Application of SNP (Single Nucleotide Polymorphism) molecular marker of rice grain cadmium content related gene YZCd3

Abstract

The invention relates to application of an SNP (Single Nucleotide Polymorphism) molecular marker of a rice grain cadmium content related gene YZCd3 in the field of nucleic acid detection. The method for identifying or assisting in identifying the cadmium content of the rice grains comprises the steps that the genotype of an SNP site in a rice genome to be detected is detected, the cadmium content of the rice grains is identified or assisting in identifying according to the genotype, the SNP site is an SNP site on a rice chromosome 12, the nucleotide type of the SNP site is G or C, and the SNP site is the 23rd nucleotide of a sequence 4 in a sequence table. The grain cadmium content of the to-be-detected rice with the genotype of CC at the SNP site is lower than that of the to-be-detected rice with the genotype of GG at the SNP site. The identification method disclosed by the invention can be applied to breeding of low-cadmium rice varieties.

Description

Technical Field

[0001] The present invention relates to the application of a SNP molecular marker of a rice grain cadmium content-related gene YZCd3 in the field of nucleic acid detection. Background Art

[0002] As one of the world's most important food crops, rice's yield and quality directly impact human food security. However, with the acceleration of industrialization and the widespread use of agricultural chemicals, heavy metal pollution in soils, particularly cadmium, is becoming increasingly serious. Cadmium is a toxic heavy metal that can enter the food chain through the soil-plant system and accumulate in the human body over long periods of time, leading to a variety of health problems such as kidney damage and osteoporosis. Rice has a strong ability to absorb and accumulate cadmium, especially in soils severely contaminated with cadmium. The problem of excessive cadmium content in rice grains is becoming increasingly prominent. Therefore, reducing the cadmium content in rice grains has become a major research topic in agricultural science.

[0003] In recent years, the rapid development of genomics and molecular biology technologies, particularly the application of genome-wide association studies (GWAS) and single nucleotide polymorphism (SNP) molecular markers, has provided new avenues for the identification and functional study of genes associated with cadmium content in rice. SNP molecular markers, due to their high density, high genetic stability, and ease of use, have become an important tool for gene mapping and molecular breeding. Using SNP markers, researchers can quickly and accurately screen rice varieties with low cadmium accumulation, thereby reducing cadmium accumulation in rice grains during the breeding process. This not only helps improve the food safety of rice but also promotes the development of environmentally friendly agriculture. Summary of the Invention

[0004] The problem to be solved by the present invention is how to identify or assist in identifying the cadmium content in rice grains.

[0005] In order to solve the above technical problems, the present invention first provides a method for identifying or assisting in identifying the cadmium content of rice grains, comprising detecting the genotype of the SNP site in the rice genome to be tested, and identifying or assisting in identifying the cadmium content of the rice grains according to the genotype, wherein the genotype is GG or CC, the GG is the homozygous type of the SNP site being G, and the CC is the homozygous type of the SNP site being C.

[0006] As an embodiment, the method for identifying or assisting in identifying the cadmium content in rice grains may include the following steps: (1) Using the genomic DNA of the rice to be tested as a template, a primer combination is used to perform KASP molecular marker detection; the primer combination consists of primer A, primer B and primer C; The primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 1 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-44 of sequence 1 in the sequence list; The primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-44 of sequence 2 in the sequence list; The primer C is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence list; (2) After completing step (1), performing fluorescence detection to determine the genotype of the SNP of the rice to be tested; (3) Identifying the cadmium content of the grains of the rice to be tested based on the genotype results: the cadmium content of the grains of the rice to be tested whose genotype at the SNP site is CC is lower than that of the rice to be tested whose genotype at the SNP site is GG.

[0007] The invention also provides a rice breeding method.

[0008] The rice breeding method provided by the present invention comprises detecting the genotype of the SNP site in the rice genome, and selecting rice with a genotype of CC at the SNP site as a parent for breeding, wherein CC is a homozygous type of the SNP site C.

[0009] As an implementation method, the rice breeding method may include the following steps: (1) Using the genomic DNA of the rice to be tested as a template, the above primer set was used for KASP molecular marker detection; (2) After completing step (1), performing fluorescence detection to determine the genotype of the SNP site of the rice to be tested; (3) Select CC genotype rice for rice breeding with low grain cadmium content.

