TW201422818A - Method for determining the rice cultivars from taiwan and foreign countries - Google Patents

Abstract

The present invention relates to a method for determining the rice cultivars from Taiwan and foreign countries by genotyping single nucleotide polymorphisms in SEQ ID NO: 1-12 of rice genomic DNA using Multiplex SNP PCR with 12 primer sets. Then identify the rice cultivars through analyzing the length of PCR products.

Description

Method for identifying domestic and foreign rice varieties

The invention relates to a method for identifying rice varieties by using molecular markers, in particular to detecting single nucleotide polymorphism by using a novel single primer polymorphic polymerase chain reaction (Multiplex SNP PCR). (single nucleotide polymorphism, SNP) to identify domestic and foreign rice varieties.

Rice is the staple food of the Chinese people, and it is also the industry with the widest area of cultivation and the largest number of farmers. Since Taiwan joined the World Trade Organization (WTO) in 2002, foreign rice can be imported and sold according to tariff quotas. There are more and more types of white rice sold in the market, which has a considerable impact on the domestic rice industry. At this stage, the government actively promotes the rice industry's three-product strategy of “variety, quality and brand” to enhance the competitiveness of domestic rice. Among the three-product strategy, the purity of “variety” of rice is the source and the most important. Therefore, at present, domestic rice is used to maintain the heritability and quality of rice varieties. The COA has a three-level breeding system to ensure the superiority of rice seeds, that is, through the original species, original species, and seed collection. Excellent seed count.

In the field, the variability within the variety may be caused by mutation, pollen pollution, intraspecific heterogeneity after multiple generations and separation of seeds, and genetic variation may occur under the tertiary reproductive system. After the land preparation, the farmers purchased the seedlings from the nursery for transplanting, and then carried out field operations such as replanting, weed control, fertilization, drainage and pest control. After the rice field is harvested, it is transported to the rice mill for low-temperature drying, low-temperature storage, ridge valley, color sorting, whitening and packaging. Finally, it is sold on the market. These man-made operations and modulation processes may cause mixed rice varieties. . In addition, some uncut traders deliberately mix high-priced rice varieties with other lower-priced varieties to increase sales.

In the past, the identification of rice varieties relied on the appearance phenotype, the composition of seed proteins, the difference in isozyme or enzyme activity. However, because these patterns, physiology, and biochemical indicators are susceptible to the effects of interlacing between varieties, environments, and even varieties and environments, reproducibility is also poor. Therefore, the molecular marker technology related to the genetic composition of varieties has better reproducibility and reproducibility, and it has become the best tool for many crops in seed purity inspection and variety identification.

With the advancement of genetics and the rise of molecular biology, molecular marker technology has evolved and improved, including restriction fragment length polymorphism (RFLP), random amplified polymorphic nucleic acid molecular analysis (random) Amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), single repeat sequence method (microsatellite or simple sequence repeat (SSR), simple sequence repeat interval (Inter-Simple Sequence Repeat) , ISSR) and sequence insertion and deletion markers (insertion/deletion, In/Del), etc., among which SSR/ISSR molecular marker correlation techniques are most common, mainly tandem repeats with 1 to 5 nucleotides as the basic unit. It is more than 100 to 200 bps in length. Microsatellite DNA is distributed at different sites throughout the genome. Due to the size and sequence of repeating units and the number of repeats, it is rich in polymorphism. However, due to the different number of core sequence repeats, the amplification of PCR products of different lengths is one of the commonly used methods for detecting DNA polymorphism. Although this technique can determine different rice varieties at home and abroad, the strains are subjected to PCR reaction using different primer pairs, and the difference in product size due to the difference in the number of simple sequence repeats of each rice variety is performed for each rice variety. In high-resolution electrophoresis analysis, since simple repeat sequences of the same type in a living body are ubiquitously present in the genome, these primers for distinguishing simple repeat sequences may be bonded to different positions of the genome at the same time in PCR, so the analysis The process is highly prone to the appearance of nonspecific PCR products that make electricity The background of the swimming gel is complicated and interferes with the judgment of the electrophoresis band. The difference in the number of repetitions between some varieties is too small or the mixed rice is less than 10%, which makes it impossible to visually distinguish and distinguish the difference in the length of the DNA fragments on the electrophoretic film. Leading to misjudgment of rice varieties. On the other hand, the domestic rice is very close to the parents used in the breeding process, especially the stalked rice. Because the early Taiwanese rice species are mostly introduced from Japan, and the main parents are limited, the genetic relationship is very close, genetic diversity ( The genetic diversity is about 4/1000 (0.4%). It is not easy to distinguish the rice varieties with similar genetic relations by the traditional SSR molecular markers, so that there are cases of misjudgment. Therefore, these molecular markers are still difficult to distinguish among the varieties that are too close in the identification of rice varieties, and cannot be 100% confirmed for the defects of the sampled rice samples.

Single nucleotide polymorphism (SNP) refers to the difference in individual nucleotides between different alleles at the same locus. This difference includes the deletion or insertion of a single base, the most common being a single Nucleotide substitutions, and often occur between purines (A and G) or pyrimidines (C and T). The SNP marker distinguishes the difference in genetic material between two individuals and is considered to be the best genetic marker for application. SNPs are rapidly replacing SSR as the technology of choice for DNA molecular markers in plant breeding and genetics applications, because SNPs are rich, stable, suitable for automation, efficient, and increasingly cost-effective. Recently, a method of high-throughput and multiple single-nucleotide polymorphism (Multiplex SNP) has been developed. Based on the technology of SNP, multiple compatible markers are combined, and multiple pairs of SNP primers are added in a single reaction. That is, multiple types of sites are simultaneously detected in a single PCR reaction, which is high-throughput and suitable for automated analysis. In the prior art, 10 SNP multiple primers (10-plex) and 15 SNP multiple primers (15-plex) have been developed, but the 10-plex primer group mainly screens SNP molecular markers for domestic indica rice varieties, so The identification of domestic japonica rice varieties is poor. The 15-plex primer set is detected by Tetra-primer ARMS-PCR, which is a four-primer amplification hindered mutant system. The PCR product obtained by detecting the SNP of the same site is detected, so that the difference in the size of the allele specific DNA electrophoresis band of the product is at least 1.5 times different to facilitate the discrimination of the SNP, but it is necessary to separately identify 15 sites. It is quite time consuming to fully identify the rice varieties to be tested after performing a polymerase chain reaction. The 15-plex primer group 15-plex primer group has unidentifiable varieties, and the two must be used together.

Accordingly, the present invention provides a method for identifying rice varieties at home and abroad, using a novel 12 SNP multiplex primer set, the primer set of which performs multiple single nucleotide dot diagram polymorphic polymerase chain reaction in a single reaction. It is more convenient and enhances the accuracy and sensitivity of rice identification technology, and can identify 50 kinds of rice varieties at home and abroad. The invention can reward the excellent rice factory to produce the high-quality variety of the high-quality variety labeled rice; at the same time, due to the development of the rice variety detection technology, with the sampling and labeling of the commercially available rice, the rice market is gradually moved to the grading sales system, and also beneficial to the rice production and marketing profession. The establishment of the district will enhance the separation and competitiveness of domestic rice.

