JP2017184651A - Variety discrimination methods of para rubber tree, and kits for variety discrimination of para rubber tree - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide variety discrimination methods of Para rubber tree, and kits for variety discrimination of Para rubber tree which can discriminate the variety of a Para rubber tree simply and with high accuracy.SOLUTION: The present invention provides a variety discrimination method of Para rubber tree, the method being characterized by discriminating the variety of a Para rubber tree subject by detecting and analyzing the single-nucleotide polymorphism of the genomic DNA of the Para rubber tree. The invention also provides a kit for variety discrimination of Para rubber tree, the kit being characterized by containing at least one primer set of a primer pair for amplification and a primer for single nucleotide extension, or at least one primer probe set of a primer pair for amplification and a probe pair for detection.SELECTED DRAWING: None

Description

  The present invention relates to a method for distinguishing para rubber tree varieties and a kit for distinguishing para rubber tree varieties.

  Para rubber tree (Hevea brasiliensis) is a typical rubber tree grown as a raw material for natural rubber. Conventionally, para rubber tree varieties have been identified by leaf, bark, tree shape, etc., but it takes a long time to actually identify and determine the variety. In addition, it takes time to master and master such discrimination methods. In addition, depending on the type of Para rubber tree, there are cases where there are varieties that are difficult to discriminate by appearance, and erroneous determination may occur in the type discrimination.

  Therefore, in recent years, methods for distinguishing varieties by applying some genetic engineering to plants have been developed. In these methods, a variety is identified by detecting a difference in base sequence between varieties, that is, a polymorphism. In other words, since an objective feature on the gene is used as an index, accurate variety discrimination is possible. Already in many commercial crops, varieties based on genetic engineering such as RAPD (Random Amplified Polymorphic DNA) method, RFLP (Restriction Fragment Length Polymorphisms) method, AFLP (Amplified Fragment Length Polymorphisms) method, microsatellite method (for example, Patent Document 1) A discrimination method has been developed.

JP 2009-165385 A

  However, since the above-mentioned variety discrimination method requires a large amount of DNA for discrimination, the discrimination operation is complicated, and polymorphisms for several adjacent bases are targeted for detection, fragmentation has advanced to a high degree due to deterioration. There may be a problem that the discrimination accuracy is lowered with DNA. Accordingly, there is a need for a para rubber tree cultivar identification method and a para rubber tree cultivar identification kit that can easily and accurately determine the varieties of para rubber tree.

  Accordingly, an object of the present invention is to provide a para rubber tree variety identification method and a variety identification kit that can easily and accurately determine the variety of para rubber tree.

  The method for discriminating the varieties of Para rubber tree of the present invention is characterized by discriminating the varieties of the Para rubber tree subject by detecting and analyzing a single nucleotide polymorphism of the Para rubber tree subject. By detecting a single nucleotide polymorphism (SNP) in a Para rubber tree specimen, the variety of Para rubber tree can be easily and accurately distinguished.

  In the para rubber tree cultivar discrimination method of the present invention, it is preferable to detect the single nucleotide polymorphism using a mass array method. With this configuration, a single nucleotide polymorphism can be detected as a difference in mass.

  In the para rubber tree cultivar discrimination method of the present invention, it is preferable to detect the single nucleotide polymorphism using a real-time PCR method. With this configuration, a single nucleotide polymorphism can be detected as a difference in fluorescence wavelength or presence / absence of fluorescence emission and intensity difference.

In the method for discriminating varieties of Para rubber tree of the present invention, it is preferable that the single nucleotide polymorphism is detected for at least one selected from the group consisting of the following bases:
201st base of SEQ ID NO: 1;
The 301st base of SEQ ID NO: 2;
The 251st base of SEQ ID NO: 3;
201st base of SEQ ID NO: 4;
The 301st base of SEQ ID NO: 5;
The 201st base of SEQ ID NO: 6;
The 101st base of SEQ ID NO: 7;
The 101st base of SEQ ID NO: 8;
The 101st base of SEQ ID NO: 9;
The 101st base of SEQ ID NO: 10;
The 101st base of SEQ ID NO: 11;
The 101st base of SEQ ID NO: 12;
101st base of SEQ ID NO: 13; and 101st base of SEQ ID NO: 14.
With this configuration, it is possible to suitably determine the types of para rubber tree such as PB260, PB330, PB340, IAN873, GT1, RRIC100, PR107, PB235, and AVROS2037.

When the mass array method is used in the method for discriminating varieties of Para rubber tree of the present invention, it is preferable to detect a single nucleotide polymorphism using at least one of the primer sets shown in the following (1) to (6):
(1) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 15 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 16, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 17 Primer set with extension primer;
(2) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 18 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 19, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 20 Primer set with extension primer;
(3) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 21 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 22, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 23 Primer set with extension primer;
(4) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 24 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 25, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 26 Primer set with extension primer;
(5) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 27 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 28, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 29 Primer set with extension primer;
(6) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 30 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 31, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 32 Primer set with extension primer.
With this configuration, a predetermined SNP can be specifically detected using the mass array method.

When using the real-time PCR method in the method for distinguishing para rubber tree varieties of the present invention, it is preferable to detect a single nucleotide polymorphism using at least one of the primer probe sets shown in the following (1) to (9):
(1) An amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 33 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 34, and a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 35 A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 36;
(2) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 37 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 38; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 39; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 40;
(3) an amplification primer pair comprising a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 41 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 42; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 43; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 44;
(4) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 45 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 46; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 47; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 48;
(5) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 49 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 50; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 51; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 52;
(6) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 53 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 54; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 55; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 56;
(7) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 57 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 58; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 59; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 60;
(8) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 61 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 62; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 63; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 64;
(9) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 65 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 66; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 67; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 68.
With this configuration, a predetermined SNP can be specifically detected using the real-time PCR method in combination with the cycling probe method.

The kit for distinguishing para rubber tree of the present invention includes at least one of the primer sets shown in the following (1) to (6):
(1) An amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 1 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 2, and a single base comprising the oligonucleotide having the base sequence of SEQ ID NO: 3 Primer set with extension primer;
(2) An amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 4 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 5, and a single base comprising the oligonucleotide having the base sequence of SEQ ID NO: 6 Primer set with extension primer;
(3) An amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 7 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 8, and a single base comprising the oligonucleotide having the base sequence of SEQ ID NO: 9 Primer set with extension primer;
(4) An amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 10 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 11, and a single base comprising the oligonucleotide having the base sequence of SEQ ID NO: 12 Primer set with extension primer;
(5) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 13 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 14, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 15 Primer set with extension primer;
(6) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 17, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 18 Primer set with extension primer.
With this configuration, it is possible to provide a para rubber tree variety discrimination kit including a primer set optimized for detection of a predetermined SNP using the mass array method.

Another kit for discriminating varieties of Para rubber tree according to the present invention includes at least one of the primer probe sets shown in the following (1) to (9):
(1) An amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 19 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 20, and a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 21 A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 22;
(2) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 23 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 24; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 25; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 26;
(3) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 27 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 28; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 29; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 30;
(4) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 31 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 32; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 33; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 34;
(5) an amplification primer pair comprising a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 35 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 36; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 37; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 38;
(6) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 39 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 40; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 41; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 42;
(7) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 43 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 44; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 45; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 46;
(8) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 47 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 48; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 49; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 50;
(9) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 51 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 52; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 53; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 54.
With this configuration, it is possible to provide a para rubber tree cultivar identification kit including a primer probe set optimized for detection of a predetermined SNP using a real-time PCR method in combination with a cycling probe method.

  ADVANTAGE OF THE INVENTION According to this invention, the para rubber tree kind discrimination | determination method and the para rubber tree kind discrimination | determination kit which can discriminate | determine the kind of para rubber tree simply and with high precision can be provided.

