MXPA05011893A - Dna fragment specific to cytoplasmic male sterile pepper and use thereof. - Google Patents

Abstract

The present invention relates to a DNA fragment specific to a cytoplasmic male sterile pepper comprising a polynucleotide of SEQ ID NO: 1, a candidate polynucleotide (named orf456) associated with cytoplasmic male sterile pepper consisting of a 223rd to 678th nucleic acid of SEQ ID NO:1, and a polynucleotide of SEQ ID NO: 2. The DNA fragment specific to cytoplasmic male sterile pepper comprising a polynucleotide of SEQ ID NOs: 1 or 2 can be used for identifying cytoplasmic type between male sterile and male fertile pepper by the PCR method. In addition, hybrid pepper breeders/seed companies could detect impurities of the maintainer line within the CMS line, and by ensuring purity of the CMS line, a major source of contamination of the hybrid seeds is removed leading to obvious benefits for the seed industry and farmers.

Description

SPECIFIC DNA FRAGMENT FOR THE CYTOPLASMIC MALE STERIL PEPPER AND USE OF M SMO [Technical Field] The present invention relates to a DNA fragment specific for the male cytoplasmic sterile capsicum, to a method for distinguishing the cytoplasmic genotype from the plant using the DNA-based markers and a method to prepare a male transgenic cytoplasmic sterile plant. [Background Art] Hybrid vigor is a phenomenon whereby the progeny of a cross between two inbred lines have a higher yield potential than either of one of the origins. Hybrids can produce up to 10-30% more than the best non-hybrid varieties, and are a favored option to increase yield. The most widely used system for the production of hybrid peppers is the three-line system: (a) a sterile male and female fertile line called the sterile male cytoplasmic line (CMS) because it carries a mutation that confers male sterility on the component cytoplasmic of the genome; (b) a maintainer line; and (c) a restorative line. The maintainer and restorative lines are fertile male as well as fertile female. The CMS and maintainer lines are virtually identical with respect to the nuclear component of the genome (and are often referred to as iso-nuclear lines) but differ from each other with respect to the cytoplasmic component of the genome. The male sterility of the CMS line is maternally inherited and is more likely due to a mutation in the mitochondrial DNA. The CMS line, which is fertile female, can be propagated by pollen fertilization that emanates from the maintainer line. Since the cytoplasmic component of the genome is not transferred through pollen, the progeny of such a cross would inherit the cytoplasm only from the CMS line and would therefore be sterile male. The nuclear component of the progeny genome would also be identical to that of the CMS line, even though half of it is inherited from the maintainer line, since there is no difference between these two lines with respect to this component of the genome. The hybrid seeds are produced in a cross of the CMS line with another inbred parent line, called the restorer line, which as indicated in the above is fertile male and female fertile. In this cross, the CMS line serves as the feminine origin while the restorative line is the masculine origin. The restorative line also carries the Rf (fertility restorer) gene (s) in its nuclear genome that will restore male fertility to a plant whose cytoplasm has been inherited from the CMS line.

The hybrid seeds would therefore be produced. The CMS and restorative lines are appropriately selected such that hybrids exhibit sufficient hybrid vigor (or heterosis) to produce substantially higher yields than inbred varieties. The CMS in the pepper (Capsicum annuum L.) was first documented by Peterson (Peterson PA (1958) "Cytoplasmically inherited male sterility in Capsicum." Amer Nat., 92: 111-119) for PI 164835, which was introduced from the India. Since then, commercial seed companies have used this attribute to produce hybrid seeds in the field. The (S-) male sterile cytoplasm of the Peterson lines is the only common source of CMS used to produce Hybrid pepper seeds. An example of a well characterized CMS system is found in corn. When classifying a mtD A library of cms-T corn with sterile and fertile mtRNA, Dewey et al. (Dewey RE, Levings III CS, Timothy DH (1986) "Novel recombination in the maize." Mitochondrial genome produces a unique transcriptional unit in the Texas male-sterile cytoplasm. "Cell 44: 439-449) identified a region specific to the T-cytoplasm. The region contains an unusual gene, designated as T-urfl3, which is predicted to encode a 13-kDa polypeptide (URF13). T-urfl3 is located upstream of orf25 and is co-transcribed.

Another example is found in the genus Petunia. The S-pcf gene has been detected in relation to CMS in Petunias. This site consists of the 5 'portion of the atp9 gene; the exon part of coxll; and an unknown open reading structure, urf-s (Young EG, Hanson MR (1987) "A fused mitochondrial gene associated with cytoplasmic male sterility is developmentally regulated." Cell 50: 41-49). Specific genes correlated with CMS have also been reported in beans (Johns C, Lu M, Lyznik A, Mackenzie S (1992) "A mitochondrial DNA sequence is associated with abnormal pollen development in cytoplasmic male sterile bean plants." The Plant Cell 4 : 435-449), Brassica (Grelon M, Budar F, Bonhomme S, Pelletier G (1994) "Ogura cytoplasmic male-sterility (CMS) -associated orfl38 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids." Mol Gen Genet 243: 540-547), radishes (Makaroff CA, Apel IJ, Palmer JD (1990) "Characterization of radish mitochondrial atpA-associated sequences and relationship with male sterility." Plant Mol Biol 15: 735-746), sunflowers ( Moneger R, Smart CJ, Leaver CJ (1994) "Nuclear restoration of cytoplasmic male sterility in sunflower is associated with the tissue-specific regulation of a novel mitochondrial gene." The EMBO J. 13 (1): 8-17), rice (Akagi H (1995) "Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice ". Theor. Appl. Genet 90: 948-951), carrots (Kanzaki H, Takeda M, Kameya T (1991) "Sequence analysis of a mitochondria1 DNA fragment isolated from cultured cells of carrot cytoplasmic male-sterile strain." Japanese J Genet 66: 719-724) , and sorghum (Tang HV (1996) "Transcript processing infernal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile Sorghum". Plant J. 10: 123-133). Although