阅读说明：本技术 一种利用番茄雄性不育基因及可见连锁标记创制雄性不育系的方法 (Method for creating male sterile line by using tomato male sterile gene and visible linkage marker ) 是由 张从省 王喜萍 公小君 张婷 于 2020-05-28 设计创作，主要内容包括：本发明公开了一种利用番茄雄性不育基因及可见连锁标记创制雄性不育系的方法。本发明克隆了新的番茄育性基因SlNP1,基于基因编辑技术可以快速精确的创制番茄稳定的SlNP1雄性不育系,且败育性彻底无其他不良性状的产生；通过双基因敲除连锁基因SlNP1和SlSGR1,使得雄性不育和滞绿两个表型连锁同步,通过观察乙烯利处理的叶片快速的筛选出雄性不育系,该方法方便、快捷、有效。本发明对于番茄雄性不育系的培育及番茄育种有重要意义。(The invention discloses a method for creating a male sterile line by using a tomato male sterile gene and a visible linkage marker. The invention clones a new tomato fertility gene SlNP1, can rapidly and accurately create a stable SlNP1 male sterile line of the tomato based on a gene editing technology, and has thorough abortion without generation of other undesirable traits; the linkage of male sterility and green retention is synchronized by knocking out the linked genes SlNP1 and SlSGR1 through double genes, and a male sterile line is rapidly screened out by observing ethephon-treated leaves. The invention has important significance for the cultivation of the tomato male sterile line and the tomato breeding.)

1. A preparation method of tomato male sterile plants is method A or method B;

the method A comprises the following steps: reducing or inhibiting the activity and/or content of SlNP1 protein in tomato to obtain male sterile plant;

the method B comprises the following steps: silencing or inhibiting the expression of a gene encoding SlNP1 protein in tomato or knocking out the gene encoding SlNP1 protein to obtain a male sterile plant:

the SlNP1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(A2) a protein derived from tomato and having 98% or more identity to (a1) and associated with male fertility in plants;

(A3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and is related to the male fertility of plants.

2. The method of claim 1, wherein:

the gene for coding the SlNP1 protein or the gene for coding the SlNP1 protein in the tomato is silenced or inhibited, and the gene for coding the SlNP1 protein in a mutation target plant can reduce the expression quantity of the gene for coding the SlNP1 protein in the tomato or cause the function of the gene for coding the SlNP1 protein in the tomato to be lost; the mutation is a homozygous mutation.

3. The preparation method of the tomato male sterile plant is the method C or the method D;

the method C comprises the following steps: simultaneously reducing or inhibiting the activity and/or content of SlNP1 protein and SlSGR1 protein in the tomato to obtain a male sterile plant;

the method D comprises the following steps: simultaneously silencing or inhibiting the expression of a gene coding a SlNP1 protein and a gene coding a SlSGR1 protein in tomato, or simultaneously knocking out the gene coding the SlNP1 protein and the gene coding the SlSGR1 protein to obtain a male sterile plant:

the SlNP1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(A2) a protein derived from tomato and having 98% or more identity to (a1) and associated with male fertility in plants;

(A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and is related to the male fertility of plants;

the SlSGR1 protein is (C1) or (C2) or (C3) as follows:

(C1) a protein consisting of the amino acid sequence shown in SEQ ID No. 7;

(C2) a protein derived from tomato and having 98% or more identity to (A1) and having the same function as (A1);

(C3) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.7 and has the same function.

4. The method of claim 3, wherein: the method is characterized in that the expression of a gene coding SlNP1 protein and a gene coding SlSGR1 protein in the tomato is silenced or inhibited at the same time, or the expression of a gene coding SlNP1 protein and the expression of a gene coding SlSGR1 protein in the tomato are knocked out at the same time, so that the expression level of the gene coding SlNP1 protein and the expression level of the gene coding SlSGR1 protein in the tomato are reduced or the functions of the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato are deleted at the same time by mutating the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato; the mutation is a homozygous mutation.

5. A method of preparing a tomato maintainer line comprising the steps of: mutating the gene encoding the SlNP1 protein in the target plant to reduce the expression level of the gene encoding the SlNP1 protein in the tomato or to cause the function deletion of the gene encoding the SlNP1 protein in the tomato to obtain a tomato maintainer line; the mutation is a heterozygous mutation;

the SlNP1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(A2) a protein derived from tomato and having 98% or more identity to (a1) and associated with male fertility in plants;

(A3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and is related to the male fertility of plants.

