A method for creating diploid seedless watermelon by gene editing ClSPO11-2

Abstract

The present invention discloses a method for creating diploid seedless watermelon by gene editing ClSPO11‑2, and belongs to the technical field of plant genetic engineering breeding. The present invention discloses a method for creating diploid seedless watermelon by gene editing ClSPO11‑2, and for the first time uses CRISPR / Cas9 technology to perform targeted editing on the watermelon ClSPO11‑2 gene, so that meiosis cannot be paired and synaptic, and male and female gametes are sterile, so that the gene function is lost, and the seeds are aborted after double fertilization, thereby creating seedless watermelon at the diploid level. Compared with wild-type materials, the edited diploid seedless watermelon plants created by this method have no obvious changes in phenotype during the vegetative growth stage, except for male and female gamete sterility, and the seedless phenotype can be infinitely propagated by plants edited by Clspo11‑2 / ClSPO11‑2 heterozygous. The method has the advantages of being simple and easy to operate, high efficiency, low cost and short cycle.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering breeding, and more specifically to a method for creating diploid seedless watermelon by gene editing ClSPO11-2. Background Art

[0002] Watermelon (Citrullus lanatus) is a major fruit and vegetable in summer. Its mature fruit is rich in water, sugar, minerals and multiple vitamins. It has important nutritional and edible value and is an important horticultural crop in the world. Seedless watermelon is popular among consumers and producers for its excellent nutritional quality, convenient consumption, good taste, stable yield and storage and transportation resistance. Traditional seedless watermelon research mainly focuses on hybridization of diploid and tetraploid watermelons to obtain triploid seedless, hormone induction and chromosome translocation. NUNEZ et al. (2008) reported that unpollinated watermelon female flowers treated with CPPU are prone to produce seedless fruits, but the small fruits, easy deformities and poor fruit setting rate seriously affect their commercial value. Tian Shujuan et al. (2021) found that the translocation of a 2.09Mb fragment on the Chr 06 chromosome of the mutant 148 material can induce the phenotype of few-seed watermelon. Due to production conditions, other technologies except triploid seedless watermelon have not been applied in actual production. Traditional triploid seedless watermelons are hybridized from diploid and tetraploid watermelons. It is difficult to obtain tetraploid mother plants during the breeding process. Triploid seeds have thick seed coats, underdeveloped embryos, low germination rates, and low seedling rates, resulting in high seed production costs. The long breeding cycle and high breeding costs have greatly limited the promotion and development of triploid seedless watermelons. In recent years, the technical research on seedless watermelons at the molecular biology level is in its infancy. With the promotion and application of molecular breeding, CRISPR / Cas9 editing of genes related to reproductive development can be performed, making it possible to create seedless watermelons at the diploid level.

[0003] The life cycle of angiosperms alternates between the diploid sporophyte generation and the haploid gametophyte generation, and the genetic material is transmitted through sexual reproduction to form seeds. Meiosis is a special division mode of reproductive cells to produce haploid gametes, which is the basis of the sexual reproduction process. Homologous chromosome pairing recombination plays a vital role in ensuring accurate and reliable chromosome separation and in the process of genetic information diversification. SPO11-2 protein is a homologous protein of type II topoisomerase VIA subunit, catalyzing the formation of DNA double-strand breaks, and is a key protein for initiating meiotic synaptic recombination (Gadelle D et al. 2003). The functional trait research of SPO11-2 in plants is limited to model plants and typical monocotyledonous crops, and has not been reported in watermelon. It is necessary to fully understand the molecular mechanism of plant meiotic recombination, compare the commonalities and characteristics of different species, and combine molecular breeding technology to transform the seedless trait of watermelon.

