Method for creating tomato cytoplasmic male sterility new germplasm through MSH1 gene editing and application

Abstract

The invention belongs to the technical field of crop breeding by a gene editing technology, and particularly relates to a method for creating a novel tomato cytoplasmic male sterility germplasm through gene editing and an application of the novel tomato cytoplasmic male sterility germplasm. In order to solve the problems that existing cytoplasmic male sterile materials in tomatoes are insufficient, production of tomato hybrid seeds is restricted and the like, gene editing is performed on MSH1 through a CRISPR / Cas9 technology, cytoplasmic male sterile single plants are identified in mutant selfing separation progenies and hybridized with wild types twice, and the tomato hybrid seeds are obtained. Finally, a novel non-transgenic tomato cytoplasmic male sterility material with the MSH1 gene recovered to a wild type is obtained, and meanwhile, the horticultural character of the original fertile material is kept unchanged.

Description

Technical Field

[0001] This invention belongs to the field of crop breeding technology using gene editing technology, and in particular, it is a method and application for creating new cytoplasmic male sterile tomato germplasm through gene editing. Background Technology

[0002] tomato( Solanum lycopersicum Tomato (L.) is a strictly self-pollinating crop with significant heterosis, and hybrid varieties are the main type of seed used in production. However, most current tomato hybrid seed production methods rely on artificial emasculation and pollination, which is time-consuming, labor-intensive, and prone to seed safety issues such as impurities in the hybrids (Zhang Yanhong. Research on the Application Technology of Long Style Trait in Tomato Seed Production [D]. Northwest A&F University, 2024.). Using cytoplasmic male sterile lines as the female parent for tomato hybrid production can effectively improve seed production efficiency, reduce seed production costs, ensure the purity of the hybrids, and prevent the loss of parental lines (Du M, Sun C, Deng L, Zhou M, Li J, Du Y, Ye Z, Huang S, Li T, Yu J, Li CB, Li C. Molecular breeding of tomato: Advances and challenges [J]. Journal of Integrative Plant Biology 2025, 67: 669-721.

[0003] Male sterility is prevalent in the plant kingdom, mainly divided into nuclear male sterility and cytoplasmic male sterility. It has been reported that approximately 55 male-sterile mutant materials of tomato have been identified (Hazra P, Roy T, Choudhury J, Atanassova B. Male Sterility in Tomato (Lycopersicon esculentum Mill.) and Brinjal (Solanum melongena)[J]. International Journal of Plant Breeding, 2007, 1(1): 41-50.). Most known male sterility in tomatoes is nuclear male sterility, with recessive nuclear sterility being the predominant type. Cytoplasmic male sterility is rarely observed in tomatoes (Gorman SW, McCormick S, Rick C. Malesterility in tomato[J]. Critical Reviews in Plant Sciences(1997, 16(1): 31-53.). Using existing tomato cytoplasmic male sterile lines for hybrid seed production presents challenges in maintaining the sterility of these lines. Specific hybridization designs are required to maintain sterility, hindering the large-scale production of sterile seeds for seed production and thus restricting the industrialization of tomatoes. Using cytoplasmic male sterile lines to produce tomato hybrids ensures hybrid purity, and the sterility of these lines is easily maintained, facilitating large-scale production of tomato hybrids. Therefore, there is an urgent need to create new methods for developing tomato cytoplasmic male sterile lines to further promote the industrialization of tomatoes.

[0004] MutS HOMOLOG1 ( MSH1 The mitochondrial gene is a highly conserved nuclear gene in plants, playing an important role in maintaining the stability of the mitochondrial genome (Abdelnoor RV, Christensen AC, Mohammed S, Munoz-Castillo B, Moriyama H, Mackenzie S A. Mitochondrial Genome Dynamics in Plants and Animals: Convergent Gene Fusions of a MutS Homologue[J]. Journal of Molecular Evolution, 2006, 63, 165-173.). In wild-type plants, MSH1 Restricting the exchange and recombination of mitochondrial DNA, mitochondrial genome recombination is maintained at a low frequency, inhibiting... MSH1 Following gene expression, high-frequency DNA exchange in mitochondria leads to increased mitochondrial genome recombination, which in turn affects the frequency of substoichiometry changes (copy number changes) in the mitochondrial genome, thereby mediating alterations in crop fertility (Yang XD, Kundariya H, Xu YZ, Sandhu A, Yu JT, Hutton SF, Zhang MF, Mackenzie S A. MutS HOMOLOG1-derive depigenetic breeding potential in tomato[J]. Plant Physiology , 2015, 168(1):222-232.).

