Molecular marker primer pair for identifying wild type or mutant of rice salt-tolerant gene OsMT9 and application of molecular marker primer pair

Abstract

The invention relates to the technical field of biology, and particularly provides a molecular marker primer pair for identifying a wild type or mutant rice salt-tolerant gene OsMT9 and application of the molecular marker primer pair. The molecular marker primer pair is composed of primer pairs as shown in SEQ ID NO. 9 to 10. The primer pair provided by the invention can be used for distinguishing the wild type or mutant of the rice salt-tolerant gene OsMT9, is applied to auxiliary breeding of rice salt-tolerant populations, can be used for identifying and accurately distinguishing heterozygous and homozygous genotypes in cross breeding, backcross separation improvement and the like, improves the breeding efficiency, and has important practical significance on high-yield, stable-yield and stress-resistant breeding of rice.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a molecular marker primer pair for identifying wild type or mutant of a salt-tolerant gene OsMT9 of rice and application thereof. BACKGROUND

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, feeding more than half of the world's population, and its production is of great significance. However, rice is often adversely affected by various abiotic stresses during its entire growth process, among which salt stress is one of the main stresses threatening rice production. In the face of the contradiction between population growth and limited arable land, developing and utilizing coastal beach and inland saline-alkali land resources is one of the effective ways to ensure food security. Because rice grows in a water environment, it can leach soluble salt and alkali in the soil, so it is considered as the preferred food crop for developing saline-alkali land. Improving the salt tolerance of rice through genetic improvement is a core strategy to expand its planting area and increase yield.

[0003] Currently, rice salt-tolerant breeding relies on QTL sites such as Saltol and qSKC-1 on chromosome 1, and the genetic resources are relatively limited. With the development of molecular marker-assisted selection (MAS) and gene editing technology, cloning salt-tolerant genes and developing molecular markers linked to them have become the key to accelerating salt-tolerant rice breeding. The function of metallothionein in plants has been studied by many predecessors (Saeed-ur-Rahman et al. 2020). There are 13 genes in the MT family of rice, and OsMT-3a can improve the Cd resistance of varieties; OsMT2c is induced to increase expression by Cu, and overexpression of OsMT2c improves copper tolerance (Liu et al. 2015); OsMT can improve the resistance of rice under drought stress, and OsMT1e-P and OsMT4 are involved in the defense mechanism of salt stress. However, there is no relevant report on the involvement of OsMT9 in adversity stress. 2+

[0004] ​With the development of molecular biology technology, using mutants to isolate and mine salt stress genes of rice and for rice genetic engineering to assist breeding and improve salt stress resistance have extremely important significance for effectively controlling the harm of salt stress to rice, improving rice yield and improving rice quality. However, its specific implementation also faces great challenges. When using CRISPR / Cas9 system for gene editing, the design of single guide RNA (sgRNA) is the core key and main technical bottleneck. The editing efficiency and specificity of sgRNA are highly uncertain, and its design needs to comprehensively consider dozens of complex factors such as sequence length, PAM adjacent sequence, GC content, polyT structure, seed sequence specificity, off-target effect and optimal action site on target gene. Although there are various sgRNA design software, the algorithms of these tools are different, the evaluation standards for off-target effect are different, and most of the prediction results lack reliable experimental verification support (Xie Shengsong, 2015). From thousands of possible sequences, successfully screening and obtaining an effective sgRNA that can efficiently and accurately edit the target gene and ultimately produce the expected agronomic traits is a process that requires creative labor and repeated practice verification.

