Rice low-cadmium excellent allele OsTF11 hap1 and applications thereof

Abstract

The application discloses rice low-cadmium excellent allele OsTF11 hap1 and application thereof. The application belongs to the technical field of biotechnology, and particularly relates to rice low-cadmium excellent allele OsTF11 hap1 and application thereof. The molecular marker provided by the application is an InDel molecular marker, SNP1 or / and SNP2, the InDel molecular marker is a DNA molecule at positions 665-673 of SEQ ID No: 2; the SNP1 site is a DNA molecule at position 707 of SEQ ID No: 2; and the SNP2 site is a DNA molecule at position 752 of SEQ ID No: 2. The above-mentioned molecular marker can be applied to: 1) identification or auxiliary identification of cadmium content in rice grains; 2) rice breeding; 3) preparation of products for identification or auxiliary identification of cadmium content in rice grains; and 4) preparation of products for rice breeding. Rice with a haplotype of OsTF11 hap1 may be selected for low-cadmium rice breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a rice low-cadmium excellent allele OsTF11 hap1 and application thereof. BACKGROUND

[0002] Rice is the main food crop in China, and cadmium pollution seriously threatens the safe production of rice. Molecular breeding is an advanced breeding method, which is carried out through molecular marker-assisted breeding and molecular design breeding. This method has significant advantages, can shorten the breeding period, improve the breeding efficiency, and maintain the stability of phenotype, and plays an important role in the breeding of low-cadmium accumulation rice.

[0003] The core of molecular marker-assisted breeding lies in the excavation and utilization of low-cadmium natural variation sites, and the genotyping is carried out by detecting genetic markers closely linked to the target traits. The introduction of excellent low-cadmium natural variation has made significant progress in reducing the cadmium content in rice grains. For example, Tang Wei successfully bred a sterile line carrying a low-cadmium gene OsLCT1 and OsHMA3 fragment by combining molecular marker-assisted and backcross breeding. The cadmium content of the improved sterile line is significantly lower than that of the receptor parent, which shows the potential of this method. Sun et al. further promoted this field. They used three-line hybridization method with Lemont, 02428 and R180 as parents, and bred "Lushan Silk Miao" by low-cadmium molecular marker qCd7 assisted screening. The cadmium content of the new variety is significantly lower than that of the conventional control group, which shows the potential of efficiently reducing the cadmium accumulation in rice grains. This achievement provides a new idea and method for breeding low-cadmium rice varieties under different soil conditions. At present, researchers have identified multiple low-cadmium natural variations from OsCd1 、 OsNramp5 and other genes. These findings provide valuable genetic resources for further improving rice varieties and reducing grain cadmium content. Identifying more excellent low-cadmium allele variations can provide more abundant genetic resources for breeding low-cadmium rice varieties and further promote the development of safe rice production. SUMMARY

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

[0005] In order to solve the above problems, the present application provides the application of a substance for detecting the polymorphism or genotype of a molecular marker in the genome of rice or a substance for detecting a haplotype in any of the following:

[0006] (1) identifying or assisting in identifying the cadmium content in rice grains;

[0007] (2) rice breeding;

[0008] (3) preparing a product for identifying or assisting in identifying the cadmium content in rice grains;

[0009] (4) preparing a product for rice breeding;

[0010] The molecular marker is an InDel molecular marker, SNP1 or / and SNP2, the InDel molecular marker is a DNA molecule at positions 665-673 of SEQ ID No: 2, the SNP1 site is a DNA molecule at position 707 of SEQ ID No: 2, and the SNP2 site is a DNA molecule at position 752 of SEQ ID No: 2;

[0011] The haplotype is a polymorphic combination of the InDel molecular marker and the SNP1 and the SNP2 on one chromosome of rice.

[0012] With Nipponbare as the reference genome, the physical positions of the InDel molecular marker, SNP1 or / and SNP2 are respectively Chr2:29238662-29238663, Chr2:29238629, and Chr2:29238584.

