Method for producing rice haploid and application of rice haploid in breeding

Abstract

The invention discloses a method for producing rice haploids and application of the method in breeding, by editing one or more of OsFEB1, OsFEB2 and OsFEB3 genes in rice, the functions of the OsFEB1, OsFEB2 and / or OsFEB3 genes are lost, a rice haploid induction line is obtained, generation of haploids is induced through sexual reproduction, the gene sequence of the OsFEB1 is as shown in SEQ ID NO.1, the gene sequence of the OsFEB2 is as shown in SEQ ID NO.2, the gene sequence of the OsFEB2 is as shown in SEQ ID NO.1, the gene sequence of the OsFEB2 is as shown in SEQ ID NO.2, and the gene sequence of the OsFEB3 is as shown in SEQ ID NO.2. And the gene sequence of the OsFEB3 is as shown in SEQ ID NO. 3. The haploid induction line obtained by the method provided by the invention has the capability of inducing generation of haploid plants for breeding. An RHG1 haploid induction line is combined with an MIME system, and an apomixis material is artificially created to fix the heterosis of rice.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and plant breeding technology, and particularly relates to a method for producing rice haploids and its application in breeding. Background Technology

[0002] Hybrid rice typically offers a 20% yield advantage over conventional rice, making a significant contribution to ensuring food security in China and the world. It has been recommended by the Food and Agriculture Organization of the United Nations as the preferred technology for addressing global food issues. However, hybrid rice offspring exhibit phenotypic segregation and cannot be directly used for seed production, requiring annual seed production. This significantly increases production costs, hindering not only the wider adoption and application of hybrid rice but also the full utilization of heterosis. Only through continuous innovation in hybrid rice breeding technology, reducing seed production costs, and increasing its accessibility can more farmers and businesses benefit from this technology.

[0003] As early as 1987, Academician Yuan Longping proposed that one-line hybrid rice, based on apomixis, could achieve the fixation of heterosis. Apomixis in plants refers to a reproductive method in which male and female gametes develop into new individuals without undergoing meiosis and fusion. The one-line method primarily utilizes apomixis to fix the heterosis of the F1 generation of hybrid rice, and it is considered the ultimate strategic goal of hybrid rice breeding. Artificially created apomixis materials do not result in phenotypic segregation in their offspring, just like conventional rice. Farmers can save seeds for their own cultivation, greatly reducing the production cost of hybrid rice. Therefore, fixing heterosis in rice is becoming a cutting-edge research area in plant genetics and breeding.

[0004] Related reports indicate that successful research on the fixation of heterosis in rice is based on the MiMe (Mitosis instead of Meiosis) technology system. The MiMe system can convert meiosis in reproductive cells into mitosis, thereby producing diploid male and female gametes that do not recombine chromosomes, do not undergo meiosis, and still possess reproductive function. It mainly involves simultaneous mutations in three genes related to meiosis: mutation in the pair1 gene inhibits crossing over and recombination of non-sister chromatids during homologous chromosome pairing in meiosis I; mutation in the rec8 gene causes sister chromatids to separate in late meiosis I; and mutation in osd1 causes skipping meiosis II. However, the offspring produced by self-pollination of MiMe plants are tetraploid, and the chromosome number doubles with each self-pollination. Therefore, the current challenge in successfully achieving apomixis lies in ensuring that the chromosome number does not double in each generation, i.e., creating haploid material.

[0005] Currently, the main pathways to induce haploid formation include: ectopic expression of the BBM1 gene or the dandelion PAR gene to induce parthenogenesis; knockout of the MTL gene to induce haploids; and editing of the centromere-specific histone cenh3 or DMP family genes to induce haploids. In recent years, domestic and international scholars have combined haploid induction technology with the MiMe system to artificially create apomixis to fix heterosis in rice, bringing hope for the breeding and application of one-line hybrid rice. However, this system still has shortcomings: low fixation frequency can severely affect rice seed setting rate, and there is a technical bottleneck that needs to be overcome to improve haploid induction rate. Therefore, screening and mining high-frequency haploid induction genes is of great significance for creating one-line hybrid rice with high fixation and high seed setting rates. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a method for generating rice haploids and its application in breeding. This invention utilizes gene editing technology to simultaneously knock out the fertilization barrier genes OsFEB1, OsFEB2, and OsFEB3 in the rice genome. The resulting homozygous knockout positive line (RHG1, Rice Haploid Induced Gene 1) is the haploid inducible line, capable of inducing the production of haploid plants for breeding. By combining the RHG1 haploid inducible line with the MIME system, apomixis are artificially created to fix heterosis in rice.

