Rice mutant gene and identification method

Abstract

The application discloses a rice mutant gene and a method for identifying the same. Compared with the OsNRAMP5 gene sequence of the genome of Shu Hui 498 indica rice, the rice OsNRAMP5 mutant gene has a base A deletion mutation at a position of +1083 downstream of an initiation codon ATG. The application creatively proposes that a population is grouped first, high-throughput targeted sequencing of seed endosperm mixed DNA is used to carry out grouping screening of OsNRAMP5 gene variants, and then only seeds of a variant group with the mixed sample DNA of the variant with the highest mutation possibility are sowed to grow into seedlings, and subsequently, single plants carrying the corresponding variant in the variant group are screened and identified. The technology can greatly save time, land, reagent and other costs, and improve screening efficiency and land utilization efficiency. The method has high efficiency, good accuracy and simple operation, can better realize high-throughput, low cost and operation convenience, and is suitable for popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of rice mutation breeding technology, and particularly relates to a rice mutant gene and its identification method. Background Technology

[0002] In recent years, environmental pollution, especially heavy metal pollution in soil, has become a significant threat to crop growth, agricultural product safety, and public health. Rice is my country's most important food crop. Cadmium pollution in soil not only causes soil degradation and dysfunction, damaging plant growth and development to varying degrees, but also accumulates in rice grains through absorption and translocation by rice plants, becoming a major pathway to harm human health. Screening for low-cadmium-accumulating rice materials from existing rice germplasm resource banks is the preferred approach for breeders. However, screening for low-cadmium rice materials from existing sources is time-consuming and labor-intensive, and it's difficult to integrate various desirable traits. With advancements in scientific and technological methods, breeders are increasingly inclined to use methods such as mutagenesis of rice materials with excellent quality traits to screen for low-cadmium mutants, or targeted editing of genes related to Cd absorption and accumulation using gene editing technology to obtain novel low-cadmium rice materials.

[0003] Mutation breeding is a breeding technique that induces gene mutations in rice materials through physical and chemical factors, then screens out mutants with desired traits from the mutant population to cultivate new varieties. Japanese scientists Ishikawa et al. used carbon ion beam irradiation on the seeds of the rice variety "Koshihikari" and screened three low-cadmium mutants of the OsNRAMP5 gene from the obtained mutant library. Field trials showed that the cadmium content in the grains of these mutants in cadmium-contaminated soil (0.05 mg / kg) was significantly lower than that of the wild type (1.73 mg / kg). Since then, the breeding value of the OsNRAMP5 gene has been recognized, and various researchers in China have successively created new OsNRAMP5 gene mutants using CRISPR-Cas9 gene editing methods to rapidly cultivate low-cadmium rice varieties. Gene editing is currently the fastest and most efficient method for targeted mutation induction. However, according to the "Regulations on the Safety Management of Agricultural Genetically Modified Organisms," gene-edited crops are still considered genetically modified crops and are subject to regulation. The time when they will be allowed to be used in production remains unclear. This forces breeders to abandon this method and turn to traditional mutagenesis to meet the urgent need for low-cadmium varieties in production. Since mutagenesis is random and non-directional, specific types of mutants need to be screened from a large mutant library. If low-cadmium rice varieties are screened by measuring the cadmium content of rice, the workload and cost are enormous. Among the offspring of mutagenesis, although M1 is the generation with the most diverse mutations, most of its mutations are repairable damage to the plant genome, which will be repaired in the next generation and cannot be stably inherited by offspring. Alternatively, the damage may be too severe, resulting in failure to grow properly or produce normal seeds. Therefore, screening is generally carried out in M2. In a typical mutation breeding progeny screening system, M2 rice needs to be planted first, and after it grows, its leaves are sampled, mixed, and genomic DNA is extracted and sent for testing. Then, the sequencing analysis results are used to trace and confirm the mutation status of individual plants. This sampling process is quite labor-intensive. Furthermore, M2 plants are often produced in large numbers, requiring significant land, manpower, resources, and financial investment for planting and sampling. In addition, from a time-sensitivity perspective, the planting of M2 rice in traditional screening methods is highly dependent on climate, limiting planting to specific times. Spatially, both sampling and planting require a certain amount of land. Therefore, traditional mutation breeding requires screening for mutants of specific genes from large populations of tens of thousands or more, which is generally difficult for research teams to handle and is not suitable for widespread application. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a rice mutant gene and an identification method. The cadmium content in grains of rice mutants containing the OsNRAMP5 mutant gene of this invention, grown in cadmium-contaminated soil, is significantly lower than that of the wild type. The screening and identification methods used are efficient, convenient, accurate, and easy to operate.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a rice OsNRAMP5 mutant gene, wherein, compared with the OsNRAMP5 gene sequence of the indica rice Shuhui 498 genome, the OsNRAMP5 mutant gene has a mutation of base A deletion at position +1083 downstream of the start codon ATG in the OsNRAMP5 gene sequence.

