WO2004099416A1

WO2004099416A1 - Dna fragment specific to cytoplasmic male sterile pepper and use thereof

Info

Publication number: WO2004099416A1
Application number: PCT/KR2004/001063
Authority: WO
Inventors: Byung Dong Kim; Dong Hwan Kim; Jeong Gu Kang
Original assignee: Seoul National University Industry Foundation
Priority date: 2003-05-07
Filing date: 2004-05-07
Publication date: 2004-11-18
Also published as: MXPA05011893A

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

DNA FRAGMENT SPECIFIC TO CYTOPLASMIC MALE STERILE PEPPERAND USE THEREOF

[Technical Field] The present invention relates to a DNA fragment specific to cytoplasmic male sterile pepper, a method for distinguishing the cytoplasmic genotype of plant using said DNA based markers, and a method for preparing a transgenic cytoplasmic male sterile plant. [Background Art] Hybrid vigor is a phenomenon by which the progeny of a cross between two inbred lines has a higher yield potential than either one of the parents. Hybrids can yield up to 10-30% more than the best non-hybrid varieties, and are a favored option for increasing yield.

The most widely used system for hybrid pepper production is the three line system: (a) a male sterile and female fertile line called the cytoplasmic male sterile (CMS) line because it carries a male sterility- conferring mutation in the cytoplasmic component of the genome; (b) a maintainer line; and (c) a restorer line. The maintainer and restorer lines are male fertile as well as female fertile. The CMS and maintainer lines are practically 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 most likely due to a mutation in the mitochondrial DNA. The CMS line, being female fertile, can be propagated by fertilization with pollen emanating 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 male sterile. The nuclear component of the genome of the progeny would also be identical to that of the CMS line, even though half of it is inherited from the maintainer line, as 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 parental line, called the restorer line, which as indicated above is male fertile and female fertile. In this cross, the CMS line serves as the female parent while the restorer line is the male parent. The restorer line also carries Rf (restorer of fertility) gene/s in its nuclear genome which will restore male fertility to a plant whose cytoplasm has been inherited from the CMS line. The hybrid seeds therefore would be produced. The CMS and restorer lines are appropriately chosen such that the hybrids exhibit sufficient hybrid vigor (or heterosis) to produce substantially higher yields than inbred varieties.

The CMS in 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 India. Since then, commercial seed companies have utilized this trait to produce hybrid Fi seeds in the field. Male sterile (S-) cytoplasm of Peterson's lines is the only common source of CMS used to produce hybrid Fi pepper seeds. One example of a well-characterized CMS system is found in maize. By screening an mtDNA library from cms-T maize 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 T-cytoplasm. Said region contains an unusual gene, designated as T-urf13, which is predicted to encode a 13-kDa polypeptide (URF13). T-urf13 is positioned upstream of orf25 and is co-transcribed.

Another example is found in the genus Petunia. The S-pcf gene has been detected in correlation with CMS in Petunias. This locus consists of the 5' portion of atp9 gene; the exon part of coxll; and an unknown open reading frame, urf-s (Young EG, Hanson MR (1987) "A fused mitochondrial gene associated with cytoplasmic male sterility is developmentally regulated." Cell 50: 41-49). The 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 orf138 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 a /4-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 mitochondrial 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 internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile Sorghum." Plant J. 10:123-133).

Although these CMS-associated genes are commonly generated by intra-rearrangement of mtDNA (Hanson MR (1991) "Plant mitochondrial mutations and male sterility." Annu Rev Genet 25:461-486), the open reading frames share no significant sequence homology. How these genes may act in CMS plants and result in mitochondrial dysfunction and non-functional pollens has not been known until now.

CMS traits are commercially very useful and important in hybrid F-i seed production. This is why transgenic male sterile plants have been attempted and 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) have developed male sterile tobacco by using a tapetum-specific promoter and barnase gene, which functions as a ribonuclease gene in plants. Several experiments have been also attempted to transform these CMS-associated genes into fertile plants. The 011239, CMS-associated mitochondrial DNA sequence in the common bean (Abad AR, Mehrtens 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 male-sterile phenotype even though targeting of the protein to mitochondria has not been made (He S, Abad AR, Gelvin SB, Mackenzie SA (1996) "A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco." Proc. Natl. Acad. Sci. USA 93:11763-11768). Another CMS-associated gene, urf-s sequence of the pcf gene which encodes the 25kDa protein in the genus Petunia, had been transformed to petunia and tobacco plants with constructs of mitochondrial targeting sequences. Even though expression of 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 tobacco." Plant Mol Biol 28:83-92).

In addition, correct identification of male fertile (N-) cytoplasm and male sterile (S-) cytoplasm in plants including food crops is very important in breeding systems. The estimation of hybrid seed purity and cytoplasmic genotype is conventionally done by the grow-out test (GOT), which is based on the assessment of morphological and floral characteristics (that 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 as they cannot be marketed until these results become available. In addition, substantial delays can result in the first growing season after hybrid seed production which is taken up by the GOT, which is also the preferred season for hybrid cultivation. In such cases, the seeds have to be stored for up to a year, i.e., until the subsequent growing season, before they can be marketed. For seed companies, large amounts of capital are therefore locked in the form of hybrid seed stocks for prolonged periods while awaiting the results of the GOT. Another disadvantage of the GOT is that it can be subjective due to environmental influences on the expression of morphological characteristics. Further, there is also the possibility that adverse climatic conditions (such as heavy wind or rain, high temperatures, drought) can damage or destroy the crop and make it difficult to collect the data. In order to solve the above problems, a technique using the CMS-associated sequence 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 Polymerase Chain Reaction (PCR), and it is ideally suited for this purpose as it is much more efficient for handling large numbers of samples than hybridization-based methods like Restriction Fragment Length Polymorphisms (RFLPs).

