Molecular marker primer pair for identifying wild type or mutant of salt-tolerant gene os sgr1 of rice and application thereof

Abstract

This invention relates to the field of biotechnology, specifically providing methods for identifying salt tolerance genes in rice. OsSGR1 Molecular marker primer pairs for wild-type or mutant organisms and their applications. The molecular marker primer pairs consist of the primer pairs shown in SEQ ID NO:6-8. The primer pairs provided by this invention can be used to distinguish rice salt tolerance genes. OsSGR1 Wild-type or mutant strains can be used in salt-tolerant rice populations for assisted breeding. They can be identified in hybridization breeding, backcross segregation improvement, etc., accurately distinguishing between heterozygous and homozygous genotypes, improving breeding efficiency, and having important practical significance for high-yield, stable-yield and stress-resistant rice breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and identifies salt tolerance genes in rice. OsSGR1 Molecular marker primer pairs for wild-type or mutant organisms and their applications. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, feeding more than half of the world's population. Rice production is of great significance to global food security. It is estimated that by 2050, food production needs to increase by about 50% to meet the needs of the world's growing population. However, rice is frequently subjected to various abiotic stresses throughout its growth cycle, such as salinity, drought, flooding, and extreme temperatures. Among these, salinity stress is one of the most significant abiotic stresses threatening rice production.

[0003] Soil salinization has shrunk arable land and is one of the causes of the food crisis. According to incomplete statistics from UNESCO and the FAO, approximately 945 million hectares of land worldwide are affected by salinity, accounting for about 20% of global agricultural land. Faced with a growing population, limited arable land, and increasingly severe secondary soil salinization due to improper irrigation, making it difficult to significantly increase rice yields, developing and utilizing coastal and inland saline-alkali land resources is one of the effective ways to ensure arable land availability. Rice is a moderately salt-sensitive crop that grows in aquatic environments; rice cultivation can leach soluble salts and alkalis from the soil. Therefore, rice is the preferred food crop for developing coastal and saline-alkali land.

[0004] Improving rice's salt tolerance through genetic modification is one of the effective ways to increase rice planting area and yield. Currently, the salt-tolerant QTLs used in breeding are mainly located at two loci on rice chromosome 1: qSKC-1 and Saltol. With the development of molecular biotechnology, using mutants to isolate and discover salt-stress-tolerant genes in rice, and using them for rice genetic engineering for assisted breeding and alkali-stress improvement, is of paramount importance for effectively controlling the damage of salt stress to rice, increasing rice yield, and improving rice quality. (Note: The last sentence about chlorosis genes seems unrelated and likely refers to a different topic.) SGR The encoded product belongs to an ancient class of proteins, a magnesium ion dechelatase, containing a predicted chloroplast transport peptide. In indica rice, CRISPR / Cas9 knockout or RNAi interference was used. OsSGR All of these can significantly delay the leaf yellowing process. However, no information has yet been found regarding... OsSGR1 Reports on improving rice's resistance to salt stress.

[0005] The applicant has already conducted research and published. OsSGR1It is a negatively regulated salt tolerance gene, according to a Chinese patent. OsSGR1 The paper "Application of Protein and its Encoding Gene in Regulating Salt Tolerance in Rice" discloses that a specific mutation in this gene (base insertion leading to functional abnormality) induces salt tolerance in rice. Currently, the applicant is using CRISPR / Cas9 technology to specifically edit the rice salt tolerance gene. OsSGR1 This results in a significant improvement in salt tolerance. OsSGR1 Homozygous mutants with loss of gene function. To improve the utilization efficiency of homozygous mutants in salt-tolerant breeding, how can a molecular marker be developed to distinguish homozygous mutants from wild-type mutants to realize the salt-tolerant gene function? OsSGR1 Efficient identification of mutant genotypes is of great value in subsequent salt-tolerant breeding using homozygous mutants. Summary of the Invention

[0006] The technical problem to be solved by this invention is: to propose a method for identifying rice salt tolerance genes. OsSGR1 Molecular marker primer pairs for wild-type or mutant organisms and their applications.