[0007] The rice OsNRAMP5 mutant gene has the nucleotide sequence shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention also provides a method for screening and identifying low-cadmium mutants, comprising the following steps:

[0009] (1) Take m seeds and divide them into n groups, each group containing m / n seeds; for each group of seeds, perform the following treatment: cut off the endosperm end of each seed and save the remaining seed embryo ends for later use; mix all the seed endosperm ends of the group to form a pooled sample, then extract pooled DNA from the pooled sample and number it; a total of n pooled DNAs are obtained, numbered 1 to n.

[0010] (2) High-depth targeted sequencing of the OsNRAMP5 gene region of rice was performed on each of the mixed pool DNA samples. The sequencing results were compared with the OsNRAMP5 gene sequence of the indica rice Shuhui 498 genome to identify the variation. Through bioinformatics analysis, the mixed sample DNA with the highest mutation probability was screened out, and the corresponding seed group was called the variant group.

[0011] (3) Plant the m / n seeds of the mutant group individually at the embryo end. When the seeds grow to the three-leaf stage, take leaf samples from each individual plant and extract genomic DNA samples from each individual plant. Perform PCR amplification on the genomic DNA samples of each individual plant and identify the PCR amplification products with |△F|>0.05 by HRM analysis, thereby identifying the individual plants in the mutant group carrying the corresponding mutation.

[0012] In some specific embodiments of the present invention, the low-cadmium mutant is a mutant containing the rice OsNRAMP5 mutant gene.

[0013] In some specific embodiments of the present invention, the mutation with the highest probability of mutation is the mutation of base A deletion occurring at position +1083 downstream of the start codon ATG in the OsNRAMP5 gene sequence.

[0014] In some specific examples of the present invention, in step (1), the value of m / n ranges from 80 to 100.

[0015] In some specific examples of the present invention, in step (1), the seed is a naturally mutated seed or an M2 seed that induces a mutant population.

[0016] In some specific examples of the present invention, in step (1), the M2 seed is obtained by mutagenesis of rice seeds by non-lethal dose physicochemical mutagenesis to obtain M1 rice seeds; the M1 rice seeds are planted and the seeds produced are the M2 seeds.

[0017] In some specific embodiments of the present invention, the physicochemical mutagenesis method is a combination of one or more of the following physical mutagenesis and chemical mutagenesis methods: the physical mutagenesis includes ultraviolet mutagenesis, X-ray mutagenesis, γ-ray mutagenesis, β-ray mutagenesis, α-ray mutagenesis, high-energy particle mutagenesis, cosmic ray mutagenesis, and microgravity mutagenesis; the chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analog mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis, and intercalation dye mutagenesis, wherein the alkylating agent mutagenesis includes EMS (ethyl methanesulfonate) mutagenesis, DES (diethyl sulfate) mutagenesis, and EI (ethyleneimine) mutagenesis.

[0018] In some specific embodiments of the present invention, the non-lethal dose refers to a dose controlled within a range of 20% above or below the median lethal dose. This dose control achieves both a certain mutation rate and a certain number of viable seeds; for example, it is a median lethal dose. By controlling the mutagenesis method and the dose, a balance can be achieved between mutation efficiency and viable seed quantity.