[0007] To address the aforementioned technical problems, this invention provides a method for generating rice haploids. This method involves editing one or more of the OsFEB1, OsFEB2, and OsFEB3 genes in rice to render the OsFEB1, OsFEB2, and / or OsFEB3 genes non-functional, thereby obtaining rice haploids. The gene sequence of OsFEB1 is shown in SEQ ID NO.1, the gene sequence of OsFEB2 is shown in SEQ ID NO.2, and the gene sequence of OsFEB3 is shown in SEQ ID NO.3.

[0008] Furthermore, the above method involves knocking out the OsFEB1, OsFEB2, and / or OsFEB3 genes in rice using the CRISPR / Cas9 system, thereby causing the OsFEB1, OsFEB2, and / or OsFEB3 genes to lose their function.

[0009] The above method, further, includes the following steps:

[0010] (1) Design primers based on the gene sequences of OsFEB1, OsFEB2 and / or OsFEB3, and construct CRISPR / Cas9 vector A with OsFEB1, OsFEB2 and / or OsFEB3 genes knocked out;

[0011] (2) The CRISPR / Cas9 vector A was transformed into rice to obtain T0 generation plants with bis-allelic homozygous knockout of OsFEB1, OsFEB2 and / or OsFEB3.

[0012] (3) Sow the T0 generation plants and self-pollinate to obtain rice haploid induction lines.

[0013] The above method further includes primers designed based on the OsFEB1 gene sequence, including FEB1-1F and FEB1-1R; primers designed based on the OsFEB2 gene sequence, including FEB1-2F and FEB1-2R; and primers designed based on the OsFEB3 gene sequence, including FEB1-3F and FEB1-3R.

[0014] The gene sequence of FEB1-1F is shown in SEQ ID NO.4;

[0015] The gene sequence of FEB1-1R is shown in SEQ ID NO.5;

[0016] The gene sequence of the FEB2-1F is shown in SEQ ID NO.6;

[0017] The gene sequence of the FEB2-1R is shown in SEQ ID NO.7;

[0018] The gene sequence of FEB3-1F is shown in SEQ ID NO.8;

[0019] The gene sequence of FEB3-1R is shown in SEQ ID NO.9.

[0020] Furthermore, in the above method, the rice is indica rice.

[0021] Based on a general technical concept, the present invention also provides an application of rice haploids obtained by the method in breeding.

[0022] The above application, further, the method of application includes the following steps:

[0023] S1. Primers were designed based on the gene sequences of PAIR1, OsREC8 and OsOSD1 to construct CRISPR / Cas9 vector B, which knocked out the three genes of PAIR1, OsREC8 and OsOSD1.

[0024] S2. CRISPR / Cas9 vector A and CRISPR / Cas9 vector B are co-transformed into rice seeds to obtain T0 generation plants with bis-allel homozygous knockout.

[0025] S3. Self-pollinate the T0 generation plants to obtain T1 generation seeds, thereby fixing the heterosis of rice.

[0026] Furthermore, in the above applications, primers designed based on the PAIR1 gene sequence include PAIR1-1F and PAIR1-1R; primers designed based on the OsREC8 gene sequence include REC8-1F and REC8-1R; and primers designed based on the OsOSD1 gene sequence include OSD1-1F and OSD1-1R.

[0027] The gene sequence of OSD1-1F is shown in SEQ ID NO.10;

[0028] The gene sequence of OSD1-1R is shown in SEQ ID NO.11;

[0029] The gene sequence of PAIR1-1F is shown in SEQ ID NO.12;

[0030] The gene sequence of PAIR1-1R is shown in SEQ ID NO.13;

[0031] The gene sequence of REC8-1F is shown in SEQ ID NO.14;

[0032] The gene sequence of REC8-1R is shown in SEQ ID NO.15.

[0033] Compared with the prior art, the advantages of the present invention are as follows:

[0034] (1) This invention provides a method for producing rice haploids. By using gene editing technology to knock out the fertilization barrier genes OsFEB1, OsFEB2, and OsFEB3 in the rice genome, the resulting homozygous knockout positive line (RHG1, RiceHaploid Induced Gene 1) is a haploid inducing line, capable of inducing the production of haploid plants for breeding. This invention is the first to demonstrate that the RHG1 inducing line can induce haploid production during sexual reproduction processes such as self-pollination, which is of great significance for creating high-frequency haploid inducing lines and optimizing and improving apomixis induction systems. It should also be noted that, in this application, knocking out one or both of the fertilization barrier genes OsFEB1, OsFEB2, and OsFEB3 can also produce rice haploids. Plants with homozygous knockout of three genes (OsFEB1, OsFEB2, and OsFEB3) simultaneously exhibited a high haploid induction rate of 6.25%-25%, which is higher than the induction rate obtained by knocking out one gene alone and significantly higher than the induction rate disclosed in existing technologies.