To achieve the above object, the present invention provides a method for identifying domestic and foreign rice varieties, the steps comprising: (1) using a primer set and increasing by a multiple single nucleotide polymorphic polymerase chain reaction (Multiplex SNP PCR) (amplify) the genomic DNA of a plurality of known varieties of rice comprising a single nucleotide polymorphism (SNP) site in the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 12. Fragment to obtain the fragment length of each PCR product obtained by increasing the amplitude of the known varieties of rice; (2) using a primer set and by multiple single nucleotide polymorphic polymerase chain reaction (Multiplex SNP PCR) Amplifying a genetic DNA of a rice to be tested to obtain a fragment length of each PCR product obtained by increasing the amplitude of the rice to be tested; and (3) obtaining PCR by the amplification of the rice to be tested by the primer set The length of the fragment of the product is compared with the length of the fragment of each PCR product obtained by the amplification of the known variety rice by the primer group in step (1), thereby identifying the variety of the rice to be tested, wherein the primer set is selected from the first primer Group (SEQ ID NO 13 and SEQ ID NO: 14), second primer set (SEQ ID NO: 15 and SEQ ID NO: 16), third primer set (SEQ ID NO: 17 and SEQ ID NO: 18), fourth primer set ( SEQ ID NO: 19 and SEQ ID NO: 20), fifth primer set (SEQ ID NO: 21 and SEQ ID NO: 22), sixth primer set (SEQ ID NO: 23 and SEQ ID NO: 24), Seven primer sets (SEQ ID NO: 25 and SEQ ID NO: 26), eighth primer set (SEQ ID NO: 27 and SEQ ID NO: 28), ninth primer set (SEQ ID NO: 29 and SEQ ID NO: 30), a tenth introduction group (SEQ ID NO: 31 and SEQ ID NO: 32), an eleventh introduction group (SEQ ID NO: 33 and SEQ ID NO: 34), and a twelfth introduction group (SEQ ID NO: 35 and SEQ ID NO: 36); wherein the known variety of rice is selected from Taikeng 2 (TK2), Taikeng 4 (TK4), Taichung No. 5 (Taikeng) 5, TK5), Taikeng No. 8 (Taikeng 8, TK8), Taichung No. 9 (Taikeng 9, TK9), Taichung No. 14 (Taikeng 14, TK14), Taichung No. 16 (Taikeng 16, TK16), Kaohsiung No. 139 (Kaohsiung 139, KH139), Kaohsiung 145 (Kaohsiung 145, KH145), Taoyuan No. 1 (Taoyuan 1, TY1), Taoyuan No. 3 (Taoyuan 3, TY3) ), Tainan 11, TN11, Tainung 71 (TNG71), Hualien 19 (HL19), Hualien 20 (HL20), Koshihikari, Japan Taoyuan glutinous 2 (TYW2), Taichung sen 2 (TS2), Taichung sen10 (T C S10), Tainung sen 22 (TNGS22), Taichung Native 1, TN1, Thailand Jasmine 85, Kaohsiung 141 (KH141), Kaohsiung 142 (KH142), Kaohsiung 143 (KH143), Kaohsiung 144 (Kaohsiung 143, KH143) Kaohsiung 144, KH144), Kaohsiung 146 (KH146), Kaohsiung 147 (Kahsiung 147, KH147), Taoyuan 4 (TY4), Tainan 13 (Tainan 13, TN13), Tainan 14 (Tainan 14) Tainan 14, TN14), Taitung 30 (TT30), Taitung 32 (Taitung 32, TT32), Tai Nong 74 (Tainung 74, TNG74), Tai Nong 75 (Tainung 75, TNG75), Tai Nong No. 84 (Tainung 84, TNG84), Taichung No. 192 (Taichung 192, TC192), Taichung No. 194 (Taichung 194, TC194), Hualien 21 (Hualien 21, HL21), Miaoli No. 1 ( Miaoli 1, ML1), Taichung No. 1, (Taikeng glutinous 1, TKW1), Taikeng glutinous 3 (TKW3), Taikeng glutinous 12 (TKW12), Taitung No. 31 (Taitung glutinous 31, TTW31), Tainung glutinous 73 (TNGW73), Taichung sen17 (T C S17), Tai Nong 籼 14, TNGS14, Tai Nong Tainung sen glutinous 21 (TNGSW21), Taichung sen glutinous 1, TCSW1, and Taichung sen glutinous 2 (TCSW2).

The invention provides a method for identifying domestic and foreign rice varieties, which utilizes a primer set and increases the genetic DNA of the rice to be tested by a multiple single nucleotide polymorphic polymerase chain reaction, wherein the concentration of the primer group is: The first primer set (SEQ ID NO: 13 and SEQ ID NO: 14) is 1.0 μM, and the second primer set (SEQ ID NO: 15 and SEQ ID NO: 16) is 1.0 μM, and the third primer set (SEQ) ID NO: 17 and SEQ ID NO: 18) are 1.6 μM, and the fourth primer set (SEQ ID NO: 19 and SEQ ID NO: 20) The system is 3.0 μM, the fifth primer set (SEQ ID NO: 21 and SEQ ID NO: 22) is 1.0 μM, and the sixth primer set (SEQ ID NO: 23 and SEQ ID NO: 24) is 2.0 μM. The seven primer set (SEQ ID NO: 25 and SEQ ID NO: 26) was 4.0 μM, and the eighth primer set (SEQ ID NO: 27 and SEQ ID NO: 28) was 2.0 μM, and the ninth primer set (SEQ ID) NO:29 and SEQ ID NO:30) are 4.0 μM, the tenth primer set (SEQ ID NO:31 and SEQ ID NO:32) is 2.0 μM, and the eleventh primer set (SEQ ID NO:33 and SEQ) ID NO: 34) is 2.0 μM and the twelfth primer set (SEQ ID NO: 35 and SEQ ID NO: 36) is 3.0 μM. The reaction conditions are first to react at 94 ° C for 15 minutes, and then at 94 ° C, 30 seconds, 55 ° C, 90 seconds, 72 ° C, 90 seconds for a cycle, and repeat this cycle 29 times, the temperature is adjusted to 60 ° C reaction 30 minutes. And the genomic DNA of rice is derived from the endosperm of rice seeds or the plants after germination of rice seeds.

The invention further provides a primer group capable of identifying domestic and foreign rice varieties, which adopts a rice DNA as a template and obtains a single nucleotide polymorphism (SNP) position by a polymerase chain reaction. Fragment length of the spot, wherein the primer set is selected from the group consisting of a first primer set (SEQ ID NO: 13 and SEQ ID NO: 14), a second primer set (SEQ ID NO: 15 and SEQ ID NO: 16), and a third Primers (SEQ ID NO: 17 and SEQ ID NO: 18), fourth primer set (SEQ ID NO: 19 and SEQ ID NO: 20), and fifth primer set (SEQ ID NO: 21 and SEQ ID NO: 22) ), sixth primer set (SEQ ID NO: 23 and SEQ ID NO: 24), seventh primer set (SEQ ID NO: 25 and SEQ ID NO: 26), eighth primer set (SEQ ID NO: 27 and SEQ) ID NO: 28), ninth primer set (SEQ ID NO: 29 and SEQ ID NO: 30), tenth primer set (SEQ ID NO: 31 and SEQ ID NO: 32), eleventh primer set (SEQ ID) NO: a population consisting of 33 and SEQ ID NO: 34) and a twelfth primer set (SEQ ID NO: 35 and SEQ ID NO: 36), and the primer sets are analyzed for several known rice varieties and one is to be tested The rice is then compared to identify the variety of the rice to be tested. Among them, the known variety of rice is the above 50 rice varieties. And the polymerase chain reaction reaction system uses the primer set to perform a polymerase chain reaction separately or simultaneously uses the primer set to perform multiple single nucleotide polymorphic polymerase chain reaction (Multiplex SNP PCR).