these genes associated with CMS are commonly generated by the intra-rearrangement of mtDNA (Hanson MR (1991) "Plant mitochondrial mutations and male sterility". Annu Rev Genet 25: 461-486), open reading structures do not share significant sequence homology. How these genes can act in CMS plants and result in mitochondrial dysfunction and non-functional pollens have not been known until now. The CMS attributes are commercially very useful and important in the production of hybrid F seed. This is why transgenic male sterile plants have been attempted to be developed by several research groups. For example, Mariani et al. (Mariani C, Beuckeleer J, Truettner J, Leemans J, Goldberg RB (1990) "Induction of male sterility in plants by a chimeric ribonuclease gene." Nature 347: 737-741) has developed sterile tobacco male when using a package-specific promoter and the barnase gene, which functions as a robonuclease gene in plants. Several experiments have also been attempted to transform these genes associated with CMS into fertile plants. The orf239, mitochondrial DNA sequence associated with CMS in common bean (Abad AR, ehrtens BJ, Mackenzie SA (1995) "Specific expression in reproductive tissues and fate of a mitochondrial sterility-associated protein in cytoplasmic male sterile beans." Plant Cell 7: 271-285) was used to transform tobacco with or without a mitochondrial targeting sequence. Transformed tobacco exhibited a semi-sterile or sterile male phenotype even when the mitochondrial protein has not been directed (He S, Abad AR, Gelvin SB, Mackenzie SA (1996) VA cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobáceo. "Proc. Nati. Acad. Sci. USA 93: 11763-11768) Another gene associated with CMS, urf-s sequence of the pcf gene that encodes the 25kDa protein in the Petunia genus, has been transformed to petunia plants and tobacco plants with mitochondrial directing sequence constructions, although the expression of the PCF protein was detected in mitochondria of transgenic petunia and tobacco plants, the fertility of the plants was not affected (Wintz H, Chen HC, Sutton CA, Conley CA, Cobb A, Ruth D, Hanson MR (1995) "Expression of the CMS-associated urf-s sequence in transgenic petunia and tobáceo" Plant Mol Biol 28: 83-92). In addition, the correct identification of the (N-) male fertile cytoplasm and the (S-) male sterile cytoplasm in plants including food crops is very important in reproduction systems. The purity estimation of the hybrid seed and the cytoplasmic genotype is conventionally made by the growth test (GOT), which is based on the estimation of morphological and floral characteristics (which distinguish the hybrid) in a representative sample of plants that are grown to maturity. For example, pepper plants take several months to reach maturity, and the seeds have to be stored under appropriate conditions since they can not be marketed until these results become available. In addition, substantial delays may result in the first growing season after the production of the hybrid seed that is taken by the GOT, which is also the preferred station for growing the hybrid. In such cases, the seeds have to be stored for up to one year, that is until the subsequent growing season, before they can be commercialized. For seed companies, large amounts of capital are therefore blocked in the form of hybrid seed supplies for extended periods while the GOT expects the results. Another disadvantage of GOT is that it can be subjective due to environmental influences on the expression of morphological characteristics. In addition, there is also the possibility that adverse weather conditions (such as strong wind or rain, high temperatures, drought) may damage or destroy the crop and make it difficult to collect the data. In order to solve the above problems, a technique using the sequence associated with CMS as a DNA marker has been developed to easily detect a male sterile cytoplasmic type. This technique uses a DNA marker that is detected by the Polymerase Chain Reaction (PCR), and is ideally suited for this purpose since it is much more efficient for handling large numbers of samples than methods based on hybridization similar to Restriction Fragment Length Polymorphisms (RFLPs). [Description] [Technical Problem] To solve the problems of the prior art, one aspect of the present invention is to provide a DNA fragment associated with male cytoplasmic sterility in the pepper. Another object of the present invention is to provide a construct for use in obtaining a transgenic male sterile plant using a polynucleotide consisting of 223vo to 678avo nucleic acid of SEQ ID NO: 1, and a DNA sequence capable of directing the protein expressed with the coding region in the mitochondrion. Another object of the present invention is to provide a method for producing a transgenic male sterile plant. Another object of the present invention is to provide a method for inhibiting pollen production in a transgenic plant. Another object of the present invention is to provide nucleotide sequences specific to CMS peppers. Another object of the present invention is to provide a method for identifying male sterility in pepper by the PCR method. Another object of the present invention is to provide a set of PCR primers for identifying male sterility in the pepper. [Technical Solution] In order to carry out the aspects of the present invention, the present invention provides a DNA fragment specific for the male sterile capsicum comprising a polynucleotide of SEQ ID NO: 1 or a polynucleotide consisting of 223nd to 678avo nucleic acid of SEQ ID NO: 1. In addition, the present invention provides a transgenic male sterile plant comprising a polynucleotide sequence consisting of the 223nd to 678th nucleic acid of SEQ ID NO: 1. In addition, the present invention provides a construct for use in obtaining a sterile plant transgenic male, comprising: a) a polynucleotide sequence (called orf456) consisting of the 223nd to the 678th nucleic acid of SEQ ID NO: 1; b) a promoter that is active in the plant and is operably linked to the polynucleotide to achieve expression thereof; and c) a DNA sequence capable of transferring the protein expressed by the polynucleotide of a) to the mitochondrion. In addition, the present invention provides a method for producing transgenic male sterile plants comprising: a) preparing a construct comprising i) a polynucleotide