Rice mutant gene and identification method

Abstract

The invention discloses a rice mutant gene and an identification method. Compared with an OsNRAMP5 gene sequence of an indica rice Shuhui 498 genome, the OsNRAMP5 mutant gene of the rice is subjected to base A deletion mutation at the downstream + 1083 site of an initiation codon ATG. The invention creatively proposes that groups are firstly grouped, grouping screening of OsNRAMP5 gene variants is carried out by utilizing high-throughput targeted sequencing of seed endosperm mixed sample DNA, and then only seeds of a variation group in which the screened variant mixed sample DNA with the highest mutation possibility is located are sown into seedlings. And subsequently screening and identifying single plants carrying corresponding variation in the variation group according to single plants. The technology can greatly save the cost of time, land, reagents and the like, and improves the screening efficiency and land utilization efficiency. The method disclosed by the invention is high in efficiency, good in accuracy, simple and convenient to operate, capable of better realizing high throughput, low cost and operation convenience, and suitable for popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of rice mutation breeding, and in particular relates to a rice mutant gene and an identification method. Background Art

[0002] In recent years, environmental pollution, especially heavy metal pollution in soil, has become an important factor threatening crop growth, agricultural product safety and national health. Rice is the most important food crop in my country. Heavy metal cadmium pollution in soil not only causes the decline of paddy field soil quality and soil dysfunction, which damages plant growth and development to varying degrees, but also accumulates in rice grains through absorption and transfer by rice plants, becoming an important channel for endangering human health. Screening low-cadmium accumulation rice materials from existing rice germplasm resource banks is the preferred option for breeders. However, screening low-cadmium rice materials from existing rice materials is time-consuming and laborious, and it is difficult to integrate various excellent traits. With the advancement of scientific and technological means, breeders are more inclined to choose to induce mutagenesis of rice materials with excellent quality traits and screen low-cadmium mutants from them or use gene editing technology to target and edit genes related to Cd absorption and accumulation to obtain new low-cadmium rice materials.

[0003] Mutation breeding is a breeding technique that uses physical, chemical and other factors to induce gene mutations in rice materials, and then screens out mutants with desired traits from the mutant population to cultivate new varieties. Japanese scientist Ishikawa et al. used carbon ion beams to irradiate seeds of the rice variety "Koshihikari" and screened out three low-cadmium mutants of the OsNRAMP5 gene from the mutant library. Field experiments found that the cadmium content in the grains of these mutants on 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 begun to be valued, and different domestic researchers have successively created new OsNRAMP5 gene mutants through the CRISPR-Cas9 gene editing method to quickly cultivate low-cadmium rice varieties. Gene editing is currently the fastest and most efficient method for directional induction of mutations. 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 regulated. The time when they will be allowed to be used in production is still unclear, which forces breeders to abandon this method and turn to traditional mutagenesis to meet the urgent demand for low-cadmium varieties in production. Since mutagenesis is random and non-directional, specific types of mutants need to be screened from a larger mutant library. If low-cadmium rice varieties are screened by measuring the cadmium content of rice, the workload and cost are very huge: among the induced offspring, although M1 is the generation with the richest mutations, most of its mutations are reparable damage to the plant genome, which will be repaired in the next generation and cannot be stably inherited to the next generation, or because the damage is too severe to grow smoothly and cannot bear fruit normally, so screening is generally carried out in M2. In the normal screening system for offspring of mutagenesis breeding, M2 needs to be planted first, and then its leaves are sampled and mixed after it grows, and the genomic DNA is extracted for testing. After that, the mutation of the single plant is traced and confirmed based on the sequencing analysis results, which is a lot of work for sampling. And M2 is often in large quantities, which requires a lot of land and manpower, material and financial resources for planting and sampling. In addition, from the perspective of timeliness, in traditional screening methods, the planting of rice M2 is more dependent on climate, and it can only be planted at specific times; from a spatial perspective, traditional screening methods require a certain site foundation for both sampling and planting. It can be seen that traditional mutagenesis breeding requires screening mutants of specific genes from large-scale populations of more than tens of thousands of plants, which is difficult for general research teams to bear and is not suitable for widespread promotion. Summary of the invention

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

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, 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 undergoes a mutation of base A deletion at position +1083 downstream of the start codon ATG of the OsNRAMP5 gene sequence.

