used for gel electrophoresis for confirmation of PCR products, and electrophoresis was performed for about 40 minutes in order to clarify separation between bands. The results are shown in FIG.
[0077]
(Example 5: Variety identification by marker)
Using the primer pair of the present invention and the STS-type primer, cultivar identification between representative varieties distributed in Japan and cultivars grown in the Hokuriku region was performed. PCR conditions for the type discrimination are as in Example 2.
[0078]
As primers of the present invention, in addition to the primers of SEQ ID NOs: 1 to 10, the following primers were used:
37P2-5-H primer: SEQ ID NO: 11: GGGTATCATGCCTTTACTGG
37P2-N3 primer: SEQ ID NO: 12: GCGACAGGAGTAGCCCTTC.
[0079]
The following primers were used as 12 sets of STS type primers:
18P1-5-1 primer: SEQ ID NO: 26: GGCTCTAGCGGTCATCAGG
18P1-3-1 primer: SEQ ID NO: 27: TTCTCATCATAACATATTGATCTTGC
18P12-52 primer: SEQ ID NO: 28: ATGGCCGTGCCTCTGTTTCC
18P12-32 primer: SEQ ID NO: 29: CAGCAGTTTGGAAAACCAACG
18P14-51 primer: SEQ ID NO: 30: AAGACATAGGCCTCGGTTGC
18P14-31 primer: SEQ ID NO: 31: TGTTGAACTTGGGGGTATGC
27P1-5-1 primer: SEQ ID NO: 32: CATGCGCCTTCACAACCACC
27P1-3-1 primer: SEQ ID NO: 33: CAATTGACTTTTGCAAGATGC
29P1-5 primer: SEQ ID NO: 34: AAGCAATCACTTCCTGCCAACC
29P1-3 primer: SEQ ID NO: 35: TCGTCTCCAGCAACATTCTG
33P1-5 primer: SEQ ID NO: 36: AGTGCTCAAGAAATCATAGCTGA
33P1-3 primer: SEQ ID NO: 37: GAAGCTTGTGAGTAACGGGC
34P1-5-H primer: SEQ ID NO: 38: TCTCCCAGATCAAGAACTAGC
34P1A-3 primer: SEQ ID NO: 39: CGGCAAGCACACGCGTC
34P2-5 primer: SEQ ID NO: 40: ATTACTGCCAAACCGCTTGC
34P2-3 primer: SEQ ID NO: 41: TGTGATCCATATTATTGCTCTAACC
34P3-5-1 primer: SEQ ID NO: 42: TCCCTCCCTTCTCACACTACTCC
34P3-3-1 primer: SEQ ID NO: 43: GACACCAGCTTCCCTTGTCC
35P1-5 primer: SEQ ID NO: 44: TCTGACACTCTTATCAACAATATACATGAC
35P1-3 primer: SEQ ID NO: 45: TGCAAGAAGGAACGAAGAGC
37P1-5 primer: SEQ ID NO: 46: GCACACTATTATCCCTTCTCTCATATGC
37P1-3 primer: SEQ ID NO: 47: CATGGCGCTCACACAGG
38P1-5 primer: SEQ ID NO: 48: ACCGAGACATCATGTTGTCTGTC
38P1-3 primer: SEQ ID NO: 49: TTTGAGCCCCATAAATGAATAATCTG.
[0080]
The comparison results using the above primers are shown in FIG. When an amplification product by PCR was confirmed, “1” was described, and when an amplification product was not confirmed by PCR, “0” was described.
[0081]
Many rice varieties that could not be identified by the above STS primers (SEQ ID NOs: 26 to 49) are genetically related to each other (for example, Koshihikari, Kinuhikari, Dontokoi), but when using the primer of the present invention Could be identified.
[0082]
Specifically, Koshihikari, Hitomebore, Kinuhikari, Haenuki, Dontokoi, Yukinosei, Koibuki, Hanaechizen, which could not be identified by the 12 sets of STS primers used in the past, were applied by the method of the present invention. It became possible to discriminate | determine by using 4 sets of created SNP discrimination | determination markers (sequence number 1-8).
[0083]
From the above results, by using the primer optimized in the present invention, it became possible to discriminate single nucleotide polymorphisms without causing false positives and false negatives. In addition, a marker for identifying genetically related rice varieties, which requires a great effort to set a marker that can be identified by the conventional method using an STS primer, can be easily created.
[0084]
Further, by combining the primer of the present invention with the conventional STS type primer, it becomes possible to identify more closely related rice.
[0085]
【The invention's effect】
By using the primer of the present invention, it became possible to discriminate a mutation at a single base level with higher accuracy than the conventional method. Further, in the discrimination of SNPs genotype, when the discrimination method using the primer of the present invention is used, it is not necessary to use a particularly expensive machine as in the prior art. Therefore, simple and stable variety identification is possible with the primer of the present invention.
[0086]
Since the primer of the present invention determines single-base substitution without causing a false positive problem, mutations at more sites on the genome can be stably identified than in the conventional method.
[0087]
The primer of the present invention is also useful as a DNA marker for selective breeding in breeding of rice and the like.
[0088]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows an upper part of FIG. 1 showing genome partial sequences of Koshihikari and other varieties. The position of the SNP when comparing the genome partial sequences of Koshihikari and other varieties is indicated by arrows.
The middle diagram of FIG. 1 shows partial sequences of SNP discrimination primers A1 to A6 and B1 to B6 that amplify the peripheral region of the genome partial sequence shown in the upper diagram. Only the portion corresponding to the genomic DNA sequence in the sequence of the SNP discrimination primer is shown in FIG. “-” Indicates the same base as the genomic DNA sequence. Bases that differ from the genomic DNA sequence are shown in single letter code. The 3 ′ terminal bases of all the SNP discrimination primers A1 to A6 are “T”. On the other hand, the 3 ′ terminal bases of all the SNP discrimination primers B1 to B6 are “C”. Therefore, in FIG. 1, the sequence at the 3 ′ end of the SNP discrimination primer is indicated by “T / C”.
The lower figure of FIG. 1 shows the result of PCR using a primer for SNP discrimination.
FIG. 2 shows the results of PCR using SNP discrimination primers having the sequences described in SEQ ID NOs: 9 and 10. The cultivar name shown below the gel photograph is the cultivar name of the rice from which the genomic DNA used as the template DNA was prepared.
FIG. 3 shows the results of PCR using SNP discrimination primers having the sequences set forth in SEQ ID NOs: 1-8. The cultivar name shown below the gel photograph is the cultivar name of the rice from which the genomic DNA used as the template DNA was prepared.
FIG. 4 is a diagram showing the results of PCR using genomic DNA prepared from each rice variety using SNP discrimination primers and STS primers. The used primer is described in SEQ ID NO. “*” Was added to the name of the breed that could not be discriminated when the STS primer was used, but became distinguishable only when the SNP discrimination primer was used.