[0035] (2) This invention provides an application of rice haploids in breeding, combining the RHG1 haploid induction line with the MIME system to artificially create apomixis to fix the heterosis of rice, laying a theoretical and applied foundation for further creating one-line hybrid rice with high fixation rate and high seed setting rate suitable for agricultural production. Attached Figure Description

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0037] Figure 1 The figure shows the gene structure and mutation type of the rhg1 knockout mutant in Embodiment 1 of the present invention. In the figure, a is a schematic diagram of the CRISPR / Cas9-RHG1 knockout vector structure; bd in the figure shows the gene structure, target sequence and mutation type of the rhg1 three-gene homozygous mutant lines OsFEB1, OsFEB2 and OsFEB3.

[0038] Figure 2 Phenotypic comparisons of Huazhan-wild-type (HZ-WT) and rhg1 mutant diploid plants in the same growing environment.

[0039] Figure 3 Phenotypes of haploid plants of the Huazhan-wild type (HZ-WT) and rhg1 mutant.

[0040] Figure 4 To identify chromosome ploidy in HZ-WT and rhg1 haploid plants by flow cytometry. Detailed Implementation

[0041] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0042] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0043] Example 1

[0044] A method for inducing haploidy according to the present invention includes the following steps:

[0045] (1) Construct a CRISPR / Cas9 knockout vector A targeting the fertilization disorder genes OsFEB1, OsFEB2, and OsFEB3. The gene sequence of OsFEB1 is shown in SEQ ID NO.1, the gene sequence of OsFEB2 is shown in SEQ ID NO.2, and the gene sequence of OsFEB3 is shown in SEQ ID NO.3. Specific steps include:

[0046] 1.1 Primers for target gene sites of OsFEB1, OsFEB2 and OsFEB3 (see Table 1 for details) were designed using the online website (E-CRISP Design) and then synthesized by Hunan Youkanglai Biotechnology Co., Ltd.

[0047] FEB1-1F:5'-GGCAGGAGATCAAGTCGTGCACGG-3' (SEQ ID NO.4);

[0048] FEB1-1R: 5'-AAACCCGTGCACGACTTGATCTCC-3' (SEQ ID NO. 5).

[0049] FEB2-1F:5'-GCCGGAAGCCGATGGAGGCGAGCA-3' (SEQ ID NO.6);

[0050] FEB2-1R: 5'-AAACTGCTCGCCTCCATCGGCTTC-3' (SEQ ID NO. 7).

[0051] FEB3-1F:5'-GTTGGACGCGCGCGGCGTCGGCAA-3' (SEQ ID NO.8);

[0052] FEB3-1R: 5'-AAACTTGCCGACGCCGCGCCGTC-3' (SEQ ID NO. 9).

[0053] Table 1: RHG1 Information Statistics Table

[0054]

[0055] 1.2. Perform the enzyme digestion and ligation reaction according to the following procedure:

[0056] 2.1 Add ddH2O to the adapter primer centrifuge tube to prepare a 100μM stock solution. Then, add 98μL of ddH2O to a 0.2mL PCR tube, followed by 1μL of each of the target gene forward and reverse primers. Gently vortex and centrifuge to mix the liquids thoroughly. This completes the pre-preparation of the adapter primer mixtures for the three target genes.

[0057] 2.2. Heat the water bath to 100℃, place the three PCR tubes from step 2.1 on a floating plate in the boiling water bath, and let them cool naturally to room temperature for later use. The adapter primer preparation is now complete.

[0058] 2.3. Prepare a 10 μL 1×Bsa I enzyme digestion and ligation reaction system. Add the corresponding amounts of reagents according to the reaction system in Table 2 and carry out the reaction to obtain 3 tubes of reaction products. Place them on ice for later use.

[0059] Table 2: Enzyme digestion and ligation reaction system

[0060]

[0061] 1.3 First round of PCR amplification.

[0062] 1.3.1. Prepare reaction systems according to Table 3 for the 3 tubes of reaction products obtained in step 2.3. After gentle mixing, perform PCR reaction to complete the first round of nested PCR amplification and obtain the first round of PCR amplification products.

[0063] Table 3: First-round nested PCR amplification system

[0064]

[0065] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3'

[0066] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3'

[0067] 1.3.2. Prepare a 2% gel. Take 4 μL of the first-round PCR amplification product and add 2 μL of 10×Loadig Buffer. Mix well and perform gel electrophoresis at 120V for 30 min. If the main band is single, proceed to the second round of nested PCR reaction.

[0068] 1.4 Second round of PCR amplification.

[0069] 1.4.1 Take 1 μL of the reaction product from step 3.2, dilute it 20 times, add the corresponding amount of reagents according to Table 4, and carry out the reaction to obtain the second round of PCR amplification products.