[0005] The inventors have previously studied and published that OsMT9 is a negative regulation salt tolerance gene, and the Chinese patent "Application of OsMT9 protein and its coding gene in regulating salt tolerance of rice" discloses that after specific mutations (single base insertion and base deletion mutation or large fragment deletion mutation) of the gene, the rice exhibits salt tolerance. The inventors have obtained an OsMT9 gene function loss homozygous mutant with significantly improved salt tolerance by CRISPR / Cas9 technology site-directed editing of the salt tolerance gene OsMT9 of rice. In order to improve the utilization efficiency of the homozygous mutant in salt tolerance breeding, how to develop a molecular marker for distinguishing homozygous mutants from wild types to achieve efficient identification of the genotype of the salt tolerance gene OsMT9 mutant has important value in subsequent utilization of homozygous mutants for salt tolerance breeding. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a molecular marker primer pair for distinguishing wild type or mutant of 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 application provides a mutant gene of rice salt tolerance gene OsMT9, and the nucleotide sequence of the mutant gene is shown in SEQ ID NO. 6 or SEQ ID NO. 11.

[0009] The second aspect of the present application provides a method for identifying wild type or mutant salt-tolerant gene OsMT9 of rice, the method comprising the following steps:

[0010] Step one, extracting genomic DNA of the tested rice sample;

[0011] Step two, using the primer pair for identifying wild type or mutant salt-tolerant gene OsMT9 of rice to perform PCR amplification on the DNA obtained in step one; the primer pair comprises a forward primer F with the nucleotide sequence shown in SEQ ID NO. 9 and a reverse primer R with the nucleotide sequence shown in SEQ ID NO. 10;

[0012] Step three, detecting the amplification product by agarose gel electrophoresis;

[0013] Step four, if only a 207 bp band can be amplified, the sample is a homozygous mutant of OsMT9 gene and exhibits salt-tolerant traits; if only a 417 bp band can be amplified, the sample is wild type and exhibits salt-sensitive traits; if both a 417 bp band and a 207 bp band can be amplified, the sample is a heterozygous mutant of OsMT9 gene and exhibits salt-sensitive traits.

[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 of the encoded protein is shown in SEQ ID NO: 3.

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

[0016] The fourth aspect of the present application provides a PCR reagent for identifying wild type or mutant salt-tolerant gene OsMT9 of rice, comprising the primer pair described above.

[0017] The fifth aspect of the present application provides the application of the molecular marker primer pair described above or the PCR reagent described above, which is any one of the following:

[0018] (1) application in identifying wild type or mutant salt-tolerant gene OsMT9 of rice;

[0019] (2) application in preparing a product for identifying wild type or mutant salt-tolerant gene OsMT9 of rice.

[0020] The present application has the following beneficial effects:

[0021] (1) A molecular marker primer pair for distinguishing the osmt9 mutant from the wild type is developed to realize 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 marker provided by the application is a PCR amplification-based functional marker designed according to the CRISPR / Cas9-specific modification site of the salt-tolerant gene OsMT9 between the mutant and the wild type, the genotype of which can directly reflect the phenotype of the plant, there is no error caused by genetic exchange, the operation is simpler than the sequencing method, and the cost is reduced.

[0023] (3) The identification method provided by the application can be applied to the assisted breeding of rice salt-tolerant trait improvement, and can be used for identification in hybrid breeding, backcross separation population, etc., to accurately distinguish the heterozygous and homozygous genotypes and improve the breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a schematic diagram of the expression transcriptome of the metal-sulfur transport protein in the above-ground part and the underground part of rice after salt stress.

[0025] Figure 2 It is a schematic diagram of the structure of OsMT9 gene and the target sequence element of CRISPR / Cas-OsMT9 carrier and the sequencing result peak diagram; wherein the upper diagram is the reference sequence of the wild type ZH11, and the lower diagram is the target point sequencing peak diagram of the mutant osmt9-ko-22-1 and osmt9-ko-23-15.

[0026] Figure 3 It is a salt-tolerant phenotype identification diagram of the osmt9 mutant 13-day seedlings, 10‰ salt treatment for 8 days, and rehydration culture for 7 days, bar = 7 cm.

