Rice low-cadmium excellent allelic OsTF11hap1 and application thereof

Abstract

The invention discloses a low-cadmium excellent allelic rice OsTF11hap1 and application of the low-cadmium excellent allelic rice The invention belongs to the technical field of biology, and particularly relates to rice low-cadmium excellent allelic OsTF11hap1 and application thereof. The molecular marker provided by the invention is an InDel molecular marker, SNP1 or / and SNP2, and the site of the InDel molecular marker is a DNA molecule at the 665-673 site of SEQ ID No: 2; the SNP1 site is a DNA molecule at the 707th site of SEQ ID No: 2; an SNP2 site is a DNA molecule at the 752th site of SEQ ID No: 2; the molecular marker can be applied to (1) identification or auxiliary identification of the cadmium content of rice grains; 2) rice breeding; (3) preparing a product for identifying or assisting in identifying the cadmium content of the rice grains; and 4) preparing a rice breeding product. And the rice of the haplotype OsTF11hap1 can be selected for low-cadmium rice breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and specifically relates to rice low-cadmium superior allele OsTF11 hap1 and its applications. Background Art

[0002] Rice is a major food crop in my country, and its safe production is crucial to national food security. However, cadmium pollution seriously threatens the safe production of rice. Molecular breeding is an advanced breeding method that is carried out through molecular marker-assisted breeding and molecular design breeding. This method has significant advantages, can shorten the breeding cycle, improve breeding efficiency, and maintain phenotypic stability, and plays an important role in the breeding process of rice with low cadmium accumulation.

[0003] The core of molecular marker-assisted breeding is to explore and utilize the low-cadmium natural variation sites, and to screen genotypes by detecting genetic markers that are closely linked to the target trait. The introduction of excellent low-cadmium natural variation has made significant progress in reducing the cadmium content of rice grains. For example, Tang Wei successfully selected sterile lines carrying the low-cadmium gene OsLCT1 and OsHMA3 fragments by combining molecular marker-assisted and backcross breeding. The cadmium content of brown rice in these improved sterile lines was significantly lower than that of the recipient parents, demonstrating the potential of this method. The research of Sun et al. further advanced this field. They used the three-line hybrid method, with Lemont, 02428 and R180 as parents, and bred "Lushan Silk Seedling" through the low-cadmium molecular marker qCd7-assisted screening. The cadmium content of the grain of this new variety was significantly lower than that of the conventional control group, showing the potential to effectively reduce cadmium accumulation in grains. This achievement provides new ideas and methods for breeding low-cadmium rice varieties under different soil conditions. At present, researchers have identified multiple low-cadmium natural variations from genes such as OsCd1 and OsNramp5. These findings provide valuable genetic resources for further improving rice varieties and reducing the cadmium content in grains. Identifying more excellent low-cadmium alleles can provide richer genetic resources for breeding low-cadmium rice varieties and further promote the development of safe rice production. Summary of the invention

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

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

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

[0007] (2) Rice breeding;

[0008] (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains;

[0009] (4) preparing rice breeding products;

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

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

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

[0013] The present invention also provides a method for identifying or assisting in identifying the cadmium content of rice grains, comprising detecting the polymorphism or genotype of a molecular marker in a rice genome to be detected or a rice haplotype, and identifying or assisting in identifying the cadmium content of rice grains according to the polymorphism or genotype, wherein the molecular marker is an InDel molecular marker, SNP1 or / and SNP2, wherein the InDel molecular marker position is a DNA molecule at position 665-673 of SEQ ID No: 2; the SNP1 position is a DNA molecule at position 707 of SEQ ID No: 2; and the SNP2 position 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 nucleotides 665-673 of SEQ ID No: 2 of the AA genotype are a gene type lacking 5'-GCGGCGGCG-3', and the nucleotides 665-673 of SEQ ID No: 2 of the BB genotype are a gene type containing 5'-GCGGCGGCG-3';

[0015] The SNP1 contains two genotypes, TT and AA, wherein the TT genotype is a genotype in which the 707th nucleotide of SEQ ID No: 2 is T, and the AA genotype is a genotype in which the 707th nucleotide of SEQ ID No: 2 is A;