[0019] In some specific examples of the present invention, in step (1), the mixed DNA is obtained by the following method: two small steel balls are added to each of the mixed samples, cooled with liquid nitrogen, and then crushed with a tissue homogenizer; preheated DNA extraction buffer is added to the crushed tissue, gently shaken, and 20% SDS and NaCl are added at the same time, mixed, and incubated in a water bath at 65°C for 30 min, gently inverted and shaken once every 20 min to promote cell lysis; then centrifuged at 14000 rpm for 10 min at room temperature; the supernatant is taken and chloroform / isoamyl alcohol is added, inverted and mixed, and placed at room temperature for 5 min, and centrifuged at 12000 rpm for 10 min; the supernatant is taken and extracted again with an equal volume of chloroform; 2 volumes of isopropanol are added, placed at -20°C for 1 h, and then centrifuged at 12000 rpm at 4°C for 10 min, the supernatant is discarded, and washed once with 1 mL of 75% ethanol and 1 mL of anhydrous ethanol; after drying at 65°C, 100 μL of ddH2O is added to dissolve and set aside for use.

[0020] In some specific examples of the present invention, in step (2), the bioinformatics analysis includes: for the annotation file obtained by comparing the mutations, firstly filtering out the mutations with a mutation frequency ≤ 0.2, then selecting and retaining the mutations with a mutation count of 1 to 3 from the remaining mutations; finally, excluding two types of false positives: ① large-scale mutations; and ② mutations with little difference in mutation frequency among the groups in the original annotation file.

[0021] In some specific examples of the present invention, in step (3), the genomic DNA samples of each individual plant are extracted using the CTAB method.

[0022] In some specific embodiments of the present invention, in step (3), the primer pairs used in the PCR amplification system include:

[0023] Forward primer F: 5'-TTCAGGCTGGACCGTGTCAAGT-3' (SEQ ID NO.2);

[0024] Reverse primer R: 5'-GGCCGGCATTAATTTACATCCTA-3' (SEQ ID NO.3);

[0025] The PCR amplification program is as follows: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 45 s, for a total of 40 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0026] In some specific examples of the present invention, step (4) is also included: performing PCR amplification and Sanger sequencing on the genomic DNA samples of the identified single plants carrying the corresponding variant, and simultaneously performing PCR amplification and Sanger sequencing on the genomic DNA samples of the identified wild-type single plants, comparing and analyzing the sequencing results of the two, thereby verifying that the mutation that occurred is the corresponding variant.

[0027] In some specific embodiments of the present invention, in step (4), the primer pairs used in the PCR amplification system include:

[0028] Forward primer F: 5'-TTCAGGCTGGACCGTGTCAAGT-3' (SEQ ID NO.2);

[0029] Reverse primer R: 5'-GGCCGGCATTAATTTACATCCTA-3' (SEQ ID NO.3);

[0030] The PCR amplification program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, for a total of 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0031] In some specific examples of the present invention, in step (4), the corresponding mutation is a mutation that occurs at position A 1083 downstream of the start codon ATG in the OsNRAMP5 gene sequence.

[0032] Furthermore, this invention further verifies that the cadmium accumulation content in grains of rice mutants with the stated OsNRAMP5 mutant gene grown on cadmium-contaminated soil is significantly lower than that in grains of wild-type rice grown on cadmium-contaminated soil. Therefore, the stated OsNRAMP5 mutant gene can be used for rice variety breeding, especially for low-cadmium rice varieties, and rice with the stated OsNRAMP5 mutant gene can be used for planting on cadmium-contaminated soil.

[0033] Compared with the prior art, the present invention has the following beneficial technical effects:

[0034] This invention creatively proposes a method of first grouping the population, then using high-throughput targeted sequencing of seed endosperm mixed DNA to screen for OsNRAMP5 gene variants. Only seeds from the variant group with the highest mutation probability are sown and seedlings are grown. Subsequently, individual plants carrying the corresponding variant are screened and identified within each variant group. Since seed processing and testing can be performed at any time, the initial screening can be completed in the laboratory without even needing to be done in the field. This technology significantly saves time, land, and reagent costs, improving screening efficiency and land use efficiency. This invention provides a method for screening and identifying low-cadmium mutants from large population samples that is highly efficient, accurate, and easy to operate. The combination of targeted sequencing and HRM analysis further enhances high throughput, low cost, and ease of operation, making it suitable for widespread application.

[0035] The OsNRAMP5 mutant gene of this invention produces grains with significantly lower cadmium accumulation than wild-type grains grown in cadmium-contaminated soil, and can be used for the breeding or preparation of low-cadmium rice varieties for planting in cadmium-contaminated soil. Attached Figure Description

[0036] Figure 1 This is the planting situation for Group 237.

