阅读说明：本技术 一种利用番茄绿茎紧密连锁标记创制雄性不育系的方法 (Method for creating male sterile line by using tomato green stem close linkage marker ) 是由 张从省 王喜萍 公小君 张婷 于 2020-08-07 设计创作，主要内容包括：本发明公开了一种利用番茄绿茎紧密连锁标记创制雄性不育系的方法。本发明利用育性基因MS15和绿茎基因F3H这对全新的组合开发了一种番茄雄性不育连锁标记,可用于不育系保持扩繁过程中不育系和保持系的筛选。本发明所使用的育性基因MS15和绿茎基因F3H的遗传距离只有1.35cM,理论上会产生更低的遗传及表型分离率,将大大降低现有番茄可见连锁标记不育系筛选出错率。(The invention discloses a method for creating a male sterile line by utilizing a tomato green stem close linkage marker. The invention develops a tomato male sterility linkage marker by using a brand new combination of a fertility gene MS15 and a green stem gene F3H, and can be used for screening a sterile line and a maintainer line in the sterile line maintaining and expanding propagation process. The genetic distance between the fertility gene MS15 and the green stem gene F3H used in the invention is only 1.35cM, which theoretically can generate lower genetic and phenotypic separation rate, and can greatly reduce the error rate of screening the existing tomato visible linkage marker sterile line.)

Use of the MS15 protein and the F3H protein in any one of the following (1) to (5):

(1) cultivating a tomato male sterile line;

(2) cultivating a tomato maintainer line;

(3) propagating tomato male sterile line;

(4) distinguishing a sterile line and a maintainer line in the process of propagating the tomato male sterile line;

(5) tomato breeding;

the MS15 protein is a1) or a2) or A3) as follows:

A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 6;

A2) a protein derived from tomato and having 98% or more identity and the same function as A1);

A3) 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.6 and has the same function;

the F3H protein is B1) or B2) or B3) as follows:

B1) a protein consisting of the amino acid sequence shown in SEQ ID No. 7;

B2) protein derived from tomato and having more than 98% identity with B1) and having the same function;

B3) 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.

2. Use of a biomaterial related to MS15 protein and a biomaterial related to F3H protein in any one of the following (1) to (5):

(1) cultivating a tomato male sterile line;

(2) cultivating a tomato maintainer line;

(3) propagating tomato male sterile line;

(4) distinguishing a sterile line and a maintainer line in the process of propagating the tomato male sterile line;

(5) tomato breeding;

the biological material related to the MS15 protein is any one of the following C1) to C8):

C1) a nucleic acid molecule encoding the MS15 protein of claim 1;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1);

C4) a recombinant vector comprising the expression cassette of C2);

C5) a recombinant microorganism comprising the nucleic acid molecule of C1);

C6) a recombinant microorganism comprising the expression cassette of C2);

C7) a recombinant microorganism comprising the recombinant vector of C3);

C8) a recombinant microorganism comprising the recombinant vector of C4).

The biological material related to the F3H protein is any one of the following D1) to D8):

D1) a nucleic acid molecule encoding the F3H protein of claim 1;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D3) a recombinant vector comprising the nucleic acid molecule of D1);

D4) a recombinant vector comprising the expression cassette of D2);

D5) a recombinant microorganism comprising the nucleic acid molecule of D1);

D6) a recombinant microorganism comprising the expression cassette of D2);

D7) a recombinant microorganism comprising the recombinant vector of D3);

D8) a recombinant microorganism comprising the recombinant vector of D4).

3. A method for cultivating a tomato male sterile line, which is X1) or X2);

x1) to make the function of MS15 protein and F3H protein in tomato lose or inhibit the activity of MS15 protein and F3H protein in tomato, to obtain tomato male sterile line;

x2) knocking out or silencing the gene coding the MS15 protein and the gene coding the F3H protein in tomato or inhibiting the expression of the gene coding the MS15 protein and the gene coding the F3H protein in tomato to obtain a tomato male sterile line.

4. A method for breeding tomato maintainer lines, which is Y1) or Y2);

y1) to make the function of MS15 protein and F3H protein in tomato lose or inhibit the activity of MS15 protein and F3H protein in tomato, to obtain tomato maintenance line;

y2) knocking out or silencing the gene coding the MS15 protein and the gene coding the F3H protein in tomato or inhibiting the expression of the gene coding the MS15 protein and the gene coding the F3H protein in tomato to obtain the tomato maintainer line.

5. The method according to claim 3 or 4, characterized in that: the X2), the gene coding for MS15 protein and the gene coding for F3H protein in the knockout tomato are used for mutating the gene coding for MS15 protein and the gene coding for F3H protein in tomato; the mutation is a homozygous mutation;

or, Y2), the gene encoding MS15 protein and the gene encoding F3H protein in the knockout tomato are such that the gene encoding MS15 protein and the gene encoding F3H protein in tomato are mutated; the mutation is a heterozygous mutation.

