Application of SNP (Single Nucleotide Polymorphism) molecular marker of rice grain cadmium content related gene HuNCd1

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) molecular marker of a rice grain cadmium content related gene HuNCd1. The invention belongs to the technical field of biology, and particularly relates to application of an SNP (Single Nucleotide Polymorphism) molecular marker of a rice grain cadmium content related gene HuNCd1. 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 7, the nucleotide type of the SNP site is A or G, and the SNP site is the 25th nucleotide of a sequence 4 in a sequence table. The substance for detecting the polymorphism and genotype of the SNP site can be combined with other substances (such as substances for detecting the single nucleotide polymorphism or genotype of other molecular markers related to the cadmium content of the rice grains) to prepare a product for identifying the rice variety with low cadmium content of the rice grains.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of a SNP molecular marker of a rice grain cadmium content-related gene HuNCd1. Background Art

[0002] In recent years, with the acceleration of industrialization and urbanization, soil cadmium contamination has become increasingly prominent, leading to frequent excessive cadmium levels in grains of crops like rice, posing a serious threat to food security and human health. Rice, a major staple crop in my country, exhibits significant genotypic variation in its ability to accumulate cadmium in its grains. Molecular breeding has become an effective approach to ensuring safe rice production and improving the quality of agricultural products.

[0003] The HuNCd1 gene is a key functional gene regulating cadmium content in rice grains. The development and application of its SNP molecular markers provides key technical support for molecular-assisted selection of low-cadmium accumulation traits in rice. Therefore, in-depth research on the HuNCd1 gene and its application of SNP molecular markers has important theoretical and practical significance for accelerating the breeding of low-cadmium rice varieties, promoting the development of green agriculture, and protecting public health. 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] To address the above technical problems, the present invention first provides a method for identifying or assisting in identifying the cadmium content in rice grains, comprising detecting the genotype of a SNP site in the rice genome to be tested, and identifying or assisting in identifying the cadmium content in the rice grains based on the genotype. The SNP site is a SNP site on rice chromosome 7, whose nucleotide type is A or G, and is the 25th nucleotide of sequence 4 in the sequence listing. The genotype is AA or GG, wherein AA is the homozygous type when the SNP site is A, and GG is the homozygous type when the SNP site is G.

[0006] As an embodiment, the method for identifying or assisting in identifying the cadmium content in rice grains may include the following steps:

[0007] (1) Using genomic DNA of 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;

[0008] 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;

[0009] 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 22-46 of sequence 2 in the sequence list;

[0010] The primer C is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence list;

[0011] (2) After completing step (1), performing fluorescence detection to determine the genotype of the SNP of the rice to be tested;

[0012] (3) Identifying the cadmium content of the rice grains to be tested based on the genotype results: the cadmium content of the rice grains to be tested whose genotype at the SNP site is AA is lower than that of the rice grains to be tested whose genotype at the SNP site is GG.

[0013] The invention also provides a rice breeding method.

[0014] 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 the genotype of the SNP site being AA as a parent for breeding, wherein AA is the homozygous type of the SNP site being A.

[0015] As an implementation method, the rice breeding method may include the following steps:

[0016] (1) Using the genomic DNA of the rice to be tested as a template, the above primer set was used to perform KASP molecular marker detection;

[0017] (2) After completing step (1), performing fluorescence detection to determine the genotype of the SNP site of the rice to be tested;

[0018] (3) Select AA genotype rice for rice breeding with low cadmium content in grains.

[0019] In the above method, the primer dissolution and preparation method can be as follows: first, dilute the three primers separately with ddH2O to 100mM, and then prepare the primer working solution as follows: primer A 12μL, primer B 12μL, primer C 30μL, ddH2O 46μL, as the KASP-labeled primer working solution, and store at -20°C until use.

[0020] 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.

[0021] 2×Master mix was purchased from LGC Company, product number: 1536Formulation V4.0TF.

[0022] In the above method, KASP labeling can be performed on a common PCR amplification instrument.

[0023] In the above method, the reaction procedure of KASP labeling can be:

[0024] Step 1: pre-denaturation at 94°C for 15 min;

[0025] 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;

[0026] 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;

[0027] Step 4: 94°C for 20s, annealing at 57°C for 60s, 26 cycles.

[0028] In the above method, the genotype of the SNP in the rice plant 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 terminal fluorescence readings. The fluorescence scanning results are graphically displayed using the R software package, with G base types showing FAM fluorescence near the x-axis; A base types showing HEX fluorescence near the y-axis; and samples without a detectable signal are distributed near the origin.

