Male fertility-related gene and use thereof

Abstract

The prevent invention belongs to the field of biotechnology. Provided are a male fertility-related gene and the use thereof. Phenotypic and genetic analysis is performed on a wheat male sterile mutant ms9 derived from EMS mutagenesis, and the wheat fertility-related gene TaMS9 is obtained by using the Mutmap analysis after re-sequencing in combination with a traditional map-based cloning method. The cloned wheat fertility gene and the male sterile mutant thereof are applied to new-generation wheat hybrid breeding technologies, and hold significant theoretical and practical importance for the utilization of heterosis in wheat.

Description

A male fertility-related gene and its application Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and specifically relates to the cloning of recessive nuclear male sterility genes in plants, a breeding method of male sterile lines thereof and the application of the same in hybrid breeding, and more specifically relates to the cloning of a recessive nuclear male sterility gene in wheat and its promoter, and the application of the same in hybrid breeding. Background Art

[0002] Wheat is one of the most important grain crops in modern agriculture, serving as a staple food for approximately 35% of the world's population. With my country's economic development and population growth, demand for wheat is also increasing. However, increasing wheat yields through conventional breeding alone is difficult to achieve. Fully leveraging hybrid vigor in wheat is the preferred approach to comprehensively enhance wheat production capacity.

[0003] Because wheat is a monoecious, self-pollinating plant, artificial de-tasseling for hybrid seed production is impractical. Therefore, male sterility is an important agronomic trait necessary to exploit hybrid vigor to increase crop yields. The identification of male sterile mutants and the cloning of male fertility genes are fundamental to the development of hybrid seed production systems. Male sterility in wheat is categorized into two types: genetic male sterility (GMS) and cytoplasmic male sterility (CMS). GMS materials are further divided into dominant and recessive GMS materials based on their inheritance. Due to their inherent genetic characteristics, GMS can only be used in conventional recurrent breeding and backcrossing and cannot serve as maternal parents for hybrid seed production. Recessive GMS materials, however, are susceptible to reversible sterility and can be used as maternal parents for wheat hybrid seed production.

[0004] Because wheat is an allohexaploid, the functional redundancy of its three subgenomes results in significantly fewer recessive mutants controlled by single genes in wheat compared to other diploid species. To date, only three recessive nuclear male fertility regulatory loci controlled by single genes have been reported in wheat: MS1, MS5, and NWMS1 (Klindworth et al., 2002; Li et al., 2019). The MS1 and MS5 genes have been cloned (Tucker et al., 2017; Wang et al., 2017; Pallotta et al., 2019). MS1 is the most widely used nuclear male sterile material in practice. Its corresponding gene encodes a lipid transfer protein, a newly evolved gene in the Poaceae family (Wang et al., 2017; Tucker et al., 2017). The MS5 gene also encodes a lipid transfer protein, but it is not of the same class as MS1 (Pallotta et al., 2019).

[0005] To obtain more stable and reliable recessive male sterile mutants and their corresponding genes for use in wheat hybrid breeding, we previously screened a large number of male sterile mutants from the wheat EMS mutant library. Among these, ms9 is a novel, single-gene-controlled, recessive nuclear male sterile material with a pollenless appearance. We conducted genetic analysis, gene cloning, and promoter analysis on this mutant, and successfully applied the ms9 mutant and its corresponding TaMS9 gene to a new generation of hybrid wheat technology systems. Summary of the Invention

[0006] All references cited 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 this 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 TaMS9, 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 under stringent conditions to a DNA sequence described in (a) or (b); or

[0012] (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has 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 will appreciate that the fertility-related genes described herein also include homologous gene sequences that are highly homologous to the nucleotide sequence or protein sequence of the TaMS9 gene and have the same fertility regulation or restoration function. The highly homologous homologous genes having fertility regulation functions include DNA sequences that can hybridize with DNA having the sequence shown in SEQ ID NO: 1 or 2 under stringent conditions. Alternatively, they may be nucleotide sequences whose encoded amino acid sequence has greater than 85% similarity to the amino acid sequence of the protein shown in SEQ ID NO: 3. The "stringent conditions" used herein are well known and include, for example, hybridization in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA at 53°C-60°C for 12-16 hours, followed by washing at 62°C-68°C with a wash solution containing 0.5×SSC and 0.1% SDS for 15-60 minutes.

[0015] The aforementioned homologous genes also include DNA sequences that have 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 have fertility regulating functions, and can be isolated from any plant. The percentage of sequence similarity can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the Karlin and Altschul algorithm. These are 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 following groups of sequences:

[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 under stringent conditions to a DNA sequence described in (a) or (b); or

[0020] (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has 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 expression cassette is further operably linked to a promoter capable of driving its expression. The promoter includes, but is not limited to, a constitutive expression promoter, an inducible promoter, a tissue-specific expression promoter, or a spatiotemporal 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:12.

