WO2008123714A1 - Procédé de production d'une semence hybride utilisant une plante de la nouvelle lignée raphanus sativus à stérilité mâle génique cytoplasmique et marqueurs d'adn utilisés pour sélectionner la plante de ladite lignée raphanus sativus - Google Patents

Procédé de production d'une semence hybride utilisant une plante de la nouvelle lignée raphanus sativus à stérilité mâle génique cytoplasmique et marqueurs d'adn utilisés pour sélectionner la plante de ladite lignée raphanus sativus Download PDF

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WO2008123714A1
WO2008123714A1 PCT/KR2008/001935 KR2008001935W WO2008123714A1 WO 2008123714 A1 WO2008123714 A1 WO 2008123714A1 KR 2008001935 W KR2008001935 W KR 2008001935W WO 2008123714 A1 WO2008123714 A1 WO 2008123714A1
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Prior art keywords
cgms
raphanus sativus
line
seq
sativus line
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PCT/KR2008/001935
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English (en)
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Young Pyo Lee
Soon Kee Sung
Sunggil Kim
Young Soon Ahn
Hyo Jeong Kim
Chae Wan Lim
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Dongbu Hitek Co., Ltd.
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Priority to CN200880016071.3A priority Critical patent/CN101679982B/zh
Priority to CA2720710A priority patent/CA2720710A1/fr
Priority to GB1111840.3A priority patent/GB2478682B/en
Priority to JP2010502028A priority patent/JP5089764B2/ja
Priority claimed from KR1020080031591A external-priority patent/KR100885075B1/ko
Publication of WO2008123714A1 publication Critical patent/WO2008123714A1/fr
Priority to GBGB1017160.1A priority patent/GB201017160D0/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A01H1/023—Male sterility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for producing a hybrid seed using a Cytoplasmic-Genic Male Sterility (CGMS) Raphanus sativus line plant and DNA markers for selecting the said Raphanus sativus line plant. More precisely, the present invention relates to a novel CGMS (D-CGMS) Raphanus sativus line plant, a method for producing a hybrid seed using the same, chloroplast DNA markers for selecting the D-CGMS Raphanus sativus line plant having the nucleotide sequence represented by SEQ. ID.
  • CGMS Cytoplasmic-Genic Male Sterility
  • SNP Single Nucleotide Polymorphism
  • Fi hybrid breeding has been continued by using heterosis.
  • Most of the vegetable seeds on market are Fi hybrid seeds.
  • the seed production method of Fi hybrids is exemplified by artificial crossing, self-incompatibility, dichogamy, male sterility and interspecific hybridization, etc.
  • these methods except the male sterility based method, not only require a long time and great effort but also have difficulty in maintaining seed purity and economic efficiency. Therefore, studies have been continued to develop male sterile resources and to use them for breeding.
  • Male Sterility indicates infertility caused by abnormality of male organs such as pollen, anther, and stamen, which is observed in various plants. This sterility is caused naturally or artificially by natural mutation, interspecific/intergeneric hybridization or mutagen treatment, etc. The induction mechanisms of male sterility are classified into two categories: one is genetic effects and the other is environmental effects. Male sterility used for breeding is largely induced by genetic causes. The genetically induced male sterility is divided into three types: Genie Male Sterility (GMS), Cytoplasmic Male Sterility (CMS) and Cytoplasmic-Genic Male Sterility (CGMS) .
  • GGS Genie Male Sterility
  • CMS Cytoplasmic Male Sterility
  • CGMS Cytoplasmic-Genic Male Sterility
  • CMS and CGMS are both attributed to the mal-functioning of mitochondria in cytoplasm, which are distinguished from each other by the presence or absence of a nuclear fertility restoration gene (Rundfeldt, J. , Z. convincedzuchtung, I960, 44: 30-62; Shivanna, K. R. and B. M. Johri . , Pollen sterility, in the angiosperm pollen, structure and function. 1985. Wiley Eastern Ltd. New Delhi) .
  • Genie Male Sterility is transmitted by recessive heredity, which means an object containing homozygous recessive gene becomes sterile. So, when Fi hybrid seeds are produced by GMS, only 50% of the obtained seeds are sterile. Therefore, to maintain and produce such male sterility, each object has to bloom and pollen generation has to be confirmed, requiring high costs and effort.
  • Cytoplasmic Male Sterility (CMS) and Cytoplasmic- Genic Male Sterility (CGMS) are characterized by maternal inheritance, and are mainly caused by mal-functioning of mitochondria in cytoplasm so that abnormal pollen is generated to lost fertility.
  • CMS does not contain a fertility restoration gene, the next generation is all sterile (100%) no matter what fertile gamete is used for crossing.
  • CMS has advantage of maintenance of such male sterility but also has disadvantages of limitation in commercial use because there is no restoration mechanism from male sterility in plants using seed products such as rape, and limitation in use to leaf and root vegetables only.
  • CGMS When CGMS is crossed with a male fertile gamete that does not contain a gene for restoration from male sterility, 100% of the progenies are male sterile.
  • CGMS is crossed with a fertile gamete containing a homozygous dominant gene for restoration from male sterility, 100% of the progenies are male fertile.
  • Fi hybrid seed production from those plants providing usable seeds is only- possible with CGMS line plants having fertility restoration mechanism but not with CMS line plants.
  • Raphanus sativus lines known so far are NWB-CMS (Raphanus sativus var NWB) , Ogura-CGMS (Raphanus sativus var Ogura) and Kosena-CGMS (Raphanus sativus cv Kosena) , etc.
  • NWB-CMS is known to be caused by a pure cytoplasmic factor whose fertility restoration gene source has not been disclosed (Korean Patent No. 0399333, Nahm et. al., Theor. Appl . Genet. Ill: 1191-1200, 2005).
  • Ogura-CGMS and Kosena-CGMS are Cytoplasmic-Genic Male sterility affected by not only an authentic cytoplasmic factor but also a nuclear factor.
  • Ogura-CGMS and Kosena-CGMS are known to be caused by the generation of new ORF in mitochondrial DNA in cytoplasm. These presumably have fertility restoration genes in their nuclei (Desloire S. et al., EMBO report 4: 588-594, 2003). Particularly, it has been reported that Ogura-CGMS and Kosena-CGMS have lots in common in the characteristics of their male sterility inducing genes and fertility restoration genes and thereby have similarity in their male sterility mechanisms (Koizuka, et. al., Theor. Appl. Genet. 100: 949-955, 2000; Koizuka et. al., Plant J.
