Application of SNP molecular markers for HuNCd1, a gene associated with cadmium content in rice grains

Abstract

This invention discloses the application of SNP molecular markers for the rice grain cadmium content-related gene HuNCd1. This invention belongs to the field of biotechnology, specifically relating to the application of SNP molecular markers for the rice grain cadmium content-related gene HuNCd1. The method for identifying or assisting in the identification of rice grain cadmium content includes detecting the genotype of an SNP site in the genome of the rice to be tested, and identifying or assisting in the identification of rice grain cadmium content based on the genotype. The SNP site is a single SNP site on rice chromosome 7, with nucleotide type A or G, and is the 25th nucleotide of sequence 4 in the sequence listing. Substances for detecting the SNP site polymorphism and genotype can be combined with other substances (such as substances for detecting single nucleotide polymorphisms or genotypes of other molecular markers related to rice grain cadmium content) to prepare a product for identifying rice varieties with low rice grain cadmium content.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of SNP molecular markers of HuNCd1, a gene related to cadmium content in rice grains. Background Technology

[0002] In recent years, with the acceleration of industrialization and urbanization, soil cadmium pollution has become increasingly prominent, leading to frequent instances of excessive cadmium content in rice and other grain crops, seriously threatening food security and human health. As a crucial staple crop in my country, rice exhibits significant genotypic differences in its cadmium accumulation capacity. Developing low-cadmium-accumulation rice varieties through molecular breeding has become an effective way to ensure safe rice production and improve the quality of agricultural products.

[0003] The HuNCd1 gene is a crucial functional gene regulating cadmium content in rice grains. The development and application of its SNP molecular markers provide key technical support for molecularly assisted selection of rice with low cadmium accumulation traits. Therefore, in-depth research on the HuNCd1 gene and the application of its SNP molecular markers is of significant theoretical and practical value for accelerating the breeding process of low-cadmium rice varieties, promoting green agriculture, and safeguarding public health. Summary of the Invention

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

[0005] To address the above technical problems, this invention first provides a method for identifying or assisting in the identification of cadmium content in rice grains. This method includes detecting the genotype of a SNP site in the genome of the rice to be tested, and identifying or assisting in the identification of cadmium content in the rice grains based on the genotype. The SNP site is a single SNP site on rice chromosome 7, and its nucleotide type is A or G, specifically the 25th nucleotide of sequence 4 in the sequence listing. The genotype is AA or GG, where AA is homozygous for the SNP site A, and GG is homozygous for the SNP site G.

[0006] As one implementation scheme, the method for identifying or assisting in the identification of cadmium content in rice grains may include the following steps:

[0007] (1) Using the genomic DNA of the rice to be tested as a template, KASP molecular marker detection was performed using a primer composition; the primer composition consisted of primer A, primer B and primer C;

[0008] Primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing or whose nucleotide sequence is the single-stranded DNA at positions 22-44 of sequence 1 in the sequence listing;

[0009] Primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing or whose nucleotide sequence is the single-stranded DNA at positions 22-46 of sequence 2 in the sequence listing;

[0010] Primer C is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing;

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

[0012] (3) The cadmium content of the rice grains to be tested was determined based on the genotype results: the cadmium content of the rice grains to be tested with the genotype AA at the SNP locus was lower than that of the rice grains to be tested with the genotype GG at the SNP locus.

[0013] This invention also provides a method for rice breeding.

[0014] The rice breeding method provided by the present invention includes detecting the genotype of the SNP locus in the rice genome, selecting rice with the genotype AA at the SNP locus as the parent for breeding, wherein AA is the homozygous type of the SNP locus being A.

[0015] As an implementation method, rice breeding methods 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 detect KASP molecular markers;

[0017] (2) After completing step (1), perform fluorescence detection to determine the genotype of the SNP site in 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 to 100mM with ddH2O, and then prepare the primer working solution as follows: 12μL of primer A, 12μL of primer B, 30μL of primer C, and 46μL of ddH2O. This solution is used as the KASP-labeled primer working solution and stored at -20℃ for later 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] Two of the Master mixes were purchased from LGC, part number: 1536 Formulation V4.0TF.

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

[0023] In the above method, the reaction procedure for KASP tags can be:

[0024] Step 1: Pre-denaturation at 94℃ for 15 min;

[0025] Step 2: 94℃ for 20s, 61℃ for 60s, 94℃ for 20s, 60.4℃ for 60s, 94℃ for 20s, 59.8℃ for 60s, 94℃ for 20s, 59.2℃ for 60s, 94℃ for 20s, 58.6℃ for 60s, 94℃ for 20s, 58℃ for 60s, 94℃ for 20s, 57.4℃ for 60s, 94℃ for 20s, 56.8℃ for 60s, 94℃ for 20s, 56.2℃ for 60s, 94℃ for 20s, 55.6℃ for 60s;

[0026] Step 3: Denaturation at 94℃ for 20 seconds, annealing at 55℃ for 60 seconds, 5 cycles; if the delamination is not obvious, add 5 more cycles to extend the process;

[0027] Step 4: 94℃ for 20s, anneal at 57℃ for 60s, 26 cycles.