[0027] Figure 4 It is the survival rate statistics of ZH11 and osmt9 before and after salt treatment. The values shown are mean ± standard deviation, n = 3. *, significant difference, P < 0.05; **, extremely significant difference, P < 0.01, and the statistical analysis method is one-way analysis of variance.

[0028] Figure 5 It is the T clone verification result of the molecular marker detection.

[0029] Figure 6 It is a schematic diagram of the backcross breeding route of the molecular marker. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0031] Example 1

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

[0033] 1. Rice OsMT9 gene sequence and expression pattern analysis

[0034] In Ensembl Plants database (http: / / plants.ensembl.org / index.html), rice OsMT9 gene was queried, the nucleotide sequence in japonica rice in Hua 11 is shown in SEQ ID NO. 1, and the CDS sequence is shown in SEQ ID NO. 2. The encoded protein contains 78 amino acids, and the sequence is shown in SEQ ID NO. 3. In order to study the function of the gene, the present application first analyzes the expression pattern of the gene in different tissue parts of rice by using gene expression database, and the expression analysis result shows that OsMT9 gene is specifically expressed in rice root.

[0035] 2. Transcriptome analysis of rice OsMT9 gene

[0036] WT and ko-1 aerial parts of leaves under salt stress for 6, 30 and 102 h were sampled, 3 biological replicates for each sample, 3 plants were randomly selected for each replicate, frozen with liquid nitrogen and stored at -80℃ for standby. The total RNA of plants was extracted by TRIzol method, the mRNA with polyA tail was enriched by Oligo(dT) magnetic beads, and the mRNA was broken into 250-300 bp fragments by ion breaking, so as to construct the mRNA library. Double-end sequencing was carried out based on Illumina NovaSeq 6000 platform. The total RNA extraction and quality detection, cDNA library construction and sequencing were completed by Beijing Baire and Kang Biotechnology Co., Ltd. After filtering the raw data, removing the adapter and low-quality reads, the high-quality reads obtained were aligned to the reference genome of rice 'Nipponbare' (IRGSP-1.0) (Mortazavi et al. 2008) using HISAT2 software. According to the alignment results, the number of reads covered in the range from the start to the end of each gene was counted, and the expression of each gene and the Pearson correlation coefficient between WT and ko-1 replicate samples were calculated by FPKM method (Trapnell et al. 2010). The DESeq2 (Love et al. 2014) software was used to analyze the difference of gene expression level between WT and ko-1, and the standard for screening differentially expressed genes (DEGs) was: expression difference fold |log2 (Fold Change)|≥1, P≤0.05. In order to further analyze the biological function and metabolic pathway of DEGs, GO enrichment analysis was carried out based on the hypergeometric distribution principle. By analyzing the transcriptome data before and after salt stress, it was found that the members of the metallothionein family all responded to salt stress Figure 1 )

[0037] 3. Functional verification of rice OsMT9 gene

[0038] In order to clarify the function of OsMT9 gene in rice, the present application adopts CRISPR / Cas9 gene editing method to site-specifically mutate the gene sequence and knock out the function of the gene in rice.

[0039] The present application selects rice Zhonghua 11 (hereinafter referred to as ZH11) as the receptor material for gene editing. The present application selects the target sequence 1 of the nucleotide sequence from the 356th base to the 375th base from the start codon ATG, as shown in SEQ ID NO. 4; and the target sequence 2 of the nucleotide sequence from the 552th base to the 571th base from the start codon ATG of the coding region of OsMT9 gene, as shown in SEQ ID NO. 5 (see Figure 2 ).

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

[0041] The gene editing vector of the present application is pEGCas9Pubi-B-OsMT9, and the basic vector of the vector is pEGCas9Pubi-B. The present application designs a target point on a primer, then obtains MT-sgRNA through PCR, and then connects it to the basic vector through one-step cloning method. The specific construction process is as follows:

[0042] i) Design of target gRNA. The gene sequence of OsMT9 is input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence is set as NGG. The DNA sequence of the target region selected by the present application is shown in SEQ ID NO. 4.