  • Ogura-CGMS has been widely used for the production of Raphanus sativus Fi hybrid seeds owing to its comparatively stable male sterility. In particular owing to its male fertility restoration mechanism favors the production of Fi hybrid seeds by introducing Ogura-CGMS in rape. Because of this, Ogura-CGMS is the most preferred male-sterility source but has difficulty in securing lines for maintaining it.
  • This source can be effective in the production of Raphanus sativus Fi hybrid seeds and also can be effective in the induction of male sterility in plants like a rape providing seed products.
  • a morphological marker has been used for the selection of a target line, but this method is limited in number of useful morphological markers and affected by various environmental factors. So, studies have been actively undergoing on molecular breeding using a DNA marker involved in a target phenotype for the breeding. DNA markers are very useful because they make the selection in early generation possible, eliminate environmental factors and facilitate the quantitative phenotype analysis. So, DNA markers have been actively developed in many plants and they have contributed to the increase of selection efficiency as a novel breeding technique. Most of DNA markers for the diagnosis of male sterility are molecular markers based on mitochondrial DNA having large genetic variations.
  • mitochondrial genome has a very complicated structure comprising various types of subgenomic DNA, because of frequent recombinations and rearrangements using short repeat sequence (M. Bellaoui, et. al . , MoI. Gen. Genet. 257: 177-185, 1998; M. Arrieta-Montiel et. al., Genetics, 158(2), 851-864, 2001).
  • mitochondrial genome can be composed of different gene structures with different quantity, so it is necessary to make sure in the development of DNA markers whether or not the difference in quantity of genome can make male sterile or fertile plants.
  • Chloroplast DNA is known to have less genetic variations, so that there is no quantitative difference in gene specific structures. So, chloroplast DNA based molecular markers for male sterility facilitates more stable and accurate determination of male sterility (M. J. Havey, et. al., Appl Genet. 90: 263-268, 1995; T. Motegi, et. al., Euphytica 129: 319-323, 2003).
  • the usability of molecular markers in plant breeding has been well recognized but a novel molecular marker facilitating more economical, fast and massive screening of samples has still been targeted by those in the art. With the advancement of molecular marker screening methods, various types of molecular markers such as RFLP
  • Length Polymorphism have been developed. But, these markers are limited in sample analysis and spend high costs.
  • SNP markers can be efficiently used in breeding because it facilitates automatic screening and massive analysis owing to the recent advancement of fluorescent technology and capillary electrophoresis.
  • the present inventors collected resources for the development of novel male sterility sources and selected CGMS (D-CGMS) Raphanus sativus line plants having male sterility mechanism that is different from those of the conventional NWB-CMS and CGMS (Ogura and Kosena) , followed by disclosure of its characteristics.
  • the present inventors further confirmed that the D-CGMS provides higher male sterility induction rate than that of the conventional Ogura-CGMS and facilitates line maintenance.
  • the present inventors completed this invention by developing stable chloroplast DNA based molecular markers and mitochondrial DNA based SNP (Single Nucleotide Polymorphism) molecular markers for the selection of the D-CGMS Raphanus sativus line plant.
  • SNP Single Nucleotide Polymorphism
  • CGMS Cytoplasmic-Genic Male Sterility
  • the present invention provides a novel Cytoplasmic-Genic Male Sterility (D-CGMS) Raphanus sativus line plant having the nucleotide sequence represented by SEQ. ID. NO: 5 and D-CGMS specific SNP (Single Nucleotide Polymorphism) on the nucleotide located at the 171 st residue of the nucleotide sequence represented by SEQ. ID. NO: 12.
  • D-CGMS Cytoplasmic-Genic Male Sterility
  • SNP Single Nucleotide Polymorphism
  • the present invention further provides a method for producing CGMS Raphanus sativus line hybrid seeds having cytoplasmic-genic male sterility containing the step of crossing the D-CGMS Raphanus sativus line plant (as a crossing mother) with a male fertile plant to induce male sterility.
  • the present invention also provides DNA markers for the selection of D-CGMS Raphanus sativus line plants which have the nucleotide sequence supposed to be hybridized with the nucleotide sequence represented by SEQ. ID. NO: 5 or DNA containing the nucleotide sequence represented by SEQ. ID. NO: 5 under the strict conditions.
  • the present invention also provides SNP markers for the selection of D-CGMS Raphanus sativus line plants which comprise oligonucleotide selected from the nucleotide sequence represented by SEQ. ID. NO: 12 and composed of the nucleotide sequence containing 15-225 continuous nucleotides including the 171 st nucleotide of the above sequence or its complementary nucleotide sequence.
  • the present invention also provides a selection kit for D-CGMS Raphanus sativus line plants containing the primer set or probe capable of detecting the nucleotide sequence of the DNA marker or the oligonucleotide of the SNP marker.
  • the present invention also provides a method for the selection of D-CGMS Raphanus sativus line plants comprising the following steps:
  • step 1) extracting DNA samples from target plants; 2) amplifying the DNA samples of step 1) by PCR using the primer set for the amplification of DNA markers or SNP markers;
  • the present invention also provides a method for the selection of D-CGMS Raphanus sativus line plants comprising the following steps:
  • step 2) amplifying the DNA samples of step 1) by using the selection kit for D-CGMS Raphanus sativus line plants;
  • step 3 detecting the DNA markers or SNP markers by electrophoresis with the amplified samples of step 2).
  • the present invention provides a use of the DNA markers or SNP markers for the selection of D-CGMS Raphanus sativus line plants.
  • Raphanus sativus line indicates the Raphanus sativus line that has been separated and identified in this invention and confirmed to have a completely different novel genotype from that of the conventional CGMS.
  • Plant in this invention includes plant organs, plant tissues, plant cells, seeds and callus.
  • the present invention provides a novel Cytoplasmic- Genic Male Sterility (D-CGMS) Raphanus sativus line plant having the nucleotide sequence represented by SEQ. ID. NO: 5 and D-CGMS specific SNP (Single Nucleotide Polymorphism) on the nucleotide located at the 171 st residue of the nucleotide sequence represented by SEQ. ID. NO: 12.
  • D-CGMS Cytoplasmic- Genic Male Sterility
  • the present inventors tried to develop a novel Cytoplasmic-Genic Male Sterility Raphanus sativus line for the improvement of Fi hybrid seed production.