Claims (5)

A set of primers comprising the following primer pairs (A) or (B) :
(A)
(1) SEQ ID NOs: 1 and 2;
(2) SEQ ID NOs: 3 and 4;
(3) SEQ ID NOs: 5 and 6; and (4) SEQ ID NOs: 7 and 8 ,
(B)
(1) SEQ ID NOs: 1 and 2;
(3) SEQ ID NOs: 5 and 6;
(4) SEQ ID NOs: 7 and 8; and
(5) SEQ ID NOs: 9 and 10. A set of primers comprising the following primer pairs (A ') or (B'):
(A ’)
  (1) SEQ ID NOs: 1 and 2;
  (2) SEQ ID NOs: 3 and 4;
  (3) SEQ ID NOs: 5 and 6;
  (4) SEQ ID NOs: 7 and 8; and
  (6) SEQ ID NOs: 11 and 12,
(B ’)
  (1) SEQ ID NOs: 1 and 2;
  (3) SEQ ID NOs: 5 and 6;
  (4) SEQ ID NOs: 7 and 8;
  (5) SEQ ID NOs: 9 and 10; and
  (6) SEQ ID NOs: 11 and 12. A method for discriminating genotypes of single nucleotide polymorphisms in rice genome using the primer set according to claim 1. A kit for determining a genotype of a single nucleotide polymorphism in a rice genome, comprising the primer set according to claim 1. 5. The kit according to claim 4 , further comprising the following primer pair:
(6) SEQ ID NOs: 26 and 27;
(7) SEQ ID NOs: 30 and 31;
(8) SEQ ID NOs: 32 and 33;
(9) SEQ ID NOs: 34 and 35;
(10) SEQ ID NOs: 36 and 37;
(11) SEQ ID NOs: 38 and 39;
(12) SEQ ID NOs: 40 and 41;
(13) SEQ ID NOs: 42 and 43;
(14) SEQ ID NOs: 44 and 45;
(15) SEQ ID NOs: 46 and 47; and (16) SEQ ID NOs: 48 and 49;
A kit for discriminating a genotype of a single nucleotide polymorphism in a rice genome, comprising a primer pair selected from the group consisting of:

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