[0016] The SNP2 contains two genotypes, TT and GG, wherein the TT genotype is a genotype in which the 752nd nucleotide of SEQ ID No: 2 is T, and the GG genotype is a genotype in which the 752nd nucleotide of SEQ ID No: 2 is G;

[0017] The cadmium content in the grain of the rice to be tested, in which the nucleotides at positions 665-673 of SEQ ID No:2 are of a gene type lacking 5'-GCGGCGGCG-3', the nucleotides at position 707 are TT, and the nucleotides at position 752 are TT, is lower than that in which the nucleotides at positions 665-673 of SEQ ID No:2 are of a gene type containing 5'-GCGGCGGCG-3', the nucleotides at position 707 are AA, and the nucleotides at position 752 are GG.

[0018] The rice haplotype may be OsTF11 hap1 and OsTF11 hap2 , wherein the haplotype OsTF11 hap1 The type is as follows: the nucleotides 665-673 of SEQ ID No: 2 of the OsTF11 gene are missing 5'-GCGGCGGCG-3', the nucleotides 707 and 752 are TT.

[0019] The haplotype OsTF11 hap2 The type is as follows: the nucleotides at positions 665-673 of SEQ ID No: 2 of the OsTF11 gene contain 5'-GCGGCGGCG-3', the nucleotide at position 707 is AA, and the nucleotide at position 752 is GG.

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

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

[0022] The haplotype OsTF11 hap1 The cadmium content of seeds was lower than that of haplotype OsTF11 hap2 .

[0023] The present invention also provides a method for rice breeding, which comprises detecting the polymorphism or genotype or haplotype of the molecular markers mentioned above in the rice genome, and selecting the following types for breeding:

[0024] 1) Selecting the rice to be tested of the gene type in which the nucleotides at positions 665-673 of SEQ ID No: 2 are missing 5'-GCGGCGGCG-3', the nucleotides at position 707 are TT, and the nucleotides at position 752 are TT as the parent for breeding;

[0025] 2) Select haplotype OsTF11 hap1 The tested rice is used as a parent for breeding, and the haplotype OsTF11 hap1 The nucleotides 665-673 of SEQ ID No: 2 of the OsTF11 gene are of a gene type lacking 5'-GCGGCGGCG-3', the nucleotide 707 is TT, and the nucleotide 752 is TT.

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

[0027] (1) Using the genomic DNA of the rice to be tested as a template, the above primer set is used to perform KASP molecular marker detection;

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

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

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

[0031] In the above method, the primer dissolution and preparation method can be: dissolve the freeze-dried primer in sterile ultrapure water to prepare a 100 μM stock solution. Before use, dilute the stock solution to 10 μM as a working solution. As a KASP-labeled primer working solution, store it at -20°C for future use.

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

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

[0034] In the above method, the reaction procedure of KASP labeling can be: 1. Initial denaturation: 94°C, 15 minutes; 2. Cycling steps (10 cycles): 94°C, 20 seconds; 61-55°C, 60 seconds (0.6°C lowered for each cycle); 3. Cycling steps (26 cycles): 94°C, 20 seconds; 55°C, 60 seconds; 4. Termination reaction: 37°C, 30 seconds.

[0035] In the above method, the method for determining the genotype of the SNP of the rice to be tested can be: after the PCR reaction is completed, the fluorescence signal is converted into an analyzable value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) to read the fluorescence data of the reaction product. Genotyping is performed using the terminal end reading fluorescence value, and the fluorescence scanning results are graphically displayed using the R software package. The TT base type carries FAM fluorescence and is distributed near the x-axis; the AA base type carries HEX fluorescence and is distributed near the y-axis; and samples without detection signals are distributed near the origin.

[0036] The application of the above-mentioned method in rice breeding also falls within the scope of protection claimed by the present invention.

[0037] The present invention also provides the following product, which contains the substance described above, and the product can be any of the following:

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

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

[0040] C3) Products for 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 chip. Among them, the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein DNA binding reaction, and a chip based on fluorescent molecule DNA binding reaction.