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

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

[0009] (1) m seeds are taken and divided into n groups, each group including m / n seeds; each group of seeds is subjected to the following treatments: the endosperm end of each seed is cut off and the remaining embryo end of the seed is kept for later use; all the endosperm ends of the seeds in the group are mixed to form a mixed sample, and then mixed pool DNA is extracted from the mixed sample and numbered; a total of n mixed pool DNAs are obtained and numbered 1 to n;

[0010] (2) performing high-depth targeted sequencing of the rice OsNRAMP5 gene region on each of the mixed pool DNAs, comparing the sequencing results with the OsNRAMP5 gene sequence of the indica rice Shuhui 498 genome for mutations, and screening out the mixed sample DNA with the highest mutation probability through bioinformatics analysis, and the corresponding seed group is called a variant group;

[0011] (3) Planting the embryo ends of m / n seeds of the variant group as single seeds, and sampling the leaves of each plant when they grow to the stage of three leaves and one heart, and extracting the genomic DNA samples of each plant; performing PCR amplification on the genomic DNA samples of each plant, and identifying the PCR amplification products with |△F|>0.05 through HRM analysis, thereby identifying the individual plants carrying the corresponding mutations in the variant group.

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

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

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

[0015] In some specific examples of the present invention, in step (1), the seeds are naturally mutated seeds or M2 seeds of an induced mutation population.

[0016] In some specific examples of the present invention, in step (1), the M2 seeds are obtained by: inducing rice seeds by a non-lethal dose of physical and chemical mutagenesis to obtain M1 rice seeds; planting the M1 rice seeds and harvesting the seeds produced, namely, the M2 seeds.

[0017] In some specific examples 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 intercalating dye mutagenesis, wherein the alkylating agent mutagenesis includes EMS (ethyl methanesulfonate) mutagenesis, DES (diethyl sulfate) mutagenesis, and EI (ethyleneimine) mutagenesis.

[0018] In some specific examples of the present invention, the non-lethal dose refers to controlling the dose within a range of 20% above and below the semi-lethal dose. This dose control can obtain both a certain mutation rate and a certain number of live seeds; for example, a semi-lethal dose. By controlling the mutagenesis method and dose, the balance between mutation efficiency and live seed quantity can be coordinated.

[0019] In some specific examples of the present invention, in step (1), the mixed pool DNA is obtained by the following method: adding two small steel balls to each of the mixed samples, first cooling with liquid nitrogen, and then crushing with a tissue crusher; adding preheated DNA extraction buffer to the crushed tissue, gently shaking, adding 20% ​​SDS and NaCl at the same time, mixing, and bathing at 65°C for 30 minutes, gently inverting and shaking once every 20 minutes to promote cell lysis; then centrifuging at room temperature at 14000rpm for 10 minutes; taking the supernatant and adding chloroform / isoamyl alcohol, inverting and mixing, and then placing at room temperature for 5 minutes, and centrifuging at 12000rpm for 10 minutes; taking the supernatant and extracting it again with an equal volume of chloroform; adding 2 times the volume of isopropanol, placing at -20°C for 1 hour, and then centrifuging at 4°C and 12000rpm for 10 minutes, discarding the supernatant, and washing once with 1mL 75% ethanol and 1mL anhydrous ethanol; after drying at 65°C, adding 100μL ddH2O to dissolve 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 variation, first filtering out the variation with a mutation frequency ≤ 0.2, then screening and retaining the variation with a mutation frequency of 1 to 3 from the remaining variation; and finally excluding two types of false positives: ① fragmented variation; and ② variation with little difference in mutation frequency among 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 strain are extracted separately using the CTAB method.

[0022] In some specific examples of the present invention, in step (3), the primer pair used in the PCR amplification system includes:

[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 procedure is as follows: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 45 s, for a total of 40 cycles; extension at 72°C for 5 min, and storage at 4°C.