[0005] Currently, tomato cytoplasmic male sterile germplasm resources are relatively scarce, which limits their application in hybrid seed production. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for... MSH1 Methods and applications of gene editing for creating novel cytoplasmic male sterile tomato germplasm.

[0007] This invention addresses the problem of insufficient cytoplasmic male sterility material in tomatoes, which restricts the production of tomato hybrids. It utilizes CRISPR / Cas9 technology to... MSH1 Gene editing was performed, and cytoplasmic male-sterile single plants were identified in the self-pollinating progeny of the mutant. These plants were then crossed twice with the wild type to ultimately obtain... MSH1 The genes were restored to wild-type non-transgenic tomato cytoplasmic male sterile new material, while the horticultural traits of the original fertile material remained unchanged.

[0008] To solve the above-mentioned technical problems, the present invention provides a method... MSH1 A method for creating new cytoplasmic male-sterile tomato germplasm through gene editing: This involves modifying the cytoplasm of fertile tomatoes... MSH1 Gene editing is performed on exon regions to mediate cytoplasmic male sterility.

[0009] As an improvement to the method of the present invention: by improving the fertile tomato... MSH1 Gene editing is performed in the exon region, utilizing the plant's own DSB repair mechanism to introduce DNA sequence deletions, thus... MSH1 Loss of gene function induces mitochondrial genome recombination, altering the substoichiometry (copy number) of potential CMS-related genes in tomatoes, thereby mediating cytoplasmic male sterility.

[0010] As a further improvement to the method of the present invention: by editing MSH1 The exon regions of the gene were analyzed to obtain T0 generation edited plants. Non-transgenic homozygous edited plants were isolated through self-pollination. Cytoplasmic male-sterile single plants were identified in the T2 generation. These were then crossed twice with wild-type plants, ultimately resulting in the isolation of… MSH1 Cytoplasmic male sterile material whose genes have been restored to wild type is a new tomato cytoplasmic male sterile germplasm.

[0011] As a further improvement to the method of the present invention: using CRISPR / Cas9 technology to... MSH1 Gene editing is performed.

[0012] As a further improvement to the method of the present invention, the method includes the following steps: (1) sgRNA target site selection in tomatoes MSH1 Based on the CRISPR / Cas9 target site design principles, the exon region of the gene was selected as the target site, with the base 20 bp upstream of the prototype spacer sequence adjacent to the motif. The target site should also have a GC content between 40% and 60%, a high target hit rate, and a low off-target rate. Forcibly changing the first base of the target site to G helps improve editing efficiency, i.e., 5'-GN. 19 -NGG-3', where NGG is a PAM sequence, and N... 19This represents a 19bp base recognition sequence excluding G; (2) Design the vector and construct the required primers based on the recognition sequence. The primer sequences are as follows: DT1-BsF: 5'-ATATATGGTCTCGATTGAAGGGACAGTGAACGCCAAGTT-3' DT1-F0: 5'-TGAAGGGACAGTGAACGCCAAGTTTTAGAGCTAGAAATAGC-3' DT2-R0: 5'-AACCCACATAATATTAACATAGCAATCTCTTAGTCGACTCTAC-3' DT2-BsR: 5'-ATTATTGGTCTCGAAACCCACATAATATTAACATAGCAA-3' (3) Using the intermediate vector pCBC-DT1T2 as a template, PCR amplification was performed using the primers from step (2), and the PCR product was purified and recovered. The purified product was used to construct a CRISPR / Cas9 recombinant vector. The successfully ligated vector was transferred into Escherichia coli DH5α competent cells. Single clones were picked for plasmid extraction and sequencing. Plasmids with correct sequencing results were transferred into Agrobacterium tumefaciens GV3101 using the heat shock method. That is, primers required for vector construction are designed based on the recognition sequence, PCR amplification is performed using pCBC-DT1T2 as a template, the purified PCR product is ligated with the pHSE401 vector using Golden Gate, transformed into E. coli DH5α competent cells, single clones are picked for plasmid extraction and sequencing, and plasmids with correct alignment results are transformed into Agrobacterium GV3101 using the heat shock method.