[0023] The expression cassette of the present invention further comprises a pollen-inactivating gene that can interfere with the function or formation of male gametes in plants containing the pollen-inactivating gene. Such pollen-inactivating genes include, but are not limited to, barnase genes, amylase genes, and DAM methylase genes. More specifically, the pollen-inactivating gene is the maize α-amylase gene, the nucleotide sequence of which is shown in SEQ ID NO:15.

[0024] The expression cassette of the present invention further comprises a screening gene, which 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 a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene, a fluorescent protein gene, etc. Specifically, the screening gene includes but is not limited to a blue grain gene, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, a glyphosate resistance gene, a glufosinate resistance gene, a bar gene, a red fluorescent gene DsRED, an mCherry gene, a cyan fluorescent protein gene, a yellow fluorescent protein gene, a luciferase gene, a green fluorescent protein gene, etc.

[0025] The present invention also discloses a method for regulating plant fertility, wherein the method restores the male fertility of the wheat ms9 mutant by transferring a fertility-related gene into the wheat ms9 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 under stringent conditions to a DNA sequence described in (a) or (b); or

[0029] (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has 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. The method uses wheat ms9 mutant plants as the transformation recipient material and transforms three tightly linked target genes into the sterile mutant recipient plants. The three target genes are the fertility-related gene TaMS9, a pollen inactivation gene, and a screening gene. The fertility-related gene TaMS9 can restore fertility to the sterile transformed recipients, the pollen inactivation gene can inactivate pollen containing the transformed exogenous gene, i.e., lose its fertilization ability, and the screening gene can be used to sort transgenic seeds or tissues from non-transgenic seeds or tissues. 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 a barnase gene, an amylase gene, a DAM methylase, and the like. More specifically, the pollen inactivation gene is the maize α-amylase gene Zm-AA, the nucleotide sequence of which is shown in SEQ ID NO:15. The pollen inactivation gene is linked to a promoter that preferentially expresses in male gametes. More specifically, the promoter preferentially expressing in male gametes includes but is not limited to the PG47 promoter and the Zm13 promoter. The screening gene can be used to select plants or vectors containing the expression cassette. The screening gene includes but is not limited to a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene, a fluorescent protein gene, and the like. 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 method for breeding male sterile lines, the method comprising the following steps:

[0034] (a) The following vector is introduced into the wheat ms9 male sterile line to obtain a maintainer line containing the following vector, wherein the vector comprises: a fertility-related gene TaMS9, which can restore the male fertility of the wheat ms9 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 above-mentioned vector is transferred to simultaneously produce wheat MS9 male sterile line without the vector and maintainer line seeds containing the vector; or driving pollen from the maintainer plants to wheat MS9 male sterile line plants, so that the wheat MS9 male sterile line plants are pollinated and reproduced to produce MS9 male sterile line or its allelic sterile line seeds.

[0036] In the above-mentioned male sterile line breeding method, the pollen inactivation gene includes but is not limited to a barnase gene, an amylase gene, a DAM methylase, and the like. More specifically, the pollen inactivation gene is the maize α-amylase gene Zm-AA, the nucleotide sequence of which is shown in SEQ ID NO:15. The pollen inactivation gene is linked to a promoter that preferentially expresses in male gametes. More specifically, the promoter preferentially expressing in male gametes includes but is not limited to the PG47 promoter and the Zm13 promoter. The screening gene can be used to select plants or vectors containing the expression cassette. The screening gene includes but is not limited to a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene, a fluorescent protein gene, and the like. 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 ms9 male sterile line, thereby obtaining a maintainer line of the wheat ms9 nuclear male sterile line or its allelic sterile line, wherein the vector comprises: a fertility-related gene TaMS9, which can restore the male fertility of the wheat ms9 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 from non-transgenic seeds.