[0042] In the above-mentioned application or product, the substance is the following D1), D2) or D3):

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

[0044] D2) the substance is a PCR reagent containing the primer combination 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-mentioned applications, methods and products, the primer composition may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be connected to a nucleic acid. Markers include but are not limited to dyes; radioactive labels, such as 32P; binding moieties, such as biotin; haptens, such as digoxin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or move the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly read the sequence) without a marker.

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

[0048] The primer A is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No: 3 or a single-stranded DNA whose nucleotide sequence is the 22nd to 41st position of SEQ ID No: 3 in the sequence list;

[0049] The primer B is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No: 4 or a single-stranded DNA whose nucleotide sequence is the 22nd to 41st position of SEQ ID No: 4 in the sequence list;

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

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

[0052] The present invention also provides the use of the DNA molecule described above in any of the following:

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

[0054] (2) Rice breeding;

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

[0056] (4) Preparing rice breeding products.

[0057] The material for detecting the InDel molecular marker or SNP site polymorphism and genotype or the material for detecting the rice haplotype can be combined with other materials (such as materials for detecting the single nucleotide polymorphism or genotype of other molecular markers related to the cadmium content in rice grains) to prepare a product for identifying rice varieties with low cadmium content in grains.

[0058] Herein, the purpose of the breeding may include cultivating rice varieties with low cadmium content in grains. The rice may be a pure line or an inbred line, such as Guichao No. 2, Nipponbare, or a hybrid of the two.

[0059] The rice may be a rice variety.

[0060] The experimental results show that OsTF11 hap1 The introduction of rice varieties can significantly reduce the cadmium content in grains. This discovery not only enriches our understanding of the cadmium accumulation regulatory network of rice, but also provides a powerful tool for the targeted improvement of low-cadmium rice varieties in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Haplotype analysis of OsTF11.

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

[0063] Figure 3 This is the cadmium accumulation phenotype analysis of OsTF11 NIL line. a. NIL rice field phenotype; b. NIL rice plant height; c. NIL rice grain cadmium content. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0065] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0066] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0067] The 520 rices and 30 rices in the following examples are recorded 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. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0068] Guichao No. 2, Guihuahuang and Nihonbare in the following embodiments have been recorded 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 amajor facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies. Nat Commun. 2019 Jun 12; 10 (1): 2562. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0069] The following examples were processed using GraphPad Prism 7.0 software, and the t test was used to compare the statistical differences in the cadmium content of grains of different combined haplotypes. The P value was calculated by Tukey's test. A P value less than 0.05 was defined as a statistically significant difference; a P value less than 0.01 was defined as a statistically extremely significant difference.

[0070] Example 1. OsTF11 haplotype analysis

[0071] HaploView software was used to perform haplotype analysis on the OsTF11 gene of 430 rice micro-core resource germplasms, and the sequence SNP and InDel sequences were concatenated for haplotype analysis. The grain cadmium content data of the haplotypes were processed using GraphPad Prism7.0 software, and the statistical differences in grain cadmium content of different combined haplotypes were compared using t test. The P value was calculated by Tukey's test. A P value less than 0.05 was defined as a statistically significant difference; a P value less than 0.01 was defined as a statistically extremely significant difference.

[0072] The coding region of the OsTF11 gene was sequenced using resequencing data from the micro-core germplasm library of 430 rice varieties. The study found three key mutation sites: the gene type of nucleotides 664-665 of SEQ ID No:1 is 5'-GT-3', the c.698T>A (p.F233Y) missense mutation at position 698 (SNP1 site), and the c.743T>G (p.V248G) missense mutation at position 743 (SNP2 site). These three mutation sites show a high degree of linkage (linkage disequilibrium coefficient r 2 =0.976)( Figure 1 ), forming two haplotypes OsTF11 hap1 and OsTF11 hap2 The corresponding physical positions in the Nipponbare reference genome sequence are Chr2:29238662-29238663, Chr2:29238629, and Chr2:29238584.

[0073] The gene type in which the nucleotides 665-673 of SEQ ID No: 2 at the InDel site lack 5'-GCGGCGGCG-3' was defined as the AA genotype, and the gene type in which the nucleotides 665-673 of SEQ ID No: 2 at the InDel site contained 5'-GCGGCGGCG-3' was defined as the BB genotype.