[0007] The technical solution proposed to solve the above technical problems is as follows:

[0008] The first aspect of this invention provides a rice salt tolerance gene. OsSGR1 The mutated gene and the biological material associated with the mutated gene, wherein the mutated gene is... OsSGR1 The gene inserts a T base at position 1363, starting from the start codon ATG. The nucleotide sequence of the mutant gene is shown in SEQ ID NO:4.

[0009] The biomaterial is any one of the following A1) to A3):

[0010] A1) An expression cassette containing the nucleic acid molecule described in SEQ ID NO:4;

[0011] A2) A recombinant vector containing the expression cassette described in A1);

[0012] A3) A recombinant microorganism containing the expression cassette described in A1), or a recombinant microorganism containing the recombinant vector described in A2), wherein the microorganism is Agrobacterium.

[0013] The second aspect of the present invention provides the application of the mutant gene or the biological material in improving the salt tolerance of rice. Specifically, the operation involves introducing the mutant gene or the biological material into rice under salt stress and then cultivating it.

[0014] The third aspect of this invention provides a method for identifying rice salt tolerance genes. OsSGR1A molecular marker primer set for wild-type or mutant organisms, the primer set comprising: a forward primer F1 with the nucleotide sequence shown in SEQ ID NO. 6, a forward primer F2 with the nucleotide sequence shown in SEQ ID NO. 7, and a reverse primer R with the nucleotide sequence shown in SEQ ID NO. 8, the identification method comprising the following steps:

[0015] Step 1: Extract genomic DNA from the rice samples used in the test;

[0016] Step 2: Perform PCR amplification on the DNA obtained in Step 1 using the primer set described above;

[0017] Step 3: Detect the amplification products by agarose gel electrophoresis;

[0018] Step 4: If only a 169 bp band can be amplified, the sample is... OsSGR1 The sample is a homozygous mutant exhibiting salt tolerance; if only a 173 bp band is amplified, the sample is wild-type and exhibits salt sensitivity; if both 169 bp and 173 bp bands are amplified simultaneously, the sample is... OsSGR1 The gene is a heterozygous mutant and exhibits salt sensitivity.

[0019] In the method, the wild type OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein is shown in SEQ ID NO:3.

[0020] In the method described, rice salt tolerance genes OsSGR1 The mutant was constructed using sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:5.

[0021] A fourth aspect of the present invention provides a method for identifying rice salt tolerance genes comprising the primer pair described above. OsSGR1 PCR reagents for wild-type or mutant strains.

[0022] The fifth aspect of this invention provides the application of the aforementioned molecular marker primer set or the aforementioned PCR reagent, wherein the application is any of the following:

[0023] (1) Identification of salt tolerance genes in rice OsSGR1 Applications in wild-type or mutant strains;

[0024] (2) In the preparation of identification of rice salt tolerance genes OsSGR1 Applications in products containing wild-type or mutant strains.

[0025] The beneficial effects of this invention are:

[0026] (1) A method for distinguishingOsSGR1 A set of molecular marker primers for mutants and wild-type genes was used to achieve salt tolerance. OsSGR1 Efficient identification of mutant genotypes is crucial for subsequent applications. OsSGR1 Mutants are of great value in salt-tolerant breeding.

[0027] (2) The molecular markers provided by this invention are based on salt tolerance genes. OsSGR1 Functional markers based on PCR amplification designed at CRISPR / Cas9-specific modified sites between mutants and wild types have genotypes that directly reflect the plant phenotype, eliminating errors caused by genetic exchange. They are simpler to operate than sequencing methods and reduce costs.

[0028] (3) The identification method provided by the present invention can be applied to auxiliary breeding for the improvement of salt tolerance traits in rice. It can be used for identification in hybridization breeding, backcross segregating populations, etc., to accurately distinguish between heterozygous and homozygous genotypes and improve breeding efficiency. Attached Figure Description

[0029] Figure 1 for OsSGR1 Schematic diagram of gene structure and target sequence elements of CRISPR / Cas-OsSGR1 vector.

[0030] Figure 2 These are peak diagrams of sequencing results; the top diagram shows the sequencing peaks of the wild-type ZH11 target, and the bottom diagram shows the peaks of the mutant. ossgr1 Target sequencing peak diagram.

