Application of SNP molecular marker of rice grain manganese content related gene HunMn1

Abstract

The application discloses application of a SNP molecular marker of a rice grain manganese content related gene HuNMn1 and belongs to the technical field of biotechnology, in particular to application of a SNP molecular marker of a rice grain manganese content related gene HuNMn1.The substance for detecting the polymorphism or genotype of the SNP in the rice genome can be applied in any one of the following aspects: (1) identification or auxiliary identification of rice grain manganese content; (2) rice breeding; (3) preparation of a product for identification or auxiliary identification of rice grain manganese content; (4) preparation of a product for rice breeding; the SNP site is an SNP site on a chromosome 7 of rice, the nucleotide type is A or T, and is the 29th nucleotide in sequence 1 in the sequence table.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of SNP molecular markers of HuNMn1, a gene related to manganese content in rice grains. Background Technology

[0002] Manganese is an essential micronutrient for plant growth and development, playing a crucial role in physiological processes such as photosynthesis, respiration, and antioxidant responses in rice. The manganese content of rice grains not only directly affects rice yield and quality but also has significant implications for human health. With increasing emphasis on nutrition and health, developing manganese-enriched rice varieties has become a key objective of modern rice breeding.

[0003] With the development of molecular biology techniques, genomics research has provided new avenues for elucidating the molecular mechanisms of manganese accumulation in rice grains. Through genome-wide association studies and QTL mapping, several gene loci associated with manganese content in rice grains have been identified. The HuNMn1 gene encodes a metal transporter protein that plays a crucial role in the transport and accumulation of manganese in rice grains. However, the lack of rapid and accurate molecular markers for identifying superior allelic variants of this gene severely restricts its application in rice variety improvement. Single nucleotide polymorphism (SNP)-based molecular markers offer advantages such as high polymorphism, good stability, and convenient detection. Developing SNP molecular markers closely linked to the HuNMn1 gene and applying them in practical breeding processes has significant theoretical and practical value for rapidly screening rice germplasm resources with high manganese content, improving the efficiency of marker-assisted selection breeding, and cultivating high-quality manganese-rich rice varieties. Summary of the Invention

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

[0005] To address the above technical problems, this invention first provides the application of a substance for detecting KASP polymorphisms or genotypes in the rice genome in any of the following situations:

[0006] (1) To identify or assist in the identification of manganese content in rice grains;

[0007] (2) Rice breeding;

[0008] (3) Prepare products for identification or auxiliary identification of manganese content in rice grains;

[0009] (4) Prepare rice breeding products;

[0010] The SNP site is a site on rice chromosome 7, and its nucleotide type is A or T, which is the 29th nucleotide of sequence 1 in the sequence listing.

[0011] Using the genome sequence of the common rice variety Nipponbare as a reference genome, the SNP site is located at position 8872318 bp on chromosome 7 of rice (specifically, position 29 of sequence 1 in the sequence listing).

[0012] The present invention also provides a method for identifying or assisting in the identification of manganese content in rice grains, comprising detecting the genotype of the SNP locus in the genome of the rice to be tested, and identifying or assisting in the identification of manganese content in rice grains based on the genotype, wherein the genotype is AA or TT, wherein AA is a homozygous type of the SNP being A, and TT is a homozygous type of the SNP being T.

[0013] As one implementation scheme, the method for identifying or assisting in the identification of manganese content in rice grains may include the following steps:

[0014] (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;

[0015] Primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing or whose nucleotide sequence is single-stranded DNA at positions 22-50 of sequence 2 in the sequence listing;

[0016] Primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing or whose nucleotide sequence is single-stranded DNA at positions 22-49 of sequence 3 in the sequence listing.

[0017] Primer C is a single-stranded DNA molecule whose nucleotide sequence is sequence 4 in the sequence listing;

[0018] (2) After completing step (1), perform fluorescence detection to determine the genotype of the SNP in the rice to be tested;

[0019] (3) The manganese content of the rice grains was identified based on the genotype results: the manganese content of the rice grains of the rice with the genotype TT of the SNP was higher than that of the rice with the genotype AA of the SNP.

[0020] This invention also provides a method for rice breeding.

[0021] 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 TT of the SNP as the parent for breeding, wherein TT is the homozygous type of the SNP being T.