[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 individual strains carrying the corresponding mutation, and at the same time, performing PCR amplification and Sanger sequencing on the genomic DNA samples of the identified wild-type individual strains, and comparing and analyzing the sequencing results of the two to verify that the mutation that occurred is the corresponding mutation.

[0027] In some specific examples of the present invention, in step (4), the primer pair used in the PCR amplification system includes:

[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 procedure is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, for a total of 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

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

[0032] In addition, the present invention further verifies that the cadmium accumulation content in the grains of the mutant having the rice OsNRAMP5 mutant gene grown on cadmium-contaminated soil is significantly lower than the cadmium accumulation content in the grains of the wild type grown on cadmium-contaminated soil. Therefore, the rice OsNRAMP5 mutant gene can be used for rice variety breeding, especially low-cadmium rice variety breeding, and rice having the rice 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] The present invention creatively proposes to first group the population, use high-throughput targeted sequencing of seed endosperm mixed DNA to carry out group screening of OsNRAMP5 gene variants, and then only sow the seeds of the variant group with the highest mutation possibility, and then screen and identify the individual plants carrying the corresponding mutations in the variant group according to individual plants. Since the processing and delivery of seeds can be carried out at any time, the preliminary screening does not even need to be carried out in the field, but can be completed in the laboratory. This technology can greatly save time, land, reagents and other costs, and improve screening efficiency and land use efficiency. The method of screening and identifying low-cadmium mutants from a large number of population samples of the present invention is high in efficiency, good in accuracy, and easy to operate; targeted sequencing combined with HRM analysis method for identification can better achieve high throughput, low cost and convenience of operation, and is suitable for promotion and application.

[0035] The cadmium accumulation content in the grains obtained by the OsNRAMP5 mutant gene of the present invention when grown in cadmium-contaminated soil is significantly lower than the cadmium accumulation content in the grains obtained by the wild type when grown in cadmium-contaminated soil, and can be used for the breeding or preparation of low-cadmium rice varieties planted in cadmium-contaminated soil. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0038] Figure 3 This is the sequencing peak diagram of the PCR amplification product of the genomic DNA sample of the wild-type individual.

[0039] Figure 4 This is the sequencing peak diagram of the PCR amplification product of the genomic DNA sample of the mutant individual strain.

[0040] Figure 5 Comparison of cadmium accumulation content in different types of brown rice samples in soils with different cadmium contents. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments and drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0042] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased on the market.

[0043] 1. Experimental materials:

[0044] The indica rice variety DR610 comes from Wuxi Huber Biological Seed Technology Research Institute Co., Ltd.

[0045] TORO qPCR Master Mix was purchased from TOROIVD.

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

[0047] 2. Experimental process:

[0048] a) taking 1 kg of indica rice DR610 rice seeds, and subjecting them to radiation mutagenesis treatment using Cs-137 gamma rays (γ rays) at a dose of 300 Gy and a dose rate of 1.0 Gy / Min, to obtain rice material M1 generation;

[0049] b) Planting the M1 seeds and harvesting the seeds produced are the M2 seeds.

[0050] c) Take about 40,000 M2 seeds, divide them into 500 groups of about 80 seeds each, and perform the following treatments on each group of seeds: cut each seed at the endosperm (about 1 / 3 of the seed length), cut off the endosperm end of each seed, and store the remaining seed embryo end at 4°C for later use; then mix the endosperm ends of all seeds in the group to obtain a mixed sample, and then extract the mixed pool DNA of the group from the mixed sample and number them; there are a total of 500 groups, corresponding to 500 mixed pool DNAs, numbered 1 to 500;