It is a cluster call plot which shows the result of having detected the genotype of the SNP base in genomic DNA using the mass array method about 270 clone specimens containing all nine varieties. (A) is the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 1 using primer set (1). (B) is the result of detecting the genotype of the 301st SNP base of SEQ ID NO: 2 using the primer set (2). (C) is the result of detecting the genotype of the 251st SNP base of SEQ ID NO: 3 using the primer set (3). It is a cluster call plot which shows the result of having detected the genotype of the SNP base in genomic DNA using the mass array method about a total of 270 clone specimens containing all nine varieties. (D) is the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 4 using the primer set (4). (E) is the result of detecting the genotype of the 301st SNP base of SEQ ID NO: 5 using the primer set (5). (F) is the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 6 using primer set (3). It is an allele discrimination plot (AD plot) which shows the result of having detected the genotype of the SNP base in genomic DNA using the real-time PCR method about a total of nine clone specimens, one for every nine varieties. (A) is the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 6 using the primer probe set (1). (B) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 7 using the primer probe set (2). (C) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 8 using the primer probe set (3). It is AD plot which shows the result of having detected the genotype of the SNP base in genomic DNA using real-time PCR method about a total of nine clone specimens, one each for all nine varieties. (A) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 9 using the primer probe set (4). (B) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 10 using the primer probe set (5). (C) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 11 using the primer probe set (6). It is AD plot which shows the result of having detected the genotype of the SNP base in genomic DNA using real-time PCR method about a total of nine clone specimens, one each for all nine varieties. (A) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 12 using the primer probe set (7). (B) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 13 using the primer probe set (8). (C) is the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 14 using the primer probe set (9). In each AD plot shown as (a), (b), and (c) in FIG. 4, plots of GT1 clones are indicated by white symbols and arrows. In each AD plot shown as (a), (b), and (c) in FIG. 5, plots of GT1 clones are indicated by white symbols and arrows. In each AD plot shown as (a), (b), and (c) in FIG. 6, plots of GT1 clones are indicated by white symbols and arrows.

  Hereinafter, the present invention will be described in detail based on the embodiments.

[Method of distinguishing para rubber tree varieties]
The method for discriminating varieties of Para rubber tree according to the present invention is characterized by detecting and analyzing a single nucleotide polymorphism (hereinafter also referred to as “SNP”) of a Para rubber tree subject, thereby discriminating the variety of the Para rubber tree subject. . Thus, predicting the phenotype of a subject by detecting and analyzing a single nucleotide polymorphism of the subject is generally called SNP typing. In the method for discriminating varieties of the present invention, a single base difference in the genomic DNA of a Para rubber tree specimen, that is, a single nucleotide polymorphism (SNP) is targeted for detection, and genomic DNA highly fragmented due to degradation is used as a sample. Even if it is used, it is possible to easily and accurately discriminate the type. In addition, compared to polymorphisms to be detected by other genetic engineering breed discrimination methods, SNPs have a higher frequency of appearance in genomic DNA, and by using a combination of multiple SNPs as a detection target, a large number of breeds can be increased. Can be identified with accuracy. Such SNPs can also be used as co-dominant markers that can distinguish between genotypes whose alleles are heterozygous and two genotypes whose alleles are homozygous by selecting an appropriate detection technique. it can.

  SNP detects genomic DNA extracted from a Para rubber tree specimen as a sample. As a Para rubber tree specimen for extracting genomic DNA, any tissue of a Para rubber tree plant may be used, and the growth stage is not limited. Extraction of genomic DNA from plant tissues can be performed by a conventional method, for example, phenol extraction method, cetyltrimethylammonium bromide (CTAB) method, alkaline SDS method and the like. In addition, crudely purified genomic DNA can be used. If necessary, the extracted genomic DNA may be purified by a known DNA purification method such as chloroform / isoamyl alcohol treatment, isopropanol precipitation, phenol / chloroform deproteinization, ethanol precipitation, or the like. Extraction and / or purification of genomic DNA may be performed using a commercially available kit or the like.

  The single nucleotide polymorphism may be detected for a base at a predetermined single nucleotide polymorphism site (hereinafter also referred to as “SNP site”) disclosed in the present specification, or known single nucleotide polymorphism identification (“SNP”). The SNP bases identified using any of the techniques (also called mapping) may be detected using SNP mapping to identify the detection target SNP site, for example, genomes extracted from clones of various varieties of Para rubber tree The SNP site to be detected can be identified by analyzing the base sequence of the DNA with a next-generation sequencer such as Hiseq2000, making SNP calls using software such as samtools, and performing sequence comparison by set operation.

  The SNP detection method is not particularly limited, and any known genetic engineering detection method capable of detecting SNP can be used, including a mass array method and a real-time PCR method described later.

  The base type or genotype of the detected SNP is analyzed by comparing it with information such as the base type and genotype of each variety detected at a predetermined SNP site disclosed in Table 1 described later in this specification. Alternatively, analysis may be performed by comparing information such as base type and genotype identified using any of the known SNP mapping techniques.

  The para rubber tree cultivar identification method of the present invention may be used alone or in combination with other cultivar identification methods. In the case of using the kind discrimination method of the present invention alone, the kind can be discriminated by setting one or a plurality of SNPs as detection targets and using the detection results alone or in combination. When using the variety identification method of the present invention in combination with other variety identification methods, for example, after narrowing the candidates to several varieties using the variety identification method in the form of leaves, bark, tree shape, etc. A variety can be identified using the method for identifying a variety of the present invention.

  The variety of para rubber tree that can be discriminated by the method for discriminating para rubber tree of the present invention is not particularly limited, and examples thereof include PB260, PB330, PB340, IAN873, GT1, RRIC100, PR107, PB235, and AVROS2037.

  The para rubber tree cultivar discrimination method of the present invention is not particularly limited, but it is preferable to detect SNPs using a mass array method, a real-time PCR method or the like.

<Detection using mass array method>
In detection using the mass array method, a difference in mass caused by SNP is detected. Specifically, using genomic DNA as a template, a region of about several tens to several hundreds of bp including the SNP to be detected is amplified by PCR, and then an extension primer is hybridized immediately before the SNP site using the amplified product as a template. By performing an extension reaction with a DNA polymerase using a dideoxynucleotide (ddNTP) mixture or a mixture of dideoxynucleotide (ddNTP) and deoxynucleotide (dNTP) as a substrate, fragments having different masses and lengths depending on the SNP can be obtained. Generated. By purifying the product and performing mass spectrometry using a MALDI-TOF mass spectrometer or the like, the type and genotype of the base can be specified. The mass array method is not particularly limited. For example, the mass array method can be performed using a mass array high-throughput SNP analysis system of Sequenom.

  In the mass array method, a primer set of an amplification primer pair and a single base extension primer is used. The amplification primer pair and the single-base extension primer are specifically designed for the SNP site to be detected on the genomic DNA.

  The amplification primer pair used in the mass array method is an oligonucleotide pair designed so that genomic DNA can be used as a template and a region of about several tens to several hundreds of bp including the SNP site to be detected can be amplified by PCR. Any combination of a forward primer and a reverse primer may be used. A person skilled in the art can appropriately design such an amplification primer pair in consideration of the template size, the base length of the primer, the GC content, the Tm value, and the like based on the base sequence information of the amplification target region. . Each primer of such an amplification primer pair has several bases that are not complementary to the template on the 5 ′ side for the purpose of stabilizing and improving the amplification reaction, suppressing degradation of the amplification product, detecting, purifying, isolating or cloning. A tag sequence having about ˜ten bases may be added.