[0013] (4) Using tomato cotyledons that have grown for 7 days as explants, T0 generation regenerated plants were obtained by using Agrobacterium-mediated genetic transformation and hygromycin resistance screening system.

[0014] As a further improvement to the method of the present invention: MSH1 gene editing target sites were designed using an online website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), and the selected target site sequences were 5'-AAAGGGACAGTGAACGCCAACGG-3' and 5'-ACTATGTTAATATTATGTGGTGG-3'. When designing primers, the first base of the two target sites was changed to G.

[0015] As a further improvement to the method of the present invention, step (3) further includes: incorporating two sgRNAs into the same vector.

[0016] As a further improvement to the method of the present invention, it also includes the following steps: (1) Identification of T0 generation mutant plants (gene editing detection steps of regenerated plants): Genomic DNA was extracted from the leaves of regenerated plants and PCR amplification was performed using vector-specific primers HYG-F / R. The amplification products were detected by agarose gel electrophoresis. Regenerated plants that could amplify HYG bands were positive transgenic plants. Primers for amplifying target sites were designed based on the sequences near the two target sites. PCR amplification was performed using the genomic DNA of positive transgenic plants as templates. The amplification products were sequenced, and the gene editing of the transgenic plants was analyzed based on the sequencing results. (2) Fertility identification steps: T0 generation mutants were self-pollinated, T1 generation seeds were harvested from single plants and sown, genomic DNA of T1 plants was extracted at the two-leaf-one-heart stage, and homozygous edited plants without transgenic fragments were screened using the method in step (1), and T2 generation seeds were harvested from self-pollinated and sown. (3) Fertility identification: The T2 generation non-transgenic homozygous edited plants obtained in step (2) are subjected to fertility identification, which mainly includes identifying pollen viability and observing the presence or absence of seeds in the fruit; Specifically, the T0 generation mutants were self-pollinated and segregated, and the T1 generation seeds were harvested and sown. Homozygous mutant lines without exogenous T-DNA insertion were detected in the T1 plants, and the T2 generation seeds were harvested and sown. Cytoplasmic male-sterile individual plants were searched in the T2 generation, primarily by pollen viability assessment and the presence or absence of seeds within the fruit to determine fertility.

[0017] (4) Propagation steps for cytoplasmic male sterility materials: using the identified tomato varieties msh1 A cytoplasmic male-sterile mutant was used as the maternal parent and crossed twice with a wild-type mutant. The offspring were then selected for... MSH1 The cytoplasmic male sterile plant whose genes are restored to wild type is the new cytoplasmic male sterile germplasm of tomato.

[0018] That is, pollinating sterile materials with wild-type pollen can maintain their sterility and... MSH1 The genes were restored to the wild type.

[0019] This invention also provides a method for... MSH1 Application of gene editing methods for creating novel cytoplasmic male-sterile tomato germplasm in tomato breeding.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a method for creating new tomato cytoplasmic male sterility germplasm through gene editing technology, by... MSH1 The exon regions of the gene were edited. Non-transgenic homozygous edited T1 generation plants were isolated through self-pollination of the T0 generation. Seeds from the T2 generation were harvested through self-pollination. Cytoplasmic male-sterile single plants were identified in the T2 generation, and these were obtained through two consecutive crosses with the wild type. MSH1 Materials whose genes are restored to wild type and retain the cytoplasmic male sterility trait are new tomato cytoplasmic male sterility germplasm; (2) The method of the present invention is different from creating male sterile materials by directly editing important genes related to the development of tomato stamens. The cytoplasmic male sterile materials created by the method of the present invention are not only a valuable resource in tomato production, but also provide important materials for the study of nucleocytoplasmic interaction. (3) The tomato cytoplasmic male sterility material created by this invention plays an important role in identifying cytoplasmic male sterility-related genes in tomatoes; (4) The cytoplasmic male sterile material created by the method of the present invention is a non-transgenic cytoplasmic male sterile material, and its sterile trait is easy to maintain, which has reference value for the creation of cytoplasmic male sterile materials for other crops; (5) The new tomato cytoplasmic male sterility germplasm created by this invention is a powerful supplement to the existing tomato cytoplasmic male sterility materials; (6) This invention has broad application prospects and economic benefits.