[0051] The specific method for extracting the mixed pool DNA is as follows: two small steel balls (2 mm in diameter) are added to the mixed sample of the endosperm end of each group of seeds, first cooled with liquid nitrogen, and then crushed with a tissue crusher (frequency of 50 Hz, time of 60 s); 400 μL of DNA extraction buffer (whose components include: 0.4 mol / L NaCl, 20 mmol / L Tris-HCl (pH 8.0) and 5 mmol / L EDTA (pH 8.0)) preheated in a 65°C water bath or oven is added to the crushed tissue, gently shaken, and 40 μL of 20% sodium dodecyl sulfate (SDS) and 300 μL of 6 mol / L NaCl, mix well, incubate at 65℃ for 30min, gently invert and shake once every 20min to promote cell lysis; then centrifuge at 14000rpm for 10min at room temperature; take the supernatant and add 700μL chloroform / isoamyl alcohol (volume ratio of 24:1), invert and mix well, place at room temperature for 5min, and centrifuge at 12000rpm for 10min; take the supernatant and extract it again with an equal volume of chloroform; add 2 times the volume of isopropanol, place at -20℃ for 1h, centrifuge at 4℃ and 12000rpm for 10min, discard the supernatant, wash once with 1mL of 75% ethanol and 1mL of anhydrous ethanol; after drying in an oven at 65℃, add 100μL ddH2O to dissolve for use, and detect the concentration and quality.

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

[0053] High-depth targeted sequencing refers to the use of multiplex PCR to amplify the OsNRAMP5 gene region for each pool of extracted DNA, and the next-generation sequencing of the PCR amplification products, with a sequencing depth of more than 100*m / n. Specifically, it includes: primer design, multiplex PCR, PCR product recovery, and sequencing library quality inspection.

[0054] Mutation information annotation refers to: comparing the sequencing results with the OsNRAMP5 gene sequence of the reference genome (Oryza Sativa R498, i.e., the indica rice Shuhui 498 (R498) genome), and using the gene variation annotation function of the snpeff software to annotate the information of all detected variation sites (including chromosome, position, 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 the coding region and non-coding region of the rice OsNRAMP5 gene.

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

[0057] e) Bioinformatics analysis of annotation files:

[0058] For the obtained annotation files, we first filter out the variants with mutation frequency ≤ 0.2, and then select and retain the variants with mutation frequency 1 to 3 from the remaining variants; finally, we exclude two types of false positives: ① fragmented 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 1 to 500; the mutation frequency refers to the percentage of reads detected at a certain site during second-generation sequencing and variation analysis that show a certain mutation, compared to the total number of reads containing the site; and block mutation refers to the situation where multiple consecutive sites upstream and downstream of a mutation site mutate, rather than a single point mutation.

[0060] Based on this, the mixed sample DNA of group No. 237 was screened to contain a mutation with the highest possibility of mutation, and the mutation type was a frameshift mutation caused by a base A deletion (as shown in Table 1 below). That is, compared with the OsNRAMP5 gene sequence of the reference genome rice R498, the OsNRAMP5 mutant gene has a 1 bp base (base A) deletion at position 8940978 of chromosome 7 of the rice genome (corresponding to position +1083 downstream of the start codon ATG of the OsNRAMP5 gene), that is, a base A deletion mutation occurred (GA becomes G). The sequence of the OsNRAMP5 mutant gene is shown in SEQ ID NO.1. By 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: positions 1084 to 1090 of the OsNRAMP5 gene sequence of the reference genome are GGGAGGC, and positions 1084 to 1089 of the OsNRAMP5 mutant gene sequence are GGGGGC. It can be seen that the OsNRAMP5 mutant gene sequence has a 1 bp base deletion: the deletion of base A at position 1087 (downstream of the start codon ATG + 1083).

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

[0062] Table 1 Annotation information of 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 GA-G mutations were detected in 138 reads, with a frequency of 138 / 16,571*100%.

[0065] f) The embryo ends of the 80 seeds of group No. 237 were planted as single seeds. When they grew to the stage of three leaves and one heart, the number of surviving single plants was 67. The growth conditions were as follows: Figure 1 As shown in the figure, leaves were sampled from individual plants, and the genomic DNA of each individual plant was extracted by the CTAB method according to the following steps: two small steel balls (2 mm in diameter) were added to the leaf samples of each individual plant, which were first cooled with liquid nitrogen and then crushed with a tissue crusher (frequency of 35 Hz, time of 60 s); 800 μL of CTAB extract preheated in a 65°C water bath or oven was added to the crushed tissue (the composition of the CTAB extract includes: 2% CTAB, 100 mmol / L Tris-HCL (pH 8.0), 20 mmol / L LEDTA, 1.4 mol / L NaCl), mix well, incubate at 65℃ for 1h, gently invert and shake once every 20min to promote cell lysis; add 1mL chloroform / isoamyl alcohol (volume ratio is 24:1); invert and mix well, place at room temperature for 5min, centrifuge at 12000rpm for 10min; take the supernatant and add 2 times volume of isopropanol, place at -20℃ for 1h, centrifuge at 4℃ and 12000rpm for 10min, discard the supernatant, wash once with 1mL 75% ethanol and 1mL anhydrous ethanol; after drying in an oven at 65℃, add 30μL ddH2O to dissolve for use, and detect the concentration and quality;

