CN110724703B

CN110724703B - Method for preparing short style tomatoes

Info

Publication number: CN110724703B
Application number: CN201910936056.0A
Authority: CN
Inventors: 周明; 李常保; 李传友; 邓磊; 李保思
Original assignee: Institute of Genetics and Developmental Biology of CAS; Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Institute of Genetics and Developmental Biology of CAS; Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2021-08-20
Anticipated expiration: 2039-09-29
Also published as: CN110724703A

Abstract

The invention discloses a method for creating tomato with short style, which uses CRISPR/Cas9 system to carry out mutation knockout on the 5 th exon or the 6 th exon of tomato PS-2 gene, the style of the obtained mutant strain is shortened, and the castration efficiency is obviously improved.

Description

Method for preparing short style tomatoes

Technical Field

The invention belongs to the technical field of crop breeding, relates to a method for creating tomato short-style pillars, and particularly relates to a method for creating tomato short-style pillar germplasm by knocking out PS-2 gene by using a CRISPR/Cas9 system.

Background

Tomatoes (Solanum lycopersicum) are full flowers and self-pollinated crops, while the tomato seeds sold on production are all the first hybrid generation (generation F1). When producing tomato hybrid seeds, manual emasculation operation is required. In the case of emasculation, the operation is generally carried out by separating the flower bud petals with a tool such as tweezers and then removing the stamens. Because the tomato flower organ is small, the operation is difficult, and the time and the labor are wasted.

The castration efficiency of the tomatoes is improved, the hybrid seed production efficiency of the tomatoes can be improved to a great extent, and the seed production cost is reduced.

Shortening the stigma helps to improve castration efficiency. If the stigma is shortened, the top end of the bud can be directly pinched by hands during castration, and the petals and anthers can be quickly pulled out without damaging the stigma (the stigma of a wild type tomato is generally level with or slightly lower than an anther cylinder, and the operation can damage the stigma to cause the incapability of pollination and seed setting).

Reference documents:

L Deng,H Wang,et al.,2017.Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system.Journal of Genetics and Genomics.1-4.doi.org/10.1016/j.jgg.2017.10.002.

Benoit Gorguet,et al.,2017.ps-2,the gene responsible for functional sterility in tomato,due to non-dehiscent anthers,is the result of a mutation in a novel polygalacturonase gene.Theor Appl Genet(2009)118:1199–1209.DOI10.1007/s00122-009-0974-9.

disclosure of Invention

According to the invention, the PS-2 gene is knocked out by using a CRISPR/Cas9 technology to obtain the tomato mutant with the shortened style.

More specifically, the invention provides a method for preparing short style tomatoes,

the method is to knock out tomato PS-2 gene or homologous gene thereof which keeps the encoding ability of polygalacturonase activity. In some embodiments, the tomato germplasm is: TB 0993.

In some embodiments, the coding sequence of the tomato PS-2 gene is set forth in SEQ ID No. 1.

Homologous genes of the present invention that retain the ability to encode polygalacturonase activity include, but are not limited to: the gene or GenBank accession number shown as SEQ ID NO.1 of the coding sequence in tomato varieties including tomato TB0993 is: a homologous gene that is homologous to the PS-2 gene of EU111748 and retains the encoding ability of polygalacturonase activity, or a mutant of the aforementioned homologous gene that retains the encoding ability of polygalacturonase activity (mutation types include, but are not limited to, insertion, deletion, variation, fragment translocation, fragment fusion; mutation routes include, but are not limited to, hybrid, natural mutant/species or artificial mutant/species; mutation sites or regions include, but are not limited to, intron regions, exon regions, non-coding regions).

In some embodiments, the tomato PS-2 gene or homologous gene thereof that retains the ability to encode a polygalacturonase activity encodes a protein having the sequence set forth in SEQ ID No. 3.

In some embodiments, the method is to prepare a mutant tomato homozygote for the knockout of the tomato PS-2 gene or a homologous gene thereof that retains the ability to encode polygalacturonase activity.

In some embodiments, the method is a knockout of exon 5 of the tomato PS-2 gene or a homologous gene thereof that retains the ability to encode a polygalacturonase activity.

In some embodiments, the method is a knockout of exon 6 of the tomato PS-2 gene or a homologous gene thereof that retains the ability to encode a polygalacturonase activity.

In some embodiments, the method is to mutate the nucleic acid coding sequence of the tomato PS-2 gene or its homologous gene that retains the ability to encode a polygalacturonase activity into a nucleic acid sequence as set forth in any one of SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, or a combination thereof.

In some embodiments, the method is to mutate the protein sequence encoded by the tomato PS-2 gene or its homologous gene that retains the ability to encode a polygalacturonase activity into a protein sequence as set forth in any one of SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, or a combination thereof.

In some embodiments, the knockout of the tomato PS-2 gene or its cognate gene that retains the polygalacturonase activity encoding ability is achieved using the CRISPR/Cas9 system.

In some embodiments, the target sequence in the sgRNA-expressing vector in the CRISPR/Cas9 system is shown in SEQ ID No.4 and/or SEQ ID No. 5.

In a second aspect, the present invention provides a sgRNA for use in carrying out the creation method of the first aspect of the invention or in preparing a tomato variety produced by the creation method of the first aspect of the invention.

In a third aspect, the present invention provides a sgRNA expression vector for implementing the method of the first aspect of the present invention, or for preparing a tomato variety prepared by the method of the first aspect of the present invention.

In a fourth aspect, the present invention provides a kit containing the sgRNA according to the second aspect of the present invention, or the vector for expressing the sgRNA according to the third aspect of the present invention.

In a fifth aspect, the present invention provides a mutein encoded by the post-mutation PS-2 gene or its homologous gene retaining the coding ability for polygalacturonase activity in the mutant strain prepared by the creation method of the first aspect of the present invention.

In some embodiments, the amino acid sequence of the mutein is as shown in any one of SEQ ID No.6, SEQ ID No.8, SEQ ID No.10 or a combination thereof.

In a sixth aspect, the present invention provides a gene the coding sequence of which is capable of encoding a mutein according to the fifth aspect of the invention.

In some embodiments, the coding sequence of the gene is as set forth in any one of SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 or a combination thereof.