  • the present inventors first investigated floral organs and pollen development of Raphanus sativus lines distributed from National Horticultural Research Institute, Korea (20 kinds of Raphanus sativus lines collected from Uzbekistan, etc) .
  • male sterility exhibiting abnormal pollen development was detected in one of those lines, which was named "D-CGMS ⁇ Raphanus sativus var D-CGMS)".
  • the newly found CGMS (D-CGMS) Raphanus line had less pollen, compared with the normal fertile plant, and has been used for the production of hybrid seeds.
  • D-CGMS had sterile pollen but a few (see Figure 1) .
  • the morphology and vitality of pollen of D-CGMS were investigated by FDA (fluorescein diacetate) staining, followed by observation under SEM. As a result, the pollen of D-CGMS was produced less and shaped abnormally and had no vitality (see Figure 2). This phenotype was different from that of the conventional Ogura-CGMS, but it is expected that it can be efficiently used for attracting insects for insect mediated mass pollination because it has a floral organ similar to that of a normal plant.
  • the present inventors further performed cytohistological observation to investigate male sterility mechanism more precisely. As a result, it was confirmed that the pollen was normally developed in a normal fertile plant, while abnormality was observed from the tetrad phase, which was the early stage of pollen development, in the Ogura-CGMS Raphanus sativus line plant. In the D-CGMS Raphanus sativus line plant, the pollen was normally developed until the tetrad phase, but abnormality was observed in the microspore maturation phase, which resulted in male sterility (see Figure 3) .
  • the male sterility mechanism of the D-CGMS Raphanus sativus line plant is completely different from that of the conventional Ogura-CGMS. That is, there is no problem in female organs and only abnormal development of pollen results in male sterility, the shape of a floral organ is similar to that of a normal fertile plant but pollen has no vitality, so that it can be effectively used for attracting insects for Fi hybrid seed production via insect mediated mass pollination.
  • the present invention also provides a D-CGMS Raphanus sativus line callus (Accession No: KCTC11101BP) .
  • the present invention also provides a method for producing CGMS Raphanus sativus line hybrid seeds having cytoplasmic-genic male sterility containing the step of crossing the D-CGMS Raphanus sativus line plant (as a female parent) with a male fertile plant to induce male sterility.
  • Fi seeds with Cytoplasmic-Genic Male Sterility can be produced by crossing the D-CGMS Raphanus sativus line with a male fertile plant.
  • the D-CGMS Raphanus sativus line was crossed as female parent with 10 lines collected from Uzbekistan and Russia. As a result, some of Fi hybrid seeds produced by crossing with "DB112 line” and "DB117 line” were fertile, indicating that DB112 and DB117 lines contained fertility restoration genes. So, the novel D-CGMS Raphanus sativus line was confirmed to be the Cytoplasmic-Genic Male Sterility (CGMS) Raphanus sativus line containing a fertile restoration gene (see Table 3) .
  • CGMS Cytoplasmic-Genic Male Sterility
  • D-CGMS was confirmed to be the Cytoplasmic-Genic Male Sterility (CGMS) Raphanus sativus line containing a fertility restoration gene and exhibit higher male sterility induction rate during breeding, compared with the conventional Ogura-CGMS, which overcomes the limitation of the conventional method for breeding using the Ogura-CGMS in bringing male sterility.
  • Fi hybrid seeds can be produced by crossing the D-CGMS Raphanus sativus line plant with a normal fertile plant which is a target to be introduced with male sterility.
  • the method of the present invention can be applied in every male fertile line plant which does not contain a restoration gene from D-CGMS restoerers.
  • the said male fertile line is preferably SamYang (R012), SungKong (R015) , JungSangYeoreum (R020) , HaChu(R029), line separated from “JungSangYeoreum” (DB002), HannongYeoreum (DB003), line separated from “HannongYeoreum” (DBlIl), DeaBuRyeongYeoreum (DB084), line separated from “HaChu” (DB092) or resources collected from foreign countries such as DB019, DB421, DB037, DB067, DB121, DB261, DB321, DB401, DB501, and DB901, etc, but not always limited thereto.
  • the present inventors differentiated seeds of the D- CGMS Raphanus sativus line to induce callus and then deposited the induced callus of the D-CGMS Raphanus sativus line at Korean Collection for Type Cultures (KCTC) , Korean Research Institute of Bioscience and Biotechnology (KRIBB) on March 27, 2007 (Accession No: KCTC11101BP) .
  • KCTC Korean Collection for Type Cultures
  • KRIBB Korean Research Institute of Bioscience and Biotechnology
  • the present invention also provides DNA markers for the selection of D-CGMS Raphanus sativus line plants which have the nucleotide sequence supposed to be hybridized with the nucleotide sequence represented by SEQ. ID. NO: 5 or DNA containing the nucleotide sequence represented by SEQ. ID. NO: 5 under the strict conditions.
  • the D-CGMS Raphanus sativus line is a completely different male sterile line from the conventional Ogura-CGMS Raphanus sativus line and is effective in production of Fi hybrid seeds.
  • the present inventors developed DNA markers for the selection of D-CGMS Raphanus sativus line plants based on stable chloroplast DNA. Based on the information of chloroplast nucleotide sequence (GenBank Accession No: DQ231548) of Brassica rapa ssp.
  • PCR was performed with the forward primer represented by SEQ. ID. NO: 1 ('5-AGGGCGGTGCTCTGACCAATTGAACTA-S') and the reverse primer represented by SEQ. ID. NO: 2 ( ' 5- GAGCGGTTAATGGGGACGGACTGTAAA-S'), and the products thereby had difference in D-CGMS (see Figure 4) .
  • D-CGMS had the genotype in which "ATATATTGATATCTATA" sequence was repeated once again.
  • PCR was performed with the forward primer represented by SEQ. ID. NO: 3 ('5-GCGGGTAGCTTACATATTCCTTCTTATG-S') and the reverse primer represented by SEQ. ID.
  • DNA marker herein is preferably the nucleotide sequence possibly hybridized with DNA containing the nucleotide sequence represented by SEQ.
  • the strict conditions can be determined during the washing process after hybridization.
  • One of the strict conditions is as follows: washing at room temperature at ⁇ x SSC with 0.2% SDS for 15 minutes, washing with 6 ⁇ SSC, 0.2% SDS at room temperature for 15 minutes, washing with 2 ⁇ SSC, 0.2% SDS at 45°C for 30 minutes, and washing with 2* SSC, 0.2% SDS at 50 ° C for 30 minutes, and this washing process is repeated twice.