[0037] Figure 2 High-resolution melting (HRM) curves of 68 PCR amplification products.

[0038] Figure 3 This is a sequencing peak diagram of PCR amplification products from a wild-type single plant genomic DNA sample.

[0039] Figure 4 This is a sequencing peak diagram of the PCR amplification products of a mutant single-plant genomic DNA sample.

[0040] Figure 5 Comparison of cadmium accumulation in different types of brown rice samples from soils with varying cadmium content. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased on the market.

[0043] I. Experimental Materials:

[0044] The indica rice variety DR610 originated from Wuxi Hubble Biological Seed Industry Technology Research Institute Co., Ltd.

[0045] TORO qPCR Master Mix, purchased from TOROIVD Diagnostics Group.

[0046] 2×Rapid Taq Master Mix, purchased from Vazyme Biotech Co., Ltd.

[0047] II. Experimental Procedure:

[0048] a) Take 1 kg of indica rice DR610 seeds and use Cs-137 gamma rays (γ rays) for radiation mutagenesis treatment at a dose of 300 Gy and a dose rate of 1.0 Gy / Min to obtain rice material M1 generation.

[0049] b) Plant the M1 seeds and harvest the resulting seeds, which are the M2 seeds.

[0050] c) Take approximately 40,000 M2 seeds and divide them into 500 groups of about 80 seeds each. For each group of seeds, perform the following treatment: cut open the endosperm of each seed (about 1 / 3 of the seed length) and cut off the endosperm end of each seed. Store the remaining embryo end of the seed at 4℃ for later use. Then mix the endosperm ends of all seeds in the group to obtain one pooled sample. Extract the pooled DNA from the pooled sample and number it. There are a total of 500 groups, corresponding to 500 pooled DNA samples, numbered 1 to 500.

[0051] The specific method for extracting DNA from the mixed pool is as follows: Two small steel balls (2 mm in diameter) are added to the endosperm end of each seed sample mixture. The mixture is first cooled with liquid nitrogen, then broken up using a tissue homogenizer (frequency 50 Hz, time 60 s). 400 μL of DNA extraction buffer (preheated in a 65°C water bath or oven, consisting of 0.4 mol / L NaCl, 20 mmol / L Tris-HCl (pH 8.0), and 5 mmol / L EDTA (pH 8.0)) is added to the homogenized tissue. The mixture is gently shaken, and simultaneously 40 μL of 20% sodium dodecyl sulfate (SDS) and 300 μL of 6 mol / L... NaCl was added, mixed, and incubated in a 65°C water bath for 30 min, gently inverting and shaking every 20 min to promote cell lysis. Then, the mixture was centrifuged at 14,000 rpm for 10 min at room temperature. The supernatant was collected and 700 μL of chloroform / isoamyl alcohol (volume ratio 24:1) was added. The mixture was inverted and mixed, and then incubated at room temperature for 5 min. The mixture was centrifuged at 12,000 rpm for 10 min. The supernatant was collected and extracted again with an equal volume of chloroform. Two volumes of isopropanol were added, and the mixture was incubated at -20°C for 1 h. The mixture was then centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was discarded, and the mixture was washed once with 1 mL of 75% ethanol and once with 1 mL of anhydrous ethanol. The mixture was dried in a 65°C oven and then dissolved in 100 μL of ddH2O for later use. The concentration and mass were then measured.

[0052] d) High-depth targeted sequencing and variant information annotation of the OsNRAMP5 gene region of rice were performed on each extracted pool DNA. The high-depth targeted sequencing and variant information annotation were both completed by Hunan Huazhi Biotechnology Co., Ltd.

[0053] High-depth targeted sequencing refers to the process of amplifying the OsNRAMP5 gene region using multiplex PCR on each extracted pool of DNA, followed by next-generation sequencing of the PCR amplification products to a sequencing depth of over 100*m / n. This includes primer design, multiplex PCR, PCR product recovery, and quality control of the sequencing library before sequencing.

[0054] Variation information annotation refers to: aligning the sequencing results with the OsNRAMP5 gene sequence of the reference genome (Oryza Sativa R498, i.e., the genome of indica rice Shuhui 498 (R498)); and using the gene variation annotation function of snpeff software to annotate the information of all detected variation sites (including chromosome, location, wild-type sequence, mutant sequence, variation type, variation degree, material group number, mutation frequency, etc.) to obtain an annotation file.