[0066] g) In a 96-well PCR plate (using a Hard-Shell low-position 96-well full-skirt PCR reaction plate adapted to the HRM detector), PCR amplification was performed on 67 individual genomic DNA samples to be tested and 1 standard sample (wild-type DNA) control according to the following settings to obtain 68 PCR amplification products; wherein, 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 for HRM analysis was as follows: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 45 s, for a total of 40 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0073] h) After the PCR amplification is completed, 20 μL of mineral oil is added to each well of the 96-well PCR plate to cover it. Then, the HRM detection system is operated. After the temperature of the machine rises to 55°C, the sample PCR plate is placed in the HRM detector (Lightscanner 96, Idaho Technology Inc, USA) and the temperature is adjusted at 0.1°C·s -1 The PCR product was scanned by heating from 55°C to 95°C at a speed of 100 °C to obtain the melting curve formed by the change of fluorescence signal intensity with the increase of temperature. The scanning results were analyzed using Light Scanner data analysis software (Call ITTM2.0, Idaho Technology Inc, USA) and the test results were given. Each PCR amplification product corresponds to a high-resolution melting curve (HRM).

[0074] The high-resolution melting curve corresponding to the standard sample (wild-type DNA) control was used as the baseline (recorded as a horizontal straight line) to compare the maximum fluorescence difference △Fluorescence (abbreviated as ΔF) between the high-resolution melting curves corresponding to the 67 individual genomic DNA samples and the high-resolution melting curves corresponding to the standard sample (wild-type DNA) control. Figure 2 shown.

[0075] from Figure 2 It can be clearly seen that the 67 HRM curves correspond to two different types: gray curves and red curves. Among them, the peak values ​​of the gray curves (the maximum difference with the standard sample control, |△F|) are all less than or equal to 0.05, and the gray curve corresponds to the wild type. The results show that 66 samples are wild type; the peak value of the red curve (the maximum difference with the standard sample control, |△F|) is greater than 0.05, and the red curve corresponds to the mutant type. The results show that 1 sample is a mutant.

[0076] 3. Sequencing Verification

[0077] The genomic DNA of the individual strains corresponding to the identified mutant samples was amplified by PCR, and the unpurified PCR amplification products were sent to Zhejiang Youkang Biotechnology Co., Ltd. for Sanger sequencing to verify the variation of the deletion mutation. Figure 4 shown. Figure 4 The base sequence marked in the figure is oriented from 3'-UTR to 5'-UTR.

[0078] The genomic DNA of the individual strain corresponding to the wild-type sample identified above was amplified by PCR, and the unpurified PCR amplification product was sent to Zhejiang Youkang Biotechnology Co., Ltd. for Sanger sequencing to verify the variation of its deletion mutation. Figure 3 shown. Figure 3 The base sequence marked in the figure is oriented from 3'-UTR to 5'-UTR.

[0079] Among them, 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 was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 15 s, for a total of 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0085] It can be seen that: Figure 3 The peak positions of the wild-type sequencing peaks are evenly distributed, while Figure 4In the mutant sequencing peak graph, at the first base T of GCCTCCT, a base T (red peak) is missing, which causes the base C (blue peak) behind to move forward, making the DNA double strand mismatched, and causing all the bases on the rear side to move forward to form a peak graph with a double peak. Therefore, the results of sequencing verification show that from the 3'-UTR to 5'-UTR base sequence, the mutation is a 1 bp deletion: a 1 base T deletion, mutated from CT to C; correspondingly, in the complementary base sequence from 5'-UTR to 3'-UTR, a base A is missing at the second base A of AGGAGGC (the reverse complementary sequence of GCCTCCT in the figure), which then leads to the appearance of a double peak graph. Therefore, the results of sequencing verification show that the mutation is a 1 bp base deletion, that is, a 1 base A deletion, mutated from GA to G.