[0043] ii) Amplification of sgRNA expression cassette by overlapping PCR and nested PCR. The primer pair containing the sgRNA target sequence described above is synthesized, and then the primer pair is annealed, and then connected with the Bsa I enzyme cut 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, the vector pEGCas9Pubi-B is donated by Long Tan of Hainan University), to obtain the recombinant vector pEGCas9Pubi-OsMT9. The recombinant vector pEGCas9Pubi-OsMT9 is transformed into E. coli DH5α, and positive clones are selected for sequencing. The specific steps refer to the method 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 / Cas9 toolkit for multiplex 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 of T0 generation plants, the following steps are taken for detection:

[0056] The present application first extracts rice leaf DNA by CTAB method, and the specific method is as follows: the DNA extraction method refers to the traditional CTAB method (Rogers and Bendich, 1985). Take 3 cm rice leaves into a sterilized 2 mL centrifuge tube, add 6 mm steel balls, use a cell crusher to break the tissue, then extract by CTAB method, finally add 200 μL of sterilized water (ddH2O) to the extracted sample to dissolve the air-dried sample DNA, and wait for use. After the DNA is completely dissolved, 2 μL of sample is taken for nucleic acid OD value (A260 / A280) and nucleic acid concentration determination by ultraviolet spectrophotometer (Nanodrop 2000), and the DNA sample is diluted to 50 ng / μL for standby.

[0057] PCR uses 5 μL of 2×PCR premix (containing Mg 2+ ; Taq DNA Polymerase; 2.5 mM dNTPs; 10×PCR Buffer) of Biomiga, 1 μL of primer (containing 0.5 μL of forward and reverse primer), 1 μL of template DNA, and ddH2O to make up 10 μL. The PCR amplification program is the conventional 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 of amplification, and finally 72℃ extension for 5 min). The amplified product is electrophoresed on an 8% non-denaturing polyacrylamide gel, stained with 0.1% AgNO3, and developed with formaldehyde and NaOH for photography.

[0058] Comparative analysis of the nucleotide sequence of mutant osmt9-ko-23-15 found that Figure 2 ), compared with the unedited wild type (WT), the mutant OsMT9 gene mutant osmt9-ko-23-15 has a 210-base deletion from the start codon ATG to position 359 to 568, the nucleotide sequence is shown as SEQ ID NO. 6, the CDS sequence is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 8. The OsMT9 gene mutant osmt9-ko-22-1 has a T base inserted from the start codon ATG to position 359, and a 5-base ATCTG deletion from position 564 to 568, the nucleotide sequence is shown as SEQ ID NO. 11, the CDS sequence is shown as SEQ ID NO. 12, and the amino acid sequence is shown as SEQ ID NO. 13. The nucleotide deletion encoded by the mutant causes a frameshift in the amino acid and leads to premature termination of amino acid translation.

[0059] Example 2

[0060] This example performs phenotype analysis on the mutant osmt9 obtained in Example 1, as follows:

[0061] 1. Salt tolerance identification of mutant osmt9

[0062] The parameters of light and dark alternating culture are as follows: light intensity is 120 μmol·m -2 ·s -1 , temperature is 28℃ / 25℃ (day / night), and light cycle is 10h light / 14h dark.

[0063] The rice seeds to be tested are OsMT9-L1 mutant T1 generation homozygous seeds, background material ZH11, and empty control. The experiment is repeated 3 times to take the average value, and the steps of each repetition are as follows:

[0064] (1) For each material, 24 rice seeds to be tested are taken and packed in kraft paper bags, and then soaked in water at 28℃-30℃ for 48h.

[0065] (2) After step 1 is completed, the seeds are germinated at 28℃-30℃ for 24h (the seeds need to be kept moist during the germination process), and germinated seeds are obtained.

[0066] (3) After step 2 is completed, 96-well plates are taken, the lower edge of each well is cut, and then 1 germinated seed is placed in each well (embryo bud upward, embryo root downward).

