KR101027924B1 - Method for detecting cytoplasm types of plant of Allium cepa line using the DNA marker developed based on novel cytoplasmic male sterility CMS-specific chimeric gene - Google Patents

Abstract

The present invention is directed to a DNA marker for selection of male sterility and normal onion-based plants developed using a cytoplasmic-genic male sterility (CMS) specific novel chimeric gene ( orf725 ) and structural features of the gene. It is about. Specifically, the novel male infertility specific chimeric gene ( orf725 ) enables the identification of two onion male infertility cytoplasms (CMS-S and CMS-T) and normal cytoplasm at the same time, and the chimeric gene ( orf725). DNA markers can be easily used for the production of onion hybrid seeds using CMS by completely replacing the existing DNA markers, which are longer than 5 years, and inefficient DNA markers. In addition, it is possible to identify all three types of cytoplasm present in onion through a single PCR, which is very useful for the selection of CMS onion system and CMS maintenance system, which enables early selection of CMS cytoplasm types. It can greatly accelerate the development period of onion hybrid varieties and contribute to systematically performing various and excellent varieties.

Onion, Cytoplasmic Male Infertility, Chimeric Gene, Hybrid Seed, DNA Marker

Description

Method for detecting cytoplasm types of plant of Allium cepa line using the DNA marker developed based on novel cytoplasmic male sterility (CMS ) -specific chimeric gene}

The present invention is a DNA marker used to determine the cytoplasmic type of onion-based plants developed by using a newly discovered chimeric gene ( orf725 ) specific to onion cytoplasmic male sterility (CMS) and the chimeric gene structural characteristics. It's about arguments.

Hybrid stress is a phenomenon in which the breeding offspring of two species have higher production potential than either parent. In the case of hybrids, the yield can be increased by about 10 to 30% compared to the best non-hybrid fixed species, which is advantageous in increasing the yield.

Upbringing of one hybrid variety using hybrid accents in plants has been going on for a long time, and most of the commercially available vegetable seeds are F ₁ hybrid seeds. F ₁ hybrid seed cultivation methods include artificial mating, self-incompatibility, male migration, male infertility, and cross-breeding but are difficult in terms of time, effort, uniformity and economy except for the use of male infertility. Because of the many problems, research continues to develop and use male infertility resources in many crops.

Male infertility refers to a phenomenon in which infertility occurs due to abnormalities in male organs such as pollen, anther, and stamen. It is caused artificially or naturally, mainly by natural mutations, interdependent hybrids, and treatment with mutant organic agents. Male infertility is caused by genetic causes and environmental causes. Most male infertility systems used for breeding are male infertility due to genetic causes. Male infertility caused by these genetic causes can be divided into three types: genetic male sterility (GMS), cytoplasmic male sterility (CMS) and cytoplasmic-male sterility (Cytoplasmic- sterility) Genic Male Sterility (CGMS). CMS and CGMS are mostly due to mitochondrial abnormalities in the cytoplasm, and may be divided between CMS and CGMS depending on the presence of a gestational recovery gene (Rundfeldt, J., Z. Pflanzenzuchtung, 1960, 44: 30-62; Shivanna, KR and BM Johri., Pollen sterility, in the angiosperm pollen, structure and function.1985. Wiley Eastern Ltd. New Delhi).

Genetic male sterility (GMS) is primarily recessive and infertile in the case of a homozygous recessive, so when producing F ₁ hybrid seeds using only 50% infertile Can be obtained. Therefore, in order to maintain and multiply male sterility, a lot of effort and cost are incurred because it is necessary to check the existence of pollen by flowering each individual. Cytoplasmic Male Sterility (CMS) and Cytoplasmic-Genic Male Sterility (CGMS), on the other hand, cause abnormalities in the mitochondria in the cytoplasm, resulting in abnormal pollen, and thus losing maternal fertility. It is characterized by heredity.