[0086] 4. Pot Plant Experiment

[0087] The selected individual plants were transplanted to Hainan for planting and harvesting to obtain the M3 generation of rice materials. The M3 seeds were planted as single seeds, and when they grew to three leaves and one heart, the leaves were sampled for each plant. The genomic DNA of each plant was extracted by referring to the above-mentioned CTAB method, and sequenced to distinguish the wild type, sister line and mutant type. Note: Sister line refers to the same inbred line with the same source as the wild type and with slight differences. The sister line and the wild type have the same blood relationship, similar genetic composition, comparable combining ability and similar traits.

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

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

[0090] The rice seedlings were grown to the three-leaf and one-heart stage (approximately 1 month) in a normal environment and transplanted into a plastic bucket (30 cm in diameter) filled with 15 kg of the above-mentioned air-dried sieved soil. Then, the flowerpots were placed in a mesh house (with a minimum temperature of 20°C and a maximum temperature of 40°C during this period), and the rice plants maintained a 2-3 cm water layer throughout the growth period.

[0091] There are 6 pots in total, each containing 6 plants.

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

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

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

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

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

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

[0098] After harvesting, the rice seeds were dried at 105°C for 30 minutes and stored at 60°C for 24 hours. The brown rice seeds were hulled and ground into powder and sieved (<0.154 mm). 0.2 grams of brown rice sample was weighed from each group of potted plants, 7 mL of nitric acid was added, and digested at 160°C for 40 minutes. The digestate was concentrated at 140°C for 3 hours until the remaining concentrate was less than 1 mL. Then 30 mL of ultrapure water was added for dilution. The cadmium content in the digestate was determined by an inductively coupled plasma mass spectrometer (model iCAP RQ), and the cadmium accumulation content in the brown rice sample was calculated. The results are shown in Tables 2 and 3 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 much different, but in soil with a cadmium concentration of 2 mg / kg, the cadmium accumulation content in the grains grown by the mutant (average value of 0.09 mg / kg) is significantly lower than that in the grains grown by the wild type (2.09 mg / kg). The situation of the sister line of the wild type is not much different from that of the wild type. This shows that the mutant is a low-cadmium mutant.

[0102] It can be seen that the above embodiment provides a method for screening and identifying low-cadmium mutants, comprising the following steps:

[0103] (1) m seeds are taken and divided into n groups, each group including m / n seeds; each group of seeds is subjected to the following treatments: the endosperm end of each seed is cut off and the remaining embryo end of the seed is kept for later use; all the endosperm ends of the seeds in the group are mixed to form a mixed sample, and then mixed pool DNA is extracted from the mixed sample and numbered; a total of n mixed pool DNAs are obtained and numbered 1 to n;

[0104] (2) performing high-depth targeted sequencing of the rice OsNRAMP5 gene region on each of the mixed pool DNAs, comparing the sequencing results with the OsNRAMP5 gene sequence of the indica rice Shuhui 498 genome for mutations, and screening out the mixed sample DNA with the highest mutation probability through bioinformatics analysis, and the corresponding seed group is called a variant group;

[0105] (3) Planting the embryo ends of m / n seeds of the variant group as single seeds, and sampling the leaves of each plant when they grow to the stage of three leaves and one heart, and extracting the genomic DNA samples of each plant; performing PCR amplification on the genomic DNA samples of each plant, and identifying the PCR amplification products with |△F|>0.05 through HRM analysis, thereby identifying the individual plants carrying the corresponding mutations in the variant group.

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

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

[0108] Wherein, in step (1), the seeds are naturally mutated seeds or M2 seeds of an induced mutation population.

[0109] The individual genomic DNA corresponding to the identified mutant sample can be further compared and analyzed with the individual genomic DNA corresponding to the identified wild-type sample through PCR amplification and Sanger sequencing to verify the mutation.