The seventh aspect of the present invention provides the use of the creation method according to the first aspect of the present invention, the sgRNA according to the second aspect of the present invention, the vector expressing the sgRNA according to the third aspect of the present invention, the kit according to the fourth aspect of the present invention, the mutein according to the fifth aspect of the present invention, or the gene according to the sixth aspect of the present invention, in constructing seed production with shortened tomato style.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, tomato PS-2 gene is knocked out through CRISPR/Cas9, and short-stub mutant material is created. The method for creating the short-stub mutant material has application prospect in tomato seed production.

According to the invention, the short stub material is obtained by knocking out the PS-2 gene through the CRISPR/Cas9, so that the manual castration operation can be simplified, the seed production efficiency is improved, and the seed production cost is reduced. The technical method for obtaining the mutant material can quickly and effectively create the short-stub mutant and is expected to be widely applied to seed production.

Drawings

FIG. 1 is a diagram of CRISPR/Cas9 knockout target sequences Guide1 and Guide2 on the PS-2 gene of the invention.

FIG. 2 is a schematic sequence diagram of a mutant obtained by knocking out PS-2 gene target sequences Guide1 and Guide2 through CRISPR/Cas 9.

FIG. 3 is a photograph of flowers in the early blooming stage of wild type material TB 0993.

FIG. 4 is a photograph of flowers at the initial blooming stage of mutant # 1.

In FIG. 5, the left picture is the photograph of the flower in full bloom stage of the wild type material TB0993, and the right picture is the photograph of the flower in full bloom stage of the mutant # 1.

In FIG. 6, the left picture is the photograph of the flower in full bloom stage of the wild type material TB0993, and the right picture is the photograph of the flower in full bloom stage of the mutant # 1.

FIG. 7 is a photograph of a wild type TB0993 plant.

FIG. 8 is a photograph of mutant #1 plants.

The left picture in fig. 9 is a photograph of leaves of the wild-type material TB 0993. The right panel is a photograph of mutant #1 leaves.

FIG. 10 is a schematic diagram of a method for emasculation of wild-type tomato flowers.

FIG. 11 is a schematic diagram of a method for emasculation of mutant #1 tomato flowers.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1. Tomato material

Tomato variety TB0993 was used. TB0993 is a fine inbred line of tomato powder, which is bred by a hybrid variety group BF001 of Neem corporation in Netherlands in 2009 through an 8-generation directional purification system (a single plant method is used for selecting plants with TYLCV Ty1 and Ty3a locus and high fruit setting rate). The invention carries out whole genome sequencing on TB0993 tomatoes, the sequencing is completed by Beijing Tianyihui-Chi biotechnology limited company, and the sequencing result is not disclosed.

2. Introduction of genes

According to the report of Benoit Gorguet et al in 2009: the PS-2 gene (GenBank: EU111748) encodes polygalacturonase. Comprises 9 exons, the sequence comprises 6877 nucleotides (wherein 32-6748 is a coding region, 32-130,1838-1969,2686-2853,3409-3429,3565-3772,192-5273,5619-5727,5819-5938 and 6509-6748 are exons respectively), and the mature protein comprises 392 amino acid residues and can control the anther dehiscence.

According to the sequencing result of the tomato variety TB0993, the DNA coding sequence of the nucleic acid coding region of the PS-2 gene of the tomato variety TB0993 is (named as SEQ ID NO. 1):

ATGGAGAAATTCAATGAAGAAGAAGATCAAGCTAAGGTTACAACAATTAATGTGGATAGCTTTGGAGCTAAAGGTGATGGAAGTATAGATGATACAAATGCATTTCAAAAAGCATGGAAAGAAGCTTGTTCATCTTCACATGTTGTGAATTTTGTGGTGTCCCAGAACAAGAAATATCTTCTCAAACCAATCAAATTTTATGGGCCATGCAAATCTTCCATTACAATGCAGATTTATGGAACCCTATTAGCATCTGATGATACTTCAGATTACAAGAAGGATAGTAGGCACTGGCTTATTTTTGATAGTGTTCAAAAATTGGTTGTTGGAGGAGCTGGAGTTATCAATGGCAATGGCAAAATTTGGTGGCAACATTCTTGCAAAATTAATAAAAAATTGCCATGCAAGGTAGCACCCACGGCCCTGACATTTTACAAGTGTAACAACTTGAAAGTGAAGGACCTTAAAATAGAAAATGCACAACAAATACATTTGCTAATTGAGAAGTGTGTTGGTGTTGAAGTTACAAAATTGGTAGTGACTTCTCCAGAAAATAGCCCTAATACTGATGGAATCCATATAACTAGCACTCAAAATATTCAAATTTCTGATTCCACCATTGCCACAGGTGATGATTGCATCTCAATTGTGGATGGATCTCAGAAGGTCTTAGCCACTGGCATTACTTGTGGACCAGGTCATGGAATTAGTATTGGAAGTTTGGGAGGTGGAAATTCAGAAGCTCATGTGTCTGATATTCATGTAAATGGAGCTAAGCTTTATGAAACTACAAATGGACTTAGGATTAAGACTTGGCCGGGAGGATTTGGAAGTGCAAGCAATATTAAGTATCAAAATGTGGTTATGAATAATGTCAAAAATCCAATAATTATAGACCAAAATTATTGTGATCAAGCTGATGGTCCATGCAAAGCTGAGACTGACTCGGCAGTTGAAGTGAAAAATGTGATTTATCAAAATATCAAAGGCACAAGTGCAACAAATGATGCAATAAGTATCAAGTGCAGCAAAAAAATTCCATGTGAAGGAATTTTGATGGAGAATGTGAAATTGTTAGGAGGAAATGGTGAAACTCCAAATGGTATTTGGGGAAATATCAATAATCTTACGTGCAAAAATGTTTTACCAGAATGTCAAAAAAACTCAAAAATTGTATAA

the mature mRNA nucleic acid coding sequence of the PS-2 gene of tomato variety TB0993 was (designated SEQ ID No. 2):

