KR102143644B1 - Method for producing male-sterile plant - Google Patents

Abstract

The present invention relates to a mutation in a microcell-specific gene induces male infertility in flowering plants, and a specific sequence mutation of MYB81 in flowering plants plays a crucial role in inducing male infertility and the induction of male infertility is organ/stage specificity. It can be used as a decisive genetic tool by identifying what has a, and can induce male sterility in flowering plants specifically for microcells. Therefore, the method of the present invention is more stable than the conventional method for producing male infertile plants, and since it is possible to manufacture male infertile plants without the need for other maintenance systems to maintain male infertility, the present invention provides easy development of new varieties and high added value. It will be useful for industrial production.

Description

Method for producing male-sterile plant}

The present invention relates to a mutation in a microcell-specific gene induces male infertility in flowering plants, and to a method for inducing male infertility by removing two amino acids at the nucleic acid binding site of the MYB81 gene specific for pollen generation.

Higher plants have a complex life cycle that alternates between the growth of diploid sporangial organisms and very reduced haplotype gametes. In flowering plants, for redundant fertilization, the male gametophyte (or pollen grain) plays an essential role in plant propagation and crop production through the generation of male gametophytes and delivery to the embryo sac (Borg et al. , 2009). Both gametophyte (potted) and sporophyte (drug and carpet) tissues participate in this process. The male gamete life history can be divided into two phases: (1) a developmental phase that occurs inside the chamber and leads to the formation of a mature pollen, and (2) begins with the influence of grains on the surface of the pistil head. The functional phase or progamic phase that continues with pollen tube growth and ends in redundant fertilization (Honys et al, 2006). Pollen of flowering plants included as part of sexual reproduction in the male gamete of seed plants is produced from the anther of flowers. Pollen development begins in a young chamber and consists of two main stages-microcell development and microembryo development. It starts from the pollen blast, which is a diploid formed in the anther, and undergoes one meiosis and two mitosis. After meiosis, four haploids are formed, and then the nuclei of microcells released by calos decomposition move to the germ cell pole. After that, the polar microcells become vegetative cells and reproductive cells with completely different cellular characteristics through'asymmetric pollen mitosis I'. Feeder cells contain most of the cytoplasm and intracellular organelles, and the nucleus is less condensed. In contrast, germ cells contain very limited cytoplasm and intracellular organelles, and their nuclei are highly condensed. Subsequently, as the carlos surrounding the germ cells is decomposed, the germ cells move into the cytoplasm of the feeder cells, becoming a two-cell pollen with a unique structure with the germ cells in the feeder cells. The reproductive cells go through the second symmetrical pollen cell division (pollen mitosis II, PMII) to create two sperm cells and finally complete the three-cell pollen (McCormick, 2004). The vegetative cells then move the sperm cells to the magnetic spouse. It functions to make a flower pot tube.

Pollen development is a reproductive generation that creates male spouses. It is an important biological process that is the basis for the existence of flowering plants in a very short cycle, and at the same time, the possibility of grafting it into the agricultural field is also great, and a regulatory gene for the asymmetric first pollen cell division during the pollen development process. The network has yet to be revealed.

Male sterile mutations due to natural and induced mutations have been reported in several higher plants. The mutations that produce non-functional pollen are associated with several different phenotypes, including pollen with structural mutations and also functional defects associated with gamete formation, particularly meiosis. Male infertility can be divided into genotypic male sterility and cytoplasmic male sterility according to heredity. Nuclear genotype male infertility is found because it adversely affects survival. Since it is mainly recessive and becomes infertile in the case of a homozygous recessive, a lot of effort is required to maintain and propagate the infertility system. Therefore, it is difficult to determine fertility because the flowers are small, and there is a disadvantage that it is difficult to apply to crops that take a lot of time until flowers bloom. In addition, since cytoplasmic male infertility produces 100% fertility strains no matter which fertile line is bred, individuals and lines capable of producing 50% or 100% fertility strains by crossing various fertile strains to infertile strains, that is, the maintenance system. It is inconvenient because it has to be found and planted, but the intended purpose of the use of infertility can be achieved. However, for hybrid breeding, artificial male infertility through radiation and chemical drugs to obtain male infertility has also been attempted, but there is a problem in that side effects must be resolved. Although a lot of investment is invested in the development and research of male sterile plants for the production of hybrid seeds, the research results on this are very poor due to the problems of male sterile plants so far. If it is possible to overcome the shortcomings of these male sterilization plants, that is, secure a stable male sterility and maintain sterility without the need for other maintenance systems, it can be used for easy development of new varieties and high value-added industrial production. I will be able to. Therefore, there has been a constant demand for a novel gene capable of securing stable male infertility and maintaining infertility without the need for other maintenance systems.

Thus, various genes related to male infertility have been identified in various plant species such as Arabidopsis thaliana, wheat, cabbage, soybean, tomato, sunflower and chive, but as in the present invention, the MYB81 gene, which is crucial for male infertility in plants, and uses thereof Nothing has been revealed about.

An object of the present invention is to provide a nucleic acid in which TACGTT is deleted from the ORF of the MYB81 gene.

In addition, an object of the present invention is to provide a vector for plant transformation comprising the nucleic acid of the present invention and a transgenic plant transformed with the vector.

In addition, an object of the present invention is to provide a male infertile plant (AP28-23 heterozygous and homozygous) in which TACGTT is deleted from the ORF of the MYB81 gene.

In addition, an object of the present invention is to provide a nucleic acid molecule that inhibits the expression of the MYB81 gene and a composition for inducing male infertility comprising the same.

In addition, it is an object of the present invention to provide a composition for determining male infertility of a plant.

In addition, an object of the present invention is to provide a method for producing a male infertile plant.

In addition, an object of the present invention is to provide a method for determining male infertility of a plant.

In order to solve the above problems, the present invention provides a nucleic acid in which TACGTT is deleted from the ORF of the MYB81 gene.

In addition, the present invention provides a vector for plant transformation comprising the nucleic acid of the present invention and a transgenic plant transformed with the vector.

In addition, the present invention provides a male infertile plant (AP28-23 heterozygous and homozygous) in which TACGTT is deleted in the ORF of the MYB81 gene.

In addition, the present invention provides a nucleic acid molecule that inhibits the expression of the MYB81 gene, and a composition for inducing male infertility comprising the same.

In addition, the present invention provides a composition for determining male infertility of a plant.

In addition, the present invention provides a method for producing a male infertile plant.

In addition, the present invention provides a method for determining male infertility of a plant.

In the present invention, since specific sequence mutations of MYB81 in flowering plants have been found to act decisively in inducing male infertility and that inducing male infertility has organ/stage specificity, this can be used as a definitive genetic tool, and microcell-specific flowering May induce male infertility in plants. Therefore, it is more stable than the conventional male infertility plant manufacturing method, and since it is possible to manufacture male infertile plants without the need for other maintenance systems to maintain male infertility, the present invention is useful for the development of new varieties and high value-added industrial production. You will be able to use it.