[0022] As an implementation method, rice breeding methods may include the following steps:

[0023] (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;

[0024] (2) After completing step (1), perform fluorescence detection to determine the genotype of the SNP site in the rice to be tested;

[0025] (3) Select TT genotype rice for rice breeding with high manganese content in grains.

[0026] In the above method, the primer dissolution and preparation method can be as follows: First, dilute the three primers to 100 μM with ddH2O, and then prepare the primer working solution as follows: 12 μL of primer A, 12 μL of primer B, 30 μL of primer C, and 46 μL of ddH2O. This solution is used as the KASP-labeled primer working solution and stored at -20℃ for later use.

[0027] 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.

[0028] Two of the Master mixes were purchased from LGC, part number: 1536 Formulation V4.0TF.

[0029] In the above method, KASP labeling can be performed on a regular PCR amplification instrument.

[0030] In the above method, the reaction procedure for KASP tags can be:

[0031] Step 1: Pre-denaturation at 94℃ for 15 min;

[0032] 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;

[0033] 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;

[0034] Step 4: 94℃ for 20s, anneal at 57℃ for 60s, 26 cycles.

[0035] 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. T-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.

[0036] The application of the above methods in rice breeding also falls within the scope of protection of this invention.

[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 manganese content in rice grains;

[0040] C2) Products used for identifying or assisting in the identification of manganese 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 22-50 of Sequence 2 in the sequence listing, single-stranded DNA with nucleotide sequences of positions 22-49 of Sequence 3 in the sequence listing, and single-stranded DNA with nucleotide sequences of Sequence 4 in the sequence listing. Alternatively, the primer composition may be a primer set consisting of single-stranded DNA shown in Sequence 2, Sequence 3, and Sequence 4 in the sequence listing. Sequence 2 in the sequence listing consists of 50 nucleotides, with nucleotides 1-21 being the FAM adapter sequence (as a marker) and nucleotides 22-50 being the specific sequence; Sequence 3 in the sequence listing consists of 49 nucleotides, with nucleotides 1-21 being the HEX adapter sequence (as a marker) and nucleotides 22-49 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 manganese content in rice grains;

[0054] (2) Rice breeding;

[0055] (3) Prepare products for identification or auxiliary identification of manganese 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 manganese content in rice grains) to prepare a product for identifying rice varieties with high manganese content in rice grains.

[0059] In this article, the breeding objective may include developing rice with high manganese content in the grains. The rice may be a pure line or an inbred line.

[0060] By detecting SNP polymorphisms or genotypes in the rice manganese content-related gene HuNMn1, it can be used to identify or assist in the identification of rice grain manganese content, rice breeding, and the preparation of related products. Compared with existing technologies, the SNP molecular markers of this invention are closely related to rice grain manganese content, enabling efficient and accurate identification of manganese accumulation characteristics in rice varieties, significantly improving the accuracy and reliability of identification. Attached Figure Description

[0061] Figure 1 Genotyping of 60 rice germplasm materials using the HuNMn1 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 161 experimental samples and 60 validation samples in the following examples are 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. The biological materials are available to the public from the applicant and are intended solely for the purpose of replicating the experiments of this invention and shall not be used for any other purpose.

[0066] Example 1: HuNMn1 Genotyping Analysis and Molecular Marker Development

[0067] 1. Planting and harvesting of materials

[0068] The experimental field was a typical paddy field in southern my country, with a soil manganese content of 390.01 mg / kg and a pH of 6.042.

[0069] 161 experimental samples and 60 validation samples were planted in 2023. The specific procedures from sowing onwards 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 per row, with a plant spacing of 30 cm. After the rice matured, grain samples were collected. To avoid boundary effects, the two plants adjacent to the aisle were discarded, and the grains from the remaining plants were harvested together.

[0070] 2. Determination of manganese content in rice grains

[0071] 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 manganese content determination.

[0072] The manganese 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 manganese content data of rice grains. All sample measurements were repeated three times. The manganese content of rice grains was determined by inductively coupled plasma atomic emission spectrometry, and the results are shown in Table 1.