6. The method of claim 5, wherein: the substance which mutates the gene coding for MS15 protein and the gene coding for F3H protein in tomato is CRISPR/Cas9 system;

or, the CRISPR/Cas9 system comprises a Cas9 nuclease and a sgRNA; the sgrnas include a sgRNA targeting a gene encoding MS15 protein and a sgRNA targeting a gene encoding F3H protein;

or, the target sequence of the sgRNA of the gene targeting the coding MS15 protein is 432-454 th site from the 5' end of SEQ ID NO. 1; the target sequence of the sgRNA targeting the gene coding the F3H protein is shown as position 331-353 from the 5' end of SEQ ID NO. 2.

7. The method for propagating the tomato male sterile line comprises the following steps: and hybridizing the tomato male sterile line obtained by the method of any one of claims 3-6 as a female parent and the tomato maintainer line obtained by the method of any one of claims 4-6 as a male parent to realize the propagation of the tomato male sterile line.

8. The method of claim 7, wherein: the method further comprises the step of distinguishing the tomato male sterile line from the tomato maintainer line in the progeny of the cross by visually observing the color of the stems.

9. Any one of the following M1) -M4):

m1) substances which either disable the function of the MS15 protein and F3H protein in tomato or inhibit the activity of the MS15 protein and F3H protein in tomato;

m2) knocking out or silencing the gene encoding MS15 protein and the gene encoding F3H protein in tomato or suppressing the expression of the gene encoding MS15 protein and the gene encoding F3H protein in tomato;

m3) the CRISPR/Cas9 system as described in claim 6;

m4) the sgRNA described in claim 6.

10. Use of the product of claim 9 in any one of the following (1) to (5):

(1) cultivating a tomato male sterile line;

(2) cultivating a tomato maintainer line;

(3) propagating tomato male sterile line;

(4) distinguishing a sterile line and a maintainer line in the process of propagating the tomato male sterile line;

(5) and (5) tomato breeding.

Technical Field

The invention relates to the field of plant breeding, in particular to a method for creating a male sterile line by utilizing a tomato green stem close 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. China 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 million 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 species 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 main methods adopted at present include manual or mechanical emasculation, chemical emasculation and the 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 have been 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 109456979a), LAP3(CN 109207505 a), and the like, and the reasons why most of these genes have not been 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 the 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, and substitution 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.

The existing tomato visible linkage marker sterile line utilizes fertility gene MS10 and green stem gene F3H/GST (Zhang et al, 2016; CN 105525016A), the genetic distances of the MS10 gene and the F3H/GST gene are respectively 5.8cM and 2.2cM, the genetic distances are in positive correlation with a recombination exchange value, the MS10 gene and the F3H/GST gene are separated during meiosis, phenotype is not linked, and about 1% -6% of fertile plants exist in a green stem, so that the operation and purity influence is brought to hybrid production.

Disclosure of Invention

The first purpose of the invention is to provide new application of MS15 protein and F3H protein.

The present invention provides the use of the MS15 protein and the F3H protein in any one of the following (1) to (5):

(1) cultivating a tomato male sterile line;

(2) cultivating a tomato maintainer line;

(3) propagating tomato male sterile line;

(4) distinguishing a sterile line and a maintainer line in the process of propagating the tomato male sterile line;

(5) tomato breeding;

the MS15 protein is a1) or a2) or A3) as follows:

A1) a protein consisting of an amino acid sequence shown in SEQ ID No. 6;

A2) a protein derived from tomato and having 98% or more identity and the same function as A1);

A3) 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.6 and has the same function;

the F3H protein is B1) or B2) or B3) as follows:

B1) a protein consisting of the amino acid sequence shown in SEQ ID No. 7;

B2) protein derived from tomato and having more than 98% identity with B1) and having the same function;

B3) 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 second purpose of the invention is to provide a new application of the biological material related to the MS15 protein and the biological material related to the F3H protein.

The invention provides application of biological materials related to MS15 protein and biological materials related to F3H protein in any one of the following (1) to (5):

(1) cultivating a tomato male sterile line;

(2) cultivating a tomato maintainer line;

(3) propagating tomato male sterile line;

(4) distinguishing a sterile line and a maintainer line in the process of propagating the tomato male sterile line;

(5) tomato breeding;

the biological material related to the MS15 protein is any one of the following C1) to C8):

C1) a nucleic acid molecule encoding the MS15 protein;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1);

C4) a recombinant vector comprising the expression cassette of C2);

C5) a recombinant microorganism comprising the nucleic acid molecule of C1);

C6) a recombinant microorganism comprising the expression cassette of C2);

C7) a recombinant microorganism comprising the recombinant vector of C3);

C8) a recombinant microorganism comprising the recombinant vector of C4).