AUGGAGAAAUUCAAUGAAGAAGAAGAUCAAGCUAAGGUUACAACAAUUAAUGUGGAUAGCUUUGGAGCUAAAGGUGAUGGAAGUAUAGAUGAUACAAAUGCAUUUCAAAAAGCAUGGAAAGAAGCUUGUUCAUCUUCACAUGUUGUGAAUUUUGUGGUGUCCCAGAACAAGAAAUAUCUUCUCAAACCAAUCAAAUUUUAUGGGCCAUGCAAAUCUUCCAUUACAAUGCAGAUUUAUGGAACCCUAUUAGCAUCUGAUGAUACUUCAGAUUACAAGAAGGAUAGUAGGCACUGGCUUAUUUUUGAUAGUGUUCAAAAAUUGGUUGUUGGAGGAGCUGGAGUUAUCAAUGGCAAUGGCAAAAUUUGGUGGCAACAUUCUUGCAAAAUUAAUAAAAAAUUGCCAUGCAAGGUAGCACCCACGGCCCUGACAUUUUACAAGUGUAACAACUUGAAAGUGAAGGACCUUAAAAUAGAAAAUGCACAACAAAUACAUUUGCUAAUUGAGAAGUGUGUUGGUGUUGAAGUUACAAAAUUGGUAGUGACUUCUCCAGAAAAUAGCCCUAAUACUGAUGGAAUCCAUAUAACUAGCACUCAAAAUAUUCAAAUUUCUGAUUCCACCAUUGCCACAGGUGAUGAUUGCAUCUCAAUUGUGGAUGGAUCUCAGAAGGUCUUAGCCACUGGCAUUACUUGUGGACCAGGUCAUGGAAUUAGUAUUGGAAGUUUGGGAGGUGGAAAUUCAGAAGCUCAUGUGUCUGAUAUUCAUGUAAAUGGAGCUAAGCUUUAUGAAACUACAAAUGGACUUAGGAUUAAGACUUGGCCGGGAGGAUUUGGAAGUGCAAGCAAUAUUAAGUAUCAAAAUGUGGUUAUGAAUAAUGUCAAAAAUCCAAUAAUUAUAGACCAAAAUUAUUGUGAUCAAGCUGAUGGUCCAUGCAAAGCUGAGACUGACUCGGCAGUUGAAGUGAAAAAUGUGAUUUAUCAAAAUAUCAAAGGCACAAGUGCAACAAAUGAUGCAAUAAGUAUCAAGUGCAGCAAAAAAAUUCCAUGUGAAGGAAUUUUGAUGGAGAAUGUGAAAUUGUUAGGAGGAAAUGGUGAAACUCCAAAUGGUAUUUGGGGAAAUAUCAAUAAUCUUACGUGCAAAAAUGUUUUACCAGAAUGUCAAAAAAACUCAAAAAUUGUAUAA

the following vector design and gene knockout operations of the present invention are based on the aforementioned nucleic acid coding sequences.

The protein sequence of the PS-2 gene of tomato variety TB0993 is (designated as SEQ ID NO. 3): MEKFNEEEDQAKVTTINVDSFGAKGDGSIDDTNAFQKAWKEACSSSHVVNFVVSQNKKYLLKPIKFYGPCKSSITMQIYGTLLASDDTSDYKKDSRHWLIFDSVQKLVVGGAGVINGNGKIWWQHSCKINKKLPCKVAPTALTFYKCNNLKVKDLKIENAQQIHLLIEKCVGVEVTKLVVTSPENSPNTDGIHITSTQNIQISDSTIATGDDCISIVDGSQKVLATGITCGPGHGISIGSLGGGNSEAHVSDIHVNGAKLYETTNGLRIKTWPGGFGSASNIKYQNVVMNNVKNPIIIDQNYCDQADGPCKAETDSAVEVKNVIYQNIKGTSATNDAISIKCSKKIPCEGILMENVKLLGGNGETPNGIWGNINNLTCKNVLPECQKNSKIV

3. Gene knock-out System vector introduction

The vector is a CRISPR/Cas9 dual-vector system PTX041, which is presented by plum friend-passing laboratory of institute of genetics and developmental biology of Chinese academy of sciences. Wherein the sgRNA is expressed with tomato U6 promoter and the maize codon optimized Cas9 is expressed with 2 × CaMV 35S promoter. For specific features of the vector system, please refer to Deng et al (2017).

4. Knockout target selection and construction of recombinant PTX041 vector

The position and sequence of the target sequence on the PS-2 gene are shown in FIG. 1.

(1) A Target point is selected from 5 th and 6 th exons of the PS-2 gene respectively, which are respectively called Guide1 and Guide2 in sequence, and the sequences are shown as Target in figure 1. In the following primers, linkers (capital letters are target sequences, lower case letters are linker sequences, contain Bal1 enzyme cutting sites and pCBC-DT1T2_ tomato 6 vector sequences, and are convenient for PCR amplification and subsequent enzyme cutting connection) are added before and after Guide1 and Guide2 to form primer pairs:

Guide1F：

atatatggtctcgtttgGTGTAACAACTTGAAAGTGAgttttagagctagaaatagc (named SEQ ID NO.12)

Guide2R：

attattggtctcgaaacGATTGCATCTCAATTGTGGAccaaactacactgttagattc (designated SEQ ID NO. 13);

wherein the target sequence for exon 5 is: GTGTAACAACTTGAAAGTGA, named: SEQ ID NO.4, the target sequence for exon 6 is: GATTGCATCTCAATTGTGGA, named: SEQ ID NO. 5.

(2) The primers Guide1F and Guide2R are subjected to PCR amplification by taking a plasmid pCBC-DT1T2_ tomato U6 as a template, so that the obtained amplification product simultaneously contains sequences aiming at two targets of Guide1 and Guide2, and after the sequences are expressed in target cells, the sequences can express and simultaneously knock out the two targets, so that the knocking-out efficiency is improved. Thus, double mutations can be caused simultaneously. The sequence of the amplification product was (black italic underlined target or reverse complement thereof) (designated SEQ ID No. 14):

(3) the amplified product was cleaved simultaneously with the PTX-041 vector (dual promoter expression vector, which can simultaneously transcribe two independent sgRNAs, Deng et al.2017) with BSaI and ligated with T4 ligase to form a ligation product, called PTX-GUIDE1,2, a recombinant vector. The recombinant vector comprises two targets and two promoters, and can be used for simultaneously transcribing two independent sgRNAs. The recombinant vector also simultaneously transcribes Cas 9.