The invention further provides a method for identifying domestic and foreign rice varieties, the steps comprising: (1) analyzing genomic DNA of several known varieties of rice, and detecting 12 single nucleotide polymorphic genes by a polymerase chain reaction; a single nucleotide polymorphism in the nucleic acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 12; (2) analyzing a genomic DNA of a rice to be tested, using a polymerase chain reaction Detecting 12 single nucleotide polytype genotypes, the single nucleotide polymorphisms are located in the nucleic acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 12; and (3) The 12 single nucleotide polymorphism genotypes obtained by measuring the rice are compared with the 12 single nucleotide polymorphism genotypes of the known varieties of rice in step 1 to identify the rice to be tested. a variety in which the 23rd base of the nucleic acid sequence shown by SEQ ID NO: 1 is the A/T genotype, and the 21st base of the nucleic acid sequence shown by SEQ ID NO: 2 is the A/G genotype, SEQ ID NO The 483th base of the nucleic acid sequence indicated by 3 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 4 is the G/A genotype. The 21st base of the nucleic acid sequence shown by SEQ ID NO: 5 is a C/T genotype, and the 21st base of the nucleic acid sequence shown by SEQ ID NO: 6 is a G/A genotype, SEQ ID NO: 7. The 367th base of the nucleic acid sequence shown is the G/A genotype, the 22nd base of the nucleic acid sequence shown by SEQ ID NO: 8 is the A/C genotype, and the nucleic acid sequence shown by SEQ ID NO: 9. 267 bases are G/A genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 10 is A/G The genotype, the 20th base of the nucleic acid sequence shown by SEQ ID NO: 11 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 12 is the T/C genotype. And the polymerase chain reaction is carried out using a primer pair complementary to the nucleic acid sequences of SEQ ID NO: 1 to SEQ ID NO: 12. And the primer pair can be used to use the genomic DNA of the rice varieties as a template to increase the nucleic acid fragments carrying the genotypes. And the genomic DNA of rice is derived from the endosperm of rice seeds or the plants after germination of rice seeds.

In the method for identifying domestic and foreign rice varieties of the present invention, the first to the twelfth introduction group can list the rice nuclear monoacids as a template, and the "single micro-test tube" or "single reaction" Multiple single nucleotide point mutation polymorphic polymerase chain reaction is performed to obtain a nucleic acid sequence fragment having a single nucleotide polymorphic site, thereby distinguishing domestic and foreign rice varieties. Therefore, the present invention can effectively identify different strains of domestic stalk/stalk or sorghum/salt rice varieties, because the genetic composition of most stalk rice varieties is very similar to the standard product Tainong 67, so the present invention specifically selects domestic varieties and SNPs of different or specific (such as single species) of Tai Nong 67, using the SNP specific sequence primer pairs designed by these SNP sites, and then further combining multiple sets of different SNP specific primer pairs to carry out Multiplex SNP PCR reaction and Variety identification.

The primer of the present invention is designed according to the nature of the SNP, and the third base at the 3' end is designed to be an incorrect base, and the specificity of the primer has been increased. The basic principles are strong mismatch: G/A and C/T; weak mismatch: C/A and G/T; medium mismatch: A/A, C/ C, G/G and T/T, in which the length of the primer is about 20~25 mers. In addition, in order to facilitate the identification of the electrophoresis band on the agarose gel, the PCR product obtained by detecting the SNP changes at the same point is deliberately designed to make the difference of the size of the final DNA electrophoresis band at least 15 bps or more, and the appropriate adhesion is adjusted. Temperature, GC base content, primer After the length and other conditions, the condition will be automatically designed by the program. Finally, the most suitable sequence of SNPs will be selected for SNP-specific primer pair synthesis to prevent the primers from competing in the PCR reaction and causing false bonding to generate noise. . Therefore, the 12-plex primer set of this case is more convenient and easy to popularize and apply, compared with the prior art 15-plex primer set, which is a four-primer amplification of the polymerase chain reaction (Tetra-primer ARMS-PCR). Improve the accuracy and sensitivity of rice identification techniques.

Another feature of the present invention is that all SNP sites are located on a gene having a functional expression, that is, an exon, and the gene marker of the present invention is located in an intron compared to the prior art SNP site. Functionality affects the external performance of rice and, therefore, can also be used in breeding selection.

The identification method of the domestic and foreign rice varieties of the present invention, all of the synthesized primers are firstly carried out by TAKARA DICE gradient polymerase chain reactor, and the gradient PCR of the SNP polymorphic variety is confirmed by the TAKARA DICE gradient polymerase chain reactor. And look for the most suitable polymerization temperature. Finally, the average temperature of each SNP-specific primer group is taken as the polymerization temperature of multiplex PCR, and the optimal conditions are optimized to facilitate the application of future varieties. In the PCR reaction, each SNP-specific primer is added to a single microtube or a single reaction in proportion, and a commercially available multiplex kit is used according to the recommended PCR reaction. Therefore, the identification method of the domestic and foreign rice varieties of the present invention only needs the equipment possessed by the general meristem laboratory, and is mainly completed by a PCR machine, which saves time, cost and convenience. Fast and easy to operate. In addition, the present invention is capable of analyzing a small amount of sample (1 ng gDNA) with high sensitivity, and can be sampled and analyzed at any stage during plant growth; in addition, the sample can be stored for a long period of time, and the stability and reproducibility of the identification result are high.

The identification method of the domestic and foreign rice varieties of the invention can be applied to the identification of domestic and foreign rice varieties after the open rice trade; the establishment of the rice history certification system, the establishment of a market rice quality control mechanism; and the food management method. In response to the city The demand for the labeling of rice varieties for sale; the provision of agricultural testing units, the general breeders to maintain the purity of new or existing rice varieties, and the operation of the government management unit, namely the seedling industry, on the breeding system of improved waterways; The identification of this variety, the detection and maintenance of seed breeding of excellent rice varieties, the determination of commercially available rice varieties, the trading of rice varieties of rice farmers, that is, the management of commercial rice by the administrative units. The identification method of the domestic and foreign rice varieties of the present invention is based on the variable number tandem repeat (VNTR) of the SSR molecular marker, but is a single nucleotide for different rice varieties. Multi-type, using the SNP combination of different rice varieties measured by each primer group, can improve the sensitivity, stability and accuracy of rice varieties. It is better than the DNA fingerprinting technology of existing rice varieties, especially the relationship. For the close domestic rice varieties (such as Taitung 32 (TT32) and Taiwanese No. 16 (TK16)), the dual gene frequency (allelic frequency) of each of the 12 SNP sites of the present invention is 0.24. Between 0.64, it is much improved compared to the prior art dual gene frequency of 0.0143 to 0.471.