[0074] The genotype in which the nucleotide type at position 707 of SEQ ID No: 2 at the SNP1 site is T is defined as a TT genotype, and the genotype in which the nucleotide type at position 707 of SEQ ID No: 2 at the SNP1 site is A is defined as an AA genotype.

[0075] The genotype in which the nucleotide at position 752 of SEQ ID No: 2 at the SNP2 site is T is defined as a TT genotype, and the genotype in which the nucleotide at position 752 of SEQ ID No: 2 at the SNP2 site is G is defined as a GG genotype.

[0076] By integrating the phenotypic data of grain cadmium content of 430 rice germplasm resources, these rice varieties were divided into two haplotypes with significant differences ( Figure 1 ): Low cadmium accumulation type OsTF11 hap1 and high cadmium accumulation type OsTF11 hap2 . Carry OsTF11 hap1 199 rice varieties with the same haplotype showed lower grain cadmium accumulation ability, with an average cadmium content of 0.28±0.22 mg / kg; hap2 The 231 varieties of the haplotype showed higher levels of grain cadmium accumulation, with an average cadmium content of 0.51±0.27 mg / kg.

[0077] Among them, haplotype OsTF11 hap1 The type is as follows: the nucleotides at positions 665-673 of SEQ ID No: 2 of the OsTF11 gene are deleted 5'-GCGGCGGCG-3' (abbreviated as Deletion), the nucleotides at positions 707 and 752 are TT.

[0078] Among them, haplotype OsTF11 hap2 The following types are used: a gene type in which the nucleotides at positions 665-673 of SEQ ID No: 2 of the OsTF11 gene contain 5'-GCGGCGGCG-3' (abbreviated as Insertion), the nucleotides at position 707 are AA, and the nucleotides at position 752 are GG.

[0079] Table 2. Haplotype detection results of 430 rice germplasms and phenotype study of cadmium content in grains

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] The results of the study showed that the cadmium content in rice grains can be identified and assisted in identification by detecting the haplotype type of rice.

[0091] Example 2: Development of OsTF11 KASP molecular markers

[0092] 1. Primer set design

[0093] The KASP molecular marker primer set was designed for the upstream and downstream sequences of the superior allele c.698T>A (p.F233Y). The KASP marker primer set consists of two upstream specific primers (primer A and primer B) and one downstream universal primer (primer C) (Table 1). 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.

[0094] Table 1. OsTF11 KASP molecular marker primer sequence information

[0095]

[0096] Note: The underlined information is the fluorescent tag sequence.

[0097] The single-stranded DNA molecules indicated by primer A and primer C amplify the fragment with SNP site A; the single-stranded DNA molecules indicated by primer B and primer C amplify the fragment with SNP site T. The fluorescent signal of the fluorescent group bound to the FAM sequence or HEX sequence in the template can be read by an enzyme marker or a fluorescent quantitative PCR instrument. The specific primer sequences were synthesized by Zhongyujin Labeling (Beijing) Biotechnology Co., Ltd.

[0098] 2. KASP reaction

[0099] Using the designed KASP markers, 30 rice materials were selected for genotype identification. The specific identification steps are as follows:

[0100] Preparation of KASP-labeled primer working solution: Dissolve the lyophilized primer in sterile ultrapure water to prepare a 100 μM stock solution. Before use, dilute the stock solution to 10 μM as a working solution. As a KASP-labeled primer working solution, store at -20°C for future use.

[0101] The PCR amplification system was as follows: 50 ng of genomic DNA, 0.02 μL of primer mixture, 0.6 μL of 1×KASP Mix (Low Rox), and the reaction system was supplemented to 3 μL with sterile ultrapure water.

[0102] The PCR reaction program is as follows: 1. Initial denaturation: 94°C, 15 minutes; 2. Cycling steps (10 cycles): 94°C, 20 seconds; 61-55°C, 60 seconds (0.6°C decreases for each cycle); 3. Cycling steps (26 cycles): 94°C, 20 seconds; 55°C, 60 seconds. 4. Termination reaction: 37°C, 30 seconds.

[0103] A blank control (NTC) in which no template DNA was added to the reaction system was set up at the same time in the experiment, and one control was set up for each plate.