[0013] The application further provides a method for identifying or assisting in identifying the cadmium content in rice grains, comprising detecting the polymorphism or genotype or haplotype of a molecular marker in the genome of the rice to be tested, and identifying or assisting in identifying the cadmium content in rice grains according to the polymorphism or genotype, wherein the molecular marker is an InDel molecular marker, SNP1 or / and SNP2, the InDel molecular marker is a DNA molecule at positions 665-673 of SEQ ID No: 2, the SNP1 site is a DNA molecule at position 707 of SEQ ID No: 2, and the SNP2 site is a DNA molecule at position 752 of SEQ ID No: 2.

[0014] In the above method or application, the InDel molecular marker contains two gene types AA and BB, the AA genotype has a nucleotide sequence of 5'-GCGGCGGCG-3' missing at positions 665-673 of SEQ ID No: 2, and the BB genotype has a nucleotide sequence of 5'-GCGGCGGCG-3' at positions 665-673 of SEQ ID No: 2.

[0015] The SNP1 contains two gene types TT and AA, the TT genotype is a gene type with a nucleotide T at position 707 of SEQ ID No: 2, and the AA genotype is a gene type with a nucleotide A at position 707 of SEQ ID No: 2.

[0016] The SNP2 contains two gene types, TT and GG. The TT genotype is the gene type with nucleotide T at position 752 of SEQ ID No:2, and the GG genotype is the gene type with nucleotide G at position 752 of SEQ ID No:2.

[0017] The rice grains of the tested rice with nucleotides 665-673 of SEQ ID No:2 having a gene type that lacks 5'-GCGGCGGCG-3', and nucleotides 707 and 752 are both nucleotides TT, have lower cadmium content than the rice grains of the tested rice with nucleotides 665-673 of SEQ ID No:2 having a gene type that contains 5'-GCGGCGGCG-3', and nucleotides AA and GG at position 707.

[0018] The rice haplotype can be... OsTF11 hap1 and OsTF11 hap2 The haplotype OsTF11 hap1 It is of the following type: OsTF11 The nucleotides 665-673 of SEQ ID No:2 of the gene are of the gene type with a deletion of 5'-GCGGCGGCG-3', and the nucleotide TT is at position 707, and the nucleotide TT is at position 752.

[0019] The haplotype OsTF11 hap2 It is of the following type: OsTF11 The gene SEQ ID No:2 has a gene type containing 5'-GCGGCGGCG-3' at nucleotides 665-673, nucleotide AA at position 707, and nucleotide GG at position 752.

[0020] Furthermore, the haplotype OsTF11 hap1 It can be the genotype of a DNA molecule containing SEQ ID No:1.

[0021] Furthermore, the haplotype OsTF11 hap2 It can be the genotype of a DNA molecule containing SEQ ID No:2.

[0022] The haplotype OsTF11 hap1 The cadmium content in the grains is lower than that of haplotypes. OsTF11 hap2 .

[0023] This invention also provides a method for rice breeding, the method comprising detecting polymorphisms, genotypes, or haplotypes of the molecular markers described above in the rice genome, and selecting the following types for breeding:

[0024] 1) Select rice varieties with nucleotides 665-673 of SEQ ID No:2 that are missing 5'-GCGGCGGCG-3', and with nucleotide TT at position 707 and nucleotide TT at position 752 as parents for breeding.

[0025] 2) Select haplotype as OsTF11 hap1 The rice species to be tested were used as parents in breeding, and the haplotypes... OsTF11 hap1 for OsTF11 The nucleotides 665-673 of SEQ ID No:2 of the gene are of the gene type with a deletion of 5'-GCGGCGGCG-3', and the nucleotide TT is at position 707, and the nucleotide TT is at position 752.

[0026] As an implementation method, rice breeding methods may include the following steps:

[0027] (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;

[0028] (2) After completing step (1), perform fluorescence detection to determine the genotype of the molecular marker site in the rice to be tested;

[0029] (3) Select the TT genotype as the superior rice breed.

[0030] Furthermore, the molecular marker may be SNP1.