(4) PTX-GUIDE1,2 was transformed into E.coli DH 5. alpha. competent cells, and a single colony was picked up and cultured overnight in liquid LB medium containing 50mg/L kanamycin (Kan) at 37 ℃ with shaking at 200 rpm. With primer TST 1: AGCGGATAACAATTTCACACAGGA (named as SEQ ID NO.15), and extracting positive plasmid to transform Agrobacterium AGL 0.

(5) A common tomato cotyledon is infected by AGL0 agrobacterium by a leaf disc method to respectively obtain a resistance bud line of a transformation PTX-GUIDE1 and 2 knockout carrier related vector, and a corresponding plant is obtained after transplanting.

5. Verification of transformed plants

The primers for detecting the mutation condition of the PS-2 gene of the 5 th exon gene knockout plant are as follows:

upstream primer Pu1 (designated SEQ ID NO. 16):

CCATGCAAGGTAGCACCCACG；

downstream primer Pd1 (designated as SEQ ID NO. 17):

CCTGTGGCAATGGTGGAATCAG。

the primers for detecting the mutation condition of the PS-2 gene of the 6 th exon gene knockout plant are as follows:

upstream primer Pu2 (designated SEQ ID NO. 18):

CACACCGAAAATCTTGAAAGTGTAG；

downstream primer Pd2 (designated as SEQ ID NO. 19):

GAGCTTCTGAATTTCCACCTCCC。

the plant genome DNA was extracted using a DNA extraction kit (high-efficiency plant genome DNA extraction kit from Tiangen Biochemical technology (Beijing) Ltd., Cat. No.: DP 350-03).

Genomic DNA was PCR amplified using Takara PrimerSTAR Max DNA polymerase from Takara, Inc., and primers Pu1, Pd1, and Pu2, Pd 2. The reaction condition is 98 ℃ for 30 s; 30 cycles of 98 ℃ for 15s,60 ℃ for 15s and 72 ℃ for 30 s; 5min at 72 ℃.

Sequencing the PCR products (delivered to Beijing Tianyihui-Yuan biotechnology, Inc.) and sequencing and splicing the PS-2 genes in a plurality of mutant strains.

Finally, the mutation of the 5 th exon of PS-2 in the mutant strain #1-2 and the mutation of the 6 th exon of PS-2 in the mutant strain #3 are found, and the mutant strains are all mutant homozygotes.

The results of the mutation position sequencing are shown in FIG. 2.

The mutated coding sequence and amino acid sequence are as follows:

the mutated nucleic acid coding sequence of the mutant #1 coding sequence was (wherein underlining indicates the mutated nucleic acid sequence to the newly formed stop codon) (designated: SEQ ID NO.6):

ATGGAGAAATTCAATGAAGAAGAAGATCAAGCTAAGGTTACAACAATTAATGTGGATAGCTTTGGAGCTAAAGGTGATGGAAGTATAGATGATACAAATGCATTTCAAAAAGCATGGAAAGAAGCTTGTTCATCTTCACATGTTGTGAATTTTGTGGTGTCCCAGAACAAGAAATATCTTCTCAAACCAATCAAATTTTATGGGCCATGCAAATCTTCCATTACAATGCAGATTTATGGAACCCTATTAGCATCTGATGATACTTCAGATTACAAGAAGGATAGTAGGCACTGGCTTATTTTTGATAGTGTTCAAAAATTGGTTGTTGGAGGAGCTGGAGTTATCAATGGCAATGGCAAAATTTGGTGGCAACATTCTTGCAAAATTAATAAAAAATTGCCATGCAAGGTAGCACCCACGGCCCTGACATTTTACAAGTGTAACAACTTGAAAGAAGGACCTTAA

the mutated protein sequence of the mutant #1 coding sequence is (wherein the amino acid sequence after mutation is underlined) (designated as SEQ ID NO.7):

MEKFNEEEDQAKVTTINVDSFGAKGDGSIDDTNAFQKAWKEACSSSHVVNFVVSQNKKYLLKPIKFYGPCKSSITMQIYGTLLASDDTSDYKKDSRHWLIFDSVQKLVVGGAGVINGNGKIWWQHSCKINKKLPCKVAPTALTFYKCNNLKEGP

the mutated nucleic acid coding sequence of mutant #2 coding sequence was (wherein underlining indicates the mutated nucleic acid sequence to the newly formed stop codon) (designated: SEQ ID NO.8):

ATGGAGAAATTCAATGAAGAAGAAGATCAAGCTAAGGTTACAACAATTAATGTGGATAGCTTTGGAGCTAAAGGTGATGGAAGTATAGATGATACAAATGCATTTCAAAAAGCATGGAAAGAAGCTTGTTCATCTTCACATGTTGTGAATTTTGTGGTGTCCCAGAACAAGAAATATCTTCTCAAACCAATCAAATTTTATGGGCCATGCAAATCTTCCATTACAATGCAGATTTATGGAACCCTATTAGCATCTGATGATACTTCAGATTACAAGAAGGATAGTAGGCACTGGCTTATTTTTGATAGTGTTCAAAAATTGGTTGTTGGAGGAGCTGGAGTTATCAATGGCAATGGCAAAATTTGGTGGCAACATTCTTGCAAAATTAATAAAAAATTGCCATGCAAGGTAGCACCCACGGCCCTGACATTTTACAAGTGTAACAACTTGAAAGGTGAAGGACCTTAA

the mutated protein sequence of the mutant #2 coding sequence is (wherein the amino acid sequence after mutation is underlined) (designated as SEQ ID NO.9):

MEKFNEEEDQAKVTTINVDSFGAKGDGSIDDTNAFQKAWKEACSSSHVVNFVVSQNKKYLLKPIKFYGPCKSSITMQIYGTLLASDDTSDYKKDSRHWLIFDSVQKLVVGGAGVINGNGKIWWQHSCKINKKLPCKVAPTALTFYKCNNLKGEGP

the nucleic acid coding sequence after mutation of the mutant #3 coding sequence was (wherein underlining indicates the mutated nucleic acid sequence to the newly formed stop codon) (designated: SEQ ID NO.10):