sequence (called orf456) consisting of the 223nd to 678th nucleic acid of the SEQ ID NO: 1. ii) a promoter that is active in the plant, and that is operably linked to the polynucleotide to achieve expression thereof; and i) a DNA sequence capable of transferring the protein expressed by the polynucleotide of a) to the mitochondrion; and b) transform the construction into a plant or plant cell. In addition, the present invention provides a method for inhibiting pollen production in a plant, comprising: a) preparing a construct comprising i) a polynucleotide sequence (called o? F456) consisting of the 223nd to 678th nucleic acid of the SEQ ID NO: 1. ii) a promoter that is active in the plant, and that is operably linked to the polynucleotide to achieve expression thereof; and iii) a DNA sequence capable of transferring the protein expressed by the polynucleotide of a) to the mitochondrion; and b) transform the construction into a plant or plant cell. In addition, the present invention provides a CMS specific DNA fragment (SEQ ID NO: 1, 1596 bp) in the 3 'flanking region of coxll in the pepper to identify a male sterile pepper by PCR, which comprises: a) driving a Polymerase Chain Reaction (PCR) with a forward primer capable of quenching a part of a cox.ll genome gene or a part of the nucleotide sequence of SEQ ID NO: 1, and a rear primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 1 on the DNA of the plant or the mitochondrial DNA of the plant; and b) observing whether the DNA fragment is amplified or not amplified, and where the presence of amplified fragments indicates that the plant is a male sterile line, and the absence thereof indicates that the plant is a male fertile line. In addition, the present invention provides a method for identifying male sterility in a plant comprising: a) conducting a Polymerase Chain Reaction (PCR) with a forward primer capable of quenching a part of an atp6 genome gene or a part of the nucleotide sequence of SEQ ID NO: 2, and a back primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 2 on the DNA of the plant or the mitochondrial DNA of the plant; and b) observing whether a DNA fragment is amplified or not amplified, and where the presence of amplified fragments indicates that the plant is a male sterile line, and the absence thereof indicates that the plant is a male fertile line. In addition, the present invention provides a set of PCR primers for identifying male sterility in a plant comprising: a) a forward primer capable of quenching a part of a coxll genome gene or a part of a nucleotide sequence of the SEQ ID NO: 1; and b) a back primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 1 onto the plant DNA or mitochondrial DNA of the plant, and wherein the size of the amplified DNA fragment is 50 bp. or above 2kbp. In addition, the present invention provides a CMS specific DNA fragment (SEQ ID NO: 2, 251 bp) in the 3 'flanking region of atp6 in the pepper to identify a male sterile pepper by PCR, comprising: a) a primer forward capable of tuning a part of a gene of the apt6 genome or a part of a nucleotide sequence of the SEO ID NO: 2; and b) a back primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 2 on the plant DNA or mitochondrial DNA of the plant, and wherein the size of the amplified DNA fragment is 50 bp. or above 1 kb kbp. [Description of the Drawings] FIG. 1 shows a Southern blot analysis of mtDNAs digested with EcoRl, Hindlll and Ba Hl hybridized with eight mitochondrial probes (genes coxl, coxll, coxlll, atpA, atp6, atp9, cob, nad9) to compare male fertile lines (F) and male sterile (S) in C. annuum L. FIG. 2 shows the Northern blot analysis of mtR s than with three mitochondrial probes (atpA, atpG, coxll). The atp6 and coxll genes show patterns of polymorphic bands of RNA transcripts (indicated by the arrow). FIG. 3 shows results of RFLP and the inverse PCR of the genes atpG and coxll in the pepper (Capslcum annuum L.). (A): Results of the Southern analysis between the N-cytoplasm and the S-cytoplasm with the probe atp6 (left) and coxll (right). (B): Reverse PCR amplification for the cloning of a DNA sequence that is specific for the pepper lines. The predicted PCR fragments are marked with an asterisk (*).

FIG. 4 shows a schematic diagram of the coxll coding and a flanking region between a maintainer line (N-cytoplasm) and the CMS line (S-cytoplasm) of the pepper. FIG. 5 shows a schematic diagram comparison of the coding of atp6 and one. flanking region between a maintainer line (N-cytoplasm) and the CMS line (S-cytoplasm) of the pepper. (A): Schematic representation of the nucleotide sequence comparison of the genes (N) atp6-l and (S) atpG-1 of C. annuum. (B): Schematic representation of nucleotide sequence comparison of (N) atp6-2 and (S)? atp6-2. The highly conserved region is indicated by the green box. The red box shows the truncated region with no nucleotide sequence homology with the 3 'region of (N) atp6-2. The arrow indicates the primer pairs for the reverse PCR ~ 456 'which is located in the 3' region of the coxll gene in a sterile line. FIG. 6 shows a schematic representation of the nucleotide sequence comparison of the coxll gene between a fertile line and a sterile line. The arrow indicates the pairs of primers for the reverse PCR, sequencing and RT-PCR experiments on the 3 'coxll region.

FIG. 7 shows the results of the RT-PCR experiments on the orf456 gene which is located in the 3 'region of the coxll gene in a sterile line. FIG. 8 shows the results of the Northern blot analysis on mtRNAs from fertile, sterile lines and restored with the orf456 probe. Approximately 15 g / strip of RNA is loaded on a 1.2% agarose gel and transferred to a nylon N + membrane. F: fertile line, S: sterile line, R: restorative line. FIG. 9 shows the results of bacterial growth inhibition tests by expressing the orf456 gene. FIG. 10 shows the construction of orf456 and the eghp-1 transgenes for transformation. The diagram shows the strategy for cloning each gene construct in the plant transformation vector pCAMBIA2300. FIG. 11 shows a GFP fluorescence image in a transient onion expression assay. FIG. 12 shows a GFP fluorescence image in an Arabidopsis transformant. FIG. 13 shows the morphology of the flower in the transformants (directed to the mitochondria, a) male sterile and the transformants (not directed to the male b) fertile mitochondria.