[0070] Table 4: Second-round nested PCR amplification system

[0071]

[0072] B1 primer: 5'-TTCAGAGGTCTCTCTCTCGCACTGGAATCGGCAGCAAAGG-3';

[0073] BL primer: 5'-AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC-3'.

[0074] 1.4.2 After the reaction, prepare a 2% gel. Add 4 μL of the second-round PCR amplification product to 2 μL of 10× Loading Buffer and mix thoroughly. Perform gel electrophoresis at 120V for 30 min. Image the gel; if a single band is observed, proceed to the next step.

[0075] 1.5 Product Purification and Recovery: The three tubes of PCR products obtained in step 4.2 were purified and recovered using the Easy Pure PCR Purification Kit. Specific steps included:

[0076] 1.5.1 Add the specified amount of anhydrous ethanol to Wash Solution and the specified amount of isopropanol to Buffer B3.

[0077] 1.5.2 Transfer the PCR product to a 1.5 mL centrifuge tube, add 5 times the volume of Buffer B3, and mix thoroughly.

[0078] 1.5.3 Transfer the product from the previous step to an adsorption column (the center of the purification column is made of silicon-based material, which can adsorb DNA in a high-salt environment and release DNA in a low-salt environment. This adsorption column is provided in the All-Gold PCR Purification and Recovery Kit), centrifuge at 8,000xg at room temperature for 30s, and discard the waste liquid in the collection tube.

[0079] 1.5.4 Add 500 μL Wash Solution, centrifuge at 9,000 x g at room temperature for 30 s, and discard the waste liquid in the collection tube.

[0080] 1.5.5 Repeat step 5.4 once.

[0081] 1.5.6. Run at room temperature for 2 minutes at 9,000 x g.

[0082] 1.5.7 Transfer the adsorption column to a clean 1.5 mL centrifuge tube and let it stand at room temperature for 3 min to allow the ethanol to evaporate completely.

[0083] 1.5.8 Add 15 μL of ddH2O to the center of the adsorption membrane, let it stand at room temperature for 1 min, centrifuge at 12,000 x g for 1 min, and store the purified DNA solution in a refrigerator at 4°C for later use.

[0084] 1.6 Target-vector linkage: According to the reaction system in Table 5, the purified product obtained in step 5.8 was linked to the pYLCRISPR / Cas9 vector to obtain the linkage product.

[0085] Table 5: Carrier-linked reaction system

[0086]

[0087] 1.7. Transform the ligation product obtained in step 6 into Escherichia coli DH5α.

[0088] 1.7.1 Take DH5α competent cells at -80℃ and incubate them on ice for 5 min;

[0089] 1.7.2 Add all the ligation product to the competent state, gently blow it a few times, and then place it in an ice bath for 30 minutes;

[0090] 1.7.3, 42℃, heat shock for 45s;

[0091] 1.7.4. Ice bath for 2 minutes;

[0092] 1.7.5. Add 0.5 mL of LB liquid to the clean bench and incubate at 37°C for 1 hour.

[0093] 1.7.6 Centrifuge at 4,000 x g at room temperature for 1 min, discard the supernatant, and repeatedly blow and mix the precipitate.

[0094] 1.7.7. Pipette the precipitate onto a kanamycin solid plate and incubate overnight at 37°C with the plate inverted position.

[0095] 1.7.8. Randomly select single colonies and culture them by shaking, then send them to Qingke Biotechnology Co., Ltd. for sequencing to detect whether the three target sites are ligated into the pYLCRISPR / Cas9 vector. Based on the sequencing results, select positive colonies to extract plasmids. Figure 1 a) refers to the CRISPR / Cas9-RHG1 knockout vector (vector A for short).

[0096] (2) Vector A was transformed into indica rice Huazhan and japonica rice TP309 by Agrobacterium-mediated transformation to obtain T0 generation positive plants.

[0097] The specific steps are as follows:

[0098] 2.1 Callus Induction. Select plump, mature seeds from Huazhan rice and remove the husks. Place them in a 37℃ oven overnight. Place the dried seeds in sterile Erlenmeyer flasks and surface sterilize with 75% ethanol for 5 min. Rinse once with sterile water, then treat with 0.15% HgCl2 for 15 min, rinse 5-7 times with sterile water, disinfect with sodium hypochlorite stock solution for 40 min, rinse 5 times with sterile water, and air dry on sterile filter paper. Inoculate the seeds onto indica rice induction medium, ensuring half of the embryo is in contact with the medium. Inoculate 6-8 seeds per flask and culture in the dark at 26±1℃ for 3 weeks to induce callus formation.

[0099] 2.2 Subculture of callus tissue. Select light yellow, dense, and relatively dry embryogenic callus tissue and inoculate it onto subculture medium. Subculture at 26±1℃ for 1-2 times, each time for 20 days.