[0021] The method of this invention is of great significance for creating novel cytoplasmic male sterile materials for tomatoes and promoting the utilization of heterosis in tomatoes. It also has reference value for creating cytoplasmic male sterile materials for other crops. Attached Figure Description

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 Vector-specific PCR detection for regenerated plants: (1-15: regenerated plants; +: positive plasmid control; -: negative wild-type control; DW: ddH2O blank control).

[0024] Figure 2 Vector-specific PCR detection for non-transgenic homozygous mutants (T1-1 and T1-2).

[0025] Figure 3 Sequencing results for target sites of non-transgenic homozygous mutants (T1-1 and T1-2).

[0026] Figure 4 For pollen viability testing: Alexander staining and in vitro germination of pollen from wild-type (WT), mutants T2-1-18 and T2-2-5.

[0027] Figure 5 For fruit development phenotype; Figure 5 middle: a: Wild-type (WT) self-pollinated fruit; b: Mutant T2-1-18 self-pollinated fruit; c: Mutant T2-2-5 self-pollinated fruit; d: Hybrid fruit with wild-type as male parent and mutant T2-2-5 as female parent; In each small image, the left image is a top view of the fruit, and the right image is a cross-section of the same fruit.

[0028] Figure 6 For comparison of floral organ phenotypes; Flowers and buds of wild-type (WT), mutants T2-1-18, and T2-2-5.

[0029] Figure 7 For comparison of leaf morphology; Figure 7 middle: a: Wild type; b: Chlorotic yellowing leaves of mutant T2-1-18; c: Chlorotic whitening leaves of mutant T2-2-5.

[0030] Figures 8-11 The combination is wild-type Ailsa Craig's MSH1 Gene sequence.

[0031] Figures 12-15 The combination is T1-1 MSH1 Gene sequence.

[0032] Figures 16-19 The combination is T1-2. MSH1 Gene sequence. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: The materials used in this invention are as follows: Fertile cultivar Ailsa Craig (AC); The editing vector pHSE401 and intermediate vector pCBC-DT1T2 used were derived from Addgene; DNA Extraction Kit: Cooler Master CTAB; Escherichia coli competent cells: DH5α (Qingke Biotechnology); Agrobacterium competent cells: GV3101 (Weidi Biotechnology); Primer synthesis and sequencing were both completed by Zhejiang Youkang Biotechnology Co., Ltd.

[0034] This invention relates to a method for creating novel cytoplasmic male-sterile tomato germplasm using gene editing technology, comprising the following steps: I. Construction of CRISPR / Cas9 vector and Agrobacterium-mediated transformation: (a) Selection of sgRNA target sites: Use the Sol Genomics Network website (https: / / solgenomics.net / ) to find tomatoes. MSH1 The gene sequence (Solyc09g090870) was used, and the CDS sequence was submitted to the CRISPR-P 2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to select target sites. The sgRNA target site was selected in the exon region of the MSH1 gene. Following the CRISPR / Cas9 target site design principles, a target site was selected 20 bp upstream of the motif adjacent to the prototype spacer sequence. The target site should also have a GC content of 40-60%, high target hit rate, and low off-target rate. The selected target site sequences are shown below; the underlined sequences are PAM sequences. Changing the first base of the target site to G during primer design helps improve gene editing efficiency.