FIG. 14 shows a phenotype of transgenic Arabidopsis in the stage of transformant seed sets directed to the mitochondria. FIG. 15 shows the transformants of transgenic T0 transgenic Arabidopsis showing both of the sterile male and male fertile phenotypes. FIG. 16 shows a PCR Amplification of 20 varieties of pepper culture or accesses with pairs of SCAR specific primers of CMS. [Mode for the Invention] In the following detailed description, only selected modalities have been shown and described, simply by way of illustration of the best mode contemplated by the inventors to carry out the invention. As will be understood, the invention can be modified in several aspects, all without departing from the invention. Accordingly, the drawings and description will be considered as illustrative and not restrictive in nature. A specific DNA fragment for the male sterile cytoplasmic pepper of the present. invention includes a polynucleotide of SEQ ID NO: 1, a polynucleotide consisting of the 223avo to 678av0 nucleic acid of SEQ ID NO: 1, or a polynucleotide of SEQ ID NO: 2. The polynucleotide of SEQ ID NO: 1 (1596 bp) is located at the 3 '-terminal of the coxll gene and contains the orf456 region at positions 223 to 678 of the nucleic acids as an open reading structure. The polynucleotide of SEQ ID NO: 2 (251 bp) is located in the 3 '-terminal of gene apt6 3' - truncated. The DNA fragment specific for the male sterile cytoplasmic pepper can be used to prepare a male sterile plant or / and distinguish a male sterile line from a line of maintenance pepper. The present invention could be applied to all kinds of plants, and preferably includes solanaceae similar, to pepper, eggplant, tobacco, tomato and petunia; Brassicaceae similar to turnip, cauliflower and broccoli; species of floral plants similar to lily and chrysanthemum; and woody plants. To prepare the transgenic male sterile plant, an expression construct with the DNA fragment. { orf456) associated with male cytoplasmic sterility in pepper, wherein the DNA fragment operably linked to, and under the regulatory control of a transcriptional and transcriptional regulatory sequence can be prepared. Transcriptional and translational regulatory sequences are those that can function in specific organisms (ie, bacteria, yeast, fungi, plants, insects, animals and humans), cells or tissues that effect the transcriptional and translational expression of the foreign gene with which they are associated, and that can be used according to the host cell. Examples of transcriptional and translational regulatory sequences include, but are not limited to, a promoter, an enhancer, a pre-sequence leader and a terminator. The promoter can be derived from a highly expressed gene to direct the transcription of a downstream structural sequence. Such promoters can be derived from a plasmid or public vector, and examples include a promoter RA8, a promoter TA29, and so on. The selection of the suitable promoter is well within the level of ordinary skill in the art. The expression construct can further include a multi-cloning site, a selectable marker, origins of replication and DNA sequences capable of transferring the protein expressed by the foreign gene to the mitochondria, for example, pre-sequence of the subunit IV of the yeast cytochrome c oxidase (coxIV) of SEQ ID NO: 3. The expression construct can be a common vector, and examples are a plasmid or viral vector including the vector pCAMBIA2300 consisting of the CaMV 35S promoter and the terminator of the nopaline synthase (us). However, any other plasmid or vector can be used as long as it is applicable and viable in the host. Transformation methods for generating transformants according to the type of host cell are well known, for example, transfection with calcium phosphates, DEAE-Dextran-mediated transfection, electroporation (Davis, L., Dibner, M., Battey , I., Basic Methods in Molecular Biology, 1986), thermal shock, transfer mediated by Agrobacterium tumefaciens, protoplast transformation, micro-injection and biolistic transfection. In one embodiment of the present invention, the recombinant pCAMBIA2300 vectors harboring the orf456 fragments were introduced into onions, and into Arabidopsis via the DNA transfer mediated by Agrobacterium tumefaciens. The transient expression tests on onion showed that the presequence of the IV subunit of yeast cytochrome c oxidase (coxIV) of the SEO ID NO: 3 is able to transfer the protein expressed by the foreign gene to the mitochondria. Transformation to Arabidopsis thaliana with the recombinant pCAMBIA2300 vector harboring an orf456 fragment showed a sterile male phenotype without pollen. The subject of the present invention is also a process for restoring male fertile plants from male transgenic sterile plants, and according to the invention, it is characterized in that it comprises the following steps: (a) transforming the selected upper plant by introducing at least one copy of the hybrid DNA construct as defined above in a recipient plant by means of a plasmid of a vector containing the sequence, in order to obtain transgenic male sterile plants (TMSP); (b) transforming the same top plant as in (a) by introducing at least one copy of a hybrid antisense DNA construct, including the antisense coding region of the orf456 gene by means of a plasmid containing the reverse off456 sequence, in order to obtain transgenic male fertile plants (TMFP); and crossing the transgenic male sterile plants obtained in (1) and the male fertile plants obtained in (2), in order to obtain vigorous hybrids whose masculine fertility has been restored and which has pre-selected characteristics. The subject of the present invention also relates to plasmids that include a hybrid antisense sequence, as defined above, associated with a promoter selected from the constitutive promoters and promoters specific for the anthers and also associated with a suitable terminator. In addition, a DNA fragment specific for the male sterile cytoplasmic pepper of the present invention can be used to identify a male sterile pepper from a maintainer pepper. The method for identifying a male sterile capsicum comprises a) conducting the polymerase chain reaction (PCR) with a forward primer capable of quenching a gene part of the genome coxll, or the genome of the apt6 genome, or a part of a nucleotide sequence of SEQ ID NOs: 1 or 2 and a rear primer capable of quenching a part of a nucleotide sequence of the SEO ID NOs: 1 or 2 for the DNA of the plant or the mitochondrial DNA of the plant as a template, and ) observe if the DNA fragment is amplified or not amplified. The presence of the amplified fragments indicated that the plant is a male sterile line, and the absence of the same indicates that the plant is a male fertile line. The size of the amplified DNA fragment can be 50 bp to above 2 kbp, and the length of the forward primer and the back primer can be from 50 bp to above 1 kbp. In the observation stage, if the DNA fragment is amplified or can not be detected by means of agarose gel electrophoresis or polyacrylamide gel followed by staining with ethidium bromide. Also, a method that employs radio-marking, colorimetry, quemiluminescence, or fluorescence can be applied to detect PCR products. In one embodiment of the present invention, a primer comprising a nucleotide sequence of SEQ ID NOs: 15 or 17 as a forward primer and a primer comprising a nucleotide sequence of SEQ ID NOs: 16 or 18 as a primer Rear are designed. The following examples are provided to further illustrate the present invention, and are not intended to limit the invention beyond the limitations set forth in the appended claims. EXAMPLES Example 1: RFLP and Northern blot analysis between sterile and male fertile peppers. The male fertile lines are almost isogenic (genotype N / rf / rf), sterile male (S / rf / rf), and restorative (S / Rf / Rf) of Capsicum annuum cv. Milyang were used in this study. These were provided by Hungnong Seed Company. 1-1. Analysis of RELP. To isolate the mtDNA from a maintainer plant of a CMS plant, young leaves of C. annuum were harvested after etiolation and homogenized in 70 ml of homogenization buffer (0.1 M Tris-HCl, pH7.2, 0.5 M mannitol , ethylene glycerol-bis (ß-aminoethyl ether) 0.001 M, acid?,?,? ', N' -tetraacetic acid (EGTA), 0.2% bovine serum albumin (BSA), 0.05% cysteine) per 10 g of samples The mitochondria were purified by gradient centrifugation in sucrose, and then the mtDMA was isolated by the DNase I procedure (Sparks RB, Dale RMK (1980) "Characterization of 3H-labeled supercoiled mitochondrial DNA from tobaceo suspension culture celia." Mol Gen Genet 180: 351-355). The mtDNA (10, pg) of the male fertile lines and CMS were separated on 0.8% agarose gels after digestion with EcoRl (Boehringer Mannheim, Gernany) and transferred to Hybond N + nylon membranes (Amersham Pharmacia Biotech, NJ , USES) . Eight mitochondrial probes (coxl, coxll, coxlll, atpA, atp6, atp9, cob, nad.9) were selected for the RFLP analysis. The mitochondrial DNA probes were radioactively labeled by random priming with [α-32P] dCTP (Amersham Pharmacia Biotech, NJ, USA). Southern blot analysis was performed in the hybridization buffer (0.75 M NaCl, 0.125 M citric acid, 0.05 M sodium phosphate, 5 x Denhardt's solution, 3% dextran sulfate, 2.5 mM EDTA, 0.6 SDS %, pH 7.2, 50% formamide) at 42 ° C for 24 h. stains were washed in 2 X SSC, 0.1% SDS at 65 ° C for 10 min; 1 X SSC, 0.05% SDS at 65 ° C for 20 rain, the spots were exposed to X-ray films (Kodak, USA). Figure 1 shows Southern blot analysis of mtDNAs digested with EcoRl, Hindlll, and BamHl hybridized with eight mitochondrial probes (genes coxl, coxll, coxlll, atpA, atp6, atp9r cob, nad9) to compare male fertile lines (N ) and male sterile (S) in C. annuum L. Three genes (atpA, atp6, coxll) showed polymorphism between fertile and sterile mtDNAs. 1-2: Northern blot analysis For Northern blot analysis, total RNA (20 μg) of sterile, fertile, restored anther was fractionated on a standard formaldehyde gel (1.2% agarose) and transferred to a Hybond N + nylon membrane (Amersham Pharmacia Biotech., USA) by capillary staining (Sambrook et al., 1989). Northern blot analysis was conducted on these three genes (atpñ, atp6, coxll). The spots were hybridized at 60 ° C for 16 hrs. and the final wash was 0.5 X SSC, 0.1% SDS. Figure 2 shows the result of Northern blot analyzes on mtRNAs with three mitochondrial probes (atpA, atp6, coxll) showing polymorphic bands in the Southern blot analysis. The atp6 and coxll gene show patterns of polymorphic bands of the RNA transcripts (indicated by the arrow).