The CMS and male infertility maintenance system are substantially the same in the nuclear component of the genome (so often called the isonuclear line), but differ in the composition of the genome in the cytoplasm. Male sterility in the CMS cell system is maternally inherited and appears to be due to mutations in mitochondrial DNA. The CMS cell system of magnetic fertility can be proliferated by fertilization due to pollen shedding from the male sterility maintenance system. Since the cytoplasmic components of the genome are not transmitted by pollen, these hybridizers inherit only the cytoplasmic properties of the CMS cell system and become male infertility. Although only half is inherited from the male sterility maintenance system, since there is no difference between the two lines with respect to the composition of the genome, the nuclear component of the genome in the offspring will be identical to the nuclear component of the CMS cell system.

Hybrid seed can be produced by crossing CMS cramps with another parental line, male and female fertility, and fertility recovery lines, as indicated above. In this mating, the reproductive recovery line acts as the male parent while the CMS line acts as the female parent. The fertility recovery line will also carry a male restorer of fertility (Rf) within the nuclear genome, restoring male fertility in plants with the cytoplasm inherited from the CMS line. Therefore, the hybrid seed is produced. The CMS and fertility recovery strains are selected with good combinations that exhibit hybrid stresses (or heterosis) that show substantially higher productivity than purely fixed species.

One example of a well characterized CMS system is found in corn. By screening a mtDNA library from cms-T corn with sterile and fertile mRNA, Dewey et al. Identified sites specific for T-cytoplasm. This site contains a special gene named T- urf13 which is predicted to encode 13-kDa polypeptide (URF13). T- urf13 is located upstream of orf25 and co-transcribed (Dewey RE, Levings III CS, Timothy DH (1986) "Novel recombination in the maize mitochondrial genome produces a unique transcriptional unit in the Texas malesterile cytoplasm." Cell 44: 439 -449).

Another example is found in petunia. The S- pcf gene was found to be associated with CMS in petunias. These locus are 5 parts of the atp9 gene; exon portion of coxII ; Unknown open reading frame that is (open reading frame) is composed of three portions of the urf -s (Young EG, Hanson MR (1987) "A fused mitochondrial gene associated with cytoplasmic male sterility is developmentally regulated." Cell 50: 41- 49).

Certain genes associated with CMS are also described in John 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 malesterility (CMS) -associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids. Mol Gen Genet 243 540-547), 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), sunflower (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.App Genet. 90: 948-951), carrot (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 sugarcane (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).

Typically, even though these CMS-related genes are generated by in-rearrangement of mtDNA, open reading frames do not share significant sequence homology (Hanson MR (1991) "Plant mitochondrial mutations and male sterility." Annu Rev Genet 25 : 461-486). It is not yet known how these genes act in CMS plants and cause mitochondrial dysfunction and disabling pollen.

CMS properties are very useful and important commercially for the generation of F ₁ seeds. This is why transgenic male sterile plants are tried and developed by some researchers. For example, Mariani et al. Developed male sterile tobacco using a taper-specific promoter and barnase gene that function as a rionuclease gene in plants (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). Some experiments have attempted to transform these CMS-related genes into fertile plants. Orf239 , a common CMS-related mitochondrial DNA sequence in kidney beans, was transformed with tobacco with or without a mitochondrial target sequence (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.Transformed tobacco showed a semi-sterile or male infertility phenotype even if the protein was not targeted to mitochondria (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).

In addition, the correct discrimination of male fertility cytoplasm and male infertility cytoplasm of plants, including edible crops, is very important for breeding systems.

Conventional sarcoma used markers according to variations in morphological characteristics (eg flower color) to select the lineage of the desired trait (Staub JE et al., Hort Scienc e, 31 (5): 729-741, In 1996, many morphological markers are not only likely to change depending on the environment (eg cucumber degenerative gene de), but also have a very limited number of markers. Accordingly, recent studies have been conducted to develop molecular markers (DNA markers) using mutations that appear at the DNA sequence level. DNA markers are present in plants so much that they cannot be compared with morphological markers, and they are advantageous for use as markers without impeding the function or physiology of plants (Jones N., et al., New Phytol., 137: 165-177, 1997).

