CN120536628A

CN120536628A - Application of SNP molecular markers for HuNCd1, a gene related to cadmium content in rice grains

Info

Publication number: CN120536628A
Application number: CN202510937280.7A
Authority: CN
Inventors: 黎妮; 王伟平; 苗雪雪; 何振艳; 闫慧莉; 郝爽楠; 许文秀; 马爱民; 李晶
Original assignee: Institute of Botany of CAS; Hunan Hybrid Rice Research Center
Current assignee: Institute of Botany of CAS; Hunan Hybrid Rice Research Center
Priority date: 2025-07-08
Filing date: 2025-07-08
Publication date: 2025-08-26

Abstract

The invention discloses an application of SNP molecular markers of rice grain cadmium content related genes HuNCd. The invention belongs to the technical field of biology, and particularly relates to application of SNP molecular markers of rice grain cadmium content related genes HuNCd. The method for identifying or assisting in identifying the cadmium content of rice grains comprises the steps of detecting genotypes of SNP loci in rice genomes to be detected, identifying or assisting in identifying the cadmium content of the rice grains according to the genotypes, wherein the SNP locus is one SNP locus on a chromosome 7 of rice, the nucleotide type of the SNP locus is A or G, and the SNP locus is 25 th nucleotide of a sequence 4 in a sequence table. The substances for detecting the SNP locus polymorphism and the genotype 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 of rice grains) to prepare a product for identifying rice varieties with low cadmium content of rice grains.

Description

Application of SNP molecular marker of rice grain cadmium content related gene HuNCd1

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of SNP molecular markers of rice grain cadmium content related genes HuNCd.

Background

In recent years, with the acceleration of industrialization and urban progress, the problem of cadmium pollution of soil is increasingly prominent, so that the phenomenon that the cadmium content in grains of grain crops such as rice exceeds the standard frequently occurs, and the grain safety and the human health are seriously threatened. Rice is used as an important staple grain crop in China, and has obvious genotype difference in grain cadmium accumulation capacity. The cultivation of rice varieties with low cadmium accumulation by means of molecular breeding is an effective way for guaranteeing the safe production of rice and improving the quality of agricultural products.

HuNCd1 gene is used as an important functional gene for regulating and controlling cadmium content of rice grains, and development and application of SNP molecular markers provide key technical support for molecular auxiliary selection of low cadmium accumulation characters of rice. Therefore, the application of HuNCd gene and SNP molecular marker thereof is studied deeply, and has important theoretical significance and practical value for accelerating the breeding process of low-cadmium rice varieties, promoting green agricultural development and guaranteeing public health.

Disclosure of Invention

The invention aims to solve the problem of how to identify or assist in identifying the cadmium content of rice kernels.

In order to solve the technical problems, the invention firstly provides a method for identifying or assisting in identifying the cadmium content of rice seeds, which comprises the steps of detecting the genotype of SNP loci in a genome of rice to be detected, identifying or assisting in identifying the cadmium content of the rice seeds according to the genotype, wherein the SNP locus is one SNP locus on chromosome 7 of the rice, the nucleotide type of the SNP locus is A or G, and the SNP locus is 25 th nucleotide of a sequence 4 in a sequence table. The genotype is AA or GG, the AA is homozygous type with the SNP locus A, and the GG is homozygous type with the SNP locus G.

As one embodiment, the method for identifying or aiding in the identification of cadmium content of rice kernels may comprise the steps of:

(1) Taking genome DNA of rice to be detected as a template, and adopting a primer composition to carry out KASP molecular marker detection, wherein the primer composition consists of a primer A, a primer B and a primer C;

the primer A is a single-stranded DNA molecule with a nucleotide sequence of a sequence 1 in a sequence table or a single-stranded DNA with a nucleotide sequence of 22 th-44 th positions of the sequence 1 in the sequence table;

The primer B is a single-stranded DNA molecule with a nucleotide sequence of sequence 2 in a sequence table or a single-stranded DNA with a nucleotide sequence of 22 th-46 th positions of sequence 2 in the sequence table;

the primer C is a single-stranded DNA molecule with a nucleotide sequence of sequence 3 in a sequence table;

(2) After the step (1) is completed, performing fluorescence detection to determine the genotype of the SNP of the rice to be detected;

(3) And (3) identifying the cadmium content of the rice seeds to be detected according to the genotype result, wherein the cadmium content of the rice seeds to be detected with the genotype of AA at the SNP locus is lower than that of the rice seeds to be detected with the genotype of GG at the SNP locus.