  The primer for single base extension used in the mass array method is about 20 bases to 1 base upstream from the 5 ′ side of the single base site to be detected using the sense strand or antisense strand of the amplification product obtained by the amplification primer pair as a template. Any oligonucleotide capable of hybridizing may be used. Those skilled in the art will appropriately use such a single-base extension primer in consideration of the template size, primer base length, GC content, Tm value, etc. based on the base sequence information 5 ′ upstream of the single base to be detected. Can be designed to

  The extension reaction in the mass array method can be performed by extending only one base to be detected by utilizing the fact that the PCR extension reaction is stopped by the incorporation of dideoxynucleotide (ddNTP). When a dideoxynucleotide mixture (ddATP, ddGTP, ddCTP, and ddTTP) is used as a substrate, the PCR extension reaction always stops at the next base (SNP site) of the primer, and the mass difference between products with one base added to the primer is eliminated. Mass spectrometry can be used to determine the base of the SNP site. The product of the elongation reaction thus obtained can be purified as necessary, mass-analyzed by MALDI-TOF MS, and the genotype can be analyzed by mass number. When the genotype of the SNP to be detected is homozygous, there is only one kind of base at the SNP site, and therefore only one peak of the mass spectrum of the extension reaction product is observed. When the genotype of the SNP to be detected is heterozygous, since there are two types of bases at the SNP site, two mass spectrum peaks of the extension reaction product are observed.

  Alternatively, in the extension reaction, a mixture of one kind of dideoxynucleotide complementary to one kind of base of SNP and the remaining three kinds of dNTPs can be used as a substrate. In this case, when there is a base complementary to ddNTP at the SNP site, only one base is extended to stop the reaction, but when there is a different base (that is, not complementary to ddNTP), then the dideoxy is present in the template strand. Since the extension reaction continues until a base complementary to the nucleotide appears, the mass difference between the extension reaction products can be increased. The extension reaction product thus obtained is also purified as necessary, and similarly mass-analyzed by MALDI-TOF MS. Based on the mass number, the type of SNP site base and the combination thereof, that is, the gene The type can be specified.

As described above, the genotypes of bases obtained for one or more SNP sites of the subject are the base genotype data obtained in advance for known varieties for the same SNP sites, for example, Table 1 shown below. The type of the subject can be discriminated in light of the test table as shown in Table 3.
Alternatively, the result of mass spectrometry can be obtained as a cluster call plot or the like by analyzing with typing software such as Type Analyzer Software manufactured by Sequenom. By comparing the plots obtained for one or more SNP sites of the subject in this way with plots obtained in advance for known varieties for the same SNP site, the type of the subject can be determined. it can.

<Detection using real-time PCR>
In detection using a real-time PCR method, hybridization of a detection probe having a base complementary to SNP is detected in real time as a reporter signal such as fluorescence. Specifically, using a genomic DNA as a template, a region of about 100 to 250 bp including the SNP to be detected is amplified by PCR, and the SNP site and the detection probe are hybridized. The detection probe has one end labeled with a reporter (for example, a fluorescent dye) and the other end labeled with a quencher (quenching substance). When the quencher is in the vicinity of the reporter, the quencher suppresses the reporter luminescence, but when the detection probe is cleaved or degraded due to nuclease activity or the like and the quencher is separated from the reporter, the reporter luminescence is detected It becomes possible. The genotype can be identified by analyzing the light emission of this reporter with a real-time PCR apparatus or the like. By using the real-time PCR method, SNP can be detected as a difference in fluorescence wavelength or presence / absence of fluorescence emission and intensity difference. As a detection probe, one probe having a base complementary to any one of a plurality of polymorphic bases may be used, or a plurality of probes having a base complementary to each type of a plurality of polymorphic bases May be used. When multiple detection probes are used, the type and combination of bases can be identified with high sensitivity by analyzing the difference in wavelengths detected using reporters with different emission and absorption wavelengths (multiplex analysis). Thus, the genotype can be determined.

  In the real-time PCR method, a primer probe set of an amplification primer pair and a detection probe is used. The amplification primer pair and the detection probe are designed specifically for the SNP to be detected on the genomic DNA.

  The amplification primer pair used in the real-time PCR method is a pair of oligonucleotides designed so that genomic DNA can be used as a template and a region of about 100 to 250 bp including the SNP site to be detected can be amplified by PCR. What is necessary is just a combination of a primer and a reverse primer. A person skilled in the art can appropriately design such an amplification primer pair in consideration of the template size, the base length of the primer, the GC content, the Tm value, and the like based on the base sequence information of the amplification target region. .

  The detection probe used in the real-time PCR method is an oligonucleotide having a base sequence that specifically hybridizes with a region of several to thirty or more bases including the SNP site to be detected, one end of which is a quencher (quenching) Substance) and the other end is labeled with a reporter (fluorescent dye). A set of detection probes that specifically hybridize to each of the polymorphic bases detected at the SNP site to be detected can also be used as a detection probe pair. The detection probe is designed according to the detection principle. Although it does not specifically limit as a detection principle, For example, the thing using a cycling probe method, the thing using TaqMan probe method, etc. are mentioned.

  In the detection principle by the cycling probe method, a chimeric probe (hereinafter also referred to as a cycling probe) in which an oligonucleotide is composed of DNA and RNA is used as a detection probe. The cycling probe is a chimeric oligonucleotide consisting of RNA and DNA consisting of several bases to several tens bases, one end of which is labeled with a fluorescent substance and the other end is labeled with a quencher. Further, only the nucleotide at the site complementary to the SNP to be detected or the 1 base 5 'side thereof is RNA, and the remaining nucleotides are DNA. A cycling probe and RNase H are added to a normal PCR reaction solution to perform a PCR reaction. When the signing probe is intact, no fluorescence is emitted because the distance between the reporter and the quencher is short. However, when the signing probe forms a hybrid with the template or the PCR amplification product, the RNA site is cleaved by RNase H contained in the reaction solution, and the distance between the reporter and the quencher is increased to emit fluorescence. Since there is no cleavage by RNase H when there is a mismatch at the RNA site of the cycling probe or its 1 base 5 'side, detection with very high sequence specificity is possible. In addition, a pair of cycling probes each having at least two cycling probes each having a complementary base specific to each SNP base and labeled with a different type of reporter (fluorescent dye) are designed for detection. By the multiplex analysis used as the base type, the type and combination of bases can be identified with high sensitivity, and the genotype can be determined.

  According to the detection principle by the TaqMan probe method, an oligonucleotide composed of DNA of 10 to 30 bases (hereinafter also referred to as TaqMan probe) is used as a detection probe. Like the cycling probe, the TaqMan probe is labeled with a fluorescent substance at one end and a quencher at the other end. A TaqMan probe is added to a normal PCR reaction solution to perform a PCR reaction. In the state where the TaqMan probe is intact, since the distance between the reporter and the quencher is short, no fluorescence is emitted. However, when the TaqMan probe forms a hybrid with the template or PCR amplification product, the TaqMan probe hybridized by the 5 ′ → 3 ′ exonuclease activity of the DNA polymerase (eg, Taq DNA polymerase) contained in the reaction solution Is hydrolyzed, and the distance between the reporter and the quencher is increased, causing fluorescence. Detection with a TaqMan probe can also be detected with high sequence specificity. Moreover, a probe for detection by designing a pair of TaqMan probes each having at least two TaqMan probes each having a complementary base specific to each SNP base and labeled with a different type of reporter (fluorescent dye) As a result, it is possible to detect the type and genotype of the base at the SNP site with high sensitivity by analyzing the difference in emission wavelength and absorption wavelength by fluorescence analysis.

  The fluorescent dye for labeling the detection probe is not particularly limited. For example, 6-carboxyfluorescein (6-FAM), 2 ′, 4 ′, 5 ′, 7 ′, 1,4-hexachlorofluorescein (HEX), 2 ', 4', 1,4-tetrachlorofluorescein (TET), 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and the like. The quencher (quenching substance) for labeling the detection probe is not particularly limited. For example, carboxytetramethylrhodamine (TAMRA), Black Hole Quencher dye (BHQ), 4-((4- (dimethylamino) phenyl) azo ) Benzoic acid (DABCYL) and the like.