Accordingly, the present inventors have discovered a novel gene specific for male infertility present in two onion male infertility cytoplasm and characterized its characteristics, and based on the characteristics of the newly discovered gene, two male infertility in onions The present invention was completed by developing stable DNA markers capable of selecting useful onion-based plants by discriminating between normal and one normal cytoplasm.

Therefore, the present invention provides an onion-specific chimeric gene having cytoplasmic male sterility (CMS).

Another object of the present invention relates to a DNA labeling factor for selecting plants of the CMS and normal cytoplasmic onion system.

The present invention provides an onion specific chimeric gene with cytoplasmic male sterility (CMS).

Another object of the present invention relates to a DNA labeling factor for selecting plants of the CMS and normal cytoplasmic onion system.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention provides a nucleotide sequence of a chimeric gene specific to male sterile onion, and provides a method for determining cytoplasm type in onion by PCR and a PCR primer set for discriminating cytoplasm type in onion.

In order to improve the productivity of F ₁ hybrid seeds, the present inventors used normal and molecularly reported molecular markers among 176 onion strains grown in Korea and Japan to find onion-specific genes with cellular male infertility. After classifying into the cytoplasm and two male infertility cytoplasms (CMS-S and CMS-T cytoplasmic types), they were used to find a novel genetic structure that was specifically observed only in the male infertility cytoplasm.

The onion male infertility specific gene is a chimeric gene in a form in which two different kinds of genes are combined. Nucleotide is a specific DNA sequence of a cytoplasmic male infertile plant comprising the nucleotide (2357bp) described in SEQ ID NO: 1, wherein the nucleotide is similar to the orfA501 gene extracted from a part of onion coxI gene and 3'-terminal leek The base sequences are present in linked form.

The DNA sequence is present in the mitochondrial genome and is related to the cytoplasmic male infertility of onions, and the CMS onion-based infertile hybrid with the cytoplasmic male infertility including the cytoplasmic male infertility can be produced. .

The present invention also provides a DNA labeling factor for selecting plants of the two CMS onion system.

The use of the novel onion cytoplasmic male infertility specific chimeric gene of the present invention enables efficient F ₁ hybrid seed production and has developed a DNA molecule label capable of selecting CMS onion lineage plants based on mitochondrial genomes.

The specific chimeric DNA sequence of the cytoplasmic male infertility onion of the present invention can be used to distinguish onions having normal cytoplasm and two male infertile cytoplasms. The method for determining the male sterile onion is a forward primer capable of annealing a part of a nucleotide sequence similar to the normal coxI gene of SEQ ID NO: 2 in the novel gene orf725 of SEQ ID NO: 1 and the normal coxI gene specific 3 'terminal, respectively. And performing PCR using two reverse primers that can anneal to the novel orf725 gene specific 3 ′ end and observing whether the DNA fragment is amplified. At this time, the type of the amplified fragment indicates what kind of male infertile cells the plant possesses, or whether it is a male genus family having a normal cytoplasm. The size of the amplified DNA fragment is 300bp to 2000bp, and the lengths of the forward primer and the reverse primer are 15bp to 30bp. See FIGS. 1 and 2.

In the observation step, the amplification of the DNA fragments can be measured by electrophoresis on agarose gel or polyacrylamide gel, followed by ethidium bromide staining. In addition, PCR products can be detected by radiation-labeling, colorimetric, chemiluminescent or fluorescence methods.

In an embodiment of the present invention, the primer consisting of the nucleotide sequence of SEQ ID NO: 3 (5'- CATAGGCGGGCTCACAGGAATA-3 ') and the reverse primer as the forward primer is SEQ ID NO: 4 (5'- AATCCTAGTGTCCGGGGTTTCT-3') and 5 (5 ') A primer consisting of the nucleotide sequence of CAGCGAACTTTCATTCTTTCGC-3 ′) is designed and constructed.

As described above, it can be very easily used to produce hybrid seeds of CMS onion lineage using cytoplasmic male infertility specific chimeric genes, and also selecting using a novel chimeric gene specific to onion CMS according to the present invention. DNA markers are very useful for the selection of CMS onion strains because PCR can be detected by PCR using primers to amplify them. There is also an advantage that can be removed.

