WO2007097574A1 - Molecular marker associated with tmv resistance and use thereof - Google Patents
Molecular marker associated with tmv resistance and use thereof Download PDFInfo
- Publication number
- WO2007097574A1 WO2007097574A1 PCT/KR2007/000920 KR2007000920W WO2007097574A1 WO 2007097574 A1 WO2007097574 A1 WO 2007097574A1 KR 2007000920 W KR2007000920 W KR 2007000920W WO 2007097574 A1 WO2007097574 A1 WO 2007097574A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tmv
- seq
- resistant
- plant
- primer
- Prior art date
Links
- 239000003147 molecular marker Substances 0.000 title abstract description 30
- 241000196324 Embryophyta Species 0.000 claims abstract description 112
- 241000723873 Tobacco mosaic virus Species 0.000 claims abstract description 94
- 239000002773 nucleotide Substances 0.000 claims abstract description 40
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 34
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 28
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 28
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 28
- 108020004414 DNA Proteins 0.000 claims description 61
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 23
- 238000004458 analytical method Methods 0.000 claims description 18
- 240000004160 Capsicum annuum Species 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 claims description 10
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 claims description 9
- 235000007862 Capsicum baccatum Nutrition 0.000 claims description 9
- 108091035242 Sequence-tagged site Proteins 0.000 claims description 9
- 239000001728 capsicum frutescens Substances 0.000 claims description 9
- 108091060211 Expressed sequence tag Proteins 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 7
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 claims description 5
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 claims description 5
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 claims description 5
- 244000241235 Citrullus lanatus Species 0.000 claims description 5
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 5
- 241000219112 Cucumis Species 0.000 claims description 5
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 claims description 5
- 240000008067 Cucumis sativus Species 0.000 claims description 5
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 5
- 244000299507 Gossypium hirsutum Species 0.000 claims description 5
- 244000061176 Nicotiana tabacum Species 0.000 claims description 5
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 5
- 240000007377 Petunia x hybrida Species 0.000 claims description 5
- 241000220317 Rosa Species 0.000 claims description 5
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 230000004544 DNA amplification Effects 0.000 claims description 4
- 108091092878 Microsatellite Proteins 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 description 15
- 238000011161 development Methods 0.000 description 12
- 230000018109 developmental process Effects 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000003550 marker Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 241000700605 Viruses Species 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000723824 Pepper mild mottle virus Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 125000005842 heteroatom Chemical group 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 208000035240 Disease Resistance Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 238000010222 PCR analysis Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241001124076 Aphididae Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 241000208293 Capsicum Species 0.000 description 1
- 241001544457 Capsicum chacoense Species 0.000 description 1
- 235000003989 Capsicum chacoense Nutrition 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 108700003861 Dominant Genes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000552068 Eucarpia Species 0.000 description 1
- 241000220485 Fabaceae Species 0.000 description 1
- 101150062031 L gene Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241000723848 Tobamovirus Species 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001390 capsicum minimum Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 229940016590 sarkosyl Drugs 0.000 description 1
- 108700004121 sarkosyl Proteins 0.000 description 1
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 description 1
- 229910000342 sodium bisulfate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 244000000006 viral plant pathogen Species 0.000 description 1
Classifications
-
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- 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
-
- 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
- A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
- A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
- A01H1/126—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for virus resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01B—CABLES; CONDUCTORS; INSULATORS; SELECTION OF MATERIALS FOR THEIR CONDUCTIVE, INSULATING OR DIELECTRIC PROPERTIES
- H01B5/00—Non-insulated conductors or conductive bodies characterised by their form
- H01B5/02—Single bars, rods, wires, or strips
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01B—CABLES; CONDUCTORS; INSULATORS; SELECTION OF MATERIALS FOR THEIR CONDUCTIVE, INSULATING OR DIELECTRIC PROPERTIES
- H01B5/00—Non-insulated conductors or conductive bodies characterised by their form
- H01B5/14—Non-insulated conductors or conductive bodies characterised by their form comprising conductive layers or films on insulating-supports
-
- H—ELECTRICITY
- H02—GENERATION; CONVERSION OR DISTRIBUTION OF ELECTRIC POWER
- H02G—INSTALLATION OF ELECTRIC CABLES OR LINES, OR OF COMBINED OPTICAL AND ELECTRIC CABLES OR LINES
- H02G5/00—Installations of bus-bars
- H02G5/005—Laminated bus-bars
-
- 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 inventive molecular marker may be used to determine the genotype of the TMV-resistant plant.