[0010] In the above method, the primer dissolution and preparation method can be as follows: first, dilute the three primers to 100 mM with ddH2O, and then prepare the primer working solution as follows: 12 μL of primer A, 12 μL of primer B, 30 μL of primer C, and 46 μL of ddH2O, which is used as the KASP-labeled primer working solution and stored at -20°C until use.

[0011] In the above method, the KASP reaction system can be: DNA 0.8 μL, 2× Master mix 0.4 μL, primer working solution 0.022 μL, ddH2O 0.4 μL.

[0012] 2×Master mix was purchased from LGC Company, product number: 1536 Formulation V4.0 TF.

[0013] In the above method, the reaction procedure of KASP labeling can be: Step 1: pre-denaturation at 94°C for 15 min; Step 2: 94℃ 20s, 61℃ 60s, 94℃ 20s, 60.4℃ 60s, 94℃ 20s, 59.8℃ 60s, 94℃ 20s, 59.2℃ 60s, 94℃ 20s, 58.6℃ 60s, 94℃ 20s, 58℃ 60s, 94℃ 20s, 57.4℃ 60s, 94℃ 20s, 56.8℃ 60s, 94℃ 20s, 56.2℃ 60s, 94℃ 20s, 55.6℃ 60s; Step 3: denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, 5 cycles; if typing is not obvious, add 5 more cycles for expansion; Step 4: 94°C for 20 s, annealing at 57°C for 60 s, 26 cycles.

[0014] A KASP molecular marker primer set was designed targeting the upstream and downstream sequences of the superior allele variant site of the YZCd3 gene. The KASP marker primer set consists of two upstream specific primers (primer A and primer B) and one downstream universal primer (primer C).

[0015] The primer set for identifying the polymorphism at SNP site 12_18413344 includes specific primers A and B and universal primer C. The 5' end of primer A is labeled with 6-carboxyfluorescein (FAM); the 5' end of primer B is labeled with hexachloro-6-methylfluorescein (HEX). The single-stranded DNA molecules indicated by primers A and C amplify the fragment with SNP site A; the single-stranded DNA molecules indicated by primers B and C amplify the fragment with SNP site C. The single-stranded DNA molecules indicated by primers A and C amplify the fragment with SNP site C; the single-stranded DNA molecules indicated by primers B and C amplify the fragment with SNP site T. The fluorescent signal from the fluorophore bound to the FAM or HEX sequence in the template can be read using a microplate reader or fluorescence quantitative PCR instrument. The SNP-specific primer sequences were synthesized by Zhongyu Gold Labeling (Beijing) Biotechnology Co., Ltd.

[0016] In the above method, the genotype of the SNP in the rice to be tested can be determined by: after the PCR reaction is completed, the fluorescence signal is converted into an analyzable value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) to read the fluorescence data of the reaction product. Genotyping is performed using end-to-end fluorescence readings. The fluorescence scanning results are graphically displayed using the R software package, with T base types emitting FAM fluorescence distributed near the x-axis; C base types emitting HEX fluorescence distributed near the y-axis; and samples without a detectable signal distributed near the origin.

[0017] The application of the above method in rice breeding also falls within the protection scope of the present invention.

[0018] The present invention also provides the use of a substance for detecting the polymorphism or genotype of KASP in the rice genome in any of the following: (1) Identify or assist in identifying the cadmium content in rice grains; (2) Rice breeding; (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains; (4) Preparation of rice breeding products; The SNP site is a site on rice chromosome 12, and its nucleotide type is G or C. It is the 23rd nucleotide of sequence 4 in the sequence list. The nucleotide sequence of sequence 4 is as follows: 5'-GGGAAGGAGGGAGTAGTAGATTsTGTGTAGGTGTAGCCACCCCATGCTATGACGTTTGCATGGTCAGC-3', where s represents c or g.

[0019] The genome sequence of the common rice variety Chinese Spring is used as the reference genome, and the SNP site is at 18413344bp on rice chromosome 12 (specifically, position 23 of sequence 4 in the sequence list).

[0020] The present invention also provides a product for detecting the polymorphism or genotype of SNP sites in the rice genome.