[0004] CRISPR / Cas9-mediated plant genome editing technology can perform site-specific modification of the seedless trait of watermelon, thereby achieving precise breeding of seedless watermelon. Therefore, providing a method for creating diploid seedless watermelon by gene editing ClSPO11-2 is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a method for creating diploid seedless watermelon by gene editing ClSPO11-2, and uses CRISPR / Cas9 to perform gene editing on the watermelon TOP VIA subunit homologous protein ClSPO11-2 (Cla97C01G002360) to create diploid seedless watermelon, thereby shortening the breeding period and reducing the breeding cost.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for creating diploid seedless watermelon by gene editing ClSPO11-2, the specific steps are as follows:

[0008] 1) Design target gRNA1, gRNA2, gRNA3, and gRNA4 according to the 5'-UTR region and ClSPO11-2 CDS sequence;

[0009] 2) Construction of ClSPO11-2 CRISPR / Cas9 dual-target knockout vector; gRNA1 and gRNA2, gRNA3 and gRNA4 were combined with dual targets respectively;

[0010] (1) Using restriction endonuclease BsaI-HF to digest the CRISPR / Cas9 vector pBSE402, and recovering the digested vector pBSE402;

[0011] (2) Using the intermediate vector pCBC-DT1T2 as a template, PCR amplification was performed using primers Target1-1F / Target1-2R and Target1-3F / Target1-4R to recover target fragment 1 and target fragment 2, respectively;

[0012] (3) homologously recombining the vector pBSE402 digested in step (1) with the target fragment recovered in step (2) to transform DH5α competent cells;

[0013] (4) shaking the colonies obtained in step (3) and extracting the recombinant plasmids, sequencing and comparing them, and transforming the recombinant plasmids with correct sequencing into Agrobacterium EHA105 competent cells;

[0014] (5) performing PCR detection on the bacterial colonies obtained in step (4), and performing genetic transformation of watermelon after verification;

[0015] 3) Genetic transformation of watermelon is carried out through infection, co-cultivation, recovery culture, selection culture, bud elongation culture and rooting culture stages to obtain transgenic plants;

[0016] 4) Performing gene editing detection on the transgenic plants obtained in step 3), and transplanting the gene-edited plants to a test field to observe the seedless phenotype.

[0017] Furthermore, the 5'-UTR region and ClSPO11-2 CDS sequence are shown in SEQ ID NO.2.

[0018] Furthermore, the target gRNA1, gRNA2, gRNA3, and gRNA4 sequences are as follows:

[0019] gRNA1: 5'-CTAGTAGAATGGAGCCAGC-3'; SEQ ID NO.3;

[0020] gRNA2: 5'-GATATCCTCACTCCTCAAG-3'; SEQ ID NO.4;

[0021] gRNA3: 5'-ATCCTCCATGAGATTTTTC-3'; SEQ ID NO.5;

[0022] gRNA4: 5'-CACTCCAGCCATCTCAAAT-3'; SEQ ID NO. 6.

[0023] Further, the sequences of the primers Target1-1F / Target1-2R and Target1-3F / Target1-4R are as follows:

[0024] Target1-1F:

[0025] 5'-TCGAAGTAGTGATTG CTAGTAGAATGGAGCCAGC GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.7;

[0026] Target1-2R:

[0027] 5'-TTCTAGCTCTAAAAC CTTGAGGAGTGAGGATATC CAATCTCTTAGTCGACTCTA-3';SEQ IDNO.8;

[0028] Target1-3F:

[0029] 5'-TCGAAGTAGTGATTG GAAAAATCTCATGGAGGAT GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.9;

[0030] Target1-4R:

[0031] 5'-TTCTAGCTCTAAAAC CACTCCAGCCATCTCAAAT CAATCTCTTAGTCGACTCTA-3'; SEQ ID NO. 10.

[0032] Furthermore, the method is applied in seedless watermelon breeding.

[0033] The material selected for genetic transformation of watermelon in the present invention is the wild variety 'YL'.

[0034] The gene editing is achieved with the help of the CRISPR / Cas9 system. The vegetative growth of the Clspo11-2 mutant is not affected, the male and female gametes are sterile, wild-type pollen cannot be normally fertilized when pollinated, and only a small amount of white-shelled seeds are produced. The Clspo11-2 / ClSPO11-2 heterozygous edited plants F1 generation seedlings were screened out to obtain Clspo11-2 / Clspo11-2 homozygous edited plants, and the seedless phenotype can be propagated, accelerating the creation process of diploid seedless watermelon. The gene editing method of the present invention can shorten the breeding period of seedless watermelon, save breeding costs, enrich breeding resources, and accelerate the breeding of seedless watermelon at the molecular level.