[0031] Figure 3 for ossgr1 Salt tolerance test of mutants. A represents ZH11 and... ossgr1 The plant's growth state before salt treatment, bar = 1 cm; B represents ZH11 and ossgr1 The growth status of plants after salt treatment, bar = 1 cm; C represents the survival rate statistics of ZH11 and ossgr1 before and after salt treatment. The values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference, P < 0.05; ** indicates extremely significant difference, P < 0.01. The statistical analysis method is one-way ANOVA.

[0032] Figure 4 Schematic diagram of competitive typing primer design for target mutation sites.

[0033] Figure 5 To validate the results of T-clone detection using molecular markers.

[0034] Figure 6 This is a schematic diagram of the backcrossing and transformation route using molecular markers. Detailed Implementation

[0035] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0036] CRISPR / Cas vector BGK03: Hangzhou Baige Biotechnology Co., Ltd., product catalog number BGK03. Example 1

[0037] This embodiment provides rice. OsSGR1 The functions and applications of genes include the following:

[0038] 1. Rice OsSGR1 Gene sequence and expression pattern analysis

[0039] The Ensembl Plants database (http: / / plants.ensembl.org / index.html) contained information on rice. OsSGR1 The nucleotide sequence of the gene in *Zhonghua 11* of japonica rice is shown in SEQ ID NO: 1, and the CDS sequence is shown in SEQ ID NO: 2. Its encoded protein contains 274 amino acids, and its sequence is shown in SEQ ID NO: 3. To study the function of this gene, this invention first used a gene expression database to analyze the expression pattern of this gene in different tissues of rice. The expression analysis results showed... OsSGR1 The gene is predominantly expressed in the rice roots, suggesting that it may be involved in the rice root development process.

[0040] 2. Rice OsSGR1 Functional verification of genes

[0041] To clarify OsSGR1 To investigate the function of a gene in rice, this invention employs the CRISPR / Cas9 gene editing method to site-directedly mutate the gene sequence and knock out its function in rice. This invention selects the conventional rice variety ZH11 as the recipient material for gene editing. This invention selects the nucleotide sequence from base 1346 to base 1365 of the gene coding region, starting from the start codon ATG (shown in SEQ ID NO. 5), as the target region for CRISPR / Cas9 gene editing (see [link to target region]). Figure 1 ).

[0042] (1) OsSGR1 Construction of CRISPR / Cas9 gene editing vector

[0043] The gene editing vector of this invention is pEGCas9Pubi-B-OsSGR1, and the base vector of this vector is pEGCas9Pubi-B. This invention involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into the base vector using a one-step cloning method. The specific construction process is as follows:

[0044] i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] OsSGR1 The gene sequence was input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence was set to NGG. The DNA sequence of the target region selected in this invention is shown in SEQ ID NO: 5.

[0045] ii) Amplify the sgRNA expression cassette by overlap PCR and nested PCR. Primer pairs containing the above-mentioned sgRNA target sequences were synthesized and annealed. Then, the primer pairs were ligated with the binary vector pEGCas9Pubi-B (see Ma X, Zhang Q, Zhu Q. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Mol Plant. 2015, 8(8):1274-1284, vector pEGCas9Pubi-B was kindly provided by Professor Long Tuan of Hainan University) to obtain the recombinant vector pEGCas9Pubi-OsSGR1. The recombinant vector pEGCas9Pubi-OsSGR1 was transformed into E. coli DH5α, and positive clones were selected for sequencing. The specific steps were as described in the reference "Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plants. BMC plant biology 14:327."

[0046] iii) Sequencing verification.

[0047] The successful construction of pEGCas9Pubi-OsSGR1 was verified by sequencing.

