A fertility-related gene and its application in hybrid breeding

Abstract

The present invention provides a wheat fertility-related gene TaMS8 and an application method thereof, and belongs to the field of biotechnology. The present invention obtains a recessive single gene-controlled male sterility material by phenotypic screening and genetic analysis of an EMS mutagenesis library, and then obtains the wheat fertility-related gene TaMS8 by a Mutmap method, and regulates the expression of the gene to regulate plant fertility, so as to produce and maintain a wheat male sterile line and prepare hybrid seeds, which has important theoretical and practical significance for establishing an efficient wheat hybrid seed production technology, and studying the mechanism of wheat male sterility and hybrid vigor.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and specifically relates to the cloning of a recessive nuclear male sterile gene of a plant, a breeding method of a male sterile line thereof and an application thereof in hybrid breeding, and more specifically relates to the cloning of a recessive nuclear male sterile gene of wheat and a promoter thereof, and an application thereof in hybrid breeding. Background Art

[0002] Hybrid vigor is a natural phenomenon that is ubiquitous in the biological world. Making full use of hybrid vigor to significantly increase crop yields and improve crop quality has achieved huge economic benefits in rice, corn, sorghum, cotton, rapeseed and other crops. Wheat is the most widely cultivated and adaptable food crop in the world, and about 40% of the world's population relies on wheat as their staple food. Like rice, wheat also has obvious hybrid vigor. However, due to its own complex genetic characteristics, hybrid wheat does not perform as well as hybrid rice, and the theoretical and practical research on hybrid vigor of wheat lags far behind that of rice, corn, rapeseed and other crops.

[0003] Wheat is a self-pollinating crop, and the core of utilizing its hybrid vigor is to establish a technical system for the efficient production of wheat hybrids. The key to wheat hybrid vigor utilization seed production technology is to have a suitable recessive nuclear male sterile female parent. Male sterility refers to the inability of plants to produce anthers, pollen or male gametes with normal functions during sexual reproduction. The revelation of the mechanism of male sterility is the basis for utilizing hybrid vigor to improve wheat yield and quality. The wheat genome is large and complex, so the data accumulated so far on the mechanism of wheat male sterility is very limited. Therefore, applying modern molecular biology and cell biology methods to the study of the mechanism of wheat male sterility has important theoretical and practical significance for strengthening the research and utilization of wheat hybrid vigor.

[0004] Wheat is an allohexaploid with three sets of similar but not nearly identical genomes. Therefore, single-gene-controlled recessive mutants in wheat are significantly less than those in other species. Currently, there are only two loci that have been publicly reported to control recessive nuclear male sterility in wheat, MS1 and MS5. MS1 is located on the short arm of wheat chromosome 4B and encodes a 220-amino acid non-specific lipid transfer protein (nsLTP) (Wang et al., 2017; Tuker et al., 2017). nsLTP is widely present in plants and is characterized by an 8CM domain consisting of eight cysteine ​​residues (Boutrot et al., 2008). MS1 is a newly evolved protein in the Poaceae family. In situ hybridization showed that the gene is specifically expressed in high amounts in the microspore mother cells of anthers. Lipid binding experiments showed that MS1 can specifically bind to phosphatidic acid (PA) and several types of inositol phospholipids (phosphoinositide [PIPs]). Sequence analysis showed that there is an MS1 ​​homologous gene in each of the ABD genomes of the wheat genome, but due to methylation modification in the promoter region of the MS1 homologous gene in the AD genome, these two genes are completely unexpressed in hexaploid wheat. MS5 is located on wheat chromosome 3A and also encodes a nonspecific lipid transport protein, but except for the 8CM domain composed of 8 cysteine ​​residues, its amino acid sequence is not highly homologous to MS1 (Pallotta et al., 2019). MS5 is only conserved in the wheat tribe.

[0005] In order to obtain more wheat male sterility resources, the present invention screened out more than 20 genetically stable recessive single gene controlled male sterility materials through phenotypic screening and genetic analysis of the EMS mutagenesis library. After allelic detection of these materials with existing ms1 and ms5 mutants, a new single gene controlled wheat recessive nuclear male sterility line was found. The allelic hybridization detection experiment confirmed that it was not allelic with both the MS1 site and the MS5 site. The present invention named it MS8. The present invention cloned the wheat TaMS8 gene using the Mutmap method. The gene and the male sterility line produced by the mutation of the gene provide necessary elements for constructing a new hybrid breeding system. Summary of the invention

[0006] All references mentioned herein are incorporated by reference.

[0007] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Unless otherwise specified, the techniques used or referred to herein are standard techniques known to one of ordinary skill in the art. The materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0008] The present invention provides a fertility-related gene TaMS8, wherein the nucleotide sequence of the fertility-related gene is selected from one of the following groups of sequences:

[0009] (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2;

[0010] (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3;

[0011] (c) a DNA sequence that can hybridize with a DNA sequence described in (a) or (b) under stringent conditions; or

[0012] (d) a DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and having the function of restoring fertility; or

[0013] (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

[0014] Those skilled in the art should know that the fertility-related genes described in the present invention also include homologous gene sequences that are highly homologous to the nucleotide sequence or protein sequence of the TaMS8 gene and have the same fertility regulation or restoration function. The highly homologous homologous genes with fertility regulation function include DNA sequences that can hybridize with DNA having the sequence shown in SEQ ID NO: 1 or 2 under stringent conditions. Or a nucleotide sequence whose encoded amino acid sequence has more than 85% similarity with the protein amino acid sequence shown in SEQ ID NO: 3. The "stringent conditions" used in this article are well known, including hybridization at 53°C-60°C for 12-16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH6.4) and 1mM EDTA, and then washing at 62°C-68°C with a washing solution containing 0.5×SSC and 0.1% SDS for 15-60 minutes.

[0015] The above homologous genes also include DNA sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to the full length of the sequence shown in SEQ ID NO: 1 or 2, and having fertility regulation function, which can be isolated and obtained from any plant. Among them, the percentage of sequence similarity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm. This is well known to those skilled in the art.

[0016] The present invention also provides an expression cassette, wherein the expression cassette contains the DNA sequence of the fertility-related gene disclosed in the present invention, and the nucleotide sequence of the fertility-related gene is selected from one of the sequences in the following groups:

[0017] (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2;

[0018] (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3;

[0019] (c) a DNA sequence that can hybridize with a DNA sequence described in (a) or (b) under stringent conditions; or

[0020] (d) a DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and having the function of restoring fertility; or

[0021] (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

[0022] Specifically, the fertility-related gene in the above expression cassette is also operably connected to a promoter that can drive its expression, and the promoter includes but is not limited to a constitutive expression promoter, an inducible promoter, a tissue-specific expression promoter, or a spatiotemporal-specific expression promoter. More specifically, the promoter is a pollen-specific expression promoter. Preferably, the nucleotide sequence of the pollen-specific expression promoter is shown in SEQ ID NO: 10.

[0023] The above expression cassette of the present invention further comprises a pollen inactivation gene, which can interfere with the function or formation of male gametes containing the pollen inactivation gene in the plant. The pollen inactivation gene includes but is not limited to barnase gene, amylase gene, DAM methylase, etc. More specifically, the pollen inactivation gene is a corn α-amylase gene, and its nucleotide sequence is shown in SEQ ID NO: 14.

[0024] The above expression cassette of the present invention further comprises a screening gene, and the screening gene can be used to screen out plants, plant tissue cells or vectors containing the expression cassette. The screening gene includes but is not limited to blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, etc. Specifically, the screening gene includes but is not limited to: blue grain genes, chloramphenicol resistance genes, hygromycin resistance genes, streptomycin resistance genes, spectinomycin resistance genes, sulfonamide resistance genes, glyphosate resistance genes, glufosinate resistance genes, bar genes, red fluorescent genes DsRED, mCherry genes, cyan fluorescent protein genes, yellow fluorescent protein genes, luciferase genes, green fluorescent protein genes, etc.

[0025] The present invention also discloses a method for regulating plant fertility, wherein the method restores the male fertility of the wheat ms8 mutant by transferring a fertility-related gene into the wheat ms8 mutant, wherein the nucleotide sequence of the fertility-related gene is selected from one of the following groups of sequences:

[0026] (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2;

[0027] (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3;

[0028] (c) a DNA sequence that can hybridize with a DNA sequence described in (a) or (b) under stringent conditions; or

[0029] (d) a DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and having the function of restoring fertility; or

[0030] (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

[0031] The present invention also discloses a method for maintaining a male sterile line, wherein the method uses a wheat ms8 mutant plant as a transformation recipient material, and transforms three tightly linked target genes into the sterile mutant recipient plant. The three target genes are respectively a fertility-related gene TaMS8, a pollen inactivation gene, and a screening gene. Among them, the fertility-related gene TaMS8 can restore the fertility of the sterile transformation recipient, the pollen inactivation gene can inactivate the pollen containing the transformed exogenous gene, that is, lose the fertilization ability, and the screening gene can be used for sorting transgenic seeds or tissues and non-transgenic seeds or tissues, and the sorted non-transgenic seeds are used as sterile lines to produce hybrids, and the transgenic seeds are used as maintenance lines to continuously and stably produce sterile lines.

[0032] In the above-mentioned method for maintaining male sterile lines, the pollen inactivation gene includes but is not limited to barnase gene, amylase gene, DAM methylase, etc. More specifically, the pollen inactivation gene is the corn a-amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO:14. The pollen inactivation gene is connected to a promoter that prefers male gamete expression. More specifically, the promoter that prefers male gamete expression includes but is not limited to PG47 promoter, Zm13 promoter, etc. The screening gene can be used to screen out plants or vectors containing the expression cassette. The screening gene includes but is not limited to blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, etc. Specifically, the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, etc.

[0033] More specifically, the present invention also discloses a breeding method for a male sterile line, the method comprising the following steps:

[0034] (a) The following vector is introduced into the wheat ms8 male sterile line to obtain a maintenance line containing the following vector, wherein the vector comprises: a fertility-related gene TaMS8, which can restore the male fertility of the wheat ms8 mutant; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, so that the active male gametes produced in the plant do not contain the vector; and a screening gene, which can be used for sorting transgenic seeds or tissues from non-transgenic seeds or tissues.

[0035] (b) self-pollinating the maintainer plants formed after the transfer of the above-mentioned vector to simultaneously produce wheat ms8 male sterile line seeds without the vector and maintainer line seeds containing the vector; or driving pollen from the maintainer plants to wheat ms8 male sterile line plants to pollinate the wheat ms8 male sterile line plants to reproduce ms8 male sterile line seeds or its allelic sterile line seeds.

[0036] In the above-mentioned method for maintaining male sterile lines, the pollen inactivation gene includes but is not limited to barnase gene, amylase gene, DAM methylase, etc. More specifically, the pollen inactivation gene is the corn a-amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO:14. The pollen inactivation gene is connected to a promoter that prefers male gamete expression. More specifically, the promoter that prefers male gamete expression includes but is not limited to PG47 promoter, Zm13 promoter, etc. The screening gene can be used to screen out plants or vectors containing the expression cassette. The screening gene includes but is not limited to blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, etc. Specifically, the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, etc.

[0037] The present invention also discloses a method for producing a retainer system, the method comprising the following steps:

[0038] (a) The following vector is introduced into the wheat ms8 male sterile line, thereby obtaining the maintenance line of the wheat ms8 nuclear male sterile line or its allelic sterile line, wherein the vector comprises: a fertility-related gene TaMS8, which can restore the male fertility of the wheat ms8 nuclear male sterile line or its allelic sterile line; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, thereby making the fertile male gametes produced in the plant free of the vector; and a screening gene, which can be used for sorting transgenic seeds and non-transgenic seeds.

[0039] In the production method of the above-mentioned maintenance line, the pollen inactivation gene includes but is not limited to barnase gene, amylase gene, DAM methylase, etc. More specifically, the pollen inactivation gene is the corn a-amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO:14. The pollen inactivation gene is connected to a promoter that prefers male gamete expression. More specifically, the promoter that prefers male gamete expression includes but is not limited to PG47 promoter, Zm13 promoter, etc. The screening gene can be used to screen out plants or vectors containing the expression cassette. The screening gene includes but is not limited to blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, etc. Specifically, the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, etc.

[0040] The present invention also discloses a method for propagating a maintainer line, the method comprising the following steps:

[0041] (a) transferring the following vector into a wheat ms8 nuclear male sterile line or its allelic sterile line, thereby obtaining a maintenance line of the wheat ms8 nuclear male sterile line or its allelic sterile line, wherein the vector comprises: a fertility-related gene TaMS8, which can restore the male fertility of the wheat ms8 nuclear male sterile line or its allelic sterile line; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, thereby making the fertile male gametes produced in the plant free of the vector; and a screening gene, which can be used for sorting transgenic seeds and non-transgenic seeds; and

[0042] (b) self-pollinating the maintainer plants formed after the transfer of the above-mentioned vector, that is, breeding in a 1:1 ratio to obtain wheat ms8 nuclear male sterile line or its allelic sterile line seeds without the vector and maintainer line seeds containing the vector.

[0043] The present invention also discloses a seed production method, which comprises:

[0044] (a) Introducing the following vector into a wheat ms8 nuclear male sterile line or its allelic sterile line to obtain a maintenance line of the wheat ms8 nuclear male sterile line or its allelic sterile line, wherein the vector comprises: a fertility-related gene TaMS8, which can restore the male fertility of the wheat ms8 nuclear male sterile line or its allelic sterile line; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, thereby making the fertile male gametes produced in the plant free of the vector.

[0045] (b) self-pollinating the maintainer plants after being transformed with the above vector; and

[0046] (c) After self-pollination, the maintainer line seeds containing the vector and the wheat ms8 nuclear male sterile line or its allelic sterile line seeds not containing the vector are obtained.

[0047] In the above-mentioned male sterile line breeding or maintenance method, maintainer line production method or breeding method, seed production method, etc. of the present invention, step (a) can also be to introduce a vector containing the fertility-related gene TaMS8, the pollen inactivation gene and the screening gene into an ordinary plant, obtain a transgenic plant containing the vector, and then hybridize it with the wheat ms8 nuclear male sterile line or its allelic sterile line, and obtain a maintainer line plant with a background of wheat ms8 nuclear male sterile line or its allelic sterile line and containing the vector through directed selection.