[0035] The present invention uses CRISPR / Cas9 technology to edit the watermelon ClSPO11-2 gene, thereby causing the gene to be functionally deficient, meiosis to be unable to undergo homologous recombination, pairing and synapsis to fail, male and female gametes to be sterile, and normal fertilization to produce a small amount of aborted seeds, thereby creating a diploid seedless watermelon. The diploid seedless watermelon created by the present invention through CRISPR / Cas9 effectively shortens the breeding cycle of seedless watermelons, and provides a theoretical basis for the study of seedless watermelons at the molecular biology level.

[0036] It can be known from the above technical scheme that compared with the prior art, the present invention discloses a method for creating diploid seedless watermelon by gene editing ClSPO11-2. For the first time, CRISPR / Cas9 technology is used to perform targeted editing on the watermelon ClSPO11-2 gene. The target point is deleted or the base is inserted so that the gene function is lost, the male and female gametes are sterile, and the wild-type pollen cannot be normally double fertilized when pollinated, and the seeds are aborted, thereby creating a diploid seedless watermelon. Compared with the wild-type material, the edited diploid seedless watermelon plant created by the method of the present invention has no obvious phenotypic changes in the vegetative growth stage except for the sterility of male and female gametes. The method has the advantages of being simple, efficient, low cost and short cycle. By gene editing ClSPO11-2 to create diploid seedless watermelon, the breeding period is shortened, the breeding cost is saved, the breeding resources of seedless watermelon are enriched, and the breeding process of seedless watermelon is accelerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0038] Figure 1 The accompanying drawing is a schematic diagram of the 5'-UTR region and the ClSPO11-2 gene structure of the present invention; black boxes represent exons, black lines represent introns, red rectangles represent 5'UTR, and the ClSPO11-2 gene target site is marked by an arrow;

[0039] Figure 2 The accompanying drawing is a schematic diagram of the recombinant construction of the pBSE402 CRISPR / Cas9 vector of the present invention;

[0040] Among them, A: dual targets of gRNA1 and gRNA2; B: dual targets of gRNA3 and gRNA4; expression frames pro35S:zCAS9, pro35S:GFP, pro35S:Basta belong to the original vector pBSE402;

[0041] Figure 3 The accompanying drawing is a schematic diagram of the Clspo11-2 CRISPR / Cas9 mutation types of the present invention;

[0042] Among them, WT is the wild material 'YL' sequence, and Clspo11-2 is the 'YL' mutant sequence; the gRNA target sequence and PAM sequence are underlined and bolded, respectively; the missing bases of the mutant are indicated by black dashed lines, and the inserted bases are indicated by lowercase bold letters;

[0043] Figure 4 The attached figure shows the observation of pollen viability of Clspo11-2-1 of the present invention;

[0044] Among them, a: wild-type WT male flowers; b: Clspo11-2-1 male flowers; c: wild-type WT pollen vitality; d: Clspo11-2-1 pollen vitality;

[0045] Figure 5 The accompanying drawing shows the phenotype observation of the seedless fruit of Clspo11-2-1 of the present invention;

[0046] Among them, a: self-pollinated mature fruit of wild type WT; b: mature fruit of Clspo11-2-1 mutant; c: cross-section of self-pollinated mature fruit of wild type WT; d: cross-section of mature fruit of Clspo11-2-1 mutant; e: number of seeds in self-pollinated mature fruit of wild type WT; f: number of seeds in mature fruit of Clspo11-2-1 mutant. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Example 1 Analysis of ClSPO11-2 (Cla97C01G002360) gene structure and design of editing sites

[0049] The BlastClSPO11-2 gene in the watermelon genome database of the Cucurbitaceae website (http: / / cucurbitgenomics.org / ) was obtained, with gene ID ClSPO11-2: Cla97C01G002360; and its gene sequence was analyzed.