[0039] In the above-mentioned method for producing a maintainer line, the pollen-inactivating gene includes, but is not limited to, a barnase gene, an amylase gene, a DAM methylase, and the like. More specifically, the pollen-inactivating gene is the maize α-amylase gene Zm-AA, the nucleotide sequence of which is shown in SEQ ID NO:15. The pollen-inactivating gene is linked to a promoter that preferentially expresses in male gametes. More specifically, the promoter preferentially expressing in male gametes includes, but is not limited to, the PG47 promoter and the Zm13 promoter. The screening gene can be used to select plants or vectors containing the expression cassette. The screening gene includes, but is not limited to, a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene, a fluorescent protein gene, and the like. 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, which comprises the following steps:

[0041] (a) introducing the following vector into a wheat ms9 nuclear male sterile line or its allelic sterile line, thereby obtaining a maintainer line of the wheat ms9 nuclear male sterile line or its allelic sterile line, wherein the vector comprises: a fertility-related gene TaMS9, which can restore the male fertility of the wheat ms9 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 from non-transgenic seeds; and

[0042] (b) self-pollinating the maintainer plants formed after the vector was introduced, i.e., breeding at a ratio of 1:1 to obtain wheat ms9 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 ms9 nuclear male sterile line or its allelic sterile line to obtain a maintainer line of the wheat ms9 nuclear male sterile line or its allelic sterile line, wherein the vector comprises: a fertility-related gene TaMS9, which can restore the male fertility of the wheat ms9 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 ensuring that the fertile male gametes produced in the plant do not contain the vector.

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

[0046] (c) After self-pollination, maintainer line seeds containing the vector and wheat ms9 nuclear male sterile line or its allelic sterile line seeds without the vector are obtained.

[0047] In the above-mentioned male sterile line propagation or maintenance method, maintainer line production method or propagation method, seed production method, etc. of the present invention, step (a) can also be to introduce a vector containing the fertility-related gene TaMS9, 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 ms9 nuclear male sterile line or its allelic sterile line, and through directed breeding, obtain a maintainer line plant with a background of the wheat ms9 nuclear male sterile line or its allelic sterile line and the vector.

[0048] In the above-mentioned male sterile line propagation method or maintenance method, maintainer 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 under stringent conditions to a DNA sequence described in (a) or (b); or

[0052] (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has 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 above-mentioned fertility-related gene TaMS9 can also be operably connected to a pollen-specific expression promoter, which can drive the expression of the TaMS9 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 above-mentioned fertility-related gene TaMS9 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: 13. In the aforementioned male sterile line propagation or maintenance methods, maintainer line production or propagation methods, and seed production methods of the present invention, the pollen inactivation gene includes, but is not limited to, barnase genes, amylase genes, DAM methylase genes, and the like. More specifically, the pollen inactivation gene is the maize α-amylase gene Zm-AA, the nucleotide sequence of which is shown in SEQ ID NO:15. The pollen inactivation gene is linked to a promoter that preferentially expresses in male gametes. More specifically, the promoter preferentially expressing in male gametes includes, but is not limited to, the PG47 promoter and the Zm13 promoter.

[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 a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene or a fluorescent gene. Specifically, the screening gene includes but is not limited to a blue grain gene, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, a glyphosate resistance gene, a glufosinate resistance gene, a bar gene, a red fluorescent gene DsRED, an mCherry gene, a cyan fluorescent protein gene, a yellow fluorescent protein gene, a luciferase gene, a green fluorescent protein gene, etc.

[0056] The present invention also provides a pollen-specific expression promoter, the nucleotide sequence of which is shown in SEQ ID NO:12. SEQ ID NO:12 was linked to the reporter gene GUS to construct a vector for transformation of rice and wheat. The GUS expression activity and expression pattern in the transgenic plants were detected and analyzed. GUS staining analysis of the roots, stems, leaves, and flowers of the transgenic plants revealed that the promoter provided by the present invention drives expression of the GUS gene in plant pollen. This indicates that SEQ ID NO:12 provided by the present invention is a pollen-specific expression promoter.

[0057] The plant pollen-specific expression promoter provided by the present invention comprises a nucleotide sequence as shown in SEQ ID NO: 12 in the sequence listing, or comprises a nucleotide sequence having 90% or more similarity to the nucleotide sequence listed in SEQ ID NO: 12, or comprises a 500 or more continuous nucleotide fragment derived from SEQ ID NO: 12, and can drive the expression of a nucleotide sequence operably linked to the promoter in plant pollen. Expression vectors, transgenic cell lines, and host bacteria containing the above sequence all fall within the scope of protection of the present invention. Primer pairs for amplifying any nucleotide fragment of the SEQ ID NO: 12 promoter disclosed in the present invention also fall within the scope of protection of the present invention.

