Molecular marker primer pair for identifying wild type or mutant of salt-tolerant gene osmt9 of rice and application thereof

Abstract

This invention relates to the field of biotechnology, specifically providing a method for identifying salt tolerance genes in wild-type or mutant rice. OsMT9 Molecular marker primer pairs and their applications. The molecular marker primer pairs consist of the primer pairs shown in SEQ ID NO. 9-10. The primer pairs provided by this invention can be used to distinguish rice salt tolerance genes. OsMT9 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

Molecular marker primer pairs for identifying wild-type or mutant rice salt tolerance gene OsMT9 and their applications Technical Field

[0001] This invention belongs to the field of biotechnology and relates to molecular marker primer pairs for identifying wild-type or mutant rice salt tolerance gene OsMT9 and their applications. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, feeding more than half the world's population, and its production is of great significance. However, rice is frequently subjected to various abiotic stresses throughout its growth process, among which salt stress is one of the major threats to rice production. Faced with the contradiction between population growth and limited arable land, developing and utilizing coastal tidal flats and inland saline-alkali land resources is one of the effective ways to ensure food security. Because rice grows in an aquatic environment, it can leach soluble salts and alkalis in the soil, thus it is considered the preferred food crop for developing saline-alkali land. Improving rice's salt tolerance through genetic modification is a core strategy for expanding its planting area and increasing yield.

[0003] Currently, rice salt-tolerant breeding relies heavily on QTL loci such as Saltol and qSKC-1 on chromosome 1, resulting in relatively limited genetic resources. With the development of marker-assisted selection (MAS) and gene editing technologies, cloning salt-tolerant genes and developing linked molecular markers has become crucial for accelerating salt-tolerant rice breeding. The function of metallothioneins in plants has been extensively studied (Saeed-ur-Rahman et al., 2020). The rice MT family contains 13 genes, and OsMT-3a can increase Cd levels in varieties. 2+ Resistance; OsMT2c expression is increased by Cu induction, and OsMT2c overexpression improves copper tolerance (Liu et al. 2015). OsMT can enhance rice resistance to drought stress, and OsMT1e-P and OsMT4 are involved in the defense mechanism against salt stress. However, there are no reports on the involvement of OsMT9 in abiotic stress.

[0004] With the development of molecular biotechnology, the use of mutants to isolate and discover salt stress-tolerant genes in rice, and their application in 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. However, its implementation also faces significant challenges. When using the CRISPR / Cas9 system for gene editing, the design of single-guide RNA (sgRNA) is the core key and major technical bottleneck determining success or failure. The editing efficiency and specificity of sgRNA are highly uncertain, and its design requires a comprehensive consideration of dozens of complex factors, including sequence length, PAM neighboring sequences, GC content, polyT structure, seed sequence specificity, off-target effects, and the optimal site of action on the target gene. Although various sgRNA design software exist, the algorithms underlying these tools vary greatly, the evaluation criteria for off-target effects differ, and most prediction results lack reliable experimental verification support (Xie Shengsong, 2015). Successfully screening and obtaining an effective sgRNA from thousands of possible sequences that can efficiently and accurately edit the target gene and ultimately produce the desired agronomic traits is a process that requires creative labor and repeated practical verification.

[0005] The inventors had previously researched and published that OsMT9 is a negatively regulating salt tolerance gene. The Chinese patent, "Application of OsMT9 Protein and its Encoding Gene in Regulating Salt Tolerance in Rice," discloses that specific mutations in this gene (single-base insertion, base deletion, or large-segment deletion mutations) induce salt tolerance in rice. Currently, the inventors have used CRISPR / Cas9 technology to specifically edit the rice salt tolerance gene OsMT9, obtaining a homozygous loss-of-function mutant with significantly enhanced salt tolerance. To improve the utilization efficiency of homozygous mutants in salt tolerance breeding, developing a molecular marker to distinguish homozygous mutants from wild-type mutants, and achieving efficient genotyping of the OsMT9 mutant, is of significant value for subsequent salt tolerance breeding using homozygous mutants. Summary of the Invention

[0006] The technical problem to be solved by this invention is to propose a molecular marker primer pair for identifying wild-type or mutant rice salt tolerance gene OsMT9 and its application.

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

[0008] The first aspect of the present invention provides a mutant gene of rice salt tolerance gene OsMT9, the nucleotide sequence of which is shown in SEQ ID NO. 6 or SEQ ID NO. 11.