Through the present invention, those skilled in the art can identify the 50 rice varieties for the 12 SNP sites described in the present invention by any known method. Specifically, the methods used include, but are not limited to, high-resolution melt (HRM), MALDI-TOF mass spectrometry (matrix assisted laser desertion/ionization time-of-flight mass spectrometry), and various Sequencing, etc.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

The first collection of domestic and foreign rice samples includes: Taikeng 2 (TK2), Taikeng 4 (Tik4, TK4), Taikeng 5 (TK5), Taichung 8 (Taikeng 8, TK8) ), Taikeng 9, TK9, Taikeng 14, TK14, Taikeng 16, TK16, Kaohsiung 139 (KH139), Kaohsiung 145 (Kaohsiung) 145, KH145), Taoyuan 1 (TY1), Taoyuan 3 (Taoyuan 3, TY3), Tainan 11 (Tainan 11, TN11), Tainong 71 (Tainung 71, TNG71), Hualien 19 ( Hualien 19, HL19), Hualien 20 (HL20), Koshihikari, Taoyuan glutinous 2 (TYW2), Taichung sen 2 (TS2), Taichung 籼 10 No. (Taichung sen10, T C S10), Tainung sen 22 (TNGS22), Taichung Native 1, TN1, Jasmine 85, Kaohsiung 141 (KH141), Kaohsiung No. 142 (Kaohsiung 142, KH142), Kaohsiung 143 (Kahsiung 143, KH143), Kaohsiung 144 (Kaohsiung 144, KH144), Kaohsiung 146 (Kaohsiung 146, KH146), Kaohsiung 147 (Kaohsiung 147) KH147), Taoyuan 4 (TY4), Tainan 13 (Tainan 13, TN13), Tainan 14 (Tainan 14, TN14), Taitung 30 (Taitung 30, TT30), Taitung 32 (Taitung 32, TT32), Tainung 74 (TNG74), Tainung 75 (TNG75), Tainong 84 (TNG84), Taichung No. 192 (Taichung 192, TC192), Taichung No. 194 ( Taichung 194, TC194), Hualien 21 (HL21), Miaoli 1, ML1, Taikeng glutinous 1, TKW1, Taikeng glutinous 3, TKW3), Taikeng glutinous 12 (TKW12), Taitung glutinous 31 (TTW31), Tainung glutinous 73 (TNGW73), Taichung sen17 (Taizhong sen17, T C S17), Tainung sen 14, TNGS14, Tainung sen glutinous 21 (TNGSW21), Taichung sen glutinous 1, TCSW1 and Taichung 籼糯 2 No. (Taichung sen glutinous 2, TCSW2), a total of 50 varieties, except for Japan's Koshihikari and Thailand's Jasmine85 are all domestic breeds.

Next, the genomic DNA of each rice sample was extracted, and the single nucleotide polymorphism of each rice sample was detected by multiple single nucleotide polymorphism polymerase chain reaction (Multiplex SNP PCR) with a specific primer set. (single nucleotide polymorphism, SNP), by determining the size of the polymerase chain reaction reaction product (that is, the length of the electrophoresis band), whether the rice sample has the single nucleotide polymorphism site, and further know the variety of the rice sample, such as Figure 1 shows. The foregoing embodiment is described in detail as follows:

Example 1: Extraction and quantification of rice genomic DNA 1.1 White rice genomic DNA extraction

Approximately 10 grains of white rice were used, and a 2 mL microcentrifuge tube of Thermo Fisher QSP was used and a steel ball (7 mm) for grinding was added, and the rice was ground to a fine powder at 14,000 rpm using a homogenizer. The DNA extraction method was modified by cetyl-trimethyl ammonium bromide (CTAB). Add 750 μL of 1% CTAB to each tube and place at 65 °C for 20 minutes. Add 10 μL of proteinase K (20 mg/mL) to each tube and shake evenly. Allow to react at 65 °C for 30 minutes (shake evenly every 10 minutes) . 800 μL of phenol/chloroform/isoamyl alcohol (25:24:1) was added, shaken for 3 minutes and centrifuged at 14,000 rpm for 10 minutes. Place the supernatant into another centrifuge tube, add 400 μL of chloroform/isoamyl alcohol (24:1), centrifuge at 14000 rpm for 5 minutes, then take the supernatant, add 2/3 volume of isopropanol, and let stand at room temperature. minute. Centrifuge at 14000 rpm for 10 minutes, dehydrate with 70% EtOH, centrifuge for a few seconds, pour off the waste and place the pellet at 40 °C. Minutes get the preliminary product. The preliminary product was added to 200 μL of 1X TE buffer (Tris-HCl 10 mM, EDTA 1 mM, pH 8.0) and completely dissolved at 40 ° C for 1 hour. Add 10 μL of RNase (500 μg/mL) and mix well. The reaction was carried out at 37 ° C for more than 40 minutes. 450 μL of 95% EtOH was added, precipitated at -20 ° C for 30 minutes or more, centrifuged at 14,000 rpm for 10 minutes, and the waste liquid was removed and air-dried to obtain a DNA product. The product was fully dissolved in 50 μL of TE buffer, quantified and stored at -20 ° C, and the quantified DNA was diluted to 1 ng/μL for subsequent PCR reaction analysis. DNA quantification was performed using a Qubit® fluorometer (Invitrogen, USA) and a Qubit® dsDNA HS Assay Kit (step 10 or step 40).

1.2 Genomic DNA extraction of rice seed germination

The rice seeds were grown to 2 weeks after germination, and the aboveground parts of the plants were cut and stored in a freeze-dried manner. The method of rice plants rapid extraction of DNA collected was dried, freeze-dried rice shear blade for about 2 ~ 3 mm 2 size, 20 mL of leaf DNA extraction solution (QuickExtract ^TM, EPICENTRE® Biotechnologies, USA), and 65 The DNA was quickly extracted by heating at ° C for 6 minutes followed by heat treatment at 98 ° C for 2 minutes. Note that this method is a fast DNA extraction method that sacrifices quality, so DNA cannot be stored for long periods of time. The DNA was also diluted to 1 ng/μL after quantification, followed by subsequent PCR reaction analysis (step 10 or step 40).

Example 2: Establishment of a single nucleotide polymorphism site that can distinguish 50 kinds of domestic and foreign rice

Single nucleotide polymorphisms of each rice sample were detected using multiple single nucleotide polymorphic polymerase chain reaction (Multiplex SNP PCR) (step 20). In the second embodiment of the present invention, SNP difference sites which can distinguish the above 50 domestic and foreign rice lines are identified, and the SNP sites comprise the 23rd base of the nucleic acid sequence represented by SEQ ID NO: 1 as the A/T gene. Type, the 21st base of the nucleic acid sequence shown by SEQ ID NO: 2 is A/G The genotype, the 483th base of the nucleic acid sequence shown by SEQ ID NO: 3 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 4 is the G/A genotype, SEQ ID NO The 21st base of the nucleic acid sequence shown in 5 is a C/T genotype, the 21st base of the nucleic acid sequence shown by SEQ ID NO: 6 is a G/A genotype, and the nucleic acid sequence shown by SEQ ID NO: 7. The 367th base is the G/A genotype, the 22nd base of the nucleic acid sequence shown by SEQ ID NO: 8 is the A/C genotype, and the 267th base of the nucleic acid sequence shown by SEQ ID NO: 9. The G/A genotype, the 20th base of the nucleic acid sequence shown by SEQ ID NO: 10 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 11 is the A/G genotype. The 20th base of the nucleic acid sequence shown by SEQ ID NO: 12 is a T/C genotype, and a total of 12 SNP sites (step 30).