[Disclosure] [Technical Problem]

To solve the problems of the prior art, it is an aspect of the present invention to provide a DNA fragment associated with cytoplasmic male sterility in pepper.

It is 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 the 223^rd to 678^th nucleic acid of SEQ ID NO: 1 , and a DNA sequence capable of targeting the protein expressed by said coding region into the mitochondrion.

It is another object of the present invention is to provide a method for producing a transgenic male sterile plant.

It is another object of the present invention is to provide a method for inhibiting the production of pollen in a transgenic plant.

It is another object of the present invention is to provide nucleotide sequences specific to CMS pepper.

It is another object of the present invention is to provide a method for identifying male sterility in pepper by the PCR method. It is another object of the present invention is to provide a PCR primer set for identifying male sterility in pepper.

[Technical Solution]

In order to accomplish the aspects of the present invention, the present invention provides a DNA fragment specific to cytoplasmic male sterile pepper comprising a polynucleotide of SEQ ID NO: 1 or a polynucleotide consisting of the 223^rd to 678^th nucleic acid of SEQ ID NO:1.

Further, the present invention provides a transgenic male sterile plant comprising a polynucleotide sequence consisting of the 223^rd to 678^th nucleic acid of SEQ ID NO:1. Further, the present invention provides a construct for use in obtaining a transgenic male sterile plant, comprising: a) a polynucleotide sequence (named orf456) consisting of the 223^rd to 678^th 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.

Further, the present invention provides a method for producing transgenic male sterile plants comprising: a) preparing a construct comprising i) a polynucleotide sequence (named orf456) consisting of the 223^rd to 678^th nucleic acid of SEQ ID NO:1 , ii) a promoter that is active in the plant, and that is operably linked to the polynucleotide so as 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) transforming the construct into a plant or plant cell. Further, the present invention provides a method for inhibiting the production of pollen in a plant, comprising: a) preparing a construct comprising i) a polynucleotide sequence (named orf456) consisting of the

223^rd to 678^th nucleic acid of SEQ ID NO:1 , ii) a promoter that is active in the plant, and that is operably linked to the polynucleotide so as 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) transforming the construct into a plant or plant cell.

Further, the present invention provides a CMS specific DNA fragment (SEQ ID NO:1 , 1596 bp) at the 3' flanking region of coxll in pepper for identifying a male sterile pepper by PCR, comprising: a) conducting a Polymerase Chain Reaction (PCR) with a forward primer capable of annealing a part of a coxll genome gene or a part of a nucleotide sequence of SEQ ID NO:1 , and a reverse primer capable of annealing a part of a nucleotide sequence of SEQ ID NO:1 on plant DNA or plant mitochondrial DNA; and b) observing whether DNA fragment is amplified or not, and wherein the presence of amplified fragments indicates that the plant is a male sterile line, and the absence of the same indicates that the plant is a male fertile line.

Further, 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 annealing a part of the atp6 genome gene or a part of a nucleotide sequence of SEQ ID NO:2, and reverse primer capable of annealing a part of a nucleotide sequence of SEQ ID NO:2 on plant DNA or plant mitochondrial DNA; and b) observing whether a DNA fragment is amplified or not, and wherein the presence of the amplified fragments indicates that the plant is a male sterile line, and the absence of the same indicates that the plant is a male fertile line.

Further, the present invention provides a PCR primer set for identifying male sterility in a plant comprising: a) a forward primer capable of annealing a part of a coxll genome gene or a part of a nucleotide sequence of SEQ ID NO:1 ; and b) a reverse primer capable annealing of a part of a nucleotide sequence of SEQ ID NO:1 on plant DNA or plant mitochondrial DNA, and wherein the size of the amplified DNA fragment is from 50 bp to over 2kbp.

Further, the present invention provides a CMS specific DNA fragment (SEQ ID NO:2, 251 bp) at the 3' flanking region of atpβ in pepper for identifying a male sterile pepper by PCR, comprising: a) a forward primer capable of annealing a part of an aptβ genome gene or a part of a nucleotide sequence of SEQ ID NO:2; and b) a reverse primer capable of annealing a part of a nucleotide sequence of SEQ ID NO:2 on plant DNA or plant mitochondrial DNA, and wherein the size of the amplified DNA fragment is from 50 bp to over 1 kb kbp.

[Description of Drawings]

FIG. 1 shows a Southern-blot analysis of mtDNAs digested with

EcoRI, Hind\\\, and SamHI hybridized with eight mitochondrial probes (coxl, coxll, coxlll, atpA, atpβ, atp9, cob, nad9 genes) to compare male

fertile (F) and male sterile (S) lines in C. annuum L.

FIG. 2 shows Northern-blot analysis of mtRNAs with three

mitochondrial probes (atpA, atpβ, coxll). The atpβ and coxll genes show

polymorphic band patterns of RNA transcripts (indicated by the arrow).

FIG. 3 shows results of RFLP and inverse PCR of atpβ and coxll

genes in pepper (Capsicum annuum L.). (A): Results of Southern-blot

analysis between N- cytoplasm and S- cytoplasm with atpβ (left) and coxll

(right) probe. (B): Inverse PCR amplification for cloning of a DNA

sequence that is specific to CMS lines of pepper. Predicted PCR

fragments are marked with an asterisk (*).