[0021] The product for detecting the polymorphism or genotype of the SNP site in the rice genome provided by the present invention contains the above-mentioned substance for detecting the polymorphism or genotype of the SNP site in the rice genome, and the product is any one of: C1) Products for detecting single nucleotide polymorphisms or genotypes related to cadmium content in rice grains; C2) Products for identifying or assisting in identifying the cadmium content in rice grains; C3) Products used in rice breeding.

[0022] In the above applications, methods, and products, the substance may be a reagent and / or instrument required for determining the polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chip. Among them, SNP chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on "one-step" reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule DNA binding reactions.

[0023] Optionally, the substance is the following D1), D2) or D3): D1) the substance is a primer composition for amplifying a rice genomic DNA fragment including the SNP site; D2) the substance is a PCR reagent containing the primer combination described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0024] Optionally, the amplification may be PCR amplification. The primer composition consists of the primer A, the primer B and the primer C.

[0025] D3) The kit may further comprise KASP Master Mix.

[0026] In the above-mentioned uses, methods, and products, the primer composition may or may not be labeled with a label. A label refers to any atom or molecule that can be used to provide a detectable effect and that can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radioactive labels such as 32P; binding moieties such as biotin; haptens such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes, alone or in combination with moieties that can inhibit or shift the emission spectrum via fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. The label can be a charged moiety (positive or negative) or, alternatively, charge-neutral. The label can comprise a nucleic acid or protein sequence, or a combination thereof, as long as the sequence comprising the label is detectable. In some embodiments, the nucleic acid is directly detected (e.g., by direct sequence read) without a label. For example, the primer composition may be a primer composition composed of a single-stranded DNA having a nucleotide sequence of positions 22-44 of Sequence 1 in the sequence listing, a single-stranded DNA having a nucleotide sequence of positions 22-44 of Sequence 2 in the sequence listing, and a single-stranded DNA having a nucleotide sequence of positions 3 in the sequence listing. The primer composition may also be a primer set consisting of the single-stranded DNA shown in Sequence 1 in the sequence listing, the single-stranded DNA shown in Sequence 2 in the sequence listing, and the single-stranded DNA shown in Sequence 3 in the sequence listing. Sequence 1 in the sequence listing consists of 44 nucleotides, nucleotides 1-21 of which are a FAM linker sequence (serving as a marker), and nucleotides 22-44 of which are a specific sequence; Sequence 2 in the sequence listing consists of 44 nucleotides, nucleotides 1-21 of which are a HEX linker sequence (serving as a marker), and nucleotides 22-44 of which are a specific sequence.

[0027] The present invention also provides a DNA molecule, the nucleotide sequence of which is shown as Sequence 1 in the sequence listing.

[0028] The application of the above-mentioned DNA molecules also falls within the scope of protection of the present invention. The application is specifically any of the following: (1) Identify or assist in identifying the cadmium content in rice grains; (2) Rice breeding; (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains; (4) Preparation of rice breeding products.

[0029] Optionally, in the above application, the DNA molecule serves as a detection target.

[0030] In the above, the rice may be an inbred line.

[0031] In the above, the rice breeding index includes the cadmium content in rice grains.

[0032] Furthermore, the purpose of rice breeding includes cultivating rice with low cadmium content in grains (the cadmium content in grains is lower than that of the parents).

[0033] The substances for detecting the polymorphism and genotype of the SNP site can be combined with other substances (such as substances for detecting single nucleotide polymorphism or genotype of other molecular markers related to the cadmium content in rice grains) to prepare products for identifying rice varieties with low cadmium content in grains.

[0034] This study provides an efficient and accurate method for screening rice varieties with low cadmium accumulation by developing SNP molecular markers based on the YZCd3 gene, which is of great significance for improving rice food safety and promoting the development of environmentally friendly agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Genotyping of 60 rice germplasm accessions using the YZCd3 molecular marker. C:C indicates the CC genotype, G:G indicates the GG genotype, and NTC indicates the control. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0037] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0038] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0039] The 379 experimental materials in the following embodiments have been recorded in: Huili Yan, Wenxiu Xu, JianyinXie, Yiwei Gao, Lulu Wu, Liang Sun, Lu Feng, Xu Chen, Tian Zhang, ChanghuaDai, Ting Li, Xiuni Lin, Zhanying Zhang, Xueqiang Wang, Fengmei Li, XiaoyangZhu, Jinjie Li, Zichao Li, Caiyan Chen, Mi Ma, Hongliang Zhang* & ZhenyanHe*. Variation of a major facilitator superfamily gene contributes todifferential cadmium accumulation between rice subspecies. NatureCommunications. 2019. 10: 2562. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0040] In the following examples, the experimental field is a typical cadmium-contaminated rice field in my country, with a soil cadmium content of 0.41 mg / kg and a pH of 5.3.