[0058] As used herein, a "promoter" refers to a DNA regulatory region that typically comprises a TATA box that directs RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site for a specific coding sequence. A promoter may also contain other recognition sequences, typically located upstream or 5' of the TATA box, commonly referred to as upstream promoter elements, which regulate transcription efficiency. Those skilled in the art will appreciate that while the nucleotide sequences for the promoter regions disclosed herein have been identified, isolating and identifying other regulatory elements upstream of the TATA box within the specific promoter regions identified herein is also within the scope of the present invention. Therefore, the promoter regions disclosed herein are generally further defined as comprising upstream regulatory elements, such as those elements, enhancers, and the like, that regulate the tissue expression and temporal expression of a coding sequence. Similarly, promoter elements that enable expression in target tissues (e.g., male tissue) can be identified and isolated and used in conjunction with other core promoters to validate male tissue-preferred expression. A core promoter refers to the minimal sequence required to initiate transcription, such as the sequence known as the TATA box, which is commonly found in promoters of protein-encoding genes. Therefore, the upstream promoter of the TaMS9 gene can be optionally used in conjunction with its own core promoter or a core promoter from another source.

[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 methods: operably linking the promoter sequence to a reporter gene to form a transformable vector, then transferring the vector into a plant, and confirming its expression characteristics by observing the expression of the reporter gene in various tissues and organs of the plant in the obtained transgenic offspring; or subcloning the above-mentioned vector into an expression vector for transient expression experiments, and detecting the function of the promoter or its regulatory region through transient expression experiments.

[0061] The selection that is used for testing the suitable expression vector of promotor 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 skill 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 property pollen, or introduces callus, to carry out functional verification.

[0062] Furthermore, the promoter of the present invention can be linked to nucleotide sequences other than the TaMS9 gene to express other heterologous nucleotide sequences. The promoter nucleotide sequences of the present invention, as well as fragments and variants thereof, can be assembled together with heterologous nucleotide sequences into an expression cassette for expression in the desired plant, more specifically, in the male organs of the plant. The expression cassettes contain suitable restriction enzyme sites for insertion of the promoter and heterologous nucleotide sequence. These expression cassettes can be used to genetically manipulate any plant to obtain a desired 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, maize α-amylase genes, auxin genes, rot B, cytotoxin genes, diphtheria toxin, DAM methylase, 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 that is operably linked to the promoter disclosed herein.

[0065] The present invention also provides a transcription terminator sequence. The nucleotide sequence of the transcription terminator is shown in SEQ ID NO: 13, and has the function of terminating gene transcription expression.

[0066] The present invention also provides an expression cassette, vector, or engineered strain comprising the pollen-specific expression promoter SEQ ID NO: 12 provided by the present invention. Specifically, the nucleotide sequence of the fertility-related gene TaMS9 provided by the present invention can be constructed downstream of the promoter SEQ ID NO: 12 provided by the present invention, thereby driving 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. It can also be used for 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 maintainer lines; and it can be applied to pollen abortion experiments, thereby avoiding biosafety issues caused by plant transgenic drift or pollen escape, which is of great significance to the creation of plant male sterile lines and maintainer lines.

[0068] The nucleotide sequence and promoter sequence or expression cassette of the TaMS9 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. The insertion can be a positional or random insertion.

[0069] The present invention relates to transferring a nucleotide sequence, vector or expression cassette into a plant, introducing the nucleotide sequence, vector or expression cassette into a plant, or transforming the plant, and refers to the transfer of the nucleotide sequence, vector or expression cassette into a recipient cell or recipient plant by conventional transgenic methods. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform the recombinant expression vector into a plant cell to produce the transgenic plant of the present invention. Transformation methods can 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 TaMS9 and its promoter, as well as a method for using the gene to propagate and maintain the ms9 male sterile line. The fertility-related gene, fertility maintenance of the nuclear male sterile line, and sterile line propagation method provided by the present invention have significant production promotion value and application value for crop hybrid breeding production. 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 a 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] 1.Klindworth DL,Williams ND,Maan S S.Chromosomal location of genetic male sterility genes in four mutants of hexaploid wheat.Crop Sci,2002,42:1447-1450.

[0073] 2.Li J, Zhang J, Li H, et al. The Major Factors Causing the Microspore Abortion of Genic Male Sterile Mutant NWMS1 in Wheat (Triticum aestivum L.). IntJ Mol Sci, 2019, 20: 6252

[0074] 3.Tucker EJ,Baumann U,Kouidri A,et al.Molecular identification of the male wheat fertility gene Ms1 and its prospects for hybrid breeding.NatCommun,2017,8:869

[0075] 4.Wang Z, Li J, Chen SX, et al. Poaceae-specific MS1 encodes a phospholipid-binding protein for male fertility inbread wheat. Proc Natl Acad Sci USA, 2017, 114: 12614-12619

[0076] 5. Pallotta MA, Warner P, Kouidri A, et al. Wheat ms5 male-sterility is induced by recessive homoeologous A and D genome non-specific lipid transfer proteins. Plant J, 2019,99:673-685 BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 shows the phenotype of the wheat ms9 mutant, where Figure A shows the whole ear of the parent Ningchun No. 4 (left) and the ms9 mutant (right) at full flowering. Figures B and C show the anther development of the parent Ningchun No. 4 and the ms9 mutant at the corresponding period in Figure A, respectively. Figures D and E show the results of iodine-potassium iodide staining of the starch grains of the mature anthers of the parent Ningchun No. 4 and the ms9 mutant, respectively.