[0035] sgRNA-1: 5'-AAAGGGACAGTGAACGCCAA CGG -3' sgRNA-2: 5'-ACTATGTTAATATTATGTGG TGG -3' (II) Construction of vectors and transformation with Agrobacterium 1. Based on the target sequence, design primers. The primer sequences are as follows: lowercase letters represent the BsaI restriction endonuclease recognition sites, and underlined bases represent the 19 bases (excluding G) at the two target sites: DT1-BsF: 5'-ATATATggtctcGATTG AAGGGACAGTGAACGCCAA GTT-3' DT1-F0: 5'-TG AAGGGACAGTGAACGCCAA GTTTTAGAGCTAGAAATAGC-3' DT2-R0: 5'-AAC CCACATAATATTAACATAG CAATCTCTTAGTCGACTCTAC-3' DT2-BsR: 5'-ATTATTggtctcGAAAC CCACATAATATTAACATAG CAA-3'.

[0036] 2. Using four primers (DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR) and the intermediate vector pCBC-DT1T2 diluted 100-fold as a template, PCR amplification was performed. The amplification product was the target fragment containing two sgRNAs. The amplification system is shown in Table 1. Table 1

[0037] The amplification program is as follows: pre-denaturation at 94℃ for 2 min; (94℃ for 15 s, 60℃ for 30 s, 68℃ for 1 min) × 30 cycles; final extension at 68℃ for 5 min; finally, stop the reaction when the temperature drops to 4℃ and remove the product.

[0038] 3. Purify and recover the PCR product, and construct a CRISPR / Cas9 recombinant vector using the purified and recovered target fragment; that is, perform enzyme digestion and ligation reactions using the Golden Gate method, which allows the enzyme digestion and ligation reactions to be carried out in the same test tube. After digestion with BsaI, the vector and target fragment are ligated by T4 DNA ligase, and finally the target fragment containing two sgRNAs is incorporated into the pHSE401 vector. The reaction system is shown in Table 2, and the amplification program is as follows: 37℃ for 5 h, 50℃ for 5 min, and 80℃ for 10 min.

[0039] Table 2

[0040] 4. Take 5 μl of the final product from the above steps and transform it into DH5α E. coli competent cells. Spread the cells on LB medium containing Kans and incubate overnight at 37°C upside down. Pick single clones for sequencing using the seq primer: 5'-TGTCCCAGGATTAGAATGATTAGGC-3'. Transform plasmids with correct sequencing results (i.e., those satisfying the target fragment obtained in step 2, into the pHSE401 vector) into Agrobacterium GV3101 using a heat shock method.

[0041] II. Genetic transformation of tomato AC (Ailsa Craig) Seed disinfection and acquisition of sterile seedlings: Tomato AC seeds were soaked in sterile water for 2 hours, then 4% sodium hypochlorite solution was added and the seeds were shaken at 40 rpm for 8 minutes on a shaker. 75% alcohol was added and shaken for 1 minute in a clean bench. The seeds were then rinsed three times with sterile water. After disinfection, the seeds were dried on sterile filter paper and then evenly spread on the sowing medium with tweezers and cultured in the dark for 2 days. Subsequently, the seeds were cultured at a constant temperature of 25°C for 5 days under 16 hours of light and 8 hours of darkness.

[0042] Pre-culture: 7 days after sowing, cut 2-3 wounds in the flat cotyledons in a clean bench and place them in a pre-culture medium lined with sterile filter paper for 1 day in the dark.

[0043] Infection: Agrobacterium tumefaciens culture was incubated overnight at 200 rpm in a shaker at 28°C. After turbidity was observed, the culture was centrifuged at 4500 rpm for 4 min at room temperature, the supernatant was discarded, and the culture was resuspended in MS liquid medium. OD 600Adjust to 0.5. Infect cotyledons in the dark for 5 min, then place the cotyledons face up in a pre-culture medium with sterile filter paper and incubate in the dark for 2 days.

[0044] Callus induction: After co-culturing for 2 days, cotyledons were placed in MS1 ​​differentiation medium to induce callus.

[0045] Induction of adventitious buds: After 2 weeks, the cotyledons from which callus was induced were transferred to MS2 budding medium for culture to induce bud formation.