6. A method of preparing a tomato maintainer line comprising the steps of: simultaneously mutating a gene coding SlNP1 protein and a gene coding SlSGR1 protein in the tomato to reduce the expression quantity of the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato or to cause the function of the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato to be lost, thus obtaining a tomato maintainer line; the mutation is a heterozygous mutation;

the SlNP1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(A2) a protein derived from tomato and having 98% or more identity to (a1) and associated with male fertility in plants;

(A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and is related to the male fertility of plants;

the SlSGR1 protein is (C1) or (C2) or (C3) as follows:

(C1) a protein consisting of the amino acid sequence shown in SEQ ID No. 7;

(C2) a protein derived from tomato and having 98% or more identity to (A1) and having the same function as (A1);

(C3) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.7 and has the same function.

7. The tomato male sterile line propagation method is method E or method F;

the method E comprises the following steps: preparing tomato male sterile plants as a tomato male sterile line according to the method of claim 1 or 2; preparing a maintainer line according to the method of claim 5; pollinating the tomato male sterile line by adopting a maintainer line to realize the propagation of the tomato male sterile line;

the method F comprises the following steps: preparing tomato male sterile plants as a tomato male sterile line according to the method of claim 3 or 4; preparing a maintainer line according to the method of claim 6; and pollinating the tomato male sterile line by adopting the maintainer line to realize the propagation of the tomato male sterile line.

8. The method of claim 7, wherein: in the method F, after the maintainer line is adopted to pollinate the tomato male sterile line, the ethephon treatment is carried out on the leaf of the offspring, and the sterile line and the maintainer line are selected and distinguished by observing the color of the leaf with naked eyes.

The application of the SlNP1 protein or related biological materials thereof in regulation of tomato fertility or tomato breeding;

the relevant biological material is any one of the following (1) to (3):

(1) a gene encoding a SlNP1 protein;

(2) an agent for silencing or inhibiting the expression of (1) or knocking out (1) in a plant of interest;

(3) a substance for reducing or inhibiting the activity and/or content of a SlNP1 protein in a plant of interest;

the SlNP1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(A2) a protein derived from tomato and having 98% or more identity to (a1) and associated with male fertility in plants;

(A3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and is related to the male fertility of plants.

The application of combined use of the SlNP1 protein and the SlSGR1 protein or the application of related biological materials of the SlNP1 protein and the SlSGR1 protein in breeding a tomato sterile line or tomato breeding;

the relevant biological material is any one of the following (1) to (3):

(1) genes encoding the SlNP1 protein and the SlSGR1 protein;

(2) an agent for silencing or inhibiting the expression of (1) or knocking out (1) in a plant of interest;

(3) a substance for reducing or inhibiting the activity and/or content of a SlNP1 protein and a SlSGR1 protein in a plant of interest;

the SlNP1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(A2) a protein derived from tomato and having 98% or more identity to (a1) and associated with male fertility in plants;

(A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and is related to the male fertility of plants;

the SlSGR1 protein is (C1) or (C2) or (C3) as follows:

(C1) a protein consisting of the amino acid sequence shown in SEQ ID No. 7;

(C2) a protein derived from tomato and having 98% or more identity to (A1) and having the same function as (A1);

(C3) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.7 and has the same function.

Technical Field

The invention relates to the field of plant breeding, in particular to a method for creating a male sterile line by using a tomato male sterile gene and a visible linkage marker.

Background

Tomatoes are one of the most widely consumed vegetables worldwide, with a yield of 1.82 million tons in 2017 and a value of over 600 million dollars. The Chinese is the country with the largest tomato cultivation area and the largest total production amount in the world, and the annual output is more than 5000 ten thousand tons. Tomato is a strict closed-flower pollination crop, the heterosis is obvious, the heterosis refers to the phenomenon that two varieties with different genetic bases or similar varieties are hybridized in the plant, and the first filial generation of the hybrid is superior to the parents in the aspects of growth vigor, vitality, adaptability, yield and the like. The key to heterosis utilization is the development and utilization of a controllable pollination system to prevent self-pollination failure due to self-pollination. In order to ensure the purity of the hybrid, the female parent must be rendered male-incompetent. The method mainly adopted at present comprises manual or mechanical emasculation, chemical emasculation and adoption of a male sterile line as a female parent. The artificial emasculation means that the male flowers of female parents are artificially removed before the pollination period, a large amount of manpower and material resources are consumed, and the artificial emasculation is more difficult for the crops with strict self-pollination, such as tomatoes and the like. Mechanical detasseling and chemical detasseling have a large impact on the normal growth of plants. Therefore, the creation of the male sterile line can reduce the cost and improve the purity of the hybrid, and has important significance for the hybrid breeding.