[0048] In the above-mentioned male sterile line propagation method or maintenance method, maintenance line production method or propagation method, seed production method, etc. of the present invention, the nucleotide sequence of the fertility-related gene is selected from one of the following groups of sequences:

[0049] (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2;

[0050] (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3;

[0051] (c) a DNA sequence that can hybridize with a DNA sequence described in (a) or (b) under stringent conditions; or

[0052] (d) a DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and having the function of restoring fertility; or

[0053] (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

[0054] The fertility-related gene TaMS8 can also be operably connected to a pollen-specific expression promoter, which can drive the expression of the TaMS8 gene in plant pollen. The pollen-specific expression promoter is selected from one of the groups consisting of promoters of fertility regulatory genes such as MS26, NP1, MSP1, PAIR1, PAIR2, ZEP1, MELL, PSS1, TDR, UDT1, GAMYB4, PTC1, API5, WDA1, CYP704B2, MS26, MS22, DPW, MADS3, OSC6, RIP1, CSA, AID1, 5126 or Ms45. More specifically, the nucleotide sequence of the pollen-specific expression promoter is shown in SEQ ID NO:10. The fertility-related gene TaMS8 can also be operably connected to a terminator, which can be the terminator of any gene that has been disclosed. Specifically, the nucleotide sequence of one of the terminators is shown in SEQ ID NO:11. In the above-mentioned male sterile line propagation or maintenance method, maintenance line production method or propagation method, seed production method, etc. of the present invention, the pollen inactivation gene includes but is not limited to barnase gene, amylase gene, DAM methylase, etc. More specifically, the pollen inactivation gene is the maize a-amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO: 14. The pollen inactivation gene is linked to a promoter that prefers male gamete expression. More specifically, the promoter that prefers male gamete expression includes but is not limited to PG47 promoter, Zm13 promoter, etc.

[0055] In the above-mentioned male sterile line breeding or maintenance method, maintenance line production method or breeding method, seed production method, etc. of the present invention, the screening gene includes but is not limited to blue grain gene, antibiotic resistance gene, herbicide resistance gene or fluorescent gene. Specifically, the screening gene includes but is not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, etc.

[0056] The present invention also provides a pollen-specific expression promoter, whose nucleotide sequence is shown in SEQ ID NO: 10. SEQ ID NO: 10 is connected to the reporter gene GUS, a vector is constructed to transform rice and wheat, and the GUS expression activity and expression pattern in the transgenic plants are detected and analyzed. By performing GUS staining analysis on the roots, stems, leaves and flowers of the transgenic plants, it is found that the promoter provided by the present invention drives the GUS gene to be expressed in plant pollen. This indicates that SEQ ID NO: 10 provided by the present invention is a pollen-specific expression promoter.

[0057] The plant pollen-specific expression promoter provided by the present invention contains a nucleotide sequence as shown in SEQ ID NO: 10 in the sequence table, or contains a nucleotide sequence having a similarity of more than 90% with the nucleotide sequence listed in SEQ ID NO: 10, or contains 500 or more continuous nucleotide fragments derived from the nucleotide sequence listed in SEQ ID NO: 10, and can drive the expression of the nucleotide sequence operably connected to the promoter in plant pollen. The expression vector, transgenic cell line and host bacteria containing the above sequence all belong to the protection scope of the present invention. The primer pair for amplifying any nucleotide fragment of the SEQ ID NO: 10 promoter disclosed in the present invention is also within the protection scope of the present invention.

[0058] The "promoter" of the present invention refers to a DNA regulatory region, which generally includes a TATA box that can guide RNA polymerase II to initiate RNA synthesis at a suitable transcription start site of a specific coding sequence. The promoter may also include other recognition sequences, which are generally located upstream or 5' of the TATA box, and are generally referred to as upstream promoter elements, which play a role in regulating transcription efficiency. It should be known to those skilled in the art that although the nucleotide sequence of the promoter region disclosed in the present invention has been identified, it is also within the scope of the present invention to separate and identify other regulatory elements in the upstream region of the TATA box of the specific promoter region identified in the present invention. Therefore, the promoter region disclosed herein is generally further defined as comprising upstream regulatory elements, such as those elements, enhancers, etc., for regulating the tissue expressivity and temporal expression function of the coding sequence. In the same manner, promoter elements that enable expression in target tissues (e.g., male tissues) can be identified and isolated, and used together with other core promoters to verify the preferential expression of male tissues. The core promoter refers to the minimum sequence required to initiate transcription, such as a sequence called a TATA box, which is generally possessed by the promoters of protein-encoding genes. Therefore, the upstream promoter of the TaMS8 gene can be used optionally in conjunction with its own core promoter or one from other sources.

[0059] The core promoter can be any known core promoter, such as the cauliflower mosaic virus 35S or 19S promoter (U.S. Pat. No. 5,352,605), the ubiquitin promoter (U.S. Pat. No. 5,510,474), the IN2 core promoter (U.S. Pat. No. 5,364,780), or the Scrophulariaceae mosaic virus promoter.

[0060] The function of the gene promoter can be analyzed by the following method: the promoter sequence is operably linked to the reporter gene to form a transformable vector, and the vector is then transferred into the plant. In the transgenic offspring obtained, the expression characteristics of the reporter gene are confirmed by observing the expression of the reporter gene in various tissues and organs of the plant; or the above-mentioned vector is subcloned into an expression vector for transient expression experiments, and the function of the promoter or its regulatory region is detected by transient expression experiments.

[0061] The selection of suitable expression vector that is used for testing promoter or regulatory region function will depend on the host and this expression vector is introduced into the method for the host, and this class method is well known to those of ordinary skills in the art.For eukaryotes, the zone in the vector comprises the zone of control transcription initiation and control processing.These zones are operably connected to reporter gene, and described reporter gene comprises YFP, UidA, GUS gene or luciferase.The expression vector that comprises the inferred regulatory region that is positioned at the genomic fragment can be introduced into complete tissue, for example stage pollen, or introduces callus, to carry out functional verification.

[0062] In addition, the promoter of the present invention can also be connected to a nucleotide sequence other than the TaMS8 gene to express other heterologous nucleotide sequences. The promoter nucleotide sequence of the present invention and its fragments and variants can be assembled in an expression cassette together with the heterologous nucleotide sequence for expression in the target plant, more specifically, in the male organs of the plant. The expression cassette has suitable restriction enzyme sites for inserting the promoter and heterologous nucleotide sequence. These expression cassettes can be used to genetically manipulate any plant to obtain the desired corresponding phenotype.

[0063] The pollen-specific expression promoter disclosed in the present invention can be used to drive the expression of the following heterologous nucleotide sequences so that the transformed plants obtain a male sterile phenotype. The heterologous nucleotide sequences can encode enzymes or modifying enzymes that promote carbohydrate degradation, amylases, debranching enzymes and pectinases, more specifically, barnase genes, corn a-amylase genes, auxin genes, rot B, cytotoxin genes, diphtheria toxins, DAM methylases, or dominant male sterility genes.

[0064] In certain embodiments, the nucleotide sequence operably linked to the downstream of the promoter of the present invention mentioned in the present invention, wherein the "nucleotide sequence" can be a structural gene, a regulatory gene, an antisense gene of a structural gene, an antisense gene of a regulatory gene, or a small RNA capable of interfering with the expression of an endogenous gene operably linked to the promoter disclosed herein.

[0065] The present invention also provides a transcription terminator sequence, the nucleotide sequence of which is shown in SEQ ID NO: 11, and has the function of terminating gene transcription expression.

[0066] The present invention also provides an expression cassette, vector or engineered strain, wherein the expression cassette, vector or engineered strain comprises the pollen-specific expression promoter SEQ ID NO: 10 provided by the present invention. Specifically, the nucleotide sequence of the fertility-related gene TaMS8 provided by the present invention can be constructed downstream of the promoter SEQ ID NO: 10 provided by the present invention, thereby driving the expression of the fertility gene in the transformed recipient plant.

[0067] The pollen-specific expression promoter provided by the present invention can be used for the specific expression of exogenous genes in pollen, thereby avoiding the adverse effects caused by the continuous expression of the exogenous genes in other tissues of the plant, and can also be used for the functional analysis and identification of genes related to plant pollen growth and development; it can be used for the creation of male sterile lines and maintenance lines; and it can be applied to pollen abortion experiments, thereby avoiding the biosafety problems caused by plant transgenic drift or pollen escape, which is of great significance to the creation of plant male sterile lines and maintenance lines.

[0068] The nucleotide sequence and promoter sequence or expression cassette of the TaMS8 gene provided by the present invention can be inserted into any vector, plasmid, yeast artificial chromosome, bacterial artificial chromosome or other vector suitable for transformation into a host cell. Preferred host cells are bacterial cells, especially bacterial cells for cloning or storing polynucleotides or for transforming plant cells, such as Escherichia coli, Agrobacterium tumefaciens and Agrobacterium rhizogenes. When the host cell is a plant cell, the expression cassette or vector can be inserted into the genome of the transformed plant cell. Insertion can be a localized or random insertion.

[0069] The present invention describes the transfer of nucleotide sequences, vectors or expression cassettes into plants or the introduction of plants or the transformation of plants, all referring to the transfer of nucleotide sequences, vectors or expression cassettes into recipient cells or recipient plants by conventional transgenic methods. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform recombinant expression vectors into plant cells to produce transgenic plants of the present invention. Transformation methods may include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection. The transformation method also includes Agrobacterium-mediated plant transformation methods, etc.

[0070] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a fertility-related gene TaMS8 and its promoter, and a method for using the gene to propagate and maintain the ms8 male sterile line. The fertility-related gene, fertility maintenance of the nuclear male sterile line and the method for propagating the sterile line provided by the present invention have great production promotion value and application value for the hybrid breeding production of crops. The fertility gene provided by the present invention and the sterile line produced by the gene mutation provide resources for wheat hybrid breeding, and also provide necessary elements for constructing the third-generation hybrid breeding system. The male sterile line produced by the gene mutation is used to produce hybrid seeds, which is of great significance for breaking through and improving the existing "three-line" and "two-line" hybridization technologies.

[0071] References

[0072] Boutrot F.,Chantret N.,Gautier MF.Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein(nsLtp)gene families and identification of wheat nsLtp genes by EST data mining.BMC Genomics 2008,9:86-104

[0073] Pallotta MA, Warner P, Kouidri A, Tucker EJ, Baes M, Suchecki R, Watson-Haigh N, Okada T, Garcia M, Sandhu A, Singh M, Wolters P, Albertsen MC, Cigan AM, Baumann U, Whitford R. Wheat ms5 male-sterility is induced by recessive homoeologous A and D genome non-specific lipid transfer proteins. Plant J 2019, 99:673-685

[0074] Tucker EJ., Baumann U., Kouidri A., Suchecki R., Baes M., Garcia M., Okada T., Dong C., Wu Y., Sandhu A., Singh M., Langridge P., Wolters P., Albertsen MC., Cigan AM., Whitford R. Molecular identification of the wheat male fertility gene Ms1 and its prospects for hybrid breeding. Nat Commun 2017, 8:869

[0075] Wang Z, Li J, Chen S, Heng Y, Chen Z, Yang J, Zhou K, Pei J, He H, Deng XW, Ma L. Poaceae-specific MS1 encodes a phospholipid-binding protein for male fertility in bread wheat. Proc Natl Acad Sci U S A. 2017, 114:12614-12619. Description of the Drawings

[0076] Figure 1This is the phenotype of the wheat ms8 mutant, where Figure A shows the ears of the parent Ningchun No. 4 (left) and the ms8 mutant (right) at full flowering, Figures B and C show the anthers of the parent Ningchun No. 4 and the ms8 mutant at full flowering, and Figures D and E show the iodine-potassium iodide staining of the mature anthers of the parent Ningchun No. 4 and the ms8 mutant, respectively.

[0077] Figure 2 This is a distribution chart of wild-type and mutant SNP index on wheat chromosome 1A.

[0078] Figure 3 These are the results of TaMS8 gene mutmap and map-based cloning. The first row shows the positioning results of 404 ms8 sterile plants, the second row shows the fine positioning results after expanding the population, the third row shows the SNP analysis results within the fine positioning interval, and the fourth row shows the candidate gene structure and mutation sites.

[0079] Figure 4 Genotype and phenotypic analysis of exchange individuals screened for fine mapping of the TaMS8 gene.

[0080] Figure 5 To analyze the expression pattern of TaMS8 gene in different tissues of Chinese spring wheat by semi-quantitative RT-PCR and fluorescence quantitative PCR, ACTIN is a housekeeping gene. The upper figure is the agarose gel electrophoresis of RT-PCR products of TaMS8 gene in different organs or tissues; the lower figure is the quantitative RT-PCR results of TaMS8 gene in different organs or tissues, 0 is anther in meiosis stage, 1 is anther in mononuclear stage, 2 is anther in binuclear stage, 3 is anther in trinuclear stage, palea is a mixed material of palea and lemma, and gDNA is genomic DNA control.

[0081] Figure 6 Schematic diagram of the construction of a plant transformation vector for functional complementation of the ms8 mutant, wherein LB and RB are the left and right boundaries of T-DNA, respectively, and the expression of the Bar resistance gene is driven by Ubip (promoter of the Ubi gene) and terminated by the Nos terminator; the TaMS8 sequence is shown in SEQ ID NO: 12. DETAILED DESCRIPTION

[0082] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0083] Example 1. Acquisition and phenotypic analysis of wheat ms8 mutants

[0084] In order to obtain more wheat male sterility resources, the present invention obtained more than one hundred wheat male sterility materials by large-scale phenotypic screening of 1 million M2 generation plants in the EMS mutagenesis library of the spring wheat variety Ningchun No. 4. Subsequently, more than 20 genetically stable recessive single gene controlled male sterility materials were screened out. After allelic detection of these materials with existing ms1 and ms5 mutants, a new single gene controlled wheat recessive nuclear male sterility line was found. The allelic hybridization detection experiment confirmed that it was not allelic with both the MS1 site and the MS5 site. The present invention named it MS8.

[0085] The results of genetic analysis showed that in the F2 generation population of the mutant hybridized with its parent Ningchun No. 4, the segregation ratio of fertile plants to sterile plants was 242:69, indicating that the locus was a recessive mutation controlled by a single gene. The phenotype of the ms8 mutant and its parent was observed. There was no visible difference between the vegetative growth period and the parent until the flowering period. The panicle type of the ms8 mutant was significantly different from that of the parent Ningchun No. 4: the angle of opening of the guard glumes and outer glumes of the ms8 mutant and the awns on the outer glumes was significantly larger than that of normal plants. The anthers of the parent plants could dehisce normally, and the pollen grains could be clearly seen to be released, while the anthers of the ms8 mutant were relatively shriveled and did not dehisce at all. Pollen activity was identified by iodine-potassium iodide (I2-KI) staining. The starch grains of mature pollen of the parent plants were darkly colored and plump and spherical, while those of the ms8 mutant were obviously wrinkled and not colored at all, indicating that its pollen was inactive and therefore did not produce seeds at all in the later stage (see Figure 1 ).