[0100] 2.3 Callus pre-culture. Select dense and relatively dry embryogenic callus and inoculate them on pre-culture medium, then incubate in the dark for 3-4 days.

[0101] 2.4 Activation and scale-up culture of Agrobacterium. Prepare LB medium containing 50 mg / L kanamycin, 34 mg / L chloramphenicol, and 50 mg / L rifampin. Stir the medium on a plate to activate Agrobacterium EHA105. After two days, pick single colonies and streak them on the plate again. Incubate at 28°C for 48 hours for later use.

[0102] 2.5 Agrobacterium suspension culture. Scrape half or one loop of Agrobacterium into the suspension using an inoculation loop, then transfer the Agrobacterium to 100 mL of liquid co-culture medium (NBM + 0.1 mM As) and incubate in a shake flask at 28°C for 30-60 min. Adjust the OD of the Agrobacterium suspension. 600 The value is 0.8-1.0.

[0103] 2.6. Agrobacterium infection of callus tissue and co-culture. Select callus tissue with a dry surface and dense structure from the pre-culture in step 3), transfer it to a sterile Erlenmeyer flask, air dry it on sterile filter paper until the surface turns white, and then immerse it in the Agrobacterium suspension from step 5) for 20-30 minutes, shaking it every 5 minutes. Discard the bacterial suspension, invert the Erlenmeyer flask into a sterile dish containing filter paper for about 15 minutes, and let it air dry. After that, transfer the callus tissue to a co-culture medium, with filter paper moistened with liquid co-culture medium on top. Note that not too much callus tissue should be placed in one culture dish to ensure that the callus tissue is in full contact with the sterile filter paper. Incubate in the dark at 25-26℃ for 3 days.

[0104] 2.7 Washing and Screening. Transfer the co-cultured callus to a sterile Erlenmeyer flask and rinse 5-7 times with sterile water until the liquid is clear. Soak the callus in sterile water containing 500 mg / L cephalosporin and 400 mg / L carbenicillin. Seal the flask with a sterile filter membrane and treat it in a shaker at 28°C and 180-200 rpm for 20-30 minutes. Discard the sterile water containing antibiotics. Invert the Erlenmeyer flask in a sterile petri dish containing filter paper for about 15 minutes. Air dry the callus on sterile filter paper and transfer it to the appropriate antibiotic selection medium. Screen for 2-3 cycles, each cycle lasting about 2 weeks.

[0105] 2.8 Callus pre-differentiation and differentiation. The selected resistant callus tissues were transferred to pre-differentiation medium (Y + 500 mg / L cephalosporin + 400 mg / L carbenicillin) and treated in the dark for 5-7 days. Bright yellow, pre-differentiated callus tissues were selected and transferred to differentiation medium (DL + 500 mg / L cephalosporin + 400 mg / L carbenicillin) and cultured in a light incubator at 25-26℃, light intensity of 1000-1500 lx, for 14 hours for approximately 40-60 days, with the medium changed every 20 days.

[0106] 2.9 Rooting. When the differentiated green seedlings are about 5-8cm tall, cut off the original roots of the plant and transfer them to rooting medium (R). Place them in a light incubator at 28℃ with a light intensity of 1000-1500lx for 14 hours.

[0107] 2.10 Transplanting. After 3-4 weeks of rooting treatment, open the cap of the conical flask and add 1cm of distilled water. Harden the seedlings in a light-cured culture room for 3-5 days. Rinse the culture medium attached to the seedlings with tap water and transplant them into trays filled with soil. Once the seedlings have survived, transplant them to the experimental field and cultivate them until the rice matures.

[0108] (3) Screening for T0 generation plants (RHG1) with homozygous knockout of three genes OsFEB1, OsFEB2 and OsFEB3 to obtain haploid induction lines, sowing and retaining self-pollinated seeds.

[0109] 3.1 Design detection primers targeting the knockout sites of the three genes respectively to identify the mutation types of the target genes:

[0110] FEB1-cj-1F:5'-GCCCACGCACCGGCACATTG-3';

[0111] FEB1-cj-1R:5'-GCTGCAGCGCCAGGCTGTTCC-3'.

[0112] FEB2-cj-1F:5'-GGACGCCACTACCATCAACAAT-3';

[0113] FEB2-cj-1R:5'-GGCGAGCTCGGCGTCGCAGAAG-3'.

[0114] FEB3-cj-1F:5'-TTATGGATTGTGGCTGTGTGGT-3';

[0115] FEB3-cj-1R:5'-CTGCGTCGTCTCGCCGTTGAGG-3'.

[0116] 3.2. Take leaves from T0 generation transgenic plants and extract plant DNA using the CTAB method. The experimental steps are as follows:

[0117] 3.2.1 Weigh 0.1g of fresh young leaves and place them in a 2mL centrifuge tube. Add a 5mm diameter steel ball, tighten the cap, and freeze in liquid nitrogen for 1min.