[0009] A second aspect of the present invention provides a method for identifying wild-type or mutant rice salt tolerance gene OsMT9, the identification method comprising the following steps:

[0010] Step 1: Extract genomic DNA from the rice samples used in the test;

[0011] Step 2: Perform PCR amplification on the DNA obtained in Step 1 using a molecular marker primer pair for identifying the wild-type or mutant rice salt tolerance gene OsMT9; the primer pair includes: forward primer F with the nucleotide sequence shown in SEQ ID NO. 9 and reverse primer R with the nucleotide sequence shown in SEQ ID NO. 10;

[0012] Step 3: Detect the amplification products by agarose gel electrophoresis;

[0013] Step 4: If only a 207 bp band can be amplified, the sample is a homozygous mutant of the OsMT9 gene and exhibits salt tolerance; if only a 417 bp band can be amplified, the sample is wild-type and exhibits salt sensitivity; if both 417 bp and 207 bp bands can be amplified simultaneously, the sample is a heterozygous mutant of the OsMT9 gene and exhibits salt sensitivity.

[0014] In the method, the nucleotide sequence of the wild-type OsMT9 gene is shown in SEQ ID NO:1, and the amino acid sequence encoding the protein is shown in SEQ ID NO:3.

[0015] In the method, the nucleotide sequence of the OsMT9 gene of the mutant, as shown in SEQ ID NO:6, is constructed using a CRISPR / Cas9 system, and the sgRNA used in the CRISPR / Cas9 system targets sequences as shown in SEQ ID NO:4 and SEQ ID NO:5.

[0016] A fourth aspect of the present invention provides a PCR reagent comprising the above-described primer pairs for identifying wild-type or mutant rice salt tolerance gene OsMT9.

[0017] The fifth aspect of the present invention provides the application of the above-described molecular marker primer pairs or the above-described PCR reagents, wherein the application is any of the following:

[0018] (1) Application in identifying wild-type or mutant rice salt tolerance gene OsMT9;

[0019] (2) Application in the preparation of products for identifying wild-type or mutant rice salt tolerance gene OsMT9.

[0020] The beneficial effects of this invention are:

[0021] (1) A molecular marker primer pair was developed to distinguish between the osmt9 mutant and the wild type, so as to achieve efficient identification of the genotype of the salt-tolerant gene OsMT9 mutant, which has important value in subsequent salt-tolerant breeding using the osmt9 mutant.

[0022] (2) The molecular markers provided by this invention are functional markers based on PCR amplification designed based on the CRISPR / Cas9-specific modification sites of the salt tolerance gene OsMT9 between mutants and wild types. Their genotypes can directly reflect the plant phenotypes, and there are no errors caused by genetic exchange. The operation is simpler than sequencing methods and reduces costs.

[0023] (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

[0024] Figure 1 is a schematic diagram of the transcriptome of metal sulfur transporter protein response to salt in rice after salt stress.

[0025] Figure 2 shows the OsMT9 gene structure, the target sequence elements of the CRISPR / Cas-OsMT9 vector, and the sequencing results peak diagram; the upper figure is the wild-type ZH11 reference sequence, and the lower figure is the sequencing peak diagram of the target sites of the mutants osmt9-ko-22-1 and osmt9-ko-23-15.

[0026] Figure 3 shows the salt tolerance phenotype of osmt9 mutant seedlings after 13 days of treatment with 10‰ salt for 8 days and rehydration culture for 7 days, bar = 7cm.

[0027] Figure 4 shows the survival rates of ZH11 and osmt9 before and after salt treatment. Values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference (P < 0.05); ** indicates highly significant difference (P < 0.01). Statistical analysis was performed using one-way ANOVA.

[0028] Figure 5 shows the results of T-clone validation using molecular marker detection.

[0029] Figure 6 is a schematic diagram of the molecular marker backcrossing and transformation route. Detailed Implementation

[0030] 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.

[0031] Example 1

[0032] This embodiment provides the function and application of the rice OsMT9 gene, specifically including the following:

[0033] 1. Analysis of rice OsMT9 gene sequence and expression pattern

[0034] The rice OsMT9 gene was found in the Ensembl Plants database (http: / / plants.ensembl.org / index.html). Its nucleotide sequence in japonica rice (Zhonghua 11) is shown in SEQ ID NO.1, and its CDS sequence is shown in SEQ ID NO.2. The encoded protein contains 78 amino acids, and its sequence is shown in SEQ ID NO.3. To study the function of this gene, this invention first analyzed its expression pattern in different tissues of rice using a gene expression database. The expression analysis results showed that the OsMT9 gene is specifically expressed in rice roots.