[0028] The method described above for determining the genotype of the SNP in the rice sample is as follows: After the PCR reaction, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) are used to convert the fluorescence signal into analyzable values ​​to read the fluorescence data of the reaction products. Genotyping is performed by reading the fluorescence values ​​at the terminal ends. The fluorescence scanning results are graphically displayed using the R software package. G base types exhibit FAM fluorescence, distributed near the x-axis; A base types exhibit HEX fluorescence, distributed near the y-axis; samples with no detected signal are distributed near the origin.

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

[0030] This invention also provides the application of a substance for detecting KASP polymorphisms or genotypes in the rice genome in any of the following:

[0031] (1) To identify or assist in the identification of cadmium content in rice grains;

[0032] (2) Rice breeding;

[0033] (3) Prepare products for identification or auxiliary identification of cadmium content in rice grains;

[0034] (4) Prepare rice breeding products;

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

[0036] Using the genome sequence of the common rice variety Nipponbare as a reference genome, the SNP site is located at 8874894 bp on chromosome 7 of rice (specifically, position 25 of sequence 4 in the sequence listing).

[0037] This invention also provides products for detecting polymorphisms or genotypes of SNP sites in the rice genome.

[0038] The product provided by this invention for detecting polymorphisms or genotypes of SNP sites in the rice genome contains the aforementioned substances for detecting polymorphisms or genotypes of SNP sites in the rice genome, wherein the product is any one of the following:

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

[0040] C2) Products used for identifying or assisting in the identification of 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 to determine 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 chips. The 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 D1), D2), or D3):

[0044] D1) The substance described is a primer composition for amplifying rice genomic DNA fragments including the SNP sites;

[0045] D2) The substance described is a PCR reagent containing the primer composition 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 primer A, primer B, and primer C.

[0048] The kit described in D3 may also include KASP Master Mix.

[0049] In the above applications, methods, and products, the primer composition may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, 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 whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading).

[0050] The primer composition described herein may be a primer composition consisting of single-stranded DNA with nucleotide sequences of positions 22-44 of Sequence 1 in the sequence listing, single-stranded DNA with nucleotide sequences of positions 22-46 of Sequence 2 in the sequence listing, and single-stranded DNA with nucleotide sequences of Sequence 3 in the sequence listing. The primer composition may also be a primer set consisting of single-stranded DNA shown in Sequence 1, Sequence 2, and Sequence 3 in the sequence listing. Sequence 1 in the sequence listing consists of 44 nucleotides, with nucleotides 1-21 being the FAM adapter sequence (as a marker) and nucleotides 22-44 being the specific sequence; Sequence 2 in the sequence listing consists of 46 nucleotides, with nucleotides 1-21 being the HEX adapter sequence (as a marker) and nucleotides 22-46 being the specific sequence.

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

[0052] The applications of the aforementioned DNA molecules also fall within the scope of protection of this invention. Specifically, the applications are those found in any of the following:

[0053] (1) To identify or assist in the identification of cadmium content in rice grains;

[0054] (2) Rice breeding;

[0055] (3) Prepare products for identification or auxiliary identification of cadmium content in rice grains;

[0056] (4) Prepare rice breeding products.

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

[0058] The substance that detects the SNP site polymorphism and genotype can be combined with other substances (such as substances that detect single nucleotide polymorphisms or genotypes of other molecular markers related to cadmium content in rice grains) to prepare a product for identifying rice varieties with low cadmium content in rice grains.

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

[0060] The SNP molecular markers provided by this invention are closely related to the cadmium content in rice grains, enabling efficient and accurate identification of cadmium accumulation characteristics in rice varieties. This method is simple to operate, suitable for large-scale sample detection, and has promising application prospects. Attached Figure Description

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

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0065] The rice germplasm resources described in the following examples are described 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 biological material is available to the public from the applicant and is intended solely for the purpose of replicating the experiments of this invention and shall not be used for any other purpose.

[0066] The rice experimental field of this invention is a typical cadmium-contaminated rice field in my country, with a soil cadmium content of 0.41 mg / kg and pH = 5.3.