The biological material related to the F3H protein is any one of the following D1) to D8):

D1) a nucleic acid molecule encoding the F3H protein;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D3) a recombinant vector comprising the nucleic acid molecule of D1);

D4) a recombinant vector comprising the expression cassette of D2);

D5) a recombinant microorganism comprising the nucleic acid molecule of D1);

D6) a recombinant microorganism comprising the expression cassette of D2);

D7) a recombinant microorganism comprising the recombinant vector of D3);

D8) a recombinant microorganism comprising the recombinant vector of D4).

The nucleic acid molecule encoding the MS15 protein is the DNA molecule described in any one of the following c1) -c 3):

c1) DNA molecule shown in SEQ ID No. 1;

c2) a DNA molecule which hybridizes under stringent conditions with the DNA molecule defined in c1) and encodes a plant fertility-associated protein;

c3) 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 by c1) or c2) and encodes a plant fertility-related protein.

The nucleic acid molecule encoding the F3H protein is a DNA molecule described in any one of the following d1) -d 3):

d1) DNA molecule shown in SEQ ID No. 2;

d2) a DNA molecule which is hybridized with the DNA molecule defined by d1) under strict conditions and codes a protein related to the synthesis of plant anthocyanin;

d3) a DNA molecule which has more than 99 percent, more than 95 percent, more than 90 percent, more than 85 percent or more than 80 percent of identity with the DNA sequence limited by d1) or d2) and codes the protein related to the synthesis of the plant anthocyanin.

The stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The third purpose of the invention is to provide a method for cultivating the male sterile line of the tomato.

The method for cultivating the tomato male sterile line provided by the invention is X1) or X2);

x1) to make the function of MS15 protein and F3H protein in tomato lose or inhibit the activity of MS15 protein and F3H protein in tomato, to obtain tomato male sterile line;

x2) knocking out or silencing the gene coding the MS15 protein and the gene coding the F3H protein in tomato or inhibiting the expression of the gene coding the MS15 protein and the gene coding the F3H protein in tomato to obtain a tomato male sterile line.

In the above method for breeding tomato male sterile line, in the step X2), the gene coding for MS15 protein and the gene coding for F3H protein in the knockout tomato are mutated in the gene coding for MS15 protein and the gene coding for F3H protein in tomato; the mutation is a homozygous mutation (mutations occurring in both chromosomes are identical).

It is a fourth object of the present invention to provide a method for breeding tomato maintainers.

The method for cultivating the tomato maintainer line provided by the invention is Y1) or Y2);

y1) to make the function of MS15 protein and F3H protein in tomato lose or inhibit the activity of MS15 protein and F3H protein in tomato, to obtain tomato maintenance line;

y2) knocking out or silencing the gene coding the MS15 protein and the gene coding the F3H protein in tomato or inhibiting the expression of the gene coding the MS15 protein and the gene coding the F3H protein in tomato to obtain the tomato maintainer line.

In the above method for breeding tomato maintainer line, in Y2), the gene encoding MS15 protein and the gene encoding F3H protein in the knockout tomato are mutated in the gene encoding MS15 protein and the gene encoding F3H protein in tomato; the mutation is a heterozygous mutation (only one of the two chromosomes is mutated, and the other chromosome is not mutated).

Further, the substance for mutating the gene encoding the MS15 protein and the gene encoding the F3H protein in the tomato is a CRISPR/Cas9 system; the CRISPR/Cas9 system includes Cas9 nuclease and sgrnas; the sgrnas include a sgRNA targeting a gene encoding MS15 protein and a sgRNA targeting a gene encoding F3H protein.

Further, the target sequence of the sgRNA targeting the gene coding the MS15 protein is 432-454 th site from the 5' end of SEQ ID NO. 1; the target sequence of the sgRNA targeting the gene coding the F3H protein is shown as position 331-353 from the 5' end of SEQ ID NO. 2.

In a specific embodiment of the invention, the CRISPR/Cas9 system is specifically a double-gene knockout vector YBK-MS 15-F3H-Target. The double-gene knockout vector YBK-MS15-F3H-Target comprises sgRNA (the Target sequence is 432 th and 454 th positions from 5 'end of SEQ ID NO. 1) driven by AtU6 promoter and targeting a gene coding MS15 protein, sgRNA (the Target sequence is 331 st and 353 th positions from 5' end of SEQ ID NO. 2) driven by AtU6 promoter and targeting a gene coding F3H protein, Cas9 nuclease coding gene driven by pYao promoter and kanamycin screening gene driven by 35S promoter.