1 is a view confirming the pollen phenotype of the wild-type Arabidopsis pollen grain, AP28-23 heterozygous or homozygous.
Figure 2 is a diagram showing the time of occurrence of the cause of the AP28-23 mutant:
TET: tetrad;
CMS: central microspore;
PMS: polarized microspore;
EBC: early bicellular pollen;
LBC: late bicellular pollen;
TC: tricellular pollen;
AbDEG: abnormally degenerating or dead pollen;
AbEBC: abnormal phenotype resembling early bicellular pollen; And
AbPMS: abnormal phenotype resembling polarized microspore.
3 is a diagram showing the results of the genetic transmission analysis of AP28-23:
A: AP28-23 in bilateral hybridization test Number of mutant (AP28-23/+) and wild type (+/+) plants;
B: Heterozygous AP28-23 frequency of mutations and wild-type alleles predicted in male and female gamete populations; And
C: Predicted/observed mutation frequency in autologous offspring of heterozygous AP28-23.
4 is a diagram showing a process of AP28-23 map-based cloning.
5 is a diagram showing the principle of SSLP mapping.
Figure 6 is a schematic diagram showing the location of the SSLP marker used in the genetic mapping (genetic mapping) analysis.
7 is a diagram showing the mutant locus of AP28-23.
8 is a diagram showing the results of sequence analysis of genomic DNA of At2g26960:
Blue highlight: start/end codon;
Blue capital letters: exons;
Lowercase purple: intron;
Gray highlight: tested sequence site;
Red highlight: polymorphism sequence of Col/Ler;
Green highlight: mutation site; And
Red box: primer binding site.
9 is a diagram confirming that 6bp (TACGTT) was deleted as a result of sequence analysis of At2g26960- gDNA.
Figure 10 is a diagram confirming the phenotype by deletion of the MYB81 gene:
A: 6 AP28-23 heterozygous plants (+/-) transformed with a complementation vector ( MYB81 ), 49 AP28-23 heterozygous plants (+/-) and 8 AP28-23 homozygous (-/-) percentage of normal and abnormal pollen grains in the mutant;
B: MYB81 transcript expression;
SD: seedlings;
RT: root;
Lf: leaf;
ST: stem;
YB: young buds;
OF: blooming flowers,
F2.1: tetraploid and microcellular mixture;
F2.2: Mixture of microcellular and early two-cell pollen;
F2.3: late two-cell pollen;
F3.0: three-cell pollen; And
MP: mature pollen.
11 is a diagram showing the results of promoter GUS analysis using a transformation line containing pMYB81-GUS (A and C) or pMYB81-CYCB1:1 ^MDB- GUS (B).
12 is a diagram showing the intracellular location of the MYB81 protein during pollen development:
A: callose dissolution;
B and C: median microcells with exine accumulation;
D: polarized microcells;
E: early ear cells;
F: metaphase-ear cell;
G: late ear cell;
H: three cell pollen;
I: root cells of transgenic plants containing pMYB81-MYB81-mRFP;
mn: microcellular nucleus;
vn: feeder cell nucleus;
gn: generative nucleus;
sn, sperm cell nucleus; And
v: vacuole.
13 is a diagram showing the root cells of a transgenic plant containing p35S-MYB81-mRFP1 (K) or pXVE-MYB81-mRFP1 (J) in the presence of estradiol.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technique or configuration well known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. However, the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of equality interpreted from the description of the claims to be described later and therefrom.

In addition, terms used in the present specification (terminology) are terms used to properly express a preferred embodiment of the present invention, which may vary according to a user, an operator's intention, or customs in the field to which the present invention pertains. Therefore, definitions of these terms should be made based on the contents throughout the present specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

All technical terms used in the present invention, unless defined otherwise, are used in the sense as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention. The contents of all publications described by reference herein are incorporated into the present invention.

In one aspect, the present invention relates to a nucleic acid in which TACGTT is deleted in the ORF of the MYB81 gene.

In one embodiment, the MYB81 gene may be derived from Arabidopsis thaliana.

In one embodiment, the nucleic acid may be specifically expressed in flower buds during the pollen development stage.

In one embodiment, the MYB81 gene may include the nucleotide sequence of SEQ ID NO: 3, the ORF of MYB81 may include the nucleotide sequence of SEQ ID NO: 2, and the nucleic acid in which TACGTT is deleted in the ORF of the MYB81 gene is SEQ ID NO: 1 It may include a nucleotide sequence of.

In one embodiment, the nucleic acid in which TACGTT is deleted in the ORF of the MYB81 gene may be the nucleotide sequence of SEQ ID NO: 1.

In one embodiment, a promoter including the nucleotide sequence of SEQ ID NO: 4 may be further included. The pMYB81 promoter including the nucleotide sequence of SEQ ID NO: 4 specifically induces expression at the time of young pollen cells occurring after meiosis, thereby enabling targeted expression of specific genes or modified genes. In one embodiment of the present invention, when the gene in which the TACGTT is deleted was widely expressed in the ORF of the MYB81 gene, the plant died, so in order to induce male sterility of the plant using the deleted gene, using the present promoter desirable.

In one embodiment, the deletion of TACGTT in the ORF of the MYB81 gene may indicate a male-specific phenotype.

In one embodiment, male infertility may be caused in plants by the deletion of the TACGTT.

In one aspect, the present invention relates to a male infertile plant comprising a nucleic acid in which TACGTT is deleted in the ORF of the MYB81 gene.

In one embodiment, the MYB81 gene includes both genomic DNA (genomic DNA) and cDNA.

In one embodiment, the deletion of the TACGTT may be performed using a known gene editing technique, for example, a method such as gene scissors.

In one embodiment, the male infertile plant of the present invention may be a plant in which valine and tyrosine (V35 and Y36) of MYB domain protein 81, which are members of the R2R3-MYB transcription factor gene family, are deleted.

In one embodiment, the plant may be a flowering plant.

In one aspect, the present invention relates to a vector for plant transformation comprising the nucleic acid of the present invention.

In one embodiment, the vector of the present invention may further include a promoter comprising the nucleotide sequence of SEQ ID NO: 4. The pMYB81 promoter including the nucleotide sequence of SEQ ID NO: 4 specifically induces expression at the time of young pollen cells occurring after meiosis, thereby enabling targeted expression of specific genes or modified genes. In one embodiment of the present invention, when the gene in which the TACGTT is deleted was widely expressed in the ORF of the MYB81 gene, the plant died, so in order to induce male sterility of the plant using the deleted gene, using the present promoter desirable.

In one embodiment, the vector of the present invention is CaMV 35S, UBQ14, actin, ubiquitin, pEMU, MAS, histone, SCP (Oh et al, 2010), MSP1 (Honys et al, 2006) or DUO1 (Rotman et al, 2005). It may include a promoter of, and may include SCP, MSP1 or DUO1 that specifically operates in the developmental stage of pollen cells.

In one embodiment, the vector of the present invention may include a promoter comprising the nucleotide sequence of SEQ ID NO: 4.

The term "MYB81 or MYB81 " of the present invention refers to the wild-type gene of MYB81, "myb81 Or myb81 " refers to the mutant gene of the present invention in which 6bp of MYB81 is deleted.

The term "promoter" of the present invention refers to a region upstream of DNA from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in a plant cell. A “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation.

The vector of the present invention may further include a leader sequence, a polyadenylation sequence, a promoter, an enhancer, an upstream activation sequence, a signal peptide sequence, and a regulatory sequence including a transcription terminator, if necessary.

The term "vector" of the present invention is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can be reproduced independently in host cells. The term “carrier” is often used interchangeably with “vector”. The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals usable in eukaryotic cells are known.

Transformation of plants refers to any method of transferring DNA into plants. Such transformation methods do not necessarily have periods of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including monocotyledons as well as dicotyledons. In principle, any transformation method can be used to introduce hybrid DNA according to the present invention into suitable progenitor cells. Methods include calcium/polyethylene glycol method for protoplasts (Krens et al, 1982, Nature 296: 72-74; Negrutiu et al, 1987, Plant Mol Biol 8: 363-373), electroporation method for protoplasts (Shillito et al, 1985, Bio/Technol 3: 1099-1102), microinjection with plant urea (Crossway et al, 1986, Mol Gen Genet 202: 179-185), particle bombardment of various plant urea (DNA or RNA-coated) (Klein et al, 1987, Nature 327: 70), Infection by viruses in Agrobacterium tumerfaciens-mediated gene transfer (incomplete) by invasion of plants or transformation of mature pollen or vesicles (EP 0 301 316 It can be appropriately selected from A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery.

The vectors of the invention may contain one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, and all genes capable of distinguishing a transformed cell from a non-transgenic cell correspond to this. Examples include herbicide resistance genes such as glyphosate, phosphinothricin and glufosinate, kanamycin, G418, bleomycin, hygromycin, There is an antibiotic resistance gene such as chloramphenicol, but is not limited thereto.

In one aspect, the present invention relates to a transgenic plant transformed with the vector of the present invention.

In one aspect, the present invention relates to a nucleic acid molecule that inhibits the expression of the MYB81 gene.

In one embodiment, it may be siRNA, shRNA or miRNA that inhibits the expression of the MYB81 gene.

As used herein, the term “expression inhibition” refers to causing a decrease in the expression (to mRNA) or translation (to a protein) of a target gene, preferably thereby making the target gene expression undetectable or present at an insignificant level. Means being done.

As used herein, the term “siRNA (small interfering RNA)” refers to a short double-stranded RNA capable of inducing RNAi (RNA interference) through cleavage of a specific mRNA. In general, siRNA is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto.

In one aspect, the present invention relates to a composition for inducing male infertility comprising a nucleic acid molecule that inhibits the expression of the MYB81 gene.