[0118] 3.2.2 Place the quick-frozen centrifuge tubes in a high-throughput grinder (Ningbo Scientz-48) and grind at a 38Hz oscillation frequency for 40 seconds;

[0119] 3.2.3 Add 600 μL of 2×CTAB buffer to the ground tissue, invert the sample several times to mix, and incubate in a 65℃ water bath for 40 min, shaking the sample once every 10 min during the process.

[0120] 3.2.4 After removing the sample and allowing it to warm to room temperature, add 600 μL of chloroform, invert the sample several times, and centrifuge at 10,000 rpm for 5 min.

[0121] 3.2.5. Pipette 500 μL of supernatant into a 1.5 mL centrifuge tube, add anhydrous ethanol cooled at -20 °C, gently invert several times, let stand at room temperature for 30 min, and then centrifuge at 12,000 rpm for 5 min.

[0122] 3.2.6 Discard the supernatant, add 400 μL of 75% ethanol to the centrifuge tube to wash the white precipitate, and centrifuge at 12,000 rpm for 5 min;

[0123] 3.2.7 Discard the supernatant, allow to air dry, then add 200 μL of ddH2O to fully dissolve the DNA;

[0124] 3.2.8. Take 5 μL of DNA extraction solution from each sample, add 1 μL of 10× Loading Buffer, and detect the concentration and integrity of the extracted DNA by 2% gel electrophoresis.

[0125] 3.3 Using the three pairs of FEB gene detection primers designed in 3.1, the PCR reaction system was prepared for the extracted plant DNA according to Table 6.

[0126] Table 6: PCR reaction system for OsFEB gene

[0127]

[0128] 3.4 The PCR products were sent to Hunan Youkanglai Biotechnology Co., Ltd. for Sanger sequencing. Using the target sequences of FEB1-1F, FEB2-1F and FEB3-1F as references, the target site sequences were analyzed, and multiple T0 generation transgenic positive mutant lines (rhg1) with simultaneous homozygous knockout of the three FEB1, FEB2 and FEB3 genes were successfully obtained.

[0129] Figure 1 The figure shows the gene structure and mutation type of the rhg1 knockout mutant. In the figure, a is a schematic diagram of the CRISPR / Cas9-RHG1 knockout vector structure; bd in the figure shows the gene structure, target sequence and mutation type of the rhg1 three-gene homozygous mutant lines OsFEB1, OsFEB2 and OsFEB3.

[0130] Two rhg1 Huazhan lines with homozygous knockout of three genes were selected from multiple positive transformant lines. These lines were planted at the Changsha Chunhua transgenic base in May 2023, in Hainan for breeding in December 2023, and at the Changsha transgenic base in May 2024, respectively. Phenotypic observation and chromosome ploidy testing were conducted on the two positive populations planted at multiple locations and in multiple seasons. The results showed that haploid plants appeared in the offspring of the rhg1 population, and the haploid-induced phenotype was stable. Furthermore, compared to other haploid-inducing genes, the haploid induction rate of the rhg1 lines remained relatively high, ranging from 6.25% to 25%. This is of great significance for creating high-frequency haploid-inducing lines and for the breeding and application of one-line hybrid rice.

[0131] Experiment 1: Morphological examination of the dwarf plants of rhg1 progeny.

[0132] Since haploid plants are significantly different from diploid plants in morphology, haploid plants can be initially screened by observing their morphology.

[0133] Figure 2 Phenotypic representations of Huazhan-wild-type (HZ-WT) and rhg1 mutant diploid plants under the same growing environment. Figure a shows the phenotype of HZ-WT and rhg1 diploid plants at maturity (scale bar = 15 cm). Figure b shows mature spikelets of HZ-WT and rhg1 diploid plants (scale bar = 5 cm). Figure c shows pollen grains of HZ-WT and rhg1 diploid plants stained with 1% KI (scale bar = 100 μm).

[0134] Figure 3Phenotypic representations of haploid plants of the Huazhan-wild type (HZ-WT) and rhg1 mutants. Figure d shows the phenotype of mature HZ-WT and rhg1 haploid plants (scale bar = 15 cm); figure e shows mature spikelets of HZ-WT and rhg1 haploid plants (scale bar = 5 cm); figure f shows pollen grains of HZ-WT and rhg1 haploid plants stained with 1% KI (scale bar = 100 μm).

[0135] Experiment 2: The SYSMEX flow cytometry assay kit was used to accurately identify haploid lines in the preliminarily screened rhg1 progeny population.