The method for breeding the tomato male sterile line or breeding the tomato maintainer line further comprises the step of screening tomato mutants with mutation of MS15 gene and F3H gene.

The tomato mutant plant in which the MS15 gene and the F3H gene are homozygously mutated is a tomato male sterile line;

the tomato mutant plant with heterozygous mutation of the MS15 gene and the F3H gene is a tomato maintainer line (tomato restorer line).

In a specific embodiment of the invention, the tomato male sterile line is mutant T1-5. The mutant T1-5 differed from the genomic DNA of the wild type tomato Ailsa Craig only in that in the gene encoding the MS15 protein, a fragment deletion had occurred in both chromosomes, the deletion fragment being located at position 444-448 of SEQ ID NO.1 and in the gene encoding the F3H protein, a fragment deletion had occurred in both chromosomes, the deletion fragment being located at position 341-347 of SEQ ID NO. 2.

The tomato maintainer line is mutant T1-1. The mutant T1-1 differed from the genomic DNA of the wild type tomato Ailsa Craig only in that in the gene coding for the MS15 protein a fragment deletion had been made in one chromosome which was located at position 444 and 448 of SEQ ID NO.1 and in the other chromosome which was not mutated, and in the gene coding for the F3H protein a fragment deletion had been made in one chromosome which was located at position 341 and 347 of SEQ ID NO.2 and in the other chromosome which was not mutated.

It is a fifth object of the present invention to provide a method for propagating tomato male sterile lines.

The method for propagating the tomato male sterile line comprises the following steps: and hybridizing the tomato male sterile line obtained by cultivating according to the method for cultivating the tomato male sterile line as a female parent and the tomato maintainer line obtained by cultivating according to the method for cultivating the tomato maintainer line as a male parent to realize the propagation of the tomato male sterile line.

Further, the method for expanding propagation of the tomato male sterile line also comprises the step of distinguishing the tomato male sterile line from the tomato maintainer line in filial generations by observing the color of the stems through naked eyes. The filial generation with green stem is male sterile line of tomato; the hybrid progeny with purple stem color is tomato maintainer line.

A sixth object of the present invention is to provide any one of the following M1) -M4):

m1) substances which either disable the function of the MS15 protein and F3H protein in tomato or inhibit the activity of the MS15 protein and F3H protein in tomato;

m2) knocking out or silencing the gene encoding MS15 protein and the gene encoding F3H protein in tomato or suppressing the expression of the gene encoding MS15 protein and the gene encoding F3H protein in tomato;

m3) the CRISPR/Cas9 system described above;

m4) the sgRNA described above.

In the product, the substance of the gene coding for MS15 protein and the gene coding for F3H protein in the knockout tomato is specifically the CRISPR/Cas9 system.

The application of the product in any one of the following (1) to (5) also belongs to the protection scope of the invention:

(1) cultivating a tomato male sterile line;

(2) cultivating a tomato maintainer line;

(3) propagating tomato male sterile line;

(4) distinguishing a sterile line and a maintainer line in the process of propagating the tomato male sterile line;

(5) and (5) tomato breeding.

Any of the above tomato maintainers can also be used as a tomato restorer line.

The tomato material may be Ailsa Craig.

The invention develops a tomato male sterility linkage marker by using a brand new combination of a fertility gene MS15 and a green stem gene F3H, and can be used for screening a sterile line and a maintainer line in the sterile line maintaining and expanding propagation process. The genetic distance between the fertility gene MS15 and the green stem gene F3H used in the invention is only 1.35cM (as shown in figure 1), which theoretically can generate lower genetic and phenotypic separation rate, and can greatly reduce the error rate of screening the existing tomato visible linkage marker sterile line.

Drawings

FIG. 1 is a schematic diagram of the genetic distance between a fertility gene and a visible linked marker gene of green stem.

FIG. 2 is a schematic diagram of a double knockout vector.

FIG. 3 shows the genotype identification of the ms15ms15/f3hf3h mutant.

FIG. 4 shows the fertility phenotype of the ms15ms15/f3hf3h mutant.

FIG. 5 shows phenotype observed in the progeny of the MS15MS15/F3Hf3h mutant in cross with the MS15MS15/F3Hf3h mutant.

FIG. 6 shows the propagation strategy of the sterile line with visible linkage markers in green stems.

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. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The CPB supports in the following examples are described in the literature: zhao et al (2016.) An alternative strategy for targeted gene replacement in using a dual-sgRNA/case 9design. scientific reports,6,23890.; the public is available from Weifang-flourishing biological species industry Co., Ltd.

The tomato variety Ailsa Craig in the examples below was purchased from the vegetable and flower institute, Chinese academy of agricultural sciences.