[0041] In the following examples, 379 experimental materials and 60 verification materials were planted in 2021. The specific operating steps are as follows: sowing is carried out when the soaked seed buds are 5 mm long, and the germinated seeds are sown in the field until they grow into seedlings. Two weeks later, transplanting is carried out with a row spacing of 25 cm; 8 plants are planted in each row with a plant spacing of 20 cm. After the rice is mature, grain samples are collected. In order to avoid the boundary effect, the two plants on the left and right adjacent to the aisle are discarded, and the grains of the remaining plants are mixed. The recovered rice grain samples are dried in the sun or placed in an oven at 60°C for 3 days. After the mass is constant, the rice is husked using a rice huller, and the obtained brown rice samples are placed in 5 ml centrifuge tubes. The brown rice samples are then crushed using a high-throughput silent tissue grinder for subsequent cadmium content determination.

[0042] In the following examples, the cadmium content of rice grains was determined by a single acid digestion method using a far-infrared temperature-controlled digestion furnace and a glass digestion tube. The specific steps are briefly described as follows: (1) Sample weighing: Accurately weigh 0.2000 g (accurate to 0.0001 g) of crushed rice grain sample and place it in a glass digestion tube to prevent the powder from sticking to the wall.

[0043] (2) Acidification: Add 1 ml of high-grade pure nitric acid and digest overnight. (3) Digestion: Cover with a bent-neck funnel and digest at 200°C for 6 h until the digestion solution is colorless and transparent or slightly yellow.

[0044] (3) Adjust the volume: Wash the digestion solution in the tube with distilled water, transfer the washing solution to a 15 ml constant volume tube, and adjust the volume to 15 ml.

[0045] (4) Filtration: After shaking, filter the quantified liquid using a 0.45 μm water filter membrane into a 10 ml centrifuge tube for testing. Quality Control: Two blank controls and three rice flour component analysis standard substances (National Standard Material, GBW100349, Steel Research Institute NAK Testing Technology Co., Ltd.) were set up during each batch digestion to ensure the accuracy and reliability of the cadmium content data of rice grains. All sample measurements were repeated three times. The cadmium content of rice grains was determined using an inductively coupled plasma mass spectrometer. The results are shown in Table 1.

[0046] The following example analyzes the YZCd3 (12_18413344) genotype of 379 rice MCC population materials using t The statistical differences in grain cadmium content between different combined haplotypes were compared using the -test. P values were calculated using the Tukey's test. A P value less than 0.05 was defined as a statistically significant difference; a P value less than 0.01 was defined as a highly significant difference.

[0047] Example 1: Discovery of SNPs Related to Cadmium Content in Rice Grains Using phenotypic and genotypic data on total cadmium content in grains from 379 rice accessions (MCC populations), genome-wide association analysis pinpointed a single-nucleotide polymorphism (SNP) site, Chr12_18413344, associated with total cadmium content in rice grains. This SNP, designated YZCd3, is located at position 18413344 on chromosome 12 in the Nipponbare genome sequence, and its nucleotides are either C or G. This SNP has two genotypes: CC and GG. Genotype CC represents the homozygous C SNP, while genotype GG represents the homozygous G SNP.

[0048] Significant differences in grain cadmium content were analyzed for different alleles of the screened SNPs. The results are as follows: Sequencing results for 379 accessions are shown in Table 1. 237 accessions had the low-cadmium genotype CC, with an average grain cadmium content of 0.398 mg / kg; 142 accessions had the high-cadmium genotype GG, with an average grain cadmium content of 0.498 mg / kg. Statistical results showed that the grain cadmium content of rice accessions corresponding to GG was significantly higher than that of rice accessions corresponding to CC (P < 0.01). The SNP YZCd3 can be used as a molecular marker for identifying or assisting in the identification of grain cadmium content in different rice lines (Table 2).