[0078] Figure 2 shows the distribution of SNPs on chromosome 2A of the wheat ms9 mutant analyzed using mutmap technology after resequencing.

[0079] Figure 3 shows the positioning results of the TaMS9 gene, where the first row is the preliminary positioning results, the second and third rows are the fine positioning results, and the fourth row is the gene structure and mutation site of the candidate gene.

[0080] Figure 4 is a semi-quantitative RT-PCR analysis of the expression pattern of the TaMS9 gene in different tissues of Chinese spring wheat. The upper figure shows the results of the TaMS9 gene in different organs or tissues, the lower figure shows the internal reference gene ACTIN, 0 is the anther in the meiotic stage, 1 is the anther in the uninucleate stage, 2 is the anther in the binucleate stage, 3 is the anther in the trinucleate stage, and the palea is a mixed material of the palea and lemma. DETAILED DESCRIPTION

[0081] Example 1. Wheat ms9 is a pollenless recessive nuclear male sterile mutant

[0082] During a large-scale phenotypic screening of the Ningchun 4 spring wheat variety EMS mutagenesis library, we obtained multiple wheat male sterile materials. Further genetic analysis identified a new recessive nuclear male sterile material controlled by a single gene. Allelic hybridization experiments confirmed that it is non-allelic with any previously reported wheat male sterility loci. We named it MS9.

[0083] In the F2 generation of hybrids between this mutant and its parent, Ningchun 4, the segregation ratio of fertile to sterile plants was 126:32. F2 populations of this mutant with wheat varieties, such as China Spring and Fielder, showed fertile to sterile plant ratios of 220:68 and 241:68, respectively. These genetic data indicate that the ms9 mutant is a recessive mutation controlled by a single gene. Compared to its parental variety, Ningchun 4, the ms9 mutant showed no significant differences during vegetative growth. However, at flowering, the ms9 mutant had fluffy ears, significantly wider awn opening angles, and thin, shrunken anthers that did not dehisce. Pollen viability was assessed using iodine-potassium iodide (I2-KI) staining. Mature pollen from the parental variety was plump and spherical, with darkly stained starch granules, whereas the ms9 mutant anthers contained no visible pollen (see Figure 1).

[0084] Example 2. Map-based cloning of the wheat MS9 gene

[0085] Because the ms9 mutant was generated by EMS chemical mutagenesis, we cloned the MS9 gene using mutmap analysis combined with map-based cloning. First, we prepared samples for resequencing of the ms9 mutant. DNA from 87 sterile plants from the F2 generation of a hybrid between the ms9 mutant and the parental variety, Ningchun 4, was combined to form a mutant DNA pool. DNA from 72 wild-type plants (as determined by the phenotype of the F3 generation) was combined to form a wild-type DNA pool. Both pools were sent to a sequencing company for resequencing, with 500 gigabytes of data generated each. Mutmap analysis revealed numerous single nucleotide polymorphisms (SNPs) in the mutant material. These SNPs were primarily concentrated within the 600-800 Mb range on the long arm of wheat chromosome 2A, with a distinct peak at 700 Mb, indicating that MS9 is likely located within this region (see Figure 2).

[0086] Based on preliminary mutmap results, we designed five SNP markers near 700 Mb on wheat chromosome 2A, initially localizing the MS9 locus within 1.37 Mb of the wheat genome. To fine-tune the MS9 locus, we developed additional SNP markers within this 1.37 Mb region. Using 543 sterile plant samples, we narrowed the localization to a 67.8 Kb interval. Five coding genes were annotated within this region, but only the second gene harbored a SNP in its coding region, resulting in a missense mutation. This suggests that the gene containing this SNP is likely the MS9 gene, which we named TaMS9 (see Figure 3).

[0087] The 3541st nucleotide after the ATG of the TaMS9 gene mutated from G to A. The genomic sequence after the mutation is shown in SEQ ID NO: 4, and the CDS sequence after the mutation is shown in SEQ ID NO: 5. As a result, the 725th amino acid of the encoded protein sequence mutated from glycine (Gly) to glutamic acid (Glu), and the amino acid sequence is shown in SEQ ID NO: 6.