[0050] 1-8 bp in SEQ ID NO.1 is the 5'-UTR region, and 9-2230 bp is the watermelon ClSPO11-2 gene sequence; Sequence analysis of SEQ ID NO.1 is as follows Figure 1 As shown, the ClSPO11-2 gene has eleven exons.

[0051] According to the 5'-UTR region and the CDS sequence of ClSPO11-2 (Cla97C01G002360) (SEQ ID NO.2), the target was designed on the CRISPRdirect website (http: / / crispr.dbcls.jp / ), including 4 targets. The three targets of ClSPO11-2 target gRNA1, gRNA2 and gRNA3 are all on the first exon, and the gRNA4 target is on the second exon. In SEQ ID NO.2, 1-8bp is the 5'-UTR region, and 9-1163bp is the ClSPO11-2 CDS sequence.

[0052] 5'-UTR region and watermelon ClSPO11-2 gene sequence:

[0053] CTAGTAGA

[0054] 5'-UTR region and ClSPO11-2 CDS sequence:

[0055] CTAGTAGA

[0056] The ClSPO11-2 target sequence is as follows:

[0057] gRNA1: 5'-CTAGTAGAATGGAGCCAGC-3'; SEQ ID NO.3;

[0058] gRNA2: 5'-GATATCCTCACTCCTCAAG-3'; SEQ ID NO.4;

[0059] gRNA3: 5'-ATCCTCCATGAGATTTTTC-3'; SEQ ID NO.5;

[0060] gRNA4: 5'-CACTCCAGCCATCTCAAAT-3'; SEQ ID NO. 6.

[0061] Example 2 Construction of ClSPO11-2 CRISPR / Cas9 dual-target knockout vector

[0062] Among them, ClSPO11-2 gRNA1 and gRNA2, gRNA3 and gRNA4 respectively combine dual targets. The main expression elements of the recombinant plasmid are shown in Figure 2 .

[0063] The CRISPR / Cas9 vector pBSE402 was digested with restriction endonuclease BsaI-HF (NEW ENGLAND BioLabs) and the digested vector pBSE402 was recovered. Enzyme digestion system (30 μL): 10x CutSmart buffer 3 μL, pBSE402 (1-2 μg) 2 μL, BsaI-HF 1 μL, ddH2O 24 μL.

[0064] Using the intermediate vector pCBC-DT1T2 as a template, Target1-1F / Target1-2R (containing gRNA1 and gRNA2, respectively) and Target1-3F / Target1-4R (containing gRNA3 and gRNA4, respectively) as primers, PCR amplification was performed using Vazyme P505 high-fidelity enzyme (Phanta Max Super-Fidelity DNA Polymerase).

[0065] The primer sequences of Target1-1F / Target1-2R and Target1-3F / Target1-4R are as follows:

[0066] Target1-1F:

[0067] 5'-TCGAAGTAGTGATTG CTAGTAGAATGGAGCCAGC GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.7;

[0068] Target1-2R:

[0069] 5'-TTCTAGCTCTAAAAC CTTGAGGAGTGAGGATATC CAATCTCTTAGTCGACTCTA-3';SEQ IDNO.8;

[0070] Target1-3F:

[0071] 5'-TCGAAGTAGTGATTG GAAAAATCTCATGGAGGAT GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.9;

[0072] Target1-4R:

[0073] 5'-TTCTAGCTCTAAAAC CACTCCAGCCATCTCAAAT CAATCTCTTAGTCGACTCTA-3'; SEQ ID NO. 10.

[0074] Amplification system (50μL): 2x Phanta Max Buffer 25μL, dNTP Mix (10mM) 1μL, upstream and downstream primers (10μM) 2μL each, Phanta Max Super-Fidelity DNA Polymerase 1μL, template DNA 2μL, ddH2O 17μL. PCR reaction program: pre-denaturation 95℃3min; denaturation 95℃15s, annealing 58℃15s, extension 72℃30s, 35 cycles; complete extension 72℃5min. Agarose gel electrophoresis was used to detect the recovery of target fragment 1 (with Target1-1F / Target1-2R as primers) and target fragment 2 (with Target1-3F / Target1-4R as primers).