Example 3: Multiple single nucleotide polymorphic polymerase chain reaction

The multiplex polymerase chain reaction uses a multiplex polymerase chain reaction reagent set (Multiplex PCR Kit, QIAGEN®, Germany), and the reaction volume of each component is adjusted according to the reagent set manual. The 12-plex introduction group (12-plex) differs in the specificity of the primers. Therefore, the dilution ratio of each primer is different. Therefore, the amount of DNA in the multiple SNP polymerase chain reaction, the addition amount of Q buffer (Q buffer) and The number of cycles has also been adjusted. Q buffer is added to the PCR reaction in this reagent group to reduce the production of non-specific product electrophoresis bands.

Multiple reference nucleotide polymorphic polymerase chain reaction of 12 sets of reference standards for each variety. The total reaction volume is 10 μL, including 5 μL 2X QIAGEN multiplex PCR master mix, 1 μL 10X primer mix (primer mix) 1 μL Q buffer, 2 μL RNase-free water, and 1 μL DNA (ng/μL) samples. The polymerase chain reaction was carried out using a thermal cycler (PCR Thermal Cycler, Takara, Japan) under the reaction conditions of 94 ° C for 15 minutes, followed by 94 ° C, 30 seconds, 55 ° C, 90 seconds, 72 ° C, 90 seconds. For a cycle, and repeat this cycle after 29 times, the temperature The reaction was adjusted to 60 ° C for 30 minutes, 1 cycle, and finally to 25 ° C to end the reaction (step 50).

The primer mixture is a mixture of forward and reverse of each primer set, and each primer needs to be diluted to a different concentration. See Table 1:

Example 4: Analysis of multiple single nucleotide polymorphic polymerase chain reaction products 4.1 Capillary Electrophoresis Analysis

After PCR, the product was subjected to capillary electrophoresis using an e-GENE HAD-GT12 ^TM DNA Analyzer (QIAGEN®, Germany) with a high-resolution capillary electrophoresis reagent kit (QIAxcel DNA High Resolution Kit) containing a QIAxcel DNA High Resolution Cartridge clip. The clips are placed in the instrument and used with the QIAxcel BioCalculator software. According to the procedure of use, it takes about 15 μL to hold the volume of the sample in a 96-well sample disk, and the total volume of the PCR is about 10 μL. Therefore, the reaction product should be diluted to 15 μL with QX DNA Dilution buffer attached to Kit for PCR product analysis. Using the 15 bp/1 kb QX alignment marker, select the OM820.mtd that the manufacturer assisted in (single sample analysis time 820 seconds), and adjust the Adjust separation time 0-120 seconds depending on the temperature. The interpretation method of the results is similar to the conventional colloidal electrophoresis interpretation method, and the rest of the details refer to the QIAxcel® DNA Handbook (step 30 or step 60).

Referring to Fig. 2, the relative position peaks of 12 sets of primers of 12-plex SNP PCR were analyzed by Qiaxcel capillary electrophoresis using the method for identifying rice varieties at home and abroad according to the present invention, and each peak represents the fragment length of the PCR product. Referring to Figure 3, the identification of the present invention is made for different rice varieties, Taikeng 16, TK16, Tainung sen glutinous 21 (TNGSW21) and Kaohsiung 142 (KH142). The method of Qiacelcel capillary electrophoresis of 12-plex SNP PCR analysis was carried out by domestic and foreign rice varieties. Taking TK16 as an example, in the twelfth primer group (117 bp), the wild type is A, so the product cannot be amplified when PCR is performed. If the 117 bp is G phenotype, the amplification reaction is performed by PCR. There is a peak performance when performing capillary electrophoresis, which means that the variety has a single nucleotide polymorphism (SNP) in the allele. Through the above results, the results of each segment are clear and easy to interpret, and the amount of noise is small, which proves that the 12 sets of primers used in the present invention have high specificity.

1.2 Colloidal electrophoresis analysis

Analyzed by electrophoresis in 3% agarose gel (AMRESCO Agarose SFR ^TM, No. AM-J234, USA). Heat in a microwave oven until the gel is completely dissolved into a completely transparent form. Shake evenly after adding 4 μL of EtBr (10 mg/mL). After standing for 10 minutes, pour into the glue tank (length 14.8 cm, width 13.8 cm), drive the bubble to the edge, and put the tooth comb that requires the number of holes. Allow to stand for more than 40 minutes to be solidified. All PCR products were mixed with 1 μL of 6X dye and uniformly injected into the alveolar. Loadingmarker uses GeneDirex®'s 2 μL 50bp DNA Ladder to perform electrophoresis at 120V. The dye is stopped by the recording dye from the top and bottom of the film to the lowest edge of the film. Finally, the DNA fingerprint on the film is observed by UV. Strips, photo archived for analysis (step 30 or step 60).

Referring to Fig. 4, the electrophoresis results of the 2012 Taiwan rice recommended varieties were analyzed by the 12-plex SNP PCR of the present invention, 1 being TCS10, 2 being TK2, 3 being TK8, 4 being TK9, 5 being TK14, and 6 being TK16, 7 It is TNG71, 8 is KH139, 9 is KH145, 10 is TN11, 11 is TC192, 12 is HL21, 13 is TT30, 14 is Koshihikari, 15 is Tai Nong 67 (TNG67), 16 is water, and 17 is mixed. Taking TCS10 as an example, the position of 556 bp in the second introduction group is usually A. Therefore, the product cannot be amplified when PCR is performed. If 556 bp is G phenotype, the cultivar has a single nucleotide polymorphism in the allele ( SNP), an allele-specific primer will bind to this site, and the product will be amplified during the PCR reaction, and the electrophoresis band will be generated by electrophoresis. Tainong 67 was selected as the wild type standard design as the control variety without strip performance; 17 was the mixture of TN11, KH139 and KH142 varieties, which was used as the control group of the relative positions of the 12 groups of primers.

Example 5: Analysis of results of rice varieties

The polymorphisms of different SNP loci among 50 cultivars were compiled in Table 2 as a reference standard for cultivar identification of multiple single nucleotide polymorphic polymerase chain reaction reactions of 12 groups of primers (step 70):

The present invention provides a method for detecting single nucleotide polymorphism by using multiple single nucleotide polymorphic polymerase chain reaction (Multiplex SNP PCR) to identify domestic and foreign rice varieties, which can be simultaneously used in a single reaction. The detection of multiple sites is fast and cost-effective, and the use of capillary electrophoresis can be a time-saving tool for automated analysis.