[0067] In the following examples, 471 experimental samples and 60 validation samples (Table 1) were planted in 2021. The specific operating steps, starting from sowing, were as follows: Sowing was carried out when the germinated seeds reached a sprout length of 5 mm. The germinated seeds were then sown in the field until they grew into seedlings. Two weeks later, transplanting was carried out, with a row spacing of 25 cm; 8 seedlings were planted in each row, with a plant spacing of 20 cm. After the rice matured, grain samples were collected. To avoid boundary effects, the two plants adjacent to the aisle on either side were discarded, and the grains from the remaining plants were harvested together.

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

[0069] The collected rice grain samples were sun-dried or placed in an oven at 60°C for 3 days to achieve constant quality. Afterward, the grains were hulled using a rice huller, and the resulting brown rice samples were placed in 5ml centrifuge tubes. The brown rice samples were then pulverized using a high-throughput silent tissue grinder for subsequent cadmium content determination.

[0070] The cadmium content of rice grains was determined by a 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 of crushed rice grain sample (accurate to 0.0001g) and put it into a glass digestion tube to avoid the powder sticking to the wall. (2) Adding acid: Add 1ml of analytical grade pure nitric acid and digest overnight in a cold environment. (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 volume adjustment tube, and adjust the volume to 15ml. (5) Filtration: After shaking well, filter the liquid after volume adjustment into a 10ml centrifuge tube using a 0.45μm aqueous filter membrane for testing. Quality control: Two blank controls and three rice flour component analysis standard materials (national standard material, GBW100349, Steel Research Institute Nake Testing Technology Co., Ltd.) were set up for 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 inductively coupled plasma mass spectrometry.

[0071] Example 1: HuNCd1 Genotyping Analysis and Molecular Marker Development

[0072] In its previous work, the applicant used phenotypic data and genotypic data of total cadmium content in rice MCC population materials (the phenotypic data of total cadmium content in rice grains was obtained from the determination of total cadmium content in rice grains harvested in the field in 2021; the genotypic data was obtained from the variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies) to locate a SNP locus, Chr7_8874894, associated with total cadmium content in rice grains through genome-wide association analysis, and named it HuNCd1.

[0073] The SNP locus is located at position 8874894 on chromosome 7 of the Nipponbare rice genome sequence information, and its nucleotide value is either A or G (the SNP locus is the 25th nucleotide of sequence 4 in the sequence listing, where r represents A or G). There are two genotypes for the SNP locus: AA and GG. Genotype AA is homozygous for SNP A, and genotype GG is homozygous for SNP G. Genotypic analysis was performed on the HuNCd1(7_8874894) genotype of 471 rice MCC population materials, and the statistical differences in grain cadmium content among different genotypes were compared using the t-test. The p-value was calculated using Tukey's test. A p-value less than 0.05 was defined as statistically significant; a p-value less than 0.01 was defined as highly statistically significant.

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

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

[0076]

[0077]

[0078]

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[0080]

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[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

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

[0093]

[0094] 2. HuNCd1 genotyping and molecular marker development

[0095] KASP molecular marker primer sets were designed for the upstream and downstream sequences of the superior allelic variant site Chr7_8874894 (HuNCd1) 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) (specific sequences are shown in Table 3). The 5' end of the designed primer FA was labeled with 6-carboxyfluorescein (FAM) dye; the 5' end of FB was labeled with hexachloro-6-methylfluorescein (HEX) dye.

[0096] Primers A and C amplify single-stranded DNA molecules with SNP sites of G; primers B and C amplify single-stranded DNA molecules with SNP sites of A. The fluorescence signal of the fluorescent group in the template that binds to the FAM or HEX sequence can be read by an ELISA reader or a quantitative PCR instrument. All specific primer sequences were synthesized by Zhongyujin Labeling (Beijing) Biotechnology Co., Ltd.

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

[0098]

[0099] 3. Establishment of a method for detecting the gene type of SNP marker Chr7_8874894 (HuNCd1) using a primer set based on KASP markers.

[0100] The different allele types at position 8874894 on chromosome 7 (SNP site Chr7_8874894) were detected using the KASP marker primer set in Table 3.

[0101] 1) PCR amplification system and procedure

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

[0103] Preparation of KASP-labeled primer working solution: First, dilute the three primers to 100mM with ddH2O, then prepare the primer working solution according to the following formula: Primer A 12μL, Primer B 12μL, Primer C 30μL, ddH2O 46μL. Store at -20℃ for later use.

[0104] The PCR amplification system consisted of: 0.8 μL DNA, 0.4 μL 2×Master mix, 0.022 μL primer working solution, and 0.4 μL ddH2O.

[0105] Two of the Master mixes were purchased from LGC, part number: 1536 Formulation V4.0TF.