[0035] 2. Transcriptome analysis of the rice OsMT9 gene

[0036] Aboveground leaves of WT and ko-1 rice plants subjected to salt stress for 6, 30, and 102 h were sampled, with three biological replicates per sample. Three plants were randomly selected from each replicate, flash-frozen in liquid nitrogen, and stored at −80℃ for later use. Total RNA was extracted from the plants using the TRIzol method. mRNA with polyA tails was enriched using Oligo(dT) magnetic beads, and the mRNA was fragmented into 250–300 bp fragments using ion fragmentation to construct mRNA libraries. Paired-end sequencing was performed using the Illumina NovaSeq 6000 platform. Total RNA extraction and quality control, cDNA library construction, and sequencing were all performed by Beijing Berry Genomics Co., Ltd. After filtering the raw data to remove adapters and low-quality reads, the high-quality reads obtained were aligned to the reference genome (IRGSP-1.0) of rice 'Nipponbare' (Mortazavi et al., 2008) using HISAT2 software. Based on the alignment results, the number of reads covered by each gene from start to end was counted. The expression level of each gene and the Pearson correlation coefficient between the WT and ko-1 replicates were calculated using the FPKM method (Trapnell et al., 2010). Differential expression levels between WT and ko-1 were analyzed using DESeq2 software (Love et al., 2014). The criteria for screening differentially expressed genes (DEGs) were: fold change |log2 (Fold Change)| ≥ 1, P ≤ 0.05. To further analyze the biological functions and metabolic pathways of DEGs, GO enrichment analysis was performed based on the hypergeometric distribution principle. Analysis of transcriptome data before and after salt stress revealed that all members of the metallothionein family responded to salt stress (Figure 1).

[0037] 3. Functional verification of the rice OsMT9 gene

[0038] To clarify the function of the OsMT9 gene in rice, this invention uses the CRISPR / Cas9 gene editing method to perform site-directed mutations in the gene sequence and knock out the function of the gene in rice.

[0039] This invention selects the conventional rice variety ZH11 as the recipient material for gene editing. The invention selects target sequence 1, as shown in SEQ ID NO.4, which is the nucleotide sequence from base 356 to base 375 starting from the start codon ATG; and target sequence 2, as shown in SEQ ID NO.5, which is the nucleotide sequence targeting the coding region of the OsMT9 gene from base 552 to base 571 starting from the start codon ATG (see Figure 2).

[0040] (1) Construction of CRISPR / Cas9 gene editing vector for OsMT9

[0041] The gene editing vector of this invention is pEGCas9Pubi-B-OsMT9, 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:

[0042] i) Design of target gRNA. The OsMT9 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. 4.

[0043] 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-OsMT9. The recombinant vector pEGCas9Pubi-OsMT9 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 multiple genome editing in plants. BMC plant biology 14:327."

[0044] iii) Sequencing verification.

[0045] A positive clone with correct sequencing results is a successfully constructed pEGCas9Pubi-OsMT9 gene editing vector.

[0046] (2) Agrobacterium-mediated genetic transformation of rice

[0047] The pEGCas9Pubi-OsMT9 gene editing vector successfully constructed above was transformed into Agrobacterium EHA105 via heat shock. After identification by PCR, the bacterial culture was stored at -80℃ with glycerol.

[0048] Freshly peeled embryos of the hybrid rice variety Zhonghua 11, approximately 1.5 mm in size, 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 washings at the bottom of the tubes were aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then left to stand in the dark for 8 minutes.

[0049] 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 any excess infection solution. Place all embryos with their scutes facing upwards and co-culture at 23°C in the dark for 3 days.

[0050] 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, be careful to remove any sprouts that grow on the embryos.

[0051] After the recovery culture was completed, the immature embryos were placed on a selection medium containing 1.5 mg / L Bialaphos for 3 rounds of selection culture, with each round lasting 2 weeks. Then, they were transferred to a selection medium containing 2 mg / L Bialaphos for 2 rounds of selection culture, with each round lasting 2 weeks.

[0052] The resistant callus was transferred to propagation medium and cultured in the dark at 28°C for 2 weeks. Then, the propagated resistant callus was transferred to induction medium and cultured in the dark at 28°C for 2 weeks. Finally, it was transferred to differentiation medium and cultured under light at 25°C and 5000 lx for 2 weeks.

[0053] After the culture is completed, the differentiated seedlings are separated into individual seedlings and placed in a rooting medium. They are cultured at 25°C, 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.