Example 2: Reverse PCR and Sequencing of the flanking region of coxll and atp6 2-1. Reverse PCR for the 3 'flanking region of the coxll gene Reverse PCR was conducted to clone the 3' region of the coxll gene in the maintainer pepper and CMS. For inverse CPR, mtDNA (5 g) was digested overnight at 37 ° C in a 100 μm reaction mixture. containing 10 units of EcoRl (Boehringer Mannheim, Germany). The digestion mixtures were extracted by phenol / chloroform and the DNA was precipitated with ethanol. The religation of the mtDNA took place during 30 min at 37 ° C in 200 μ? using 3 units of T4 DNA ligase (BRL, USA). Subsequently, the ligation mixtures were inactivated for 20 min at 65 ° C. Then, from the extraction with phenol / chloroform, the DNA was precipitated with ethanol and dissolved in 50 μ? of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4). The PCR was performed on approximately 500 ng of DNA in a thermal cycler (Perkin Elmer 9600), the PCR reaction mixture consisted of 25 pmol of each primer (set of primers of the SEO ID NOs: 5 and 6), 200 uM of each dNTP, 2.5 units of ^ Taq DNA polymerase (TakaRa, Japan), and 5 μ? of buffer solution of 10 X ExTag DNA polymerase in a total volume of 50 μ ?. The PCR amplification was at 94 ° C (1 min), 60 ° C (1 min), and 72 ° C (2 min) for 35 cycles. The PCR products were separated on a 1% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. 2-2. Reverse PCR for the 5 'and 3' flanking region of the atp6 gene The PCR was performed on approximately 500 ng of DNA in a thermal cycler (Perkin Elmer 9600). The PCR reaction mixture consisted of 25 pmol of each primer (set of primers of SEQ ID NOs: 7 and 8), 200 μ? of each dNTP, 2.5 units of ExTaq DNA polymerase (TakaRa, Japan), and 5 μ? of buffer solution of 10 X ExTaq DNA polymerase in a total volume of 50 μ? . PCR amplification was at 94 ° C (1 min), 60 ° C (1 min), and 72 ° C (2 min) for 35 cycles. The PCR products were separated in one of the 1% agarose, stained with ethidium bromide, and visualized under ultraviolet light. Figure 3 shows the results of RFLP and inverse PCR of the genes atpG and coxll in chile pepper (Capsicum annuum L.). "A" is the result of Southern blot analysis between the N-cytoplasm and S-cytoplasm with the atp6 (left) and coxll (right) probes. The mtDNA (10,] ig) of the maintainer lines (N-cytoplasm) and CMS (S-cytoplasm) were separated on 0.8% agarose gels after digestion with an EcoRl enzyme. "B" is the result of the inverse PCR amplification for the cloning of a DNA sequence is specific to the pepper CMS line, and "Mi" and "M2" indicate the size markers K / HindIII and lkb DNA plus the link, respectively (Promega Co. USA). The predicted PCR fragments were marked with an asterisk (*). 2-3. Determination of a nucleotide that is specific for the CMS lines of chili pepper The amplified products of experiments 2-1 and 2-2 were separated on 0.8% agarose gel and purified using gel extraction kits (Qiagen, Germany) , pGEM-T easy vectors (Promega, USA) were cloned and sequenced with a Perkin Elmer 9600 PCR machine and an AB1377 automatic sequencer (Applied Biosystems, USA). Figure 4 shows a schematic comparison of the coxll coding and flanking region between a maintainer line (M-cytoplasm) and a CMS line (3-cytoplasm) of the pepper. The arrow indicates the primer pairs for the inverse PCR of the 3 'coxll region. The size of the EcoRI-ScoRI fragment of each coxll is indicated on the right side. The sequence specified for CMS of the 3 'flanking region of coxll is revealed as 1596 bases (SEQ ID No. 1). Figure 5 shows a schematic comparison of the coding and flanking region atp6 between a maintainer line and (N-cytoplasm) and a CMS line (S-cytoplasm) of the pepper. "A" is a schematic structural comparison of the genes (N) atp6-l and (S) atp6-l of C. annuum, and "B" is a schematic structural comparison of (N) atp6-2 and (S)? atp6-2. The highly conserved regions are indicated by the green box. The red box shows the truncated region that has no homology of nucleotide sequences in the 3 'region of (N) to tp6-2. The arrow indicates the primer pairs for the inverse PCR of the 3 'atp6 region. The sizes of the EcoRI-EcoRl fragment of each of the atp6 copies are indicated on the right side. The sequence specified for CMS of the 3 'flanking region of the atp6 gene is revealed as 251 bases (SEQ ID No. 2). 4-4. Identification of the candidate open reading structure associated with CMS in pepper A new open reading structure was found through the ORF Finder program on the NCBI page. Through the results of the inverse PCR and sequencing, the difference in the structure of the coxll gene between the fertile and sterile peppers was plotted and the presence of a new open reading structure, called orf456r, was detected in the 3 'region of the coxll gene. in the sterile line (figure 6). In the case of the atp6 gene, the new open reading structure or the chimeric gene were not detected in the coding and flanking regions atp6. Example 3: Cloning for the 3 'region of the coxll 3-1 gene. Reverse transcriptase PCR experiments To investigate whether the open reading structure assumed by the ORF Finder program was actually transcribed in a CMS plant, RT-PCR was performed with specific primer pairs (SEQ ID NOs: 6, 9 and 10) Three micrograms of total anther RNA were used in a 10 μ? Reaction. of the cDNA synthesis of the first strand induced by M-MLV reverse transcriptase (Gibco BRL, USA), according to the protocols provided by the manufacturer. The PCR was performed in 1 μ? of cDNA in a thermal cycler (Perkin Elmer 9600) using 10 pmol of each primer, 100 μ? of each dNTP, 1.5 units of E >;: Tag DNA polymerase (TaKaRa, Japan), and 2.5 μ? of buffer 10 X ExTaq DNA polymerase in 25 μ ?. PCR amplification was at 94 ° C (1 min), 50 ° C (1 min), and 72 ° C (2 min) for 30 cycles. The RT-PCR products