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[0066] Example 2: Development of a primer set for identifying YZCd3 molecular markers and establishment of an identification method 1. Design of KASP molecular marker primer set A KASP molecular marker primer set was designed targeting the upstream and downstream sequences of the superior allele variant site 12_18413344. The KASP marker primer set consists of two upstream specific primers (primer A and primer B) and one downstream universal primer (primer C) (Table 3).

[0067]

[0068] Note: The underline indicates the fluorescent tag sequence. The 5' end of primer A is linked to a 6-carboxyfluorescein (FAM) fluorescent tag, and the 5' end of primer B is linked to a hexachloro-6-methylfluorescein (HEX) fluorescent tag.

[0069] The single-stranded DNA molecules indicated by primers A and C amplify fragments containing the nucleotide G at the SNP site, and the fluorescent signal of the FAM group can be read using a microplate reader or a fluorescence quantitative PCR instrument. The single-stranded DNA molecules indicated by primers B and C amplify fragments containing the nucleotide C at the SNP site, and the fluorescent signal of the HEX group can be read using a microplate reader or a fluorescence quantitative PCR instrument. Specific primer sequences were synthesized by Zhongyujin Labeling (Beijing) Biotechnology Co., Ltd.

[0070] 2. KASP-labeled PCR amplification system and procedure Rice genomic DNA was extracted using the CTAB method and dissolved in 400 μL of TE. DNA quality was assessed by electrophoresis on a 1% agarose gel. The extracted DNA was required to be free of obvious impurities, exhibit clear bands, and be free of degradation. After DNA concentration was measured, the DNA was uniformly diluted to 28.3 ng / μL. PCR amplification was performed using the diluted rice genomic DNA as a template.

[0071] Preparation of KASP-labeled primer working solution: Dilute each of the three primers to 100 mM in ddH2O. Then prepare the primer working solution as follows: 12 μL of primer A, 12 μL of primer B, 30 μL of primer C, and 46 μL of ddH2O. Store at -20°C until needed.

[0072] The PCR amplification system was as follows: DNA 0.8 μL, 2× Master mix 0.4 μL, primer working solution 0.022 μL, and ddH2O 0.4 μL.

[0073] The PCR reaction procedure was as follows: Step 1: pre-denaturation at 94°C for 15 min; Step 2: 94℃ 20s, 61℃ 60s, 94℃ 20s, 60.4℃ 60s, 94℃ 20s, 59.8℃ 60s, 94℃ 20s, 59.2℃ 60s, 94℃ 20s, 58.6℃ 60s, 94℃ 20s, 58℃ 60s, 94℃ 20s, 57.4℃ 60s, 94℃ 20s, 56.8℃ 60s, 94℃ 20s, 56.2℃ 60s, 94℃ 20s, 55.6℃ 60s; Step 3: denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, 5 cycles; if typing is not obvious, add 5 more cycles for expansion; Step 4: 94°C for 20 s, annealing at 57°C for 60 s, 26 cycles.

[0074] A blank control (NTC) in which no template DNA was added to the reaction system was also set up in the experiment, with one control set for each plate.

[0075] 3. Genotyping After the PCR reaction is completed, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) are used to convert the fluorescence signal into analyzable values and read the fluorescence data of the reaction products. Fluorescence scanning results are displayed graphically using the R software package. G base types with FAM fluorescence are distributed near the x-axis; C base types with HEX fluorescence are distributed near the y-axis; samples with no detectable signal are distributed near the origin.

[0076] The excitation wavelength for FAM is 485 nm, and the emission wavelength is 520 nm. The excitation wavelength for HEX is 535 nm, and the emission wavelength is 556 nm. The excitation wavelength for the system reference fluorescence, ROX, is 575 nm, and the emission wavelength is 610 nm.

[0077] Example 3: Application of YZCd3 molecular marker in identifying cadmium content in rice Rice to be tested: 60 different rice germplasm resources, detailed information is shown in Table 4.