FIG. 4 shows a schematic diagram of coxll coding and a flanking

region between a maintainer line (N-cytoplasm) and CMS line (S-

cytoplasm) of pepper.

FIG. 5 shows a schematic diagram comparison of atpβ coding and a

flanking region between a maintainer line (N-cytoplasm) and a CMS line

(S-cytoplasm) of pepper. (A): Schematic representation of the nucleotide

sequence comparison of (N) atpβ-1 and (S) atpβ-1 genes of C. annuum.

(B): Schematic representation of the nucleotide sequence comparison of the (N) atpβ-2 and (S) ψ atpβ-2. The highly conserved region is indicated

by the green box. The red box shows the truncated region without

nucleotide sequence homology with the 3' region of (N) atpβ-2. The arrow

indicates the primer pairs for inverse PCR of the atpβ 3' region. EcoRI-

EcoRI fragment sizes of each atpβ copies are indicated at the right side.

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 primer pairs for inverse PCR, sequencing,

and RT-PCR experiments on the coxll 3' region.

FIG. 7 shows the results of RT-PCR experiments on the orf456

gene which is located on the 3' region of the coxll gene in a sterile line.

FIG. 8 shows the results of Northern-blot analysis on mtRNAs of

fertile, sterile, and restored lines with the orf456 probe. About 15 /zg/lane

of RNA is loaded in 1.2% agarose gel and is transferred to a N⁺ nylon

membrane. F: fertile line, S: sterile line, R: restorer line.

FIG. 9 shows the results of bacterial growth inhibition tests by

orf456 gene expression. FIG. 10 shows the construction of orf456 and egfp-1 transgenes for

transformation. The diagram shows the strategy for cloning each gene

construct into the pCAMBIA2300 plant transformation vector.

FIG. 11 shows an image of GFP fluorescence in an onion transient

expression assay.

FIG. 12 shows an image of GFP fluorescence in an Arabidopsis

transformant.

FIG. 13 shows flower morphology of the male sterile (mitochondria-

targeted, a) transformants and male fertile (mitochondria-non-targeted, b)

transformants.

FIG. 14 shows a transgenic Arabidopsis phenotype at the stage of

seed sets in the mitochondrial-targeted transformants.

FIG. 15 shows heterogenic T₀ transgenic Arabidopsis transformants

which show both the male sterile and male fertile phenotypes.

FIG. 16 shows a PCR Amplification of 20 pepper cultivars or

accessions with CMS-specific SCAR primer pairs.

[Mode for Invention]

In the following detailed description, only selected embodiments of the invention have been shown and described, simply by way of illustration of the best mode contemplated by the inventors of carrying out the invention. As will be realized, the invention may be modified in various respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive.

A DNA fragment specific to cytoplasmic male sterile pepper of the present invention includes a polynucleotide of SEQ ID NO: 1 , a polynucleotide consisting of the 223^rd to 678^th 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 coxll gene and contains the orf456 region at positions 223 to 678 of the nucleic acids as an open reading Frame. The polynucleotide of SEQ ID NO: 2 (251 bp) is located at the 3'-terminal of the 3'-truncated aptβ gene.

The DNA fragment specific to cytoplasmic male sterile pepper can be used for preparing a male sterile plant or/and distinguishing a male sterile line from a maintainer pepper line.

The present invention could be applied to all kinds of plants, and it preferably includes solanaceae like pepper, eggplant, tobacco, tomato, and petunia; Brassicaceae like turnip, cauliflower, and broccoli; floral plant species like lily and chrysanthemum; and woody plants.

For preparing the transgenic male sterile plant, an expression construct with the DNA fragment (orf456) associated with cytoplasmic male sterility in pepper, wherein the DNA fragment is operably linked to and under the regulatory control of a transcriptional and translational regulatory sequence can be prepared. The transcriptional and translational regulatory sequences are those which can function in specific organisms (i.e., bacteria, yeast, fungi, plant, 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 employed according to the host cell. Examples of transcriptional and translational regulatory sequences include a promoter, an enhancer, a targeting presequence, and a terminator, but they are not limited thereto.

The promoter can be derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from a public plasmid or vector, and examples include a RA8 promoter, a TA29 promoter, and so on. Selection of the appropriate 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 a DNA sequences capable of transferring the protein expressed by the foreign gene to the mitochondrion, for example, a presequence of subunit IV of the yeast cytochrome c oxidase (coxlV) of SEQ ID NO: 3. The expression construct may be a common vector, and examples are a plasmid or viral vector including the pCAMBIA2300 vector which consists of the CaMV 35S promoter and the terminator of the nopaline synthase (nos) gene. However, any other plasmid or vector may be used as long as it is replicable and viable in the host.

Transformation methods for generating transformants according to the host cell type are well known, e.g. calcium phosphate transfection, DEAE-Dextran mediated transfection, electrophoration (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, 1986), heat-shock, Agrobactehum tumefaciens-mediated DNA transfer, protoplast transformation, microinjection, and biolistic transfection.

In one embodiment of the present invention, recombinant pCAMBIA2300 vectors harboring orf456 fragments were introduced into onions, and into Arabidopsis by Agrobactehum tumefaciens-med^'iaied DNA transfer. Onion transient expression tests showed that the presequence of subunit IV of the yeast cytochrome c oxidase (cox IV) of SEQ ID NO: 3 is capable of transferring the protein expressed by the foreign gene to the mitochondrion. Transformation to Arabidopsis thaliana with recombinant pCAMBIA2300 vector harboring an orf456 fragment showed a male sterile phenotype without pollen.