[0088] In the wheat varieties Ningchun No. 4 and China Spring, the genomic DNA sequence of the TaMS9 gene coding region is completely consistent without any difference, as 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.

[0089] Example 3. Expression analysis of TaMS9 gene

[0090] Based on the genetic structure of the TaMS9 gene, we designed trans-intron semi-quantitative RT-PCR primers TaMS9-RTF and TaMS9-RTR (SEQ ID NOs: 7 and 8) using the cDNA sequence of this gene as a template. Total RNA was extracted from roots, stems, leaves, inflorescences, lemmas, pistils, and anthers of Chinese spring wheat at different developmental stages, and reverse-transcribed into cDNA. The expression of the TaMS9 gene was detected using the wheat ACTIN gene as an internal control. The results showed that the TaMS9 gene was widely expressed in various tissues and organs of wheat, including anthers at all developmental stages (see Figure 4).

[0091] The primer sequences used are as follows:

[0092] TaMS9-RTF:5'-GTGCTTGTCAAGAATACCGACAAC-3'(SEQ ID NO:7)

[0093] TaMS9-RTR:5'-GATGATGGATGCTAACTGCTCCACT-3'(SEQ ID NO:8)

[0094] ACTIN-RTF:5'-TCAGCCATACTGTGCCAATC-3'(SEQ ID NO:9)

[0095] ACTIN-RTR:5'-CTTCATGCTGCTTGGTGC-3'(SEQ ID NO:10)

[0096] Example 4. Transgenic Functional Complementation of Wheat ms9 Mutant

[0097] First, a stable transformation vector was constructed for functional complementation of wheat ms9 mutants. Using the binary expression vector P1300-BAR as the backbone, the 9411bp TaMS9 genomic sequence (the sequence is shown in SEQ ID NO: 11, comprising a 2984bp promoter sequence, a 4704bp genomic sequence, and a 1723bp terminator sequence) was inserted into the vector using the in-fusion method using the HindIII and SacI restriction endonuclease sites, thereby forming the plant expression vector p1300-BAR-TaMS9. The promoter sequence is shown in SEQ ID NO: 12, the genomic sequence is shown in SEQ ID NO: 1, and the terminator sequence is shown in SEQ ID NO: 13. The plant expression vector was transformed into immature embryos of heterozygous plants of the ms9 mutant using Agrobacterium-mediated genetic transformation technology, and a total of 23 transgenic positive T0 generation plants were obtained. Six T0 plants were identified as homozygous for the TaMS9 site using adjacent SNP markers. The pollen fertility of these 6 T0 plants was observed and iodine-potassium iodide stained, and it was found that the pollen fertility of 4 of them was completely restored.

[0098] Example 5. Functional analysis of the TaMS9 gene promoter

[0099] First, the 2984bp promoter sequence of the TaMS9 gene (SEQ ID NO:12) and the GUS reporter gene (SEQ ID NO:14) were inserted into the binary expression vector P1300-BAR using an in-fusion method to form the p1300-BAR-TaMS9::GUS expression vector. This vector was then transformed into immature wheat embryos using Agrobacterium-mediated genetic transformation, yielding 20 T0-generation transgenic-positive plants. GUS staining was used to analyze the expression of the TaMS9 gene promoter in various tissues and organs of these T0-generation transgenic-positive plants. The results were consistent with the semi-quantitative RT-PCR pattern of the TaMS9 gene, demonstrating that the TaMS9 gene promoter is ubiquitously expressed.

[0100] Example 6. Application of TaMS9 gene in new generation wheat hybrid breeding technology

[0101] Next-generation hybrid breeding technology utilizes recessive nuclear male sterile wheat mutants for hybrid seed production. The principle is to transfer three tightly linked genes—a fertility restorer gene, a pollen inactivation gene, and a seed marker gene—into the recessive nuclear male sterile wheat mutant, thereby creating a male sterility maintainer line that combines restoration and maintenance functions. Selfing of this maintainer line allows for the continuous reproduction of sterile lines for hybrid seed production. This new generation of hybrid breeding technology utilizes the wheat ms9 male sterile mutant and the TaMS9 gene.