[0073] Table 1. Manganese content in rice grains and HuNMn1 genotypes of 161 rice varieties

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] 3. HuNMn1 genotyping and molecular marker development

[0081] Genotypic analysis of the HuNMn1(7_8872318) genotype in 161 rice MCC accessions was performed, and t-tests were used to compare the statistical differences in grain manganese content among different genotypes. P-values ​​were 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.

[0082] Table 2. Manganese content in grains of rice germplasm materials of different genotypes

[0083]

[0084] In summary, using phenotypic and genotypic data of total manganese content in grains from 161 rice MCC population materials, genome-wide association analysis located a SNP locus Chr7_8872318 associated with total manganese content in rice grains, named HuNMn1. This locus is located at position 8872318 on chromosome 7 of the rice Nipponbare genome sequence information, and its nucleotide is A or T (the SNP locus is the 29th nucleotide of sequence 1 in the sequence listing, where w represents A or T). The SNP locus has two genotypes: AA and TT. Genotype AA is homozygous for SNP A, and genotype TT is homozygous for SNP T.

[0085] The significant differences in grain manganese content corresponding to different allelic genotypes of the screened SNP loci were analyzed, and the results are as follows: Based on the sequencing results of 161 germplasm resources in the previous study (Table 1), the low manganese genotype of 113 materials was AA, with an average grain manganese content of 26.216 mg / kg; the high manganese genotype of 48 materials was TT, with an average grain manganese content of 41.933 mg / kg. The grain manganese content of rice materials corresponding to AA was significantly lower than that of rice materials corresponding to TT (P<0.01). The SNP locus HuNMn1 can be used as a molecular marker for identifying or assisting in the identification of grain manganese content in different rice varieties.

[0086] Example 2: Development of KASP molecular marker primer set for SNP locus HuNMn1 and establishment of SNP locus genotyping method

[0087] KASP molecular marker primer sets were designed for the upstream and downstream sequences of the superior allelic variant site 7_8872318. The KASP-labeled primer sets consisted of two upstream specific primers (primer A and primer B) and one downstream universal primer (primer C) (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.

[0088] Primers A and C amplify single-stranded DNA molecules with SNP site T; primers B and C amplify single-stranded DNA molecules with SNP site A. The fluorescence signal of the fluorescent group in the template binding 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.

[0089] Table 3. Primer sequences for HuNMn1 molecular markers

[0090]

[0091] The specific steps for detecting the gene type of SNP marker Chr7_8872318 (HuNMn1) using the KASP marker primer set are as follows:

[0092] 1) PCR amplification system and procedure

[0093] DNA template extraction: DNA 0.8 μL, 2×Master mix 0.4 μL, primer working solution 0.022 μL, ddH2O 0.4 μL.

[0094] PCR amplification system: DNA 0.8 μL, 2×Master mix 0.4 μL, primer working solution 0.022 μL, ddH2O 0.4 μL.

[0095] Two of the Master mixes were purchased from LGC, part number: 1536 Formulation V4.0TF.

[0096] PCR reaction procedure: Step 1: Pre-denaturation at 94℃ for 15 min;

[0097] 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;

[0098] 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;

[0099] Step 4: 94℃ for 20s, anneal at 57℃ for 60s, 26 cycles.

[0100] 2) Genotyping

[0101] After the PCR reaction, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) were used to convert the fluorescence signal 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. T-type samples showed FAM fluorescence, distributed near the x-axis; A-type samples showed HEX fluorescence, distributed near the y-axis; and samples with no detected signal were distributed near the origin.

[0102] 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.

[0103] The results are as follows:

[0104] If only the FAM group shows a fluorescent signal, then the genotype of the SNP site HuNMn1 of the rice being tested is TT (i.e., the SNP site HuNMn1 in the rice genome is a homozygous T).

[0105] If only the HEX group shows a fluorescent signal, then the HuNMn1 genotype of the rice being tested is AA (i.e., the HuNMn1 SNP site in the rice genome is homozygous for A).

[0106] Example 3: Application of HuNMn1 molecular marker and its identification primer set in the identification of manganese content in rice.

[0107] Using the HuNMn1 marker developed in Example 1, genotyping was performed on 60 different types of rice germplasm resources. The identification method was the same as in Example 2. The results are shown in Table 4.