were cloned into the pGEM-T easy vectors (Promega, USA), and sequenced with the T7 and SP6 primers (SEQ ID NOs: 11-14). Figure 7 shows the results of the RT-PCR experiments on the orf456 gene that is located in the 3 'gel coxll gene region in the sterile line. The RT-PCR with the pair of primers (SEO ID NOs: 9 and 10) is performed to detect the fact that the newly made orf456 is real and only transcribed in the sterile line. The RT-PCR with the set of primers (SEQ ID NOs: 6 and 10) is performed to detect if the orf456 gene is co-transcribed with coxll located in the upstream region. Figure 8 shows the results of Northern blot analysis on mlRNAs from sterile, sterile lines and restored with the orf456 probe. Approximately 15 μ? / Fran a of RNA was loaded on 1.2% agarose gel and transferred to a nylon N + membrane. F: fertile line, S: sterile line, R: restorative line. Figure 3 shows the results of Northern blot analysis on mtRNAs from fertile, sterile, and restorative lines with the crf456 probe. The open reading structure oxf456 is really and only transcribed in the sterile and restorative line that carries the S-cytoplasm. Example 4: Bacterial Growth Inhibition Test To investigate how orf456 affects the mitochondria of plants and results in mitochondrial dysfunction and male sterility, a heterologous system, ie a bacterial cell, was adopted. The possible toxicity was examined on a bacterial cell of the orf456 gene. The orf456 gene was cloned into an expression vector pTrcHis2-T0P0 and transfected into a cell of the ToplO strain of E. coli (Invitrogen, USA). As controls, cells carrying pTrcHis2-T0P0 + LacZ genes were cultured under the same conditions and induced by 1 mM IPTG. Cloning and transformation were conducted according to the manufacturer's protocols. ToplO cells containing the LacZ gene (control) and the orf456 gene were precultured in 3 ml of LB medium with 50 μg / ml ampicillin at 37 ° C for 16 rs. 50 μ? from a primary culture was transferred to 20 ml of the medium and cultivated at 37 ° C for 2-3 hrs. At the time of O. D.60o = 0.6, lmM of IPTG was added and the growth rate of each transformant was inspected for its absorbance every hour. The growth of E. coli cells was markedly impaired as soon as the expression of £ 456 was induced. Figure 9 shows the results of bacterial growth inhibition tests by expressing the orf456 gene. The proportion of growth of E. coli that carries constructions with orf456 and induced by IPTG lmM severely deteriorated compared with other constructions. Example 5: Test for the Management of the Foreign Gene to the Mitochondria and Preparation of Transformants 5-1. Preparation of constructions for transformation by Arajidopsis The fragment egfp-1 (SEO ID NO: 4, GFP variants acquired from Clontech) were amplified from the vector pEGFP-1 (Clontech, Palo Alto, USA). The amplified coxlV target sequences (SEO ID NO: 3, yeast DNA fragment for the cytochrome c oxidase subunit IV precursor) and the egfp-1 gene were ligated by T4 DNA ligase (Promega, USA), then cloned to the pCAMBIA2300 vector (MJC). Also, a construction coxlV-orf456 and the construction of non-address-orf4 6 as shown in Figure 9 were prepared and ligated into vector pCAMBIA2300. In Figure 10, the terminator sequence of the nopaline synthase (nos) gene was fused to the 3 'end of the sequences orf456 and egfp-1. In the first construct (referred to as coxlV-orf456), the sequence of orf456 was fused to the transit peptide sequence of the nuclear coxlV gene presequences of yeast strain Y187 (Stratagene, USES) . The second construct (referred to as no-address-orf456) does not have the mitochondrial targeting peptide sequences. The third construct (referred to as coxlV-egfp-1) was made using the coxlV and egfp-1 presequences of the pEGFP-1 vector (Clontech, USA). 5-2. Transient Expression Experiments on Onion The fusion constructs (coxlV presequence fused to the gene egfp-1) in the pCAMBIA2300 vector were expressed transiently in the epidermal cells of the onion after transfection. Internal epidermal peels (2 X 2 cm) of onion were placed on agar plates containing 1 X MS salts, 30 g / L sucrose, and 2% agar, pH 5.7. The shells were bombarded with 1 h of transfer to the agar plates. Particle bombardments are described by Scott et al. (1999). After incubation of 20-22 hrs in light, mitochondrial localization was examined by confocal laser scanning microscopy using the Radiance 2000 Multi-Photon Imangen Formation System (Bio-Rad, Hercules, CA) in the National Instrumentation Center for Environmental Management (NICEM, Su eon, South Korea). Figure 11 shows an image of the GFP fluorescence in the onion transient expression assay. the left picture shows the fluorescence of GFP in the onion by the construction coxlV + egfp-1. The Mitotracker CMSRox dye (Molecular Probé Co., USA) was used for the detection of mitochondria (right photograph). The GFP image and the Mitotracker dye images were completely matched. 5-3. Transformation of the Arabidopsis plant The Columbra ecotype plants of Arabidopsis thaliana were used for the transformation experiments, a strong CaMV 35S promoter and the terminator were used in the construction of the vector. The mitochondrial target sequence used was derived from the yeast coxlV presequences, and the orf456 and egfp-1 sequences used in the constructs were amplified by PCR to facilitate cloning. The inserts were confirmed by enzyme digestion and sequencing. The inserts were digested with an appropriate restriction enzyme to be cloned into the plant transformation vector pCAMBIA2300. Clones that have correct inserts were selected in the LB kanamycin medium. Transformations in LBA4404 of Agrobacterium tumefaciens were made by the thermal shock method (Sambrook et al., 1989). The plants of Arabidopsis thaliana were transformed by the Agrobacterium tumefaciens that