At present, there is no method for effectively identifying different rice varieties in Taiwan and distinguishing between Taiwan rice varieties and foreign rice varieties. The invention can establish an accurate identification and discrimination method for rice varieties in China, as a basis for market management, and at the same time solve farmers and The controversy over the variety identification of rice seedlings to ensure the purity of the variety is conducive to the sustainable development of the rice industry. In addition, the commercially available small package white rice will move toward high quality and high unit price. The invention can also be used as a commodity certification to strengthen the confidence of Chinese people in domestic brands and improve the quality of rice in China.

10~70‧‧‧ Flowchart steps

Figure 1 is a flow chart of a method for identifying rice varieties at home and abroad according to the present invention.

Fig. 2 is a peak diagram of 12-plex SNP PCR using Qiaxcel capillary electrophoresis using the method for identifying domestic and foreign rice varieties of the present invention.

Fig. 3 is a peak diagram of Qiaxcel capillary electrophoresis of 12-plex SNP PCR analysis using different rice varieties (TK16, TCSW21 and KH142) by the method for identifying domestic and foreign rice varieties.

Figure 4 is a diagram showing the electrophoresis pattern of 12-plex SNP PCR by colloidal electrophoresis using the method for identifying domestic and foreign rice varieties; 1 is TCS10, 2 is TK2, 3 is TK8, 4 is TK9, and 5 is TK14, 6 TK16, 7 is TNG71, 8 is KH139, 9 is KH145, 10 is TN11, 11 is TC192, 12 is HL21, 13 is TT30, 14 is Koshihikari, 15 is TNG67, 16 is water, and 17 is mixed.

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Claims (20)