The invention also provides a rice breeding method.

The rice breeding method provided by the invention comprises the steps of detecting the genotype of the SNP locus in a rice genome, and selecting rice with the genotype of the SNP locus of AA as a parent for breeding, wherein the AA is homozygous with the SNP locus of A.

As an implementation method, the rice breeding method can comprise the following steps:

(1) Taking genome DNA of rice to be detected as a template, and adopting the primer group to carry out KASP molecular marker detection;

(2) After the step (1) is completed, performing fluorescence detection to determine the genotype of the SNP locus of the rice to be detected;

(3) And selecting AA genotype rice to carry out rice breeding with low cadmium content in grains.

In the method, the method for dissolving and preparing the primers comprises the steps of diluting 3 primers with ddH ₂ O to 100mM, respectively, and preparing primer working solutions according to the following steps of 12 mu L of primer A, 12 mu L of primer B and 30 mu L, ddH ₂ O46 mu L of primer C as KASP marked primer working solutions, and preserving at-20 ℃ for standby.

In the above method, the KASP reaction system may be DNA 0.8. Mu.L, 2 XMaster mix 0.4. Mu.L, primer working solution 0.022. Mu. L, ddH ₂ O0.4. Mu.L.

Wherein 2 XMaster mix is available from LGC company under the trade designation 1536Formulation V4.0TF.

In the above method, the KASP labeling can be performed on a common PCR amplification unit.

In the above method, the KASP labeling reaction procedure may be:

The first step, pre-denaturation at 94 ℃ for 15min;

second step ：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;

Thirdly, denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s and 5 cycles, and if parting is not obvious, adding 5 cycles for expansion;

And the fourth step, 94 ℃ for 20s, and 57 ℃ for 60s and 26 cycles.

In the method, the method for determining the genotype of the SNP of the rice to be detected can be that after the PCR reaction is completed, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) are utilized to convert the fluorescence signal into an analyzable value to read fluorescence data of a reaction product. The fluorescence value is read by the terminal end to carry out genotyping, the fluorescence scanning result is graphically displayed by using an R software package, the G base type has FAM fluorescence and is distributed near the x axis, the A base type has HEX fluorescence and is distributed near the y axis, and the sample without detected signals is distributed near the origin.

The application of the method in rice breeding also belongs to the protection scope of the invention.

The invention also provides the application of the substances for detecting the polymorphism or genotype of KASP in rice genome,

(1) Identifying or assisting in identifying the cadmium content of rice kernels;

(2) Breeding rice;

(3) Preparing a product for identifying or assisting in identifying the cadmium content of rice grains;

(4) Preparing a rice breeding product;

The SNP locus is one SNP locus on a rice chromosome 7, the nucleotide type of the SNP locus is A or G, and the SNP locus is 25 th nucleotide of a sequence 4 in a sequence table.

The genome sequence of a common rice variety Japanese sunny is taken as a reference genome, and the SNP locus is 8874894bp of a rice chromosome 7 (specifically the 25 th locus of a sequence 4 in a sequence table).

The invention also provides a product for detecting polymorphism or genotype of SNP locus in rice genome.

The product for detecting the polymorphism or genotype of the SNP locus in the rice genome provided by the invention contains the substance for detecting the polymorphism or genotype of the SNP locus in the rice genome, and the product is any one of the following substances:

c1 A product for detecting single nucleotide polymorphism or genotype related to cadmium content of rice grains;

C2 Identifying or assisting in identifying a product of rice grain cadmium content;

C3 A product for rice breeding.

In the above applications, methods and products, the substance may be a reagent and/or instrument required to determine the polymorphism or genotype of the SNP site by at least one of DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. The SNP chip comprises a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele specific primer extension reaction, a chip based on one-step method reaction, a chip based on primer connection reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and a chip based on fluorescent molecule DNA binding reaction.

Alternatively, the substance is D1), D2) or D3) as follows:

d1 The substance is a primer composition for amplifying rice genome DNA fragments including the SNP locus;

d2 The substance is a PCR reagent containing the primer composition of D1);

d3 The substance is a kit containing the primer composition of D1) or the PCR reagent of D2).