In crop breeding, not only the effort and expense are low, but also the detection of DNA markers using high-speed, high-accuracy and sensitive PCR technology enables more accurate selection of target traits.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Example 1 onion CMS (cytoplasmic Male Sterility ) Isolation of cytoplasmic specific gene DNA .

In order to isolate onion genomic DNA, 176 onion strains grown in Korea and Japan were classified into two male infertility and one normal cytoplasmic type by using a child test through crossbreeding and known molecular markers. It was used as a material for searching for infertility specific genes. Chives male infertility specific sequences ( orfA501 , see Figure 1) have been reported to exist in onion male infertility (Engelke T, Terefe D, Tatlioglu T (2003) “A PCR-based marker system monitoring CMS- ( S), CMS- (T) and (N) -cytoplasm in the onion ( Allium cepa L. ). ”Theor Appl Genet 107: 162-167) The genome walking technique was used to find peripheral sequences and gene structures that were completely different from the western leek sequences (see FIG. 1). Genome walking was performed using a kit of Clontech Co., Ltd. (Universal Genome Walker kit, Clontech, Palo Alto, CA, USA).

In order to investigate the copy number in the mitochondrial genome of the above-mentioned male infertility specific gene, PCR was performed on each of three lines having two male infertility cytoplasms and one normal cytoplasm. In order to observe the relative ratio of the newly discovered gene ( orf725 ) and the normal coxI gene, the PCR cycle was run 30 and 40 times (see FIG. 2). After separating the DNA from the leaf tissue of the plant, it was used to amplify the orf725 gene using a primer having a nucleotide sequence represented by SEQ ID NO: 6 and SEQ ID NO: 7 as a template and a base represented by SEQ ID NO: 8 and SEQ ID NO: 11 A primer having a sequence was used to amplify the normal coxI gene of SEQ ID NO: 2. As a result, the newly discovered orf725 gene was found in large amounts in only two male infertility cytoplasm, and the normal coxI gene was very small in CMS-S type and large in CMS-T and normal cytoplasmic type. I could see. Based on the results that the relative proportions of these two genes in the mitochondrial genomes differ depending on the cytoplasmic type, DNA markers capable of identifying three cytoplasmes by a single PCR were developed. PCR was performed using Clontech's PCR kit (Advantage 2 Polymerase Mix, Clontech, Palo Alto, CA, USA). The reaction conditions were denatured for 5 minutes at 95 ° C., followed by 30 seconds at 95 ° C .; 30 seconds at 65 ° C .; And 40 cycles of 1 minute 30 seconds at 72 ° C., followed by final reaction at 72 ° C. for 10 minutes.

[Example 2] DNA to determine the top of two cytoplasmic male sterile cytoplasm and at least in the onion Cover factor development

Based on the relative ratios between the orf725 gene and the normal coxI gene found in Example 1, DNA markers were developed that can distinguish three cytoplasmes. In order to check whether the following experiment was carried out. As an experimental material for this, 24 varieties or breeding plant plants of known cellular type were used (see FIG. 3).

PCR was performed using DNAs isolated from the 24 plants described above as templates, and simultaneously using three primers having the nucleotide sequences represented by SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. PCR reaction conditions were 5 minutes at 95 ° C after per-denaturation at 95 ° C; 30 seconds at 60 ° C .; And 40 cycles of 1 minute 30 seconds at 72 ° C., followed by final reaction at 72 ° C. for 10 minutes. Thereafter, 1% agarose gel electrophoresis was performed. As a result, as can be seen in Figure 3, the normal cytoplasm was detected a band of size 833bp amplified from the coxI gene, and in the CMS-S type cytoplasm was detected a band of 628bp amplified from the orf725 gene. However, it was confirmed that both bands were amplified in the cytoplasm of CMS-T type.