[0106] The PCR reaction procedure is as follows: Step 1: Pre-denaturation at 94℃ for 15 min;

[0107] Step 2: 94℃ for 20s, 61℃ for 60s, 94℃ for 20s, 60.4℃ for 60s, 94℃ for 20s, 59.8℃ for 60s, 94℃ for 20s, 59.2℃ for 60s, 94℃ for 20s, 58.6℃ for 60s, 94℃ for 20s, 58℃ for 60s, 94℃ for 20s, 57.4℃ for 60s, 94℃ for 20s, 56.8℃ for 60s, 94℃ for 20s, 56.2℃ for 60s, 94℃ for 20s, 55.6℃ for 60s;

[0108] Step 3: Denaturation at 94℃ for 20 seconds, annealing at 55℃ for 60 seconds, 5 cycles; if the delamination is not obvious, add 5 more cycles to extend the process;

[0109] Step 4: 94℃ for 20s, anneal at 57℃ for 60s, 26 cycles.

[0110] 2) Genotyping

[0111] After the PCR reaction, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) were used to convert the fluorescence signals into analyzable numerical values ​​to read the fluorescence data of the reaction products. The fluorescence scanning results were graphically displayed using the R software package. G bases showed FAM fluorescence, distributed near the x-axis; A bases showed HEX fluorescence, distributed near the y-axis; samples with no detected signal were distributed near the origin.

[0112] The FAM excitation wavelength is 485 nm, and the emission wavelength is 520 nm. The HEX excitation wavelength is 535 nm, and the emission wavelength is 556 nm. The system reference fluorescence ROX excitation wavelength is 575 nm, and the emission wavelength is 610 nm.

[0113] The results are as follows:

[0114] If only the FAM group shows a fluorescent signal, then the genotype of the SNP site HuNCd1 in the rice to be tested is GG (that is, the SNP site HuNCd1 in the rice genome is a homozygous G).

[0115] If only the HEX group shows a fluorescent signal, then the HuNCd1 genotype of the rice being tested is AA (i.e., the HuNCd1 SNP site in the rice genome is homozygous for A).

[0116] Example 2: Application of HuNCd1 molecular marker and its identification primer set in the identification of cadmium content in rice.

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

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

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[0120]

[0121]

[0122] The results showed that 30 materials had the genotype AA, and 30 materials had the genotype GG. 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 HuNCd1, a gene related to grain cadmium content, in different rice germplasm resources.

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

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

[0125] Table 5. Significant differences in cadmium content and genotype among 60 rice varieties.

[0126]

[0127] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for identifying or assisting in the identification of cadmium content in rice grains, characterized in that: This includes detecting the genotype of SNP sites in the genome of the rice to be tested, and identifying or assisting in the identification of cadmium content in rice grains based on the genotype. The SNP site is an SNP site on chromosome 7 of rice, and its nucleotide type is A or G, which is the 25th nucleotide of sequence 4 in the sequence listing.

2. A method for rice breeding, characterized by: The method includes detecting the genotype of the SNP locus in claim 1 in the rice genome, selecting rice with the genotype AA at the SNP locus as a parent for breeding, wherein AA is a homozygous type of the SNP locus being A.

3. The method according to claim 1 or 2, characterized in that: The genotype of the SNP locus is AA or GG, where AA is the homozygous type of the SNP locus being A, and GG is the homozygous type of the SNP locus being G; the cadmium content in the grains of the tested rice with the genotype AA at the SNP locus is lower than that of the tested rice with the genotype GG at the SNP locus.

4. The application of the method according to claim 1 or 2 in the breeding of rice with low cadmium content in grains.

5. Application of substances for detecting SNP polymorphisms or genotypes in the rice genome in any of the following: (1) To identify or assist in the identification of cadmium content in rice grains; (2) Selecting and breeding rice varieties with low cadmium content in grains; (3) Prepare products for identification or auxiliary identification of cadmium content in rice grains; (4) Prepare and breed rice products with low cadmium content in grains; The SNP site is an SNP site on rice chromosome 7, and its nucleotide type is A or G, which is the 25th nucleotide of sequence 4 in the sequence listing.

6. The application according to claim 5, characterized in that: The genotype of the SNP locus is AA or GG, where AA is the homozygous type of the SNP locus being A, and GG is the homozygous type of the SNP locus being G; the cadmium content in the grains of the tested rice with the genotype AA at the SNP locus is lower than that of the tested rice with the genotype GG at the SNP locus.

7. The application according to claim 6, characterized in that: The substance is either D1), D2), or D3). D1) The substance is a primer composition for amplifying rice genomic DNA fragments including the SNP sites; D2) The substance is a PCR reagent containing the primer composition 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 application according to claim 7, characterized in that: The primer composition consists of primer A, primer B and primer C; Primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing or whose nucleotide sequence is the single-stranded DNA at positions 22-44 of sequence 1 in the sequence listing; Primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing or whose nucleotide sequence is the single-stranded DNA at positions 22-46 of sequence 2 in the sequence listing; Primer C is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing.

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