As described above, genotypes determined for one or more SNP sites of the subject are genotype data obtained in advance for known varieties for the same SNP site, for example, Tables 1, 4 and 5 shown below. The type of the subject can be discriminated in light of the test table as described above.
Alternatively, the results of the fluorescence analysis can be obtained as a genotype allele discrimination plot (AD plot) or the like by analyzing with typing software, for example, Applied Biosystems StepOne Software ForStepOne and StepOnePlus-Time PCR Systems. . By comparing the plots obtained for one or more SNP sites of the subject in this way with plots obtained in advance for known varieties for the same SNP site, the type of the subject can be determined. it can.

<Detection target SNP site on genomic DNA>
The SNP site to be detected in the method for discriminating varieties of Para rubber tree of the present invention is not particularly limited, but it is preferable to detect at least one selected from the group consisting of the following bases:
201st base of SEQ ID NO: 1;
The 301st base of SEQ ID NO: 2;
The 251st base of SEQ ID NO: 3;
201st base of SEQ ID NO: 4;
The 301st base of SEQ ID NO: 5;
The 201st base of SEQ ID NO: 6;
The 101st base of SEQ ID NO: 7;
The 101st base of SEQ ID NO: 8;
The 101st base of SEQ ID NO: 9;
The 101st base of SEQ ID NO: 10;
The 101st base of SEQ ID NO: 11;
The 101st base of SEQ ID NO: 12;
101st base of SEQ ID NO: 13; and 101st base of SEQ ID NO: 14.
By detecting SNP using at least one of these bases as a marker, varieties such as PB260, PB330, PB340, IAN873, GT1, RRIC100, PR107, PB235, and AVROS2037 can be suitably identified.

  For example, when the cultivar is a subject, one base detected for the SNP site is as shown in Table 1 below. A, G, C, and T represent adenine, guanine, cytosine, and thymine bases, respectively. When only one base symbol is described, only one type of base is detected, indicating that the allele is a homozygous form of the single base. When two base symbols are described with diagonal lines, two types of bases are detected, indicating that the allele is a heterozygous type of the two bases.

<Primer set that can be used for mass array method>
In the present invention, a primer set that can be used for SNP detection using the mass array method can be appropriately designed by a person skilled in the art based on the base sequence information of the genomic DNA region containing the SNP site to be detected using well-known conventional techniques. . Although it does not specifically limit as a suitable specific example of this primer set, For example, the primer set shown to following (1)-(6) is mentioned.
(1) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 15 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 16, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 17 Primer set with extension primer;
(2) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 18 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 19, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 20 Primer set with extension primer;
(3) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 21 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 22, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 23 Primer set with extension primer;
(4) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 24 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 25, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 26 Primer set with extension primer;
(5) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 27 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 28, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 29 Primer set with extension primer;
(6) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 30 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 31, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 32 Primer set with extension primer.

  As long as each primer of the amplification primer pair of the primer sets (1) to (6) can amplify the genomic DNA region containing the SNP site to be detected, 1 to the number of oligonucleotides of the above base sequence Those in which bases are point-mutated, deleted or duplicated, and those in which nucleotides of 1 to several bases are added to the 5 ′ and / or 3 ′ side of the oligonucleotide of the above base sequence can also be used. The added nucleotide may be complementary to the genomic DNA, or may be one that functions as a tag sequence rather than being complementary to the genomic DNA.

  As long as a single base extension primer of the primer set of (1) to (6) is capable of a single base extension reaction of a predetermined SNP, one to several bases of the oligonucleotide of the above base sequence is point-mutated or deleted. Or what overlapped and what added the nucleotide of 1 to several bases to the 5 'side of the oligonucleotide of the said base sequence can also be used. The added nucleotide may be complementary to the genomic DNA, or may be one that functions as a tag sequence rather than being complementary to the genomic DNA.

In addition, the base sequence of each sequence number is as follows.
SEQ ID NO: 15: 5′-AAGATCACGAATCCCTCTGG-3 ′
SEQ ID NO: 16: 5′-ATGATGGTCTTCTAGGCAGCTTC-3 ′
SEQ ID NO: 17: 5′-CGAATCCCTCTGGGAATAAC-3 ′
SEQ ID NO: 18: 5′-ACCTTGGGGTCCAAAATTGAG-3 ′
SEQ ID NO: 19: 5′-GCAAGATGCTCATTGATGG-3 ′
SEQ ID NO: 20: 5′-CCCTCAAAGGAATCCCC-3 ′
SEQ ID NO: 21: 5′-CTTGAACTGTGATTTGCAGG-3 ′
SEQ ID NO: 22: 5′-CTCCAAGGAGGTCTGTAAGC-3 ′
SEQ ID NO: 23: 5′-AGACTGTTTATTGAAAGAGATG-3 ′
SEQ ID NO: 24: 5′-TCGAGGCAGAGAAGAATTTC-3 ′
SEQ ID NO: 25: 5′-AGCATCACAAGGACAATGGC-3 ′
SEQ ID NO: 26: 5′-GGCAGAGAAGAATTTCACATTG-3 ′
SEQ ID NO: 27: 5′-AGACCTGGCAAAGTCACACC-3 ′
SEQ ID NO: 28: 5′-GTGGGAGGTCAGGTTGTTTG-3 ′
SEQ ID NO: 29: 5′-GCAGTTGTTTTGGTTGAT-3 ′
SEQ ID NO: 30: 5′-GGAAGACTCGGTGTTTTGTAG-3 ′
SEQ ID NO: 31: 5′-GTGAATGAGCAGCTGATACCG-3 ′
SEQ ID NO: 32: 5′-TAAATGCCGTTTGCCCGT-3 ′

  By using the primer sets (1) to (6) above, it is possible to suitably detect the SNP at the predetermined SNP site described above. The SNP sites to be detected by the primer sets (1) to (6) are as shown in Table 2 below. Further, by analyzing the detection results using the primer sets (1) to (6) described above by the mass array method based on the test table of Table 3 below, the variety of Para rubber tree can be determined.

<Primer probe set that can be used for real-time PCR>
In the present invention, a primer probe set that can be used for SNP detection using the real-time PCR method should be appropriately designed by a person skilled in the art based on the base sequence information of the genomic DNA region containing the SNP site to be detected using well-known conventional techniques. Can do. Although it does not specifically limit as a suitable specific example of this primer probe set, When using combining a cycling probe method with real-time PCR method, the primer probe set shown to following (1)-(9) is mentioned, for example. In each probe of the detection probe pair shown in the following (1) to (9), the nucleotide complementary to the SNP to be detected or the nucleotide at the 1-base 5 ′ side is RNA, and the other is DNA. It is a cycling probe.

(1) An amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 33 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 34, and a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 35 A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 36;
(2) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 37 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 38; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 39; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 40;
(3) an amplification primer pair comprising a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 41 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 42; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 43; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 44;
(4) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 45 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 46; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 47; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 48;
(5) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 49 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 50; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 51; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 52;
(6) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 53 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 54; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 55; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 56;
(7) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 57 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 58; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 59; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 60;
(8) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 61 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 62; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 63; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 64;
(9) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 65 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 66; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 67; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 68.

  Each primer of the primer pair for amplification of the primer probe set of (1) to (9) above is an oligo of the above base sequence as long as it can amplify the genomic DNA region containing the SNP site to be detected by the real-time PCR method. Those in which 1 to several bases of nucleotides are point mutations, deletions or duplications, or those in which 1 to several bases of nucleotides are added to the 5 ′ and / or 3 ′ side of the oligonucleotides of the above base sequences can be used it can. The added nucleotide may be complementary to the genomic DNA, or may be one that functions as a tag sequence rather than being complementary to the genomic DNA.

  As long as each probe of the probe pair for detection of the primer probe set of (1) to (9) can be used for detection of the base of the SNP site to be detected by the real-time PCR method, 1 to 1 of the oligonucleotide having the above base sequence is used. Those in which several bases are point-mutated, deleted or duplicated, and those in which nucleotides of 1 to several bases are added to the 5 ′ and / or 3 ′ side of the oligonucleotide of the above base sequence can also be used. The added nucleotide may be complementary to the genomic DNA, or may be one that functions as a tag sequence rather than being complementary to the genomic DNA.