The subject of the present invention is also a process for restoring male-fertile plants from transgenic male-sterile plants, and in accordance with the invention, is characterized in that it comprises the following steps:

(a) transforming the selected higher plant by introducing at least one copy of the hybrid DNA construct as defined above into a recipient plant by means of a vector plasmid containing said sequence, in order to obtain transgenic male-sterile plants (TMSP); (b) transforming the same higher plant as in (a) by introducing at least one copy of an antisense hybrid DNA construct, including the antisense coding region of the orf456 gene by means of a plasmid containing the reverse orf456 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 male fertility has been restored and which have pre-selected characteristics. The subject of the present invention also concerns plasmids including an antisense hybrid sequence, as defined above, associated with a promoter chosen from among the constitutive promoters and the promoters specific for the anthers and also associated with a suitable terminator.

In addition, a DNA fragment specific to cytoplasmic male sterile pepper of the present invention can be used for identifying a male sterile pepper from a maintainer pepper. The method for identifying a male sterile pepper comprises a) conducting polymerase chain reaction (PCR) with a forward primer capable of annealing a part of the coxll genome gene, or the aptβ genome gene, or a part of a nucleotide sequence of SEQ ID NOs:1 or 2 and a reverse primer capable of annealing a part of a nucleotide sequence of SEQ ID NOs:1 or 2 for plant DNA or plant mitochondrial DNA as a template, and b) observing whether the DNA fragment is amplified or not, The presence of the amplified fragments indicates 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 may be from 50 bp to over 2 kbp, and the length of the forward primer and the reverse primer may be from 50 bp to over 1 kbp. In the observing step, whether the DNA fragment is amplified or not can be detected by electrophoresis on agarose gel or polyacrylamide gel followed by ethidium bromide staining. Also, a method employing radio-labeling, colorimetry, chemiluminescence, or fluorescence can be applied to detect PCR products. In an 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 reverse primer 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 in the appended claims

EXAMPLES

Example 1 : RFLP and Northern-blot analysis between male sterile

and fertile pepper

The near isogenic male fertile (N/rf/rf genotype), male sterile

(S/rf/rf), and restorer (S/Rf/Rf) lines of Capsicum annuum cv. Milyang were used in this study. These were provided by Hungnong Seed Company.

1-1. RELP analysis

To isolate the mtDNA from a maintainer and a CMS plant, young

leaves of C. annuum were harvested after etiolation and homogenized in

70 ml of homogenizing buffer (0.1 M Tris-HCI pH7.2, 0.5 M mannitol, 0.001

M ethylene glycerol-bis (β-aminoethyl ether), N,N,N',N'-tetraacetic acid

(EGTA), 0.2% bovine serum albumin (BSA), 0.05% cysteine) per 10 g of

samples. Mitochondria were purified by sucrose gradient centrifugation,

and then mtDNA was isolated by the DNase I procedure (Sparks RB, Dale RMK (1980) "Characterization of ³H-labelled supercoiled mitochondrial

DNA from tobacco suspension culture cells." Mol Gen Genet \ 80:351 -355).

MtDNA (10 g) of male fertile and CMS lines were separated on

0.8% agarose gels after digestion with EcoRI (Boehringer Mannheim,

Germany) and were transferred to Hybond N⁺ nylon membranes

(Amersham Pharmacia Biotech, NJ, USA). Eight mitochondrial probes

(coxl, coxll, coxlll, atpA, atpβ, atp9, cob, nad9) were selected for RFLP

analysis. Mitochondrial DNA probes were radioactively labeled by random

priming with [α-32P] dCTP (Amersham Pharmacia Biotech, NJ, USA).

Southern-blot analysis was performed in hybridization buffer (0.75 M NaCI,

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. The blots 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 min. The blots were

exposed to X-ray films (Kodak, USA).

Figure 1 shows Southern-blot analysis of mtDNAs digested with

EcoRI, Hind\\\, and BamH\ hybridized with eight mitochondrial probes

(coxl, coxll, coxlll, atpA, atpβ, atp9, cob, nad9 genes) to compare male fertile (N) and male sterile (S) lines in C. annuum L. Three genes (atpA,

atpβ, coxll) showed polymorphism between fertile and sterile mtDNAs.

1-2: Northern-blot analysis

For Northern-blot analysis, total RNA (20 βg) from sterile, fertile, and

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 blotting (Sambrook et al., 1989).

Northern-blot analysis was conducted on these three genes (atpA, atpβ,

coxll). The blots were hybridized at 60 ^°C for 16 hrs and the final wash

was in 0.5 X SSC, 0.1% SDS.

Figure 2 shows the result of Northern-blot analysis of mtRNAs with

three mitochondrial probes (atpA, atpβ, coxll) which show polymorphic

bands in Southern-blot analysis. The atpβ and coxll gene show

polymorphic band patterns of RNA transcripts (indicated by the arrow).

Example 2: Inverse PCR and Sequencing of flanking region of coxll

2-1. Inverse PCR for 3' flanking region of coxll gene Inverse PCR was conducted for cloning the 3' region of coxll gene

in maintainer and CMS pepper. For inverse PCR, mtDNA (5 μg) was

digested overnight at 37 ^°C in a 100 μl reaction mixture containing 10 units

of EcoRI (Boehringer Mannheim, Germany). The digestion mixtures were

extracted by phenol/chloroform and the DNA was ethanol-precipitated.