[0110] Wherein, in step (1), M2 seeds are obtained by: inducing rice seeds by a non-lethal dose of physical and chemical mutagenesis to obtain M1 rice seeds; planting the M1 rice seeds and harvesting the resulting seeds, namely, M2 seeds.

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

[0112] The above-mentioned 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 above-mentioned chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analog mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis, and intercalating dye mutagenesis.

[0114] The above-mentioned alkylating agent mutagenesis includes EMS (ethyl methanesulfonate) mutagenesis, DES (diethyl sulfate) mutagenesis, and EI (ethyleneimine) mutagenesis.

[0115] The above non-lethal dose refers to controlling the dose within a range of 20% above and below the semi-lethal dose. This dose control can obtain both a certain mutation rate and a certain number of live seeds; for example, a semi-lethal dose. By controlling the dose of the mutagenesis method, the balance between mutation efficiency and live seed quantity can be coordinated.

[0116] It can be seen that the purpose of the present invention has been completely and effectively achieved. The method and principle of the present invention have been demonstrated and explained in the embodiments. Without departing from the principles, the implementation methods can be modified arbitrarily. Therefore, the present invention includes all modified implementation methods based on the spirit and scope of the claims.

Claims

1. A rice OsNRAMP5 mutant gene, characterized in that: 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.

2. The rice OsNRAMP5 mutant gene according to claim 1, characterized in that: It has the nucleotide sequence shown in SEQ ID NO.

1.

3. A method for screening and identifying low-cadmium mutants, comprising the following steps: (1) m seeds are taken and divided into n groups, each group including m / n seeds; each group of seeds is subjected to the following treatments: the endosperm end of each seed is cut off and the remaining embryo end of the seed is kept for later use; all the endosperm ends of the seeds in the group are mixed to form a mixed sample, and then mixed pool DNA is extracted from the mixed sample and numbered; a total of n mixed pool DNAs are obtained and numbered 1 to n; (2) performing high-depth targeted sequencing of the rice OsNRAMP5 gene region on each of the mixed pool DNAs, comparing the sequencing results with the OsNRAMP5 gene sequence of the indica rice Shuhui 498 genome for mutations, and screening out the mixed sample DNA with the highest mutation probability through bioinformatics analysis, and the corresponding seed group is called a variant group; (3) Planting the embryo ends of m / n seeds of the variant group as single seeds, and sampling the leaves of each plant when they grow to the stage of three leaves and one heart, and extracting the genomic DNA samples of each plant; performing PCR amplification on the genomic DNA samples of each plant, and identifying the PCR amplification products with |△F|>0.05 through HRM analysis, thereby identifying the individual plants carrying the corresponding mutations in the variant group.

4. The method according to claim 3, characterized in that The low-cadmium mutant is a mutant containing the rice OsNRAMP5 mutant gene.

5. The method according to claim 3, characterized in that The variation with the highest mutation probability is the variation of base A deletion occurring at position +1083 downstream of the start codon ATG in the OsNRAMP5 gene sequence.

6. The method according to claim 3, characterized in that In step (1), the seeds are naturally mutated seeds or M2 seeds of an induced mutation population.

7. The method according to claim 6, characterized in that In step (1), the M2 seeds are obtained by: inducing rice seeds to obtain M1 rice seeds by a non-lethal dose of physical and chemical mutagenesis; planting the M1 rice seeds and harvesting the resulting seeds, which are the M2 seeds.

8. The method according to claim 3, characterized in that In step (3), the primer pairs used in the PCR amplification system include: Forward primer F: 5'-TTCAGGCTGGACCGTGTCAAGT-3' (SEQ ID NO. 2); Reverse primer R: 5'-GGCCGGCATTAATTTACATCCTA-3' (SEQ ID NO. 3); The PCR amplification procedure is as follows: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 45 s, for a total of 40 cycles; extension at 72°C for 5 min, and storage at 4°C.

9. Use of the rice OsNRAMP5 mutant gene according to claim 1 or 2 in rice variety breeding.

10. Use of rice having the rice OsNRAMP5 mutant gene as claimed in claim 1 or 2 in planting on cadmium-contaminated soil.

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