The base sequences of the respective sequence numbers shown for the primer probe sets of (1) to (9) above are as follows. The nucleotide sequences of oligonucleotides contained in the detection probe (SEQ ID NOs: 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67 and In 68), a character (for example, C ^* ) marked with ^{* in} the upper right represents a base corresponding to the SNP site on the sense strand of genomic DNA, and a character in parentheses represents RNA. For example, the detection probe containing the oligonucleotide of SEQ ID NO: 35 is a cytosine (C) detection probe, and when the base of the SNP site in the sense strand of genomic DNA and / or its amplified product is cytosine (C), It hybridizes to the antisense strand of genomic DNA and / or its amplification product. As a result, the detection probe is cleaved or decomposed by RNase H, and the fluorescent substance bound to the detection probe emits light.

SEQ ID NO: 33: 5′-TTGCTGATTTGTGTAAGTGATTAG-3 ′
SEQ ID NO: 34: 5′-ATTCACATGCCTTTGAACATA-3 ′
SEQ ID NO: 35: 5′-CCTGTC ^* (G) GA-3 ′
SEQ ID NO: 36: 5′-GCCTGT (A ^* ) GG-3 ′
SEQ ID NO: 37: 5′-AGTCTCCATGGTGGGTATC-3 ′
SEQ ID NO: 38: 5′-GTGCCCACAATGCAGTAA-3 ′
SEQ ID NO: 39: 5′-GC (G ^* ) AAAAGGA-3 ′
SEQ ID NO: 40: 5′-CA ^* (A) AAAGGAAAA-3 ′
SEQ ID NO: 41: 5′-AAGAGCAATGCCTGAGAG-3 ′
SEQ ID NO: 42: 5′-CTTTATCCGGAATTTGTAATGGA-3 ′
SEQ ID NO: 43: 5′-CAGCTAT ^* (G) GC-3 ′
SEQ ID NO: 44: 5′-CTAC ^* (G) GCG-3 ′
SEQ ID NO: 45: 5′-CGTTCCTTGCTGTTGTT-3 ′
SEQ ID NO: 46: 5′-TTCCATTGAGTTCATGC-3 ′
SEQ ID NO: 47: 5′-TCCTTGTA (A ^* ) AAC-3 ′
SEQ ID NO: 48: 5′-TCCTTGTA (G ^* ) AAC-3 ′
SEQ ID NO: 49: 5′-TCTTTGTAGAGAAATCACACAGTA-3 ′
SEQ ID NO: 50: 5′-AAGCTCAAGCTCATGGA-3 ′
SEQ ID NO: 51: 5′-AAGCAAATA (A ^* ) TAG-3 ′
SEQ ID NO: 52: 5′-AGCAAATA (G ^* ) TAG-3 ′
SEQ ID NO: 53: 5′-GCGGCTGCCACAACCT-3 ′
SEQ ID NO: 54: 5′-CAACCTCTCATACAGCAACAAC-3 ′
SEQ ID NO: 55: 5′-CCCGCC ^* (A) ATT-3 ′
SEQ ID NO: 56: 5′-TCCGCT ^* (A) ATT-3 ′
SEQ ID NO: 57: 5′-GTAATCTTTGTGTATCCGTCGTA-3 ′
SEQ ID NO: 58: 5′-GGATTGATCGCAAGAACTGA-3 ′
SEQ ID NO: 59: 5′-CCTCC (G ^* ) TCA-3 ′
SEQ ID NO: 60: 5′-GCCTCC (A ^* ) TC-3 ′
SEQ ID NO: 61: 5′-GGAATATGGATGGTGAAATG-3 ′
SEQ ID NO: 62: 5′-ATACAACACAGATTCTTCTCACTCC-3 ′
SEQ ID NO: 63: 5′-TAC (A ^* ) GTTAAGTC-3 ′
SEQ ID NO: 64: 5′-TACCATAC (G ^* ) GT-3 ′
SEQ ID NO: 65: 5′-CTCATCGAATGGGTGAAGG-3 ′
SEQ ID NO: 66: 5′-AGTAGGAGGTACACCAGCAA-3 ′
SEQ ID NO: 67: 5′-AG (G ^* ) AGTGTC-3 ′
SEQ ID NO: 68: 5′-AAG (A ^* ) AGTGGGTC-3 ′

  By using the primer probe set of (1) to (9) above, the base of the predetermined SNP site described above can be suitably detected. The SNP sites to be detected by the sets (1) to (9) are as shown in Table 4 below. By analyzing the detection results using the primer probe sets (1) to (18) described above by the real-time PCR method based on the test table of Table 5 below, the variety of Para rubber tree can be discriminated.

[Para rubber tree variety discrimination kit]
The kit for discriminating varieties of Para rubber tree according to one embodiment of the present invention includes at least one of the primer sets of (1) to (6) above. By including at least one of the primer sets of (1) to (6) above, it is suitably used for detection of the predetermined SNP disclosed in Table 1 above in the method for distinguishing para rubber tree varieties using the mass array method Can do. In addition to at least one of the primer sets of (1) to (6) above, this variety discrimination kit is capable of assigning a genotype detected using the primer set to a Para rubber tree variety ( For example, the information shown in Table 2 or 3 above may be included as, for example, a test table. Furthermore, reagents, containers, experimental protocols, and the like that can be used for detection by the mass array method may be included.

  Another embodiment of the present invention is a kit for distinguishing para rubber tree varieties, which includes at least one of the primer probe sets of (1) to (9) above. Thereby, in the method for discriminating varieties of Para rubber tree using the real-time PCR method in combination with the cycling probe method, it can be suitably used for detection of the predetermined SNP disclosed in Table 1 above. In addition to at least one of the primer probe sets described in (1) to (9) above, this variety discrimination kit can assign the genotype detected using the primer probe set to a Para rubber tree variety. Information (for example, information shown in Table 4 or 5 above) may be included as, for example, a test table. Furthermore, reagents, containers, experimental protocols, and the like that can be used for detection using the cycling probe method in combination with the real-time PCR method may be included.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to the following Example at all.

[Preparation of Para rubber tree sample and extraction and purification of genomic DNA]
Nine varieties of Para rubber tree, PB260, PB330, PB340, IAN873, GT1, RRIC100, PR107, PB235, and AVROS2037 clone leaves were collected from the Para rubber tree plantation. The above SNP was detected using genomic DNA extracted and purified from these leaves as a material. Extraction and purification of genomic DNA from leaves was performed using DNeasy Plant Mini Kit manufactured by Qiagen.

[Detection of SNP by mass array method]
About genomic DNA extracted and purified from the clone, SNP was detected by mass array method using iPLEX (registered trademark) Gold system of SEQUENOM. The experimental procedure followed the SEQUENOM iPLEX Gold application guide. The specific experimental procedure is as follows.

<Amplification of genomic DNA region including SNP site>
Using the extracted and purified genomic DNA as a template, the genomic DNA region including the detection target SNP site was amplified using the amplification primer pairs of the primer sets of (1) to (6) above. A PCR thermal cycler Dice manufactured by Takara Bio Inc. was used as a thermal cycler. The amplification primer pair used was obtained by adding a 10 base tag (5′-ACGTTGGATG-3 ′: SEQ ID NO: 69) to the 5 ′ side of each oligonucleotide encoded by the base sequence of the above SEQ ID NO. . A PCR reaction solution was prepared in a 96-well sample microtiter plate, and a PCR reaction was performed. The reaction solution composition and reaction conditions were as follows according to the iPLEX Gold application guide.