carries the constructions coxlV (directed) -orf456 and non-directed orf456. The transformation mediated by Agrobacterium tumefaciens was carried out by means of a modified floral immersion method (Clough and Bent, 1998). The transgenic plants were selected in a medium containing kanamycin sulfate (50 g mi-1). The green plants that survive the antibiotic treatment were retained for further analysis. Figure 12 shows an image of the GFP fluorescence expressed in Arabidopsis thaliana roots. The vegetative growth of transformants was uniform and similar to that of untransformed control plants or non-targeted transformants. In flowering, the 31 plants of every 51 Arabidopsis transformants transfected with the mitochondrial targeting signal showed male sterile phenotype in the Ti generation. This classification was based on the morphology of the flower and the set of seeds. The 3 Arabidopsis plants of every 50 transformants transfected with non-targeted constructs showed male sterile phenotype. This was a rather unexpected result in the case of non-directional experiments. However, it was postulated that this product orf456 in the cytoplasm could not have any prejudicial effect on plant cells since the results tested in bacterial growth inhibition tests have not yet been expressed in the mitochondria. Figure 13 shows the morphology of the flower of the male sterile transformants (directed to the mitochondria) and the male fertile transformants (not directed to the mitochondria). "a" is a floral photograph of the sterile male transformants that carry the orf456 to the mitochondria, and, b "is a floral photograph of the male fertile transformants that carry orf456 not directed to the mitochondria. Arabidopsis in the stage of seed pools in the mitochondria-directed transformants (a) and in the non-mitochondrial transformants (b) Figure 15 is a photograph of the Arabidopsis transformants showing the sterile masculine and male fertile phenotypes in the same provisions, which provide the vacuum infiltration transformation methods, the arrow indicates the normal silicea produced from normal pollination, and the tip of the arrow indicates the flower that is not pollinated and did not produce normal seed sets. -4- Confirmation of transgenic plants Total sheet DNA for PCR and Southern blot analysis were extracted (Kim and collaborators, 2001). PCR amplification was performed on 100 ng of total DNA with a PTC-200 thermal cycler (MJ Research, USA) using thirty cycles of 30 s at 94 ° C, 45 s at 55 ° C, and 90 s at 12 ° Z. The total DNA digested with restriction enzymes was electrophoresed on a 0.8% agarose gel, transferred onto a nylon membrane, and hybridized with the probes labeled with [cc-32P] dCTP (Amersham Pharmacia Biotech, NJ, USES) . Table 2 shows the evaluation of male fertility based on a pollen production in confirmed transgenic Arabidopsis Ti plants that contain the orf456 transgene. (Table 2) Example 6: Determination of fertile or male sterile by genotype 6-1. Primer The specific oligonucleotide primers that can be used in a PCR assay to distinguish the maintainer lines (N-cytoplasm) and CMS (S-cytoplasm) from pepper chili pepper (Capsicum annuum L.) are designed. PCR primer coxll SCAR Front primer (SEQ ID No: 15) - a part of the coxll rear coding gene coding region (SEQ ID No: 16) - a part of the sequence that is unique to the CMS lines. PCR primer apt6 SCAR Front primer (SEQ ID No: 17) - a part of the coding region of the atp6 rear primer gene (SEO ID No: 18) - a part of the sequence that is unique in the CMS lines. Positive control coxll Front primer: SEQ ID NO: 19 Rear primer: SEQ ID NO: 20 6-2. PCR assay to distinguish the CMS and maintainer lines in the chilli peppers Using the primer sets, the PCR was performed as described below. The total DNA (200 ng) was mixed with 200 μ? A ?? of dNTP, 20 pM of each primer, 5 μ? of the 10 X reaction buffer, and 2.5 units of Taq polymer (Takara, Japan) in a reaction volume of 50 μ? . PCR amplification for the atp6 SCAR marker was carried out at 94 ° C (1 min), 52 ° C (1 min), and 72 ° C (2 min), for 35 cycles. The amplified DNA was subjected to 0.8% agarose gel electrophoresis. In the case of the coxll SCAR marker, the annealing was carried out at 56 ° C. Figure 16 is a photograph showing the PCR results for the 20 varieties of pepper culture, where "N" indicates a fertile phenotype, "S" indicates a sterile phenotype, and "M" indicates an NHindlll DNA marker. Using the pair of coxll SCAR primers, there was an amplification of a 708 bp DNA fragment in the CMS lines, but no PCR amplification was observed in the maintainer lines. In the case of the pair of atp6 SCAR primers, there was an amplification of a 607 bp DNA fragment in the CMS lines, but PCR amplification was not observed in the maintainer lines. To verify if the PCR reactions were done well, a pair of PCR primers encompassing the coding region of the coxll gene was used as a control. The fragment size of the PCR amplification was approximately 1.5 kb. The Capsicum annuum used in the PCR experiment is listed in Table 1. (Table 1) Variety of Genotype Phenotype Variety of Phenotype Genotype Cultivation 0 Cytoplasmic * Culture or Cytoplasmic * Access Access 80-2 S Sterile 11 Subicho-1 H Fertile 80-5 S Sterile 12 Milyang-B N Fertile KC268-1-1 S Sterile 13 FC-2 (CMS? Sterile of China) KC268-1-3 S Sterile 14 FC-3 (CMS S Sterile of Europe) C268-2-1 S Sterile 15 FC-4 s Sterile CMS-A S Sterile 16 4570? Sterile Chilsungcho-A s Sterile 17 4578 S Sterile Milyang-A s Sterile 18 TF68 N Fertile CMS-B N Fertile 19 Anciao N Fertile Chilsungcho-1 15 Fertile 20 CM334 N Fertile * N: N-cytoplasm, 5: S-cytoplasm

Claims (27)

1. CLAIMS 1. A DNA fragment specific for a male sterile citcplasmic pepper, characterized in that it comprises a polynucleotide of 223- to 678-nucleic acid of SEQ ID NO: 1.