[0086] Example 2. Cloning of wheat MS8 gene

[0087] Since genetic analysis of the ms8 mutant showed that it was a recessive mutation controlled by a single gene, we chose the mutmap technology, i.e., mutation site mapping, to clone its gene.

[0088] First, a hybrid F2 population of the ms8 mutant and the parent Ningchun No. 4 was constructed, and the DNA of the sterile plants in the F2 population was mixed into a mutant DNA pool. After tracking the offspring phenotype of the fertile plants in the F2 population, the DNA of the individual plants with wild-type genotypes at the MS8 locus in the F2 population was mixed into a wild-type DNA pool. The two pools were sent to a sequencing company for DNA sequencing, and 500G of data were measured for each pool. The sequencing data of each pool was aligned to the wheat Chinese Spring reference sequence for mutmap analysis to obtain the single nucleotide polymorphism sites (SNPs) of the two samples. According to the distribution of the SNP Index on each chromosome, it can be seen that among the 21 pairs of chromosomes of wheat, there is an obvious peak at 400-600Mb on chromosome 1A, indicating that MS8 is likely to be located in this area (see Figure 2 ).

[0089] Primers were designed for the SNPs in this section and molecular verification was performed. The peak of the SNP index is located approximately at 480-490 Mb on the long arm of wheat chromosome 1A. We selected five high-confidence SNPs in this interval and named them 1A-1 to 1A-5. Except for 1A-4, the other four SNPs were developed as CAPs (Cleaved Amplified Polymorphic Sequences) molecular markers. The verification results in parents and mutants showed that these five SNPs are real. Linkage analysis of the five SNP markers and the MS8 locus was performed using 404 ms8 sterile plants isolated from the F2 generation population, and MS8 was located within the range of 3.65 Mb between 481,704,309 bp (1A-1) and 485,352,404 bp (1A-3), 0.12 cM from 1A-1 and 0.25 cM from 1A-3DYZ8 (see Figure 3 ).

[0090] To further precisely locate MS8, we first screened 2160 F2 populations using CAPs markers 1A-1 (481,704,309 bp) and 1A-5 (488,109,008 bp), obtaining a total of 37 exchange plants, and then used CAPs marker 1A-3 (485,352,404 bp) to identify the genotypes of these 37 plants, obtaining 21 exchange plants between 1A-1 and 1A-3. Phenotypic observation of the 21 exchange plants combined with the genotypic identification results further narrowed the location interval of the MS8 locus to 1.33 Mb between 481,704,309 bp (1A-1) and 483,032,932 bp (1A-2) (see Figure 3 ), and there are 13 exchange strains remaining in this range. New SNP markers 1A-6 (482,337,315bp), 1A-13 (482,081,042bp), 1A-16 (482,661,640bp), 1A-17 (482,830,475bp) and 1A-20 (482,184,426bp) were developed between 1A-1 and 1A-2. After genotyping these 13 exchange strains, combined with their MS8 locus genotypes (inferred from the phenotyping results of their offspring), the MS8 locus was narrowed down to the 646Kb segment between 1A-20 (482,184,426bp) and 1A-17 (482,830,475bp) (see Figure 3 and Figure 4). Thirteen coding genes were annotated in this 646Kb genomic sequence. After analyzing the SNPs in this segment, only one SNP 1A-6 fell within the gene coding segment, resulting in a missense mutation, suggesting that the gene where the SNP is located is likely to be the MS8 gene, which we named TaMS8. The 1664th nucleotide of the genomic DNA sequence after the ATG of the gene where 1A-6 is located mutated from G to A, wherein the mutated genomic sequence is shown in SEQ ID NO: 23, and the mutated CDS sequence is shown in SEQ ID NO: 24, resulting in the 208th amino acid of the encoded protein sequence mutated from glycine (Gly) to aspartic acid (Asp), and the amino acid sequence is shown in SEQ ID NO: 25 (see Figure 3 ).

[0091] In the Chinese Spring variety of wheat, the genomic DNA sequence of the TaMS8 gene coding region is shown in SEQ ID NO:1, the CDS sequence is shown in SEQ ID NO:2, and the protein sequence is shown in SEQ ID NO:3.

[0092] Example 3. Expression specificity of TaMS8 gene

[0093] In order to analyze the expression pattern of TaMS8 gene, the cDNA sequence of the gene was used as a template to design semi-quantitative RT-PCR primers TaMS8-RTF and TaMS8-RTR (SEQ ID NO: 4 and 5) and fluorescent quantitative PCR primers TaMS8-QF and TaMS8-RTR (SEQ ID NO: 5 and 6). The wheat ACTIN gene was used as an internal reference to design semi-quantitative RT-PCR primers ACTIN-RTF and ACTIN-RTR (SEQ ID NO: 7 and 8) and fluorescent quantitative PCR primers ACTIN-QF and ACTIN-RTR (SEQ ID NO: 8 and 9). Total RNA was extracted from various tissue materials of Chinese spring wheat and cDNA templates were synthesized. The expression of TaMS8 gene was detected by semi-quantitative RT-PCR method and fluorescent real-time quantitative RT-PCR method. The results are shown in Figure 2. Figure 5 As shown, the gene expression peaks in anthers during the meiotic stage and the mononucleate stage, and the expression in anthers during the binucleate stage is significantly weakened, and the expression is even lower during the trinucleate stage. The results of quantitative PCR are consistent with those of semi-quantitative RT-PCR. The expression in other tissues and organs, such as roots, stems, leaves, lemma / lemma, pistil, and ears of various sizes before meiosis is relatively low.

[0094] The primer sequences used are as follows:

[0095] TaMS8-RTF:5'-ACTGCACAGACCACCATTGAGATT-3'(SEQ ID NO:4)

[0096] TaMS8-RTR:5'-ATCAAGTAGCGCGCAGACATTG-3'(SEQ ID NO:5)

[0097] TaMS8-QF:5'-CGACGACAAGAAGAAGGTTGAGGAG-3'(SEQ ID NO:6)

[0098] ACTIN-RTF:5'-TCAGCCATACTGTGCCAATC-3'(SEQ ID NO:7)

[0099] ACTIN-RTR:5'-CTTCATGCTGCTTGGTGC-3'(SEQ ID NO:8)

[0100] ACTIN-QF:5'-TTCCAGCCATCTTTCATTG-3'(SEQ ID NO:9)

[0101] Example 4. Transgenic Functional Complementation of Wheat ms8 Mutant

[0102] In order to verify the function of the TaMS8 gene, we constructed a stable transformation vector for functional complementation of the wheat ms8 mutant. Using the binary expression vector pCAMBIA1300 as the backbone, the hygromycin resistance expression cassette driven by the plant resistance screening gene 35S promoter on pCAMBIA1300 was first replaced with the Bar resistance gene expression cassette driven by the Ubi promoter on the pAHC20 vector. On this basis, the 6783bp TaMS8 genomic sequence (shown in SEQ ID NO: 12, which contains a 2907bp promoter sequence (SEQ ID NO: 10), a 2988bp genomic sequence (SEQ ID NO: 1) and an 888bp terminator sequence (SEQ ID NO: 11)) was inserted into the multiple cloning site by the in-fusion method to form the plant expression vector p1300-TaMS8 (see Figure 6 ).

[0103] Using Agrobacterium-mediated genetic transformation technology, the functional complementation vector p1300-TaMS8 vector was transformed into the young embryos of the heterozygous plants of the wheat ms8 mutant, and a total of 45 transgenic positive plants were obtained. Since the genotype of the young embryos used as transformation explants may be the wild type, heterozygous or homozygous mutation state of the MS8 locus, 9 of the transgenic positive plants were found to be the ms8 homozygous mutant genotype through adjacent SNP marker genotype identification. The pollen fertility of these 9 transgenic positive plants was observed, and it was found that the pollen was fertile. The above results indicate that the mutation of the TaMS8 gene leads to the male sterility phenotype of the ms8 mutant.

[0104] Example 5. Construction and functional analysis of TaMS8 gene promoter expression vector

[0105] Using the genomic DNA of wheat Ningchun No. 4 variety as a template, a 2907bp TaMS8 gene promoter fragment (SEQID NO: 10) was amplified and connected to the pCAMBIA1300 vector of the Bar gene driven by the Ubi promoter through the in-fusion technology, and then the GUS reporter gene (SEQ ID NO: 11) was inserted behind the 2907bp TaMS8 gene promoter (SEQ ID NO: 10) using the in-fusion technology (see Figure 8). The above plasmid was transformed into wheat embryos using Agrobacterium-mediated genetic transformation technology to obtain 15 transgenic positive plants. The GUS staining analysis results of different tissues and organs of the transgenic positive plants were consistent with the expression pattern analysis results of the TaMS8 gene, indicating that the TaMS8 gene promoter is an anther-specific expression promoter.

[0106] Example 6. Application of TaMS8 gene in new generation wheat hybrid breeding technology

[0107] The present invention applies the wheat ms8 male sterile mutant and TaMS8 gene to the new generation of wheat hybrid breeding technology. The core of the new generation of hybrid breeding technology is to use the wheat recessive nuclear male sterile mutant as the transformation receptor material, and transform the fertility restoration gene, pollen inactivation gene and seed marker gene into the male sterile mutant to form a transgenic strain with a restoration line and a maintenance line. The maintenance line is used to pollinate the sterile line, thereby breeding the sterile line.

[0108] The present invention uses TaMS8 gene as fertility restoration gene, maize gene (ZmBT1-ZmAA) as pollen inactivation gene and blue grain gene as seed marker gene, and constructs three expression cassettes together on pCAMBIA1300 vector in which resistance gene is transformed into Bar gene driven by Ubi promoter.

[0109] 1) Based on the pCAMBIA1300 vector in which the resistance gene is modified into the Bar gene driven by the Ubi promoter;

[0110] 2) TaMS8 gene expression cassette, the target gene TaMS8 and its promoter and terminator are all from the wheat variety Ningchun No. 4, the full-length nucleotide sequence is shown in SEQ ID NO: 12, wherein the promoter sequence of the TaMS8 gene is shown in SEQ ID NO: 10, the terminator sequence is shown in SEQ ID NO: 11, the genomic DNA sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the nucleotide sequence is shown in SEQ ID NO: 3;

[0111] 3) Gene expression cassette PG47: ZmBT1-ZmAA-IN2-1, the target gene is ZmAA, the transit peptide is ZmBT1, and the open reading frame of ZmBT1-ZmAA (whose nucleotide sequence is shown in SEQ ID NO: 14) is connected to the downstream of the promoter PG47 (whose nucleotide sequence is shown in SEQ ID NO: 15) and the upstream of the terminator IN2-1 (whose nucleotide sequence is shown in SEQ ID NO: 16).

[0112] 4) Blue grain marker gene expression cassette see patent WO2019090496A1. The target gene is 3215bp ThMYB1 genomic sequence (including 1952bp promoter sequence (shown in SEQ ID NO: 17), 822bp genomic sequence (shown in SEQ ID NO: 18) and 441bp terminator sequence (shown in SEQ ID NO: 19)) and 4422bp ThR1 genomic sequence (including 2084bp promoter sequence (shown in SEQ ID NO: 20), 1720bp CDS sequence (shown in SEQ ID NO: 21) and 618bp terminator sequence (shown in SEQ ID NO: 22)).