  Subsequently, in order to neutralize unreacted dNTP in the reaction solution, it was treated with shrimp alkaline phosphatase (SAP). Specifically, 2 μL of SAP solution per well (solution composition: 10 × SAP buffer 0.17 μL, 1.7 U / μL SAP enzyme 0.3 μL, HPLC grade water 1.53 μL) was added and vortexed. Spin down with a centrifuge (1000 rpm, 1 minute). The temperature was maintained at 37 ° C for 40 minutes, then maintained at 85 ° C for 5 minutes, and then cooled to 4 ° C.

<SNP extension reaction>
To the sample microtiter plate, 1.06 μL of a cocktail containing a primer for single base extension was added per well, and an extension reaction was performed by PCR. The cocktail composition and reaction conditions were as follows.

<Identification of base and identification of variety>
According to the iPLEX Gold application guide, the sample microtiter plate was set on a dimple plate pasted with resin, and mass spectrometry was performed with MALDI-TOF MS (MassARRAY Compact System, manufactured by Sequenom). Cluster call plots were obtained by analyzing the results of mass spectrometry with typing software (Type Analyzer Software from Sequenom).
The cluster call plots obtained for each primer set of (1) to (6) for a total of more than 270 clone samples including all 9 varieties are shown in FIGS. 1 (a) to 1 (c) and FIG. 2 (d), respectively. ) To (f).

  In the cluster call plots of FIGS. 1 and 2, one plot point (indicated by a black circle, black triangle, black square, or inverted black triangle symbol) corresponds to one clone sample. The horizontal axis (Low Mass Allele) and the vertical axis (High Mass Allele) of the plot graph are the ratio of the low mass PCR product and the high mass PCR product of the total PCR products obtained for each clone sample, respectively. Indicates the percentage. FIG. 1A shows the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 1 using the primer set (1). FIG. 1B shows the result of detecting the genotype of the 301st SNP base of SEQ ID NO: 2 using the primer set (2). FIG.1 (c) is the result of having detected the genotype of the 251st SNP base of sequence number 3 using the primer set (3). FIG.2 (d) is the result of having detected the genotype of the 201st SNP base of sequence number 4 using primer set (4). FIG. 2 (e) shows the result of detecting the genotype of the 301st SNP base of SEQ ID NO: 5 using the primer set (5). FIG. 2 (f) shows the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 6 using the primer set (3).

  The genotypes shown in these cluster call plots will be described with reference to FIGS. 1 (a), 1 (c) and 2 (b). FIG. 1 (a) is the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 1 using the primer set (1), so the types of bases detected are guanine and adenine, which are identified. The genotypes are a guanine homozygous type (G), an adenine homozygous type (A), and a heterozygous type (G / A) of adenine and guanine (Tables 1 and 3). Since the molecular weight of guanine is about 151 and the molecular weight of adenine is about 135, a PCR product having a guanine base is detected as a high mass PCR product, and a PCR product having an adenine base is detected as a low mass PCR product. The In the clone specimen in which the genotype of the SNP site is a homozygous type (G) of guanine, the ratio of the low mass number PCR product is theoretically 0 out of the total PCR products obtained, and the high mass number PCR product Is theoretically 1, so that the scale of the horizontal axis is 0.0 and the scale of the vertical axis is near 1.0, that is, as a point of an inverted black triangle near the upper left corner of the graph. In the clone specimen in which the genotype of the SNP site is a homozygous type (A) of adenine, the ratio of the low mass number PCR product is theoretically 1 out of the total PCR products obtained, and the high mass number PCR product Is theoretically 0, so that the scale of the horizontal axis is 1.0 and the scale of the vertical axis is around 0.0, that is, plotted as black triangle points near the lower right corner of the graph. In a clone specimen in which the genotype of the SNP site is a heterozygous type (G / A) of guanine and adenine, the ratio of the low mass number PCR product is theoretically 0.5 out of the total PCR products obtained. The ratio of the PCR product with a high mass number is theoretically 0.5, so that the scale of the horizontal axis is 0.5 and the scale of the vertical axis is near 0.5, that is, a black square point near the center of the graph Is plotted as A black dot (No Call) represents a specimen in which the genotype of the SNP site could not be determined due to poor signal. “Other” indicates the number of specimens for which the genotype of the SNP site was determined manually, that is, by an experimenter rather than a computer.

  Since FIG. 1 (c) shows the result of detecting the genotype of the 251st SNP base of SEQ ID NO: 3 using the primer set (3), the types of bases detected are cytosine and thymine, and are identified. The genotypes are cytosine homozygous type (C), thymine homozygous type (T), and cytosine and thymine heterozygous type (C / T) (Tables 1 and 3). Since the molecular weight of cytosine is about 111 and the molecular weight of thymine is about 126, a PCR product having a thymine base is detected as a high mass PCR product, and a PCR product having a cytosine base is detected as a low mass PCR product. The In the clone specimen in which the genotype of the SNP site is a thymine homozygous type (T), the ratio of the low mass number PCR product is theoretically 0 out of the total PCR products obtained, and the high mass number PCR product Is theoretically 1, so that the scale of the horizontal axis is 0.0 and the scale of the vertical axis is near 1.0, that is, as a point of an inverted black triangle near the upper left corner of the graph. In the clone specimen in which the genotype of the SNP site is cytosine homozygous (C), the ratio of the low mass number PCR product is theoretically 1 out of the total PCR products obtained, and the high mass number PCR product is Is theoretically 0, so that the scale of the horizontal axis is 1.0 and the scale of the vertical axis is around 0.0, that is, plotted as black triangle points near the lower right corner of the graph. In a clone sample in which the genotype of the SNP site is a heterozygous type (C / T) of cytosine and thymine, the ratio of the low mass number PCR product is theoretically 0.5 out of the total PCR products obtained. The ratio of the PCR product with a high mass number is theoretically 0.5, so that the horizontal scale is 0.5 and the vertical scale is 0.5, that is, a black square symbol near the center of the graph. Is plotted as A black dot (No Call) represents a specimen in which the genotype of the SNP site could not be determined due to poor signal. “Other” indicates the number of specimens for which the genotype of the SNP site was determined manually, that is, by an experimenter rather than a computer.

  Since FIG. 2 (e) shows the result of detecting the genotype of the 301st SNP base of SEQ ID NO: 5 using the primer set (5), the types of detected bases are cytosine and adenine, which are identified. The genotypes are cytosine homozygous (C), adenine homozygous (A), and cytosine and adenine heterozygous (C / A) (Tables 1 and 3). Since the molecular weight of cytosine is about 111 and the molecular weight of adenine is about 135, a PCR product having an adenine base is detected as a high mass PCR product, and a PCR product having a cytosine base is detected as a low mass PCR product. The In the clone specimen in which the genotype of the SNP site is a homozygous type (A) of adenine, the ratio of the low-mass PCR product is theoretically 0 out of the total PCR products obtained, and the high-mass PCR product Is theoretically 1, so that the scale of the horizontal axis is 0.0 and the scale of the vertical axis is near 1.0, that is, as a point of an inverted black triangle near the upper left corner of the graph. In the clone specimen in which the genotype of the SNP site is cytosine homozygous (C), the ratio of the low mass number PCR product is theoretically 1 out of the total PCR products obtained, and the high mass number PCR product is Is theoretically 0, so that the scale of the horizontal axis is 1.0 and the scale of the vertical axis is around 0.0, that is, plotted as black triangle points near the lower right corner of the graph. In a clone specimen in which the genotype of the SNP site is a heterozygous type (C / A) of cytosine and adenine, the ratio of the low mass number PCR product is theoretically 0.5 out of the total PCR products obtained. The ratio of the PCR product with a high mass number is theoretically 0.5, so that the scale of the horizontal axis is 0.5 and the scale of the vertical axis is near 0.5, that is, a black square point near the center of the graph Is plotted as A black dot (No Call) represents a specimen in which the genotype of the SNP site could not be determined due to poor signal. “Other” indicates the number of specimens for which the genotype of the SNP site was determined manually, that is, by an experimenter rather than a computer.