[0046] Rooting culture: The regenerated shoots are cut from the callus tissue and transferred to MS3 rooting medium to induce root formation.

[0047] Hardening-off and transplanting: After the regenerated seedlings have rooted, they were hardened off for 3 days with the cover off before being transplanted into the substrate. A total of 15 regenerated plants were obtained. The culture medium formula is shown in Table 3. Table 3

[0048] Note: Adjust the pH to 5.8~6.0, and culture the culture medium in an autoclave at 120℃ for 20 min. Add antibiotics and plant growth regulators in a clean bench after sterilization and once the culture medium has cooled to 60℃.

[0049] III. Identification of T0 generation mutant plants DNA was extracted from leaves of wild-type and 15 regenerated plants. Positive transgenic lines were detected using vector-specific primers HYG-F / R. Agarose gel electrophoresis showed that 9 regenerated plants amplified bands of approximately 500 bp, consistent in size with the positive plasmid control, indicating that these were positive transgenic plants. Figure 1 As shown in Table 4, primers for amplifying the two target sites were designed based on sequences near the two target sites. The primer names are Target1-F / R and Target2-F / R, respectively. PCR amplification of the two target sites was performed using DNA from nine transgenic plants as templates. The PCR amplification system and procedure are shown in Table 4. Sequencing of the PCR products revealed that six plants showed a double peak at the edited site, indicating that these six plants were edited, thus yielding six T0 generation mutant plants. The vector-specific primer sequences and the primer sequences for the two target sites are as follows: HYG-F: 5'-CAAAGATCGTTATGTTTATCGGCACT-3' HYG-R: 5'-TTGGCGACCTCGTATTGGGAA-3' Target1-F: 5'-ATGTATTGGGTTACGGCA-3' Target1-R: 5'-ATTCTTGCCTTCTGAGCT-3' Target2-F: 5'-ATCCCTCGTATCAACTACTG-3' Target2-R: 5'-ATATGTAAGCCTCTTAGCCAG-3' Table 4

[0050] IV. Screening of T1 generation non-transgenic homozygous mutants Six T0 generation mutant plants were self-pollinated, and T1 generation seeds were harvested from each plant and sown. Two homozygous mutant plants without exogenous T-DNA insertion were isolated from the T1 generation plants; these were designated T1-1 and T1-2, respectively. Figure 2 As shown in the diagram. Specifically, T1-1 has a 2bp deletion at the first sgRNA position; T1-2 has a 6bp deletion at the first sgRNA position and a 1bp deletion at the second sgRNA position, as shown in the diagram. Figure 3 As shown. T1-1 and T1-2 were self-pollinated, and seeds of the T2 generation were harvested from individual plants. Cytoplasmic male sterile plants were then identified in the T2 generation plants.

[0051] Wild-type Ailsa Craig MSH1 Gene sequences such as Figures 8-11 The combination, T1-1 MSH1 The gene sequence is Figures 12-15 The combination of T1-2 MSH1 The gene sequence is Figures 16-19 The combination of .

[0052] V. Identification of the athleticism of T2 generation mutants Pollen from wild-type and T2 generation mutants was collected for Alexander staining and in vitro germination experiments to observe the seed setting of wild-type and mutant fruits. The specific procedures are as follows: Alexandrite staining of pollen: Place 1-2 drops of Alexandrite stain on a glass slide, use tweezers to pick up pollen and spread it evenly in the stain, gently cover with a coverslip, let stand for 10 minutes, and observe the color of the pollen under a microscope. Pollen that is stained purple-red is viable pollen, and pollen that is blue-green is inactive pollen.

[0053] Pollen germination in vitro: Place 1-2 drops of pollen germination medium on a concave glass slide. The medium mainly consists of sucrose and boric acid. Place the anthers on the medium and squeeze them with pointed tweezers to release the pollen. Remove any obvious anther tissue. Place the slide in a petri dish containing moistened filter paper and incubate in the dark at 28°C for 1 hour. Observe and photograph the results under a microscope. A pollen tube length greater than half the diameter of the pollen grain is considered germination.