[0031] In the above method, the primer dissolution and preparation method can be as follows: dissolve the lyophilized primers in sterile ultrapure water to prepare a 100 μM stock solution. Before use, dilute the stock solution to 10 μM as the working solution. Store the KASP-labeled primer working solution at -20 ℃ for later use.

[0032] In the above method, the KASP reaction system can be: 50 ng genomic DNA, 0.02 μL primer mixture, 0.6 μL 1×KASPMix (Low Rox), and the reaction system can be supplemented with sterile ultrapure water to 3 μL.

[0033] In the above method, KASP labeling can be performed on a regular PCR amplification instrument.

[0034] In the above method, the reaction procedure for KASP labeling can be as follows: 1. Initial denaturation: 94℃, 15 minutes; 2. Cyclic steps (10 cycles): 94℃, 20 seconds; 61-55℃, 60 seconds (decreasing by 0.6℃ per cycle); 3. Cyclic steps (26 cycles): 94℃, 20 seconds; 55℃, 60 seconds; 4. Termination of reaction: 37℃, 30 seconds.

[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. TT base types exhibit FAM fluorescence and are distributed near the x-axis; AA base types exhibit HEX fluorescence and are distributed near the y-axis; samples with no detected signal are distributed near the origin.

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

[0037] The present invention also provides a product containing the substance described above, wherein the product may be any of the following:

[0038] C1) Products that detect molecular marker polymorphisms, genotypes, or haplotypes related to cadmium content in rice grains;

[0039] C2) Products used for identifying or assisting in the identification of cadmium content in rice grains;

[0040] C3) Products used in rice breeding.

[0041] 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.

[0042] In the applications or products described above, the substance is either D1), D2), or D3).

[0043] D1) The substance is a primer composition for amplifying rice genomic DNA fragments including the molecular marker;

[0044] D2) The substance is a PCR reagent containing the primer composition described in D1);

[0045] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0046] 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).

[0047] In the above applications or products, the primer composition may consist of primer A, primer B, and primer C;

[0048] Primer A is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No:3 or a single-stranded DNA molecule whose nucleotide sequence is positions 22-41 of SEQ ID No:3 in the sequence listing;

[0049] Primer B is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No:4 or a single-stranded DNA molecule whose nucleotide sequence is positions 22-41 of SEQ ID No:4 in the sequence listing;

[0050] The primer C nucleotide sequence is a single-stranded DNA molecule of SEQ ID No:5.

[0051] The present invention also provides a DNA molecule, the nucleotide sequence of which is SEQ ID No:1 or SEQ ID No:2. Optionally, in the above applications, the DNA molecule serves as a detection target.

[0052] This invention also provides the use of the DNA molecule described above 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] Substances that detect the InDel molecular marker or SNP site polymorphism and genotype, or substances that detect rice haplotypes, 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 products for identifying rice varieties with low cadmium content in grains.

[0058] In this document, the breeding objective may include developing rice varieties with low cadmium content in the grains. The rice may be a pure line or an inbred line. For example, it may be Guichao No. 2, Nipponbare, or a hybrid of both.

[0059] The rice mentioned can be a rice variety.

[0060] Experimental results show that, OsTF11 hap1 The discovery that introducing rice varieties can significantly reduce cadmium content in grains not only enriches our understanding of the regulatory network of cadmium accumulation in rice, but also provides a powerful tool for the targeted improvement of low-cadmium rice varieties in the future. Attached Figure Description

[0061] Figure 1 for OsTF11 Haplotype analysis.

[0062] Figure 2 for OsTF11 Development of KASP molecular markers.

[0063] Figure 3 for OsTF11 Phenotypic analysis of cadmium accumulation in NIL-based rice. This included: a. field phenotype of NIL-based rice; b. plant height of NIL-based rice; and c. cadmium content in NIL-based rice grains. Detailed Implementation

[0064] 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.

[0065] 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.