  As can be seen by comparing the cluster call plots of FIGS. 1 (a) to (c) and FIGS. 2 (d) to (f) with Tables 1 to 3, the mass array method using primer sets (1) to (6) was used. By detecting the single nucleotide polymorphism (SNP) of the subject, it is possible to determine the variety of Para rubber tree.

[Detection of SNPs using real-time PCR]
For genomic DNA extracted and purified from the clones, SNP was detected by real PCR using a StepOnePlus real PCR system manufactured by Applied Biosystems. The specific experimental procedure is as follows.

  The primer probe sets of (1) to (9) above were used. Of the pair of detection probes, one cycling probe is FAM labeled and the other cycling probe is HEX labeled. As a PCR reagent, Cycle (registered trademark) PCR ReactionMix manufactured by Takara Bio Inc. was used. The reaction solution composition and reaction conditions for real-time PCR were as follows.

<Identification of base and identification of variety>
For convenience of equipment used, HEX fluorescence was detected as VIC fluorescence. The base attached to the detection target was identified and analyzed using software attached to the StepOnePlus real PCR system to obtain an allele discrimination plot (AD plot).
The AD plots obtained for each of the primer probe sets (1) to (9) for 9 clone samples, one for each of all 9 varieties, are shown in FIGS. 3 (a) to 3 (c) and FIG. It shows as a)-(c) and FIG. 5 (a)-(c).
In these AD plots, plot points of GT1 clone specimens are indicated by white symbols and arrows, as shown in FIGS. 6 (a) to (c), FIGS. 7 (a) to (c) and FIGS. 8 (a) to (a). c).

  In the AD plots of FIGS. 3 to 8, one point (indicated by a black circle, black triangle, inverted black triangle, cross mark, white circle, white triangle, or inverted white triangle symbol) corresponds to one clone specimen. . The horizontal axis and vertical axis of the plot graph indicate the intensity (relative value) of FAM fluorescence and VIC (HEX) fluorescence measured for the PCR product of one clone sample, respectively, and the symbol of the base detected by the fluorescence It is added. Fig. 3 (a) shows the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 6 using the primer probe set (1). Fig. 6 (a) is the result of Fig. 3 (a) in the AD plot. The plot points of GT1 clones are indicated by white symbols (white circles) and arrows. FIG. 3 (b) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 7 using the primer probe set (2). FIG. 6 (b) is the result of FIG. The plot points of the GT1 clone are indicated by white symbols (white triangles) and arrows. FIG. 3 (c) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 8 using the primer probe set (3). FIG. 6 (c) is the result of FIG. 3 (c) in the AD plot. The plot points of the GT1 clone are indicated by white symbols (white triangles) and arrows. FIG. 4 (a) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 9 using the primer probe set (4). FIG. 7 (a) is shown in FIG. 4 (a) in the AD plot. The plot points of GT1 clones are indicated by white symbols (white circles) and arrows. FIG. 4 (b) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 10 using the primer probe set (5). FIG. 7 (b) is the result of FIG. The plot points of GT1 clones are indicated by white symbols (white circles) and arrows. FIG. 4 (c) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 11 using the primer probe set (6). FIG. 7 (c) is the result of FIG. The plot points of the GT1 clone are indicated by white symbols (white triangles) and arrows. FIG. 5 (a) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 12 using the primer probe set (7). FIG. 8 (a) is the result of FIG. 5 (a) in the AD plot. The plot points of the GT1 clone are indicated by white symbols (white triangles) and arrows. FIG. 5 (b) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 13 using the primer probe set (8). FIG. 8 (b) is the result of FIG. 5 (b) in the AD plot. The plot points of the GT1 clone are indicated by white symbols (white triangles) and arrows. FIG. 5 (c) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 14 using the primer probe set (9). FIG. 8 (c) is the result of FIG. 5 (c) in the AD plot. , GT1 clone plot points are indicated by white symbols (reverse white triangles) and arrows.

The genotypes shown in these AD plots will be described with reference to FIGS. 3 (a), 3 (b) and 3 (c). FIG. 3 (a) shows the result of detecting the genotype of the 201st SNP base of SEQ ID NO: 6 using the primer probe set (1), and the types of bases detected are cytosine and adenine. The genotypes are cytosine homozygous (C), adenine homozygous (A), and cytosine and adenine heterozygous (C / A) (Tables 1 and 5). The probe pair used was a detection probe for cytosine in which a quencher (Eclipse) was bound to the 5 ′ end of the oligonucleotide of SEQ ID NO: 35 and FAM bound to the 3 ′ end, and a quencher at the 5 ′ end of the oligonucleotide of SEQ ID NO: 36. This is an adenine detection probe in which HEX is bound to the 3 ′ end of Char (Eclipse). Therefore, when the genotype of the base at the SNP site is cytosine homozygous (C), theoretically, only FAM fluorescence is detected, and HEX fluorescence (detected as VIC fluorescence) is not detected. Plotted as a black dot near the upper left corner of the graph. When the base of the SNP site is adenine homozygous (A), theoretically, only HEX fluorescence is detected (as VIC fluorescence), and FAX fluorescence is not detected. Plotted as triangular points. When the base of the SNP site is a heterozygous type (C / A) of cytosine and adenine, theoretically, both FAM fluorescence and HEX fluorescence are detected, so the lower left corner of the graph (vertical axis and horizontal axis) It is plotted as a black triangle point near the diagonal line (starting point of the axis) and the upper right corner (near the upper right corner in FIG. 3A). A cross mark indicates a negative control in which sterilized water (dH ₂ O) was added instead of the genomic DNA as a template in the reaction solution, and neither FAM fluorescence nor HEX fluorescence was detected.

FIG. 3 (b) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 7 using the primer probe set (2), so the types of bases detected are guanine and adenine. The genotypes are guanine homozygous (G), adenine homozygous (A) and guanine and adenine heterozygous (G / A) (Tables 1 and 5). The probe used was a detection probe for guanine in which a quencher (Eclipse) was bound to the 5 ′ end of the oligonucleotide of SEQ ID NO: 39 and HEX was bound to the 3 ′ end, and a quencher at the 5 ′ end of the oligonucleotide of SEQ ID NO: 40. (Eclipse) is a probe for detecting adenine in which FAM is bound to the 3 ′ end. Therefore, when the base genotype of the SNP site is adenine homozygous (A), theoretically, only FAM fluorescence is detected, and HEX fluorescence (detected as VIC fluorescence) is not detected. Plotted as a black dot near the upper left corner of the graph. When the base of the SNP site is guanine homozygous type (G), theoretically, only HEX fluorescence is detected (as VIC fluorescence), and FAX fluorescence is not detected. Plotted as triangular points. When the base of the SNP site is a heterozygous type (G / A) of guanine and adenine, theoretically, both FAM fluorescence and HEX fluorescence are detected. It is plotted as a black triangle point near the diagonal between the axis start point) and the upper right corner. The cross mark indicates a negative control in which sterilized water (dH ₂ O) was added instead of the template genomic DNA in the reaction solution, and neither FAM fluorescence nor HEX fluorescence was detected.

FIG. 3 (c) shows the result of detecting the genotype of the 101st SNP base of SEQ ID NO: 8 using the primer probe set (3), and the types of bases detected are cytosine and thymine. The genotypes are cytosine homozygous (C), thymine homozygous (T), and cytosine and thymine heterozygous (C / T) (Tables 1 and 5). The probe used was a thymine detection probe in which a quencher (Eclipse) was bound to the 5 ′ end of the oligonucleotide of SEQ ID NO: 43 and HEX bound to the 3 ′ end, and a quencher at the 5 ′ end of the oligonucleotide of SEQ ID NO: 44. (Eclipse) is a cytosine detection probe in which FAM is bound to the 3 ′ end. Therefore, when the genotype of the base at the SNP site is cytosine homozygous (C), theoretically, only FAM fluorescence is detected, and HEX fluorescence (detected as VIC fluorescence) is not detected. Plotted as a black dot near the upper left corner of the graph. When the base of the SNP site is thymine homozygous (T), theoretically, only HEX fluorescence is detected (as VIC fluorescence), and FAX fluorescence is not detected. Plotted as triangular points. When the base of the SNP site is a heterozygous type (C / T) of cytosine and thymine, theoretically, both FAM fluorescence and HEX fluorescence are detected, so the lower left corner of the graph (vertical axis and horizontal axis) It is plotted as a black triangle point near the diagonal between the axis start point) and the upper right corner. The cross mark indicates a negative control in which sterilized water (dH ₂ O) was added instead of the template genomic DNA in the reaction solution, and neither FAM fluorescence nor HEX fluorescence was detected.