[0055] The rice OsNRAMP5 gene region includes both the coding and non-coding regions of the rice OsNRAMP5 gene.

[0056] The OsNRAMP5 gene sequence of the reference genome is shown in SEQ ID NO.4. This sequence is also recorded in NCBI (GenBank: LC196140.1), Oryza sativa Indica Group OsNRAMP5 gene for metaltransporter, complete cds, cultivar:Cho-Kou-Koku.

[0057] e) Perform bioinformatics analysis on the annotation files:

[0058] For the obtained annotation files, firstly filter out the variants with a mutation frequency ≤ 0.2, then select and retain the variants with a mutation count of 1 to 3 from the remaining variants; finally exclude two types of false positives: ① large-scale variants; ② variants with little difference in mutation frequency among the groups in the original annotation files.

[0059] Among them, the number of mutations refers to the number of times a certain mutation occurs in all groups from group 1 to group 500; the mutation frequency refers to the percentage of reads that have a certain mutation at a certain site detected during next-generation sequencing and mutation analysis, out of the total number of reads containing that site; and patchy mutations refer to the situation where multiple consecutive sites upstream and downstream of a certain mutation site have mutations, rather than a single-point mutation.

[0060] Accordingly, the mixed DNA sample from group 237 was screened and found to contain the variant with the highest mutation probability. The variant type was a frameshift mutation caused by a deletion of a single base A (as shown in Table 1 below). Specifically, compared to the OsNRAMP5 gene sequence of the reference genome rice R498, this OsNRAMP5 mutant gene has a 1 bp deletion (base A) at position 8940978 on chromosome 7 of the rice genome (corresponding to position +1083 downstream of the start codon ATG of the OsNRAMP5 gene), that is, a mutation of base A deletion (GA to G) occurs. The sequence of this OsNRAMP5 mutant gene is shown in SEQ ID NO.1. Comparing the OsNRAMP5 mutant gene sequence (SEQ ID NO.1) with the OsNRAMP5 gene sequence of the reference genome (SEQ ID NO.4), it can be found that: in the OsNRAMP5 gene sequence of the reference genome, positions 1084-1090 are GGGAGGC, while positions 1084-1089 of the OsNRAMP5 mutant gene sequence are GGGGGC. This indicates that the OsNRAMP5 mutant gene sequence has a 1 bp deletion: the deletion of base A at position 1087 (downstream of start codon ATG + 1083).

[0061] The corresponding M2 seed group (group number 237) is called the variant group.

[0062] Table 1. Annotations on the variants with the highest mutation probability.

[0063]

[0064] * indicates that the number of reads containing this site during next-generation sequencing was 16,571, of which 138 reads detected the GA-G mutation, with a frequency of 138 / 16,571*100%.

[0065] f) The 80 seeds from group 237 were planted individually at the embryo tip. When the seed reached the three-leaf stage, 67 individual plants survived. Their growth was as follows: Figure 1 As shown, leaf samples were taken from individual plants, and genomic DNA was extracted from each plant using the CTAB method (single sample) according to the following steps: Two small steel balls (2 mm in diameter) were added to each leaf sample from each plant. The sample was first cooled with liquid nitrogen, and then crushed using a tissue homogenizer (frequency 35 Hz, time 60 s). 800 μL of CTAB extraction buffer (preheated in a 65°C water bath or oven) was added to the crushed tissue. The CTAB extraction buffer consisted of 2% CTAB, 100 mmol / L Tris-HCl (pH 8.0), 20 mmol / L EDTA, and 1.4 mol / L... Add NaCl, mix well, incubate at 65℃ for 1 hour, gently invert and shake every 20 minutes to promote cell lysis; add 1 mL chloroform / isoamyl alcohol (volume ratio 24:1); invert and mix well, incubate at room temperature for 5 minutes, centrifuge at 12000 rpm for 10 minutes; take the supernatant, add 2 volumes of isopropanol, incubate at -20℃ for 1 hour, centrifuge at 12000 rpm at 4℃ for 10 minutes, discard the supernatant, wash once with 1 mL 75% ethanol and once with 1 mL anhydrous ethanol; dry in an oven at 65℃, add 30 μL ddH2O to dissolve and set aside for later use, and test the concentration and mass;