[0113] The vector was transformed into young embryos of heterozygous plants of wheat ms8 mutants by using Agrobacterium-mediated genetic transformation technology, and 15 transgenic positive plants with genotypes of ms8 homozygous mutations were obtained. The transgenic positive plants were stained with iodine-potassium iodide to detect pollen activity, and the results showed that the proportion of sterile pollen in multiple transgenic plants was about 50%, which indicated that firstly, the fertility restoration gene worked to restore the male sterility phenotype of the ms8 mutant, and secondly, the pollen lethal gene inactivated half of the transgenic pollen. In the seeds of the T1 generation transgenic plants harvested from a single plant, it can be observed that the ratio of normal color seeds to blue grain seeds is about 1:1, that is, the seeds carrying exogenous transgenes present a blue grain phenotype because of the seed marker gene. The above results show that the three expression boxes of the fertility restoration gene, the pollen inactivation gene and the seed marker gene in the present invention can all function correctly and can be successfully applied to the new generation of wheat hybrid breeding technology. SEQUENCE LISTING <110> Modern Agricultural Research Institute of Peking University Beijing Zhiyu Wheat Biotechnology Co., Ltd. <120> A fertility-related gene and its application in hybrid breeding <130> 1 <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 2988 <212> DNA <213> Triticum aestivum <400> 1 atggccaagg gcgaggggcc ggcgatcggc atcgacctcg ggacgaccta ctcctgcgtc 60 ggcgtgtggc agcatgaccg cgtcgagatc atcgccaacg accagggcaa ccgcaccacg 120 ccctcctacg tcgccttcac cgacaccgag cgcctcatcg gcgacgccgc taagaaccag 180 gtcgccatga accccaccaa caccgtcttc ggtacgtgcg ccccccctcc atccctccct 240 ctcgacccgc tgtgtagtat catgcgtcgt ctgttagatg agatctccgt ggttgcctgc 300 ggtccctgtt ctgttcttac gcgcacgatg cgatatgatt tgctctgaat aatcggatgt 360 ggctgttgcc gaatccgtca cgcagtccct tgtctctgag attaattaca gatttataat 420 tggttcatat ctgcatgtct gttctctgtt gtttggcatg ctgtagtccc ctgtttctga 480 gatttgttct atgtcagatt gtcaaattag atccttagga atggatggct tgagttaagt 540 tgggatccta ccttatgatt gaaaatacgc tatagatcta gtcttattta aattagttgc 600 agcagctgct gtcgattgtg tgccgcagtc ccgtgtttat ctgatctatc acagaattta 660 ttattagttt attggcatgg ctgctctttg tttatactgg tcgaaatgct gttatgtttt 720 ttgttctgaa ggtgctcagt tgtttgttct gcatggttca gtagtagatc cttcagaatg 780 gagggttaga attaagttgg ttggttgaaa ttatgctgta gatgaaaatt agttatactg 840 Foottactaga ttcttaagtt atacatgctt cagaaaagaa tactcgaatt atgtccacag 900 gctacctat gattgaaagt gttgaatcat gttttgctat tgattggatg atattccgaa 960 ttgaacatgg gtttcgctgt atcgttgcat cctgtaaatc cttcagaaaa tggtgtttga 1020 ttgaaattat gcttgttaat tttaaatttc tgttctcggt tatccttctg gaacagtccc 1080 agtctgcagt agcttactga atgtacctct tgttctgaag aaacagcttg cttgcattat 1140 taatctctgt ggaaccttga aatgttctgt tctgcttcat ttcagtaaac catcgcagtt 1200 cattgctgaa ccatgcaact caaatgttct gttcctctgt ttctggttgc agatgcgaag 1260 cggctgattg gcaggaggtt ctctgacccc tccgtacaga gtgacatgaa gctgtggccc 1320 ttcaaggtca tccctggccc ggctgacaag cctatgatcg tcgtcaacta caagggcgag 1380 gagaagcagt ttgctgcgga ggagatctcc tccatggtgc tcatcaagat gagggagatc 1440 gctgaggcct tcctcggcaa ctccgtcaag aacgccgtgg tcaccgtccc ggcctacttc 1500 aacgactctc agcgccaggc caccaaggac gctggtgcca tcgctgggct gaacgtgctg 1560 cgcataatca acgagcccac tgctgctgcc attgcctatg gccttgacaa gaaggcgacc 1620 agcacgggcg agaagaacgt cctcatcttc gaccttggtg gtggcacctt cgatgtgtct 1680 ctcctgacca tcgaggaggg catcttcgag gtcaaggcca ccgctggtga cactcacctg 1740 ggaggggagg acttcgacaa ccgcatggtg aaccacttcg tccaggagtt caagcgcaag 1800 cacaagaagg acatcaccgg caacccgcgt gctctgcgtc gtctccgtac ggcgtgcgag 1860 cgtgccaagc gcacgctgtc gtcgactgca cagaccacca ttgagattga ctcgctgtac 1920 gagggtgtgg acttctacac caccatcacc agggctcgct tcgaggagct caacatggac 1980 ctcttccgca agtgcatgga gccggtggag aagtgcctcc gtgacgccaa gatggacaag 2040 agcacggtgc acgacgtggt gctggtcggt ggctccaccc gtatccccaa ggtgcagcag 2100 ttgctccagg acttcttcaa cggcaaggag ctgtgcaaga gcatcaaccc cgacgaggca 2160 gtggcgtatg gcgcctccgt ccaggctgcc atcctgagcg gcgagggcaa cgagaaggtg 2220 caggacctgc tgctgctcga cgtgacgccg ctgtccctgg gcctggagac cgccggcggc 2280 gtcatgacga cgctcatccc gaggaacacc accatcccca ccaagaagga gcaggtcttc 2340 tccacctact cggacaacca gccgggcgtc ctgatccagg tgtacgaggg cgagcgcgcc 2400 aggaccaagg acaacaacct cctgggcaag ttcgagctct ctggcatccc gccggcgccc 2460 cgcggcgtgc cccagatcac ggtgtgcttc gacatcgacg ccaacggcat cctgaacgtg 2520 tcggcggagg acaagacgac tgggcagaag aacaagatca ccatcaccaa cgacaagggg 2580 cggctgagca aggaggagat cgagaagatg gtgcaggagg cggagcggta caaggcggag 2640 gacgaggagc acaagaagaa ggtggacgcc aagaacgcgc tggagaacta cgcctacaac 2700 atgcgcaaca cggtcaagga cgacaagatc gcctccaagc tcggcgccga cgacaagaag 2760 aaggttgagg aggcgatcga gggcaccatc agctggctgg acgccaacca gctcgccgag 2820 gccgacgagt tcgaggacaa gatgaggag ctggagggca tctgcaaccc catcatcgcc 2880 aagatgtacc agggcgccgc gccggacatg ggcggcggca tgggcatgga cgaggacatg 2940 cccgccggtg ctggcggcgc cggccccaag atcgaggagg tggactaa 2988 <210> 2 <211> 1947 <212> DNA <213> Triticum aestivum <400> 2 atggccaagg gcgaggggcc ggcgatcggc atcgacctcg ggacgaccta ctcctgcgtc 60 ggcgtgtggc agcatgaccg cgtcgagatc atcgccaacg accagggcaa ccgcaccacg 120 ccctcctacg tcgccttcac cgacaccgag cgcctcatcg gcgacgccgc taagaaccag 180 gtcgccatga accccaccaa caccgtcttc gatgcgaagc ggctgattgg caggaggttc 240 tctgacccct ccgtacagag tgacatgaag ctgtggccct tcaaggtcat ccctggcccg 300 gctgcaagc ctatgatcgt cgtcaactac aagggcgagg agaagcagtt tgctgcggag 360 gagatctcct ccatggtgct catcaagatg agggagatcg ctgaggcctt cctcggcaac 420 tccgtcaaga acgccgtggt caccgtcccg gcctacttca acgactctca gcgccaggcc 480 accaagggacg ctggtgccat cgctgggctg aacgtgctgc gcataatcaa cgagcccact 540 gctgctgcca ttgcctatgg ccttgacaag aaggcgacca gcacgggcga gaagaacgtc 600 ctcatcttcg accttggtgg tggcaccttc gatgtgtctc tcctgaccat cgaggagggc 660 atcttcgagg tcaaggccac cgctggtgac actcacctgg gaggggaga cttcgcaac 720 cgcatggtga accacttcgt ccaggaggttc aagcgcaagc aagaagga catcaccggc 780 aacccgcgtg ctctgcgtcg tctccgtacg gcgtgcgagc gtgccaagcg cacgctgtcg 840 tcgactgcac agaccaccat tgagattgac tcgctgtacg agggtgtgga cttctacacc 900 accatcacca gggctcgctt cgaggagctc aacatggacc tcttccgcaa gtgcatggag 960 ccggtggaga agtgcctccg tgacgccaag atggagaaga gcacggtgca cgacgtggtg 1020 ctggtcggtg gctccacccg tatccccaag gtgcagcagt tgctccagga cttcttcaac 1080 ggcaaggagc tgtgcaagag catcaacccc gacgaggcag tggcgtatgg cgctcccgtc 1140 caggctgcca tcctgagcgg cgagggcaac gagaaggtgc aggacctgct gctgctcgac 1200 gtgacgccgc tgtccctggg cctggagacc gccggcggcg tcatgacgac gctcatcccg 1260 aggaacacca ccatccccac caagaaggag caggtcttct ccacctactc ggacaaccag 1320 ccgggcgtcc tgatccaggt gtacgagggc gagcgcgcca ggaccaagga caacaacctc 1380 ctgggcaagt tcgagctctc tggcatcccg ccggcgcccc gcggcgtgcc ccagatcacg 1440 gtgtgcttcg acatcgacgc caacggcatc ctgaacgtgt cggcggagga caagacgact 1500 gggcagaaga acaagatcac catcaccaac gacaaggggc ggctgagcaa ggaggagatc 1560 gagaagatgg tgcaggaggc ggagcggtac aaggcggagg acgaggagca caagaagaag 1620 gtggacgcca agaacgcgct ggagaactac gcctacaaca tgcgcaacac ggtcaaggac 1680 gacaagatcg cctccaagct cggcgccgac gacaagaaga aggttgagga ggcgatcgag 1740 ggcaccatca gctggctgga cgccaaccag ctcgccgagg ccgacgagtt cgaggacaag 1800 atgaaggagc tggagggcat ctgcaacccc atcatcgcca agatgtacca gggcgccgcg 1860 ccggacatgg gcggcggcat gggcatggac gaggacatgc ccgccggtgc tggcggcgcc 1920 ggccccaaga tcgaggaggt ggactaa 1947 <210> 3 <211> 648 <212> PRT <213> Triticum aestivum <400> 3 Met Ala Lys Gly Glu Gly Pro Ala Ile Gly Ile Asp Leu Gly Thr Thr 1 5 10 15 Tyr Ser Cys Val Gly Val Trp Gln His Asp Arg Val Glu Ile Ile Ala 20 25 30 Asn Asp Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp 35 40 45 Thr Glu Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Met Asn 50 55 60 Pro Thr Asn Thr Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Arg Phe 65 70 75 80 Ser Asp Pro Ser Val Gln Ser Asp Met Lys Leu Trp Pro Phe Lys Val 85 90 95 Ile Pro Gly Pro Ala Asp Lys Pro Met Ile Val Val Asn Tyr Lys Gly 100 105 110 Glu Glu Lys Gln Phe Ala Ala Glu Glu Ile Ser Ser Met Val Leu Ile 115 120 125 Lys Met Arg Glu Ile Ala Glu Ala Phe Leu Gly Asn Ser Val Lys Asn 130 135 140 Ala Val Val Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala 145 150 155 160 Thr Lys Asp Ala Gly Ala Ile Ala Gly Leu Asn Val Leu Arg Ile Ile 165 170 175 Asn Glu Pro Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Ala 180 185 190 Thr Ser Thr Gly Glu Lys Asn Val Leu Ile Phe Asp Leu Gly Gly Gly 195 200 205 Thr Phe Asp Val Ser Leu Leu Thr Ile Glu Glu Gly Ile Phe Glu Val 210 215 220 Lys Ala Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn 225 230 235 240 Arg Met Val Asn His Phe Val Gln Glu Phe Lys Arg Lys His Lys Lys 245 250 255 Asp Ile Thr Gly Asn Pro Arg Ala Leu Arg Arg Leu Arg Thr Ala Cys 260 265 270 Glu Arg Ala Lys Arg Thr Leu Ser Ser Thr Ala Gln Thr Thr Ile Glu 275 280 285 Ile Asp Ser Leu Tyr Glu Gly Val Asp Phe Tyr Thr Thr Ile Thr Arg 290 295 300 Ala Arg Phe Glu Glu Leu Asn Met Asp Leu Phe Arg Lys Cys Met Glu 305 310 315 320 Pro Val Glu Lys Cys Leu Arg Asp Ala Lys Met Asp Lys Ser Thr Val 325 330 335 His Asp Val Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Val Gln 340 345 350 Gln Leu Leu Gln Asp Phe Phe Asn Gly Lys Glu Leu Cys Lys Ser Ile 355 360 365 Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ser Val Gln Ala Ala Ile 370 375 380 Leu Ser Gly Glu Gly Asn Glu Lys Val Gln Asp Leu Leu Leu Leu Asp 385 390 395 400 Val Thr Pro Leu Ser Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr 405 410 415 Thr Leu Ile Pro Arg Asn Thr Thr Ile Pro Thr Lys Lys Glu Gln Val 420 425 430 Phe Ser Thr Tyr Ser Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr 435 440 445 Glu Gly Glu Arg Ala Arg Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe 450 455 460 Glu Leu Ser Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Thr 465 470 475 480 Val Cys Phe Asp Ile Asp Ala Asn Gly Ile Leu Asn Val Ser Ala Glu 485 490 495 Asp Lys Thr Thr Gly Gln Lys Asn Lys Ile Thr Ile Thr Asn Asp Lys 500 505 510 Gly Arg Leu Ser Lys Glu Glu Ile Glu Lys Met Val Gln Glu Ala Glu 515 520 525 Arg Tyr Lys Ala Glu Asp Glu Glu His Lys Lys Lys Val Asp Ala Lys 530 535 540 Asn Ala Leu Glu Asn Tyr Ala Tyr Asn Met Arg Asn Thr Val Lys Asp 545 550 555 560 Asp Lys Ile Ala Ser Lys Leu Gly Ala Asp Asp Lys Lys Lys Val Glu 565 570 575 Glu Ala Ile Glu Gly Thr Ile Ser Trp Leu Asp Ala Asn Gln Leu Ala 580 585 590 Glu Ala Asp Glu Phe Glu Asp Lys Met Lys Glu Leu Glu Gly Ile Cys 595 600 605 Asn Pro Ile Ile Ala Lys Met Tyr Gln Gly Ala Ala Pro Asp Met Gly 610 615 620 Gly Gly Met Gly Met Asp Glu Asp Met Pro Ala Gly Ala Gly Gly Ala 625 630 635 640 Gly Pro Lys Ile Glu Glu Val Asp 645 <210> 4 <211> twenty four <212> DNA <213> Artificial sequence <400> 4 actgcacaga ccaccattga gatt 24 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <400> 5 atcaagtagc gcgcagacat tg 22 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <400> 6 cgacgacaag aagaaggttg aggag 25 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 tcagccatac tgtgccaatc 20 <210> 8 <211> 18 <212> DNA <213> Artificial sequence <400> 8 cttcatgctg cttggtgc 18 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <400> 9 ttccagccat ctttcattg 19 <210> 10 <211> 2907 <212> DNA <213> Triticum aestivum <400> 10 cacaagtttt gagcgagatt acacctgact aattggcatg agaaaataga ggagcttgcc 60 taatataagg aaatgtaatc aaatccctaa aaaatatcca aacgagtggt gcaatgcaat 120 acaccttata ttttgggttt tttctcaaaa atctatacac cttatatcaa ggaacggagg 180 gagtagcata attggatcat ataactatcc ttcaacatgc aacaaagagt cactccaaag 240 tcactaatag cggagaacaa acgaagagat tatggtaggg tacgaaacca cctcaaagtt 300 atcatttctg atcgatctat tcaaaagtcc gtagtaaaat aatataaagc tattctttcc 360 gttcaatcta tcctagagtt cgtactagaa taacacctta agacacaaat caaccaaaac 420 cctaatgtca cctagatacc ccaatgtcac ctcaagtatc cgtggatatg attatatgat 480 atgcatcaca caatctcaga ttcatctatt caaccaacac aaagaacttc aaagagtgcc 540 ccaaagttc tatcggagag tcaagacgaa aacgtgtgcc aacccctatg cataagttca 600 cgaggtcacg caagttgatc accaaaacat acatcaagtg gatcacgtga tatcccattg 660 tcaccacaga taagcacatg caagacatac atcaagtgtt ctcaaatcct taagactca 720 atccgataag ataacttcaa aggaaaactc aaaccattac aagagagtag agagggggaa 780 atattataag atccaactat atagcaaag ctcgcgatac atcaagatcg tatcacctca 840 agaacatgag agagagagag agatcaaaca catagctact ggtacatatc ctcagccccg 900 agggtggact actccctctt cgtcatggag agcaccggga tgatgaagat ggccatcgga 960 gagggattcc ccctatgga agggtgccgg aacgggtcta gattggtttt