[0048] (2) Agrobacterium-mediated genetic transformation of rice

[0049] The constructed pEGCas9Pubi-OsSGR1 vector was transformed into Agrobacterium EHA105 via heat shock. After PCR identification, the bacterial culture was stored at -80 °C with glycerol. Freshly peeled embryos of rice hybrid Zhonghua 11 (approximately 1.5 mm in diameter) were used as recipient materials. The peeled rice embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension and left for no more than 1 hour. Approximately 100 embryos were placed in each centrifuge tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount of suspension at the bottom of the tube to submerge the embryos. The tubes were then heat-shocked at 43 °C for 2 minutes, followed by an ice bath for 1 minute. The remaining wash solution at the bottom of the tube was aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then incubated in the dark for 8 minutes. Next, pour the embryos and infection solution from the centrifuge tubes onto the co-culture medium, shake well, and then use a pipette to remove excess infection solution. Ensure all embryos have their scutellaria facing upwards and co-culture at 23°C in the dark for 3 days. After co-culture, use sterile forceps to transfer the embryos to recovery medium and culture at 28°C for 7-14 days. During this process, carefully remove any sprouts that appear on the embryos. After recovery culture, place the embryos on 1.5 mg / L Bialaphos selection medium for 3 rounds of selection, each round lasting 2 weeks. Then transfer them to 2 mg / L Bialaphos selection medium for 2 rounds of selection, each round lasting 2 weeks. Transfer the resistant callus to propagation medium and culture at 28°C in the dark for 2 weeks. Then transfer the propagated resistant callus to induction medium and culture at 28°C in the dark for 2 weeks. Finally, transfer it to differentiation medium and culture at 25°C, 5000 lx under light for 2 weeks. After the culture is completed, the differentiated seedlings are separated into individual seedlings and placed in a rooting medium. They are cultured at 25 ℃, 5000 lx, and under light until they root. The seedlings are then transferred to small nutrient pots for growth. After they have survived, they are transplanted into a greenhouse. The offspring seeds are harvested 3-4 months later.

[0050] (3) Detection of CRISPR / Cas9 mutation results in T0 generation plants

[0051] To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken for detection:

[0052] This invention first employs the CTAB method to extract DNA from rice leaves. The specific method is as follows: DNA extraction is performed according to the traditional CTAB method (Rogers and Bendich, 1985). A 3 cm rice leaf is placed in a sterilized 2 mL centrifuge tube, a 6 mm steel ball is added, and the tissue is disrupted using a cell disruptor. Then, CTAB extraction is performed. Finally, 200 μL of sterile water (ddH2O) is added to dissolve the air-dried DNA sample, which is then set aside. After the DNA is completely dissolved, 2 μL of the sample is taken and the nucleic acid OD value (A260 / A280) and nucleic acid concentration are determined using a UV spectrophotometer (Nanodrop 2000). The DNA sample is then diluted to 50 ng / μL for later use.

[0053] PCR was performed using 5 μL of Biomiga 2×PCR premix (containing Mg2+, Taq DNA Polymerase, 2.5 mM dNTPs, and 10×PCR Buffer), 1 μL of primers (containing 0.5 μL each of forward and reverse primers), 1 μL of template DNA, and ddH2O to a final volume of 10 μL. The PCR amplification program was a standard SSR program (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 5 min). The amplified products were analyzed by 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after color development with formaldehyde and NaOH.

[0054] For mutants ossgr1 Nucleotide sequence alignment analysis revealed that ( Figure 2 Compared to the unedited wild type (WT), the mutated ossgr1 A T base is inserted at position 1363 starting from the start codon ATG, as shown in SEQ ID NO: 4. The deletion of the nucleotide encoded by the mutant causes a frameshift of the amino acid and leads to premature termination of amino acid translation. Example 2

[0055] This embodiment describes the mutant obtained in Example 1. ossgr1 Phenotypic analysis was performed, as follows:

[0056] 1. Mutant ossgr1 Salt tolerance assessment

[0057] The parameters for alternating light and dark culture are as follows: light intensity is 120 μmol·m⁻¹. -2 ·s -1 The temperature is 28℃ / 25℃ (day / dark), and the photoperiod is 10h light / 14h darkness.

[0058] The rice seeds to be tested are ossgr1- L1 Homozygous seeds of mutant T1 generation, along with its background material ZH11 and empty vector control. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows:

[0059] 1. For each material, take 12 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48 hours.

[0060] 2. After completing step 1, germinate the seeds at 28℃~30℃ for 24 hours (keep the seeds moist during germination) to obtain germinated seeds.

[0061] 3. After completing step 2, take a 96-well plate, cut off part of the lower edge of each well, and then put one germinated seed into each well (embryo facing up, radicle facing down).