[0066] g) On a 96-well PCR plate (using a Hard-Shell 96-well full-skirt PCR reaction plate compatible with the HRM analyzer), PCR amplification was performed on 67 single-plant genomic DNA samples to be tested and 1 standard sample (wild-type DNA) control according to the following settings, yielding 68 PCR amplification products; among which, the PCR amplification system used for HRM analysis is as follows:

[0067]

[0068] in,

[0069] Forward primer F: 5'-TTCAGGCTGGACCGTGTCAAGT-3' (SEQ ID NO.2);

[0070] Reverse primer R: 5'-GGCCGGCATTAATTTACATCCTA-3' (SEQ ID NO.3);

[0071] Primers or primer pairs were synthesized by Zhejiang Qingke Biotechnology Co., Ltd.

[0072] The PCR program used for HRM analysis is as follows: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 45 s, for a total of 40 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0073] h) After PCR amplification, add 20 μL of mineral oil to each well of the aforementioned 96-well PCR plate to cover it. Then, run the HRM detection system. Once the machine temperature reaches 55°C, place the sample PCR plate into the HRM detector (Lightscanner 96, Idaho Technology Inc., USA) at 0.1°C·s⁻¹. -1 The PCR products were scanned by heating from 55°C to 95°C, and melting curves were obtained showing the change in fluorescence signal intensity as the temperature increased. The scanning results were analyzed using Light Scanner data analysis software (Call ITTM 2.0, Idaho Technology Inc., USA), and the detection results are provided. Each PCR amplification product corresponds to a high-resolution melting curve (HRM).

[0074] Using the high-resolution melting curve corresponding to the standard sample (wild-type DNA) control as a baseline (denoted as a horizontal straight line), the maximum fluorescence difference ΔF (abbreviated as ΔF) between the high-resolution melting curves corresponding to the 67 single-plant genomic DNA samples and the high-resolution melting curve corresponding to the standard sample (wild-type DNA) control was compared. Figure 2 As shown.

[0075] from Figure 2 It is evident that the 67 HRM curves correspond to two different types: gray curves and red curves. The peak value of the gray curve (the maximum difference from the standard sample control, |△F|) is less than or equal to 0.05, corresponding to the wild type, and the results show that 66 samples are wild type. The peak value of the red curve (the maximum difference from the standard sample control, |△F|) is greater than 0.05, corresponding to the mutant type, and the results show that 1 sample is mutant.

[0076] III. Sequencing Validation

[0077] The genomic DNA of the individual mutant samples identified above was amplified by PCR. The unpurified PCR products were sent to Zhejiang Youkang Biotechnology Co., Ltd. for Sanger sequencing to verify the mutation status. The sequencing peak diagram is shown below. Figure 4 As shown. Figure 4 The base sequence directions marked in the figure range from 3'-UTR to 5'-UTR.

[0078] The genomic DNA of the single plants corresponding to the wild-type samples identified above was amplified by PCR. The unpurified PCR amplification products were sent to Zhejiang Youkang Biotechnology Co., Ltd. for Sanger sequencing to verify the mutation status. The sequencing peak diagram is shown below. Figure 3 As shown. Figure 3 The base sequence directions marked in the figure range from 3'-UTR to 5'-UTR.

[0079] The PCR amplification system used for sequencing analysis is as follows:

[0080]

[0081] Forward primer F: 5'-TTCAGGCTGGACCGTGTCAAGT-3' (SEQ ID NO.2)

[0082] Reverse primer R: 5'-GGCCGGCATTAATTTACATCCTA-3' (SEQ ID NO.3)

[0083] Primers or primer pairs were synthesized by Zhejiang Qingke Biotechnology Co., Ltd.