[0108] The results showed that the genotype of 30 materials was AA and the genotype of 30 materials was TT. The detection results of HuNMn1 marker were consistent with the sequencing results (Table 1), indicating that the KASP marker identification primer set could accurately identify the genotype of HuNMn1, a gene related to grain manganese content, in different rice germplasm resources.

[0109] Table 4. Manganese content in rice grains and genotype of TagSNP-8872318 in 60 rice varieties

[0110]

[0111]

[0112]

[0113] Of the 60 rice germplasm resources, 30 materials had the AA genotype with an average grain manganese content of 19.472 mg / kg, and 30 materials had the TT genotype with an average grain manganese content of 29.350 mg / kg (Table 5). Significance analysis of the 60 validation materials showed that the grain manganese content of the rice materials corresponding to the AA genotype was significantly lower than that of the rice materials corresponding to the TT genotype (P < 0.001). This marker can effectively classify germplasm materials with the same grain manganese content into corresponding genotypes, and the phenotypic grain manganese content data are correlated with the genotype identification results. Therefore, the developed molecular marker can accurately identify the genotype of the grain manganese content-related SNP locus HuNMn1 in rice germplasm resources.

[0114] Table 5. Manganese content in grains of 60 rice varieties

[0115]

[0116] 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. Application of substances used to detect SNP polymorphisms or genotypes in the rice genome in any of the following: (1) To identify or assist in the identification of manganese content in rice grains; (2) Rice breeding; (3) Prepare products for identification or auxiliary identification of manganese content in rice grains; (4) Prepare rice breeding products; The SNP site is a SNP site on rice chromosome 7, and its nucleotide type is A or T, which is the 29th nucleotide of sequence 1 in the sequence listing. The genotype of the SNP is AA or TT, where AA is the homozygous type of the SNP being A, and TT is the homozygous type of the SNP being T; the grain manganese content of the rice tested with the genotype TT of the SNP is higher or candidate higher than that of the rice tested with the genotype AA of the SNP. The purpose of the breeding is to select rice varieties with high manganese content in rice grains.

2. The application according to claim 1, 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).

3. The application according to claim 2, 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 2 in the sequence listing or whose nucleotide sequence is single-stranded DNA at positions 22-50 of sequence 2 in the sequence listing; Primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing or whose nucleotide sequence is single-stranded DNA at positions 22-49 of sequence 3 in the sequence listing. The primer C nucleotide sequence is the single-stranded DNA molecule of sequence 4 in the sequence listing.

4. A method for identifying or assisting in the identification of manganese content in rice grains, characterized in that: This includes detecting the genotype of SNP sites in the genome of rice to be tested, and identifying or assisting in the identification of manganese 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 T, which is the 29th nucleotide of sequence 1 in the sequence listing. The SNP has a genotype of AA or TT, where AA is a homozygous form of the SNP being A, and TT is a homozygous form of the SNP being T; the grain manganese content of the rice tested with the SNP genotype TT is higher or candidate higher than that of the rice tested with the SNP genotype AA.

5. A method for rice breeding, characterized in that: The method includes detecting the genotype of the SNP in claim 1 in the rice genome, selecting rice with the genotype TT of the SNP as a parent for breeding, wherein TT is a homozygous type of the SNP being T; the purpose of the breeding is to select rice varieties with high manganese content in rice grains.

6. The application of the method according to claim 4 or 5 in rice breeding; the purpose of the breeding is to select rice varieties with high manganese content in rice grains.

7. A nucleotide sequence is the application of sequence 1 of the sequence listing in any of the following ways: (1) To identify or assist in the identification of manganese content in rice grains; (2) Rice breeding; (3) Prepare products for identification or auxiliary identification of manganese content in rice grains; (4) Prepare rice breeding products; The purpose of the breeding is to select rice varieties with high manganese content in rice grains; The manganese content of rice grains can be identified by identifying the genotype of the SNP site in sequence 1 of the sequence listing. The SNP site is a SNP site on rice chromosome 7, and its nucleotide type is A or T, which is the 29th nucleotide of sequence 1 in the sequence listing. The genotype of the SNP locus is AA or TT, where AA is the homozygous type of the SNP being A, and TT is the homozygous type of the SNP being T; the grain manganese content of the tested rice with the SNP genotype TT is higher or candidate higher than that of the tested rice with the SNP genotype AA.

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