[0066] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0067] The 430 and 30 rice samples in the following examples are described in: Yan H, Xu W, Xie J, Gao Y, Wu L, Sun L, Feng L, Chen X, Zhang T, Dai C, Li T, Lin X, Zhang Z, Wang X, LiF, Zhu X, Li J, Li Z, Chen C, Ma M, Zhang H, He Z. Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies. Nat Commun. 2019 Jun 12;10(1):2562. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0068] The Guichao No. 2, Guihua Huang, and Nihonbashi used in the following examples are described in: Yan H, Xu W, Xie J, Gao Y, Wu L, Sun L, Feng L, Chen X, Zhang T, Dai C, Li T, Lin X, Zhang Z, Wang X, Li F, Zhu X, Li J, Li Z, Chen C, Ma M, Zhang H, He Z. Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies. Nat Commun. 2019 Jun 12;10(1):2562. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and may not be used for any other purpose.

[0069] The following examples were processed using GraphPad Prism 7.0 software, and the t-test was used to compare the statistical differences in cadmium content in grains of different co-haplotypes. 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.

[0070] Example 1 OsTF11 Haplotype analysis

[0071] HaploView software was used to analyze 430 rice microcore germplasm resources. OsTF11 Haplotype analysis was performed on the genes by tandemly connecting the SNP and InDel sequences. The grain cadmium content data for each haplotype were processed using GraphPad Prism 7.0 software, and a t-test was used to compare the statistical differences in grain cadmium content among different combined haplotypes. 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.

[0072] Using resequencing data from a microcore germplasm bank of 430 rice varieties, the... OsTF11 Sequence analysis was performed on the coding region of the gene. Three key mutation sites were identified: a 5'-GT-3' gene type at nucleotides 664-665 of SEQ ID No:1; a c.698T>A (p.F233Y) missense mutation at position 698 (SNP1); and a c.743T>G (p.V248G) missense mutation at position 743 (SNP2). These three mutation sites showed high linkage (linkage disequilibrium coefficient r² = 0.976). Figure 1 This results in two haplotypes. OsTF11 hap1 and OsTF11 hap2 The corresponding physical locations in the Nipponbare reference genome sequence are Chr2:29238662-29238663, Chr2:29238629, and Chr2:29238584.

[0073] The gene type with nucleotides 665-673 of SEQ ID No:2 at the InDel site lacking 5'-GCGGCGGCG-3' is defined as the AA genotype, and the gene type with nucleotides 665-673 of SEQ ID No:2 at the InDel site containing 5'-GCGGCGGCG-3' is defined as the BB genotype.

[0074] The gene type with nucleotide T at SEQ ID No:2 of SNP1 is defined as TT genotype, and the gene type with nucleotide A at SEQ ID No:2 of SNP1 is defined as AA genotype.

[0075] The gene type with nucleotide T at SEQ ID No:2 of SNP2 is defined as TT genotype, and the gene type with nucleotide G at SEQ ID No:2 of SNP2 is defined as GG genotype.

[0076] By integrating the phenotypic data of grain cadmium content from 430 rice germplasm resources, these rice varieties were divided into two significantly different haplotypes ( Figure 1 Low cadmium accumulation type OsTF11 hap1 and high cadmium accumulation type OsTF11 hap2 .carry OsTF11 hap1 The 199 rice varieties with haplotypes showed lower cadmium accumulation capacity in their grains, with an average cadmium content of 0.28 ± 0.22 mg / kg; while those carrying the haplotype... OsTF11 hap2 The 231 haplotype varieties showed higher levels of cadmium accumulation in the grains, with an average cadmium content as high as 0.51 ± 0.27 mg / kg.

[0077] Among them haplotype OsTF11 hap1 It is of the following type: Yes OsTF11 The 665th to 673rd nucleotides of SEQ ID No:2 of the gene are a gene type with a deletion of 5'-GCGGCGGCG-3' (abbreviated as Deletion), and the 707th position is nucleotide TT, and the 752nd position is also a gene type with nucleotide TT.

[0078] Among them haplotype OsTF11 hap2 It is of the following type: Yes OsTF11 The gene SEQ ID No:2 has a gene type containing 5'-GCGGCGGCG-3' (abbreviated as Insertion) at nucleotides 665-673, nucleotide AA at position 707, and nucleotide GG at position 752.