Religation of the mtDNA took place for 30 min at 37 °C in 200 μl using 3

units of T4 DNA ligase (BRL, USA). Subsequently, the ligation mixtures

were inactivated for 20 min at 65 ^°C. After phenol/chloroform extraction,

The DNA was ethanol-precipitated and dissolved in 50 μl TE (10 mM

Tris-HCI, 1mM EDTA, pH 7.4).

PCR was performed on about 500 ng of DNA in a thermocycler

(Perkin Elmer 9600) PCR reaction mixture consisted of 25 pmol of each

primer (primer set of SEQ ID NOs:5 and 6), 200 μM of each dNTP, 2.5

units of _Ex aq DNA polymerase (TakaRa, Japan), and 5 μl of 10 X _E aq

DNA polymerase buffer in a total volume of 50 μl. 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. Inverse PCR for 5' and 3' flanking region of atpβ gene PCR was performed on about 500 ng of DNA in a thermocycler

(Perkin Elmer 9600). The PCR reaction mixture consisted of 25 pmol of

each primer (primer set of SEQ ID NOs:7 and 8), 200 μM of each dNTP,

2.5 units of _ExTaq DNA polymerase (TakaRa, Japan), and 5 μl of 10 X

_ExTaq DNA polymerase buffer in a total volume of 50 μl. 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.

Figure 3 shows results of RFLP and inverse PCR of atpβ and coxll

genes in chili pepper (Capsicum annuum L.). "A" is results of Southern-

blot analysis between N- cytoplasm and S- cytoplasm with atpβ (left) and

coxll (right) probes. MtDNA (10 μg) of maintainer (N-cytoplasm) and CMS

(S-cytoplasm) lines were separated on 0.8% agarose gels after digestion

with an EcoRI enzyme. "B" is results of inverse PCR amplification for

cloning of a DNA sequence that is specific to CMS lines of pepper, and

"M1" and "M2" indicates the size markers λ /Hindtt and 1kb DNA puls

ladder, respectively (Promega Co. USA). Predicted PCR fragments were

marked with an asterisk (*). 2-3. Determination of a nucleotide that is specific to CMS lines of

chili pepper

The amplified products of the 2-1 and 2-2 experiments were

separated on 0.8% agarose gel and purified using Gel extraction kits

(Qiagen, Germany), cloned to pGEM-T easy vectors (Promega, USA), and

sequenced with a Perkin Elmer 9600 PCR machine and an ABI377

automatic sequencer (Applied Biosystems, USA).

Figure 4 shows a schematic comparison of coxll coding and

flanking region between a maintainer line (N-cytoplasm) and a CMS line

(S-cytoplasm) of pepper. The arrow indicates the primer pairs for inverse

PCR of the coxll 3' region. EcoRI-EcoRI fragment size of each coxll is

indicated on the right side. The CMS specific sequence of the 3' flanking

region of coxll is revealed as 1596 bases (SEQ ID No. 1).

Figure 5 shows a schematic comparison of atpβ coding and flanking

region between a maintainer line (N-cytoplasm) and a CMS line (S-

cytoplasm) of pepper. "A" is a schematic structural comparison of (N) atpβ-

1 and (S) atpβ-1 genes of C. annuum, and "B" is a schematic structural

comparison of the (N) atpβ-2 and (S) ψ atpβ-2. The highly conserved

regions are indicated by the green box. The red box shows the truncated region which has no nucleotide sequences homology with the 3' region of

(N) atpβ-2. The arrow indicates the primer pairs for the inverse PCR of the

atpβ 3' region. EcoRI-EcoRI fragment sizes of each of the atpβ copies are

indicated on the right side. The CMS specific sequence of the 3' flanking

region of atpβ gene is revealed as 251 bases (SEQ ID No. 2).

4-4. Identification of candidate open reading frame associated to

CMS in pepper

A new open reading frame was found by the program ORF Finder

in the NCBI homepage. By inverse PCR and sequencing results, the

difference in the structure of coxll gene between fertile and sterile pepper

was drawn and the presence of a new open reading frame, named orf456,

was detected at the 3' region of the coxll gene in the sterile line (figure 6).

In the case of the atpβ gene, no new open reading frame or chimeric gene

were detected in atpβ coding and flanking regions.

Example 3: Cloning for 3' region of coxll gene

3-1. Reverse-transcriptase PCR experiments

To investigate whether the open reading frame presumed by the

ORF Finder program was really transcribed in a CMS plant, RT-PCR with specific primer pairs (SEQ ID NOs: 6, 9 and 10) was performed. Three

micrograms of total anther RNA was used in a 10 μi reaction of first

strand cDNA synthesis driven by M-MLV reverse transcriptase (Gibco

BRL, USA), according to the protocols given by the manufacturer. PCR

was performed on 1 μl cDNA in a thermocycler (Perkin Elmer 9600) using

10 pmol of each primer, 100 μM of each dNTP, 1.5 units of _ExTaq DNA

polymerase (TaKaRa, Japan), and 2.5 μl of 10 X _ExTaq DNA polymerase

buffer in a 25 μl. The PCR amplification was at 94 ^°C (1 min), 50 °C (1 min),

and 72 °C (2 min) for 30 cycles. RT-PCR products were cloned to pGEM-T

easy vectors (Promega, USA), and sequenced with T7 and SP6 primers

(SEQ ID NOs:11-14).