[0054] (3) Detection of CRISPR / Cas9 mutation results in T0 generation plants

[0055] To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken for detection:

[0056] 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.

[0057] PCR was performed using Biomiga's 2×PCR premix (containing Mg). 2+ The following reagents were used: 5 μL of 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 subjected to 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after formaldehyde and NaOH staining.

[0058] Nucleotide sequence alignment analysis of the mutant osmt9-ko-23-15 revealed (Figure 2) that, compared to the unedited wild-type (WT), the mutated OsMT9 gene mutant osmt9-ko-23-15 exhibits a large deletion of 210 bases from position 359 to 568 of the start codon ATG. The nucleotide sequence is shown in SEQ ID NO. 6, the CDS sequence in SEQ ID NO. 7, and the amino acid sequence in SEQ ID NO. 8. The OsMT9 gene mutant osmt9-ko-22-1 inserts a T base at position 359 of the start codon ATG, and simultaneously deletes 5 bases (ATCTG) from position 564 to 568. The nucleotide sequence is shown in SEQ ID NO. 11, the CDS sequence in SEQ ID NO. 12, and the amino acid sequence in SEQ ID NO. 13. The deletion of the nucleotides encoded by the mutants causes frameshifts in amino acids, leading to premature termination of amino acid translation.

[0059] Example 2

[0060] This embodiment performs phenotypic analysis on the mutant osmt9 obtained in Example 1, as detailed below:

[0061] 1. Salt tolerance identification of mutant osmt9

[0062] 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.

[0063] The rice seeds tested were homozygous seeds of the OsMT9-L1 mutant T1 generation, along with background material ZH11 and an empty vector control. The experiment was repeated three times, and the average value was taken. The steps for each repetition were as follows:

[0064] (1) For each material, take 24 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48h.

[0065] (2) After completing step 1, germinate the seeds at 28℃~30℃ for 24 hours (keep the seeds moist during germination) to obtain germinated seeds.

[0066] (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).

[0067] (4) After completing step 3, place the 96-well plate (with germinated seeds on it) on a plastic box containing Yoshida rice culture solution and immerse the germinated seeds in the culture solution. Culture in alternating light and dark for 3 weeks to obtain rice seedlings that have grown to the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture solution needs to be replaced every 7 days.

[0068] (5) After completing step 4, place the 96-well plate (on which rice seedlings that have grown to the three-leaf stage) on a plastic box containing 10% NaCl Yoshida rice culture solution and immerse the roots completely in the culture solution. Under high salt stress for 8 days under alternating light and dark conditions (during the high salt stress period, the Yoshida rice culture solution is replaced every 2 days).

[0069] (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 recover for 7 days under alternating light and dark culture.

[0070] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0071] The growth status of rice seedlings before treatment is shown in Figure 3, and the survival rate statistics are shown in Figure 4.

[0072] The results showed that before salt treatment, the growth of osmt9 and ZH11 was basically the same; the survival rate of the freshwater control ZH11 and the mutant osmt9 was 100%. After salt solution treatment, the survival rate of ZH11 was about 6%, while the survival rate of the mutant osmt9 was about 43%. Statistical analysis showed that the survival rate of osmt9 was significantly higher than that of ZH11, indicating that the salt tolerance of the osmt9 mutant was significantly improved. The phenotype and survival rate of the empty vector control were basically the same as those of the background material ZH11, with no statistically significant difference.

[0073] Example 3

[0074] This embodiment provides a co-segregating molecular marker for identifying the mutant osmt9-ko-23-15 and its application. The specific process is as follows:

[0075] 1. Development of co-separated molecular markers

[0076] In this invention, primers were designed using Primer 5.0 software to develop molecular markers targeting mutation sites in sterile lines. These markers consist of two primers: MT9-F1 (SEQ ID NO. 9) and MT9-R1 (SEQ ID NO. 10). By combining PCR with agarose gel electrophoresis and non-denaturing polyacrylamide gel electrophoresis (PAGE) or agarose gel electrophoresis, the genotype of the mutant can be identified based on the presence and size of the amplified bands.

[0077] As shown in Figure 5, the molecular markers can specifically detect the osmt9-ko-23-15 mutant gene in homozygous osmt9 mutants and rice materials bred from them, and can simultaneously distinguish between the wild-type OsMT9 gene and the mutant osmt9-ko-23-15 gene; a 207 bp band was amplified for the mutant osmt9-ko-23-15 gene, while a 417 bp band was amplified for the wild-type OsMT9 gene.