[0102] The transgenic vector of the present invention is based on the pCAMBIA1300 vector. First, the resistance gene is transformed into the Bar gene driven by the Ubi promoter. Then, the fertility restorer gene TaMS9, the pollen inactivation gene ZmBT1-ZmAA from maize, and the blue seed marker gene are co-constructed into the above vector. The specific information is as follows:

[0103] 1) A fertility restorer gene expression cassette, comprising a TaMS9 gene promoter, coding region genomic sequence, and terminator, all from the wheat variety Ningchun No. 4, the full-length nucleotide sequence of which is shown in SEQ ID NO: 11, wherein the promoter sequence is shown in SEQ ID NO: 12, the coding region genomic sequence is shown in SEQ ID NO: 1, the terminator sequence is shown in SEQ ID NO: 13, and the amino acid sequence of the protein encoded by the nucleotide sequence is shown in SEQ ID NO: 3;

[0104] 2) Pollen inactivation gene expression cassette PG47: ZmBT1-ZmAA-IN2-1, the target gene is ZmAA, the transit peptide is ZmBT1, ZmBT1-ZmAA (the nucleotide sequence of which is shown in SEQ ID NO: 15) is driven by the promoter PG47 (the nucleotide sequence of which is shown in SEQ ID NO: 16), and the terminator is IN2-1 (the nucleotide sequence of which is shown in SEQ ID NO: 17).

[0105] 3) Seed marker gene expression cassette, see patent WO2019090496A1. This expression cassette consists of two tandem genes, ThMYB1 and ThR1. The 3215bp ThMYB1 genomic sequence consists of a 1952bp promoter sequence (shown in SEQ ID NO: 18), an 822bp genomic sequence (shown in SEQ ID NO: 19), and a 441bp terminator sequence (shown in SEQ ID NO: 20). The 4422bp ThR1 sequence contains a 2084bp promoter sequence (shown in SEQ ID NO: 21), a 1720bp CDS sequence (shown in SEQ ID NO: 22), and a 618bp terminator sequence (shown in SEQ ID NO: 23).

[0106] The vector was transformed into immature embryos of wheat plants harboring the ms9 mutant using Agrobacterium-mediated genetic transformation, yielding 83 transgenic plants. Molecular identification revealed that 21 plants were homozygous for the ms9 mutation. Pollen viability was assessed using iodine-potassium iodide staining, revealing that 16 of the 21 plants with the ms9 homozygous mutation had approximately 50% sterile pollen, demonstrating that the fertility restorer gene expression cassette restored the male sterility phenotype of the ms9 mutant. Simultaneously, the pollen inactivation gene expression cassette inactivated half of the transgene-bearing pollen. Among the T1 transgenic plant seeds harvested from individual plants, 10 lines had a 1:1 ratio of normal-colored seeds to blue-colored seeds, indicating that the blue-colored seed marker gene expression cassette was expressing normally, marking transgenic seeds as blue. These results demonstrate that the three expression cassettes of the present invention—the fertility restorer gene, the pollen inactivation gene, and the seed marker gene—are functioning correctly and can be successfully applied to the next generation of wheat hybrid breeding.

Claims

1. A fertility-related gene TaMS9, characterized in that The nucleotide sequence of the fertility-related gene TaMS9 is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3; (c) a DNA sequence that can hybridize under stringent conditions to a DNA sequence described in (a) or (b); or (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has the function of restoring fertility; or (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

2. An expression cassette, expression vector or engineered bacteria, characterized in that The expression cassette, expression vector or engineered bacteria comprises the fertility-related gene according to claim 1.

3. Use of a fertility-related gene, expression cassette, expression vector or engineered bacteria in regulating plant 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) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3; (c) a DNA sequence capable of hybridizing under stringent conditions to a DNA sequence described in (a) or (b); or (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has the function of restoring fertility; or (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

4. A method for regulating plant fertility, wherein the method affects the expression level of the fertility-related gene TaMS9 in the plant by overexpressing, inhibiting or mutating the gene, thereby regulating plant fertility, wherein: The nucleotide sequence of the fertility-related gene TaMS9 is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3; (c) a DNA sequence that can hybridize under stringent conditions to a DNA sequence described in (a) or (b); or (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has the function of restoring fertility; or (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

5. The method according to claim 4, wherein the mutation comprises substitution, deletion or addition of one or more nucleotides in the nucleotide sequence of the fertility-related gene.

6. The method according to any one of claims 4-5, wherein the "mutation" includes but is not limited to the following methods, such as gene mutation caused by physical or chemical methods, chemical methods include mutagenesis caused by treatment with mutagens such as EMS, or gene silencing methods such as RNAi or gene editing methods, and the gene site-directed mutagenesis method includes but is not limited to gene editing methods such as ZFN, TALEN, and / or CRISPR / Cas9.

7. The method according to claim 4, characterized in that The method comprises using the nucleotide sequence of the TaMS9 gene to complement the male sterility phenotype caused by the TaMS9 gene mutation, so as to restore the ms9 male sterile line to fertility.