[0067] (4) After step 3 is completed, the 96-well plates (with germinated seeds thereon) are placed on a plastic box containing Yoshida rice culture solution and the germinated seeds are immersed in the culture solution, and light and dark alternating culture is performed for 3 weeks to obtain rice seedlings grown to the three-leaf stage. During the light and dark alternating culture, the Yoshida rice culture solution needs to be replaced every 7d.

[0068] (5) After step 4 is completed, the 96-well plates (with rice seedlings grown to the three-leaf stage thereon) are placed on a plastic box containing 10% NaCl Yoshida rice culture solution and the roots are completely immersed in the culture solution, and high-salt stress is performed under light and dark alternating culture for 8d (during the high-salt stress, the Yoshida rice culture solution is replaced every 2d).

[0069] (6) After step 5 is completed, the 96-well plates (with rice seedlings thereon) are placed on a plastic box containing Yoshida rice culture solution, and recovery is performed under light and dark alternating culture for 7d.

[0070] The growth state of the rice seedlings is observed and the survival rate is counted. Survival rate = number of surviving rice seedlings / 12 x 100%.

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

[0072] The results show that before salt treatment, osmt9 and ZH11 have basically the same growth trend; the survival rates of fresh water control ZH11 and mutant osmt9 are 100%. After salt solution treatment, the survival rate of ZH11 is about 6%, while the survival rate of mutant osmt9 is about 43%, and the statistical analysis result shows that the survival rate of osmt9 is significantly higher than that of ZH11, which shows that the salt tolerance of osmt9 mutant has been significantly improved. The phenotype and survival rate of the empty control are basically the same as those of the background material ZH11, and there is no statistical difference.

[0073] Example 3

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

[0075] 1. Development of co-segregation molecular marker

[0076] In the present application, the primer is designed by Primer5.0 software aiming at the mutation site of the sterile line, and the molecular marker is developed, which comprises two primers: MT9-F1 (SEQ ID NO. 9) and MT9-R1 (SEQ ID NO. 10), combined with the method of PCR and agarose and non-denaturing polyacrylamide gel electrophoresis (PAGE) or agarose gel electrophoresis detection, the genotype of the mutant can be identified according to the presence or absence and size of the amplified band.

[0077] As shown in Figure 5 , the molecular marker can specifically detect the mutant gene osmt9-ko-23-15 in the homozygous mutant of rice osmt9 and the rice material transformed therefrom, and can also distinguish the wild type OsMT9 gene and the mutant osmt9-ko-23-15 gene at the same time; a 207 bp band is amplified in the mutant gene osmt9-ko-23-15, while a 417 bp band is amplified in the wild type OsMT9 gene:

[0078] 2. Application of co-segregation molecular marker

[0079] In theory, MT9-F1 / MT9-R1 can amplify 417 bp band in OsMT9 / OsMT9 homozygous wild type (AA) DNA, 207 bp band in osmt9-ko-23-15 / osmt9-ko-23-15 homozygous mutant material (aa) DNA, and two bands of corresponding sizes in OsMT9 / osmt9-ko-23-15 heterozygous (Aa) material, respectively. The T-cloning verification results of the molecular marker are shown in Figure 5 As shown in the results, the detection results of the designed functional molecular marker on the separation population completely meet the expectations, and the bands of corresponding sizes are 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 an ideal marker for OsMT9 allele detection.

[0080] Example 4

[0081] In this embodiment, the transformation experiment of osmt9 mutant gene is carried out, and the mutant osmt9-ko-23-15 is crossed, backcrossed and selfed with a receptor with excellent agronomic traits, such as R3261, and OsMT9 gene and genetic prospect selection are carried out by using a molecular marker in the process, and finally a restoration line with homozygous mutant gene in R3261 background is obtained. The technical route of hybrid transformation is shown in Figure 6 The specific implementation steps are as follows:

[0082] 1. The mutant osmt9-ko-23-15 is crossed with the receptor parent, such as R3261, as the male parent and the female parent to obtain F1.