[0054] The results are as follows: Among the T2 plants, individuals with significantly reduced fertility (e.g., T2-1-18) and completely sterile individuals (e.g., T2-2-5) were observed. Alexandrite staining showed that pollen from the wild type was stained purplish-red, while only a portion of the pollen from T2-1-18 and T2-2-5 was stained, indicating that the fertility of T2-1-18 and T2-2-5 was reduced compared to the wild type. Figure 4 As shown in the figure. Pollen germination in vitro showed that, within the same timeframe, wild-type pollen grains had already germinated significant pollen tubes, while T2-1-18 showed less germination, and T2-2-5 showed no germination at all. This further indicates that the fertility of T2-1-18 was significantly reduced, while T2-2-5 was completely sterile. Figure 4 As shown. Staining and in vitro germination also revealed that, unlike the round and plump pollen grains of the wild type, the pollen grains of T2-1-18 and T2-2-5 exhibited more shriveled and wrinkled characteristics, indicating a defect in pollen development in these two mutants. The seed set of self-pollinated fruits of T2-1-18 and T2-2-5 verified the above fertility results. The self-pollution rate of T2-1-18 was significantly reduced, with the number of seeds in the fruit being only 1%-2% of that in the wild type. Figure 5 As shown in b; however, T2-2-5 does not produce seeds at all after self-pollination, exhibiting parthenocarpy, as shown in... Figure 5 As shown in c. Using the wild-type as the male parent to pollinate T2-2-5 plants, the seed setting rate of the hybrid fruit was normal, indicating that the female is fertile. Figure 5 As shown in d. In addition, MSH1 Gene-edited mutants differ significantly from wild-type mutants in floral organ morphology and leaf color. The mutants exhibit abnormal phenotypes such as exposed stigmas in their floral organs, and chlorotic phenotypes such as yellowing and whitening in their leaves. Leaf color can serve as an early phenotypic marker for detecting whether editing has occurred in regenerated plants. Floral organs and leaves of T2-1-18 and T2-2-5 were selected for demonstration. Figure 6 , 7 As shown.

[0055] In summary, this invention provides a method and application for creating novel cytoplasmic male-sterile tomato germplasm using gene editing technology. MSH1 The exon regions of the gene were analyzed to obtain T0 generation edited plants. Non-transgenic homozygous edited plants were isolated through self-pollination. Cytoplasmic male-sterile single plants were identified in the T2 generation. These were then crossed twice with wild-type plants, ultimately resulting in the isolation of… MSH1 Cytoplasmic male sterile material whose genes have been restored to wild type is a new tomato cytoplasmic male sterile germplasm.

[0056] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All modifications that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method of... MSH1 A method for creating novel cytoplasmic male-sterile tomato germplasm through gene editing, characterized by: Through the study of fertile tomatoes MSH1 Gene editing is performed on exon regions to mediate cytoplasmic male sterility.

2. The method according to claim 1, characterized in that: Through the study of fertile tomatoes MSH1 Gene editing is performed on exon regions, utilizing the plant's own DSB repair mechanism to introduce DNA sequence deletions, thereby... MSH1 Loss of gene function induces mitochondrial genome recombination, altering the substoichiometry of potential CMS-related genes in tomatoes, thereby mediating cytoplasmic male sterility.

3. The method according to claim 1 or 2, characterized in that: By editing MSH1 The exon regions of the gene were analyzed to obtain T0 generation edited plants. Non-transgenic homozygous edited plants were isolated through self-pollination. Cytoplasmic male-sterile single plants were identified in the T2 generation. These were then crossed twice with wild-type plants, ultimately resulting in the isolation of… MSH1 Cytoplasmic male sterile material whose genes have been restored to wild type is a new tomato cytoplasmic male sterile germplasm.

4. The method according to claim 3, characterized in that: Using CRISPR / Cas9 technology MSH1 Gene editing is performed.