[0075] The digested vectors were connected with the recovered target fragments 1 and 2 by homologous recombination using Vazyme C112ClonExpress II One Step Cloning Kit. Ligation reaction system (10 μL): 1 μL of linearized vector, 2 μL of target fragment, 2 μL of 5x CE II Buffer, 1 μL of Exnase II, 4 μL of ddH2O, reacted at 37°C for 30 min, and transformed into DH5α competent cells respectively. The transformation method was carried out according to the DH5α competent cell instruction manual of Shanghai Weidi Biotechnology Co., Ltd.

[0076] Primers U626-IDF and U629-IDR were used for colony PCR detection, and the primer sequences were as follows:

[0077] U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3'; SEQ ID NO.11;

[0078] U629-IDR: 5'-AGCCCTCTTTCTTTCGATCCATCAAC-3'; SEQ ID NO. 12.

[0079] The recombinant plasmids were extracted by shaking the bacteria, sequenced and compared, and the recombinant plasmids with correct sequencing were transformed into Agrobacterium EHA105 competent cells. The transformation method was carried out according to the instructions for Agrobacterium EHA105 competent cells of Shanghai Weidi Biotechnology Co., Ltd.

[0080] The primers U626-IDF and U629-IDR were used to perform PCR detection on Agrobacterium and verify the colonies.

[0081] Example 3 Watermelon Genetic Transformation

[0082] Sowing: Take the seeds of watermelon material 'YL', soak them in a 55℃ waterbath for 30 minutes, peel off the seed shells. Enter the clean bench, disinfect the peeled kernels with 75% alcohol for 30 seconds, soak and disinfect in 3% sodium hypochlorite for 15 minutes, wash with sterile water 5 times, then dry them on sterile filter paper, spread them flat on the sowing medium (BM, breeding medium; H2O, Agar 6.64g / L), and culture them in the dark at 28℃ for 2 days.

[0083] Dip infection: After the seeds germinate, cut off the two ends of the cotyledons, and divide the remaining explants into 8 equal pieces in a cross shape for dip infection. At the same time, pick the EHA105 single colony verified by PCR into LB liquid culture medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and shake the bacterial solution until the OD 600 =0.8, resuspend the bacterial solution in MS culture medium (M519 4.43 g / L, sucrose 30 g / L, 6-BA 1.5 mg / L) to a final concentration of OD 600=0.2. The cut cotyledons were inoculated with a 20 mL syringe containing the resuspended bacterial solution under vacuum for 15 min, the explants were taken out and dried on sterile filter paper, and then spread on co-culture medium (CM; M519 4.43 g / L, sucrose 30 g / L, G3251 3 g / L, 6-BA 1.5 mg / L) padded with filter paper for co-culture, and cultured in the dark at 28°C for 3 days; M519 and G3251 were purchased from Phytotech, and 6-BA was purchased from Yuanye Company.

[0084] Recovery culture: After 3 days of co-cultivation, the cotyledon blocks were transferred to recovery medium (RM; M5194.43 g / L, sucrose 30 g / L, G32513 g / L, 6-BA 1.5 mg / L, 200 mg / L Timentin) and cultured for 7 days at 28°C, with a light intensity of 20,000 lux and a light duration of 16 h / d.

[0085] Selective culture: After the recovery culture is completed, the explants are transferred to selective medium (SM; M519 4.43g / L, sucrose 30g / L, Agar 6.4g / L, 6-BA 1.5mg / L, 1.4mg / L Basta and 200mg / LTimentin) for selective culture, and subcultured at 28℃ for 3-4 weeks, with subculture every 12 days.

[0086] Shoot elongation culture: The explants with obvious buds carrying GFP tags were transferred to shoot elongation medium (SE, seedling elongation; M519 4.43 g / L, sucrose 30 g / L, Agar 6.4 g / L, 6-BA 0.5 mg / L and Timentin 200 mg / L) for culture.