Alternatively, the amplification may be PCR amplification. The primer composition consists of the primer A, the primer B and the primer C.

D3 The kit may further comprise KASP MASTER Mix.

In the above applications, methods and products, the primer composition may or may not be labeled with a label. The 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, radiolabels such as 32P, binding moieties such as biotin (biotin), haptens such as Digoxigenin (DIG), luminescent, phosphorescent or fluorogenic 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 label may provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like. The label may be a charged moiety (positive or negative) or alternatively may be charge neutral. The label may comprise or be a combination of nucleic acid or protein sequences, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence).

The primer composition can be a primer composition consisting of single-stranded DNA of which the nucleotide sequence is 22-44 th positions of the sequence 1 in the sequence table, single-stranded DNA of which the nucleotide sequence is 22-46 th positions of the sequence 2 in the sequence table and single-stranded DNA of which the nucleotide sequence is the sequence 3 in the sequence table, and can also be a primer set of single-stranded DNA shown by the sequence 1 in the sequence table, single-stranded DNA shown by the sequence 2 in the sequence table and single-stranded DNA shown by the sequence 3 in the sequence table. The sequence 1 in the sequence table consists of 44 nucleotides, nucleotides 1 to 21 are FAM linker sequences (serving as markers), nucleotides 22 to 44 are specific sequences, the sequence 2 in the sequence table consists of 46 nucleotides, nucleotides 1 to 21 are HEX linker sequences (serving as markers), and nucleotides 22 to 46 are specific sequences.

The invention also provides a DNA molecule, and the nucleotide sequence is shown as a sequence 1 in a sequence table.

The use of the DNA molecules described above is also within the scope of the present invention. The application is specifically an application in any one of the following:

(2) Breeding rice;

(4) And (5) preparing a rice breeding product.

Alternatively, in the above application, the DNA molecule serves as a detection target.

The substances for detecting the SNP locus polymorphism and the genotype 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 of rice grains) to prepare a product for identifying rice varieties with low cadmium content of rice grains.

Herein, the purpose of the breeding may include rice with low cadmium content in the grain. The rice may be either a inbred or a pure line.

The SNP molecular marker provided by the invention is closely related to the cadmium content of rice grains, and can efficiently and accurately identify the cadmium accumulation characteristic of rice varieties. The method is simple and convenient to operate, is suitable for detecting large-scale samples, and has good application prospect.

Drawings

FIG. 1 is a diagram showing genotyping of HuNCd molecular markers on 60 parts of rice germplasm material.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

The rice germplasm resources in the examples described below have been described in :Huili Yan,et al.Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies.Nature Communications.2019.10:2562. public availability of this biomaterial from the applicant, which was used only for repeated experiments of the invention and was not available for other uses.

The rice experimental field disclosed by the invention is a typical cadmium-polluted rice field in China, the cadmium content of soil is 0.41mg/kg, and the pH=5.3.

471 Parts of the test material and 60 parts of the verification material (Table 1) were planted in 2021 in the following examples, and from the start of sowing, the specific operation steps were as follows, sowing was performed when the bud length of the seed for seed soaking was 5mm, and the germinated seed was sown into the field until it was grown into seedlings. After two weeks, transplanting is carried out, the row spacing is 25cm, 8 plants are planted in each row, and the plant spacing is 20cm. And after the rice is mature, collecting seed samples, discarding left and right plants adjacent to the corridor in order to avoid boundary effects, and mixing and collecting seed of other plants.

The method for measuring the cadmium content of the rice grains comprises the following steps:

the recovered rice grain sample is dried in the sun or is placed in an oven for drying at 60 ℃ for 3 days, after the quality is constant, a rice huller is used for hulling, and the obtained brown rice sample is placed in a 5ml centrifuge tube. And then crushing the brown rice sample by using a high-flux mute tissue grinder for subsequent cadmium content measurement.