ATGGAGAAATTCAATGAAGAAGAAGATCAAGCTAAGGTTACAACAATTAATGTGGATAGCTTTGGAGCTAAAGGTGATGGAAGTATAGATGATACAAATGCATTTCAAAAAGCATGGAAAGAAGCTTGTTCATCTTCACATGTTGTGAATTTTGTGGTGTCCCAGAACAAGAAATATCTTCTCAAACCAATCAAATTTTATGGGCCATGCAAATCTTCCATTACAATGCAGATTTATGGAACCCTATTAGCATCTGATGATACTTCAGATTACAAGAAGGATAGTAGGCACTGGCTTATTTTTGATAGTGTTCAAAAATTGGTTGTTGGAGGAGCTGGAGTTATCAATGGCAATGGCAAAATTTGGTGGCAACATTCTTGCAAAATTAATAAAAAATTGCCATGCAAGGTAGCACCCACGGCCCTGACATTTTACAAGTGTAACAACTTGAAAGTGAAGGACCTTAAAATAGAAAATGCACAACAAATACATTTGCTAATTGAGAAGTGTGTTGGTGTTGAAGTTACAAAATTGGTAGTGACTTCTCCAGAAAATAGCCCTAATACTGATGGAATCCATATAACTAGCACTCAAAATATTCAAATTTCTGATTCCACCATTGCCACAGGTGATGATTGCATCTCAATTGTTGGATGGATCTCAGAAGGTCTTAGCCACTGGCATT ACTTGTGGACCAGGTCATGGAATTAG

the mutated protein sequence of the mutant #3 coding sequence is (wherein the amino acid sequence after mutation is underlined) (designated as SEQ ID NO.11).

MEKFNEEEDQAKVTTINVDSFGAKGDGSIDDTNAFQKAWKEACSSSHVVNFVVSQNKKYLLKPIKFYGPCKSSITMQIYGTLLASDDTSDYKKDSRHWLIFDSVQKLVVGGAGVINGNGKIWWQHSCKINKKLPCKVAPTALTFYKCNNLKVKDLKIENAQQIHLLIEKCVGVEVTKLVVTSPENSPNTDGIHITSTQNIQISDSTIATGDDCISIVGWISEG LSHWHYLWTRSWN

6. Phenotypic characterization of knockout plants

The obtained tomato #1-3 homozygous mutant is subjected to selfing and seed reservation, planted on a farm of agriculture and forestry academy of sciences in Beijing, and simultaneously planted with a contrast wild-type material TB0993, the cultivation and management conditions are basically consistent, the conventional operation is adopted, and 3 plants are planted in each selfing line material. The length of the flower column of the flower which is free of lesions and is in the early flowering stage (calyx is opened, petals are yellow and white) is measured, 5 flowers are taken from each plant, and the average value is recorded and calculated. The traits of the tomato wild type and mutant inbred line stigma are shown in table 1, and the photos of the wild type material TB0993 and the mutant #1 are shown in fig. 3-6, and the recording periods are the early flowering period and the full flowering period of the tomato.

TABLE 1 wild type material TB0993 vs. mutant #1-3 tomato traits

	Length of flower column (mm)
		TB0993 (wild type)	7.08±0.25
Mutant #1	6.05±0.17
		Mutant #2	6.01±0.15
Mutant #3	6.11±0.18

Through field character investigation, as shown in fig. 7-9, no obvious difference is seen between the wild type material TB0993 and the mutant #1 in the whole plant and leaves, and the main economic characters of the 3 mutant materials are not changed significantly. However, the mutant materials all have the phenomenon that the stigma is obviously shortened (the style is shortened), and the average style length is about 1mm shorter than that of the TB0993 wild type tomato material stigma.

7. Analysis of potential off-target sites

The tomato whole genome sequence is subjected to Blast search according to the Guide1 and Guide2 target point sequences, and meanwhile, the off-target sites are evaluated by combining the website http:// www.genome.arizona.edu/criprpr/CRISSPRsearch. html, and potential theoretical off-target sites are not found.

8. Artificial castration and seed production application

Because the stigma of the mutant material is shortened, the top end of the bud can be directly pinched by hands during castration, and the petals and anthers can be quickly pulled out without damaging the stigma (the stigma of the wild type tomato is generally level with or slightly lower than an anther cylinder, and the stigma can be damaged by the operation, so that pollination and seed setting cannot be realized). Under the condition that the stigma is shortened, the petals and the stamens can be pinched by hands and taken down at one time, and compared with the prior art that the petals and the stamens need to be taken down by tweezers in sequence, the efficiency is much higher. As can be understood from FIG. 10, detasseling of wild type tomato flowers requires the removal of petals and stamens one by one with tweezers; as can be understood from FIG. 11, the mutants can rapidly remove petals and stamens by hand at one time. Therefore, the tomatoes with shortened flower columns prepared by the method are beneficial to improving the artificial emasculation efficiency and improving the tomato breeding efficiency.

It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.

Sequence listing

<110> agriculture and forestry academy of sciences of Beijing City

INSTITUTE OF GENETICS AND DEVELOPMENTAL BIOLOGY, CHINESE ACADEMY OF SCIENCES

<120> a method for breeding short style tomatoes

<130> C1CNCN190786

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1179

<212> DNA

<213> tomato (tomato)