  • This kind of strict conditions can be established by those in the art.
  • the DNA markers of the present invention based on comparatively stable chloroplast DNA can be effectively used for the selection of D-CGMS Raphanus sativus line plants and development of a novel cultivated variety.
  • the present invention also provides SNP markers for the selection of D-CGMS Raphanus sativus line plants which comprise oligonucleotide selected from the nucleotide sequence represented by SEQ. ID. NO: 12 and composed of the nucleotide sequence containing 15-225 continuous nucleotides including the 171 st nucleotide of the above sequence or its complementary nucleotide sequence.
  • the 171 st residue of the nucleotide sequence represented by SEQ. ID. NO: 12 is preferably 1 bp of Guanine but not always limited thereto.
  • the present inventors developed D-CGMS Raphanus sativus line specific SNP markers facilitating economical, fast and massive screening of samples.
  • the primary forward primer represented by SEQ. ID. NO: 6 (5'- AATCAAATAGGGCTGGTGGCGCAGT-S') and the secondary forward primer represented by SEQ. ID. NO: 7 (5 ? - CGCAGTCCCCACTTGACCAATTTGA-3' ) were constructed with 5'- direction of the atp ⁇ ORF gene, followed by 3' -genome walking.
  • SEQ. ID. NO: 6 5'- AATCAAATAGGGCTGGTGGCGCAGT-S'
  • SEQ. ID. NO: 7 5 ? - CGCAGTCCCCACTTGACCAATTTGA-3'
  • nucleotide sequence represented by SEQ. ID. NO: 10 5'-TAACCGGCCTCAACCATGGTCTAGC-3'
  • SEQ. ID. NO: 8 5'-TAACCGGCCTCAACCATGGTCTAGC-3'
  • 5'-RACE Rapid Amplification of cDNA Ends
  • the sequence was compared with the normal atp ⁇ gene (GenBank accession number; M24671) of Raphanus sativus.
  • the 5'- region of the novel Atp ⁇ -containing chimeric gene of the D-CGMS plant was identical with the nucleotide sequence of the normal apt ⁇ gene, 1 bp nucleotide was inserted in ORF and 3' -region was transformed by recombination.
  • 1 bp nucleotide and recombination the formation of the novel Atp ⁇ -containing chimeric ORF was confirmed and this gene was named "orf225" and represented by SEQ. ID.
  • orf225 gene is composed of 225 nucleotides and encodes 75 protein codons.
  • the region from the first residue to the 603 rd residue on the nucleotide sequence represented by SEQ. ID. NO: 14 was identical with the normal atp ⁇ gene Originating from Raphanus sativus. But, SNP in which the 519 th residue (the 171 st residue on the nucleotide sequence represented by SEQ. ID. NO: 12) was added with lbp of Guanine was confirmed.
  • the recombination on the 604 th residue made this gene completely different from the normal atp ⁇ gene (see Figure 10) .
  • the present inventors constructed the forward primer represented by SEQ. ID. NO: 15 (5'- ATACCTCGGGGAAGAAGCGGGGT-S' ) and the reverse primer represented by SEQ. ID. NO: 16 (5 1 - TAGCCATTTGGTGTGACCTCTGACCG-S 1 ) , followed by PCR and nucleotide sequencing.
  • SEQ. ID. NO: 15 5'- ATACCTCGGGGAAGAAGCGGGGT-S'
  • the reverse primer represented by SEQ. ID. NO: 16 5 1 - TAGCCATTTGGTGTGACCTCTGACCG-S 1
  • the PCR products of the lines were not different on agarose gel.
  • nucleotide sequences of the normal fertile lines (DBRMFl, DBRMF2 ) , Ogura-CGMS and NWB-CMS Raphanus satlvus lines were not different. But, SNP (Single Nucleotide Polymorphism) with the insertion of 1 bp nucleotide was confirmed in the D-CGMS Raphanus sativus line (see Figures 11 and 12).
  • the D-CGMS Raphanus sativus exhibited changed characteristics of protein codon because of SNP (Single Nucleotide Polymorphism) generated by the insertion of lbp Guanine in the normal apt ⁇ gene ORF.
  • SNP Single Nucleotide Polymorphism
  • the changes of C-terminal end region of the normal fertile lines (DBRMFl, DBRFM2), Ogura-CGMS, NWB-CMS and D-CGMS lines were also observed (see Figure 14) .
  • the present inventors performed following experiments using three of each normal fertile lines (DBRMFl, DBRMF2), Ogura-CGMS, NWB-CMS and D-CGMS lines.
  • the normal fertile lines (DBRMFl, DBRMF2), Ogura-CGMS, NWB-CMS, and D- CGMS lines all produced PCR products and the D-CGMS specific SNP was confirmed by nucleotide sequencing with the same (see Figure 15) .
  • the normal fertile lines (DBRMFl, DBRMF2), Ogura-CGMS and NWB-CMS lines had the sequence "GGG” and the D-CGMS Raphanus sativus line had the sequence "GGGG”, indicating that lbp of Guanine was additionally inserted, resulting in SNP.
  • orf225 gene was generated in the normal fertile lines (DBRMFl, DBRMF2), Ogura-CGMS and NWB-CMS lines, and orf267 gene was generated in the D-CGMS because of the SNP.
  • the normal fertile lines (DBRMFl, DBRMF2 ) , Ogura-CGMS and NWB-CMS lines did not contain D-CGMS specific SNP, suggesting that the D-CGMS specific SNP located at the 171 st residue of the nucleotide sequence represented by SEQ. ID. NO: 12 can be effectively used as a molecular marker for the selection of the D-CGMS Raphanus sativus line plants (see Figure 16) .
  • the present invention also provides a selection kit for D-CGMS Raphanus sativus line plants.
  • the selection kit herein contains the primer set or probe and PCR reaction mixture capable of detecting the nucleotide sequence of the DNA marker or the oligonucleotide of the SNP marker.
  • Raphanus sativus line specific DNA markers can amplify specifically the DNA marker represented by SEQ. ID. NO: 5, which can be any primer designed by those in the art, but the primer set composed of sequences represented by SEQ. ID.
  • the primer set for the amplification of the D-CGMS SNP markers can amplify the SNP markers specific to D-CGMS, which can be any primer designed by those in the art, but the primer set composed of sequences represented by SEQ. ID. NO: 15 and NO: 16 is preferred.