[0029] The application of the above method in rice breeding also falls within the protection scope of the present invention.

[0030] 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:

[0031] (1) Identify or assist in identifying the cadmium content in rice grains;

[0032] (2) Rice breeding;

[0033] (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains;

[0034] (4) preparing rice breeding products;

[0035] The SNP site is a SNP site on rice chromosome 7, the nucleotide type of which is A or G, and is the 25th nucleotide of sequence 4 in the sequence list.

[0036] The genome sequence of the common rice variety Nipponbare is used as the reference genome, and the SNP site is at 8874894bp on rice chromosome 7 (specifically, position 25 of sequence 4 in the sequence list).

[0037] The present invention also provides a product for detecting the polymorphism or genotype of SNP sites in the rice genome.

[0038] 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:

[0039] C1) Products for detecting single nucleotide polymorphisms or genotypes related to cadmium content in rice grains;

[0040] C2) Products for identifying or assisting in identifying the cadmium content in rice grains;

[0041] C3) Products used in rice breeding.

[0042] 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.

[0043] Optionally, the substance is the following D1), D2) or D3):

[0044] D1) the substance is a primer composition for amplifying a rice genomic DNA fragment including the SNP site;

[0045] D2) the substance is a PCR reagent containing the primer combination described in D1);

[0046] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0047] Optionally, the amplification may be PCR amplification. The primer composition consists of the primer A, the primer B and the primer C.

[0048] D3) The kit may further comprise KASP Master Mix.

[0049] In the above-mentioned applications, methods and products, the primer composition may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Markers 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 by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or alternatively, it can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence comprising the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a marker.

[0050] 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 22-46 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 a single-stranded DNA shown in Sequence 1 in the sequence listing, a single-stranded DNA shown in Sequence 2 in the sequence listing, and a single-stranded DNA shown in Sequence 3 in the sequence listing. Sequence 1 in the sequence listing consists of 44 nucleotides, nucleotides 1-21 are a FAM linker sequence (as a marker), and nucleotides 22-44 are a specific sequence; Sequence 2 in the sequence listing consists of 46 nucleotides, nucleotides 1-21 are a HEX linker sequence (as a marker), and nucleotides 22-46 are a specific sequence.

[0051] The present invention also provides a DNA molecule, the nucleotide sequence of which is shown as Sequence 1 in the sequence listing.

[0052] 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:

[0053] (1) Identify or assist in identifying the cadmium content in rice grains;

[0054] (2) Rice breeding;

[0055] (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains;

[0056] (4) Prepare rice breeding products.

[0057] Optionally, in the above application, the DNA molecule serves as a detection target.

[0058] 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 a product for identifying rice varieties with low cadmium content in rice grains.

[0059] Herein, the breeding objective may include rice with low cadmium content in grains. The rice may be a pure line or an inbred line.

[0060] The SNP molecular markers provided by the present invention are closely related to the cadmium content in rice grains and can efficiently and accurately identify the cadmium accumulation characteristics of rice varieties. The method is simple to operate, suitable for large-scale sample testing, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Genotyping of 60 rice germplasm materials using the HuNCd1 molecular marker. DETAILED DESCRIPTION

[0062] 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.

[0063] 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.

[0064] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0065] The rice germplasm resources in the following embodiments have been recorded in: Huili Yan, et al. Variation of amajor facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies. Nature Communications. 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.

[0066] The rice experimental field of the present invention 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.

[0067] The 471 experimental materials and 60 validation materials (Table 1) in the following examples were planted in 2021. Starting with sowing, the specific operation steps were as follows: sowing was performed when the soaked seed buds were 5 mm long. The germinated seeds were sown in the field until they grew into seedlings. Two weeks later, rice seedlings were transplanted, with 25 cm spacing between rows; 8 plants were planted per row, with 20 cm spacing between plants. After the rice matured, grain samples were collected. To avoid boundary effects, the two plants adjacent to the aisle were discarded, and the grains of the remaining plants were mixed.

[0068] The method for determining cadmium content in rice grains is as follows:

[0069] The collected rice grain samples were dried in the sun or in an oven at 60°C for 3 days. After reaching a constant mass, they were hulled using a rice husker. The resulting brown rice samples were placed in 5ml centrifuge tubes. The brown rice samples were then ground using a high-throughput silent tissue grinder for subsequent cadmium content determination.