[0136] The specific operating steps are as follows:

[0137] 1) Prepare RNase A Stock Solution. Add 1.5 mL of sterile, enzyme-free water to an EP tube containing RNase A powder, gently shake to completely dissolve the powder, and store at -20°C.

[0138] 2) Prepare the staining solution. Add 120 μL of Propidium Iodide (PI fluorescent dye) and 60 μL of RNase A Stock Solution to 20 mL of Staining Buffer, and keep on ice, protected from light.

[0139] 3) Take 0.5cm 2 Place the tender leaves in a clean glass petri dish and add 100 μL of Nuclei Extraction Buffer;

[0140] 4) Use a sharp single-edged blade to thoroughly chop the leaves for 60 seconds, then add 400 μL of Nuclei Extraction Buffer and let stand at room temperature for 3 minutes to fully lyse the leaves;

[0141] 5) Filter the dissociation solution in the culture dish into a 1.5 mL centrifuge tube using a 400-mesh filter membrane, add 1.5 mL of the staining solution prepared in step 2), and let it stand at room temperature in the dark for 30 min.

[0142] 6) Use a laminar flow cytometer to analyze the samples.

[0143] Figure 4 To identify chromosome ploidy in HZ-WT and rhg1 haploid plants by flow cytometry. The left figure shows HZ-WT (Huazhan wild type, control material) diploid plants, and the right figure shows haploid plants in the rhg1 population.

[0144] Example 2

[0145] An application of rice haploids produced by the method in Example 1 in achieving heterosis fixation in hybrid rice. The application method includes:

[0146] (1) Construct a CRISPR / Cas9 knockout vector B targeting PAIR1, OsREC8, and OsOSD1. The gene sequence of OsPAIR1 is a publicly available sequence (>LOC_Os03g01590 (Genomic Sequence)), the gene sequence of OsREC8 is a publicly available sequence (>LOC_Os05g50410 (Genomic Sequence)), and the gene sequence of OsOSD1 is a publicly available sequence (e>LOC_Os02g37850 (Genomic Sequence)). Specific steps include:

[0147] Primers for target gene sites of PAIR1, OsREC8 and OsOSD1 (see Table 7 for details) were designed using the online website (E-CRISP Design) and synthesized by Hunan Youkanglai Biotechnology Co., Ltd.

[0148] OSD1-1F: 5'-GGCACCTGCCGCCGACGAGCAACA-3' (SEQ ID NO. 10);

[0149] OSD1-1R: 5'-AAACTGTTGCTCGTCGGCGGCAGG-3' (SEQ ID NO. 11).

[0150] PAIR1-1F:5'-GCCGCAAGCAACCCAGTGCACCGC-3' (SEQ ID NO.12);

[0151] PAIR1-1R: 5'-AAACGCGGTGCACTGGGTTGCTTG-3' (SEQ ID NO. 13).

[0152] REC8-1F: 5'-GTTGTGTGGCGATCGTTGTACGAG-3' (SEQ ID NO. 14);

[0153] REC8-1R: 5'-AAACCTCGTACACGATCGCCACA-3' (SEQ ID NO. 15).

[0154] Table 7. MiMe Genetic Information Statistics

[0155]

[0156] The carrier construction process is the same as in Example 1.

[0157] (2) The co-transformation of vectors A and B into high-quality hybrid rice seeds via Agrobacterium-mediated transformation specifically includes:

[0158] 2.1. The high-yielding indica hybrid rice combination Xiangliangyou 900 was selected as the transformation background material. The CRISPR / Cas9-RHG1 knockout vector and CRISPR / Cas9-MiMe knockout vector of Example 1 were co-transformed by Agrobacterium-mediated transformation. The operation method was the same as in Example 1.

[0159] 2.2. Select T0 generation plants (Fix) with homozygous knockout of six genes (OsFEB1, OsFEB2, OsFEB3, PAIR1, OsREC8, and OsOSD1), sow them, and retain self-pollinated seeds.

[0160] The primers designed to detect the knockout targets of the three MiMe genes include:

[0161] PAIR1-cj-1F:5'-CTGTACCTGTGCATCTAATTACAG-3'

[0162] PAIR1-cj-1R:5'-CCCCATCTTATGTACTGAGCTTGCCAG-3';

[0163] REC8-cj-1F:5'-GTCGGTACCCATGGCACTAAGG-3'

[0164] REC8-cj-1R:5'-CCAGAAACCGAGACACATCATC-3';

[0165] OSD1-cj-1F:5'-ATTCTCCAGGATGCCTGAAGTGAG-3'

[0166] OSD1-cj-1R:5'-CCTAGACTGCTACTCTTGCTAGTGAT-3'.