The male sterility includes cytoplasmic sterility and nuclear sterility, and the instability of cytoplasmic sterility is easily affected by environment, so that the creation of stable nuclear sterile line is the development direction of male sterile line. With the development of molecular biology, more and more nuclear fertility genes are cloned, including Lat52 (tween et al, 1989), Ps-2(Gorguet et al, 2009), Style 2.1(Chen et al, 2007), Ms10-35(Jeong et al, 2014), MPK20(Chen et al, 2018), Ms15(Cao et al, 2019), PIF3(CN 109456979 a), LAP3(CN 109207505 a), etc., and the reasons why most of these genes are not used include: 1) the sterility is not thorough, which can lead to impure hybrid seeds; 2) there is no efficient maintainer line or simple method of distinguishing between sterile and maintainer lines.

The visible marker is a phenotype which can be effectively distinguished from a control strain by naked eyes or simple treatment, and comprises the characteristics of leaf shape, leaf color, stem color, epidermal villi, seed size, seed villi and the like. The linkage visible marker refers to that the marker is tightly linked with a target gene, and offspring can indirectly judge the genotype of the target gene through the visible marker.

Third generation gene editing (CRISPR/Cas9) is a new technology aiming at site-directed modification of genome, and is a technology which can generate modification such as base deletion, insertion, substitution and the like in a genome DNA sequence of a cell living body by artificially constructed engineered nuclease, specifically recognizing and cutting a target interval sequence of the genome, generating double-strand break, and connecting a DNA damage repair mechanism through homologous recombination or non-homologous end of a cell endogenous gene (Hsuet et al, 2014). The gene editing technology can theoretically operate any gene of any variety, realize the rapid and accurate improvement of target characters of core parents without the problems of linkage drag and the like common in traditional backcross breeding, and can realize simultaneous mutation of multiple genes, thereby greatly improving the efficiency of multi-character polymerization.

Disclosure of Invention

The invention aims to provide a method for creating a male sterile line by using a tomato male sterile gene and a visible linkage marker.

In a first aspect, the method for preparing a plant protected from male sterility of tomato of the present invention is method A or method B.

The method A comprises the following steps: reducing or inhibiting the activity and/or content of SlNP1 protein in tomato to obtain male sterile plant;

the method B comprises the following steps: silencing or inhibiting the expression of a gene encoding SlNP1 protein in tomato or knocking out the gene encoding SlNP1 protein to obtain a male sterile plant:

the SlNP1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(A2) a protein derived from tomato and having 98% or more identity to (a1) and associated with male fertility in plants;

(A3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and is related to the male fertility of plants.

The 'reduction or inhibition of the activity and/or content of the SlNP1 protein' in tomato is to cause the frame shift mutation of amino acid residues of the SlNP1 protein or the premature termination of amino acid translation.

The method is characterized in that the expression of a gene coding the SlNP1 protein in the tomato is silenced or inhibited or a gene coding the SlNP1 protein is knocked out, so that the expression quantity of the gene coding the SlNP1 protein in the tomato is reduced or the function of the gene coding the SlNP1 protein in the tomato is deleted for mutating the gene coding the SlNP1 protein in a target plant; the mutation is a homozygous mutation.

The mutation can be achieved using gene editing techniques. The gene editing technology can be CRISPR/Cas9 gene editing technology. The target sequence edited by the CRISPR/Cas9 gene can be specifically located in the exon of the gene, and more specifically can be the 966-988 th site from the 5' end of SEQ ID NO. 1.

The mutation may specifically be insertion of A between bases 982 and 983 from the 5' end of SEQ ID NO. 1.

In a second aspect, the method for preparing a plant protected from male sterility of tomato of the present invention is method C or method D.

The method C comprises the following steps: simultaneously reducing or inhibiting the activity and/or content of SlNP1 protein and SlSGR1 protein in the tomato to obtain a male sterile plant;

the method D comprises the following steps: simultaneously silencing or inhibiting the expression of a gene coding a SlNP1 protein and a gene coding a SlSGR1 protein in tomato, or simultaneously knocking out the gene coding the SlNP1 protein and the gene coding the SlSGR1 protein to obtain a male sterile plant:

the SlNP1 protein is the SlNP1 protein.