A method for identifying domestic and foreign rice varieties, the steps comprising: (1) using a primer set and amplifying a plurality of known varieties by multiple single nucleotide polymorphism polymerase chain reaction (Multiplex SNP PCR) The genomic DNA of rice comprises a fragment having a single nucleotide polymorphism (SNP) site in the nucleic acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 12 to obtain the known The length of the fragment of each PCR product obtained by increasing the amplitude of the rice by the primer group; (2) using a primer set and increasing the DNA of the rice to be measured by multiple single nucleotide polymorphic polymerase chain reaction a length of a fragment of each PCR product obtained by increasing the amplitude of the rice to be tested; and (3) comparing the lengths of the fragments of the PCR products obtained by increasing the amplitude of the rice to be tested, and step (1) the rice varieties of the known varieties The length of the fragment of each PCR product obtained by the amplification of the primer set is used to identify the rice variety to be tested, wherein the primer set is selected from the first primer set (SEQ ID NO: 13 and SEQ ID NO: 14), Two primer sets (SEQ ID NO: 15 and SEQ ID) NO: 16), third primer set (SEQ ID NO: 17 and SEQ ID NO: 18), fourth primer set (SEQ ID NO: 19 and SEQ ID NO: 20), fifth primer set (SEQ ID NO: 21 and SEQ ID NO: 22), sixth primer set (SEQ ID NO: 23 and SEQ ID NO: 24), seventh primer set (SEQ ID NO: 25 and SEQ ID NO: 26), eighth primer set ( SEQ ID NO:27 and SEQ ID NO:28), ninth primer set (SEQ ID NO:29 and SEQ ID NO:30), tenth primer set (SEQ ID NO:31 and SEQ ID NO:32), A population consisting of eleven primers (SEQ ID NO: 33 and SEQ ID NO: 34) and a twelfth primer set (SEQ ID NO: 35 and SEQ ID NO: 36). The method of claim 1, wherein the known variety is selected Free Taichung No. 2 (Taikeng 2, TK2), Taichung No. 4 (Taikeng 4, TK4), Taichung No. 5 (Taikeng 5, TK5), Taichung No. 8 (Taikeng 8, TK8), Taichung No. 9 ( Taikeng 9, TK9), Taikeng 14, TK14, Taikeng 16, TK16, Kaohsiung 139 (KH139) No. (Taoyuan 1, TY1), Taoyuan 3 (TY3), Tainan 11 (Tainan 11, TN11), Tai Nong 71 (Tainung 71, TNG71), Hualien 19 (Hualien 19, HL19), Hualien No. 20 (Hualien 20, HL20), Koshihikari, Taoyuan glutinous 2 (TYW2), Taichung sen 2 (TS2), Taichung sen 10 (TCS10) , Tainung sen 22 (TNGS22), Taichung Native 1, TN1, Jasmine 85, Kaohsiung 141 (KH141), Kaohsiung 142 (KH142), Kaohsiung 142 (KH142), Kaohsiung 143 (KH143), Kaohsiung 144 (KH144), Kaohsiung 146 (KH146), Kaohsiung 147 (KH147), Taoyuan 4 (TY4) Tainan 13, TN13, Tainan 14, TN14, Taitung 30 (TT30), Taitung 32 (TT32), Tainong 74 (TNG74) , Tainung 75 (TNG75), Tainong 84 (TNG84), Taichung 192 (Taichung 192, TC192), Taichung 194 (Taichung 194, TC194), Hualien 21 (Hualien 21, HL21), Miaoli 1, ML1, Taikeng glutinous 1, TKW1, Taikeng glutinous 3 (TKW3), Taikeng glutinous 12 (Taikeng glutinous 12, TKW12), Taitung glutinous 31 (TTW31), Tainung glutinous 73 (TNGW73), Taichung sen17 (TCS17), Tainong sen 14, TNGS14 ), Tai Nong No. 21 (Tainung sen glutinous 21, TNGSW21), Taichung sen glutinous 1, TCSW1 and Taichung sen glutinous 2 (TCSW2). The method of claim 1, wherein the concentration of the primer set is: the first primer set (SEQ ID NO: 13 and SEQ ID NO: 14) is 1.0 μM, and the second primer set (SEQ ID NO) :15 and SEQ ID NO: 16) are 1.0 μM, the third primer set (SEQ ID NO: 17 and SEQ ID NO: 18) is 1.6 μM, and the fourth primer set (SEQ ID NO: 19 and SEQ ID NO : 20) is 3.0 μM, the fifth primer set (SEQ ID NO: 21 and SEQ ID NO: 22) is 1.0 μM, and the sixth primer set (SEQ ID NO: 23 and SEQ ID NO: 24) is 2.0. μM, seventh primer set (SEQ ID NO: 25 and SEQ ID NO: 26) is 4.0 μM, and eighth primer set (SEQ ID NO: 27 and SEQ ID NO: 28) is 2.0 μM, ninth primer set (SEQ ID NO: 29 and SEQ ID NO: 30) is 4.0 μM, and the tenth primer set (SEQ ID NO: 31 and SEQ ID NO: 32) is 2.0 μM, and the eleventh introduction group (SEQ ID NO: 33 is SEQ ID NO: 34) is 2.0 μM and the twelfth primer set (SEQ ID NO: 35 and SEQ ID NO: 36) is 3.0 μM. The method of claim 1, wherein the reaction condition of the multiplex single nucleotide polymorphic polymerase chain reaction is first reacted at 94 ° C for 15 minutes, and then at 94 ° C, 30 seconds, 55 ° C, 90 seconds, 72 ° C, 90 seconds for a cycle, and repeated this cycle 29 times, the temperature was adjusted to 60 ° C reaction for 30 minutes. The method of claim 1, wherein the genomic DNA of the rice is derived from an endosperm of rice seeds or a plant after germination of rice seeds. A primer group capable of identifying domestic and foreign rice varieties is obtained by using a genomic DNA of a rice as a template and obtaining a fragment length having a single nucleotide polymorphism (SNP) site by a polymerase chain reaction. Wherein the primer set is selected from the group consisting of a first primer set (SEQ ID NO: 13 and SEQ ID NO: 14), a second primer set (SEQ ID NO: 15 and SEQ ID NO: 16), and a third primer set (SEQ ID NO: 17 and SEQ ID NO: 18), fourth primer set (SEQ ID NO: 19 and SEQ ID NO: 20), fifth primer set (SEQ ID NO: 21 and SEQ ID NO: 22), sixth Primers (SEQ ID NO: 23 and SEQ ID NO: 24), seventh primer set (SEQ ID NO: 25 and SEQ ID NO: 26), and eighth primer set (SEQ ID NO: 27 and SEQ ID NO: 28) ), a ninth primer set (SEQ ID NO: 29 and SEQ ID NO: 30), a tenth primer set (SEQ ID NO: 31 and SEQ ID NO: 32), and an eleventh primer set (SEQ ID NO: 33) SEQ ID NO: 34) and the population consisting of the twelfth primer set (SEQ ID NO: 35 and SEQ ID NO: 36). For example, the introduction group described in claim 6 of the patent scope, wherein the known variety of rice is selected from Taikeng 2 (TK2), Taikeng 4 (Tik4, TK4), and Taikeng No. 5 (Taikeng). 5, TK5), Taikeng No. 8 (Taikeng 8, TK8), Taichung No. 9 (Taikeng 9, TK9), Taichung No. 14 (Taikeng 14, TK14), Taichung No. 16 (Taikeng 16, TK16), Kaohsiung No. 139 (Kaohsiung 139, KH139), Kaohsiung 145 (Kaohsiung 145, KH145), Taoyuan No. 1 (Taoyuan 1, TY1), Taoyuan No. 3 (Taoyuan 3, TY3), Tainan No. 11 (Tainan 11, TN11), Taiwan No. 71 (Tainung 71, TNG71), Hualien 19 (Hualien 19, HL19), Hualien 20 (Hualien 20, HL20), Koshihikari, Taoyuan glutinous 2 (TYW2), Taiwan Ich2 (Taichung sen 2, TS2), Taichung sen 10 (TCS10), Tai Nong 22 (TNGS22), Taichung Re, Taichung No. 1 (Taichung Native 1, TN1), Thai Jasmine 85, Kaohsiung 141 (KH141), Kaohsiung 142 (KH142), Kaohsiung 143 (KH143), Kaohsiung 144 (Kaohsiung 144) , KH144), Kaohsiung 146 (KH146), Kaohsiung 147 (Kahsiung 147, KH147), Taoyuan 4 (TY4), Tainan 13 (Tainan 13, TN13), Tainan 14 (Tainan 14) , TN14), Taitung 30 (Taitung 30, TT30), Taitung 32 (Taitung 32, TT32), Tai Nong 74 (Tainung 74, TNG74), Tai Nong 75 (Tainung 75, TNG75), Tai Nong 84 (Tainung 84, TNG84), Taichung No. 192 (Taichung 192, TC192), Taichung No. 194 (Taichung 194, TC194), Hualien 21 (Hualien 21, HL21), Miaoli 1 (Miaoli 1, ML1), Taiwan No. 1, (Taikeng glutinous 1, TKW1), Taikeng glutinous 3 (TKW3), Taikeng glutinous 12 (TKW12), Taitung glutinous 31 (TTW31), Taiwan Tainung glutinous 73 (TNGW73), Taichung sen17 (TCS17), Tainung sen 14, TNGS14, Taiwan Tainung sen glutinous 21 (TNGSW21), Taichung sen glutinous 1, TCSW1, and Taichung sen glutinous 2 (TCSW2). The primer group described in claim 6 wherein the polymerase chain reaction reaction group uses the primer set to perform a polymerase chain reaction reaction or simultaneously uses the primer set to perform multiple single nucleotide polymorphic polymerase chain reaction. (Multiplex SNP PCR). The primer set described in claim 8 wherein a plurality of single nucleotide polymorphic polymerase chain reaction is performed, and the concentration of the primer set is: the first primer set (SEQ ID NO: 13 and SEQ ID NO) :14) is 1.0 μM, second primer set (SEQ ID NO: 15 and SEQ ID NO: 16) are 1.0 μM, the third primer set (SEQ ID NO: 