[Example 3] A male sterile specific genes from onion (orf725) and normal coxI reverse transcriptase for the investigation of gene expression of genes RT - PCR performed

RT-PCR was performed with a novel male infertility specific gene ( orf725 ) and a normal coxI gene specific primer pair (SEQ ID NOS: 9, 10 and 11) to analyze the expression patterns in the three cytoplasmes (see FIG. 4). . Experimental materials were selected from four male and female fertility strains from the flower buds of four male and female fertility strains. Was extracted to synthesize cDNA was used as a template of RT-PCR. In normal cytoplasm, RNA was extracted from flowers of four breeding lineage plants and cDNA was synthesized and used as a template.

CDNA was synthesized from the flower RNA of the plant using Invitrogen's reverse transcriptase kit (Superscript Reverse Transcriptase (RT) II kit, Invitrogen, Carlsbad, Calif.), And the expression of genes was analyzed by RT-PCR.

RT-PCR conditions were 30 minutes at 94 ° C. after per-denaturation at 94 ° C. for 3 minutes; 30 seconds at 65 ° C .; And 30 cycles of two minutes at 72 ° C., followed by a final reaction at 72 ° C. for 10 minutes. Thereafter, 1% agarose gel electrophoresis was performed. As a result, as shown in Figure 4, the orf725 gene was confirmed only in CMS-S and CMS-T, but not in the normal cytoplasm, whereas normal coxI gene is expressed only in CMS-T and normal cytoplasm and CMS-S cytoplasm It was confirmed that is not expressed in. And since the expression of the two genes does not differ between male and female infertile individuals in the isolated generation, it was confirmed that the recovery gene did not affect the expression of the two genes.

Figure 1 shows a PCR result confirming the cytoplasm of the plant using a schematic diagram for comparing the nucleotide sequence structure of the coxI gene and chimeric gene orf725 in the onion mitochondrial genome and onion cytoplasm type discriminator. (1: HT201, 2: HT202, 3: HT204, 4: Sharom, 5: Ocean, 6: Giant, 7: Sasuki, 8: Gangsukhwang, 9: Hiro)

Figure 2 shows the PCR results confirming the presence and relative ratio of the coxI gene and chimeric gene orf725 in the onion genome in three types of onion cytoplasm. (1: HT201, 2: HT202, 3: HT204, 4: Sharom, 5: Ocean, 6: Giant, 7: Sasuki, 8: Gangsukhwang, 9: Hiro)

Figure 3 shows the PCR results confirming the reliability of the DNA markers developed for various onion varieties and breeding lines. (1: OB201, 2: OB202, 3: OB204, 4: OB217, 5: OB225, 6: OB245, 7: OB278, 8: OB359, 9: Gukjosangball, 10: Hantur420, 11: Guygum, 12: PI-Gilimsung , 13: Swaro, 14: Nice, 15: Juokhwang, 16: Red Ring Ball, 17: Nurihwang, 18: Rucy, 19: Chunryukgwon, 20: Wolgwang, 21: Tobball, 22: Katamaru, 23: POP, 24: Saboroki )

4 shows the results of confirming the expression of normal coxI gene and orf725 gene by RT-PCR targeting three cytoplasmic types.