<400> 1

atggagaaat tcaatgaaga agaagatcaa gctaaggtta caacaattaa tgtggatagc 60

tttggagcta aaggtgatgg aagtatagat gatacaaatg catttcaaaa agcatggaaa 120

gaagcttgtt catcttcaca tgttgtgaat tttgtggtgt cccagaacaa gaaatatctt 180

ctcaaaccaa tcaaatttta tgggccatgc aaatcttcca ttacaatgca gatttatgga 240

accctattag catctgatga tacttcagat tacaagaagg atagtaggca ctggcttatt 300

tttgatagtg ttcaaaaatt ggttgttgga ggagctggag ttatcaatgg caatggcaaa 360

atttggtggc aacattcttg caaaattaat aaaaaattgc catgcaaggt agcacccacg 420

gccctgacat tttacaagtg taacaacttg aaagtgaagg accttaaaat agaaaatgca 480

caacaaatac atttgctaat tgagaagtgt gttggtgttg aagttacaaa attggtagtg 540

acttctccag aaaatagccc taatactgat ggaatccata taactagcac tcaaaatatt 600

caaatttctg attccaccat tgccacaggt gatgattgca tctcaattgt ggatggatct 660

cagaaggtct tagccactgg cattacttgt ggaccaggtc atggaattag tattggaagt 720

ttgggaggtg gaaattcaga agctcatgtg tctgatattc atgtaaatgg agctaagctt 780

tatgaaacta caaatggact taggattaag acttggccgg gaggatttgg aagtgcaagc 840

aatattaagt atcaaaatgt ggttatgaat aatgtcaaaa atccaataat tatagaccaa 900

aattattgtg atcaagctga tggtccatgc aaagctgaga ctgactcggc agttgaagtg 960

aaaaatgtga tttatcaaaa tatcaaaggc acaagtgcaa caaatgatgc aataagtatc 1020

aagtgcagca aaaaaattcc atgtgaagga attttgatgg agaatgtgaa attgttagga 1080

ggaaatggtg aaactccaaa tggtatttgg ggaaatatca ataatcttac gtgcaaaaat 1140

gttttaccag aatgtcaaaa aaactcaaaa attgtataa 1179

<210> 2

<211> 1179

<212> RNA

<213> tomato (tomato)

<400> 2

auggagaaau ucaaugaaga agaagaucaa gcuaagguua caacaauuaa uguggauagc 60

uuuggagcua aaggugaugg aaguauagau gauacaaaug cauuucaaaa agcauggaaa 120

gaagcuuguu caucuucaca uguugugaau uuuguggugu cccagaacaa gaaauaucuu 180

cucaaaccaa ucaaauuuua ugggccaugc aaaucuucca uuacaaugca gauuuaugga 240

acccuauuag caucugauga uacuucagau uacaagaagg auaguaggca cuggcuuauu 300

uuugauagug uucaaaaauu gguuguugga ggagcuggag uuaucaaugg caauggcaaa 360

auuugguggc aacauucuug caaaauuaau aaaaaauugc caugcaaggu agcacccacg 420

gcccugacau uuuacaagug uaacaacuug aaagugaagg accuuaaaau agaaaaugca 480

caacaaauac auuugcuaau ugagaagugu guugguguug aaguuacaaa auugguagug 540

acuucuccag aaaauagccc uaauacugau ggaauccaua uaacuagcac ucaaaauauu 600

caaauuucug auuccaccau ugccacaggu gaugauugca ucucaauugu ggauggaucu 660

cagaaggucu uagccacugg cauuacuugu ggaccagguc auggaauuag uauuggaagu 720

uugggaggug gaaauucaga agcucaugug ucugauauuc auguaaaugg agcuaagcuu 780

uaugaaacua caaauggacu uaggauuaag acuuggccgg gaggauuugg aagugcaagc 840

aauauuaagu aucaaaaugu gguuaugaau aaugucaaaa auccaauaau uauagaccaa 900

aauuauugug aucaagcuga ugguccaugc aaagcugaga cugacucggc aguugaagug 960

aaaaauguga uuuaucaaaa uaucaaaggc acaagugcaa caaaugaugc aauaaguauc 1020

aagugcagca aaaaaauucc augugaagga auuuugaugg agaaugugaa auuguuagga 1080

ggaaauggug aaacuccaaa ugguauuugg ggaaauauca auaaucuuac gugcaaaaau 1140

guuuuaccag aaugucaaaa aaacucaaaa auuguauaa 1179

<210> 3

<211> 392

<212> PRT

<213> tomato (tomato)