[0078] 2. Application of co-separated molecular markers

[0079] Theoretically, MT9-F1 / MT9-R1 can amplify a 417 bp band in OsMT9 / OsMT9 homozygous wild-type (AA) DNA, a 207 bp band in osmt9-ko-23-15 / osmt9-ko-23-15 homozygous mutant (aa) DNA, and two corresponding bands can be amplified simultaneously in OsMT9 / osmt9-ko-23-15 heterozygous (Aa) material. The T-clone validation results of the molecular markers are shown in Figure 5. The results show that the designed functional molecular markers fully meet the expectations for the detection of segregating populations. The corresponding bands were amplified in OsMT9 / OsMT9 homozygous wild type (AA), OsMT9 / osmt9-ko-23-15 heterozygous (Aa), and osmt9-ko-23-15 / osmt9-ko-23-15 homozygous mutant material (aa), respectively, which can be used as ideal markers for the detection of OsMT9 alleles.

[0080] Example 4

[0081] This embodiment conducts a transgenic experiment using the osmt9 mutant gene. The mutant osmt9-ko-23-15 is crossed, backcrossed, and self-crossed with a recipient gene exhibiting excellent agronomical traits, such as R3261. Molecular markers are used for OsMT9 gene and genetic prospect selection during this process, ultimately obtaining a restorer line carrying the homozygous mutant gene in the R3261 background. The technical route for transgenic hybridization is shown in Figure 6, and the specific implementation steps are as follows:

[0082] 1. F1 is obtained by crossing the recipient parent, such as R3261 as the male parent, with the female mutant osmt9-ko-23-15.

[0083] 2. Use F1 as the female parent and the recipient parent, such as R3261, to backcross and obtain BC1F1.

[0084] 3. Plant BC1F1 and use primer pairs such as SEQ ID No. 9-10 to detect the OsMT9 genotype. Select the OsMT9 heterozygous genotype, that is, the PCR amplification product shows both 417 bp and 207 bp bands.

[0085] 4. Using a set of molecular markers (including but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, and SCAR type markers) that show polymorphism between the osmt9-ko-23-15 mutant and the recurrent parent and are evenly distributed, a genetic background identification is performed on the single plants selected in step 3. Plants with high genotypic similarity to the recurrent parent (e.g., greater than 88% similarity, or 2% selection rate) are selected.

[0086] 5. Use the plants selected in step 4 and the recipient parent, such as R3261, to backcross and obtain BC2F1.

[0087] 6. Plant BC2F1, repeat steps 3 and 4, select plants that are heterozygous for OsMT9 genotype and have a high genetic background recovery rate (e.g., greater than 98%, or a selection rate of 2%), and harvest them from the crossbred BC2F2.

[0088] 7. Plant BC2F2, repeat steps 3 and 4, and select the plants with the highest OsMT9 genotype heterozygosity and genetic background homozygosity, which are then harvested from the cross BC2F3. The OsMT9 homozygous plants that segregate from the offspring of BC2F3 are the restorers of the OsMT9 gene in the R3261 background.

[0089] The above example uses R3261 as a breeding example, but it is not limited to R3261 and can be any rice material.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended 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. Such 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 wild-type or mutant rice salt tolerance gene OsMT9, characterized in that, The procedure includes the following steps: Step 1, extracting genomic DNA from the tested rice sample; Step 2, performing PCR amplification on the DNA obtained in Step 1 using the forward primer F (nucleotide sequence shown in SEQ ID NO. 9) and the reverse primer R (nucleotide sequence shown in SEQ ID NO. 10); Step 3, detecting the amplification products by agarose gel electrophoresis; Step 4, if only a 207 bp band is amplified, the sample is a homozygous mutant of the OsMT9 gene and exhibits salt tolerance; if only a 417 bp band is amplified, the sample is wild-type and exhibits salt sensitivity. If two bands of 417 bp and 207 bp can be amplified simultaneously, the sample is a heterozygous mutant of the OsMT9 gene and exhibits salt sensitivity; the nucleotide sequence of the wild-type rice salt-tolerant gene OsMT9 is shown in SEQ ID NO. 1, and the nucleotide sequence of the mutant rice salt-tolerant gene osmt9-ko-23-15 is shown in SEQ ID NO.

6. The rice is japonica rice Zhonghua 11.

2. The method according to claim 1, characterized in that, The mutant OsMT9 gene was constructed using a CRISPR / Cas9 system, the sgRNA used in which the CRISPR / Cas9 system targets sequences as shown in SEQ ID NO.4 and SEQ ID NO.5.

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