[0079] Table 1. Haplotype detection results and cadmium content phenotypic study of 430 rice germplasm accessions

[0080]

[0081] The results show that the cadmium content in rice grains can be identified and used for auxiliary identification by detecting haplotype types.

[0082] Example 2 OsTF11 KASP molecular marker development

[0083] 1. Primer design

[0084] KASP molecular marker primer sets were designed for the upstream and downstream sequences of c.698T>A (p.F233Y) at the superior allelic variant site. The KASP-labeled primer sets consisted of two upstream specific primers (primer A and primer B) and one downstream universal primer (primer C) (Table 2). 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.

[0085] Table 2 OsTF11 KASP molecular marker primer sequence information

[0086]

[0087] Note: Underlined information represents fluorescent tag sequence information.

[0088] Primers A and C amplify single-stranded DNA molecules with SNP site A; primers B and C amplify single-stranded DNA molecules with SNP site T. 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.

[0089] 2. KASP reaction

[0090] Using the designed KASP marker, genotyping was performed on 30 rice accessions. The specific identification steps are as follows:

[0091] Preparation of KASP-labeled primer working solution: Dissolve the lyophilized primers in sterile ultrapure water to prepare a 100 μM stock solution. Before use, dilute the stock solution to 10 μM to prepare the working solution. Store the KASP-labeled primer working solution at -20 ℃ for later use.

[0092] The PCR amplification system consisted of 50 ng of genomic DNA, 0.02 μL of primer mixture, 0.6 μL of 1×KASP Mix (Low Rox), and sterile ultrapure water to bring the reaction volume to 3 μL.

[0093] The PCR reaction program was as follows: 1. Initial denaturation: 94℃, 15 minutes; 2. Cycling steps (10 cycles): 94℃, 20 seconds; 61-55℃, 60 seconds (decreasing by 0.6℃ per cycle); 3. Cycling steps (26 cycles): 94℃, 20 seconds; 55℃, 60 seconds; 4. Termination reaction: 37℃, 30 seconds.

[0094] The experiment also included a blank control (NTC) in the reaction system without template DNA, with one control per plate.

[0095] 3. Genotyping

[0096] 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. TT base types showed FAM fluorescence, distributed near the x-axis; AA base types showed HEX fluorescence, distributed near the y-axis; samples with no detected signal were distributed near the origin.

[0097] 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.

[0098] result( Figure 2 The results showed that 15 materials had red fluorescent signals, indicating that their primers had HEX sequences attached to their 5' ends, and the genotype was AA; 15 materials had green fluorescent signals, indicating that their primers had FAM sequences attached to their 5' ends, and the genotype was TT; the negative control NTC (no template control) showed no fluorescent signal. The genotyping results of the KASP molecular marker were consistent with the previous sequencing results, proving the accuracy of the KASP molecular marker.

[0099] Table 3. Genotype and Grain Cadmium Content Data Identified Using KASP Molecular Marker Primers

[0100]

[0101] Example 3, OsTF11 hap1 Field phenotypic study of recombinant inbred lines

[0102] The hybrid rice breeding was completed between 2018 and 2022 at the Hainan Lingshui Breeding Base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the Changsha Beishan Breeding Base of the Institute of Subtropical Agriculture Ecology, Chinese Academy of Sciences. Guichao No. 2, a rice variety with good overall traits and breeding potential but high cadmium accumulation in its grains requiring improvement, was selected as the recipient parent. It was then hybridized with Nipponbare, a donor parent with low cadmium accumulation in its grains, and backcrossed multiple times to obtain the hybrid rice.