[0167] (3) Bag the spikelets of the Fix line, harvest and sow the T1 generation self-pollinated seeds. Observe whether the phenotypic segregation of the T1 generation plants occurs during the heading and maturity stages to preliminarily determine whether heterosis fixation is successful. By developing SSR molecular detection markers, molecular detection or whole-genome resequencing is performed on the progeny plants to identify whether the heterozygous sites in Xiangliangyou 900 have segregated, thereby accurately determining the frequency of heterosis fixation in this MiMe+rhg1 technology system.

[0168] In this embodiment, a total of 43 transgenic plants of Xiangliangyou 900 were obtained. Through molecular identification and sequence analysis of the target gene, four successfully homozygous knockout Fix mutants were screened. After the rice matured, 304 T1 generation self-pollinated seeds were harvested and planted in the field. It was observed that the offspring exhibited certain phenotypic segregation. A total of 23 T1 generation plants with consistent growth and similar agronomic traits to the hybrid Xiangliangyou 900 were screened. Using SSR molecular marker detection and whole-genome resequencing, it was found that these 23 plants completely maintained the heterozygous site in Xiangliangyou 900. These results indicate that the high-frequency haploid induction line RHG1 combined with the MiMe technology system can successfully fix heterosis in rice.

[0169] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for producing rice haploids, characterized in that, By modifying one or more of the OsFEB1, OsFEB2, and OsFEB3 genes in rice to cause the loss of function of the OsFEB1, OsFEB2, and / or OsFEB3 genes, a rice haploid inducible line is obtained. The rice haploid inducible line induces the production of haploids through sexual reproduction. The gene sequence of OsFEB1 is shown in SEQ ID NO.1, the gene sequence of OsFEB2 is shown in SEQ ID NO.2, and the gene sequence of OsFEB3 is shown in SEQ ID NO.

3.

2. The method according to claim 1, characterized in that, One or more of the OsFEB1, OsFEB2 and OsFEB3 genes in rice are knocked out using the CRISPR / Cas9 system, resulting in the loss of function of the OsFEB1, OsFEB2 and / or OsFEB3 genes.

3. The method according to claim 2, characterized in that, The method includes the following steps: (1) Design primers based on the gene sequences of OsFEB1, OsFEB2 and / or OsFEB3, and construct CRISPR / Cas9 vector A with OsFEB1, OsFEB2 and / or OsFEB3 genes knocked out. (2) The CRISPR / Cas9 vector A was transformed into rice to obtain T0 generation plants with bis-allelic homozygous knockout of OsFEB1, OsFEB2 and / or OsFEB3. (3) Sow the T0 generation plants and self-pollinate to obtain rice haploid induction lines.

4. The method according to claim 3, characterized in that, Primers designed based on the OsFEB1 gene sequence include FEB1-1F and FEB1-1R; primers designed based on the OsFEB2 gene sequence include FEB1-2F and FEB1-2R; primers designed based on the OsFEB3 gene sequence include FEB1-3F and FEB1-3R. The gene sequence of FEB1-1F is shown in SEQ ID NO.4; The gene sequence of FEB1-1R is shown in SEQ ID NO.5; The gene sequence of the FEB2-1F is shown in SEQ ID NO.6; The gene sequence of the FEB2-1R is shown in SEQ ID NO.7; The gene sequence of FEB3-1F is shown in SEQ ID NO.8; The gene sequence of FEB3-1R is shown in SEQ ID NO.

9.

5. The method according to any one of claims 1 to 4, characterized in that, The rice mentioned is indica rice.

6. The application of a rice haploid obtained by the method of any one of claims 1 to 5 in breeding.

7. The application according to claim 6, characterized in that, The method of application includes the following steps: S1. Primers were designed based on the gene sequences of PAIR1, OsREC8 and OsOSD1 to construct CRISPR / Cas9 vector B, which knocked out the three genes of PAIR1, OsREC8 and OsOSD1. S2. CRISPR / Cas9 vector A and CRISPR / Cas9 vector B are co-transformed into rice seeds to obtain T0 generation plants with bis-allel homozygous knockout. S3. Self-pollinate the T0 generation plants to obtain T1 generation seeds, thereby fixing the heterosis of rice.

8. The application according to claim 7, characterized in that, Primers designed based on the PAIR1 gene sequence include: PAIR1-1F and PAIR1-1R; primers designed based on the OsREC8 gene sequence include: REC8-1F and REC8-1R; primers designed based on the OsOSD1 gene sequence include: OSD1-1F and OSD1-1R. The gene sequence of OSD1-1F is shown in SEQ ID NO.10; The gene sequence of OSD1-1R is shown in SEQ ID NO.11; The gene sequence of PAIR1-1F is shown in SEQ ID NO.12; The gene sequence of PAIR1-1R is shown in SEQ ID NO.13; The gene sequence of REC8-1F is shown in SEQ ID NO.14; The gene sequence of REC8-1R is shown in SEQ ID NO.15.

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