[0070] The cadmium content in rice grains was determined by the single acid digestion method. The instrument used was a far-infrared temperature-controlled digestion furnace and the container was a glass digestion tube. The specific steps are briefly described as follows: (1) Weighing: Accurately weigh 0.2000g (accurate to 0.0001g) of crushed rice grain sample and place it in a glass digestion tube to prevent the powder from sticking to the wall. (2) Adding acid: Add 1ml of high-grade pure nitric acid and cold digest overnight. (3) Digestion: Cover with a bent-neck funnel and digest at 200℃ for 6h until the digestion liquid is colorless and transparent or slightly yellow. (4) Volume adjustment: Wash the digestion liquid in the tube with distilled water, transfer the washing liquid to a 15ml volumetric tube, and adjust the volume to 15ml. (5) Filtration: After shaking, filter the liquid after volume adjustment using a 0.45μm water filter membrane into a 10ml centrifuge tube for testing. Quality Control: Two blank controls and three rice flour component analysis standards (National Standard Material, GBW100349, Steel Research Institute NAK Testing Technology Co., Ltd.) were used during each digestion to ensure the accuracy and reliability of rice grain cadmium content data. All samples were assayed in triplicate. Cadmium content in rice grains was determined using inductively coupled plasma mass spectrometry.

[0071] Example 1. HuNCd1 genotype analysis and molecular marker development

[0072] In the applicant's previous work, the applicant used the phenotypic data of total cadmium content in grains of rice MCC population materials and its genotype data (the phenotypic data of total cadmium content in grains came from the determination of total cadmium content in rice grains harvested in the field in 2021; the genotype data came from the Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies.) Through whole-genome association analysis, a SNP site Chr7_8874894 related to the total cadmium content in rice grains was located and named HuNCd1.

[0073] The SNP site is located at position 8874894 on chromosome 7 of the Nipponbare genome sequence information of rice, and its nucleotide is A or G (the SNP site is the 25th nucleotide of sequence 4 in the sequence table, and in sequence 4, r represents A or G). The SNP site has two genotypes, AA and GG. Genotype AA is the homozygous type of SNP A, and genotype GG is the homozygous type of SNP G. The HuNCd1 (7_8874894) genotype of 471 rice MCC population materials was analyzed, and the t test was used to compare the statistical differences in cadmium content in grains of different genotypes. The P value was calculated by Tukey's test. A P value of less than 0.05 was defined as a statistically significant difference; a P value of less than 0.01 was defined as a statistically extremely significant difference.

[0074] The results are as follows: Based on the sequencing results of 471 rice germplasm resources, 327 accessions had the low-cadmium genotype AA, with an average grain cadmium content of 0.385 mg / kg; 144 accessions had the high-cadmium genotype GG, with an average grain cadmium content of 0.598 mg / kg. The results showed that the grain cadmium content of rice accessions corresponding to AA was significantly lower than that of rice accessions corresponding to GG (P < 0.01). This SNP locus can be used as a molecular marker to identify or assist in the identification of cadmium content in the grains of different rice lines.

[0075] Table 1. Cadmium content in rice grains of 471 rice varieties and HuNCd1 genotype data

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] Table 2. Cadmium content in grains of different genotype rice germplasm materials

[0093]

[0094] 2. HuNCd1 genotype analysis and molecular marker development

[0095] A KASP molecular marker primer set was designed based on the upstream and downstream sequences of Chr7_8874894 (HuNCd1), the superior allelic variant site obtained in Step 1. The KASP marker primer set consists of two upstream specific primers (primer A and primer B) and one downstream universal primer (primer C) (see Table 3 for specific sequences). The 5' end of the designed primer FA was labeled with 6-carboxyfluorescein (FAM); the 5' end of the designed primer FB was labeled with hexachloro-6-methylfluorescein (HEX) dye.

[0096] The single-stranded DNA molecules represented by primers A and C amplify the fragment where the SNP site is G; the single-stranded DNA molecules represented by primers B and C amplify the fragment where the SNP site is A. The fluorescent signal from the fluorescent group bound to the FAM sequence or HEX sequence in the template can be read by a microplate reader or fluorescence quantitative PCR instrument. The specific primer sequences were synthesized by Zhongyujin Labeling (Beijing) Biotechnology Co., Ltd.