Figure 7 shows the results of RT-PCR experiments on the orf456

gene which is located on the 3' region of the coxll gene in the sterile line.

The RT-PCR with the primer pair (SEQ ID NOs: 9 and 10) is performed to

detect the fact that the newly-made orf456 is really and uniquely

transcribed in the sterile line. The RT-PCR with the primer set (SEQ ID

NOs:6 and 10) is performed to detect whether the orf456 gene is co-

transcribed with coxll located on the upstream region. Figure 8 shows the

results of Northern-blot analysis on mtRNAs of fertile, sterile, and restored lines with the orf456 probe. About 15 g/lane of RNA was loaded in 1.2%

of agarose gel and transferred to a N+ nylon membrane. F: fertile line, S:

sterile line, R: restorer line. Figure 3 shows the results of Northern-blot

analysis on mtRNAs of fertile, sterile, and restorer lines with the orf456

probe. The orf456 open reading frame is really and uniquely transcribed in

the sterile and restorer line carrying S-cytoplasm.

Example 4: Bacterial growth inhibition test

To investigate how orf456 affects plant mitochondria and results in

mitochondrial dysfunction and male sterility, a heterologous systems, i.e. a

bacterial cell, was adopted. The possible toxicity on a bacterial cell of the

orf456 gene was examined.

The orf456 gene was cloned into a pTrcHis2-TOPO expression

vector and transfected into an E. coli strain Top10 cell (Invitrogen, USA).

As controls, cells carrying pTrcHis2-TOPO+LacZ genes were cultured

under the same conditions and induced by 1 mM IPTG. Cloning and

transformation were conducted according to the manufacturers protocols.

Top10 cells containing the LacZ gene (control) and orf456 gene were

precultured in 3 ml of LB medium with 50 #g/ml of ampicillin at 37 ^°C for 16 hrs. 50 βS. of a primary culture was transferred to 20ml of the medium and

cultured at 37 °C for 2-3 hrs. At the time of O.D.₆₀o= 0.6, 1mM of IPTG was

added and the growth rate of each transformant was monitored by its

absorbance every hour. The growth of E. coli cells was dramatically

impaired as soon as expression of orf456 was induced. Figure 9 shows

the results of bacterial growth inhibition tests by orf456 gene expression.

The growth rate of E. coli carrying constructs with orf456 and induced by

1mM IPTG is severely impaired compared to other constructs.

Example 5: Test for Targeting Foreign gene to Mitochondria and

Transformants preparation

5-1. Constructs preparation for Arabidopsis transformation

The egfp-1 fragment (SE ID NO: 4, GFP variants purchased from

Clontech) were amplified from the pEGFP-1 vector (Clontech, Palo Alto,

USA). The amplified coxlV target sequences (SEQ ID NO:3, Yeast DNA

fragment for cytochrome c oxidase subunit IV precursor) and egfp-1 gene

were ligated by T4 DNA ligase (Promega, USA), then cloned to the

pCAMBIA2300 vector (MJC). Also, a coxlV-orf456 construct and nontargeting-otf456 construct as shown in figure 9 were prepared and

ligated into the pCAMBIA2300 vector.

In figure 10, the nopaline synthase (nos) gene terminator sequence

was fused to the 3' end of the orf456 and egfp-1 sequences. In the first

construct (referred to as coxlV-orf456), the orf456 sequence was fused to

the transit peptide sequence of the presequences of the nuclear coxlV

gene of yeast strain Y187, (Stratagene, USA). The second construct

(referred to as nontargeting-orf456) did not have the mitochondrial

targeting peptide sequences. The third construct (referred to as coxlV-

egfp-1) was made using coxlV presequences and egfp-1 from pEGFP-1

vector (Clontech, USA).

5-2. Onion transient expression experiments

The fusion constructs (coxlV presequence fused to egfp- gene) in

the pCAMBIA2300 vector were transiently expressed in onion epidermal

cells after transfection. Inner 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. Peels were bombarded within 1 h of transfer to agar plates.

Particle bombardments were done as 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 Imaging System (Bio-Rad, Hercules, CA) at the

National Instrumentation Center for Environmental Management (NICEM,

Suweon, South Korea).

Figure 11 shows an image of GFP fluorescences in the onion

transient expression assay. The left picture shows the GFP fluorescences

in the onion by coxlV+egfp-1 construct. Mitotracker CMSRox dye

(Molecular Probe Co., USA) was used for detection of mitochondria (right

picture). The GFP image and Mitotracker dye images completely matched.

5-3. Arabidopsis plant transformation

The Arabidopsis thaliana ecotype Columbia plants were used for

transformation experiments. A strong CaMV 35S promoter and nos

terminator were used in vector construction. The mitochondrial target

sequence used was derived from the Yeast coxlV presequences, and the

orf456 and egfp-1 sequences used in the constructions were amplified by

PCR to facilitate cloning. Inserts were confirmed by enzyme digestion and

sequencing. The inserts were digested with an appropriate restriction

enzyme to clone into the plant transformation vector pCAMBIA2300.

Clones which had correct inserts were selected on the kanamycin LB medium. Transformations into Agrobactehum tumefaciens LBA4404 were

done by the heat-shock method (Sambrook et al., 1989). The Arabidopsis

thaliana plants were transformed by Agrobactehum tumefaciens carrying

the coxlV (targeted)-o/f456 and non-targeted orf456 constructs. The

Agrobactehum tumefaciens-medlated transformation was performed by a

modified floral-dip method (Clough and Bent, 1998). Transgenic plants

were selected on a medium containing kanamycin sulfate (50 μg ml^"1).