2. 2. The DNA fragment according to claim 1, characterized because the DNA fragment comprises a polynucleotide of SEQ ID NO: 1.
3. 3. The DNA fragment according to claim 1 or 2, characterized in that the polynucleotide is located at the 3'-terminal of a coxll gene.
4. 4. A DNA fragment specific for a sterile cytoplasmic male capsule, characterized in that it comprises a polynucleotide of the SEQ ID NO: 2.
5. 5. The DNA fragment according to claim 4, characterized in that the polynucleotide is located at 3 '. -terminal of a gene atp5 3 '-truncated.
6. 6. A transgenic male sterile plant, characterized in that it comprises a polynucleotide of 223- to 678-nucleic acid of the SEO ID NO: 1.
7. 7. A construction for the use in obtaining a transgenic male sterile plant, characterized in that it comprises: a) a polynucleotide consisting of a nucleotide sequence consisting of 223- to 678-nucleic acid of SEQ ID NO: 1; b) a promoter that is active in the plant, and operably linked to the polynucleotide to achieve expression thereof; and c) a DNA sequence capable of transferring a protein expressed by the polynucleotide of a) to the mitochondrion.
8. The construction according to claim 7, characterized in that the c) DNA sequence comprises a nucleotide sequence of the SEO ID NO: 3.
9. 9. The construction according to claim 7, characterized in that the plant is one or most selected from the group consisting of Solanaceae similar to chili pepper, eggplant, tobacco, tomato and petunia; Brassicaceae similar to turnip, cauliflower and broccoli; and species of floral plants similar to lily and chrysanthemum; and woody plants.
10. A method for producing male sterile transgenic plants, characterized in that it comprises transforming the construction of claim 7 into plants or plant cells.
11. The method according to claim 10, characterized in that the plant is one or more selected from the group consisting of Solanaceae similar to chili pepper, eggplant, tobacco, tomato and petunia; Brassicaceae similar to turnip, cauliflower and broccoli; species of floral plants similar to lily and chrysanthemum; and woody plants.
12. 12. A method for inhibiting pollen production in plants, characterized in that it comprises transforming the construction of claim 7 into plants or plant cells.
13. The method according to claim 12, characterized in that the plant is one or more selected from the group consisting of Solanaceae similar to chili pepper, eggplant, tobacco, tomato and petunia; Brassicaceae similar to turnip, cauliflower and broccoli; species of floral plants similar to lily and chrysanthemum; and woody plants.
14. 14. A method for identifying a sterile cytoplasmic male capsule, characterized in that it comprises: a) conducting the Polymerase Chain Reaction (PCR) with a forward primer capable of quenching a part of the coxll genomic DNA or a part of a nucleotide sequence of SEQ ID NO: 1 and a rear primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 1 on the genomic DNA of the plant or mitochondrial DNA of the plant; and b) observing whether the DNA fragment is amplified or not amplified, where the presence of the amplified fragments indicates that the plant is a male sterile line, and the absence thereof indicates that the plant is a male fertile line.
15. 15. The method according to claim 14, characterized in that the size of the amplified DNA fragment is 50 bp to 2 kbp.
16. The method according to claim 14, characterized in that the forward primer and the rear primer comprise from about 15 nucleic acids to 35 nucleic acids for PCR, respectively.
17. 17. The method according to claim 14, characterized in that the forward primer comprises a nucleotide sequence of SEQ ID NO: 15 and the rear primer comprises a nucleotide sequence of SEQ ID NO: 16.
18. 18. The method of according to claim 14, characterized in that the step of observing whether a DNA fragment is amplified or not amplified, comprises conducting the electrophoresis with agarose gel followed by staining with ethidium bromide.
19. 19. The method according to claim 14, characterized in that the step of observing whether a DNA fragment is amplified or not amplified, is conducted by radiolabeling, colorimetry, chemiluminescence or fluorescence.
20. 20. A method for identifying male sterility in plants, characterized in that it comprises: a) conducting the Polymerase Chain Reaction (PCR) with a forward primer capable of quenching a part of a genomic DNA of apt6 or a part of a nucleotide sequence of SEQ ID NO: 2, and a back primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 2 on the genomic DNA of the plant or the mitochondrial DNA of the plant; and b) observing whether the DNA fragment is amplified or not amplified, where the presence of the amplified fragments indicates that the plant is a male sterile line and the absence thereof indicates that the plant is a male fertile line.
21. 21. The method according to claim 20, characterized in that the size of the amplified DNA fragment is 50 bp to 1 kbp.
22. 22. The method according to claim 20, characterized in that the forward primer and the forward primer could comprise from about 15 nucleic acids to 35 nucleic acids for PCR, respectively.
23. 23. The method according to claim 20, characterized in that the forward primer comprises a nucleotide sequence of SEQ ID NO: 17 and the rear primer comprises a nucleotide sequence of SEQ ID NO: 18.
24. 24. A set of PCR primers for identifying male sterility in plants, characterized in that it comprises: a) a forward primer capable of quenching a part of a gene of the coxJI genome or a part of a nucleotide sequence of SEQ ID NO: 1; and b) a back primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 1 into the plant DNA or mitochondrial DNA of the plant, and wherein the size of the amplified DNA fragment could be 50 bp to above 2kbp.
25. 25. The set of PCR primers according to claim 24, characterized in that the forward primer comprises a nucleotide sequence of SEQ ID NO: 15, and the rear primer comprises a nucleotide sequence of SEQ ID NO: 16.
26. 26. A set of PCR primers for identifying sterility in a plant, characterized in that it comprises: a) a forward primer capable of quenching a part of a genome of the apt6 genome or a part of a nucleotide sequence of the SEO ID NO : 2; and b) a home primer capable of quenching a part of a nucleotide sequence of SEQ ID NO: 2 on the plant DNA or mitochondrial DNA of the plant, and wherein the size of the amplified DNA fragment could be 50 bp to above 1 kbp.
27. 27. The set of PCR primers according to claim 26, characterized in that the forward primer comprises a nucleotide sequence of SEQ ID NO: 17, and the back primer comprises a nucleotide sequence of SEQ ID NO: 18.