5. The method according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) sgRNA target site selection in tomatoes MSH1 Based on the CRISPR / Cas9 target site design principles, the exon region of the gene was selected as the target site, with the bases 20 bp upstream of the motif adjacent to the prototype spacer sequence. Forcibly changing the first base of the target site to G helps improve editing efficiency, i.e., 5'-GN. 19 -NGG-3', where NGG is a PAM sequence, and N... 19 This represents a 19bp base recognition sequence excluding G; (2) Design the required primers for the vector based on the recognition sequence. The primer sequences are as follows: DT1-BsF: 5'-ATATATGGTCTCGATTGAAGGGACAGTGAACGCCAAGTT-3' DT1-F0:5'-TGAAGGGACAGTGAACGCCAAGTTTTAGAGCTAGAAATAGC-3' DT2-R0: 5'-AACCCACATAATATTAACATAGCAATCTCTTAGTCGACTCTAC-3' DT2-BsR: 5'-ATTATTGGTCTCGAAACCCACATAATATTAACATAGCAA-3'; (3) Using the intermediate vector pCBC-DT1T2 as a template, PCR amplification was performed using the primers from step (2), and the PCR product was purified and recovered. The purified product was used to construct a CRISPR / Cas9 recombinant vector. The successfully ligated vector was transferred into Escherichia coli DH5α competent cells. Single clones were picked for plasmid extraction and sequencing. Plasmids with correct sequencing results were transferred into Agrobacterium tumefaciens GV3101 using the heat shock method. (4) Using tomato cotyledons that have grown for 7 days as explants, T0 generation regenerated plants were obtained by using Agrobacterium-mediated genetic transformation and hygromycin resistance screening system.

6. The method according to claim 5, characterized in that, MSH1 gene editing target sites were designed using an online website. The selected target site sequences were 5'-AAAGGGACAGTGAACGCCAACGG-3' and 5'-ACTATGTTAATATTATGTGGTGG-3'. When designing primers, the first base of the two target sites was changed to G.

7. The method according to claim 6, characterized in that, Step (3) also includes: incorporating two sgRNAs into the same vector.

8. The method according to claim 7, characterized in that It also includes the following steps: (1) Identification of T0 generation mutant plants: Genomic DNA was extracted from the leaves of regenerated plants and PCR amplification was performed using vector-specific primers HYG-F / R. The amplification products were detected by agarose gel electrophoresis. Regenerated plants that could amplify HYG bands were identified as positive transgenic plants. Primers for amplifying target sites were designed based on the sequences near the two target sites. PCR amplification was performed using the genomic DNA of positive transgenic plants as templates. The amplification products were sequenced, and the gene editing of the transgenic plants was analyzed based on the sequencing results. (2) T0 mutants were self-pollinated, T1 generation seeds were harvested from single plants and sown, and genomic DNA of T1 plants was extracted at the two-leaf-one-heart stage. Homozygous edited plants without transgenic fragments were screened using the method in step (1), and T2 generation seeds were harvested from self-pollinated and sown. (3) Fertility identification: The T2 generation non-transgenic homozygous edited plants obtained in step (2) are subjected to fertility identification, which mainly includes identifying pollen viability and observing the presence or absence of seeds in the fruit; (4) Based on the identified tomatoes msh1 A cytoplasmic male-sterile mutant was used as the maternal parent and crossed twice with a wild-type mutant. The offspring were then selected for... MSH1 The cytoplasmic male sterile plant whose genes are restored to wild type is the new cytoplasmic male sterile germplasm of tomato.

9. The method according to any one of claims 1 to 8 MSH1 Application of gene editing methods for creating novel cytoplasmic male-sterile tomato germplasm in tomato breeding.

10. Primers required for vector construction, characterized in that, The primer sequences are as follows: DT1-BsF:5'-ATATATGGTCTCGATTGAAGGGACAGTGAACGCCAAGTT-3' DT1-F0:5'-TGAAGGGACAGTGAACGCCAAGTTTTAGAGCTAGAAATAGC-3' DT2-R0:5'-AACCCACATAATTAACATAGCAATCTCTTAGTCGACTCTAC-3' DT2-BsR:5'-ATTATTGGTCTCGAAACCCACATAATTAACATAGCAA-3'。

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