In one aspect, the present invention relates to a composition for determining male infertility of plants, comprising an agent capable of confirming the deletion of TACGTT in the ORF of the MYB81 gene.

In one embodiment, the agent may be a probe or a primer set, and may include a primer set represented by SEQ ID NOs: 8 and 9, 10 and 11, 10 and 12, 10 and 13, 14 and 15, and 14 and 16. In addition, the primer set represented by SEQ ID NOs: 14 and 15 can specifically detect the wild-type MYB81 gene without the deletion mutation, and the primer sets represented by SEQ ID NOs: 14 and 16 are specific for the myb81 gene with the deletion mutation. Can be detected.

In one embodiment, by using the primer set, wild type (MYB81 +/+), heterozygous (myb81 +/-) and homozygous (myb81 -/-) plants can be distinguished.

In one aspect, the present invention relates to a method for producing a male infertile plant comprising the step of deleting TACGTT in the ORF of the MYB81 gene.

In one aspect, the present invention 1) extracting genomic DNA from a plant; And 2) It relates to a method for determining male infertility of a plant comprising the step of confirming the gene mutation in the ORF of the MYB81 gene.

In one embodiment, the genetic variation may be a TACGTT deletion.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

Example 1. Selection of male-embryonic mutant lines

In order to isolate the genes important for pollen development, a genetically-labeled mutant group was obtained from Arabidopsis thaliana groups (Ler-O wild type) using a herbicide-resistant T-DNA vector, and then herbicide-resistant plants were selected from the next generation to produce mature pollen. Pollen development-related mutants (giant pollen, one- or two-nucleated pollen, two-cell pollen, dead pollen, etc.) are selected by the method of observing pollen phenotype after collecting and staining with DAPI. The AP28-23 line (male-embryonic mutation) was selected. To confirm the pollen phenotype of the AP28-23 line after selection, wild-type Arabidopsis (n=4601), AP28-23 heterozygous mutant (n=7,186) and homozygous mutant (n=1,191) Arabidopsis flowers (n= 6349) from each of the mature pollen samples, and 250 µl of DAPI (4',6'-diamidino-2-phenylindole) staining solution (0.1 M sodium phosphate (pH 7), 1 mM EDTA, 01% Triton X-100 and 0,4 μl/ml DAPI; high grade, Sigma) was placed in a microfuge tube, vortexed, and centrifuged to obtain a pollen pellet. The obtained pollen pellet was transferred to an optical microscope slide and confirmed by bright field and UV epi-illumination.

As a result, in the case of the wild type, 96.7% of pollen grains appeared as three-cell pollen (Fig. 1A), and 3.3% were abnormal pollen grains, of which 3.2% of dead pollen grains (Figs. 1B and C) And 0.1% mononuclear or double nuclei (Fig. 1G and J). On the other hand, AP28-23 heterozygous and homozygous showed 48.5% and 0% of normal three-cell pollen grains, respectively (Fig. 1A), among abnormal pollen grains, and apoptotic pollen grains, respectively, 45.2% and 99.5% (Figs. 1B and C ), other abnormal pollen was found to be 6.2% and 0.5% (Figs. 1D to L). In addition, some abnormal pollen grains contained an irregularly sized vacuole-like structure inside the plant cytoplasm (Figs. 1F, I and L).

Example 2. Determine when the cause of the AP28-23 mutant occurs

In order to determine at which stage the defect occurs when pollen of AP28-23 occurs, pollen in the development process was observed from the anther of the DAPI-stained wild type, AP28-23 heterozygous and homozygous Arabidopsis buds.

As a result, in the case of the wild type (Fig. 2A to H), meiosis of diploid microsporocytes produced tetraploids, which are four haploids surrounded by thick callose walls (Fig. 2A), and then in the enzyme complex. It was separated into each microspore (Fig. 2B). Thereafter, the growth and development of microcells was accompanied by an increase in fusion of the outer membrane wall and progressive vacuole biogenesis (FIGS. 2C and D). The polarized microcells were polarized to an eccentric position from the wall of microcells (FIG. 2D), and the polarized microcells became bicellular pollen grains through asymmetric division in PMI (FIG. 2E). The resulting cells were internalized in the vegetative cytoplasm after calose digestion (FIG. 2F), entered the second cell cycle for PMII (FIG. 2G), and produced a three-cell pollen with two sperm cells (FIG. 2H). In the AP28-23 heterozygote, from tetraploid to microcellular polarization stage normally occurred (Figs. 2I to L), but abnormal phenotypes began to appear from the next bud stage. When the PMI producing two unequal daughter cells in about half of the developing spores was completed (Figure 2E), the other half of the mutant spores were found to have polarized nuclei and large vacuole retention (Figure 2M). These undivided microcells continued to grow as the size of the single vacuoles increased, but at the same time, they gradually degenerated and the nuclei stained with DAPI decreased (Fig. 2T, indicated by AbDEG in Figs. 2M to P). In only some cases, mutant microcells were found to divide at a delayed stage to become a much larger two-cell-like pollen with two nuclei, and DAPI-stained organelle DNA was found to be abundant compared to normal two-cell pollen grains (Figure 2E) (2.7 %-4.2%, denoted AbEBC in FIGS. 2T and 2Q-S). In addition, the incidence of abnormal spores in the late-two-cell and three-cell stage, similar to the early-two-cell stage, was 1.9-2.3% for wild type, 51.3- 51.6% for AP28-23 heterozygote, and AP28-23 isotype. It was found to be 100% conjugate (Table 1). In AP28-23 heterozygous and homozygous, a normal phenotype was observed in the polarized microcellular stage, but abnormal pollen was observed in the early-two cell stage (Table 1). At this stage, wild-type showed abnormal pollen with a frequency of 1.7%, but AP28-23 heterozygous showed 49.2% and AP28-23 homozygous showed 100% abnormal pollen (Table 1). Through this, it was found that the normal function of the mutated gene in the AP28-23 line is critically important for the progression of PMI by polarized microcells.

Therefore, it can be seen that the polarized microcellular vesicles normally occur until, but after that, microcellular dysplasia (first mitotic mitosis) does not occur and gradual apoptosis proceeds.

Example 3. Confirmation of genetic transfer and infertility of AP28-23 mutants

AP28-23's genetic transmission through male and female germ cells Heterozygous mutations were confirmed by performing a hybridization test between the Landsberg ecological type (Landsberg erecta, L er -0) and the pollen phenotype of the F ₁ progeny was screened. The transmission efficiency (TE) of AP28-23 through male and magnetic germ cells was calculated by Equation 1. For example, if the mutant allele is delivered with 100% efficiency, the hybrid test progeny will separate into a 1:1 mutant:wild type.

Cross +/+ AP28-23 ^+/- Total AP28-23 ^+/- (%) TE (%) AP28-23 ^+/- x +/+ 158 142 300 47.3 89.9 +/+ x AP28-23 ^+/- 298 2 300 0.7 0.7

The transduction efficiency (TE) of the mutant AP28-23 allele through male and female germ cells was shown in FIG. 3. TE, which represents the percentage of mutant alleles that successfully transferred the mutation to the next generation, only 0.7% of the male germ cells with the mutant allele (n=300) successfully transferred, and with the mutant allele. 89.9% of the female germ cells (n=300) were successfully delivered (Fig. 3A and Table 2). In addition, the presence of the AP28-23 homozygous at a very low frequency of 0.3% among AP28-23/+ autologous progeny is inferred as a result of decreased transmission of AP28-23 (Fig. 3C). Therefore, it is confirmed that the genetic material that causes the mutant pollen phenotype of AP28-23 is normally transmitted to the next generation through the female spouse itself, but is transmitted at an extremely low frequency through the male spouse itself, so that the function of the corresponding gene is transmitted to the male spouse itself. It was found that it was specifically required for development.

Example 4. Identification of genes causing infertility in AP28-23 mutants

To identify the causative gene that causes the mutant pollen phenotype of AP28-23, a map-based cloning method was used (FIG. 4). Specifically, F ₁ obtained by fertilizing the heterozygous AP28-23 mutant ( Ler ) with wild-type pollen grains (Col-0) was self-fertilized, and their F ₂ seeds were grown and used as a mapping population. The leaves of 2908 F ₂ plants were collected, and genomic DNA was extracted according to the CTAB (Cetyl Trimethyl Ammonium Bromide) method (Murray et al, 6 1980). Then, PCR-based mapping was performed using 10 known SSLP markers and 13 DNA samples distributed on 5 chromosomes (FIG. 5).