The SlSGR1 protein is (C1) or (C2) or (C3) as follows:

(C1) a protein consisting of the amino acid sequence shown in SEQ ID No. 7;

(C2) a protein derived from tomato and having 98% or more identity to (A1) and having the same function as (A1);

(C3) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.7 and has the same function.

The 'reducing or inhibiting the activity and/or content of the SlNP1 protein and the SlSGR1 protein in tomato simultaneously' causes the frame shift mutation of amino acid residues of the SlNP1 protein or the premature termination of amino acid translation and causes the frame shift mutation of the amino acid residues of the SlSGR1 protein or the premature termination of amino acid translation.

The method is characterized in that the expression of a gene coding SlNP1 protein and a gene coding SlSGR1 protein in the tomato is silenced or inhibited at the same time, or the expression of a gene coding SlNP1 protein and the expression of a gene coding SlSGR1 protein in the tomato are knocked out at the same time, so that the expression level of the gene coding SlNP1 protein and the expression level of the gene coding SlSGR1 protein in the tomato are reduced or the functions of the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato are deleted at the same time by mutating the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato; the mutation is a homozygous mutation.

The mutation can be achieved using gene editing techniques. The gene editing technology can be CRISPR/Cas9 gene editing technology. The target sequence edited by the CRISPR/Cas9 gene may specifically be located at an exon of the gene. The target sequence of the gene coding the SlNP1 protein can be the 966-988 th site from the 5' end of SEQ ID NO. 1. The target sequence of the gene encoding the SlSGR1 protein can be SEQ ID No. 5.

The mutation may specifically be insertion of A between bases 982 and 983 from the 5 'end of SEQ ID NO.1 and deletion of base 504 from the 5' end of SEQ ID NO. 6.

In a third aspect, the present invention provides a process for the preparation of a protected tomato maintainer line comprising the steps of: mutating the gene encoding the SlNP1 protein in the target plant to reduce the expression level of the gene encoding the SlNP1 protein in the tomato or to cause the function deletion of the gene encoding the SlNP1 protein in the tomato to obtain a tomato maintainer line; the mutation is a heterozygous mutation;

the SlNP1 protein is the SlNP1 protein.

The maintainer line is a maintainer line of sterile plants prepared by the method of the first aspect.

The mutation can be achieved using gene editing techniques. The gene editing technology can be CRISPR/Cas9 gene editing technology. The target sequence edited by the CRISPR/Cas9 gene can be specifically located in the exon of the gene, and more specifically can be the 966-988 th site from the 5' end of SEQ ID NO. 1.

The mutation may specifically be insertion of A between bases 982 and 983 from the 5' end of SEQ ID NO. 1.

In a fourth aspect, the present invention provides a process for the preparation of a protected tomato maintainer line comprising the steps of: simultaneously mutating a gene coding SlNP1 protein and a gene coding SlSGR1 protein in the tomato to reduce the expression quantity of the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato or to cause the function of the gene coding SlNP1 protein and the gene coding SlSGR1 protein in the tomato to be lost, thus obtaining a tomato maintainer line; the mutation is a heterozygous mutation;

the SlNP1 protein is the SlNP1 protein.

The SlSGR1 protein is the SlSGR1 protein.

The maintainer line is a maintainer line of sterile plants prepared by the method of the second aspect.

The mutation can be achieved using gene editing techniques. The gene editing technology can be CRISPR/Cas9 gene editing technology. The target sequence edited by the CRISPR/Cas9 gene may specifically be located at an exon of the gene. The target sequence of the gene coding the SlNP1 protein can be the 966-988 th site from the 5' end of SEQ ID NO. 1. The target sequence of the gene encoding the SlSGR1 protein can be SEQ ID No. 5.

The mutation may specifically be insertion of A between bases 982 and 983 from the 5 'end of SEQ ID NO.1 and deletion of base 504 from the 5' end of SEQ ID NO. 6.

In a fifth aspect, the method for expanding propagation of tomato male sterile line is method E or method F.

The method E comprises the following steps: preparing a tomato male sterile plant as a tomato male sterile line according to the method described in the first aspect; preparing a holding system according to the method described in the third aspect; and pollinating the tomato male sterile line by adopting the maintainer line to realize the propagation of the tomato male sterile line.