- the present invention provides a method for determining the genotype of a TMV-resistant plant, which comprises analyzing the genomic DNA of a plant using the inventive nucleic acid or primer. This method may be performed by various DNA polymorphism analyses known in the art. Concretely, this method is performed as described for the method for detecting the TMV-resistant plants.
- TE buffer 10 mM Tris-Cl, 1 mM EDTA
- the DNA solution was incubated at 65 0 C for 1 hour.
- the culture solution was centrifuged three times.
- the resulting DNA was transferred into a 1.5-ml tube and added with 100 ⁇ g/ml of RNase so as to remove RNA.
- the final concentration of the purified DNA was measured with a fluorometer.
- the experiment was performed on resistant plants using the primers set forth in Table 1 above and a DNA working kit (Seegene; K-1502) kwon in the art.
- the first PCR was performed using the nucleotide sequence of SEQ ID NO:5.
- the PCR reaction composition was prepared by mixing lOOng of a template DNA, 4 ⁇ l of 2.5 ⁇ M DW-ACP, 10 ⁇ M primer, 25 ⁇ l of 2 X SeeAMPTM ACPTM, and 18 ⁇ l of triple distilled water, and its total volume was regulated to 50 ⁇ l.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Immunology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a' tobacco mosaic virus (TMV) resistance- associated molecular marker and the use thereof, and more particularly to a nucleic acid consisting of the nucleotide sequence having a very high association with the TMV-resistant trait of plants, a primer comprising a part of the nucleotide sequence of the nucleic acid, and a method for detecting TMV-resistant plants using the nucleic acid or the primer. The inventive molecular marker has advantages in that it can detect TMV-resistant plants in a rapid and precise manner without inoculating TMV directly into plants, and also can determine the genotype of TMV-resistant plants.
Description
MOLECULAR MARKER ASSOCIATED WITH TMV RESISTANCE AND USE THEREOF
TECHNICAL FIELD The present invention relates to a tobacco mosaic virus (TMV) resistance- associated molecular marker and the use thereof, and more particularly to a nucleic acid consisting of nucleotide sequences having a very high association with the TMV- resistant trait of plants, a primer comprising a portion of the nucleotide sequence of the nucleic acid, and a method for detecting TMV -resistant plants using the nucleic acid or the primer.
BACKGROUND ART
Tobacco Mosaic Virus (hereinafter, referred to as "TMV") is a rod shaped plant-pathogenic virus belonging to a tobamovirus group and having the widest host range among plant viruses, and infects more than 22 families, 200 kinds of plants including dicotyledonous plants such as Solanaceae, Compositae and pea family, thereby causing great economic damage to plants. Plants infected with TMV show typical mosaic symptoms on leaves, newly born leaves with yellow, and long brown spots on stems and petioles. Additionally, the affected fruits show spindle streak pattern and deformed fruits.
TMV infection is mediated by seeds. Therefore, contaminated seeds or affected young plants in the soil repeatedly cultured are the first infection source. TMV infection is not mediated by aphids. TMV infection is mediated by contact. Because natural infection hosts are always present in the environment as infection source, pathogenesis of TMV is often.
Meanwhile, in order to breed a disease-resistant variety against TMV, a disease-resistant factor should be introduced by successive backcrossing from other varieties having the factor. Examples of genes having TMV resistance include a L gene of red pepper. It is known that this gene is inherited by a single dominant gene, and has several homozygotes. It is also reported that until now, 5 homozygotes (L+, Ll, L2, L3, L4) are known, and have the superiority and inferiority relationship of L+<LKL2<L3<L4 (Am. J. Hum. Genet. 32:314-331, 1980; Proc Fourth Capsicum Eucarpia Meeting, Wageningen 44-48, 1980). In each of the introduction steps of the disease-resistant factor, the disease-resistant factor should be selected through a resistance test. This makes the selection inconvenient. In this selection step, the use of a molecular marker having a close association with the disease-resistant factor will make the selection very convenient. Thus, methods for the diagnosis of TMV using the molecular marker and various technologies for the development of TMV- resistant varieties by transformation have been developed. For example, Korean Patent Application No. 1994-0027614 discloses a method for development of an expression vector containing a coat gene of TMV-t cloned from the genomic RNA of Korean tobacco mosaic virus tomato (TMV-t) species, and a gene capable of conferring resistance against TMV-t on plants by transformation. Korean Patent Application No. 1994-0029486 discloses a method for development of a plant having resistance by cloning a coat gene of pepper mild mottle virus (PMMV) and introducing it into the plant to develop an antiviral red pepper variety and an expression vector containing a gene capable of conferring the PMMV resistance to plants. Additionally, Korean Patent Application No. 1995-0043992 discloses a method for introducing a replication enzyme gene (nucleotide sequence of domain 1)
of tobacco mosaic virus into a plant to breed a virus-resistant plant, and a method for development of a plant having a new virus-resistant trait developed therewith, and Korean Patent Application No. 1993-7003916 discloses a method for manufacturing a plant having resistance against RNA virus by introducing the DNA sequence encoding a protein having enzyme activity specifically degrading RNA of plant virus causing stresses in plants into the chromosome of a- plant and expressing it, and a method for development of the RNA virus-resistant plant using the manufacturing method.