As a result, 3% of the recombination events with the marker nga168 showed that the mutant locus was closely related to At2g39010 on chromosome 2 (FIG. 6 ). Another 20 DNA samples from wild-type progeny from the same F ₂ population, with new 8 SSLP markers designed around nga168, also showed a low recombination rate, indicating the genetic association of the mutant locus with the tested markers on chromosome 2. Show. Through the analysis, the mutation locus (AP28-23 mutant pollen phenotype of AP28-23 between the two markers 26940 and 27030 between the ~37Kb region (Fig. 7 and 8, SEQ ID NO: 3) of chromosome 2 containing 11 candidate genes Including the causative genes).

Thereafter, as a result of sequencing the coding regions of 11 candidate genes, it was confirmed that 6 bases (TACGTT) of At2g26960 among 11 candidate genes were deleted at this site (Table 3 and FIG. 9). These are the genetic codes that encode valine and tyrosine (V35 and Y36), through BLAST search, the At2g26960 gene encodes the R2R3 MYB domain of the transcription factor MYB81, and the region where the gene mutation occurs is TACGTT of 106-111 bp of the R2 domain. Appeared to be. Through this, it was inferred that the function as a MYB transcriptional regulatory factor is disturbed due to the mutation located at the R2R3 DNA binding site.

Example 5. Verification of genes causing infertility

5-1. Phenotypic identification of deletion mutations

The MYB81 cDNA fragment amplified by PCR from Col wild-type plants consisted of 3 exons and 2 introns, unlike 2 exons and 1 intron predicted from Arabidopsis information. In order to confirm the exact structure of MYB8 , as a result of sequencing the full-length cDNA without deletion in both wild-type and mutant plants, respectively, it consists of the same structure (3 exons and 2 introns) (Fig. 7), 405 instead of 427 amino acids. It was deduced to encode dog amino acids (1215 bp). Accordingly, in order to confirm whether the 6bp deletion mutation identified above is the cause of the defective pollen phenotype of the AP28-23 mutant, a vector containing 5'upstream of the wild-type MYB81 gene and full-length cDNA was constructed. Specifically, by amplifying the ~1.6-kb upstream promoter (1,619kb) of the start codon ATG (1635bp including 16bp of Asci and NotI restriction enzyme recognition sites, SEQ ID NO: 4) and the full length cDNA fragment (1,215kb, SEQ ID NO: 7) After constructing a vector containing this, each of Agrobacterium tumefaciens (GV3101) was AP28-23 by the floral dipping method using It was introduced into heterozygous mutants. Transgenic plants were selected on MS (Murashige and Skoog) agar containing 20 mg/L of hygromycin. For genotyping analysis of plants, genomic DNA was extracted from the leaf tissue of each transgenic plant, homogenized with TissueLyser (Qiagen, http://wwwqiagencom), and used for PCR amplification.

As a result, as a result of genotyping T1 plants with primers specific for the wild-type MYB81 and mutant myb81 alleles, 49 heterozygous and 8 homozygous mutants bearing the complementary T-DNA vector were isolated, and non-transformed The mutant pollen phenotype was decreased compared to the control mutant plants (FIG. 10A). Through this, it was found that the MYB81 gene is the cause of the mutant pollen in AP28-23.

5-2. MYB81 gene expression site and timing analysis

In order to confirm the expression of MYB81 gene for each spore tissue and pollen development stage, seedlings, roots, leaves, stems, closed bud closters and blooming flowers, and RNA derived from developing pollen grains, respectively Was used to perform a semi-quantitative RT-PCR analysis. Specifically, using the RNeasy Plant Mini kit (Qiagen, http://www qiagencom/), seedlings, roots, leaves, stems, closed bud closters, and blooming flowers are used as wild-type spore tissues, and pollen development Stages include tetraploid and microcellular pollen mixture (F2.1), microcellular and early two-cell pollen mixture (F2.2), late two-cell pollen (F2.3), three-cell pollen (F3.0) and mature pollen ( MP), each of the total RNAs was extracted and converted into cDNA, and then a reaction product of 20 µl containing 1 µl of a single-stranded cDNA sample, e-taq polymerase, and 10 pmol of each primer was used for 30 seconds at 95° C., 95 RT-PCR (Reverse transcriptase-PCR) was performed under the conditions of 35 cycles at 15 seconds at ℃, 15 seconds at 55 ℃ and 50 seconds at 72 ℃. The histone gene was used as a control. PCR amplification products were loaded on 1% agarose gel, electrophoresed in 1X TAE buffer, and stained with EtBr (ethidium bromide).

As a result, the MYB81 transcript was detected only in young buds among spore tissues, and not found in other spore tissues (FIG. 10B). In addition, as a result of testing with RNA samples concentrated for a specific stage of pollen development, the MYB81 transcript was not expressed in mature pollen grains, but expression was detected in other developing pollen grains. It was found to be the largest in two-cell pollen grains (Fig. 10C).

5-3. Confirmation of specific MYB81 gene expression pattern

In order to confirm the more specific MYB81 expression pattern, GUS reporter analysis was performed using inflorescence and seedlings. Specifically, the vectors pMYB81-GUS and pMYB81-MDB-GUS (pMYB81-CYCB1: 1 ^MDB- GUS) containing MYB81 promoter fused with a GUS reporter with or without CYCB1;1 gene-derived MDB (mitotic destruction box) Each transformed line was generated to confirm the expression pattern of MYB81 in 50 T1 plants for each vector. The pMYB81-GUS reporter vector described above is a pMYB81 fragment identified in the above example, Oh et al. , In the intermediate vector used in 2016, it was introduced between AscI and NotI in front of the GUS coding region located between NotI and SpeI. After cutting with AscI and SpeI, the pMYB81-GUS fragment was produced by cloning the transgenic plant into pER10, which can be selected with kanamycin.

As a result, GUS signals were shown in young buds in 42 inflorescences among the pMYB81-GUS transformants and 45 among the pMYB81-MDB-GUS transformants. The use of MDB induced GUS signaling even in a smaller group of younger buds that are likely to contain developing microcells (Figures 11A and C). On the other hand, no GUS signal was observed in the seedlings derived from 20 T2 lines of pMYB81-GUS (Fig. 11B). From this, it was found that the myb81 mutant phenotype is male-specific and begins at an early stage of pollen development.

5-4. Confirmation of the intracellular expression site and timing of MYB81 protein

Using the transformant line generated with the pMYB81-MYB81-mRFP1 vector, the intracellular expression position of the MYB81 protein at each stage of pollen development was confirmed. Specifically, to construct pMYB81-MYB81, a 1,628 bp gene fragment containing a sequence immediately above the start codon was amplified with forward and reverse primers tagged with AscI and NotI positions, respectively. Full-length MYB81 cDNA with a stop codon was amplified with forward and reverse primers tagged with sequences recognized by NotI and SpeI, respectively. These two fragments were subcloned into a modified pBluscript-based vector backbone containing AscI, NotI and SpeI. The pMYB81-MYB81 fragment was cut with AscI and SpeI, and then cloned into a pER8 vector capable of selecting a transgenic plant with hygromycin. In order to construct pMYB81-MYB81-mRFP1 for partial analysis of intracellular positions, a full-length MYB81 cDNA fragment that does not contain a stop codon was amplified and inserted between NotI and BamHI positions under the MYB81 promoter. Then, the mRFP1 coding region including the stop codon was determined by Oh et al. , Sub-cloned between the BamHI and SpeI positions of the intermediate vector used in 2016. The entire expression cassette was cloned into pER10 to produce pMYB81-MYB81cDNA-mRFP1 (SEQ ID NO: 5), and transgenic plants (50) were prepared to confirm the expression of MYB81 in cells at each stage of pollen development.