  • the PCR reaction mixture herein can contain general materials for PCR, for example, Tag polymerase, reaction buffer, dNTP, MgCl 2 , BSA and distilled water, and can additionally include agarose for the detection of PCR product and electrophoresis buffer.
  • general materials for PCR for example, Tag polymerase, reaction buffer, dNTP, MgCl 2 , BSA and distilled water, and can additionally include agarose for the detection of PCR product and electrophoresis buffer.
  • the present invention also provides a method for the selection of D-CGMS Raphanus sativus line plants comprising the following steps:
  • step 2) amplifying the DNA samples of step 1) by PCR using the primer set for the amplification of DNA markers or SNP markers; and 3) detecting the D-CGMS specific DNA markers or SNP markers by electrophoresis with the amplified products of step 2) .
  • the present invention also provides a method for the selection of D-CGMS Raphanus sativus line plants comprising the following steps:
  • step 2) amplifying the DNA samples of step 1) by using the selection kit for D-CGMS Raphanus sativus line plants; and 3) detecting the D-CGMS specific DNA markers or SNP markers by electrophoresis with the amplified products of step 2) .
  • the present invention provides a use of the D-CGMS specific DNA markers or SNP markers for the selection of D-CGMS Raphanus sativus line plants.
  • the D-CGMS Raphanus sativus line plants of the invention have high Cytoplasmic-Genic Male Sterility induction rate in Fi plants, suggesting that the D-CGMS can be effectively used for the production of CGMS line hybrid seeds.
  • the D-CGMS Raphanus sativus line plants can also be selected by using the D-CGMS specific SNP (Single Nucleotide Polymorphism) located at the 171 st nucleotide of the nucleotide sequence represented by SEQ. ID. NO: 12 using the primers represented by SEQ. ID. NO: 15 and NO: 16.
  • the above primers can also be effectively used for the preparation of the selection kit for D-CMGS Raphanus sativus line plants.
  • Figure 1 is a set of photographs illustrating the morphology of floral organs and stamens of the normal fertile line, Ogura-CGMS Raphanus sativus line and novel CGMS (D-CGMS) Raphanus sativus line plants:
  • A Floral organ of the normal fertile line
  • B Floral organ of the Ogura-CGMS Raphanus sativus line
  • C Floral organ of the D-CGMS Raphanus sativus line
  • D Stamen of the normal fertile line
  • E Stamen of the Ogura-CGMS Raphanus sativus line
  • F Stamen of the D-CGMS Raphanus sativus line.
  • FIG. 2 is a set of photographs illustrating the pollens of the normal fertile line and D-CGMS Raphanus sativus line plants:
  • Figure 3 is a set of photographs illustrating the cytohistological observation on the normal fertile line, Ogura-CGMS Raphanus sativus line and D-CGMS Raphanus sativus line plants:
  • A, D, G, J and M photographs showing the cytohistological observation on each pollen development stage of the normal fertile line
  • B, E, H, K and N photographs showing the cytohistological observation on each pollen development stage of the Ogura-CGMS Raphanus sativus line
  • C, F, I, L and 0 photographs showing the cytohistological observation on each pollen development stage of the D-CGMS Raphanus sativus line.
  • Figure 4 is a set of photographs illustrating the nucleotide sequences of PCR products amplified by using the primers represented by SEQ. ID. NO: 1 and NO: 2.
  • Figure 5 is a set of photographs showing the PCR products on agarose gel amplified by using the primers represented by SEQ. ID. NO: 3 and NO: 4.
  • Figure 6 is a set of photographs illustrating the results of PCR with the D-CGMS Raphanus sativus line and 30 other lines on market using the D-CGMS specific DNA markers:
  • Figure 8 is a set of photographs illustrating the comparison of the nucleotide sequences obtained from 5'-
  • RACE Rapid Amplification of cDNA Ends
  • Raphanus sativus
  • ORF Open Reading Frame
  • Figure 9 is a set of photographs illustrating the expression of the novel Atp ⁇ -containing chimeric gene with the normal fertile line and D-CGMS Raphanus sativus line plant cDNA.
  • Figure 10 is a set of photographs illustrating the nucleotide sequence of the novel Atp ⁇ -containing chimeric gene obtained from the D-CGMS Raphanus sativus line plant by 3' -genome walking and 5'-RACE PCR: Arrows (102-124 and 781-806) : forward primer and reverse primer respectively represented by SEQ. ID. NO: 15 and NO: 16 for the development of the D-CGMS specific SNP markers .
  • Figure 11 is a photograph illustrating the comparison of the PCR products on agarose gel amplified by using the primers represented by SEQ. ID. NO: 15 and NO: 16.
  • Figure 12 is a photograph illustrating the comparison of the nucleotide sequences of the PCR products amplified by using the primers represented by SEQ. ID. NO: 15 and NO: 16.
  • Figure 13 illustrates the structures of the normal atp ⁇ gene of the conventional Raphanus sativus line, the novel Atp ⁇ -containing chimeric ORF(orf267 gene) of the normal fertile lines (DBRMFl, DBRM2), Ogura-CGMS and NWB- CMS, and the D-CGMS Raphanus sativus line specific novel Atp ⁇ -containing chimeric ORF(orf225 gene).
  • Figure 14 is a photograph illustrating the difference among the normal lines (DBRMFl, DBRMF2), Ogura-CGMS and
  • FIG. 15 is a photograph illustrating each PCR product of the normal fertile lines (DBRMFl, DBRMF2 ) , Ogura-CGMS, NWB-CMS and D-CGMS Raphanus sativus line amplified by using the primers represented by SEQ. ID. NO: 15 and NO: 16, compared on agarose gel (three plants per each line) .
  • Figure 16 is a set of photographs illustrating the chromatogram peak of each PCR product of the normal fertile lines (DBRMFl, DBRMF2), Ogura-CGMS, NWB-CMS, and D-CGMS Raphanus sativus line plants amplified by using the primers represented by SEQ. ID. NO: 15 and NO: 16, confirmed by nucleotide sequencing: A: DBRMFl line;
  • B DBRMF2 line
  • C Ogura-CGMS line
  • D NWB-CMS line
  • E D-CGMS line
  • Example 1 Development of a novel D-CGMS Raphanus sativus line and investigation of characteristics of the same ⁇ !-!> Selection of a novel D-CGMS
  • the present inventors collected novel male sterility resources which would be expected to improve the productivity of Fi hybrid seeds. Then, the present inventors investigated floral organ tissues and pollen development of those 20 kinds of Raphanus sativus line plants collected from Uzbekistan, etc, distributed from National Horticultural Research Institute, Korea As a result, a line exhibiting male sterility (Cytoplasmic-Genic Male Sterility; CGMS) by the inhibition of pollen development was selected and named "D-CGMS (Raphanus sativus var D-CGMS)".