[0087] Rooting culture: Create new incisions on the selected buds and transfer them to MS medium containing 0.5 mg / L IAA and 200 mg / L Timentin for rooting culture. Culture at 28°C until roots form.

[0088] Transplanting: When the regenerated seedlings take root and grow 4-5 true leaves, take them out of the culture bottle, rinse with clean water to remove the culture medium on the roots, transplant them into a flowerpot with a substrate: vermiculite ratio of 2:1, cover with plastic wrap to keep warm and moist for 3-4 days, gradually remove the film to harden the seedlings, and move them into the field for normal management.

[0089] Example 4 Editing detection of Clspo11-2 transgenic plants

[0090] CTAB method was used to extract DNA from watermelon regeneration seedlings carrying GFP tags of Clspo11-2 (including different target sites). Cut young leaves and place them in a 2ml centrifuge tube containing steel balls, precool them with liquid nitrogen, and grind them into powder by rapid shaking; add 800μL CTAB extraction buffer preheated at 65℃, and place them in a 65℃ water bath for 30min, turning them over every 10min; add an equal volume of 24:1 (chloroform: isoamyl alcohol) solution, mix them upside down, and centrifuge them at 8000r / min for 10min; transfer the supernatant to a 1.5ml centrifuge tube, add an equal volume of isopropanol, and mix them gently upside down; centrifuge them at 10000r / min for 10min, discard the supernatant, rinse the precipitate twice with 75% ethanol, pour it out and absorb the remaining liquid, and dissolve it with 100μL ddH2O (containing 0.1% RNase) after drying, and store it at 4℃ for later use.

[0091] Using the DNA extracted from Clspo11-2 watermelon regeneration seedlings carrying GFP tags as a template, the sequences containing the two target sites of ClSPO11-2 were PCR amplified using primers Crispr SPO11-2 detection primer F and Crispr SPO11-2 detection primer R, respectively, and the negative control was non-transgenic plant DNA. Crispr SPO11-2 detection primer F was sent to the test.

[0092] The primer sequences of ClSPO11-2 Crispr detection primer F and ClSPO11-2 Crispr detection primer R are as follows:

[0093] Crispr SPO11-2 detection primer F: 5'-AGGGTAAGCAAGAAAGGTTGTT-3'; SEQ ID NO.13;

[0094] Crispr SPO11-2 detection primer R: 5'-CGATCAATCTGTCCAATCGG-3'; SEQ ID NO.14.

[0095] Amplification system (25 μL) and reaction procedure: 2x T5 SuperMIX 12.5 μL, upstream and downstream primers (10 μM) 1 μL each, template DNA 1 μL, ddH2O 9.5 μL. PCR reaction procedure: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 58°C for 10 s, extension at 72°C for 15 s, 35 cycles; complete extension at 72°C for 5 min. Detection by agarose gel electrophoresis.

[0096] The target editing comparison results of Clspo11-2 mutant are shown in Figure 3, where Clspo11-2-1 lacked 74 bp between the gRNA1 and gRNA2 target sites, and Clspo11-2-2 inserted 1 bp at the gRNA4 target site.

[0097] Example 5 Phenotypic observation of Clspo11-2 transgenic plants The phenotypic observation is described using Clspo11-2-1 as an example.

[0098] The edited plants Clspo11-2-1 and Clspo11-2-2 were planted in the Caoxinzhuang experimental field of Northwest Agriculture and Forestry University and managed normally to observe the phenotypes.

[0099] The Clspo11-2-1 plant showed normal growth and sterile gametes in the reproductive stage. The stamens of Clspo11-2-1 were small and shrunken, and the anthers were degenerate and did not shed pollen. The pollen viability of Clspo11-2-1 was as shown by Alexander staining. Figure 4 As shown, Clspo11-2-1 pollen aborted.

[0100] The female flowers of Clspo11-2-1 were pollinated with wild-type pollen on the day of their opening (Clspo11-2-1♀X ), because the male and female gametes of Clspo11-2-1 were sterile, the fertilization was abnormal when wild-type pollen was pollinated, and the fruit expanded normally; the homologous chromosome pairing of the Clspo11-2-1 mutant failed, the male and female gametes were sterile, and a small number of empty seeds were produced in the mature watermelon fruits of Clspo11-2-1, such as Figure 5 .