As a result, no RFP signal was detected in mature pollen, but RFP signals specifically located in the nucleus were observed in 36 out of 40 T1 lines in young drugs at the mixed bud stage including developing spores or pollen. . In addition, in order to confirm the more accurate MYB81 protein expression location and timing, as a result of confirming the expression location at each stage of pollen development using continuous stage bud samples derived from several lines in T1 and T2 generations, MYB81-mRFP1 was released from the tetraploid. Immediately after, it was not detected in the youngest microcells (Fig. 12A), but soon began to be seen in the nucleus and remained detectable until the polarized microcell stage (Figs. 12B to D). Thereafter, from the later PMI stage, the RFP signal was significantly reduced and was no longer detected (Figs. 12E to H). For reference, as a result of confirming the expression of MYB81 in the roots of the seedlings, the same as the results in the promoter-GUS reporter line, RFP signals were not observed in the root cells of the seedlings for ~10 days (Fig. 12I). Through this, it was found that the expression of MYB81 is subject to strict temporal and spatial regulation, and is specific to the microcellular stage during pollen development.

5-5. MYB81 Phenotype identification upon ectopic expression of genes

Based on the fact that the expression of MYB81 confirmed through the above examples very restricts the development of pollen grains, in order to confirm whether ectopic expression of deleted MYB81 has other effects on plant development, an overexpression analysis of MYB81 of 405aa identified in the present invention was performed. For this purpose, pUBQ14-MYB81 and p35S-MYB81-dHA were prepared using p35S or pUBQ14, which are broadly expressed promoters, and introduced into wild Arabidopsis thaliana. Specifically, pUBQ14-MYB81 and p35S-MYB81 were replaced with pUBQ14 and p35S fragments amplified using primers tagged with AscI and NotI sites for the pMYB81 fragment in the pMYB81-MYB81 intermediate vector, respectively. In addition, pUBQ14-myb81 and p35S-myb81 expressing the mutant myb81 in which two amino acids were deleted by the above method were also constructed using the same restriction enzyme site. Expression cassettes were cloned into pER8 or pER10, each vector was prepared, and then transformants were selected by introducing each into wild-type Arabidopsis thaliana. In addition, in order to confirm whether MYB81 and myb81 are located in the same nucleus when overexpressed in spore cells, a constitutive promoter could not be used for MYB81 overexpression, so estradiol inducible MYB81-mRFP1 (pXVE-MYB81-mRFP1 ) And p35S-myb81-mRFP1 vector were prepared to prepare a transformant. Seedlings of the pXVE-MYB81-mRFP1 transformant were grown in MS medium with or without 5 μM estradiol, and seedlings of the p35S-myb81-mRFP1 transformant were grown in MS medium.

As a result, transformants could not be selected when transformed with p35S-MYB81-RFP and pUBQ14-MYB81-RFP vectors containing myb81 without the deletion mutation. On the other hand, when overexpressing with a vector constructed using a mutant myb81 cDNA in which 6bp for V ₃₅ Y ₃₆ was deleted, abundant transformation lines were generated at a normal transformation frequency, and 50 T1 plants selected from each vector None of them showed phenotypic changes.