[0104] 3. Genotyping

[0105] After the PCR reaction was completed, the fluorescence signal was converted into an analyzable value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) to read the fluorescence data of the reaction product. The fluorescence scanning results were graphically displayed using the R software package. The TT base type had FAM fluorescence, which was distributed near the x-axis; the AA base type had HEX fluorescence, which was distributed near the y-axis; and samples with no detected signal were distributed near the origin.

[0106] The excitation wavelength of FAM is 485nm, and the emission wavelength is 520nm. The excitation wavelength of HEX is 535nm, and the emission wavelength is 556nm. The excitation wavelength of the system reference fluorescence ROX is 575nm, and the emission wavelength is 610nm.

[0107] result( Figure 2 ) showed that the fluorescent signal points of 15 materials were red, indicating that the 5' end of the primer was connected to the HEX sequence and the genotype was AA; the fluorescent signal points of 15 materials were green, indicating that the 5' end of the primer was connected to the FAM sequence and the genotype was TT; the negative control NTC (no template control) had no fluorescent signal. The genotyping results of KASP molecular markers were consistent with the previous sequencing results, proving the accuracy of KASP molecular markers.

[0108] Table 3. Data on genotypes and grain cadmium content identified by KASP molecular marker primer set

[0109]

[0110]

[0111] Example 3, OsTF11 hap1 Study on the phenotype of recombinant inbred lines in the field

[0112] Rice hybrid breeding was completed 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 and Ecology, Chinese Academy of Sciences from 2018 to 2022. Guichao No. 2, which has good comprehensive traits and breeding application potential but high grain cadmium accumulation to be improved, was selected as the recipient parent, and hybridized with the donor parent Nipponbare with low grain cadmium accumulation as the donor parent and obtained after multiple generations of backcrossing.

[0113] To study OsTF11 hap1 Effect of gene to carry high cadmium haplotype OsTF11 hap2 The OsTF11 gene segment of Guichao 2 was replaced by the japonica rice variety Guihuahuang (carrying the low-cadmium haplotype OsTF11) using a molecular marker-assisted method. hap1 ) and successfully constructed two near-isogenic lines (NIL-L1 and NIL-L2). The specific construction method is as follows: hybridize with Guichao No. 2 as the recurrent parent and Guihuahuang as the donor parent to obtain the F1 generation. Backcross the F1 with Guichao No. 2 as the male parent to obtain BC1F1. The KASP molecular marker in Example 2 was used to identify the genotype of BC1F1, and the individual plants that were heterozygous in the target segment and had a higher background recovery rate were selected to continue backcrossing with Guichao No. 2. This was done 5 times in a row to BC5F1, and molecular markers were used to assist in the selection of individual plants with heterozygous target segments after each backcross. In the BC5F1 generation, the individual plants with heterozygous target segments and genetic backgrounds closest to the recurrent parent were selected for self-pollination to obtain the BC5F2 population, and the two near-isogenic lines NIL-L1 and NIL-L2 with homozygous target segments were screened and identified by molecular markers.

[0114] Planting experiments were carried out in cadmium-contaminated fields (cadmium content was 1.3 mg / kg), and the phenotypic characteristics of the near-isogenic lines and the cadmium content in their grains were systematically evaluated.

[0115] The results showed that the plant height of Guichao 2 was 96±2.828 cm, while that of NIL-L1 and NIL-L2 was 103.8±5.315 cm and 91.13±4.09 cm, respectively. hap1 The introduction of ferrous metals had no significant effect on the growth of rice.

[0116] The cadmium content of its grains was tested. The results showed that the cadmium content of Guichao 2 was 2.813±0.096 mg / kg, while the cadmium content of NIL-L1 and NIL-L2 was 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% ( Figure 3 This result strongly confirms that the low-cadmium haplotype OsTF11hap1 It is an important superior allele variation for genetic improvement of cadmium accumulation in rice grains.