  In each AD plot shown as FIGS. 3 (a) to 3 (c), GT1 clones indicated by white symbols and arrows are shown as FIGS. 6 (a) to 6 (c), respectively. Looking at the genotype shown in the AD plot, the GT1 clone is shown as a white circle in FIG. 6 (a), which shows the result using the primer probe set (1), that is, a cytosine homozygous type (C). FIG. 6 (b) showing the result using the primer probe set (2), which is shown as a white triangle point, ie, a heterozygous type (A / G) of adenine and guanine, and the primer probe set (3) In FIG. 6 (c), which shows the results of using the above, it is shown as a white triangle point, that is, a heterozygous type (C / T) of cytosine and thymine. On the other hand, the genotypes detected using the primer probe sets (1) to (3) for the GT1 clone are cytosine homozygous (C), adenine and guanine, respectively, as shown in Table 5. Heterojunction type (A / G), and cytosine and thymine heterojunction type (C / T). That is, the genotype shown by AD plot and the genotype shown by Table 5 corresponded. Similarly, even when the primer probe sets (4) to (6) were used, the genotypes shown in the AD plots of FIGS. 7 and 8 matched the genotypes shown in Table 5. From these results, it can be seen that the GT1 analyte can be discriminated using the primer probe sets (1) to (9) by the real-time PCR method.

  As described above, using the method for determining the type of the present invention, it was possible to determine the type of 10 types of para rubber tree (PB260, PB330, PB340, IAN873, GT1, RRIC100, PR107, PB235, and AVROS2037). .

  The method for discriminating varieties of Para rubber tree and the kit for discriminating varieties of Para rubber tree of the present invention are useful for appropriate management and tree planting of commercial varieties of Para rubber tree, and can contribute to improving the productivity of Para rubber tree plantations.

SEQ ID NO: 35: DNA / RNA binding molecule SEQ ID NO: 36: DNA / RNA binding molecule SEQ ID NO: 39: DNA / RNA binding molecule SEQ ID NO: 40: DNA / RNA binding molecule SEQ ID NO: 29: DNA / RNA binding molecule SEQ ID NO: 30: DNA / RNA binding molecule SEQ ID NO: 33: DNA / RNA binding molecule SEQ ID NO: 34: DNA / RNA binding molecule SEQ ID NO: 37: DNA / RNA binding molecule SEQ ID NO: 38: DNA / RNA binding molecule SEQ ID NO: 41: DNA / RNA binding molecule sequence No. 42: DNA / RNA binding molecule SEQ ID NO: 45: DNA / RNA binding molecule SEQ ID NO: 46: DNA / RNA binding molecule SEQ ID NO: 49: DNA / RNA binding molecule SEQ ID NO: 50: DNA / RNA binding molecule SEQ ID NO: 53: DNA / RNA binding molecule SEQ ID NO: 54: DNA / RNA binding molecule

Claims (8)

  A method for discriminating varieties of Para rubber tree, wherein the varieties of the Para rubber tree subject are discriminated by detecting and analyzing a single nucleotide polymorphism of the Para rubber tree subject.   2. The method for distinguishing para rubber tree varieties according to claim 1, wherein the single nucleotide polymorphism of the Para rubber tree specimen is detected using a mass array method.   2. The method for distinguishing para rubber tree varieties according to claim 1, wherein the single nucleotide polymorphism of the Para rubber tree specimen is detected using a real-time PCR method. The method for discriminating varieties of Para rubber tree according to any one of claims 1 to 3, wherein the single nucleotide polymorphism is detected for at least one selected from the group consisting of bases at the following sites:
201st base of SEQ ID NO: 1;
The 301st base of SEQ ID NO: 2;
The 251st base of SEQ ID NO: 3;
201st base of SEQ ID NO: 4;
The 301st base of SEQ ID NO: 5;
The 201st base of SEQ ID NO: 6;
The 101st base of SEQ ID NO: 7;
The 101st base of SEQ ID NO: 8;
The 101st base of SEQ ID NO: 9;
The 101st base of SEQ ID NO: 10;
The 101st base of SEQ ID NO: 11;
The 101st base of SEQ ID NO: 12;
101st base of SEQ ID NO: 13; and 101st base of SEQ ID NO: 14. The method for distinguishing para rubber tree varieties according to claim 2, wherein the single nucleotide polymorphism is detected using at least one of the primer sets shown in (1) to (6) below:
(1) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 15 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 16, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 17 Primer set with extension primer;
(2) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 18 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 19, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 20 Primer set with extension primer;
(3) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 21 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 22, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 23 Primer set with extension primer;
(4) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 24 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 25, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 26 Primer set with extension primer;
(5) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 27 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 28, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 29 Primer set with extension primer;
(6) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 30 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 31, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 32 Primer set with extension primer. The method for discriminating varieties of Para rubber tree according to claim 3, wherein the single nucleotide polymorphism is detected using at least one of the primer probe sets shown in the following (1) to (9):
(1) An amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 33 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 34, and a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 35 A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 36;
(2) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 37 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 38; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 39; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 40;
(3) an amplification primer pair comprising a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 41 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 42; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 43; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 44;
(4) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 45 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 46; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 47; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 48;
(5) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 49 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 50; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 51; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 52;
(6) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 53 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 54; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 55; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 56;
(7) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 57 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 58; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 59; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 60;
(8) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 61 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 62; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 63; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 64;
(9) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 65 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 66; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 67; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 68. A kit for discriminating varieties of Para rubber tree, comprising at least one of the primer sets shown in the following (1) to (6):
(1) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 15 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 16, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 17 Primer set with extension primer;
(2) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 18 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 19, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 20 Primer set with extension primer;
(3) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 21 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 22, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 23 Primer set with extension primer;
(4) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 24 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 25, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 26 Primer set with extension primer;
(5) An amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 27 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 28, and a single base comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 29 Primer set with extension primer;
(6) An amplification primer pair consisting of a primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 30 and a primer containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 31, and a single base containing the oligonucleotide having the nucleotide sequence of SEQ ID NO: 32 Primer set with extension primer. A kit for distinguishing para rubber tree varieties comprising at least one of the primer probe sets shown in the following (1) to (9):
(1) An amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 33 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 34, and a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 35 A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 36;
(2) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 37 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 38; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 39; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 40;
(3) an amplification primer pair comprising a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 41 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 42; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 43; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 44;
(4) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 45 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 46; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 47; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 48;
(5) an amplification primer pair consisting of a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 49 and a primer comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 50; a probe comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 51; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 52;
(6) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 53 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 54; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 55; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 56;
(7) an amplification primer pair consisting of a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 57 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 58; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 59; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 60;
(8) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 61 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 62; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 63; A primer probe set with a probe pair for detection consisting of a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 64;
(9) an amplification primer pair comprising a primer comprising an oligonucleotide having the base sequence of SEQ ID NO: 65 and a primer comprising the oligonucleotide having the base sequence of SEQ ID NO: 66; a probe comprising the oligonucleotide having the base sequence of SEQ ID NO: 67; A primer probe set with a detection probe pair comprising a probe comprising an oligonucleotide having the base sequence of SEQ ID NO: 68.