[0062] 4. After completing step 3, place the 96-well plate (containing the germinated seeds) on a plastic box containing Yoshida rice culture medium, immersing the germinated seeds in the medium. Culture in alternating light and dark conditions for 3 weeks to obtain rice seedlings that have reached the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture medium should be replaced every 7 days.

[0063] 5. After completing step 4, place the 96-well plate (containing rice seedlings that have grown to the three-leaf stage) on a plastic box containing 150 mM NaCl Yoshida rice culture solution and ensure that the roots are completely immersed in the culture solution. After 3 days of treatment, replace the Yoshida rice culture solution with 200 mM NaCl and treat again. After 3 days, replace the Yoshida rice culture solution with 150 mM NaCl and treat again. Perform high salt stress for 11 days under alternating light and dark conditions (during the high salt stress period, replace the Yoshida rice culture solution every 2 days).

[0064] 6. After completing step 5, place the 96-well plate (with rice seedlings on it) on a plastic box containing Yoshida rice culture medium and allow it to recover for 7 days under alternating light and dark conditions.

[0065] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0066] See the growth status of rice seedlings before treatment. Figure 3 The growth status after treatment A is shown in the figure. Figure 3 The survival rate statistics for B are shown in the table below. Figure 3 C.

[0067] The results showed that before salt treatment, ossgr1 Slightly shorter than ZH11; freshwater control ZH11 and mutant ossgr1The survival rate was 100%. After salt solution treatment, the survival rate of ZH11 was 8.33%, while that of the mutant... ossgr1 The survival rate was 75%, and statistical analysis results showed that... ossgr1 The survival rate of [the strain] was significantly higher than that of ZH11, indicating that... ossgr1 The salt tolerance of the mutant was significantly improved. The phenotype and survival rate of the empty vector control were basically consistent with those of the background material ZH11, with no statistical difference. Example 3

[0068] This embodiment provides a method for identifying mutants. ossgr1 The co-separation molecular markers and their applications are described in the following process:

[0069] 1. Development of co-separated molecular markers

[0070] In this invention, primers were designed using Primer 5.0 software to target mutation sites in sterile lines, and a pair of co-segregating molecular markers were developed. Figure 4 The mutant genotype can be identified by combining PCR with agarose gel electrophoresis and non-denaturing polyacrylamide gel electrophoresis (PAGE) or agarose gel electrophoresis with 36200-WF2 (SEQ ID NO: 6), 36200-MF1 (SEQ ID NO: 7), and a common reverse primer 36200-R1 (SEQ ID NO: 8). The presence and size of the amplified bands can be used to identify the mutant genotype.

[0071] like Figure 5 As shown, co-separated molecular markers can specifically detect rice ossgr1 Homozygous mutants and mutant genes in rice materials derived from them OsSGR1 And can distinguish between wild type at the same time. OsSGR1 Genes and mutants ossgr1 Genes; targeting mutated genes ossgr1 A 169 bp band was amplified in the middle, while in the wild type... OsSGR1 The gene amplified into a 173 bp band:

[0072] 2. Application of co-separated molecular markers

[0073] Theoretically, 36200-WF2 / 36200-MF1 / 36200-R1 in OsSGR1 / OsSGR1 A 173 bp band can be amplified in homozygous wild-type (AA) DNA. ossgr1 / ossgr1 A 169 bp band can be amplified in the DNA of the homozygous mutant material (aa), while... OsSGR1 / ossgr1In hybrid (Aa) materials, the corresponding two bands can be amplified simultaneously. The T-cloning verification results of the 36200-WF2 / 36200-MF1 / 36200-R1 molecular markers are as follows: Figure 5 As shown, the results indicate that the designed functional molecular markers achieved the expected detection results for the segregating population. OsSGR1 / OsSGR1 Homozygous wild type (AA) OsSGR1 / ossgr1 Heterozygous (Aa) and ossgr1 / ossgr1 The homozygous mutant material (aa) amplified bands of corresponding sizes, which can be used as... OsSGR1 An ideal marker for allele detection. Example 4

[0074] This embodiment performs OsSGR1 Transgenic experiments using mutant genes, using mutants ossgr1 Hybridization, backcrossing, and self-pollination with recipients exhibiting excellent agronomic traits, such as R3261, were performed, with molecular markers used throughout the process. OsSGR1 Genetic and genetic prospect selection ultimately yields a restorer line with a homozygous mutant gene in the R3261 background. The technical route for hybridization and conversion is as follows: Figure 6 As shown, the specific implementation steps are as follows:

[0075] 1. Using the recipient parent, such as R3261, as the paternal and maternal mutants. ossgr1 F1 was obtained through hybridization.