  • CGMS Chroplasmic-Genic Male Sterility
  • Ogura-CGMS Ogura Raphanus sativus line
  • Example ⁇ 1-1> the morphology and viability of pollen of the novel male sterile line were observed thoroughly under electron microscope (Scanning Electron Microscope; SEM) and by FDA staining (fluorescein diacetate) . Pollens were taken from the normal fertile line and D-CGMS Raphanus sativus line in full bloom, followed by FDA staining and observation under electron microscope. But, since there was no pollen in the Ogura- CGMS, this line was excluded from the observation. To investigate the viability of pollen, the pollens taken from flowers of the normal fertile line and D-CGMS Raphanus sativus line were stained with 0.002% FDA (fluorescein diacetate) solution, followed by observation under fluorescent microscope.
  • the present inventors performed cytohistological observation on the D-CGMS to investigate the male sterility mechanism thereof more precisely.
  • anthers of the normal fertile line, Ogura-CGMS and D-CGMS Raphanus sativus line plants were obtained at the same development stage, which was fixed in a fixative (Pipes 50 raM, 4% p- formaldehyde) at 4 ° C for 24 hours.
  • dehydration was performed at room temperature in 25%, 50%, 75% and 100% alcohol stepwise for 2 hours each, followed by dehydration in 100% alcohol once again for 24 hours.
  • Xylene was treated thereto at different concentrations of 25%, 50%, 75% and 100% at room temperature for 2 hours each, followed by treatment with 100% xylene twice for 2 hours each.
  • the samples were treated with liquid paraffin for 2 hours three times and embedded in paraffin.
  • paraffin block thin sections (10 ⁇ m in thickness) were prepared by using rotary microtome, which were adhered on the slide glass coated with albumin. The slide was dried on a slide warmer (Fisher Scientific, USA) at 40 ⁇ 3 ° C. Paraffin on the dried slide was eliminated by using xylene and the slide was dehydrated using alcohol, followed by staining with 0.25% toluidine blue and 0.05% aniline blue.
  • the male fertile plant selected from the group consisting of SamYang (R012), SungKong (R015) , JungSangYeoreum (R020) and HaChu (R029) was used as a male parents for crossing.
  • the male fertile plants were selected from the group consisting of DeaHyeongChooSeok (R049), BeakBong (R106) and resources collected from foreign countries and retained by the present inventors such as "R122", "R126” and "R127”.
  • the D-CGMS Raphanus line plant selected in Example 1 was used as a female parent for crossing with the male fertile line plant to produce CGMS Raphanus sativus line Fi hybrid seeds having Cytoplasmic-Genic Male Sterility.
  • To compare the CGMS induction rate of the D-CGMS Raphanus sativus line with that of the Ogura-CGMS Raphanus sativus line the male fertile plant used for crossing with the D- CGMS Raphanus sativus line was crossed with the Ogura- CGMS as a female parent. Pollen development and viability were investigated to confirm if male sterility was introduced in the produced Fi hybrid seeds.
  • the male fertile line plant was selected from the group consisting of line separated from “JungSangYeoreum” (DB002), HannongYeoreum (DB003) , line separated from “HannongYeoreum” (DBlIl), SamYang (DB021), DeaBuRyeongYeoreum (DB084), line separated from “HaChu” (DB092), and those male fertile line plants collected by the present inventors such as “DB019” and "DB421”.
  • DB019 DB421
  • the Fi hybrid seeds produced from 6 combinations among the cross-combinations of the 8 different male fertile lines with the Ogura-CGMS were 100% male fertile progenies, and in one of the two remaining cross-combinations (R106 x DB421), male fertile progenies and male sterile progenies were separated approximately by the ratio of 1:1.
  • the produced Fi hybrid seeds were 100% male sterile progenies.
  • the cross-combinations of the D-CGMS Raphanus line plant with the 8 different male fertile lines produced 100% male sterile Fi hybrid seeds (Table 2) .
  • the method for producing Fi hybrid seeds using Cytoplasmic-Genic Male Sterility from the D-CGMS Raphanus sativus line was confirmed to have higher male sterility induction efficiency than the conventional Ogura-CGMS Raphanus sativus line, and thus the D-CGMS Raphanus sativus line plant can be effectively used as a female parent for the production of Fi hybrid seeds.
  • Example 3 Development of male fertility restoration gene resources of D-CGMS Raphanus sativus line To screen male fertility restored lines of the D-CGMS
  • Raphanus sativus line selected in Example 1 10 male fertile lines collected from Uzbekistan and Russia were crossed with the D-CGMS Raphanus sativus line plant as a female parent. Those 10 male fertile lines used as paternal lines were collected from foreign countries
  • the D-CGMS Raphanus sativus line seeds were dipped in 70% (v/v) ethanol for 1 minute, followed by washing with sterilized water twice.
  • the seeds were dipped in 2% sodium hypochloride (NaOCl) solution for 15 minutes, followed by washing with sterilized water three times.
  • the sterilized seeds were distributed on germinating medium (1/2 MS salts, 3% sucrose, 0.8% agar) and germinated.
  • germinating medium 1/2 MS salts, 3% sucrose, 0.8% agar
  • the growing hypocotyl was cut at 5 mm intervals, which was differentiated on MS medium (containing BAP 4 mg/liter and NAA 2 mg/liter), leading to the induction of callus.
  • the callus was sub-cultured on MS medium (containing BAP 1 mg/liter and IBA 0.5 mg/liter) at 4 weeks intervals.
  • the callus growing stably thereon was selected (callus of D- CGMS Raphanus sativus line) and deposited at Korean Collection for Type Cultures (KCTC) , Korean Research Institute of Bioscience and Biotechnology (KRIBB) on March 27, 2007 (Accession No: KCTC11101BP) .
  • Examples 1 - 3 the present inventors confirmed that the D-CGMS Raphanus sativus line was completely different male sterile line from the conventional Ogura- CGMS Raphanus sativus line and Fi hybrid seeds could be efficiently produced by using the D-CGMS Raphanus sativus line.