<110> Industry Foundation of Chonnam National University <120> Method for detecting cytoplasm types of plant of Allium cepa line          using the DNA marker developed based on novel cytoplasmic male          sterility (CMS) -specific chimeric gene <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 2772 <212> DNA <213> Allium cepa <400> 1 aataagaatt atattcaatt tcagaagcaa agatggctga tctgtatcaa gaacctgatg 60 ataatatgct acgccccaga agaagaagat tcagaataaa cggaggctaa tcatggaaaa 120 aggttgacgt tcaccccctt tcttgtttgt gagtaggtgg gtgctattga gatattgggc 180 ccttaaagag tcttccttta gaaagaacgg aacgagtttg tgattcgatc ttctaaagcg 240 taaagcgaaa tcagtaggac aagatccaat ctttcgtcaa cgcaggttgc ttattctcct 300 acttaagtat attaattggg tagcgtagct ggaatttctt tttggtccct ttcgtccagt 360 ggttaggaca tcgtcttttc atgtcgaaga cacgggttcg attcccgtaa gggataggta 420 cttctcggta ctccgggata tacaccagcg ggtggaccgg ttggcgttat gccacttctc 480 cttcctcata tggtggttcg atggctcttc tctactaacc acaaagatat cggtactctc 540 tatttcatct ttgctgccat tgctggagtg atgggcacat gcttctccgt actgattcgt 600 atggaattag cccgacccgg cgatcaaatt cttggcggga atcatcagct ttataatgtt 660 ttaataactg ctcacgcttt tttaatgatc ttttttatgg tgatgccagc gatgataggt 720 ggatttggta attggtttgt tccgattctg atcggtgcac ctgacatggc atttccacga 780 ttaaataata tatcattctg gttgttgcca ccaagtctct tgctcctatt aagctccgcc 840 ttagtagaag tgggtagcgg cactgggtgg acggtctatc cgcctttaag tggtattacc 900 agccattctg gaggagcagt tgattcagcc atttttagtc ttcatctatc aggtgtttca 960 tcaattttag gttctatcaa ttttataacc actatcttca acatgcgtgg acctggaatg 1020 actatgcata gattacccct ttttgtgtgg tccgttctag tgacagcatt tctactttta 1080 ttatcacttc cggtactggc gggggcaatt acaatgttat taaccgatcg aaactttaat 1140 acaacctttt ttgatcctgc aggaggggga gaccccatat tataccagca tctctttcgg 1200 ttcttcggtc atccagaggt gtatattctc attctgcccg gattcggtat tattagtcat 1260 atcgtatcga ccttttcggg aaaaccggtc ttcgggtatc taggcatggt ttacgccatg 1320 atcagtatag gtgttcttgg atttcttgtt tgggctcatc atatgtttac tgtgggctta 1380 gacgttgata cgcgtgctta cttcaccgca gctaccatga tcatagctgt ccccactgga 1440 atcaaaatct ttagttggat cgctaccatg tggggaggtt cgatacaata caaaacaccc 1500 atgttatttg ctgtagggtt catctttttg ttcaccatag gcgggctcac aggaataatt 1560 ctagcgaatt ctgggctaga cattgctcta catgatactt attatgtggt ggcacatttc 1620 cattatgtac tttctatggg agcggttttt gctttatttg caggatttta ctattgggtg 1680 ggtaaaatct ttggtcggac atacccggaa actttaggtc aaatccattt ttggatcact 1740 tctttcgggg tgaatcttac cttctttccc atgcatttct tagggctttc gggtatgcca 1800 cgtcgcattc cagattatcc agatgcttac gccggatgga atgctctgag cagtttcggc 1860 tcttatatat ccgtagttgg gattcgttct ttcttcgtgg tcgtcacaat cactttaagc 1920 agtggaaaca acactaaatg tgctccaagc ccttgggctg ttgaagagaa gaattcaacc 1980 acacttgaat ggatggtaca aagtcctcca gcttttcata cttttggaga acttccagct 2040 atcaaagaga cgaaaagcta tgcgaatgta tcggggattg cacgagtccg cggacgggcc 2100 acttcgccca ccaggcgggg tttctccaca actccggacc cgctcgcctt gaaagagagc 2160 atgctctctt acaagaaaag tggaattcct cgttctgggg ctttctatgc ttgttctcaa 2220 gcagattccg caccttgttt atcgtcttgt tcgcttcctt tttactgctt ttgtggggac 2280 atttagtacc aatttggtac tacagcgagc ggctttccgt tgccatggat tttttgatgt 2340 cgaaaggaat tccttttgag ttggagttcg ggtgggacaa agtggtgatt cgcccagttc 2400 agtcggtggc aacttttctc aaagcagggg aagtacctcc tgttccacca gtgacgggta 2460 tagtaccacc cgtagaatcc cactgcccac tagagtgcca cttattcaca aatccatcct 2520 tgtctatgct gctcactctc ggtttggtcc tactgatgat tttttttttg ttacgaaaaa 2580 gggaggggga aagtcagtgc caaatgcttg gcaatccttg gtagagctta ttcatgattt 2640 cgtgccgaaa ggctttgctc taaccgaagc tattgcattg tttgccctaa tgatggcctt 2700 tctgatctca ttcgtcttcc aaagtgaacc agatgcttct ttaaaagaaa gcgctttgcc 2760 ttacctatga tg 2772 <210> 2 <211> 2767 <212> DNA <213> Allium cepa <400> 2 gagtcttcct ttagaaagaa cggaacgagt ttgtgattcg atcttctaaa gcgtaaagcg 60 aaatcagtag gacaagatcc aatctttcgt caacgcaggt tgcttattct cctacttaag 120 tatattaatt gggtagcgta gctggaattt ctttttggtc cctttcgtcc agtggttagg 180 acatcgtctt ttcatgtcga agacacgggt tcgattcccg taagggatag gtacttctcg 240 