[0103] For research OsTF11 hap1 The effect of genes to carry high cadmium haplotypes OsTF11 hap2 Guichao 2 was used as the recipient parent, and the gene was transferred from Guichao 2 using a molecular marker-assisted method. OsTF11 Gene segment replacement with that from the japonica rice variety Guihuahuang (carrying a low-cadmium haplotype) OsTF11 hap1 Two near-isogenic lines (NIL-L1 and NIL-L2) were successfully constructed using the corresponding fragments from the original gene. The specific construction method is as follows: Guichao 2 was used as the recurrent parent and Guihua Huang as the donor parent for hybridization to obtain the F1 generation. Guichao 2 was used as the male parent to backcross the F1 generation to obtain BC1F1. The genotype of BC1F1 was identified using the KASP molecular markers from Example 2. Individual plants that were heterozygous in the target segment and had a high background recovery rate were selected and backcrossed with Guichao 2. This backcrossing was repeated five times until BC5F1. After each backcross, molecular markers were used to select heterozygous individuals in the target segment. In the BC5F1 generation, individuals that were heterozygous in the target segment and had a genetic background most similar to the recurrent parent were selected for self-pollination to obtain the BC5F2 population. Molecular marker identification was used to screen from this population to obtain the two homozygous near-isogenic lines NIL-L1 and NIL-L2 in the target segment.

[0104] A planting trial was conducted in a cadmium-contaminated field (cadmium content of 1.3 mg / kg) to systematically evaluate the phenotypic characteristics and grain cadmium content of near-isogenic lines.

[0105] The results showed that the plant height of Guichao 2 was 96±2.828 cm, while the plant heights of NIL-L1 and NIL-L2 were 103.8±5.315 cm and 91.13±4.09 cm, respectively, indicating that... OsTF11 hap1 The introduction of [the substance] had no significant effect on the growth of rice.

[0106] The cadmium content in the grains of Guichao 2 was tested, and the results showed that the cadmium content in the grains of Guichao 2 was 2.813±0.096 mg / kg, while the cadmium contents in the grains of NIL-L1 and NIL-L2 were 1.583±0.049 and 1.433±0.016 mg / kg, respectively, which were significantly lower than those of the recipient parent Guichao 2 by 43.7% and 49.0%, respectively. Figure 3 (a, b, and c). This finding strongly confirms the presence of low-cadmium haplotypes. OsTF11 hap1 It is an important superior allelic variation for the genetic improvement of cadmium accumulation in rice grains.

[0107] 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. Use of a substance for detecting polymorphism or genotype of a molecular marker in a rice genome in any one of the following: (1) identifying or assisting in identifying cadmium content in rice grains; (2) low-cadmium-content-in-rice-grains assisted breeding; (3) preparing a product for identifying or assisting in identifying cadmium content in rice grains; (4) preparing a product for low-cadmium-content-in-rice-grains assisted breeding; the molecular marker is a combination of an InDel molecular marker, SNP1 and SNP2, the InDel molecular marker is a DNA molecule at positions 665-673 of SEQ ID No: 2; the SNP1 site is a DNA molecule at position 707 of SEQ ID No: 2; and the SNP2 site is a DNA molecule at position 752 of SEQ ID No: 2; the InDel molecular marker contains two genotypes AA and BB, the AA genotype of SEQ ID No: 2 at positions 665-673 is a genotype with a deletion of 5'-GCGGCGGCG-3'; and the BB genotype of SEQ ID No: 2 at positions 665-673 is a genotype containing 5'-GCGGCGGCG-3'; the SNP1 contains two genotypes TT and AA, the TT genotype is a genotype with a nucleotide T at position 707 of SEQ ID No: 2, and the AA genotype is a genotype with a nucleotide A at position 707 of SEQ ID No: 2; the SNP2 contains two genotypes TT and GG, the TT genotype is a genotype with a nucleotide T at position 752 of SEQ ID No: 2, and the GG genotype is a genotype with a nucleotide G at position 752 of SEQ ID No: 2; the cadmium content in the grains of the rice to be tested with a genotype of SEQ ID No: 2 at positions 665-673 being a genotype with a deletion of 5'-GCGGCGGCG-3', and at position 707 being a nucleotide TT, and at position 752 being a nucleotide TT is lower than that with a genotype of SEQ ID No: 2 at positions 665-673 being a genotype containing 5'-GCGGCGGCG-3', and at position 707 being a nucleotide AA, and at position 752 being a nucleotide GG; the substance is D1), D2) or D3) as follows: D1) the substance is a primer composition for amplifying a fragment of rice genomic DNA containing the molecular marker; 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).