<400> 3

Met Glu Lys Phe Asn Glu Glu Glu Asp Gln Ala Lys Val Thr Thr Ile

1 5 10 15

Asn Val Asp Ser Phe Gly Ala Lys Gly Asp Gly Ser Ile Asp Asp Thr

20 25 30

Asn Ala Phe Gln Lys Ala Trp Lys Glu Ala Cys Ser Ser Ser His Val

35 40 45

Val Asn Phe Val Val Ser Gln Asn Lys Lys Tyr Leu Leu Lys Pro Ile

50 55 60

Lys Phe Tyr Gly Pro Cys Lys Ser Ser Ile Thr Met Gln Ile Tyr Gly

65 70 75 80

Thr Leu Leu Ala Ser Asp Asp Thr Ser Asp Tyr Lys Lys Asp Ser Arg

85 90 95

His Trp Leu Ile Phe Asp Ser Val Gln Lys Leu Val Val Gly Gly Ala

100 105 110

Gly Val Ile Asn Gly Asn Gly Lys Ile Trp Trp Gln His Ser Cys Lys

115 120 125

Ile Asn Lys Lys Leu Pro Cys Lys Val Ala Pro Thr Ala Leu Thr Phe

130 135 140

Tyr Lys Cys Asn Asn Leu Lys Val Lys Asp Leu Lys Ile Glu Asn Ala

145 150 155 160

Gln Gln Ile His Leu Leu Ile Glu Lys Cys Val Gly Val Glu Val Thr

165 170 175

Lys Leu Val Val Thr Ser Pro Glu Asn Ser Pro Asn Thr Asp Gly Ile

180 185 190

His Ile Thr Ser Thr Gln Asn Ile Gln Ile Ser Asp Ser Thr Ile Ala

195 200 205

Thr Gly Asp Asp Cys Ile Ser Ile Val Asp Gly Ser Gln Lys Val Leu

210 215 220

Ala Thr Gly Ile Thr Cys Gly Pro Gly His Gly Ile Ser Ile Gly Ser

225 230 235 240

Leu Gly Gly Gly Asn Ser Glu Ala His Val Ser Asp Ile His Val Asn

245 250 255

Gly Ala Lys Leu Tyr Glu Thr Thr Asn Gly Leu Arg Ile Lys Thr Trp

260 265 270

Pro Gly Gly Phe Gly Ser Ala Ser Asn Ile Lys Tyr Gln Asn Val Val

275 280 285

Met Asn Asn Val Lys Asn Pro Ile Ile Ile Asp Gln Asn Tyr Cys Asp

290 295 300

Gln Ala Asp Gly Pro Cys Lys Ala Glu Thr Asp Ser Ala Val Glu Val

305 310 315 320

Lys Asn Val Ile Tyr Gln Asn Ile Lys Gly Thr Ser Ala Thr Asn Asp

325 330 335

Ala Ile Ser Ile Lys Cys Ser Lys Lys Ile Pro Cys Glu Gly Ile Leu

340 345 350

Met Glu Asn Val Lys Leu Leu Gly Gly Asn Gly Glu Thr Pro Asn Gly

355 360 365

Ile Trp Gly Asn Ile Asn Asn Leu Thr Cys Lys Asn Val Leu Pro Glu

370 375 380

Cys Gln Lys Asn Ser Lys Ile Val

385 390

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtgtaacaac ttgaaagtga 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gattgcatct caattgtgga 20

<210> 6

<211> 465

<212> DNA

<213> tomato (tomato)

<400> 6

atggagaaat tcaatgaaga agaagatcaa gctaaggtta caacaattaa tgtggatagc 60

tttggagcta aaggtgatgg aagtatagat gatacaaatg catttcaaaa agcatggaaa 120

gaagcttgtt catcttcaca tgttgtgaat tttgtggtgt cccagaacaa gaaatatctt 180

ctcaaaccaa tcaaatttta tgggccatgc aaatcttcca ttacaatgca gatttatgga 240

accctattag catctgatga tacttcagat tacaagaagg atagtaggca ctggcttatt 300

tttgatagtg ttcaaaaatt ggttgttgga ggagctggag ttatcaatgg caatggcaaa 360

atttggtggc aacattcttg caaaattaat aaaaaattgc catgcaaggt agcacccacg 420

gccctgacat tttacaagtg taacaacttg aaagaaggac cttaa 465

<210> 7

<211> 154

<212> PRT

<213> tomato (tomato)

<400> 7

Met Glu Lys Phe Asn Glu Glu Glu Asp Gln Ala Lys Val Thr Thr Ile

1 5 10 15

Asn Val Asp Ser Phe Gly Ala Lys Gly Asp Gly Ser Ile Asp Asp Thr

20 25 30

Asn Ala Phe Gln Lys Ala Trp Lys Glu Ala Cys Ser Ser Ser His Val

35 40 45

Val Asn Phe Val Val Ser Gln Asn Lys Lys Tyr Leu Leu Lys Pro Ile

50 55 60

Lys Phe Tyr Gly Pro Cys Lys Ser Ser Ile Thr Met Gln Ile Tyr Gly

65 70 75 80

Thr Leu Leu Ala Ser Asp Asp Thr Ser Asp Tyr Lys Lys Asp Ser Arg

85 90 95

His Trp Leu Ile Phe Asp Ser Val Gln Lys Leu Val Val Gly Gly Ala

100 105 110

Gly Val Ile Asn Gly Asn Gly Lys Ile Trp Trp Gln His Ser Cys Lys

115 120 125

Ile Asn Lys Lys Leu Pro Cys Lys Val Ala Pro Thr Ala Leu Thr Phe

130 135 140

Tyr Lys Cys Asn Asn Leu Lys Glu Gly Pro

145 150

<210> 8

<211> 468

<212> DNA

<213> tomato (tomato)

<400> 8

atggagaaat tcaatgaaga agaagatcaa gctaaggtta caacaattaa tgtggatagc 60

tttggagcta aaggtgatgg aagtatagat gatacaaatg catttcaaaa agcatggaaa 120

gaagcttgtt catcttcaca tgttgtgaat tttgtggtgt cccagaacaa gaaatatctt 180

ctcaaaccaa tcaaatttta tgggccatgc aaatcttcca ttacaatgca gatttatgga 240

accctattag catctgatga tacttcagat tacaagaagg atagtaggca ctggcttatt 300

tttgatagtg ttcaaaaatt ggttgttgga ggagctggag ttatcaatgg caatggcaaa 360

atttggtggc aacattcttg caaaattaat aaaaaattgc catgcaaggt agcacccacg 420

gccctgacat tttacaagtg taacaacttg aaaggtgaag gaccttaa 468

<210> 9

<211> 155

<212> PRT

<213> tomato (tomato)

<400> 9

Met Glu Lys Phe Asn Glu Glu Glu Asp Gln Ala Lys Val Thr Thr Ile

1 5 10 15

Asn Val Asp Ser Phe Gly Ala Lys Gly Asp Gly Ser Ile Asp Asp Thr

20 25 30

Asn Ala Phe Gln Lys Ala Trp Lys Glu Ala Cys Ser Ser Ser His Val

35 40 45

Val Asn Phe Val Val Ser Gln Asn Lys Lys Tyr Leu Leu Lys Pro Ile

50 55 60

Lys Phe Tyr Gly Pro Cys Lys Ser Ser Ile Thr Met Gln Ile Tyr Gly

65 70 75 80

Thr Leu Leu Ala Ser Asp Asp Thr Ser Asp Tyr Lys Lys Asp Ser Arg

85 90 95

His Trp Leu Ile Phe Asp Ser Val Gln Lys Leu Val Val Gly Gly Ala

100 105 110

Gly Val Ile Asn Gly Asn Gly Lys Ile Trp Trp Gln His Ser Cys Lys

115 120 125

Ile Asn Lys Lys Leu Pro Cys Lys Val Ala Pro Thr Ala Leu Thr Phe

130 135 140

Tyr Lys Cys Asn Asn Leu Lys Gly Glu Gly Pro

145 150 155

<210> 10

<211> 711

<212> DNA

<213> tomato (tomato)