17 and SEQ ID NO: 18) is 1.6 μM, and the fourth primer set (SEQ ID NO: 19 and SEQ ID NO: 20) is 3.0 μM, the fifth primer set (SEQ ID NO: 21 and SEQ ID NO: 22) is 1.0 μM, and the sixth primer set (SEQ ID NO: 23 and SEQ ID NO: 24) is 2.0 μM. The seventh primer set (SEQ ID NO: 25 and SEQ ID NO: 26) is 4.0 μM, and the eighth primer set (SEQ ID NO: 27 and SEQ ID NO: 28) is 2.0 μM, and the ninth primer set ( SEQ ID NO:29 and SEQ ID NO:30) are 4.0 μM, the tenth primer set (SEQ ID NO:31 and SEQ ID NO:32) is 2.0 μM, and the eleventh introduction group (SEQ ID NO:33) It is 2.0 μM with SEQ ID NO: 34) and 3.0 μM for the twelfth primer set (SEQ ID NO: 35 and SEQ ID NO: 36). The primer group described in claim 8 is characterized in that a multiple single nucleotide polymorphic polymerase chain reaction is carried out, and the reaction conditions are firstly reacted at 94 ° C for 15 minutes, and then at 94 ° C, 30 seconds, 55. °C, 90 seconds, 72 ° C, 90 seconds is a cycle, and after repeating this cycle 29 times, the temperature is adjusted to 60 ° C for 30 minutes. The primer set according to claim 6, wherein the genomic DNA of the rice is derived from an endosperm of rice seeds or a plant after germination of rice seeds. A method for identifying domestic and foreign rice varieties, the steps comprising: (1) analyzing genomic DNA of several known varieties of rice, and detecting 12 single nucleotide polymorphism genotypes by a polymerase chain reaction; Single nucleotide polymorphism in the nucleic acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 12; (2) analysis of genomic DNA of a rice to be tested, detection of 12 singles by a polymerase chain reaction a genotype of nucleotide polymorphisms in the nucleic acid sequence consisting of SEQ ID NOS: 1-12; (3) The 12 single nucleotide polymorphism genotypes obtained by the rice to be tested are compared with the 12 single nucleotide polymorphism genotypes of the known varieties of rice in step 1 to identify The rice to be tested, wherein the 23rd base of the nucleic acid sequence shown by SEQ ID NO: 1 is A/T genotype, and the 21st base of the nucleic acid sequence shown by SEQ ID NO: 2 is A/G gene The 483th base of the nucleic acid sequence shown by SEQ ID NO: 3 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 4 is the G/A genotype, SEQ ID NO: The 21st base of the nucleic acid sequence shown in Figure 5 is the C/T genotype, the 21st base of the nucleic acid sequence shown by SEQ ID NO: 6 is the G/A genotype, and the nucleic acid sequence shown by SEQ ID NO: 7. The 367th base is the G/A genotype, the 22nd base of the nucleic acid sequence shown by SEQ ID NO: 8 is the A/C genotype, and the 267th base of the nucleic acid sequence shown by SEQ ID NO: 9 is The G/A genotype, the 20th base of the nucleic acid sequence shown by SEQ ID NO: 10 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 11 is the A/G genotype, The 20th base of the nucleic acid sequence shown in SEQ ID NO: 12 is the T/C gene. . The method of claim 12, wherein the known variety of rice is selected from Taikeng 2 (TK2), Taikeng 4 (TK4), and Taichung 5 (Taikeng 5). ,TK5),Taikeng 8,TK8,Taikeng 9,TK9,Taikeng 14,TK14,Taikeng 16,TK16,Kaohsiung 139 No. (Kaohsiung 139, KH139), Kaohsiung 145 (Kaohsiung 145, KH145), Taoyuan No. 1 (Taoyuan 1, TY1), Taoyuan No. 3 (Taoyuan 3, TY3), Tainan No. 11 (Tainan 11, TN11), Tai Nong No. 71 (Tainung 71, TNG71), Hualien 19 (Hualien 19, HL19), Hualien 20 (Hualien 20, HL20), Koshihikari, Taoyuan glutinous 2 (TYW2), Taiwan No. 2 (Taichung sen 2, TS2), Taichung sen10 (T C S10), Tainung sen 22 (TNGS22), Taichung Native 1, TN1, Thailand Jasmine85, Kaohsiung 141 (KH141), Kaohsiung 142 (KH142), Kaohsiung 143 (KH143), Kaohsiung 144 (KH143), Kaohsiung 144 (KH144), Kaohsiung 146 (Kaohsiung 146, KH146), Kaohsiung 147 (KH147), Taoyuan 4 (TY4), Tainan 13 (Tainan 13, TN13), Tainan 14 (Tainan 14, TN14), Taitung 30 (Taitung 30, TT30), Taitung 32 (Taitung 32, TT32), Tai Nong 74 (Tainung 74, TNG74), Tai Nong 75 (Tainung 75, TNG75), Tai Nong 84 (Tainung 84, TNG84), Taichung 192 No. (Taichung 192, TC192), Taichung No. 194 (Taichung 194, TC194), Hualien 21 (Hualien 21, HL21), Miaoli 1 (Miaoli 1, ML1), Taichung No. 1, (Taikeng glutinous 1, TKW1 ), Taikeng glutinous 3 (TKW3), Taikeng glutinous 12 (TKW12), Taitung glutinous 31 (TTW31), Tainung glutinous 73 (Tainung glutinous 73, TNGW73), Taichung sen17 (T C S17), Tainung sen 14, TNGS14, Tainung sen glutinous 21 (TNGSW21), Taichung No. 1 (Taichung sen glutinous 21, TNGSW21) Taichung sen glutinous 1, TCSW1) and Taichung sen glutinous 2 (TCSW2). The method of claim 12, wherein the polymerase chain reaction is carried out using a primer pair complementary to the nucleic acid sequences of SEQ ID NO: 1 to SEQ ID NO: 12. The method of claim 14, wherein the primer pair is used to increase the nucleic acid fragments carrying the genotypes using the genomic DNA of the rice varieties as a template. The method according to claim 12, wherein the genomic DNA of rice is derived from the endosperm of rice seeds or the plants after germination of rice seeds. An isolated nucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12. The nucleotide according to claim 17, wherein the 23rd base of the nucleic acid sequence shown by SEQ ID NO: 1 is the A/T genotype, and the 21st of the nucleic acid sequence shown by SEQ ID NO: The base is the A/G genotype, the 483th base of the nucleic acid sequence shown by SEQ ID NO: 3 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: 4 is G/A. The 21st base of the nucleic acid sequence shown in SEQ ID NO: 5 is a C/T genotype, and the 21st base of the nucleic acid sequence shown by SEQ ID NO: 6 is a G/A genotype, SEQ ID NO The 367th base of the nucleic acid sequence shown in Figure 7 is the G/A genotype, the 22nd base of the nucleic acid sequence shown by SEQ ID NO: 8 is the A/C genotype, and the nucleic acid sequence shown in SEQ ID NO: 9. The 267th base is the G/A genotype, the 20th base of the nucleic acid sequence shown by SEQ ID NO: 10 is the A/G genotype, and the 20th base of the nucleic acid sequence shown by SEQ ID NO: The 20th base of the A/G genotype and the nucleic acid sequence shown by SEQ ID NO: 12 is the T/C genotype. The nucleotide according to claim 18, wherein the nucleotides are analyzed by comparing several known rice varieties and one rice to be tested, thereby identifying the rice varieties to be tested. The nucleotide according to claim 19, wherein the known variety of rice is selected from Taikeng 2 (TK2), Taikeng 4 (TK4), and Taichung No. 5 ( Taikeng 5, TK5), Taikeng 8, TK8, Taikeng 9, TK9, Taikeng 14, TK14, Taikeng 16, TK16, Kaohsiung 139 (KH139), Kaohsiung 145 (Kahsiung 145, KH145), Taoyuan 1 (Taoyuan 1, TY1), Taoyuan 3 (TY3), Tainan 11 (Tainan 11, TN11), Tainung 71 (TNG71), Hualien 19 (Hualien 19, HL19), Hualien 20 (Hualien 20, HL20), Koshihikari, and Taoyuan glutinous 2 (TYW2), Taichung sen 2 (TS2), Taichung sen10 (T C S10), Tainung sen 22 (TNGS22), Taichung Native 1, TN1 , Thailand Jasmine85, Kaohsiung 141 (KH141), Kaohsiung 142 (Kaohsiung 142, KH142), Kaohsiung 143 (Kaohsiung 143, KH143), Kaohsiung 144 (Kaohsiung 144, KH144), Kaohsiung 146 (Kaohsi Ung 146, KH146), Kaohsiung 147 (KH147), Taoyuan 4 (TY4), Tainan 13 (Tainan 13, TN13), Tainan 14 (Tainan 14, TN14), Taitung 30 ( Taitung 30, TT30), Taitung 32 (Taitung 32, TT32), Tai Nong 74 (Tainung 74, TNG74), Tai Nong 75 (Tainung 75, TNG75), Tai Nong No. 84 (Tainung 84, TNG84), Taichung No. 192 (Taichung 192, TC192), Taichung No. 194 (Taichung 194, TC194), Hualien 21 (Hualien 21, HL21), Miaoli 1, ML1, Taichung glutinous 1, TKW1), Taikeng glutinous 3 (TKW3), Taikeng glutinous 12 (TKW12), Taitung glutinous 31 (TTW31), Tainung glutinous 73 , TNGW73), Taichung sen17 (T C S17), Tainung sen 14, TNGS14, Tainung sen glutinous 21 (TNGSW21), Taichung 籼糯 1 (Taichung sen glutinous 1, TCSW1) and Taichung sen glutinous 2 (TCSW2).