[0078] The rice to be tested was planted in a typical cadmium-contaminated paddy field, where the soil cadmium content was 0.41 mg / kg and the pH was 5.3. After the rice matured, grain samples were collected and tested for cadmium content. The results are shown in Table 4.

[0079] The genomic DNA of each test material was extracted, and the genotype of the test rice was detected using the above-mentioned YZCd3 molecular marker. The results are shown in Table 4. Figure 1In the figure, GG indicates that the genotype of the SNP site Chr12_18413344 of the rice material is GG, CC indicates that the genotype of the SNP site Chr12_18413344 of the rice material is CC, and NTC is a blank control in which no template DNA is added to the reaction system.

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[0083] The results showed that the genotype of 30 materials was CC and the genotype of 30 materials was GG. The detection results of YZCd3 marker were consistent with the sequencing results, indicating that KASP markers can accurately identify the genotype of the gene YZCd3 related to grain cadmium content in different rice germplasm resources (Table 4 and Figure 1 ).

[0084] Statistical results (Table 5) show that among the 60 rice germplasm resources, 30 materials with the CC genotype had an average grain cadmium content of 0.13 mg / kg, while 30 materials with the GG genotype had an average grain cadmium content of 0.25 mg / kg (Table 4). A significance analysis of the 60 verified accessions showed that the grain cadmium content of rice materials corresponding to the GG genotype was extremely significantly higher than that of rice materials corresponding to the CC genotype (P < 0.001).

[0085] In summary, the SNP marker YZCd3 developed in the present invention can accurately identify the genotype of the gene YZCd3 related to the cadmium content in rice germplasm resources, and can be applied to rice breeding, such as breeding low-cadmium rice varieties.

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[0087] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for identifying or assisting in identifying the cadmium content in rice grains, characterized in that: The method includes detecting the genotype of a SNP site in the rice genome to be tested, and identifying or assisting in identifying the cadmium content of rice grains based on the genotype. The SNP site is a SNP site on rice chromosome 12, the nucleotide type of which is G or C, and is the 23rd nucleotide of sequence 4 in the sequence table.

2. The method according to claim 1, wherein: The genotype of the SNP site is GG or CC, the CC is the homozygous type of the SNP site is C, and the GG is the homozygous type of the SNP site is G; the cadmium content of the grains of the tested rice whose genotype of the SNP site is CC is lower than that of the tested rice whose genotype of the SNP site is GG.

3. A rice breeding method, characterized in that: The method comprises detecting the genotype of the SNP site in claim 1 in the rice genome, and selecting rice with a genotype of CC at the SNP site as a parent for breeding, wherein CC is a homozygous type of C at the SNP site.

4. Use of a substance for detecting polymorphism or genotype of a SNP site in a rice genome in any of the following: (1) Identify or assist in identifying the cadmium content in rice grains; (2) Rice breeding; (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains; (4) Preparation of rice breeding products; The SNP site is a SNP site on rice chromosome 12, the nucleotide type of which is G or C, and is the 23rd nucleotide of sequence 4 in the sequence list.

5. The use according to claim 4, characterized in that: The genotype of the SNP site is GG or CC, the CC is the homozygous type of the SNP site is C, and the GG is the homozygous type of the SNP site is G; the cadmium content of the grains of the tested rice whose genotype of the SNP site is CC is lower than that of the tested rice whose genotype of the SNP site is GG.

6. The use according to claim 4 or 5, characterized in that: The substance is as follows D1), D2) or D3): D1) the substance is a primer composition for amplifying a rice genomic DNA fragment including the SNP site; D2) the substance is a PCR reagent containing the primer combination described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

7. The use according to claim 6, characterized in that: The primer composition consists of primer A, primer B and primer C; The primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 1 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-44 of sequence 1 in the sequence list; The primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-44 of sequence 2 in the sequence list; The nucleotide sequence of primer C is a single-stranded DNA molecule of sequence 3 in the sequence table.

8. The primer composition according to claim 7.

9. A DNA molecule characterized by: The nucleotide sequence of the DNA molecule is Sequence 4 in the sequence listing.

10. Use of the DNA molecule according to claim 9 in any of the following: (1) Identify or assist in identifying the cadmium content in rice grains; (2) Rice breeding; (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains; (4) Preparation of rice breeding products.

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