The method F comprises the following steps: preparing a tomato male sterile plant as a tomato male sterile line according to the method described in the second aspect; preparing a maintainer line according to the method described in the fourth aspect; and pollinating the tomato male sterile line by adopting the maintainer line to realize the propagation of the tomato male sterile line.

In the method F, after the maintainer line is adopted to pollinate the tomato male sterile line, the ethephon treatment is carried out on the leaf of the offspring, and the sterile line and the maintainer line are selected and distinguished by observing the color of the leaf with naked eyes.

In a sixth aspect, the invention protects the application of the SlNP1 protein or related biological materials thereof in regulation of tomato fertility or tomato breeding;

the relevant biological material is any one of the following (1) to (3):

(1) a gene encoding a SlNP1 protein;

(2) an agent for silencing or inhibiting the expression of (1) or knocking out (1) in a plant of interest;

(3) a substance for reducing or inhibiting the activity and/or content of a SlNP1 protein in a plant of interest;

the SlNP1 protein is the SlNP1 protein.

The (2) or (3) can be specifically a gene editing vector, and can be specifically a CRISPR/Cas9 gene editing vector. The target sequence of the gene editing is 966-988 th site from the 5' end of SEQ ID NO. 1.

In a seventh aspect, the invention protects the application of combined use of the SlNP1 protein and the SlSGR1 protein or related biological materials of the two in culturing a tomato sterile line or tomato breeding;

the relevant biological material is any one of the following (1) to (3):

(1) genes encoding the SlNP1 protein and the SlSGR1 protein;

(2) an agent for silencing or inhibiting the expression of (1) or knocking out (1) in a plant of interest;

(3) a substance for reducing or inhibiting the activity and/or content of a SlNP1 protein and a SlSGR1 protein in a plant of interest;

the SlNP1 protein is the SlNP1 protein.

The SlSGR1 protein is the SlSGR1 protein.

The (2) or (3) may specifically be a gene editing vector, and specifically may be a Cas9 gene editing vector. The target sequence of the gene editing is 966-988 th site from the 5' end of SEQ ID NO.1 and SEQ ID NO. 5.

Any of the above homozygous mutations is a mutation in which both homologous chromosomes are identical.

Any of the above described heterozygous mutations is such that only one homologous chromosome is mutated.

Any one of the genes encoding the SlNP1 protein is a DNA molecule described in any one of the following items:

(B1) DNA molecule shown in SEQ ID No. 1;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence defined in (B1) or (B2) and encodes the protein.

Any one of the genes encoding the SlSGR1 protein is a DNA molecule described in any one of the following items:

(B1) DNA molecule shown in SEQ ID No. 6;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence defined in (B1) or (B2) and encodes the protein.

The stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridizing with 1mM EDTA, rinsing in 2 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.5 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridization with a mixed solution of 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 65 ℃ or 6 × SSC, 0.5% SDS at 65 ℃ followed by washing once each with 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS.

The tomato material may be Ailsa Craig.

The invention has the following obvious effects: 1) a new tomato fertility gene SlNP1 is cloned, a stable SlNP1 male sterile line of the tomato can be rapidly and accurately created based on a gene editing technology, and abortion is complete without generation of other bad characters; 2) the linkage of male sterility and green retention is synchronized by knocking out the linked genes SlNP1 and SlSGR1 through double genes, and a male sterile line is rapidly screened out by observing ethephon-treated leaves.

The invention has important significance for the cultivation of the tomato male sterile line and the tomato breeding.

Drawings

Fig. 1 shows the tissue expression pattern of the SlNP1 gene.

FIG. 2 is a schematic diagram of a fertility gene verification vector and a double gene knockout vector.

FIG. 3 is the genotype identification of the slnp1 mutant.

FIG. 4 is a phenotypic characterization of the slnp1 mutant.

FIG. 5 shows the genotype identification of the double-gene mutant.

FIG. 6 is a visual marker phenotypic identification.

FIG. 7 is a schematic diagram of the propagation strategy of the sterile line with the visible marker.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Tomato material Ailsa Craig: institute of vegetable and flower, academy of agricultural sciences, china.

CPB supports are described in the literature: zhao et al (2016.) An alternative sequence for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. The public is available from Weifang-flourishing biological species industry Co., Ltd.