gtactccggg atatacacca gcgggtggac cggttggcgt tatgccactt ctccttcctc 300 atatggtggt tcgatggctc ttctctacta accacaaaga tatcggtact ctctatttca 360 tctttgctgc cattgctgga gtgatgggca catgcttctc cgtactgatt cgtatggaat 420 tagcccgacc cggcgatcaa attcttggcg ggaatcatca gctttataat gttttaataa 480 ctgctcacgc ttttttaatg atctttttta tggtgatgcc agcgatgata ggtggatttg 540 gtaattggtt tgttccgatt ctgatcggtg cacctgacat ggcatttcca cgattaaata 600 atatatcatt ctggttgttg ccaccaagtc tcttgctcct attaagctcc gccttagtag 660 aagtgggtag cggcactggg tggacggtct atccgccttt aagtggtatt accagccatt 720 ctggaggagc agttgattca gccattttta gtcttcatct atcaggtgtt tcatcaattt 780 taggttctat caattttata accactatct tcaacatgcg tggacctgga atgactatgc 840 atagattacc cctttttgtg tggtccgttc tagtgacagc atttctactt ttattatcac 900 ttccggtact ggcgggggca attacaatgt tattaaccga tcgaaacttt aatacaacct 960 tttttgatcc tgcaggaggg ggagacccca tattatacca gcatctcttt cggttcttcg 1020 gtcatccaga ggtgtatatt ctcattctgc ccggattcgg tattattagt catatcgtat 1080 cgaccttttc gggaaaaccg gtcttcgggt atctaggcat ggtttacgcc atgatcagta 1140 taggtgttct tggatttctt gtttgggctc atcatatgtt tactgtgggc ttagacgttg 1200 atacgcgtgc ttacttcacc gcagctacca tgatcatagc tgtccccact ggaatcaaaa 1260 tctttagttg gatcgctacc atgtggggag gttcgataca atacaaaaca cccatgttat 1320 ttgctgtagg gttcatcttt ttgttcacca taggcgggct cacaggaata attctagcga 1380 attctgggct agacattgct ctacatgata cttattatgt ggtggcacat ttccattatg 1440 tactttctat gggagcggtt tttgctttat ttgcaggatt ttactattgg gtgggtaaaa 1500 tctttggtcg gacatacccg gaaactttag gtcaaatcca tttttggatc acttctttcg 1560 gggtgaatct taccttcttt cccatgcatt tcttagggct ttcgggtatg ccacgtcgca 1620 ttccagatta tccagatgct tacgccggat ggaatgctct gagcagtttc ggctcttata 1680 tatccgtagt tgggattcgt tctttcttcg tggtcgtcac aatcacttta agcagtggaa 1740 acaacactaa atgtgctcca agcccttggg ctgttgaaga gaagaattca accacacttg 1800 aatggatggt acaaagtcct ccagcttttc atacttttgg agaacttcca gctatcaaag 1860 agacgaaaag ctatgtgaag taaaagaaga aaaggtcgcg ctactaagaa cctaacagaa 1920 gaaagatcag aatgaatggt tggatcaaac ggagaatgca taaataagca gcgatgacga 1980 tgaatgagaa tgacttacta tccaaaaaaa aaaagaatgg acagattttt ttagatatga 2040 ctccgccagt atattaattt cgtcgaggca attatccaaa tttttattcg ccttcggaac 2100 ggaaaaagaa cgacccccgg atcggatgta gcttacaggt gaaccgggta accaaagtcg 2160 cgaaagaatg aaagttcgct gggtcccaga agtgcaggtt cgagtccagc tcgtgacaag 2220 gcctttttgg tcacttggtc ttgcttgtta ttgaaatatt ccgttctcat cctccctgga 2280 accatggtct tgttgcttgg cttgctttca tgtcagtact tattctcttt ctctgaactt 2340 tcagacggaa gcttaagaca tgggattgtc tggaaagaag gggcacctta cgccttggct 2400 caggcgagcg aagtgaggaa agaaacaaat ttccttagac aacagcctgt atactgactc 2460 caggggggct cgcactattc cgggaagaga aacggacaag ggctgaacat gctgaatctt 2520 tcctttattg ccggctaggc tgctagacta gaagaatccc ctgcacatga aaaagagaaa 2580 gaggcggagt aacggctttg agggagaggc cgaaattagg atagccttca gggacatgat 2640 ttggctcgga cgtccgtgtt agagcaccag catgtactcc ttgagatagc attgtcttag 2700 gattggtact gaccaagtga tcagaggtgc ttgcgggtgc aacgtgctag acctaaaact 2760 tcaacag 2767 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Aense sequence <400> 3 cataggcggg ctcacaggaa ta 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Antisense sequence <400> 4 aatcctagtg tccggggttt ct 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Antisense sequense <400> 5 cagcgaactt tcattctttc gc 22 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Sense sequence <400> 6 ttatccagat gcttacgccg gatggaa 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Antisense sequence <400> 7 aggccatcat tagggcaaac aatgcaa 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Sense sequences <400> 8 tgggtgggta aaatctttgg tcggaca 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Sences sequence <400> 9 tgggtgggta aaatctttgg tcggaca 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Antisense sequence <400> 10 cactttgtcc cacccgaact ccaactc 27 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Antisense sequence <400> 11 ccgttccgaa ggcgaataaa aatttgg 27  