In addition, after induction of estradiol, MYB81-mRFP1 (Fig. 13J) and myb81-mRFP1 (Fig. 13K) were detected in the nuclei of root cells. Through this, it could be inferred that both normal MYB81 and mutant myb81 proteins are confined to the nucleus in somatic cells, but ectopic overexpression of functional MYB81 under a constitutive promoter causes lethality during the seedling phase. Therefore, it can be seen that the expression timing and location of the MYB81 gene are very important for plant development.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Method for producing male-sterile plant <130> PN1807-261 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> myb81 mutant orf <400> 1 atggggaaag ttcgtcaaga ttcaggatca gacgatgaca attcaataaa gaaatctttt 60 actaaaggtc cttggacaca agcagaagac aatcttttga tagctgataa acatggtgat 120 ggaaactgga acgctgttca aaacaactcg ggcctttctc gttgtggtaa aagttgtcgc 180 cttcgttggg taaatcatct gagaccagat ttgaaaaaag gcgcttttac agagaaagaa 240 gagaaacgtg tcatcgaact tcatgctttg ttagggaaca aatgggcacg aatggctgaa 300 gaagtatagc tcacgtttcc ttctttaacc ctaatcttga tttttttttt cgtcttctaa 360 tcttttgttg tttctttcag ctaccgggac gaacagataa tgagatcaag aacttctgga 420 atacaagact taagagactg caacgacttg gtttacctgt ttaccccgat gaagttcgag 480 agcatgcgat gaatgcagcg acacattccg gtctaaacac tgattcattg gatggtcatc 540 atagtcaaga atatatggag gcagatactg ttgagattcc tgaagtggat ttcgagcatt 600 taccactcaa tcgatcttct tcatactacc agtcaatgct tcgacatgtt cctcccacta 660 acgtctttgt gagacaaaaa ccgtgtttct ttcagccgcc taatgtgtat aacttgatac 720 caccatctcc ttacatgtct accggaaaac gtcctagaga accagaaacc gcgtttcctt 780 gcccgggtgg atataccatg aacgagcaaa gccctcggct ctggaactat ccttttgttg 840 agaacgtttc agaacagtta ccggatagtc atttgcttgg taatgctgct tattcttctc 900 ctcctggacc tcttgttcac ggggttgaga atttcgagtt cccttcattc caatatcatg 960 aagagcctgg cggttgggga gcagatcaac ctaacccaat gccggaacat gagtcagata 1020 acactttggt tcaatctcct ctgacagctc aaacaccatc agattgtcca tcatcatcac 1080 tctatgatgg actgttggaa tcagttgtct atggatcatc aggggagaaa ccggcaaccg 1140 ataccgattc agaatcttca ctgtttcagt catttacacc agccaatgaa aacataaccg 1200 gtaaaacttg ttttcttact ctttatgcat tacatgcact tcattgtttg tgtaatcaat 1260 tcaagaagtc tcctcttctc catctgcatg acaaacttaa ttggtgtaac aaatttaggt 1320 tcaactcctt caaatcaggg acacacatcc tttgatggtg attggataag attacttctc 1380 ggtgaggata gatataaaac cgagtag 1407 <210> 2 <211> 1413 <212> DNA <213> Arabidopsis sp. <400> 2 atggggaaag ttcgtcaaga ttcaggatca gacgatgaca attcaataaa gaaatctttt 60 actaaaggtc cttggacaca agcagaagac aatcttttga tagcttacgt tgataaacat 120 ggtgatggaa actggaacgc tgttcaaaac aactcgggcc tttctcgttg tggtaaaagt 180 tgtcgccttc gttgggtaaa tcatctgaga ccagatttga aaaaaggcgc ttttacagag 240 aaagaagaga aacgtgtcat cgaacttcat gctttgttag ggaacaaatg ggcacgaatg 300 gctgaagaag tatagctcac gtttccttct ttaaccctaa tcttgatttt ttttttcgtc 360 ttctaatctt ttgttgtttc tttcagctac cgggacgaac agataatgag atcaagaact 420 tctggaatac aagacttaag agactgcaac gacttggttt acctgtttac cccgatgaag 480 ttcgagagca tgcgatgaat gcagcgacac attccggtct aaacactgat tcattggatg 540 gtcatcatag tcaagaatat atggaggcag atactgttga gattcctgaa gtggatttcg 600 agcatttacc actcaatcga tcttcttcat actaccagtc aatgcttcga catgttcctc 660 ccactaacgt ctttgtgaga caaaaaccgt gtttctttca gccgcctaat gtgtataact 720 tgataccacc atctccttac atgtctaccg gaaaacgtcc tagagaacca gaaaccgcgt 780 ttccttgccc gggtggatat accatgaacg agcaaagccc tcggctctgg aactatcctt 840 ttgttgagaa cgtttcagaa cagttaccgg atagtcattt gcttggtaat gctgcttatt 900 cttctcctcc tggacctctt gttcacgggg ttgagaattt cgagttccct tcattccaat 960 atcatgaaga gcctggcggt tggggagcag atcaacctaa cccaatgccg gaacatgagt 1020 cagataacac tttggttcaa tctcctctga cagctcaaac accatcagat tgtccatcat 1080 catcactcta tgatggactg ttggaatcag ttgtctatgg atcatcaggg gagaaaccgg 1140 caaccgatac cgattcagaa tcttcactgt ttcagtcatt tacaccagcc aatgaaaaca 1200 taaccggtaa aacttgtttt cttactcttt atgcattaca tgcacttcat tgtttgtgta 1260 atcaattcaa gaagtctcct cttctccatc tgcatgacaa acttaattgg tgtaacaaat 1320 ttaggttcaa ctccttcaaa tcagggacac acatcctttg atggtgattg gataagatta 1380 cttctcggtg aggatagata taaaaccgag tag 1413 <210> 3 <211> 3700 <212> DNA <213> Arabidopsis sp. <400> 3 cgatattgta gggatcgctt ttgattagca catttttccg atttttatta aaccctaata 60 tattagatac cttttaaagc ctcaaaatac tcagacgcta attctctatc aaattttgag 120 atttttggtg agtttttcct aaacattttt ttaatttctc ctcttcagat ttttgtaact 180 ttttcggcaa ttcattatca gaattttttg catgtttagg attagacgtt cgcctctgtt 240 attagttctc tatcaccgat caggtaagct tcattttttt ctttgttcaa tttttaatct 300 cactccaaga aagtgagatt taagcttttt aatgttgtct atattttcag ggatattaat 360 gggttgatct atacttcttt aaatctgatc gatcatacaa atccaccgcc gttgatcacg 420 aggacaaagt ctaggtagat ttttggatat ttcctagcaa aaaatcggga ggtttcgttc 480 ttgatgtttt gagattgagt ttggactttg gtaatagaga gtctatcaaa ctagtgaaat 540 cagtttatca acccataaat atcattggtc atctatcatt tctaaaccga ctataagtac 600 taaaccatac atgtctagat acggatttat ttcgtcgaaa cctttcaaga ttgtttacta 660 aacacaattt tgttatcaat ggtataggtg aattatttcg taaaaccatt gctagattca 720 gattgatgtt ttagcacacc ccaatatcct ctcaactgcg cgcggagtgc ttatggaata 780 tcctgttgag tgggtttaag acgtctgtaa tcatttccta tgtaagttct aactatagta 840 atatttacag taaaacctaa gaaattgatt aatctaaaaa ggtattaatt tatcgataaa 900 ttaataaaat tgtacaactt ttgtaaattc ttaaaaaaat tcgatataaa atgtattttt 960 ttctttataa caatagtaat taaaagtaga aagcaaatat taagtttaaa aaacaataaa 1020 acttttttta catgttgtta aatattagaa ttgaacacta caacaaatta ataaaaattg 1080 aagaaaaaaa ttactacaga tattttacat atgatattta taatatatat tagtaaattt 1140 ataaaattat taatttatac tattgatgag accatatatt attgatttgt atcaattttc 1200 acattggtct aaactcggga ccggaaaaat ttattaattt atagagatta ttaatttatc 1260 gatattaatt tatagaggtt ttactgtatt atgcattgat atatcattct taaagatgaa 1320 ttgtgtaata tactaatttt atctgattta acctaattaa tctggactaa catattcagc 1380 ataatcaatc ttgtatacat attattctag cagaattaat cttctcgata tgttgttatt 1440 tttgtgagtt ttgagcaatc tattaagaaa aagtgctgtc tatatgatgc tcaatcatag 1500 aggttatggt caaaactaac atttttaaca tttaagtgta gaggagaaaa gtttatagtt 1560 aagtctcaga aaaaatcatg tcaacttatt ccattgatag ataccatcca tcgtaccacc 1620 actgcccgcg acatgcaaac cttctccaaa agtaaagcgt taagtctctt aggagtttaa 1680 ccaccttttg tgaattttgg tgtactccat tttgcgtctt ccatagattt ctttcaataa 1740 gaaagattat gataaaaaaa attaggattt gctttggact tatcgcttgc aatttaacat 1800 gttcaaatac gcaaaaccta tatatagtta acaaataaag ccacatgagt attgattact 1860 aatttctttg tttgcttgtt tgaataccaa gaaaacttta gtgactgtaa aacgctaaaa 1920 tacacttttc ttagagaaaa tttcactgaa aaacaaaaag atatacatag agagatttca 1980 tgttagaata aggtttctac gagtatttca taagttatat tatgtatgaa ttaattaact 2040 attgcacgaa aggagtttat cttcaatatt tttttaattc cggaattata taggttaatt 2100 ttgttggcta gagagaaact tgtatatata tatatatata taaataatta catattattt 2160 tacagatgac acaagatggg gaaagttcgt caagattcag gatcagacga tgacaattca 2220 ataaagaaat cttttactaa aggtccttgg acacaagcag aagacaatct tttgatagct 2280 tacgttgata aacatggtga tggaaactgg aacgctgttc aaaacaactc gggcctttct 2340 cgttgtggta aaagttgtcg ccttcgttgg gtaaatcatc tgagaccaga tttgaaaaaa 2400 ggcgctttta cagagaaaga agagaaacgt gtcatcgaac ttcatgcttt gttagggaac 2460 aaatgggcac gaatggctga agaagtatag ctcacgtttc cttctttaac cctaatcttg 2520 attttttttt tcgtcttcta atcttttgtt gtttctttca gctaccggga cgaacagata 2580 atgagatcaa gaacttctgg aatacaagac ttaagagact gcaacgactt ggtttacctg 2640 tttaccccga tgaagttcga gagcatgcga tgaatgcagc gacacattcc ggtctaaaca 2700 ctgattcatt ggatggtcat catagtcaag aatatatgga ggcagatact gttgagattc 2760 ctgaagtgga tttcgagcat ttaccactca atcgatcttc ttcatactac cagtcaatgc 2820 ttcgacatgt tcctcccact aacgtctttg tgagacaaaa accgtgtttc tttcagccgc 2880 ctaatgtgta taacttgata ccaccatctc cttacatgtc taccggaaaa