[0084] The PCR amplification program used for sequencing analysis is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, for a total of 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0085] It can be seen that: Figure 3 The peak positions in the wild-type sequencing peak diagram are uniformly distributed, while... Figure 4In the mutation sequencing peak diagram, at the first T base of GCCTCCT, a T base is deleted (red peak), causing the subsequent C base to shift forward (blue peak). This results in DNA double-strand mismatch and the forward shift of all subsequent bases, forming a double peak. Therefore, sequencing verification results show that the mutation is a 1 bp deletion in the 3'-UTR to 5'-UTR sequence: a deletion of 1 T base, mutated from CT to C. Correspondingly, in its complementary sequence from 5'-UTR to 3'-UTR, at the second A base of AGGAGGC (the reverse complementary sequence of GCCTCCT in the diagram), a base A is deleted, leading to the double peak. Therefore, sequencing verification results show that the mutation is a 1 bp deletion, that is, a deletion of 1 A base, mutated from GA to G.

[0086] IV. Pot Experiment

[0087] Selected individual plants were transplanted to Hainan for planting and seed harvesting to obtain the M3 generation of rice material. M3 seeds were planted individually, and when they reached the three-leaf stage, leaf samples were taken from each plant. Genomic DNA was extracted from each plant using the aforementioned CTAB method, and sequenced to distinguish wild-type, sister lines, and mutants. Note: Sister lines refer to inbred lines of the same origin as the wild-type, exhibiting slight differences. Sister lines and wild-types share the same pedigree, similar gene composition, comparable combining ability, and similar traits.

[0088] The pot experiment was conducted in 2024 in the network room of Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang Province, as follows:

[0089] After applying chemical fertilizers (4.0g urea, 2.4g KH2PO4, and 3.9g KCl per 10kg of soil) and CdCl2·5 / 2H2O, the soil was mixed, watered, and left to stand for one month. Before use, the soil was air-dried and sieved to serve as the soil for pot experiments, and placed in plastic buckets. Two concentrations of CdCl2·5 / 2H2O were set at 0 and 2 mg / kg for comparison.

[0090] Rice seedlings are grown to the three-leaf and one-heart stage (about 1 month) in a normal environment, and then transplanted into plastic buckets (30 cm in diameter) containing 15 kg of air-dried and sieved soil. The pots are then placed in a net house (with a minimum temperature of 20°C and a maximum temperature of 40°C). The rice plants are kept in a 2-3 cm water layer throughout the growing period.

[0091] There are 6 potted plants in total, and each pot contains 6 plants. That is:

[0092] Potted plant 1: The concentration of CdCl2·5 / 2H2O in the soil was 0 mg / kg, and the rice seedlings were 6 wild-type plants.

[0093] Potted plant 2: The concentration of CdCl2·5 / 2H2O in the soil was 2 mg / kg, and the rice seedlings were 6 wild-type plants.

[0094] Potted plant 3: The concentration of CdCl2·5 / 2H2O in the soil was 0 mg / kg, and the rice seedlings were 6 mutant plants.

[0095] Potted plant 4: The concentration of CdCl2·5 / 2H2O in the soil was 2 mg / kg, and the rice seedlings were 6 mutant plants.

[0096] Potted plant 5: The concentration of CdCl2·5 / 2H2O in the soil was 0 mg / kg, and the rice seedlings were 6 sister lines.

[0097] Potted plant 6: The concentration of CdCl2·5 / 2H2O in the soil was 2 mg / kg, and the rice seedlings were 6 sister lines.

[0098] After harvesting, rice seeds were dried at 105℃ for 30 minutes and stored at 60℃ for 24 hours. Brown rice seeds were dehulled, ground into powder, and sieved (<0.154 mm). 0.2 g of brown rice sample was weighed from each potted plant, and 7 mL of nitric acid was added. The mixture was digested at 160℃ for 40 minutes. The digest was concentrated at 140℃ for 3 hours until less than 1 mL of concentrate remained. Then, 30 mL of ultrapure water was added for dilution. The cadmium content in the digest was determined using an inductively coupled plasma mass spectrometer (iCAP RQ), thus calculating the cadmium accumulation in the brown rice samples. The results are shown in Table 2 below. Figure 5 As shown:

[0099] Table 2. Cadmium accumulation content in brown rice samples

[0100]

[0101] It can be seen that in soil with a cadmium concentration of 0, the cadmium accumulation content of the wild type and the mutant is not significantly different. However, in soil with a cadmium concentration of 2 mg / kg, the cadmium accumulation content in the grains obtained from the mutant (average 0.09 mg / kg) is significantly lower than that in the grains obtained from the wild type (2.09 mg / kg). The situation in the sister lines of the wild type is not significantly different from that of the wild type. This indicates that this mutant is a low-cadmium mutant.