The rice grain cadmium content is determined by a monoacid digestion method, an instrument is a far infrared temperature control digestion furnace, a container is a glass digestion tube, and the specific steps are (1) weighing the sample, namely accurately weighing 0.2000g (accurate to 0.0001 g) of crushed rice grain sample, and putting the sample into the glass digestion tube to avoid powder sticking to walls. (2) adding acid, namely adding 1ml of high-grade pure nitric acid, and carrying out cold digestion overnight. (3) Digestion, namely covering a neck funnel for digestion for 6 hours at 200 ℃ until the digestion liquid is colorless and transparent or is slightly yellowish. (4) And (3) constant volume, namely cleaning digestion liquid in the tube by distilled water, transferring the cleaning liquid into a 15ml constant volume tube, and keeping the constant volume to 15ml. (5) Filtering, namely filtering the liquid with the constant volume into a 10ml centrifuge tube by using a 0.45 mu m water-based filter membrane after shaking evenly, and measuring. And the quality control is that 2 blank controls and 3 rice powder component analysis standard substances (national standard substances, GBW100349, steel grinding Nake detection technology company) are arranged during digestion of each batch, so that accurate and reliable rice grain cadmium content result data is ensured. All sample assays were repeated 3 times. The cadmium content of rice grains is measured by an inductively coupled plasma mass spectrometer.

Examples 1, huNCd genotype analysis and molecular marker development

In the earlier work of the applicant, the phenotype data of the total cadmium content of the seeds and the genotype data of the rice MCC group material are utilized (the phenotype data of the total cadmium content of the seeds are derived from the measurement of the total cadmium content of the rice seeds harvested in 2021 field, and the genotype data are derived from Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies.), and are positioned to a SNP locus Chr7_8874894 related to the total cadmium content of the rice seeds through whole genome association analysis, and the SNP locus is named as HuNCd1.

The SNP locus is positioned at 8874894 th position on chromosome 7 of genome sequence information of rice Japanese sunny, the nucleotide is A or G (the SNP locus is 25 th nucleotide of sequence 4 in a sequence table, and r in the sequence 4 represents A or G). The SNP locus has two genotypes, namely AA and GG, the genotype AA is homozygous with the SNP of A, and the genotype GG is homozygous with the SNP of G. For the genotyping analysis of HuNCd (7_8874894) of 471 rice MCC population materials, t test was used to compare the statistical differences in grain cadmium content for different genotypes. The P value is calculated from Tukey's test. A P value of less than 0.05 was defined as having a statistically significant difference, and a P value of less than 0.01 was defined as having a statistically significant difference.

The result is that according to the sequencing result of 471 rice germplasm resources in the earlier stage, the low-cadmium genotype of 327 materials is AA, the average value of the cadmium content of grains is 0.385mg/kg, the high-cadmium genotype of 144 materials is GG, and the average value of the cadmium content of grains is 0.598mg/kg. The result shows that the grain cadmium content of the rice material corresponding to AA is extremely lower than that of the rice material corresponding to GG (P < 0.01). The SNP locus can be used as a molecular marker for identifying or assisting in identifying the cadmium content of rice kernels of different strains.

Table 1, genotype data for cadmium content and HuNCd1 of rice grain of 471 varieties

TABLE 2 seed cadmium content of Rice germplasm Material of different genotypes

2. HuNCd1 genotyping and molecular marker development

A KASP molecular marker primer set was designed for the sequence upstream and downstream of chr7_8874894 (HuNCd 1) of the excellent allelic variation site obtained in step 1. The KASP-labeled primer set consisted 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 F-A was labeled with 6-carboxyfluorescein (FAM) dye, and the 5' -end of F-B was labeled with hexachloro-6-methylfluorescein (HEX) dye.

The single-stranded DNA molecules shown by the primer A and the primer C amplify fragments with SNP loci of G, and the single-stranded DNA molecules shown by the primer B and the primer C amplify fragments with SNP loci of A. The fluorescent signal of the fluorescent group bound to the FAM sequence or the HEX sequence in the template can be read by a microplate reader or a fluorescent quantitative PCR instrument. Specific primer sequences were each synthesized by the well-known Yujin labeled (Beijing) Biotechnology Co., ltd.

TABLE 3 HuNCd molecular marker KASP primer sequences

3. Establishment of method for detecting SNP marker Chr7_8874894 (HuNCd 1) gene type by using primer set of KASP marker

The KASP-labeled primer set of Table 3 was used to detect the different allele types at position 8874894 (SNP site Chr7_ 8874894) on chromosome 7 of the genomic sequence information of rice.