Green plants surviving the antibiotic treatment were retained for further

analyses.

Figure 12 shows an image of GFP fluorescences expressed in roots

of Arabidopsis thaliana.

The vegetative growth of transformants was uniform and similar to

that of non-transformed control plants or non-targeted transformants. At

flowering, the 31 plants out of 51 Arabidopsis transformants transfected

with mitochondrial targeting signal showed male-sterile phenotype in the

Ti generation. This classification was based on flower morphology and

seed set. The 3 Arabidopsis plants out of 50 transformants transfected

with non-targeted constructs showed male-sterile phenotype. This was a

rather unexpected result in the case of non-targeting experiments. However, it was postulated that this orf456 product in the cytoplasm could

not have any deleterious effect in plant cells as the results proven at the

bacterial growth inhibition tests even if it did not express in the

mitochondria.

Figure 13 shows flower morphology of the male sterile

(mitochondria-targeted) transformants and male fertile (mitochondria-non-

targeted) transformants. "a" is a floral picture of male sterile transformants

carrying mitochondria-targeted orf456, and "b" is a floral picture of male

fertile transformants carrying mitochondria-non-targeted orf456.

Figure 14 shows an Arabidopsis phenotype at the stage of seed

sets in the mitochondrial-targeted transformants (a) and in the

mitochondrial-non-targeted transformants (b).

Figure 15 is a picture of Arabidopsis transformants which shows the

male sterile and male fertile phenotypes in the same stocks, which come

from the vacuum infiltration transformation methods. The arrow indicates

the normal silique produced from normal pollination, and the arrowhead

indicates the flower which is not pollinated and did not produce normal

seed sets.

5-4. Confirmation of transgenic plants Total leaf DNA for PCR and Southern-blot analysis was extracted

(Kim et al., 2001). PCR amplification was performed on 100 ng of total

DNA with a thermal cycler PTC-200 (MJ Research, USA) using thirty

cycles of 30 s at 94 °C, 45 s at 55 TJ , and 90 s at 72 °C . The total DNA

digested with restriction enzymes were electrophoresed on a 0.8%

agarose gel, transferred onto a nylon membrane, and hybridized with [α-

³²P] dCTP (Amersham Pharmacia Biotech, NJ, USA)- labeled probes.

Table 2 shows the male fertility evaluation based on pollen

production in transgenic T-i Arabidopsis plants confirmed to contain the

orf456 transgene.

(Table 2)

Example 6: Determination of male fertile or sterile by genotyping

6-1. Primer

The specific oligonucleotide primers that can be used in a PCR

assay to distinguish maintainer (N-cytoplasm) and CMS (S-cytoplasm)

lines of chili pepper (Capsicum annuum L.) are designed. coxll SCAR PCR primer

Forward primer (SEQ ID No: 15) - a part of coding region of coxll

gene

Reverse primer (SEQ ID No: 16) - a part of the sequence that is

unique to the CMS lines.

aptβ SCAR PCR primer

Forward primer (SEQ ID No: 17) - a part of coding region of atpβ

gene

Reverse primer (SEQ ID No: 18) - a part of the sequence that is

unique to the CMS lines.

coxll positive Control

Forward primer: SEQ ID NO: 19

Reverse primer: SEQ ID NO: 20

6-2. PCR assay for distinguishing CMS and maintainer lines in chili

pepper

Using the primer sets, PCR was performed as described below.

Total DNA (200 ng) was mixed with 200 μmol of dNTP, 20 pM of

each primer, 5 μl of 10 X reaction buffer, and 2.5 unit of Taq polymerase

(Takara, Japan) in a 50 μl reaction volume. PCR amplification for atpβ SCAR marker was carried out at 94 ^°C (1 min), 52°C (1min), and 72^°C (2

min), for 35 cycles. Amplified DNA was electrophoresed in 0.8% agarose

gel. In the case of coxll SCAR marker, annealing was performed at 56 °C .

Figure 16 is a picture showing PCR results for the 20 pepper

cultivars, where "N" indicates a fertile phenotype, "S" indicates a sterile

phenotype, and "M" indicates a hlHind\\\ DNA marker.

Using the coxll SCAR primer pair, 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 case of the atpβ SCAR primer pair,

there was an amplification of a 607 bp DNA fragment in the CMS lines, but

no PCR amplification was observed in the maintainer lines. To check

whether PCR reactions were done well, a PCR primer pair spanning the

coding region of the coxll gene was used as a control. The fragment size

of PCR amplification was about 1.5 kb. The Capsicum annuum used in the

PCR experiment is listed in Table 1.

(Table 1)

*N: N-cytoplasm, S: S-cytoplasm

Claims

WHAT IS CLAIMED IS:

1. A DNA fragment specific to a cytoplasmic male sterile pepper

comprising a polynucleotide of 223^rd to 678^th nucleic acid of SEQ ID

NO:1.

2. The DNA fragment according to claim 1 , wherein the DNA fragment

comprises a polynucleotide of SEQ ID NO: 1.

3. The DNA fragment according to claims 1 or 2, wherein the

polynucleotide is located at the 3'-terminal of a coxll gene.

4. A DNA fragment specific to a cytoplasmic male sterile pepper

comprising a polynucleotide of SEQ ID NO: 2.