cgtcctagag 2940 aaccagaaac cgcgtttcct tgcccgggtg gatataccat gaacgagcaa agccctcggc 3000 tctggaacta tccttttgtt gagaacgttt cagaacagtt accggatagt catttgcttg 3060 gtaatgctgc ttattcttct cctcctggac ctcttgttca cggggttgag aatttcgagt 3120 tcccttcatt ccaatatcat gaagagcctg gcggttgggg agcagatcaa cctaacccaa 3180 tgccggaaca tgagtcagat aacactttgg ttcaatctcc tctgacagct caaacaccat 3240 cagattgtcc atcatcatca ctctatgatg gactgttgga atcagttgtc tatggatcat 3300 caggggagaa accggcaacc gataccgatt cagaatcttc actgtttcag tcatttacac 3360 cagccaatga aaacataacc ggtaaaactt gttttcttac tctttatgca ttacatgcac 3420 ttcattgttt gtgtaatcaa ttcaagaagt ctcctcttct ccatctgcat gacaaactta 3480 attggtgtaa caaatttagg ttcaactcct tcaaatcagg gacacacatc ctttgatggt 3540 gattggataa gattacttct cggtgaggat agatataaaa ccgagtagaa gaggtttctt 3600 tggaaggcct aatttataaa gtgcttgttc tctttcgtct gcataatgtc tttttatgcg 3660 ttgagtagat tagtcatgtc tttagatata tcagtatagt 3700 <210> 4 <211> 1635 <212> DNA <213> Artificial Sequence <220> <223> pMYB81 promoter <400> 4 ggcgcgccca acccataaat atcattggtc atctatcatt tctaaaccga ctataagtac 60 taaaccatac atgtctagat acggatttat ttcgtcgaaa cctttcaaga ttgtttacta 120 aacacaattt tgttatcaat ggtataggtg aattatttcg taaaaccatt gctagattca 180 gattgatgtt ttagcacacc ccaatatcct ctcaactgcg cgcggagtgc ttatggaata 240 tcctgttgag tgggtttaag acgtctgtaa tcatttccta tgtaagttct aactatagta 300 atatttacag taaaacctaa gaaattgatt gatctaaaaa ggtattaatt tatcgataaa 360 ttaataaaat tgtacaactt ttgtaaattc ttacaaaaat tcgatataaa atgtattttt 420 ttctttataa caatagtaat taaaagtaga aagcaaatat taagtttaaa aaacaataaa 480 acttttttta catgttgtta aatattagaa ttgaacacta caacaaatta ataaaaattg 540 aagaaaaaaa ttactacaga tattttacat atgatattta taatatatat tagtaaattt 600 ataaaattat taatttacac tattgatgag accatatatt attgatttgt atcaattttc 660 acattggtct aaactcggga ccggaaaaat ttattaattt atagagatta ttaatttatc 720 gatattaatt tatagaggtt ttactgtatt atgcattgat atatcattct taaagatgaa 780 ttgtgtaata tactaatttt atctgattta acctaattaa tctggactaa catattcagc 840 ataatcaatc ttgtatacat attattctag cagaattaat cttctcgata tgttgttatt 900 tttgtgagtt ttgagcaatc tattaagaaa aagtgctgtc tatatgatgc tcaatcatag 960 aggttatggt caaaactaac atttttaaca tttaagtgta gaggagaaaa gtttatagtt 1020 aagtctcaga aaaaatcatg tcaacttatt ccattgatag ataccatcca tcgtaccacc 1080 actgcccgcg acatgcaaac cttctccaaa agtaaagcgt taagtctctt aggagtttaa 1140 ccaccttttg tgaattttgg tgtactccat tttgcgtctt ccatagattt ctttcaataa 1200 gaaagattat gataaaaaaa attaggattt gctttggact tatcgcttgc aatttaacat 1260 gttcaaatac gcaaaaccta tatatagtta acaaataaag ccacatgagt attgattact 1320 aatttctttg tttgcttgtt tgaataccaa gaaaacttta gtgactgtaa aacgctaaaa 1380 tacacttttc ttagagaaaa tttcactgaa aaacaaaaag atatacatag agagatttca 1440 tgttagaata aggtttctac gagtatttca taagttatat tgtgtatgaa ttaattaact 1500 attgcacgaa aggagtttat cttcaatatt tttttaattc cggaattata taggttaatt 1560 ttgttggcta gagagaaact tgtatatata tataaataat tacatattat tttacagatg 1620 acacaaggcg gccgc 1635 <210> 5 <211> 6732 <212> DNA <213> Artificial Sequence <220> <223> pMYB81-MYB81cDNA-mRFP1 vector <400> 5 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctcggc gcgcccaacc 2220 cataaatatc attggtcatc tatcatttct aaaccgacta taagtactaa accatacatg 2280 tctagatacg gatttatttc gtcgaaacct ttcaagattg tttactaaac acaattttgt 2340 tatcaatggt ataggtgaat tatttcgtaa aaccattgct agattcagat tgatgtttta 2400 gcacacccca atatcctctc aactgcgcgc ggagtgctta tggaatatcc tgttgagtgg 2460 gtttaagacg tctgtaatca tttcctatgt aagttctaac tatagtaata tttacagtaa 2520 aacctaagaa attgattgat ctaaaaaggt attaatttat cgataaatta ataaaattgt 2580 acaacttttg taaattctta caaaaattcg atataaaatg tatttttttc tttataacaa 2640 tagtaattaa aagtagaaag caaatattaa gtttaaaaaa caataaaact ttttttacat 2700 gttgttaaat attagaattg aacactacaa caaattaata aaaattgaag aaaaaaatta 2760 ctacagatat tttacatatg atatttataa tatatattag taaatttata aaattattaa 2820 tttacactat tgatgagacc atatattatt gatttgtatc aattttcaca ttggtctaaa 2880 ctcgggaccg gaaaaattta ttaatttata gagattatta atttatcgat attaatttat 2940 agaggtttta ctgtattatg cattgatata tcattcttaa agatgaattg tgtaatatac 3000 taattttatc tgatttaacc taattaatct ggactaacat attcagcata atcaatcttg 3060 tatacatatt attctagcag aattaatctt ctcgatatgt tgttattttt gtgagttttg 3120 agcaatctat taagaaaaag tgctgtctat atgatgctca atcatagagg ttatggtcaa 3180 aactaacatt tttaacattt aagtgtagag gagaaaagtt tatagttaag tctcagaaaa 3240 aatcatgtca acttattcca ttgatagata ccatccatcg taccaccact gcccgcgaca 3300 tgcaaacctt ctccaaaagt aaagcgttaa gtctcttagg agtttaacca ccttttgtga 3360 attttggtgt actccatttt gcgtcttcca tagatttctt tcaataagaa agattatgat 3420 aaaaaaaatt aggatttgct ttggacttat cgcttgcaat ttaacatgtt caaatacgca 3480 aaacctatat atagttaaca aataaagcca catgagtatt gattactaat ttctttgttt 3540 gcttgtttga ataccaagaa aactttagtg actgtaaaac gctaaaatac acttttctta 3600 gagaaaattt cactgaaaaa caaaaagata tacatagaga gatttcatgt tagaataagg 3660 tttctacgag tatttcataa gttatattgt gtatgaatta attaactatt gcacgaaagg 3720 agtttatctt caatattttt ttaattccgg aattatatag gttaattttg ttggctagag 3780 agaaacttgt atatatatat aaataattac atattatttt acagatgaca caaggcggcc 3840 gcatggggaa agttcgtcaa gattcaggat cagacgatga caattcaata aagaaatctt 3900 ttattaaagg tccttggaca caagcagaag acaatctttt gatagcttac gttgataaac 3960 atggtgatgg aaactggaac gctgttcaaa acaactcggg cctttctcgt tgtggtaaaa 4020 gttgtcgcct tcgttgggta aatcatctga gaccagattt gaaaaaaggc gcttttacag 4080 agaaagaaga gaaacgtgtc atcgaacttc atgctttgtt agggaacaaa tgggcacgaa 4140 tggctgaaga actaccggga cgaacagata atgagatcaa gaacttctgg aatacaagac 4200 ttaagagact gcaacgactt ggtttacctg tttaccccga tgaagttcga gagcatgcga 4260 tgaatgtagc gacacattcc ggtctaaaca ctgattcatt ggatggtcat catagtcaag 4320 aatatatgga ggcagatact gttgagattc ctgaagtgga tttcgagcat ttaccactca 4380 atcgatcttc ttcatactac cagtcaatgc ttcgacatgt tcctcccact aacgtctttg 4440 tgagacaaaa accgtgtttc tttcagccgc ctaatgtgta taacttgata ccaccatctc 4500 cttacatgtc taccggaaaa cgtcctagag aaccagaaac cgcgtttcct tgcccgggtg 4560 gatataccat gaacgagcaa agccctcggc tctggaacta tccttttgtt gagaacgttt 4620 cagaacagtt accggatagt catttgcttg gtaatgctgc ttattcttct cctcctggac 4680 ctcttgttca cggggttgag aatttcgagt tcccttcatt ccaatatcat gaagagcctg 4740 gcggttgggg agcagatcaa cctaacccaa tgccggaaca tgagtcagat aacactttgg 4800 ttcaatctcc tctgacagct caaacaccat cagattgtcc atcatcatca ctctatgatg 4860 gactgttgga atcagttgtc tatggatcat caggggagaa accggcaacc gataccgatt 4920 cagaatcttc actgtttcag tcatttacac cagccaatga aaacataacc ggttcaactc 4980 cttcaaatca gggacacaca tcctttgatg gtgattggat aagattactt ctcggtgagg 5040 atagatataa aaccgaggga tccatggcct cctccgagga cgtcatcaag gagttcatgc 5100 gcttcaaggt gcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg 5160 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc 5220 ccctgccctt cgcctgggac atcctgtccc ctcagttcca gtacggctcc aaggcctacg 5280 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt 5340 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc 5400 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg 5460 gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgg atgtaccccg 5520 aggacggcgc cctgaagggc gagatcaaga tgaggctgaa gctgaaggac ggcggccact 5580 acgacgccga ggtcaagacc acctacatgg ccaagaagcc cgtgcagctg cccggcgcct 5640 acaagaccga catcaagctg gacatcacct cccacaacga ggactacacc atcgtggaac 5700 agtacgagcg cgccgagggc cgccactcca ccggcgccta actagttacc catacgacgt 5760 tccagactac gctggttacc catacgacgt tccagactac gcttgactgc agatcgttca 5820 aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 5880 atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 5940 tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 6000 aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 6060 gatccgttaa ttaaggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg 6120 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 6180 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 6240 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc 6300 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 6360 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 6420 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 6480 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 6540 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 6600 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 6660 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 6720 cttacaattt ag 6732 <210> 6 <211> 632 <212> PRT <213> Artificial Sequence <220> <223> AtMYB81-mRFP1 vector <400> 6 Met Gly Lys Val Arg Gln Asp Ser Gly Ser Asp Asp Asp Asn Ser Ile 1 5 10 15 Lys Lys Ser Phe Ile Lys Gly Pro Trp Thr Gln Ala Glu Asp Asn Leu 20 25 30 Leu Ile Ala Tyr Val Asp Lys His Gly Asp Gly Asn Trp Asn Ala Val 35 40 45 Gln Asn Asn Ser Gly Leu Ser Arg Cys Gly Lys Ser Cys Arg Leu Arg 50 55 60 Trp Val Asn His Leu Arg Pro Asp Leu Lys Lys Gly Ala Phe Thr Glu 65 70 75 80 Lys Glu Glu Lys Arg Val Ile Glu Leu His Ala Leu Leu Gly Asn Lys 85 90 95 Trp Ala Arg Met Ala Glu Glu Leu Pro Gly Arg Thr Asp Asn Glu Ile 100 105 110 Lys Asn Phe Trp Asn Thr Arg Leu Lys Arg Leu Gln Arg Leu Gly Leu 115 120 125 Pro Val Tyr Pro Asp Glu Val Arg Glu His Ala Met Asn Val Ala Thr 130 135 140 His Ser Gly Leu Asn Thr Asp Ser Leu Asp Gly His His Ser Gln Glu 145 150 155 160 Tyr Met Glu Ala Asp Thr Val Glu Ile Pro Glu Val Asp Phe Glu His 165 170 175 Leu Pro Leu Asn Arg Ser Ser Ser Tyr Tyr Gln Ser Met Leu Arg His 180 185 190 Val Pro Pro Thr Asn Val Phe Val Arg Gln Lys Pro Cys Phe Phe Gln 195 200 205 Pro Pro Asn Val Tyr Asn Leu Ile Pro Pro Ser Pro Tyr Met Ser Thr 210 215 220 Gly Lys Arg Pro Arg Glu Pro Glu Thr Ala Phe Pro Cys Pro Gly Gly 225 230 235 240 Tyr Thr Met Asn Glu Gln Ser Pro Arg Leu Trp Asn Tyr Pro Phe Val 245 250 255 Glu Asn Val Ser Glu Gln Leu Pro Asp Ser His Leu Leu Gly Asn Ala 260 265 270 Ala Tyr Ser Ser Pro Pro Gly Pro Leu Val His Gly Val Glu Asn Phe 275 280 285 Glu Phe Pro Ser Phe Gln Tyr His Glu Glu Pro Gly Gly Trp Gly Ala 290 295 300 Asp Gln Pro Asn Pro Met Pro Glu His Glu Ser Asp Asn Thr Leu Val 305 310 315 320 Gln Ser Pro Leu Thr Ala Gln Thr Pro Ser Asp Cys Pro Ser Ser Ser 325 330 335 Leu Tyr Asp Gly Leu Leu Glu Ser Val Val Tyr Gly Ser Ser Gly Glu 340 345 350 Lys Pro Ala Thr Asp Thr Asp Ser Glu Ser Ser Leu Phe Gln Ser Phe 355 360 365 Thr Pro Ala Asn Glu Asn Ile Thr Gly Ser Thr Pro Ser Asn Gln Gly 370 375 380 His Thr Ser Phe Asp Gly Asp Trp Ile Arg Leu Leu Leu Gly Glu Asp 385 390 395 400 Arg Tyr Lys Thr Glu Gly Ser Met Ala Ser Ser Glu Asp Val Ile Lys 405 410 415 Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly Ser Val Asn Gly His 420 425 430 Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr 435 440 445 Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala 450 455 460 Trp Asp Ile Leu Ser Pro Gln Phe Gln Tyr Gly Ser Lys Ala Tyr Val 465 470 475 480 Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu 485 490 495 Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val 500 505 510 Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys 515 520 525 Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln 530 535 540 Lys Lys Thr Met Gly Trp Glu Ala Ser Thr Glu Arg Met Tyr Pro Glu 545 550 555 560 Asp Gly Ala Leu Lys Gly Glu Ile Lys Met Arg Leu Lys Leu Lys Asp 565 570 575 Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Met Ala Lys Lys 580 585 590 Pro Val Gln Leu Pro Gly Ala Tyr Lys Thr Asp Ile Lys Leu Asp Ile 595 600 605 Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala 610 615 620 Glu Gly Arg His Ser Thr Gly Ala 625 630 <210> 7 <211> 1215 <212> DNA <213> Artificial Sequence <220> <223> MYB8 cDNA <400> 7 atggggaaag ttcgtcaaga ttcaggatca gacgatgaca attcaataaa gaaatctttt 60 attaaaggtc cttggacaca agcagaagac aatcttttga tagcttacgt tgataaacat 120 ggtgatggaa actggaacgc tgttcaaaac aactcgggcc tttctcgttg tggtaaaagt 180 tgtcgccttc gttgggtaaa tcatctgaga ccagatttga aaaaaggcgc ttttacagag 240 aaagaagaga aacgtgtcat cgaacttcat gctttgttag ggaacaaatg ggcacgaatg 300 gctgaagaac taccgggacg aacagataat gagatcaaga acttctggaa tacaagactt 360 aagagactgc aacgacttgg tttacctgtt taccccgatg aagttcgaga gcatgcgatg 420 aatgtagcga cacattccgg tctaaacact gattcattgg atggtcatca tagtcaagaa 480 tatatggagg cagatactgt tgagattcct gaagtggatt tcgagcattt accactcaat 540 cgatcttctt catactacca gtcaatgctt cgacatgttc ctcccactaa cgtctttgtg 600 agacaaaaac cgtgtttctt tcagccgcct aatgtgtata acttgatacc accatctcct 660 tacatgtcta ccggaaaacg tcctagagaa ccagaaaccg cgtttccttg cccgggtgga 720 tataccatga acgagcaaag ccctcggctc tggaactatc cttttgttga gaacgtttca 780 gaacagttac cggatagtca tttgcttggt aatgctgctt attcttctcc tcctggacct 840 cttgttcacg gggttgagaa tttcgagttc ccttcattcc aatatcatga agagcctggc 900 ggttggggag cagatcaacc taacccaatg ccggaacatg agtcagataa cactttggtt 960 caatctcctc tgacagctca aacaccatca gattgtccat catcatcact ctatgatgga 1020 ctgttggaat cagttgtcta tggatcatca ggggagaaac cggcaaccga taccgattca 1080 gaatcttcac tgtttcagtc atttacacca gccaatgaaa acataaccgg ttcaactcct 1140 tcaaatcagg gacacacatc ctttgatggt gattggataa gattacttct cggtgaggat 1200 agatataaaa ccgag 1215 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 26960P-AscIF1 forward primer <400> 8 aaaaggcgcg cccaacccat aaatatcatt ggtc 34 <210> 9 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> MYB81-CGNR reverse primer <400> 9 agaagcggcc gccttgtgtc atctgtaaaa taatatg 37 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 26960P-NotIF forward primer <400> 10 ttaggcggcc gcatggggaa agttcgtcaa gattcag 37 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> MYB81-SR405 reverse primer <400> 11 ttggactagt ctactcggtt ttatatctat cc 32 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MYB81-SRnostop reverse primer <400> 12 gccaactagt ctcggtttta tatctatcct c 31 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MYB81-BR405 reverse primer <400> 13 gccaggatcc ctcggtttta tatctatcct c 31 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 26960P-For1 forward primer <400> 14 ctattgatga gaccatatat tattg 25 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 26960-Rev4 reverse primer <400> 15 ccatcaccat gtttatcaac gta 23 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 26960-Rev6 reverse primer <400> 16 ccatgtttat cagctatcaa aa 22

Claims (18)

delete delete delete delete delete delete delete delete delete delete delete delete delete A primer set consisting of SEQ ID NO: 14 and SEQ ID NO: 15, and a primer set consisting of SEQ ID NO: 14 and SEQ ID NO: 15, capable of detecting the deleted nucleic acid of TACGTT, the base sequence of the 2276-2282 position of the MYB81 gene consisting of the base sequence of SEQ ID NO: 2 derived from Arabidopsis A composition for determining male sterility of Arabidopsis, including a primer set consisting of SEQ ID NO: 16. A kit for determining male infertility in Arabidopsis thaliana comprising the composition of claim 14. delete 1) extracting genomic DNA from Arabidopsis thaliana; And
2) A gene mutation that is a deletion of TACGTT, a nucleotide sequence of 2276-2282 of the MYB81 gene consisting of the nucleotide sequence of SEQ ID NO: 2 derived from Arabidopsis, is a primer set consisting of SEQ ID NO: 14 and SEQ ID NO: 15, and SEQ ID NO: 14 and SEQ ID NO: A method for determining male infertility in Arabidopsis including a step of determining using a primer set consisting of 16.
delete

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