[0117] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a substance for detecting polymorphism or genotype of a molecular marker in a rice genome or a substance for detecting haplotype in any of the following: (1) Identify or assist in identifying the cadmium content in rice grains; (2) Rice breeding; (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains; (4) preparing rice breeding products; The molecular markers are InDel molecular markers, SNP1 and / or SNP2, the InDel molecular marker position is the DNA molecule at position 665-673 of SEQ ID No: 2; the SNP1 site is the DNA molecule at position 707 of SEQ ID No: 2; the SNP2 site is the DNA molecule at position 752 of SEQ ID No: 2; The haplotype is a polymorphic combination of three molecular markers, namely, the InDel molecular marker, the SNP1 and the SNP2 on a chromosome of rice.

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

2.

3. The method according to claim 1 or the use according to claim 2, characterized in that: The InDel molecular marker contains two gene types AA and BB, the nucleotides 665-673 of SEQ ID No: 2 of the AA genotype are a gene type lacking 5'-GCGGCGGCG-3', and the nucleotides 665-673 of SEQ ID No: 2 of the BB genotype are a gene type containing 5'-GCGGCGGCG-3'; The SNP1 contains two genotypes, TT and AA, wherein the TT genotype is a genotype in which the 707th nucleotide of SEQ ID No: 2 is T, and the AA genotype is a genotype in which the 707th nucleotide of SEQ ID No: 2 is A; The SNP2 contains two genotypes, TT and GG, wherein the TT genotype is a genotype in which the 752nd nucleotide of SEQ ID No: 2 is T, and the GG genotype is a genotype in which the 752nd nucleotide of SEQ ID No: 2 is G; The cadmium content in the grain of the rice to be tested, in which the nucleotides at positions 665-673 of SEQ ID No:2 are of a gene type lacking 5'-GCGGCGGCG-3', the nucleotides at position 707 are TT, and the nucleotides at position 752 are TT, is lower than that in which the nucleotides at positions 665-673 of SEQ ID No:2 are of a gene type containing 5'-GCGGCGGCG-3', the nucleotides at position 707 are AA, and the nucleotides at position 752 are GG.

4. A method for rice breeding, characterized in that: The method comprises detecting the polymorphism or genotype or haplotype of the molecular marker described in claim 1 in the rice genome, selecting 1) the tested rice of the gene type in which the nucleotides at positions 665 to 673 of SEQ ID No: 2 are missing 5'-GCGGCGGCG-3', the nucleotides at position 707 are TT, and the nucleotides at position 752 are TT as parents for breeding; 2) Haplotype is OsTF11 hap1 The tested rice is used as a parent for breeding, and the haplotype OsTF11 hap1 The nucleotides 665-673 of SEQ ID No: 2 of the OsTF11 gene are of a gene type in which 5'-GCGGCGGCG-3' is missing, the nucleotide 707 is AA, and the nucleotide 752 is GG.

5. Use of the method according to claim 1 or 4 in rice breeding.

6. The product is characterized by: The product contains the substance described in claim 1, and the product is any one of the following: C1) Products for detecting molecular marker polymorphisms or genotypes or haplotypes related to cadmium content in rice grains; C2) Products for identifying or assisting in identifying the cadmium content in rice grains; C3) Products for rice breeding.

7. The use according to claim 2 or the product according to claim 6, characterized in that: The substance is the following D1), D2) or D3): D1) the substance is a primer composition for amplifying a rice genomic DNA fragment including the molecular marker; D2) the substance is a PCR reagent containing the primer combination described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

8. The use or product according to claim 7, characterized in that: The primer composition consists of primer A, primer B and primer C; The primer A is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No: 3 or a single-stranded DNA whose nucleotide sequence is the 22nd to 40th position of SEQ ID No: 2 in the sequence list; The primer B is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No: 4 or a single-stranded DNA whose nucleotide sequence is the 22nd to 40th position of SEQ ID No: 3 in the sequence list; The nucleotide sequence of primer C is a single-stranded DNA molecule of SEQ ID No:

5.

9. A DNA molecule, characterized in that: The nucleotide sequence of the DNA molecule is SEQ ID No: 1 or SEQ ID No:

2.

10. Use of the DNA molecule according to claim 9 in any of the following: (1) Identify or assist in identifying the cadmium content in rice grains; (2) Rice breeding; (3) Preparation of products for identifying or assisting in identifying the cadmium content in rice grains; (4) Preparing rice breeding products.

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