  • PCR and nucleotide sequencing with chloroplasts of the normal fertile line, Ogura-CGMS and D- CGMS were performed, and as a result, differences in chloroplast nucleotide sequences were confirmed.
  • PCR was performed with 50 ng of the extracted DNA as a template using a kit (Advantage 2 PCR kit, Clontech, USA) as follows: predenaturation at 94°C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 65 ° C for 30 seconds, polymerization at 72 ° C for 2 minutes, 40 cycles from denaturation to polymerization, and final extension at 72 ° C for 10 minutes .
  • nucleotide sequence of the PCR product was analyzed. As a result, the difference among chloroplast nucleotide sequences of the normal fertile line, Ogura-CGMS line and D-CGMS line was observed.
  • the nucleotide sequencing was performed by Genotech Co., Ltd.
  • Nucleotide sequences of the chloroplast DNA based PCR products were analyzed. As a result, it was confirmed that the normal fertile line is divided into two different lines having different chloroplast nucleotide sequences, which were named respectively "DBRMFl" and "DBRMF2".
  • the PCR product generated by using the forward primer represented by SEQ. ID. NO: 1 and the reverse primer represented by SEQ. ID. NO: 2 exhibited different characteristics in D-CGMS.
  • the nucleotide sequence of this PCR product was analyzed and as a result, it was confirmed that 17 bp nucleotide sequence (ATATATTGATATCTATA) was inserted only in D-CGMS ( Figure 4) .
  • PCR was performed with the forward primer represented by SEQ. ID. NO: 3 and the reverse primer represented by SEQ. ID. NO: 4.
  • PCR was performed with 50 ng of the DNA extracted from the normal fertile line, Ogura-CGMS line and D-CGMS line by the same manner as described in Example ⁇ 5-l> as a template using a kit (Advantage 2 PCR kit, Clontech, USA) as follows: predenaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 25 seconds, annealing at 68 ° C for 25 seconds, polymerization at 72 ° C for 25 seconds, 40 cycles from denaturation to polymerization, and final extension at 72 ° C for 1 minute. Upon completion of the reaction, the size of each PCR product was investigated on agarose gel.
  • Example ⁇ 5-2> For the novel D-CGMS specific selection, 30 kinds of the conventional varieties on market were used for PCR using the forward primer represented by SEQ. ID. NO: 3 and the reverse primer represented by SEQ. ID. NO: 4 by the same manner as described in Example ⁇ 5-2>.
  • the DNA markers of the present invention can be effective in the selection of the novel D- CGMS Raphaus sativus line plants ( Figure 6) .
  • Nucleotide sequence of the PCR product (156 bp) was analyzed. As a result, the sequence was identified as the sequence represented by SEQ. ID. NO: 5.
  • Example 6 Confirmation of D-CGMS specific Atp6-containing chimeric ORF and development of mitochondrial DNA based SNP markers for the selection of D-CGMS ⁇ 6-l> Confirmation of Atp ⁇ -containing chimeric QRF by genome walking in D-CGMS
  • Atp ⁇ gene is the gene found in mitochondrial genome, which is the gene encoding ATPase subunit ⁇ protein involved in ATP synthesis.
  • the male fertile line and male sterile line have different atp ⁇ gene structures (Makaroff CA et . al . , J. Biol. Chem. 264: 11706-11713, 1989; Kim et. al . , Theor. Appl . Genet. 115: 1137-1145, 2007) .
  • male sterile line had unique atp ⁇ -containing gene organization with partial deletion of 3' -region.
  • hot pepper male sterility specific SCAR Sequence-Characterized Amplified Region
  • the primary forward primer represented by SEQ. ID. NO: 6 and the secondary forward primer represented by SEQ. ID. NO: 7 for genome walking from 5' to 3' of atp ⁇ ORF were constructed based on the nucleotide sequence (GenBank accession number; M24671) of the conventional Raphanus sativus atp ⁇ gene.
  • 0.1 g of leaves of the D-CGMS Raphanus sativus line was completely crushed by using liquid nitrogen, from which DNA was extracted using a kit (DNeasy plant kit, Qiagen, USA) .
  • Genome walking library was constructed with 3 ug of the extracted DNA using a genome walking library kit
  • PCR was performed with the 1 ul of the genome walking library as a template using the primer represented by SEQ.
  • ID. NO: 6 as follows: predenaturation at 94 "C for 3 minutes; at 94 ° C for 25 seconds; at 72 ° C for 3 minutes (7 cycles) and then at 94 "C for 25 seconds; at 67 °C for 3 minutes (32 cycles); and final extension at 67 ° C for 7 minutes.
  • the reactant was 50 times diluted.
  • the second PCR (nested PCR) was performed with the 1 ul of the diluent as a template using the primer represented by SEQ. ID.
  • NO: 7 as follows: predenaturation at 94 ° C for 3 minutes; at 94 ° C for 25 seconds; at 72 ° Cfor 3 minutes (5 cycles) and then at 94 ° C for 25 seconds; at 67 ° C for 3 minutes (20 cycles); and final extension at 67 ° Cfor 7 minutes.
  • RNA used for the construction of RACE cDNA was prepared as follows: 0.1 g of flowers obtained from the D-CGMS was completely crushed by using liquid nitrogen, from which total RNA was extracted using a Tri Reagent RNA Isolation Kit (Sigma, USA) .
  • mRNA was isolated from the total RNA extracted above by using PolyATtract mRNA Isolation System IV (Promega, USA) . 1 ug of the isolated mRNA was used for the construction of RACE cDNA using a RACE cDNA amplification kit (SMARTTM RACE cDNA Amplification Kit, Clontech, USA) . Based on the nucleotide sequence represented by SEQ. ID. NO: 8, the reverse primers specific to the novel atp ⁇ gene represented by SEQ. ID. NO: 9 and NO: 10 were constructed. The first 5'-RACE PCR was performed with 1 ul of the RACE cDNA as a template using the reverse primer represented by SEQ. ID.
  • nucleotide sequence shown in Figure 10 was obtained and represented by SEQ. ID. NO: 14.
  • the nucleotide sequence represented by SEQ. ID. NO: 14 was composed of 1540 bp nucleotides and as shown in Figure 10 orf225 gene was composed of 225 bp nucleotides ranging from the 349 th nucleotide to the 573 rd nucleotide and encodes 75 protein codons ( Figure 10).