8. The method according to claim 7, wherein the sequence of the TaMS9 gene mutation is shown in SEQ ID NO: 4 or 5, and the amino acid is shown in SEQ ID NO:

6.

9. Use of the method according to any one of claims 4 to 8 in regulating plant fertility.

10. A method for producing or breeding a male sterile line, comprising the following steps: (a) transferring the following vector into the ms9 male sterile line to obtain a maintainer line containing the following vector, wherein the vector comprises: a fertility-related gene TaMS9, wherein the fertility-related gene TaMS9 can restore the male fertility of the ms9 male sterile line; and a pollen inactivation gene, wherein when expressed, the pollen inactivation gene interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, thereby causing the fertile male gametes produced in the plant to be free of 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 transfer of the above-mentioned vector to simultaneously produce ms9 male sterile seeds without the vector and maintainer seeds containing the vector; or using pollen from the maintainer plants to pollinate ms9 sterile plants, so that the ms9 sterile plants are pollinated and reproduced into ms9 sterile seeds.

11. The production or propagation method according to claim 10, wherein the nucleotide sequence of the fertility-related gene TaMS9 is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3; (c) a DNA sequence that can hybridize under stringent conditions to a DNA sequence described in (a) or (b); or (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has the function of restoring fertility; or (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

12. The production or propagation method according to claim 11, wherein the fertility-related gene TaMS9 is driven to express by a pollen-specific expression promoter, and preferably the nucleotide sequence of the pollen-specific expression promoter is shown in SEQ ID NO:

12.

13. The production or propagation method according to any one of claims 10-12, wherein the pollen inactivation gene includes but is not limited to a barnase gene, an amylase gene, a DAM methylase, etc., preferably the pollen inactivation gene is a maize α-amylase gene, and more preferably the nucleotide sequence thereof is shown in SEQ ID NO:

15.

14. The production or propagation method according to claim 13, wherein the pollen inactivation gene is linked to a promoter preferentially expressed in male gametes, preferably the promoter is a PG47 promoter or a Zm13 promoter.

15. The breeding method according to any one of claims 10 to 14, wherein the screening gene includes but is not limited to a blue grain gene, an antibiotic resistance gene, a herbicide resistance gene, a fluorescent protein gene, etc., and preferably the screening gene includes but is not limited to a blue grain gene, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, a glyphosate resistance gene, a glufosinate resistance gene, a bar gene, a red fluorescent gene DsRED, an mCherry gene, a cyan fluorescent protein gene, a yellow fluorescent protein gene, a luciferase gene, a green fluorescent protein gene, etc.

16. A method for producing or propagating a maintainer line, the method comprising the steps of: (a) transferring the following vector into the ms9 male sterile line, thereby obtaining a maintainer line of the ms9 male sterile line, wherein the vector comprises: a fertility-related gene TaMS9, wherein the fertility-related gene TaMS9 can restore the male fertility of the ms9 male sterile line; and a pollen inactivation gene, wherein when expressed, the pollen inactivation gene interferes with the function or formation of male gametes containing the pollen inactivation gene in the plant, thereby causing the fertile male gametes produced in the plant to be free of 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-mentioned vector is transferred, thereby producing ms9 male sterile seeds without the vector and maintainer seeds containing the vector.

17. The production or propagation method according to claim 16, wherein the nucleotide sequence of the fertility-related gene TaMS9 is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID NO: 1 or 2; (b) the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 3; (c) a DNA sequence that can hybridize under stringent conditions to a DNA sequence described in (a) or (b); or (d) a DNA sequence that has at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and has the function of restoring fertility; or (e) A DNA sequence complementary to any one of the sequences described in (a) to (d).

18. The production or propagation method according to claim 17, wherein the fertility restorer gene TaMS9 is driven to express by a pollen-specific expression promoter, and preferably the nucleotide sequence of the pollen-specific expression promoter is shown in SEQ ID NO:

12.

19. The production or propagation method according to any one of claims 16 to 18, wherein the pollen inactivation gene includes but is not limited to a barnase gene, an amylase gene, a DAM methylase, etc., preferably the pollen inactivation gene is a maize α-amylase gene, and more preferably the nucleotide sequence thereof is shown in SEQ ID NO:

15.

20. The production or propagation method according to claim 19, wherein the pollen inactivation gene is linked to a promoter preferentially expressed in male gametes, preferably the promoter is a PG47 promoter or a Zm13 promoter.

21. The breeding method according to any one of claims 16 to 20, wherein the screening genes include but are not limited to blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, etc., and preferably the screening genes include but are 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.

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