[0101] The phenotype of the edited plant Clspo11-2-2 is consistent with that of Clspo11-2-1, so it will not be described here.

[0102] In summary, the present invention provides a method for creating diploid seedless watermelon by gene editing ClSPO11-2, using CRISPR / Cas9 technology to perform gene editing and targeted knockout on watermelon ClSPO11-2, target deletion or insertion of bases to make its gene function missing, male and female gametes sterile, wild-type pollinated fruits swell normally, and mature fruits present a seedless phenotype, thereby creating seedless watermelon at the diploid level. The diploid seedless watermelon created by CRISPR / Cas9 in the present invention effectively shortens the breeding period of seedless watermelon, saves breeding costs, promotes the breeding of seedless watermelon varieties, and provides a theoretical basis for the study of seedless watermelon at the molecular biology level.

[0103] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for creating diploid seedless watermelon by gene editing ClSPO11-2, characterized in that: The specific steps are as follows: 1) Design target gRNA1, gRNA2, gRNA3, and gRNA4 according to the 5'-UTR region and ClSPO11-2 CDS sequence; 2) Construction of ClSPO11-2 CRISPR / Cas9 dual-target knockout vector; gRNA1 and gRNA2, gRNA3 and gRNA4 were combined with dual targets respectively; (1) Using restriction endonuclease BsaI-HF to digest the CRISPR / Cas9 vector pBSE402, and recovering the digested vector pBSE402; (2) Using the intermediate vector pCBC-DT1T2 as a template, PCR amplification was performed using primers Target1-1F / Target1-2R and Target1-3F / Target1-4R to recover target fragment 1 and target fragment 2, respectively; (3) homologously recombining the vector pBSE402 digested in step (1) with the target fragment recovered in step (2) to transform DH5α competent cells; (4) shaking the colonies obtained in step (3) and extracting the recombinant plasmids, sequencing and comparing them, and transforming the recombinant plasmids with correct sequencing into Agrobacterium EHA105 competent cells; (5) performing PCR detection on the bacterial colonies obtained in step (4), and performing genetic transformation of watermelon after verification; 3) Genetic transformation of watermelon is carried out through infection, co-cultivation, recovery culture, selection culture, bud elongation culture and rooting culture stages to obtain transgenic plants; 4) performing gene editing detection on the transgenic plants obtained in step 3), and transplanting the gene-edited plants to a test field to observe the seedless phenotype; The 5'-UTR region and ClSPO11-2 CDS sequence are shown in SEQ ID NO.2; The target gRNA1, gRNA2, gRNA3, and gRNA4 sequences are as follows: gRNA1: 5'-CTAGTAGAATGGAGCCAGC-3'; SEQ ID NO.3; gRNA2: 5'-GATATCCTCACTCCTCAAG-3'; SEQ ID NO.4; gRNA3: 5'-ATCCTCCATGAGATTTTTC-3'; SEQ ID NO.5; gRNA4: 5'-CACTCCAGCCATCTCAAAT-3'; SEQ ID NO.6; The sequences of the primers Target1-1F / Target1-2R and Target1-3F / Target1-4R are as follows: Target1-1F: 5’-TCGAAGTAGTGATTG CTAGTAGAATGGAGCCAGC GTTTTAGAGCT AGAAATAGC-3’;SEQ IDNO.7; Target1-2R: 5'-TTCTAGCTCTAAAAC CTTGAGGAGTGAGGATATC CAATCTCTTAGT CGACTCTA-3';SEQ ID NO.8; Target1-3F: 5’-TCGAAGTAGTGATTG GAAAAATCTCATGGAGGAT GTTTTAGAGCT AGAAATAGC-3’;SEQ IDNO.9; Target1-4R: 5'-TTCTAGCTCTAAAAC CACTCCAGCCATCTCAAAT CAATCTCTTAGT CGACTCTA-3';SEQ ID NO.10。 2. Application of the method described in claim 1 in seedless watermelon breeding.

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