cggtggctac 1020 gaaggcttct ggcggcggaa ctcccgatct attctgctct ccgaagtttt tagggtatat 1080 ggatatatat atatatgagg aagaagtacg tcggtggatc tccgggctgt ccacgaggca 1140 gggggcgcac ccaggggggt gggcgcgcc cccaccctcg tgggcagccc gggactcttc 1200 tggttcatct ctgatactct gtgggcttct tctggtccaa aaataagtttt cgtgaagtttt 1260 caggtcaatt ggactccgtt tgatttcct tttctgcgat actctaaaac aaggaaaaaa 1320 cagaaactgg cactgggctc taggttaata ggttagtccc aaaaatcata taaatagta 1380 tataaatgca tataaaacat ccaagattga tatatataa gcatggaaaa aatccaactt 1440 cgtgctaatg gttcagtggt tgcgatcata tggtcaagga ggtcgtgatc taccaaaacc 1500 cagacacatc gcgtcatgtt gcactcaagc aggttgtgac accaagagtc ccgcgcgccg 1560 catacaaagc aagcatcatg gtgagacatc tttatgtgct tgagaacatc ttcaattgat 1620 agtgagtttt ttgctaagca ccataaaaat gtccttagct ttgccggcac ggctaccttc 1680 cacggagtgt cccaagcttt ccattcgct ttttattatt cgatgaccca actctttctt 1740 ctaaccagtc ctccatcctc ctatttgttg caactatcat cttgtatgct gatcgaacga 1800 aaaatatgca gttcttttca aaatcccacg accagaagtc atctatcaat cgtgtactca 1860 ttggaatctg tagtgtagcc gaagcatcag agggcagaaa aacttgctgg atgaattgtg 1920 tattccatag tgcattggct tgatccaaca attcggaaac gaactggggt ggctaggcaa 1980 ttagcgagac aaatgggccc catgttctcc ttcctgggga tccagttcat atttcagatg 2040 tttgtagagt tgccattgcc tatcggcctt attagtctca tcttcatagt gtcccttcct 2100 tcaattactc ctctccaaat ttgagaaagg tggcccccaa gctccgctga aagaaaatcc 2160 gtgtcaagat agtatgcggc ttccaaaatt ctagcattga gagatccggg attttcaaga 2220 atcctccacg cctgactagc cagaagggcc aagttgaaaa tttcaaattc tctgaagccc 2280 aagcctttca tatattttgg catactcatg aagatttttt tatttttagc gaacgacatg 2340 tactgacgta cgtgggccga gactaaggcc tgggccaatt acccatgcag ggcaaggcag 2400 gcagaagcta aggacgatgt gatggtaaaa aattgctaaa aaaaatgtga tgatatattt 2460 tttagaatcc taggtttttc cttccgcgcg tgctatccac ccgacgcgtg ctcctcccac 2520 cgtctctcca fotagccta ccatgctcgt gcacattgta gaacctacca gcaccaccac 2580 cagaagcgcc cgcgcgtggg ccaccccacc ccaccccacc ccaccgccac cccgccccgc 2640 cccgcacact ccggaccttt ccagaaccat cacccctccc ccgatccgac ggccgggatc 2700 gcacgcacca gaacattcca gacccggccg cctccgccat aaaaccctcc cctctccggc 2760 cctccctccc cacttccgtt cgaaatcaaa tcagagagag gcaaagcaaa tcgcaccagg 2820 caaatcacac agagagcgcc gccgtcccaa tattccggcg aaccccaaag cgagcgagcg 2880 attcccagga gaggagagga ggcggag 2907 <210> 11 <211> 888 <212> DNA <213> Triticum aestivum <400> 11 gctgttcgtc caatgtctgc gcgctacttg atgggtgttc tgtctcggtg tcctctggat 60 gccgctcggt ggtgccagtc gtctgtcgtc gtgtcatcta gctgcaagta gtagtagtgg 120 ttcgttggag tcgtttgatc tgaaagacat gatggatgat gatgtttgct tgttggtttg 180 gaacccttga tgcttagagc ttctcgtttt ttaagtttaa aatttggtgt tcccctactt 240 ttgatcttct ctggctctgt ttattttttt gttcatctgt ggttctgcta tgttcattga 300 tgttcatctg tggcacacgg ctgacgcctt tgcccttcgg catggtggag cttgcaggac 360 tgacccacca tggtgagtct gcaacacaca atgctgttga ctgaggagtc tagtaaatag 420 tcaccgtctt cgggatgccg ctctcgtcgg ctagcagcaa gcgattatga tgttcccttg 480 cttgtttcgg ttttgaagtg cagttcgatc atgatgtttg ttgcttgctt gttctgctt 540 cggtgctccc ctaattgatc atgattagct gctatgttct gcttttattt tatttttttt 600 gaattgctgc cacttttagt agttcaacat ttttgtcatg tgctgtatct ccattctttt 660 ttcttactac tattttttga atacacttaa actttatagc acgtatctgc aacgccatcc 720 agaagttatc acttgcaaga tggtgatttt tacaggatcc caagcaccat tcaaaattat 780 attcatgata agatgcaaat tgaacacact aataggctac tcatgttttt ttaggggatc 840 aacaagtgag cccttgatat cgtgcgttgt tgtttgtctg caggttct 888 <210> 12 <211> 6783 <212> DNA <213> Triticum aestivum <400> 12 cacaagtttt gagcgagatt acacctgact aattggcatg agaaaataga ggagcttgcc 60 tatataagg aaatgtaatc aaatccctaa aaaatatcca aacgagtggt gcaatgcaat 120 acaccttata ttttgggttt tttctcaaaa atctatacac cttatatcaa ggaacggagg gagtagcata attggatcat ataactatcc ttcaacatgc aacaaagagt cactccaaag 300. tcactaatag cggagaacaa acgaagagat tatggtaggg tacgaaacca cctcaaagtt atcatttctg atcgatctat tcaaaagtcc gtagtaaaat father tattctttcc gttcaatcta tcctagagtt cgtactagaa taacacctta agacacaaat caaccaaaac cctaatgtca cctagatacc ccatgtcac ctcaagtatc cgtggatatg attatatgat atgcatcaca caatctcaga ttcatctatt caaccaacac aaagaacttc aaagagtgcc ccaaagtttc tatcggagag tcaagacgaa aacgtgtgcc aacccctatg cataagttca cgaggtcacg caagttgatc accaaaacat acatcaagtg gatcacgtga tatcccattg tcaccacaga tagcacatg caagacatac atcaagtgtt ctcaaatcct tagcatca atccgataag ataacttcaa aggaaaactc aaaccattac aagagagtag aggaggggga atttataag atccaactat aatagcaaag ctcgcgatac atcaagatcg tatcacctca agaacatgag agagagagag agatcaaaca catagctact ggtacatatc ctcagccccg 900 agggtggact actccctctt cgtcatggag agcaccggga tgatgaagat ggccatcgga 960 gagggattcc ccctatgga agggtgccgg aacgggtcta gattggtttt cggtggctac 1020 gaaggcttct ggcggcggaa ctcccgatct attctgctct ccgaagtttt tagggtatat 1080 ggatatatat atatatgagg aagaagtacg tcggtggatc tccgggctgt ccacgaggca 1140 gggggcgcac ccaggggggt gggcgcgcc cccaccctcg tgggcagccc gggactcttc 1200 tggttcatct ctgatactct gtgggcttct tctggtccaa aaataagtttt cgtgaagtttt 1260 caggtcaatt ggactccgtt tgatttcct tttctgcgat actctaaaac aaggaaaaaa 1320 cagaaactgg cactgggctc taggttaata ggttagtccc aaaaatcata taaatagta 1380 tataaatgca tataaaacat ccaagattga tatatataa gcatggaaaa aatccaactt 1440 cgtgctaatg gttcagtggt tgcgatcata tggtcaagga ggtcgtgatc taccaaaacc 1500 cagacacatc gcgtcatgtt gcactcaagc aggttgtgac accaagagtc ccgcgcgccg 1560 catacaaagc aagcatcatg gtgagacatc tttatgtgct tgagaacatc ttcaattgat 1620 agtgagtttt ttgctaagca ccataaaaat gtccttagct ttgccggcac ggctaccttc 1680 cacggagtgt cccaagcttt ctcattcgct ttttattatt cgatgaccca actctttctt 1740 ctaaccagtc ctccatcctc ctatttgttg caactatcat cttgtatgct gatcgaacga 1800 aaaatatgca gttcttttca aaatcccacg accagaagtc atctatcaat cgtgtactca 1860 ttggaatctg tagtgtagcc gaagcatcag agggcagaaa aacttgctgg atgaattgtg 1920 tattccatag tgcattggct tgatccaaca attcggaaac gaactggggt ggctaggcaa 1980 ttagcgagac aaatgggccc catgttctcc ttcctgggga tccagttcat atttcagatg 2040 tttgtagagt tgccattgcc tatcggcctt attagtctca tcttcatagt gtcccttcct 2100 tcaattactc ctctccaaat ttgagaaagg tggcccccaa gctccgctga aagaaaatcc 2160 gtgtcaagat agtatgcggc ttccaaaatt ctagcattga gagatccggg attttcaaga 2220 atcctccacg cctgactagc cagaagggcc aagttgaaaa tttcaaattc tctgaagccc 2280 aagcctttca tatattttgg catactcatg aagatttttt tatttttagc gaacgacatg 2340 tactgacgta cgtgggccga gactaaggcc tgggccaatt acccatgcag ggcaaggcag 2400 gcagaagcta aggacgatgt gatggtaaaa aattgctaaa aaaaatgtga tgatatattt 2460 tttagaatcc taggtttttc cttccgcgcg tgctatccac ccgacgcgtg ctcctcccac 2520 cgtctctcca fotagccta ccatgctcgt gcacattgta gaacctacca gcaccaccac 2580 cagaagcgcc cgcgcgtggg ccaccccacc ccaccccacc ccaccgccac cccgccccgc 2640 cccgcacact ccggaccttt ccagaaccat cacccctccc ccgatccgac ggccgggatc 2700 gcacgcacca gaacattcca gacccggccg cctccgccat aaaaccctcc cctctccggc 2760 cctccctccc cacttccgtt cgaaatcaaa tcagagagag gcaaagcaaa tcgcaccagg 2820 caaatcacac agagagcgcc gccgtcccaa tattccggcg aaccccaaag cgagcgagcg 2880 attcccagga gaggagaga ggcggagatg gccaagggcg aggggccggc gatcggcatc 2940 gacctcggga cgacctactc ctgcgtcggc gtgtggcagc atgaccgcgt cgagatcatc 3000 gccaacgacc agggcaaccg caccacgccc tcctacgtcg ccttcaccga caccgagcgc 3060 ctcatcggcg acgccgctaa gaaccaggtc gccatgaacc ccaccaacac cgtcttcggt 3120 acgtgcgccc cccctccatc cctccctctc gacccgctgt gtagtatcat gcgtcgtctg 3180 ttagatgaga tctccgtggt tgcctgcggt ccctgttctg ttcttacgcg cacgatgcga 3240 tatgatttgc tctgaataat cggatgtggc tgttgccgaa tccgtcacgc agtcccttgt 3300 ctctgagatt aattacagat ttataattgg ttcatatctg catgtctgtt ctctgttgtt 3360 tggcatgctg tagtcccctg tttctgagat ttgttctatg tcagattgtc aaattagatc 3420 cttaggaatg gatggcttga gttaagttgg gatcctacct tatgattgaa aatacgctat 3480 agatctagtc ttatttaaat tagttgcagc agctgctgtc gattgtgtgc cgcagtcccg 3540 tgtttatctg atctatcaca gaatttatta ttagtttatt ggcatggctg ctctttgttt 3600 atactggtcg aaatgctgtt atgttttttg ttctgaaggt gctcagttgt ttgttctgca 3660 tggttcagta gtagatcctt cagaatggag ggttagaatt aagttggttg gttgaaatta 3720 tgctgtagat gaaaattagt tatactgcag tactagattc ttaagttata catgcttcag 3780 aaaagaatac tcgaattatg tccacaggct accttatgat tgaaagtgtt gaatcatgtt 3840 ttgctattga ttggatgata ttccgaattg aacatgggtt tcgctgtatc gttgcatcct 3900 gtaaatcctt cagaaaatgg tgtttgattg aaattatgct tgttaatttt aaatttctgt 3960 tctcggttat ccttctggaa cagtcccagt ctgcagtagc ttactgaatg tacctcttgt 4020 tctgaaa cagcttgctt gcatttata tctctgtgga accttgaaat gttctgttct 4080 gcttcatttc agtaaaccat cgcagttcat tgctgaacca tgcaactcaa atgttctgtt 4140 cctctgtttc tggttgcaga tgcgaagcgg ctgattggca ggaggttctc tgacccctcc 4200 gtacagagtg acatgaagct gtggcccttc aaggtcatcc ctggcccggc tgacaagcct 4260 atgatcgtcg tcaactacaa gggcgaggag aagcagtttg ctgcggagga gatctcctcc 4320 atggtgctca tcaagatgag ggagatcgct gaggccttcc tcggcaactc cgtcaagaac 4380 gccgtggtca ccgtcccggc ctacttcaac gactctcagc gccaggccac caaggacgct 4440 ggtgccatcg ctgggctgaa cgtgctgcgc ataatcaacg agcccactgc tgctgccatt 4500 gcctatggcc ttgacaagaa ggcgaccagc acgggcgaga agaacgtcct catcttcgac 4560 cttggtggtg gcaccttcga tgtgtctctc ctgaccatcg aggagggcat cttcgaggtc 4620 aaggccaccg ctggtgacac tcacctggga ggggaggact tcgacaaccg catggtgaac 4680 cacttcgtcc aggagttcaa gcgcaagcac aagaaggaca tcaccggcaa cccgcgtgct 4740 ctgcgtcgtc tccgtacggc gtgcgagcgt gccaagcgca cgctgcgtc gactgcacag 4800 accaccattg agattgactc gctgtacgag ggtgtggact tctacaccac catcaccagg 4860 gctcgcttcg aggagctcaa catggacctc ttccgcaagt gcatggagcc ggtggagaag 4920 tgcctccgtg acgccaagat ggacaagagc acggtgcacg acgtggtgct ggtcggtggc 4980 tccacccgta tccccaaggt gcagcagttg ctccaggact tcttcaacgg caaggagctg 5040 tgcaagagca tcaaccccga cgaggcagtg gcgtatggcg cctccgtcca ggctgccatc 5100 ctgagcggcg agggcaacga gaaggtgcag gacctgctgc tgctcgacgt gacgccgctg 5160 tccctgggcc tggaccgc cggcggcgtc atgacgacgc tcatcccgag gaacaccacc 5220 atccccacca agaggagca gtctctcc acctactcgg acaccagcc gggcgtccctg 5280 atccaggtgt acgaggggcga gcgcgccagg accaaggaca acaacctcct gggcaagttc 5340 gagctctctg gcatcccgcc ggcgccccgc ggcgtgcccc agatcacggt gtgcttcgac 5400 atcgacgcca acggcatccct gaacgtgtcg gcggaggaca agacgactgg gcagagaac 5460 aagatcacca tcaccacga caggggcgg ctgagcagg aggagatcga gagatggtg 5520 caggaggcgg agcggtaca ggcggaggac gaggagcaca agagaaggt ggacggccaag 5580 aacgcgctgg agaactacgc ctacacatg cgcacacgg tcaggacga cagatcgcc 5640 tccaagctcg gcgccgacga caggaag gttgaggagg cgatcgaggg caccacagc 5700 tggctggacg caccagct cgccgaggcc gacgagttcg aggacagat gaggagctg 5760 gagggcatct gcaaccccat catcgccaag atgtaccagg gcgccgcgcc ggacatgggc 5820 ggcggcatgg gcatggacga ggacatgcc gccggtgctg gcggcggccgg cccagatc 5880 gaggaggtgg actaagctgt tcgtccaatg tctgcgcgct acttgatggg tgttctgtct 5940 cggtgtcctc tggatgccgc tcggtggtgc cagtcgtctg tcgtcgtgtc atctagctgc 6000 aagtagtagt agtggttcgt tggagtcgtt tgatctgaaa gacatgatgg atgatgatgt 6060 ttgcttgttg gtttggaacc cttgatgctt agagcttctc gttttttaag tttaaaattt 6120 ggtgttcccc tacttttgat cttctctggc tctgtttatt tttttgttca tctgtggttc 6180 tgctatgttc attgatgttc atctgtggca cacggctgac gcctttgccc ttcggcatgg 6240 tggagcttgc aggactgacc caccatggtg agtctgcaac acacaatgct gttgactgag 6300 gagtctagta aatagtcacc gtcttcggga tgccgctctc gtcggctagc agcaagcgat 6360 tatgatgttc ccttgcttgt ttcggttttg aagtgcagtt cgatcatgat gtttgttgct 6420 tgcttgtttc tgcttcggtg ctcccctaat tgatcatgat tagctgctat gttctgcttt 6480 tattttattt tttttgaatt gctgccactt ttagtagttc aacatttttg tcatgtgctg 6540 tatctccatt cttttttctt actactattt tttgaataca cttaaacttt atagcacgta 6600 tctgcaacgc catccagaag ttatcacttg caagatggtg atttttacag gatcccaagc 6660 accattcaaa attatattca tgataagatg caaattgaac acactaatag gctactcatg 6720 tttttttagg ggatcaacaa gtgagccctt gatatcgtgc gttgttgttt gtctgcaggt 6780 tct 6783 <210> 13 <211> 1812 <212> DNA <213> Escherichia coli <400> 13 atgttacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60 ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120 gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180 cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240 ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300 aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360 tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420 cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac 480 ttccatgatt tctttaacta tgccggaatc catcgcagcg taatgctcta caccacgccg 540 aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg 600 tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat 660 caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt gaatccgcac 720 ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa aagccagaca 780 gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 840 ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga agatgcggac 900 ttgcgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg 960 attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 1020 gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt taacctctct 1080 ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga agaggcagtc 1140 aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc gcgtgacaaa 1200 aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg tccgcaaggt 1260 gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320 atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380 gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440 gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca tcagccgatt 1500 atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560 tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620 agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata 1680 ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc gaagtcggcg 1740 gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800 ggcaaacaat ga 1812 <210> 14 <211> 1487 <212> DNA <213> Artificial Sequence <400> 14 atggcggcga caatggcagt gacgacgatg gtgacgagga gcaaggagag ctggtcgtca 60 ttgcaggtcc cggcggtggc attcccttgg aagccacgag gtggcaagac cggcggcctc 120 gagttccctc gccgggcgat gttcgccagc gtcggcctca acgtgtgccc gggcgtcccg 180 gcggggcgcg acccgcggga gcccgatccc aaggtcgtcc gggcggcctg cggcctggtc 240 caggcacaag tcctcttcca ggggtttaac tgggagtcgt gcaagcagca gggaggctgg 300 tacaacaggc tcaaggccca ggtcgacgac atcgccaagg ccggcgtcac gcacgtctgg 360 ctgcctccac cctcgcactc cgtctcgcca caaggctaca tgccaggccg cctatacgac 420 ctggacgcgt ccaagtacgg cacggcggcg gagctcaagt ccctgatagc ggcgttccac 480 ggcaggggcg tgcagtgcgt ggcggacatc gtcatcaacc accggtgcgc ggaaaagaag 540 gacgcgcgcg gcgtgtactg catcttcgag ggcgggactc ccgacgaccg cctggactgg 600 ggccccggga tgatctgcag cgacgacacg cagtactcgg acgggacggg gcaccgcgac 660 cgggcgaggg gttcgcggcg gcgcccgaca tcgaccacct caacccgcgc gtgcagcggg 720 agctctccgc ctggctcaac tggctcaggt ccgacgccgt ggggttcgac ggctggcgcc 780 tcgacttcgc caagggctac tcgccggccg tcgccagaat gtacgtggag agcacggggc 840 cgccgagctt cgtcgtcgcg gagatatgga actcgctgag ctacagcggg gacggcaagc 900 cggcgcccaa ccaggaccag tgccggcagg agctgctgga ctggacgcgg gccgtcggcg 960 ggcccgccat ggcgttcgac ttccccacca agggcctgct gcaggcgggc gtgcaggggg 1020 agctgtggcg gctgcgcgac agctccggca acgcggccgg cctgatcggg tgggcgcccg 1080 agaaggccgt caccttcgtc gacaaccatg acaccgggtc gacgcagaag ctctggccgt 1140 tcccatccga caaggtcatg cagggctacg cctacatcct cacccatcca ggagtcccct 1200 gcattttcta cgaccacatg ttcgactgga acctgaagca ggagatatcc acgctgtctg 1260 ccatcagggc gcggaacggc atccgcgccg ggagcaagct gcggatcctc gtggcggacg 1320 cggacgcgta cgtggccgtc gtcgacgaga aggtcatggt gaagatcggg acaaggtacg 1380 gcgtgagcag cgtggtcccg tcggatttcc acccggcggc gcacggcaag gactactgcg 1440 tctgggagaa agcgagcctc cgcgtcccgg cggggcgcca cctctag 1487 <210> 15 <211> 2736 <212> DNA <213> Zea mays <400> 15 tgcaccggac actgtctggt ggcataccag acagtccggt gtgccagatc agggcaccct 60 tcggttcctt tgctccttg cttttgaacc ctaactttga tcgtttattg gtttgtgttg 120 aacctttatg cacctgtgga atatataatc tagaacaaac tagttagtcc aatcatttgt 180 gttgggcatt caaccaccaa aattatttat aggaaaaggt taaaccttat ttccctttca 240 atctccccct ttttggtgat tgatgccaac acaaaccaaa gaaaatatat aagtgcagaa 300 ttgaactagt ttgcataagg taagtgcata ggttacttag attaaatca attatactt 360 ttacttgata tgcatggttg ctttcttta ttttaacatt ttggaccaca tttgcaccac 420 ttgttttgtt ttttgcaaat ctttttggaa attctttttc aaagtctttt gcaaatagtc 480 aaaggtatat gaataagatt gtaagaagca ttttcaagat ttgaaatttc tccccctgtt 540 tcaaatgctt ttcctttgac taaacaaaac tccccctgaa taaattctc ctcttagctt 600 tcaagagggt tttaataga tatcaattgg aaatatattt agatgctaat tttgaaaata 660 taccaattga aaatcaacat accaattga aattaaacat accaatttaa aaaatttcaa 720 aaagtggtgg tgcggtcctt ttgctttggg cttaatatttt ctcccccttt ggcattaatc 780 gccaaaaacg gagactttgt gagccattta tactttctcc ccattggtaa atgaaatatg 840 agtgaaagat tataccaaat ttggacagtg atgcggagtg acggcgaagg ataaacgata 900 ccgttagagt ggagtggaag ccttgtcttc gccgaagact ccatttccct ttcaatctac 960 gacttagcat agaaatacac ttgaaaacac attagtcgta gccacgaaag agatatgatc 1020 aaaggtatac aaatgagcta tgtgtgtaat gtttcaatca aagtttcgag aatcaagaat 1080 atttagctca ttcctaagtt tgctaaaggt tttatcatct aatggtttgg taaagatatc 1140 gactaattgt tctttggtgc taacataagc aatctcgata tcaccccttt gttggtgatc 1200 cctcaaaaag tgataccgaa tgtctatgtg cttagtgcgg ctgtgttcaa cgggattatc 1260 cgccatgcag atagcactct cattgtcaca taggagagg actttgctca atttgtagcc 1320 atagtcccta aggttttgcc tcatccaaag taattgcaca caacaatgtc ctgcggcaat 1380 atacttggct tcggcggtag aaagagctat tgagttttgt ttctttgaag tccaagacac 1440 cagggatctc cctagaaact gacaagtccc tgatgtgctc ttcctatcaa ttttacaccc 1500 tgcccaatcg gcatctgaat atcctattaa atcaaaggtg gatcccttgg ggtaccaaag 1560 accaaattta ggagtgtaaa ctaaatatct catgattctt ttcacggccc taaggtgaac 1620 ttccttagga tcggcttgga atcttgcaca catgcatata gaaagcatac tatctggtcg 1680 agatgcacat aaatagagta aagatcctat catcgaccgg tatacctttt ggtctacgga 1740 tttacctccc gtgtcgaggt cgagatgccc attagttccc atgggtgtcc tgatgggctt 1800 ggcatcctt attccaaact tgttgagtat gtcttgaatg tactttgttt ggctgatgaa 1860 ggtgccatct tggagttgct tgacttgaaa tcctagaaaa tatttcaact tccccacatcat 1920 agacatctcg aatttcggaa tcatgatcct actaaactct tcacaagtag atttgttagt 1980 agacccaaat atatatcat cacacaat ttggcataca aacaaactt ttgaatggt 2040 tttagtaag agtaggat cggctttact gactctgaag ccattagtga tagaaaatc 2100 tcttaggcat tcataccatg ctgttggggc tgcttgagc ccataaagcg cctttgagag 2160 tttataaaca tggttaggt actcactatc ttcaagccg agaggttgct cacatagac 2220 ctattcaccc cattgatca cttttttggt ctaatagtta tgtataattt 2280 agagtctct gtttaatggc cagatatttc taattaatct aagaatttat gatatttt 2340 aatttttat catgtctgat gagaattac aattggctc aattgggtcc tgaatttaata 2400 atagagtgaa atttaatcca gaggctctat tagaccttc attacktaat accagatat 2460 atataagata gtagagtata gtttaatgt tggcattgtt cattctttct ttgttattt 2520 aatttatgct ttccacggtg gttagtggtt acttctgaag ggtccaaata atgcatgaag 2580 agtttgagga caagagtct gccctaaaaa tagcgatgca aaggcatggt gtccaagcca 2640 tacatatagc gcactaattt tatcagcaga acaatggtat ttataggtcc tagtgcccag 2700 gcaacaagag acacgaataa agcatcgatc acgaca 2736 <210> 16 <211> 344 <212> DNA <213> Zea mays <400> 16 tgacaaagca gcattagtcc gttgatcggt ggaagaccac tcgtcagtgt tgagttgaat 60 gttgatcaa taaaatacgg caatgctgta agggttgttt tttatgccat tgataataca 120 ctgtactgtt cagttgttga actctatttc ttagccatgc caagtgcttt tcttattttg 180 aataacatta cagcaaaaag ttgaaagaca aaaaaaaaa cccccgaaca gagtgctttg 240 ggtcccaagc tactttagac tgtgttcggc gttcccccta aatttctccc ctatatctc 300 actcacttgt cacatcagcg ttctctttcc ctatatctc cacg 344 <210> 17 <211> 1952 <212> DNA <213> Elytrigia elongata <400> 17 ctccgctatg tctatggcac tatggatctt ggcgtcacgc ttcacgcctc cgccgacacc 60 gccctcaccg cctactccga tgcagactg gcgggctacc ctgacactca tcgctccacc 120 tcgggctatt gtgtctacct tggaccctca cttattcgt ggtcgtccaa gcggcagcct 180 acggtctctc gttccagtgc tgaggctgag tatcgtgcgg tggccaacgc cgtcgccgag 240 tgttcgtggc ttcgccagct gcttcaggag ctttcctgcc ctgttgactg tgccacggtg 300 gtctactgcg acaacgtctc ggcggtctac ctctccgcta acccggtgca tcatcgacgg 360 accaagcata ttgagttgga tattcatttt gttcgggaac aggtggccct tggtcatatt 420 cgtgttttac accaggggcg gaactgggcc tggggcaact ggggctatag ccccaggcat 480 ggcccatcta gtaggctacc cagcaggaat tttcccatat attagtacac ttgtagaggc 540 ccagccccag gcctcagccc acagagctct ccagtttgga cgatcgctcc aaagaatcac 600 cagcagccac gatgcaagag cgctccgatc ctcacgcttc gtctcggtag atcgaccgaa 660 aagtctcacg ctgcgccgcc aactctctcg actgctcgac tccagcagac gctcgcccag 720 gcggccggcg ctcggaaccg cggatctatc gcgccgtttg aataaagcaa tctccatgcc 780 tgatctcctg tgatccacac ccgattgaaa aggtaatgct aaatacctag gtttgtcgta 840 tcatctaata tttcccccta gttccttttt atcagctgaa atcaatgaat tggtgatgtt 900 aaattgtatt gctttagtca attttattcc atcgttcttt gttaacaggg gaaaataga 960 aatccaagat gaagtggacc cggacagttt ttcgtgatca tggtataata ttttcttgct 1020 tcccaaaatt tcatatgaac aataacttgt ctatgtctag ttcttgattt taatactca 1080 actttatgtt tacagctccg ggtcctagct actattgctt gtgaagaaac gatggcctca 1140 agtgagacaa gccttcatta atttggtacg actgtttgta atgatgcact ttgttatatt 1200 gatgagatat ttattaaatt tgctcttatg gtaataagta attcatattc tattcttaat 1260 ctgaatgtgt ggtgtacttc taacatgcat tggtacgtag agtatttggc attttatttc 1320 cttaggttg tgatatttgg cgttatttgt agtggtttag cttagtcct aggcttcgag 1380 aaatcctggc tccgcctctg ttttacacgt tcctactttt caacaatttg cagatattat 1440 gaccaaaggc ttgcctacgg cgtcatttaa ggagttccgg tccagtcttt gcgtcagccg 1500 cggtgccgct tcgactgcgg ggggtgttga gtacatgtgt tatgtgtata ttgtgtattg 1560 ggtccgtctc ctagttcttt gtatagttga ggtctatggc ccaccgttgt acatcatata 1620 tacgtgccta tgcacgagag caatacatca tgcaatcata gtctcataca ctagcgatct gaagctttac accaggttat aggttttctt cctcgacttt ggacggatat catcatcggt 1740 gccgccggtg agatgtatag gcggcggaac ggcggtgctg aacgtccgtg ttcattgcac agatggatcc tttgctagcc gtttggtgta cctaactcgg acttaggtgt tgcttgcggt gaccacatgg tgtattaata aagcacaacg gccttctgcc cacggatcaa acacagtaca cacgggcagt tgggaaaaag ataacgggga gg <210> 18 <211> 822 <212> DNA <213> Elytrigia elongata <400> 18 atggcgaagg aaggggtgaa gagaggggca tggactggca aggaggacga aaccttggct tcctacgtca aggcgcatgg cgaaggcaga tggaacgaag tccctcagag agctggtaag tccctagcta ggtaaacaga atcaatctag agaatggagt gctaaacgta atttacaggt cttcggcggt gcggcaagag ctgtcggctg cggtggctga actacctccg gccgaacatc 240 aagcgggga acatatccaa tgatgaggag gagatcatcg tcaggctcca cgctctcctt ggcaacaggt ggtcgctcat cgctggcagg ttgcctggcc gaacagacaa cgaaatcaag 360 aactactgga acagcaccct tggccggaag gtgcttcccg caccacattc cgccaccagg 420 atggttgcca cgccagacac ctccgccggc tccggatctt acaaagaggc gtcggcaagt 480 ctgtcaagct ctggacctgg tacaagcgac aaggctgcga cgccgtcgcc gctgccggcc 540 acgctgtgg cgccaaagcc tgtgaggcac acggggcacc ccttcttcct ccgggatagg 600 ccgccgccct tgccggttgc ggagacgcga accgtggcca acggggatgc ctgcagcggc 660 agcagctcgg tgacatcgga gttcccggct gtaccgccct cgttaggcag cgacgactgg 720 atggacgaag tgagagcctt ggaatcgttt ctcgagtccg acgaagactg ggtaaactct 780 gtggagatgc caacgcccaa ctctcacaca tcatcgagtt aa 822 <210> 19 <211> 441 <212> DNA <213> Elytrigia elongata <400> 19 gtacccagta taaccgtaca tgcacccaac caactatata gtgtaaggaa taaaacgacg 60 tcattagcac tggagggctg ccacgtcggc cggcggatct ttggctcgcg agtcgttgga 120 tgtggcacaa ttcagcagct caacgtctcc ctctacttcg caacagagtt tttgttttt 180 atttgcgctg gtaaaggagt tttattccat atgcataggg ttacaatcaa gaggcaggag 240 atcctccaca cacggcggac ctcgaccaag ccatacagca gtagtgctct ctgtacgact 300 atacagggcc aaacgatctg ctaccctatt ctgcagacga ctaattttca tgggaataaa 360 aactctatct accataagat gacgaatctc agcaactata tgtccatagg ctgatctggt 420 taaactgtca ccagacaaag c 441 <210> 20 <211> 2084 <212> DNA <213> Elytrigia elongata <400> 20 tttcttaggg cattatgatg atgacttggc atcactagtg cggagttact gtagcatgat 60 tctcggggtg tcacaacagt acatcctaga gatcttggat cgcgccaata tgaccaactg 120 taaccccact gctactcctg tcgacaccaa atccaaacta tccgctactg acagtccacc 180 agttcccgat cctacttat gtcgtagcat tgctggagcc ttccagtgcc tcacccgcac 240 tcaccccgat atctccatg cggtacagca ggcctgccta tttatgcacg atcctcgcgc 300 ccccaccttc agttcgtcaa aaggatctta cgctatctca aggccacatc acattatgga 360 ctgcagctta ccacttctcc tggacatgat ctcgtggctt attccgacgc agattgggcc 420 ggctgtcccg ctacactgaa gtccacctct ggtttctgtg tgttccttgg ccccaacttg 480 gtatcttggt cctccaaacg ccaacacacc gtctcttgat cgagcgccga agctgagtat 540 agggcggttg caaattgcgt cgctgaatcc tgctggcttc gccagcttct acaggagcta 600 catcaacctt cgtctagcgc cacaatcgtt tacactgaca acatcagtgg gacgtacctc 660 tcctccaacc ccgtctagca ccaacgcacc aagcacgtca agatcgactt gcattttgtc 720 cgagatcggg tggcacttgg agaggctcgg atcctgcacg tttcgtccaa ctcccaattt 780 gcggacgtgt tcaccaaggg cctgcctaca tccatcttcc atgagtcttg ttccagtctg 840 aacatccacc gtgtggagct ccgactgcgg ggtgctgtta aagtgtgttg taatattgta 900 catagacttg tcacgttgag gcctgcatgc ccatctgttg aaactacctc cacctctctg 960 ggatcacgat cccccttctc aatttttttt ttgaacacag tacagacgca agcactcata 1020 tacacgcgca tacactcacc cctatgaacg cacacacgca caccctaccc ctatgagcac 1080 ccccgaaaga ctgagccagc atatcatctt gaaatttacg aaatcaccgt agtcacctcg 1140 tcgtcgacgg gaacgtctcc tccactgaa tgcacatcgc cggaaatcct gaatgaatc 1200 caggaataa tgcgagcacc aggatttgaa ccctggtggg ttggggatac cacagtccct 1260 ctaaccatcc aaccacaggt tggttcgcat cccccttctc agttgatggc ctgcgtgcct 1320 acttcttcgg footgttgc agctctcaat atatgtactc tgactcccct aaaatatata 1380 tatatatact ctgaccactg gttgtaatgt aaccaaca cacagtgagg aattccaaac 1440 cataacttgg ttgtggggtg accattccac agcgttttat gcaacgtggc ggcagactag 1500 gttatcaca caaaaaagat ccatgcaagt ttccacgtgc tgctgcttcc aacgttgaag 1560 tgttttctgt gtcgtgcgtc gatcgtattt ggatcttctc agcaacgata gcggcgagat 1620 ttcgttgtat ctatgtcttc agagcggttc gagtagattt tttccttcat ttttcggact 1680 gaaaaag aggaaatgct accttccatc gttcgtactg tgtagttgtt ggtgcaacag 