2. Use according to claim 1, characterized in that: the primer composition consists of primer A, primer B and primer C; the primer A is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No: 3 or a single-stranded DNA with a nucleotide sequence of positions 22-41 of SEQ ID No: 3 in the sequence listing; The primer B is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No: 4 or a single-stranded DNA with a nucleotide sequence of 22-41 of SEQ ID No: 4 in the sequence listing; The primer C is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No:

5.

3. A method for identifying or aiding in the identification of the cadmium content of a rice grain, characterized in that: The method comprises detecting the polymorphism or genotype of the molecular marker in the genome of the rice to be tested, and identifying or assisting in identifying the cadmium content in the rice grain according to the polymorphism or genotype, wherein the molecular marker is a combination of an InDel molecular marker, SNP1 and SNP2, the InDel molecular marker is a DNA molecule at positions 665-673 of SEQ ID No: 2, the SNP1 site is a DNA molecule at position 707 of SEQ ID No: 2, and the SNP2 site is a DNA molecule at position 752 of SEQ ID No:

2. The InDel molecular marker contains two genotypes AA and BB, the AA genotype is a genotype with a deletion of 5'-GCGGCGGCG-3' at positions 665-673 of SEQ ID No: 2, and the BB genotype is a genotype containing 5'-GCGGCGGCG-3' at positions 665-673 of SEQ ID No:

2. The SNP1 contains two genotypes TT and AA, the TT genotype is a genotype with a nucleotide T at position 707 of SEQ ID No: 2, and the AA genotype is a genotype with a nucleotide A at position 707 of SEQ ID No:

2. The SNP2 contains two genotypes TT and GG, the TT genotype is a genotype with a nucleotide T at position 752 of SEQ ID No: 2, and the GG genotype is a genotype with a nucleotide G at position 752 of SEQ ID No:

2. The cadmium content in the grain of the rice to be tested with a genotype of a deletion of 5'-GCGGCGGCG-3' at positions 665-673 of SEQ ID No: 2, and nucleotides TT at positions 707 and 752 is lower than that of a genotype with 5'-GCGGCGGCG-3' at positions 665-673 of SEQ ID No: 2, and nucleotides AA at position 707 and nucleotides GG at position 752.

4. A method for low cadmium content in rice grain assisted breeding, characterized by: The method comprises detecting the polymorphism or genotype of the molecular marker in the genome of the rice to be tested, and selecting the rice to be tested with a genotype of a deletion of 5'-GCGGCGGCG-3' at positions 665-673 of SEQ ID No: 2, and nucleotides TT at positions 707 and 752 as the parent for breeding.

5. The method of claim 3 or 4 is used in low cadmium content in rice grain assisted breeding; the rice to be tested is selected as the parent for breeding, which has the nucleotide sequence of SEQ ID No: 2 at 665-673, the gene type of 5'-GCGGCGGCG-3' is deleted, and the nucleotide TT at 707 and the nucleotide TT at 752.

6. A product characterized by: The product contains the substance described in claim 1, and the product is any one of the following: C1) a product for detecting polymorphism or genotype of a molecular marker related to cadmium content in rice grain; C2) a product for identifying or assisting in identifying cadmium content in rice grain; C3) a product for low cadmium content in rice grain assisted breeding; The substance is D1), D2) or D3) as follows: D1) the substance is a primer composition for amplifying a fragment of rice genomic DNA containing the molecular marker, which is composed of primer A, primer B and primer C, the primer A is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No: 3 or the nucleotide sequence of 22-41 in SEQ ID No: 3 in the sequence listing, the primer B is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No: 4 or the nucleotide sequence of 22-41 in SEQ ID No: 4 in the sequence listing, the primer C is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No: 5; 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).

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