1) PCR amplification system and program

Extracting genome DNA of common rice leaves by CTAB method, adding 400 mu L TE for dissolution. The DNA is subjected to quality detection by 1% agarose gel electrophoresis, and the diluted rice genome DNA is used as a template for PCR amplification.

The KASP labeled primer working solution was prepared by diluting 3 primers with ddH ₂ O to 100mM, respectively, and preparing the primer working solution according to the following formula, primer A12. Mu.L, primer B12. Mu.L, and primer C30. Mu. L, ddH ₂ O46. Mu.L. And (3) storing at-20 ℃ for standby.

The PCR amplification system was 0.8. Mu.L of DNA, 0.4. Mu.L of 2 XMaster mix, 0.022. Mu. L, ddH2O 0.4. Mu.L of primer working solution.

The PCR reaction procedure is that the first step is 94 ℃ pre-denaturation for 15min;

And the fourth step, 94 ℃ for 20s, and 57 ℃ for 60s and 26 cycles.

2) Genotyping

After the PCR reaction is completed, the reaction products are read for fluorescence data by converting the fluorescence signal into an analyzable value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya). The fluorescence scanning result is graphically displayed by using an R software package, wherein the G base type has FAM fluorescence and is distributed near the x axis, the A base type has HEX fluorescence and is distributed near the y axis, and a sample without detected signals is distributed near the origin.

The FAM excitation wavelength is 485nm and the emission wavelength is 520nm. The HEX excitation wavelength was 535nm and the emission wavelength was 556nm. The excitation wavelength of the system reference fluorescence ROX is 575nm, and the emission wavelength is 610nm.

The results were determined as follows:

If only fluorescence signals of FAM groups are displayed, the genotype of the SNP locus HuNCd of the rice to be detected is GG (namely, the SNP locus HuNCd in the genome of the rice is homozygote of G);

If only fluorescence signals of HEX groups are displayed, the HuNCd genotype of the rice to be detected is AA (namely SNP locus HuNCd1 in the genome of the rice is homozygous type A).

Example 2, huNCd1 molecular marker and application of identification primer group in identification of cadmium content in rice

Using HuNCd markers developed in example 1, 60 different types of rice germplasm resources were selected for genotyping and cadmium content determination, and the results are shown in Table 4.

Table 4, cadmium content of 60 varieties of Rice grains and genotype of TagSNP-8874894

The results showed that 30 materials had genotype AA and 30 materials had genotype GG. The detection result of HuNCd mark developed in the research is consistent with the sequencing result (table 4), which shows that KASP mark can accurately identify the genotypes of genes HuNCd related to the cadmium content of seeds in different rice germplasm resources.

In 60 parts of rice germplasm resources, the average grain cadmium content of 30 materials with genotype AA is 0.159mg/kg, and the average grain cadmium content of 30 materials with genotype GG is 0.396mg/kg (table 4).

The 60 verification materials were subjected to significance analysis (Table 5), and the grain cadmium content of the rice material corresponding to genotype AA was very significantly lower than that of the rice material corresponding to genotype GG (P < 0.001). The marker can effectively divide the germplasm materials with the same grain cadmium content type into corresponding genotypes, and the phenotype data is matched with the genotype identification result. Therefore, the developed HuNCd molecular marker can be accurately used for identifying the cadmium content of seeds in rice germplasm resources, and can be used for breeding low-cadmium rice.

Table 5, 60 varieties of rice grain cadmium content and genotype difference significance analysis

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. A method for identifying or assisting in the identification of cadmium content in rice grains, comprising: detecting the genotype of a single nucleotide polymorphism (SNP) site in the genome of a rice to be tested, and identifying or assisting in the identification of the cadmium content in the rice grains based on the genotype; wherein 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 listing.

2. A method for rice breeding, characterized in that: the method comprises detecting the genotype of the SNP site described in claim 1 in the rice genome, and selecting rice with a genotype of AA at the SNP site as a parent for breeding, wherein the AA is a homozygous type of the SNP site 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; and the cadmium content in the grains of the rice to be tested whose genotype of the SNP site is AA is lower than that of the rice to be tested whose genotype of the SNP site is GG.

6. A product, characterized in that: the product contains the substance described in the application of 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, wherein 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 in that the nucleotide sequence of the DNA molecule is sequence 4 in the sequence list.

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;

(4) Prepare rice breeding products.