[0097] Table 3. Primer sequences for HuNCd1 molecular marker KASP

[0098]

[0099] 3. Establishment of a KASP-labeled primer set for detecting the genotype of the SNP marker Chr7_8874894 (HuNCd1)

[0100] The KASP marker primer set in Table 3 was used to detect different allele types at position 8874894 on chromosome 7 (SNP site Chr7_8874894) of the rice genome sequence information.

[0101] 1) PCR amplification system and procedure

[0102] Genomic DNA from common rice leaves was extracted using the CTAB method and dissolved in 400 μL of TE. DNA quality was checked by electrophoresis on a 1% agarose gel. PCR amplification was performed using the diluted rice genomic DNA as a template.

[0103] 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.

[0104] 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.

[0105] 2×Master mix was purchased from LGC Company, product number: 1536Formulation V4.0TF.

[0106] The PCR reaction procedure was as follows: Step 1: pre-denaturation at 94°C for 15 min;

[0107] 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;

[0108] 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;

[0109] Step 4: 94°C for 20s, annealing at 57°C for 60s, 26 cycles.

[0110] 2) Genotyping

[0111] After the PCR reaction is complete, 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 graphically displayed using the R software package. G base types with FAM fluorescence are distributed near the x-axis; A base types with HEX fluorescence are distributed near the y-axis; samples with no detectable signal are distributed near the origin.

[0112] 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.

[0113] The results are as follows:

[0114] If only the fluorescent signal of the FAM group is displayed, the genotype of the SNP site HuNCd1 of the rice to be tested is GG (i.e., the SNP site HuNCd1 in the rice genome is homozygous for G);

[0115] If only the fluorescence signal of the HEX group is displayed, the HuNCd1 genotype of the rice to be tested is AA (ie, the SNP site HuNCd1 in the rice genome is a homozygous type of A).

[0116] Example 2: Application of HuNCd1 Molecular Marker and Its Identification Primer Set in Identifying Cadmium Content in Rice

[0117] Using the HuNCd1 marker developed in Example 1, 60 rice germplasm resources of different types were selected for genotype identification and cadmium content determination. The results are shown in Table 4.

[0118] Table 4. Cadmium content in grains of 60 rice varieties and genotypes of TagSNP-8874894

[0119]

[0120]

[0121]

[0122] The results showed that 30 accessions had an AA genotype and 30 accessions had a GG genotype. The detection results of the HuNCd1 marker developed in this study were consistent with the sequencing results (Table 4), indicating that the KASP marker can accurately identify the genotype of the HuNCd1 gene related to grain cadmium content in different rice germplasm resources.

[0123] Among the 60 rice germplasm resources, the average grain cadmium content of 30 materials with genotype AA was 0.159 mg / kg; the average grain cadmium content of 30 materials with genotype GG was 0.396 mg / kg (Table 4).

[0124] Significance analysis of 60 validated accessions (Table 5) revealed that grain cadmium content in rice accessions corresponding to genotype AA was significantly lower than that in accessions corresponding to genotype GG (P < 0.001). This marker effectively categorized germplasm accessions with the same grain cadmium content into corresponding genotypes, with phenotypic data matching genotypic identification results. Therefore, the developed HuNCd1 molecular marker can accurately identify grain cadmium content in rice germplasm resources and could be used in the breeding of low-cadmium rice.

[0125] Table 5. Significance analysis of differences in cadmium content in rice grains and genotypes among 60 rice varieties

[0126]

[0127] 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 7, the nucleotide type of which is A or G, and is the 25th nucleotide of sequence 4 in the sequence table.

2. 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 the genotype of the SNP site being AA as a parent for breeding, wherein the AA is the homozygous type of the SNP site being A.

3. Application of the method according to claim 1 or 2 in rice breeding.

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) preparing rice breeding products; The SNP site is a SNP site on rice chromosome 7, the nucleotide type of which is A or G, and is the 25th nucleotide of sequence 4 in the sequence list.

5. The method according to claim 1 or 2, or the use according to claim 4, characterized in that: The genotype of the SNP site is AA or GG, wherein AA is the homozygous type of the SNP site A, and GG is the homozygous type of the SNP site G; the cadmium content in the grains of the tested rice whose genotype of the SNP site is AA is lower than that of the tested rice whose genotype of the SNP site is GG.

6. The product is characterized by: The product contains the substance in the application according to claim 4, 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.

7. The use according to claim 4 or the product according to claim 6, characterized in that: 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).

8. The use or product according to claim 7, 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 22-46 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.

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) Prepare rice breeding products.

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