[0083] 2. The F1 is backcrossed with the receptor parent, such as R3261, as the female parent to obtain BC1F1.

[0084] 3. The BC1F1 is planted, and the primer sequences such as SEQ ID No. 9-10 primer pairs are used to detect the OsMT9 genotype, and the OsMT9 heterozygous genotype is selected, that is, the bands of 417 bp and 207 bp appear simultaneously in the PCR amplification product.

[0085] 4. A group of genotypes (such as 200) are used to identify the genetic background of the single plant selected in step 3, which are polymorphic between the osmt9-ko-23-15 mutant and the recurrent parent, and are evenly distributed molecular markers (including but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR type markers), and the plants with high similarity (such as greater than 88% similarity, or 2% selection rate, etc.) to the genotype of the recurrent parent are selected.

[0086] 5. Backcross the selected plant in step 4 with the recipient parent, such as R3261, to obtain BC2F1.

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

[0088] 7. Plant the BC2F2, repeat steps 3 and 4, and select plants that are heterozygous for the OsMT9 genotype and have the highest rate of recovery of the genetic background, and collect the selfed BC2F3. The OsMT9 homozygous plants separated in the BC2F3 progeny are the restoration lines of the OsMT9 gene in the R3261 background.

[0089] The above only takes R3261 as a transformation example, but is not limited to R3261, and can be any rice material.

[0090] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solution to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A pair of molecular marker primers for identifying wild type or mutant salt-tolerant genes of rice, characterized in that, OsMT9 The primer pair comprises a forward primer F having the nucleotide sequence of SEQ ID NO. 9 and a reverse primer R having the nucleotide sequence of SEQ ID NO.

10. ​​ 2. A method for identifying salt tolerance genes in wild-type or mutant rice. OsMT9 The method is characterized by, The application further provides a method for detecting OsMT9 gene in rice, comprising the following steps: Step one, extracting genomic DNA of the test rice sample; Step two, performing PCR amplification on the DNA obtained in step one by using the primer pair of claim 1; Step three, detecting the amplification product by agarose gel electrophoresis; Step four, if only 207 bp band can be amplified, the sample is OsMT9 homozygous mutant, and shows salt tolerance trait; if only 417 bp band can be amplified, the sample is wild type, and shows salt sensitive trait; If 417 bp and 207 bp bands are amplified at the same time, the sample is OsMT9 a gene hybrid mutant and exhibits salt-sensitive traits; Among them, wild-type rice salt-tolerant genes OsMT9 The nucleotide sequence is shown in SEQ ID NO. 1, mutant rice salt tolerance gene. osmt9-ko-23-15 The nucleotide sequence is shown in SEQ ID NO.

6.

3. The method of claim 2, wherein, The mutants of the application are characterized by the presence of a mutation in the gene encoding the protein of interest, said mutation being selected from the group consisting of: OsMT9 The gene is constructed by CRISPR / Cas9 system using sgRNA targeting the sequence as shown in SEQ ID NO. 4 and SEQ ID NO.

5.

4. A gene for identifying salt tolerance in wild-type or mutant rice OsMT9 The PCR reagent comprises the primer pair as described in claim 1.

5. Use of a molecular marker primer pair according to claim 1 or PCR reagents according to claim 4, characterized in that, The application is any one of the following: (1) Use of the wild-type or mutant salt-tolerant gene in rice OsMT9 ; (2) use in the manufacture of a product of a wild-type or mutant salt-tolerant gene of rice OsMT9 ​ Among them, wild-type rice salt-tolerant genes OsMT9 The nucleotide sequence is shown in SEQ ID NO. 1, mutant rice salt tolerance gene. osmt9-ko-23-15 The nucleotide sequence is shown in SEQ ID NO. 6.

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