[0076] 2. Use F1 as the female parent and the recipient parent, such as R3261, to backcross and obtain BC1F1.

[0077] 3. Plant BC1F1 and detect them using primer sequences as shown in SEQ ID NO: 6-8. OsSGR1 Genotype, selection OsSGR1 Heterozygous genotype, meaning the PCR amplification product shows both 169 bp and 173 bp bands.

[0078] 4. Use a set of genotypes (e.g., 200) in ossgr1 Polymorphism exists between mutants and recurrent parents, and molecular markers (including but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, and SCAR type markers) are evenly distributed. Genetic background identification is performed on the single plants selected in step 3, and plants with high genotypic similarity to recurrent parents (e.g., greater than 88% similarity, or 2% selection rate) are selected.

[0079] 5. Use the plants selected in step 4 and the recipient parent, such as R3261, to backcross and obtain BC2F1.

[0080] 6. Plant BC2F1, repeat steps 3 and 4, and select... OsSGR1 Plants with heterozygous genotypes and high genetic background reversion rates (e.g., greater than 98%, or a selection rate of 2%) were collected from cross BC2F2.

[0081] 7. Plant BC2F2, repeat steps 3 and 4, and select... OsSGR1 The plant with the highest genotypic heterozygosity and the highest homozygous genetic background was collected from the cross BC2F3. Segregation occurred in the progeny of BC2F3. [[ID=9 homozygous strain, i.e., under the background of R3261 ​ Gene restoration line.

[0082] The above example uses R3261 as a breeding example, but it is not limited to R3261 and can be any rice material.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying rice salt tolerance genes OsSGR1 A set of molecular marker primers for wild-type or mutant organisms, characterized in that, The primer set includes: forward primer F1 with nucleotide sequence SEQ ID NO. 6, forward primer F2 with nucleotide sequence SEQ ID NO. 7, and reverse primer R with nucleotide sequence SEQ ID NO.

8.

2. A method for identifying salt tolerance genes in rice. OsSGR1 The method using wild-type or mutant strains is characterized by, Includes the following steps: Step 1: Extract genomic DNA from the rice samples used in the test; Step 2: Perform PCR amplification on the DNA obtained in Step 1 using the primer set described in claim 1; Step 3: Detect the amplification products by agarose gel electrophoresis; Step 4: If only a 169 bp band can be amplified, the sample is... OsSGR1 The sample is a homozygous mutant exhibiting salt tolerance; if only a 173 bp band is amplified, the sample is wild-type and exhibits salt sensitivity; if both 169 bp and 173 bp bands are amplified simultaneously, the sample is... OsSGR1 A heterozygous mutant of the gene that exhibits salt sensitivity; The wild type OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the mutant... OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. The method according to claim 2, characterized in that, Rice salt tolerance gene OsSGR1 The mutant was constructed using sgRNA, the nucleotide sequence of which is shown in SEQ ID NO.

5.

4. A method for identifying rice salt tolerance genes OsSGR1 PCR reagents for wild-type or mutant organisms, comprising the primer set as described in claim 1.

5. The application of the molecular marker primer set according to claim 1 or the PCR reagent according to claim 4, characterized in that, The application is any one of the following: (1) Identification of salt tolerance genes in rice OsSGR1 Applications in wild-type or mutant strains; (2) In the preparation of identification of rice salt tolerance genes OsSGR1 Applications in products containing wild-type or mutant strains; If the PCR product of the tested rice sample can only amplify a single 169 bp band, then the sample is... OsSGR1 The sample is a homozygous mutant exhibiting salt tolerance; if only a 173 bp band is amplified, the sample is wild-type and exhibits salt sensitivity; if both 169 bp and 173 bp bands are amplified simultaneously, the sample is... OsSGR1 The heterozygous mutant exhibits salt sensitivity; the wild-type OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the mutant... OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 4.

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