1740 cccgcgcatt tgcataaggt gccttgtata tatgtggaaa cgttcagttg ctacgtacct 1800 cttcgtggtc tgcacacgca tacagtctgc acactgatac cttaaagtaa gctttaattt 1860 tgcttcagtt tacttctcca gttcttggct agctgaatct cgacttctag tttggtcgga 1920 atttatataa ctttcctgcg tgtacagtac acagcgtatg taccatgcat ataactttat 1980 atacctttct tgatttcttt tgctgcttgt ttcctcgacc tagcgtctca ccttgtgagt 2040 tgtgcatgcg ggaaatagct actcagcggt gtggtaatcg atca 2084 <210> 21 <211> 1683 <212> DNA <213> Elytrigia elongata <400> 21 atggcgctat cagctcctcc cagtcaggaa cagccgtcgg ggaagcaatt cggctaccag 60 ctcgctgctg ctgtgaggag catcaactgg acttatggca tattttggtc catttccgcc 120 agcccgcgcc caggccactc ctcagttctg gcgtggaagg atgggttcta caacggcgag 180 ataaagacta gaaagattac cggctcgacc actacggagc ttacagcgga cgagcgcgtc 240 atgcacagaa gcaagcaact gagggagctc tacgaatcgc tcttgcccgg caactccaac 300 aaccgggcaa ggcgaccaac cgcctcactg tcaccggagg atctcgggga cggcgagtgg 360 tattacacca taagcatgac ttacaccttc caccctaatc aagggttgcc aggcaaaagc 420 tttgcgagca atcaacatgt ttggctgtac aacgctcaat acgcaaacac cagagttttc 480 ccccgcgcgc tcttagcaaa gactgcttct attcagacaa tcgtttgcat tcccttcatg 540 ggcggtgtgc ttgagctcgg aacgtcggat caggtgttgg aggacccgag catggtgaag 600 cggatcagca cgtctttctg ggagctgcac ttgccgtcat ccttggagtc gaaggatccg 660 agctccagca catcagcaaa cgataccagg gaggccaccg acatcatctt gttcgaggat 720 ttcgaccaca acgacacagt tgagggggtg atctctgagc aaagggaggt ccagtgcccg 780 tccaacgtca atctggagcg cctcacaaag cagatggacg agttccacag ccttctcggt 840 ggactggacg tgcatcctct cgaagacaga tggatcatgg acgagccctt tgagtttacg 900 ttttccccag aagtggcgcc ggctatggat atgccgagca ccgacgatgt catcgtcact 960 ttaagtaggt ccgaaggctc tcgtccatcc tgcttcacag cgtggaaggg atcatccgag 1020 tcgaaatacg tggctggcca ggtcgttggg gagtcacaga agttgctgaa taaagttgtg 1080 gctggtggtg catgggcgag caattatggc ggtcgcacca tggtgagagc tcagggaatt 1140 aacagcaaca cccatgtcat gacagagaga agacgccggg agaaactcaa cgagatgttc 1200 ctggttctca agtcactggt cccgtccatt cacaaggtag acaaagcatc catcctcaca 1260 gaaacgatag gttatcttag agaactgaag caaagggtag atcagctaga atccagccgg 1320 tcaccgtctc acccaaaaga aacaacagga ccgagcagaa gccatgtcgt cggcgctagg 1380 aagaagatag tctcggccgg atccaagagg aaggcgccag ggctggagag cccgagcaat 1440 gtcgtgaacg tgacgatgct ggacaaggtg gtgctgttgg aggtgcagtg cccgtggaag 1500 gagctgctga tgacacaagt gtttgacgcc atcaagagcc tctgtctgga cgttgtctcc 1560 gtgcaggcat ccacatcagg tggccgtctt gacctcaaga tacgagctaa tcagcagctt 1620 gcggtcggtt ctgctatggt ggcacctggg gcaatcaccg aaacacttca gaaagctata 1680 tag 1683 <210> 22 <211> 618 <212> DNA <213> Elytrigia elongata <400> 22 gcaagacttt tttgacacta gtgtattgtt aaaaacgtct tatattacga gacgaaggga 60 gtacttaaca actcaatcta caaaaaatca tgagttgggc tctatagtct gcttcgccca 120 acaaccaggt cggactagtg tgaattactg tacaccaaaa aaattctgaa ataattactg 180 aacacgtcat gacgtgcta cttccctcca aaagtatttc agatctacat tccgcctgtc 240 aattaagcac gaaatttgaa gccgattgtg ctgcccggcc ctactcacca ctacagaaat 300 gcctagctag cggtggcggc actaccgttc ggattctttc cagcttcctg aatagccag 360 cttcccgcga tgccagccgc agccagcttc tgacgggagt agcgattcgt tcggcagcag 420 agagtagctc tagacaccgt acgaacttga atagcccag gaaggaagcc ggcgtgaggg 480 agggagctag gaggagtgag ctctctggcg tggatacggc ggcaagctcg atggagcagg 540 tcgaccaggt gccgacggcg agcaacagag gaagttgagg cagggtcggg cgtccgggtg 600 cgctgaggat gggccatg 618 <210> 23 <211> 2988 <212> DNA <213> Elytrigia elongata <400> 23 atggccaagg gcgaggggcc ggcgatcggc atcgacctcg ggacgaccta ctcctgcgtc 60 ggcgtgtggc agcatgaccg cgtcgagatc atcgccaacg accagggcaa ccgcaccacg 120 ccctcctacg tcgcttcac cgacaccgag cgcctcatcg gcgacgccgc taagaaccag 180 gtcgccatga accccaccaa caccgtcttc ggtacgtgcg ccccccctcc atccctccct 240 ctcgacccgc tgtgtagtat catgcgtcgt ctgttagatg agatctccgt ggttgcctgc 300 ggtccctgtt ctgttcttac gcgcacgatg cgatatgatt tgctctgaat aatcggatgt 360 ggctgttgcc gaatccgtca cgcagtccct tgtctctgag attaattaca gatttataat 420 tggttcatat ctgcatgtct gttctctgtt gtttggcatg ctgtagtccc ctgtttctga 480 gatttgttct atgtcagatt gtcaaattag atccttagga atggatggct tgagttaagt 540 tgggatccta ccttatgatt gaaaatacgc tatagatcta gtcttattta aattagttgc 600 agcagctgct gtcgattgtg tgccgcagtc ccgtgtttat ctgatctatc acagaattta 660 ttattagttt attggcatgg ctgctctttg tttatactgg tcgaaatgct gttatgtttt 720 ttgttctgaa ggtgctcagt tgtttgttct gcatggttca gtagtagatc cttcagaatg 780 gagggttaga attaagttgg ttggttgaaa ttatgctgta gatgaaaatt agttatactg 840 Foottactaga ttcttaagtt atacatgctt cagaaaagaa tactcgaatt atgtccacag 900 gctacctat gattgaaagt gttgaatcat gttttgctat tgattggatg atattccgaa 960 ttgaacatgg gtttcgctgt atcgttgcat cctgtaaatc cttcagaaaa tggtgtttga 1020 ttgaaattat gcttgttaat tttaaatttc tgttctcggt tatccttctg gaacagtccc 1080 agtctgcagt agcttactga atgtacctct tgttctgaag aaacagcttg cttgcattat 1140 taatctctgt ggaaccttga aatgttctgt tctgcttcat ttcagtaaac catcgcagtt 1200 cattgctgaa ccatgcaact caaatgttct gttcctctgt ttctggttgc agatgcgaag 1260 cggctgattg gcaggaggtt ctctgacccc tccgtacaga gtgacatgaa gctgtggccc 1320 ttcaaggtca tccctggccc ggctgacaag cctatgatcg tcgtcaacta caagggcgag 1380 gagaagcagt ttgctgcgga ggagatctcc tccatggtgc tcatcaagat gagggagatc 1440 gctgaggcct tcctcggcaa ctccgtcaag aacgccgtgg tcaccgtccc ggcctacttc 1500 aacgactctc agcgccaggc caccaaggac gctggtgcca tcgctgggct gaacgtgctg 1560 cgcataatca acgagcccac tgctgctgcc attgcctatg gccttgacaa gaaggcgacc 1620 agcacgggcg agaagaacgt cctcatcttc gaccttggtg gtgacacctt cgatgtgtct 1680 ctcctgacca tcgaggaggg catcttcgag gtcaaggcca ccgctggtga cactcacctg 1740 ggaggggagg acttcgacaa ccgcatggtg aaccacttcg tccaggagtt caagcgcaag 1800 cacaagaagg acatcaccgg caacccgcgt gctctgcgtc gtctccgtac ggcgtgcgag 1860 cgtgccaagc gcacgctgtc gtcgactgca cagaccacca ttgagattga ctcgctgtac 1920 gagggtgtgg acttctacac caccatcacc agggctcgct tcgaggagct caacatggac 1980 ctcttccgca agtgcatgga gccggtggag aagtgcctcc gtgacgccaa gatggacaag 2040 agcacggtgc acgacgtggt gctggtcggt ggctccaccc gtatccccaa ggtgcagcag 2100 ttgctccagg acttcttcaa cggcaaggag ctgtgcaaga gcatcaaccc cgacgaggca 2160 gtggcgtatg gcgcctccgt ccaggctgcc atcctgagcg gcgagggcaa cgagaaggtg 2220 caggacctgc tgctgctcga cgtgacgccg ctgtccctgg gcctggagac cgccggcggc 2280 gtcatgacga cgctcatccc gaggaacacc accatcccca ccaagaagga gcaggtcttc 2340 tccacctact cggacaacca gccgggcgtc ctgatccagg tgtacgaggg cgagcgcgcc 2400 aggaccaagg acaacaacct cctgggcaag ttcgagctct ctggcatccc gccggcgccc 2460 cgcggcgtgc cccagatcac ggtgtgcttc gacatcgacg ccaacggcat cctgaacgtg 2520 tcggcggagg acaagacgac tgggcagaag aacaagatca ccatcaccaa cgacaagggg 2580 cggctgagca aggaggagat cgagaagatg gtgcaggagg cggagcggta caaggcggag 2640 gacgaggagc acaagaagaa ggtggacgcc aagaacgcgc tggagaacta cgcctacaac 2700 atgcgcaaca cggtcaagga cgacaagatc gcctccaagc tcggcgccga cgacaagaag 2760 aaggttgagg aggcgatcga gggcaccatc agctggctgg acgccaacca gctcgccgag 2820 gccgacgagt tcgaggacaa gatgaaggag ctggagggca tctgcaaccc catcatcgcc 2880 aagatgtacc agggcgccgc gccggacatg ggcggcggca tgggcatgga cgaggacatg 2940 cccgccggtg ctggcggcgc cggccccaag atcgaggagg tggactaa 2988 <210> 24 <211> 1947 <212> DNA <213> Elytrigia elongata <400> 24 atggccaagg gcgaggggcc ggcgatcggc atcgacctcg ggacgaccta ctcctgcgtc 60 ggcgtgtggc agcatgaccg cgtcgagatc atcgccaacg accagggcaa ccgcaccacg 120 ccctcctacg tcgcttcac cgacaccgag cgcctcatcg gcgacgccgc taagaaccag 180 gtcgccatga accccaccaa caccgtcttc gatgcgaagc ggctgattgg caggaggttc 240 tctgacccct ccgtacagag tgacatgaag ctgtggccct tcaaggtcat ccctggcccg 300 gctgcaagc ctatgatcgt cgtcaactac aagggcgagg agaagcagtt tgctgcggag 360 gagatctcct ccatggtgct catcaagatg agggagatcg ctgaggcctt cctcggcaac 420 tccgtcaaga acgccgtggt caccgtcccg gcctacttca acgactctca gcgccaggcc 480 accaagggacg ctggtgccat cgctgggctg aacgtgctgc gcataatcaa cgagcccact 540 gctgctgcca ttgcctatgg ccttgacaag aaggcgacca gcacgggcga gaagaacgtc 600 ctcatcttcg accttggtgg tgacacctt gatgtgtctc tcctgaccat cgaggagggc 660 atcttcgagg tcaaggccac cgctggtgac actcacctgg gaggggaga cttcgcaac 720 cgcatggtga accacttcgt ccaggaggttc aagcgcaagc aagaagga catcaccggc 780 aacccgcgtg ctctgcgtcg tctccgtacg gcgtgcgagc gtgccaagcg cacgctgtcg 840 tcgactgcac agaccaccat tgagattgac tcgctgtacg agggtgtgga cttctacacc 900 accatcacca gggctcgctt cgaggagctc aacatggacc tcttccgcaa gtgcatggag 960 ccggtggaga agtgcctccg tgacgccaag atggagaaga gcacggtgca cgacgtggtg 1020 ctggtcggtg gctccacccg tatccccaag gtgcagcagt tgctccagga cttcttcaac 1080 ggcaaggagc tgtgcaagag catcaacccc gacgaggcag tggcgtatgg cgctcccgtc 1140 caggctgcca tcctgagcgg cgagggcaac gagaaggtgc aggacctgct gctgctcgac 1200 gtgacgccgc tgtccctggg cctggagacc gccggcggcg tcatgacgac gctcatcccg 1260 aggaacacca ccatccccac caagaaggag caggtcttct ccacctactc ggacaaccag 1320 ccgggcgtcc tgatccaggt gtacgagggc gagcgcgcca ggaccaagga caacaacctc 1380 ctgggcaagt tcgagctctc tggcatcccg ccggcgcccc gcggcgtgcc ccagatcacg 1440 gtgtgcttcg acatcgacgc caacggcatc ctgaacgtgt cggcggagga caagacgact 1500 gggcagaaga acaagatcac catcaccaac gacaaggggc ggctgagcaa ggaggagatc 1560 gagaagatgg tgcaggaggc ggagcggtac aaggcggagg acgaggagca caagaagaag 1620 gtggacgcca agaacgcgct ggagaactac gcctacaaca tgcgcaacac ggtcaaggac 1680 gacaagatcg cctccaagct cggcgccgac gacaagaaga aggttgagga ggcgatcgag 1740 ggcaccatca gctggctgga cgccaaccag ctcgccgagg ccgacgagtt cgaggacaag 1800 atgaaggagc tggagggcat ctgcaacccc atcatcgcca agatgtacca gggcgccgcg 1860 ccggacatgg gcggcggcat gggcatggac gaggacatgc ccgccggtgc tggcggcgcc 1920 ggccccaaga tcgaggaggt ggactaa 1947 <210> 25 <211> 648 <212> PRT <213> Elytrigia elongata <400> 25 Met Ala Lys Gly Glu Gly Pro Ala Ile Gly Ile Asp Leu Gly Thr Thr 1 5 10 15 Tyr Ser Cys Val Gly Val Trp Gln His Asp Arg Val Glu Ile Ile Ala 20 25 30 Asn Asp Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp 35 40 45 Thr Glu Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Met Asn 50 55 60 Pro Thr Asn Thr Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Arg Phe 65 70 75 80 Ser Asp Pro Ser Val Gln Ser Asp Met Lys Leu Trp Pro Phe Lys Val 85 90 95 Ile Pro Gly Pro Ala Asp Lys Pro Met Ile Val Val Asn Tyr Lys Gly 100 105 110 Glu Glu Lys Gln Phe Ala Ala Glu Glu Ile Ser Ser Met Val Leu Ile 115 120 125 Lys Met Arg Glu Ile Ala Glu Ala Phe Leu Gly Asn Ser Val Lys Asn 130 135 140 Ala Val Val Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala 145 150 155 160 Thr Lys Asp Ala Gly Ala Ile Ala Gly Leu Asn Val Leu Arg Ile Ile 165 170 175 Asn Glu Pro Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Ala 180 185 190 Thr Ser Thr Gly Glu Lys Asn Val Leu Ile Phe Asp Leu Gly Gly Asp 195 200 205 Thr Phe Asp Val Ser Leu Leu Thr Ile Glu Glu Gly Ile Phe Glu Val 210 215 220 Lys Ala Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn 225 230 235 240 Arg Met Val Asn His Phe Val Gln Glu Phe Lys Arg Lys His Lys Lys 245 250 255 Asp Ile Thr Gly Asn Pro Arg Ala Leu Arg Arg Leu Arg Thr Ala Cys 260 265 270 Glu Arg Ala Lys Arg Thr Leu Ser Ser Thr Ala Gln Thr Thr Ile Glu 275 280 285 Ile Asp Ser Leu Tyr Glu Gly Val Asp Phe Tyr Thr Thr Ile Thr Arg 290 295 300 Ala Arg Phe Glu Glu Leu Asn Met Asp Leu Phe Arg Lys Cys Met Glu 305 310 315 320 Pro Val Glu Lys Cys Leu Arg Asp Ala Lys Met Asp Lys Ser Thr Val 325 330 335 His Asp Val Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Val Gln 340 345 350 Gln Leu Leu Gln Asp Phe Phe Asn Gly Lys Glu Leu Cys Lys Ser Ile 355 360 365 Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ser Val Gln Ala Ala Ile 370 375 380 Leu Ser Gly Glu Gly Asn Glu Lys Val Gln Asp Leu Leu Leu Leu Asp 385 390 395 400 Val Thr Pro Leu Ser Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr 405 410 415 Thr Leu Ile Pro Arg Asn Thr Thr Ile Pro Thr Lys Lys Glu Gln Val 420 425 430 Phe Ser Thr Tyr Ser Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr 435 440 445 Glu Gly Glu Arg Ala Arg Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe 450 455 460 Glu Leu Ser Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Thr 465 470 475 480 Val Cys Phe Asp Ile Asp Ala Asn Gly Ile Leu Asn Val Ser Ala Glu 485 490 495 Asp Lys Thr Thr Gly Gln Lys Asn Lys Ile Thr Ile Thr Asn Asp Lys 500 505 510 Gly Arg Leu Ser Lys Glu Glu Ile Glu Lys Met Val Gln Glu Ala Glu 515 520 525 Arg Tyr Lys Ala Glu Asp Glu Glu His Lys Lys Lys Val Asp Ala Lys 530 535 540 Asn Ala Leu Glu Asn Tyr Ala Tyr Asn Met Arg Asn Thr Val Lys Asp 545 550 555 560 Asp Lys Ile Ala Ser Lys Leu Gly Ala Asp Asp Lys Lys Lys Val Glu 565 570 575 Glu Ala Ile Glu Gly Thr Ile Ser Trp Leu Asp Ala Asn Gln Leu Ala 580 585 590 Glu Ala Asp Glu Phe Glu Asp Lys Met Lys Glu Leu Glu Gly Ile Cys 595 600 605 Asn Pro Ile Ile Ala Lys Met Tyr Gln Gly Ala Ala Pro Asp Met Gly 610 615 620 Gly Gly Met Gly Met Asp Glu Asp Met Pro Ala Gly Ala Gly Gly Ala 625 630 635 640 Gly Pro Lys Ile Glu Glu Val Asp 645