  • NO: 16 as follows: predenaturation at 94 ° C for 5 minutes, denaturation at 94 ° Cfor 30 seconds, annealing at 66 ° Cfor 30 seconds, polymerization at 72 ° C for 1 minute, 45 cycles from denaturation to polymerization, and final extension at 72 ° Cfor 5 minutes.
  • the 171 st nucleotide of ORF of the normal atp ⁇ gene had the additional lbp Guanine, generating SNP (Single Nucleotide Polymorphism) and recombination occurred at the 255 th nucleotide, resulting in the generation of the novel OFR in 225 bp size (orf225) ( Figure 13) .
  • SNP Single Nucleotide Polymorphism
  • protein codon was changed by SNP (Single Nucleotide Polymorphism) generated by the insertion of lbp Guanine at the 171 st nucleotide of ORF of the normal atp ⁇ gene.
  • DRMFl normal fertile lines
  • DBRMF2 normal fertile lines
  • D-CGMS-2 and D-CGMS-3 were progenies generated by crossing the D-CGMS Raphanus sativus line as a female parent with the normal fertile line. It was also investigated whether or not the D-CGMS specific
  • SNP was transmitted to next generations.
  • 0.1 g of leaves obtained from 15 plants of the normal fertile lines (DBRMFl, DBRMF2), Ogura-CGMS, NWB-CMS and D-CGMS line plants were completely crushed by using liquid nitrogen, from which DNA was extracted using a kit (DNeasy plant kit, Qiagen, USA) .
  • PCR was performed using 1 ul of the extracted DNA as a template using the forward primer represented by SEQ. ID. NO: 15 and the reverse primer represented by SEQ. ID. NO: 16 by the same conditions as described in Example ⁇ 6-3>.
  • the D-CGMS was used as a female parent for crossing with the normal fertile line, and as a result, D-CGMS-2 and D-CGMS-3 were generated. In those progenies, the D-CGMS specific SNP was confirmed, suggesting that SNP was maternally transmitted.
  • the D-CGMS specific SNP confirmed above was not found in the normal fertile lines (DBRMFl, DBRMF2), Ogura-CGMS and NWB-CMS Raphanus sativus lines. Therefore, the D-CGMS specific SNP can be effectively used as a molecular marker for the selection of D-CGMS Raphanus sativus plants.

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Abstract

La présente invention concerne un procédé de production de semences hybrides utilisant une plante de la nouvelle lignée Raphanus sativus à stérilité mâle génétique cytoplasmique (CGMS) et des marqueurs d'ADN utilisés pour sélectionner ladite plante, plus précisément une plante de la nouvelle lignée Raphanus sativus CGMS (D-CGMS). Cette invention concerne également un procédé de production de semences hybrides utilisant cette plante, des marqueurs à base d'ADN chloroplastique utilisés pour sélectionner la plante de la lignée Raphanus sativus D-CGMS comprenant la séquence nucléotidique représentée par SEQ.ID.NO:5, des marqueurs SNP (polymorphisme simple nucléotide) à base d'ADN mitochondrial utilisés pour sélectionner la plante de la lignée Raphanus sativus D-CGMS et situés sur le 171ième nucléotide de la séquence nucléotidique représentée par SEQ.ID.NO:12. La plante de la lignée Raphanus sativus D-CGMS de cette invention peut être utilisée efficacement pour produire des semences hybrides du fait de sa grande stabilité de stérilité mâle. Les marqueurs à base d'ADN chloroplastique et les marqueurs SNP à base d'ADN mitochondrial utilisés pour sélectionner les plantes de la lignée Raphanus sativus D-CGMS peuvent s'avérer très efficaces dans la sélection de plantes de la lignée Raphanus sativus D-CGMS au cours du processus de reproduction.
PCT/KR2008/001935 2007-04-06 2008-04-04 Procédé de production d'une semence hybride utilisant une plante de la nouvelle lignée raphanus sativus à stérilité mâle génique cytoplasmique et marqueurs d'adn utilisés pour sélectionner la plante de ladite lignée raphanus sativus WO2008123714A1 (fr)

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CN200880016071.3A CN101679982B (zh) 2007-04-06 2008-04-04 利用新型核质互作雄性不育型萝卜系植物以及选育该植物的dna标记生产杂交种子的方法
CA2720710A CA2720710A1 (fr) 2007-04-06 2008-04-04 Procede de production d'une semence hybride utilisant une plante de la nouvelle lignee raphanus sativus a sterilite male genique cytoplasmique et marqueurs d'adn utilises pour selectionner la plante de ladite lignee raphanus sativus
GB1111840.3A GB2478682B (en) 2007-04-06 2008-04-04 A method for producing a hybrid seed using plant of novel cytoplasmic-genic male sterility raphanus sativus line and dna markers for selecting the plant...
JP2010502028A JP5089764B2 (ja) 2007-04-06 2008-04-04 新規な細胞質−遺伝子的雄性不稔（ｃｇｍｓ）大根系統植物体を使用した雑種種子生産方法及び前記大根系統植物体選抜用ｄｎａ標識因子
GBGB1017160.1A GB201017160D0 (en) 2007-04-06 2010-10-05 A method for producing a hybrid seed using plant of novel cytoplasmic-genic male sterility raphanus sativus line and DNA markers for selecting the plant

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CN104004853A (zh) * 2014-06-18 2014-08-27 吉林省农业科学院 叶绿体dna的snp标记鉴定大豆雄性不育细胞质方法
CN106521004A (zh) * 2016-12-26 2017-03-22 山东省农作物种质资源中心 与萝卜核雄性不育基因连锁的Indel标记及其应用
CN107828909A (zh) * 2017-11-15 2018-03-23 云南省农业科学院花卉研究所 一种分子标记检测东方百合特定群体雄性不育的方法
CN111349716A (zh) * 2020-04-24 2020-06-30 云南省农业科学院园艺作物研究所 一种鉴定云萝卜2号杂交种子纯度的ssr引物及方法
CN111349716B (zh) * 2020-04-24 2022-08-19 云南省农业科学院园艺作物研究所 一种鉴定云萝卜2号杂交种子纯度的ssr引物及方法
WO2024050761A1 (fr) * 2022-09-08 2024-03-14 中国农业科学院蔬菜花卉研究所 Marqueur de pcr pour détecter un fragment de radis exogène dans une lignée d'introgression de brassica oleracea-radis, amorce et utilisation de celui-ci

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