Claims (10)

A cytoplasmic male infertility plant specific DNA sequence consisting of the nucleotides set forth in SEQ ID NO: 1. The method of claim 1, The plant is a specific chimeric DNA sequence of the plant, characterized in that to give the cytoplasmic male sterility of onion. The method of claim 1, The DNA sequence is a specific chimeric DNA sequence of a cellular male infertility plant, characterized in that present in the mitochondrial genome. delete delete a) a forward primer capable of annealing a portion of the sequence that is commonly present between the novel orf725 gene described in SEQ ID NO: 1 and the normal coxI gene nucleotide described in SEQ ID NO: 2, and the orf725 gene specific nucleotide sequence and sequence described in SEQ ID NO: 1 Performing PCR using a reverse primer capable of annealing a portion of the coxI gene specific sequence as set forth in No. 2; And b) checking whether the DNA fragment is amplified; To include, the presence and type of the fragment amplified in step b) is a method for determining the type of male sterility and cytoplasm of the plant. The method of claim 6, Onion cytoplasmic type determination method, characterized in that the amplified DNA fragment is 300bp to 2000bp. The method of claim 6, The forward primer for PCR consists of a nucleotide sequence of SEQ ID NO: 3, the reverse primer is a cytoplasmic male sterile onion discrimination method, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 4 and SEQ ID NO: 5, respectively. a) a forward primer capable of annealing a portion of the orf725 gene nucleotides of SEQ ID NO: 1 and a portion of the nucleotides of SEQ ID NO: 2; b) a set of PCR primers for determining male sterility of a plant comprising a reverse primer capable of annealing a portion of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 of the plant DNA or plant mitochondrial DNA, and amplified in step b) PCR primer set having a DNA fragment size of 300bp to 2000bp. The method of claim 9, The forward primer is composed of the nucleotide sequence of SEQ ID NO: 3, the reverse primer is a PCR primer set, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 4 and SEQ ID NO: 5.

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