[0102] Therefore, the above embodiments provide a method for screening and identifying low-cadmium mutants, including the following steps:

[0103] (1) Take m seeds and divide them into n groups, each group containing m / n seeds; for each group of seeds, perform the following treatment: cut off the endosperm end of each seed and save the remaining seed embryo ends for later use; mix all the seed endosperm ends of the group to form a pooled sample, then extract pooled DNA from the pooled sample and number it; a total of n pooled DNAs are obtained, numbered 1 to n.

[0104] (2) High-depth targeted sequencing of the OsNRAMP5 gene region of rice was performed on each of the mixed pool DNA samples. The sequencing results were compared with the OsNRAMP5 gene sequence of the indica rice Shuhui 498 genome to identify the variation. Through bioinformatics analysis, the mixed sample DNA with the highest mutation probability was screened out, and the corresponding seed group was called the variant group.

[0105] (3) Plant the m / n seeds of the mutant group individually at the embryo end. When the seeds grow to the three-leaf stage, take leaf samples from each individual plant and extract genomic DNA samples from each individual plant. Perform PCR amplification on the genomic DNA samples of each individual plant and identify the PCR amplification products with |△F|>0.05 by HRM analysis, thereby identifying the individual plants in the mutant group carrying the corresponding mutation.

[0106] In some specific embodiments of the present invention, the low-cadmium mutant is a mutant containing the rice OsNRAMP5 mutant gene.

[0107] In some specific embodiments of the present invention, the mutation with the highest probability of mutation is the mutation of base A deletion occurring at position +1083 downstream of the start codon ATG in the OsNRAMP5 gene sequence.

[0108] In step (1), the seed is a naturally mutated seed or an M2 seed that induces a mutant population.

[0109] For the single-plant genomic DNA corresponding to the identified mutant samples, the mutation can be further verified by comparing and analyzing it with the single-plant genomic DNA corresponding to the identified wild-type samples through PCR amplification and Sanger sequencing.

[0110] In step (1), M2 seeds are obtained by mutagenesis of rice seeds using a non-lethal dose of physicochemical mutagenesis to obtain M1 rice seeds; M1 rice seeds are planted and the resulting seeds are M2 seeds.

[0111] The above-mentioned physicochemical mutagenesis methods include one or more of the following physical mutagenesis and chemical mutagenesis methods:

[0112] The aforementioned physical mutagenesis includes ultraviolet mutagenesis, X-ray mutagenesis, gamma-ray mutagenesis, beta-ray mutagenesis, alpha-ray mutagenesis, high-energy particle mutagenesis, cosmic ray mutagenesis, and microgravity mutagenesis.

[0113] The aforementioned chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analog mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis, and intercalation dye mutagenesis.

[0114] The aforementioned alkylating agents for mutagenesis include EMS (ethyl methanesulfonate) mutagenesis, DES (diethyl sulfate) mutagenesis, and EI (ethyleneimine) mutagenesis.

[0115] The aforementioned non-lethal dose refers to a dose controlled within 20% of the median lethal dose. This dose control achieves both a certain mutation rate and a certain number of viable seeds; for example, it is a median lethal dose. By controlling the dose of the mutagenesis method, a balance can be achieved between mutation efficiency and viable seed quantity.

[0116] It is evident that the objective of this invention has been fully and effectively achieved. The method and principle of this invention have been demonstrated and explained in the embodiments; however, modifications may be made to the implementation methods without departing from the stated principles. Therefore, this invention includes all modified embodiments based on the spirit and scope of the claims.

Claims

1. A type of rice OsNRAMP5 Mutant genes are characterized by, Compared to the genome of indica rice Shuhui 498 OsNRAMP5 The gene sequence, located 1083 positions downstream of the start codon ATG, is described as follows: OsNRAMP5 The mutant gene has a mutation involving the deletion of the base A, and the sequence has A at position +1 in the start codon ATG. The rice... OsNRAMP5 The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

1.

2. The rice as described in claim 1 OsNRAMP5 Application of mutant genes in the breeding of low cadmium rice varieties.

3. Rice as described in claim 1 OsNRAMP5 Application of mutant rice in cadmium-contaminated soil.

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