<400> 10

atggagaaat tcaatgaaga agaagatcaa gctaaggtta caacaattaa tgtggatagc 60

tttggagcta aaggtgatgg aagtatagat gatacaaatg catttcaaaa agcatggaaa 120

gaagcttgtt catcttcaca tgttgtgaat tttgtggtgt cccagaacaa gaaatatctt 180

ctcaaaccaa tcaaatttta tgggccatgc aaatcttcca ttacaatgca gatttatgga 240

accctattag catctgatga tacttcagat tacaagaagg atagtaggca ctggcttatt 300

tttgatagtg ttcaaaaatt ggttgttgga ggagctggag ttatcaatgg caatggcaaa 360

atttggtggc aacattcttg caaaattaat aaaaaattgc catgcaaggt agcacccacg 420

gccctgacat tttacaagtg taacaacttg aaagtgaagg accttaaaat agaaaatgca 480

caacaaatac atttgctaat tgagaagtgt gttggtgttg aagttacaaa attggtagtg 540

acttctccag aaaatagccc taatactgat ggaatccata taactagcac tcaaaatatt 600

caaatttctg attccaccat tgccacaggt gatgattgca tctcaattgt tggatggatc 660

tcagaaggtc ttagccactg gcattacttg tggaccaggt catggaatta g 711

<210> 11

<211> 236

<212> PRT

<213> tomato (tomato)

<400> 11

Met Glu Lys Phe Asn Glu Glu Glu Asp Gln Ala Lys Val Thr Thr Ile

1 5 10 15

Asn Val Asp Ser Phe Gly Ala Lys Gly Asp Gly Ser Ile Asp Asp Thr

20 25 30

Asn Ala Phe Gln Lys Ala Trp Lys Glu Ala Cys Ser Ser Ser His Val

35 40 45

Val Asn Phe Val Val Ser Gln Asn Lys Lys Tyr Leu Leu Lys Pro Ile

50 55 60

Lys Phe Tyr Gly Pro Cys Lys Ser Ser Ile Thr Met Gln Ile Tyr Gly

65 70 75 80

Thr Leu Leu Ala Ser Asp Asp Thr Ser Asp Tyr Lys Lys Asp Ser Arg

85 90 95

His Trp Leu Ile Phe Asp Ser Val Gln Lys Leu Val Val Gly Gly Ala

100 105 110

Gly Val Ile Asn Gly Asn Gly Lys Ile Trp Trp Gln His Ser Cys Lys

115 120 125

Ile Asn Lys Lys Leu Pro Cys Lys Val Ala Pro Thr Ala Leu Thr Phe

130 135 140

Tyr Lys Cys Asn Asn Leu Lys Val Lys Asp Leu Lys Ile Glu Asn Ala

145 150 155 160

Gln Gln Ile His Leu Leu Ile Glu Lys Cys Val Gly Val Glu Val Thr

165 170 175

Lys Leu Val Val Thr Ser Pro Glu Asn Ser Pro Asn Thr Asp Gly Ile

180 185 190

His Ile Thr Ser Thr Gln Asn Ile Gln Ile Ser Asp Ser Thr Ile Ala

195 200 205

Thr Gly Asp Asp Cys Ile Ser Ile Val Gly Trp Ile Ser Glu Gly Leu

210 215 220

Ser His Trp His Tyr Leu Trp Thr Arg Ser Trp Asn

225 230 235

<210> 12

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atatatggtc tcgtttggtg taacaacttg aaagtgagtt ttagagctag aaatagc 57

<210> 13

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

attattggtc tcgaaacgat tgcatctcaa ttgtggacca aactacactg ttagattc 58

<210> 14

<211> 605

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atatatggtc tcgtttggtg taacaacttg aaagtgagtt ttagagctag aaatagcaag 60

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 120

tgcaaaattt tccagatcga tttcttcttc ctctgttctt cggcgttcaa tttctggggt 180

tttctcttcg ttttctgtaa ctgaaaccta aaatttgacc taaaaaaaat ctcaaataat 240

atgattcagt ggttttgtac ttttcagtta gttgagtttt gcagttccga tgagataaac 300

caataataaa tctttttaat ttatagtata tttatgtaag ttttcacgtt gagtaaatag 360

cgaagaagtt gggcccaacc aagtaaaata agaaggccgg gccattacaa ttaagtcgtc 420

acacaactgg gcttcattga aaaaagcgca aaaccgattc caggcccgtg ttagcatgaa 480

gactcaactc aaccagagat ttctccctca tcgcttacag aaaaaagcta tatgctgttt 540

atattgcgaa tctaacagtg tagtttggga ttgcatctca attgtggagt ttagagacca 600

ataat 605

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agcggataac aatttcacac agga 24

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccatgcaagg tagcacccac g 21

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cctgtggcaa tggtggaatc ag 22

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cacaccgaaa atcttgaaag tgtag 25

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gagcttctga atttccacct ccc 23

Claims

1. A method for preparing short style tomatoes,

the method is knockout tomatoPS-2A gene;

the method comprises mixing the tomatoPS-2The nucleic acid coding sequence of the gene is mutated into a nucleic acid sequence shown in any one of SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.10 or a combination thereof; or;

the method comprises mixing the tomatoPS-2The protein sequence coded by the gene is mutated into a protein sequence shown in any one of SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.11 or a combination thereof.

2. The method of creating of claim 1, wherein:

the tomato isPS-2The coding sequence of the gene is shown in SEQ ID NO. 1.

3. The method of creating of claim 1, wherein:

the tomato isPS-2The protein sequence of the gene code is shown in SEQ ID NO. 3.

4. The method of creating of claim 1, wherein:

the creation method is to knock out the tomatoPS-2The tomato mutant of the gene is prepared into a mutant homozygote.

5. The creation method of any one of claims 1-4, wherein:

the method is knockout tomatoPS-2Exon 5 of the gene.

6. The creation method of any one of claims 1-4, wherein:

the method is knockout tomatoPS-2Exon 6 of the gene.

7. The method of creating of any of claims 1-4, wherein:

knock-out of said tomatoPS-2The genes were implemented using the CRISPR/Cas9 system.

8. The authoring method of claim 7 wherein:

the target sequence in the vector for expressing sgRNA in CRISPR/Cas9 system is shown as SEQ ID number 4 and/or SEQ ID number 5.

9. Use of the creation method as described in any one of claims 1 to 8 for seed production with shortened tomato style.