Claims

1. A use of a fertility-related gene, an expression cassette, an expression vector or an engineered bacterium in regulating wheat fertility, characterized in that The fertility-related gene, expression cassette, expression vector, and engineered bacteria contain one of the following nucleotide sequences: (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

3.

2. A method for regulating wheat fertility, wherein the method suppresses or mutates fertility-related genes in wheat to affect their expression levels, thereby regulating wheat fertility, characterized in that: The fertility-related gene TaMS8 The nucleotide sequence is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

3.

3. The method according to claim 2, wherein the mutation is performed using ZFN, TALEN and / or CRISPR / Cas9.

4. The method according to claim 2, characterized in that The method comprises using TaMS8 The nucleotide sequence complement of a gene is determined by TaMS8 The male sterility phenotype caused by gene mutation ms8 The male sterile line was restored to fertile.

5. The method according to claim 4, TaMS8 The sequence of the gene mutation is shown in SEQ ID NO: 23 or 24, and the amino acid is shown in SEQ ID NO:

25.

6. Use of the method according to any one of claims 2 to 5 in regulating wheat fertility.

7. A method for producing or breeding wheat male sterile lines, the method comprising the following steps: (a) To ms8 The following vector is transferred into the male sterile line to obtain a maintenance line containing the following vector, wherein the vector contains: fertility-related genes TaMS8 , the fertility-related gene TaMS8 Can be restored ms8 male fertility of male sterile lines; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, so that the fertile male gametes produced in the plant do not contain the vector; and screening genes, which can be used to sort transgenic seeds from non-transgenic seeds; and (b) self-pollinating the maintainer plants formed after the above vector is introduced, and producing vector-free plants ms8 Male sterile seeds and maintainer seeds containing vectors; Or use pollen from maintainer plants to ms8 In the pollination of sterile plants, ms8 Sterile line pollination ms8 Sterile seeds; The fertility-related genes TaMS8 The nucleotide sequence is selected from one of the following groups of sequences: (i) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (ii) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

3.

8. The production or breeding method according to claim 7, wherein the fertility-related gene TaMS8 Expression is driven by a pollen-specific promoter. 9 . The production or propagation method according to claim 8 , wherein the nucleotide sequence of the pollen-specific expression promoter is as shown in SEQ ID NO:

10.

10. The production or propagation method according to claim 7, wherein the pollen inactivation gene comprises a barnase gene, an amylase gene, and a DAM methylase.

11. The production or propagation method according to claim 10, wherein the pollen inactivation gene is a maize α-amylase gene. 12 . The production or breeding method according to claim 11 , wherein the nucleotide sequence of the corn α-amylase gene is shown in SEQ ID NO:

14.

13. The production or propagation method according to claim 10, wherein the pollen inactivation gene is linked to a promoter that is preferentially expressed in male gametes.

14. The production or propagation method according to claim 13, wherein the promoter is a PG47 promoter or a Zm13 promoter.

15. The breeding method according to any one of claims 7 to 14, wherein the screening gene comprises a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene, and a fluorescent protein gene.

16. The production or breeding method according to claim 15, wherein the screening gene comprises: Blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene.

17. A method for producing or propagating a wheat maintainer line, the method comprising the following steps: (a) To ms8 The following vector was transferred into the male sterile line to obtain ms8 A maintenance line of a male sterile line, wherein the vector comprises: a fertility-related gene TaMS8 , the fertility-related gene TaMS8 Can be restored ms8 male fertility of male sterile lines; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, so that the fertile male gametes produced in the plant do not contain the vector; and screening genes, which can be used to sort transgenic seeds from non-transgenic seeds; and (b) self-pollinating the maintainer plants formed after the above vector is introduced, and producing vector-free plants ms8 Male sterile seeds and maintainer seeds containing vectors; The fertility-related genes TaMS8 The nucleotide sequence is selected from one of the following groups of sequences: (i) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (ii) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

3.

18. The production or propagation method according to claim 17, wherein the fertility restorer gene TaMS8 Expression is driven by a pollen-specific promoter.

19. The production or propagation method according to claim 18, wherein the nucleotide sequence of the pollen-specific expression promoter is shown as SEQ ID NO:

10.

20. The production or propagation method according to claim 17, wherein the pollen inactivation gene comprises a barnase gene, an amylase gene, and a DAM methylase.

21. The production or propagation method according to claim 20, wherein the pollen inactivation gene is a maize alpha amylase gene.

22. The production or breeding method according to claim 21, wherein the nucleotide sequence of the corn alpha-amylase gene is shown as SEQ ID NO:

14.

23. The production or propagation method according to claim 20, wherein the pollen inactivation gene is linked to a promoter that is preferentially expressed in male gametes.

24. The production or propagation method according to claim 23, wherein the promoter is a PG47 promoter or a Zm13 promoter.

25. The breeding method according to any one of claims 17-24, wherein the screening gene comprises a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene, and a fluorescent protein gene.

26. The production or breeding method according to claim 25, wherein the screening gene comprises: Blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene.

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