5. The DNA fragment according to claim 4, wherein the polynucleotide is

located at the 3'-terminal of a 3'-truncated atpβ gene.

6. A transgenic male sterile plant comprising a polynucleotide of a 223^rd to

678^th nucleic acid of SEQ ID NO:1.

7. A construct for use in obtaining a transgenic male sterile plant,

comprising:

a) a polynucleotide consisting of a nucleotide sequence consisting of a

223^rd to 678^th nucleic acid of SEQ ID NO:1. b) a promoter that is active in the plant, and operably linked to the

polynucleotide so as 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 construct according claim 7, wherein the c) DNA sequence

comprises a nucleotide sequence of SEQ ID NO: 3.

9. The construct according claim 7, wherein the plant is one or more

selected from the group consisting of Solanaceae like chili pepper,

eggplant, tobacco, tomato, and petunia; Brassicaceae like turnip,

cauliflower, and broccoli; and floral plant species like lily and

chrysanthemum, and woody plants.

10. A method for producing male sterile transgenic plants comprising

transforming the construct of claim 7 into plants or plant cells.

11. The method according claim 10, wherein the plant is one or more

selected from the group consisting of Solanaceae like chili pepper,

eggplant, tobacco, tomato, and petunia; Brassicaceae like turnip,

cauliflower, and broccoli; floral plant species like lily and

chrysanthemum; and woody plants.

12. A method for inhibiting the production of pollen in plants, comprising

transforming the construct of claim 7 into plants or plant cells.

13. The method according claim 12, wherein the plant is one or more

selected from the group consisting of Solanaceae like chili pepper,

eggplant, tobacco, tomato, and petunia; Brassicaceae like turnip,

cauliflower, and broccoli; floral plant species like lily and

chrysanthemum; and woody plants.

14. A method for identifying a cytoplasmic male sterile pepper comprising:

a) conducting Polymerase Chain Reaction (PCR) with a forward primer

capable of annealing a part of coxll genomic DNA or a part of a

nucleotide sequence of SEQ ID NO:1 and a reverse primer capable of

annealing a part of a nucleotide sequence of SEQ ID NO:1 on plant

genomic DNA or plant mitochondrial DNA; and

b) observing whether the DNA fragment is amplified or not,

wherein the presence of the amplified fragments indicates that the plant is

a male sterile line, and the absence of the same indicates that the plant

is a male fertile line.

15. The method according to claim 14, wherein the size of the amplified

DNA fragment is from 50 bp to 2 kbp.

16. The method according to claim 14, wherein the forward primer and the

reverse primer comprise from about 15 nucleic acids to 35 nucleic

acids for PCR, respectively.

17. The method according to claim 14, wherein the forward primer

comprises a nucleotide sequence of SEQ ID NO: 15 and the reverse

primer comprises a nucleotide sequence of SEQ ID NO: 16.

18. The method according to claim 14; wherein the step of observing

whether a DNA fragment is amplified or not comprises conducting

agarose gel electrophoresis followed by ethidium bromide staining.

19. The method according to claim 14, wherein the step of observing

whether a DNA fragment is amplified or not is conducted by radio-

labeling, colorimetry, chemiluminescence, or fluorescence.

20. A method for identifying a male sterility in plant comprising

a) conducting Polymerase Chain Reaction (PCR) with a forward primer

capable of annealing a part of an aptβ genomic DNA or a part of a

nucleotide sequence of SEQ ID NO:2, and a reverse primer capable of

annealing a part of a nucleotide sequence of SEQ ID NO:2 on plant

genomic DNA or plant mitochondrial DNA; and

b) observing whether the DNA fragment is amplified or not, wherein the presence of the amplified fragments indicates that the plant is

a male sterile line, and the absence of the same indicates that the plant

is a male fertile line.

21. The method according to claim 20, wherein the size of the amplified

DNA fragment is from 50 bp to 1 kbp

22. The method according to claim 20, wherein the forward primer and the

reverse primer could comprise from about 15 nucleic acids to 35

nucleic acids for PCR, respectively.

23. The method according to claim 20, wherein the forward primer

comprises a nucleotide sequence of SEQ ID NO: 17, and the reverse

primer comprises a nucleotide sequence of SEQ ID NO: 18.

24. A PCR primer set for identifying a male sterility in plant comprising:

a) a forward primer capable of annealing a part of a coxll genome gene or

a part of a nucleotide sequence of SEQ ID NO:1 ; and

b) a reverse primer capable of annealing a part of a nucleotide sequence

of SEQ ID NO:1 on plant DNA or plant mitochondrial DNA, and

wherein the size of the amplified DNA fragment could be from 50 bp to

over 2 kbp.

25. The PCR primer set according to claim 24, wherein the forward primer

comprises a nucleotide sequence of SEQ ID NO: 15, and the reverse

primer comprises a nucleotide sequence of SEQ ID NO: 16.

26. A PCR primer set for identifying a male sterility in plant comprising:

a) a forward primer capable of annealing a part of an aptβ genome gene

or a part of a nucleotide sequence of SEQ ID NO:2; and

b) a reverse primer capable of annealing a part of a nucleotide sequence

of SEQ ID NO:2 on plant DNA or plant mitochondrial DNA, and

wherein the size of the amplified DNA fragment could be from 50 bp to

over 1 kbp.

27. The PCR primer set according to claim 26, wherein the forward primer

comprises a nucleotide sequence of SEQ ID NO: 17, and the reverse

primer comprises a nucleotide sequence of SEQ ID NO: 18.