As described above, the prior art on the diagnosis of TMV resistance and on the development of TMV-resistant plants targets the TMV coat protein gene, and there is no report of the development of technology concerning a TMV-resistant factor which is inherent in plants.
Accordingly, there has been an urgent need for the development of a TMV resistance-associated molecular marker from plant lines having the TMV-resistant factor and for the development of a method for diagnosing TMV infection in plants using the developed molecular marker.
Disclosure of the Invention
Therefore, the present inventors have studied on development of TMV resistance-associated molecular marker from the plant family having TMV-resistant factor and a method for diagnosis of TMV infection in plants using the developed TMV resistance associated molecular marker for many years. As a result, they found that the molecular marker according to the present invention can rapidly and precisely detect the TMV-resistant plants without direct vaccination of TMV into the plants, and moreover, the genotype of TMV-resistant plant can be determined.
Accordingly, it is an object of the present invention to provide a TMV- resistant molecular maker and the use thereof.
To achieve the above object, in one aspect, the present invention provides an isolated nucleic acid consisting of a nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
In another aspect, the present invention provides a primer for the detection of TMV-resistant plants, which comprises consecutive nucleotides selected from a nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. In still another aspect, the present invention provides a kit for detection of
TMV-resistant plant, which comprises the nucleic acid or the primer.
In yet another aspect, the present invention provides a method for detecting a TMV-resistant plant and a method for determining the genotype of the plant, the methods comprising analyzing the genomic DNA of a plant in the presence of the nucleic acid or the primer.
The further another aspect, the present invention provides a TMV-resistant plant which reproduce asexually by tissue culture and comprises a nucleic acid consisting of a nucleotide sequence shown SEQ ID NO: 3 or SEQ ID NO: 4, as well as a seed obtained therefrom.
Hereinafter, the present invention will be described in detail. The present invention provides a molecular marker having a high association with the TMV-resistant trait of plants (hereinafter, referred to as "a TMV resistance- associated molecular marker"). The TMV resistance-associated molecular marker provided in the present invention comprises a nucleic acid consisting of the nucleotide
sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. The nucleic acid includes RNA, DNA and cDNA, and preferably means DNA. The inventive molecular marker consists of nucleotide sequences having a close association with a TMV- resistant trait, and is placed significantly close to the TMV -resistant gene. For this reason, the use of any polymorphism shown by the nucleotide sequence allows the presence or absence of the TMV resistance in plants to be determined.
Also, the inventive molecular marker comprises a primer for the detection of TMV-resistant plants, which comprises a series of nucleotide sequences selected from the nucleotide sequences of the nucleic acids. The primer may be a series of the nucleotide sequences selected from the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, the primer may have 10-22 consecutive nucleotide sequences.
The inventive primer can be designed so as to be suitable for various DNA polymorphism analyses known in the art, such as RFLP (restriction fragment length polymorphism), RAPD (randomly amplified polymorphic DNA), DAF (DNA amplification fingerprinting), AP-PCR (arbitrarily primed PCR), STS (sequence tagged site), EST (expressed sequence tag), SCAR (sequence characterized amplified regions), ISSR (inter-simple sequence repeat amplification), AFLP (amplified fragment length polymorphism), CAPS (cleaved amplified polymorphic sequence), PCR-SSCP (single-strand conformation polymorphism) and the like (Jordan et al, Theor. Appl. Genet., 106:559-567, 2003; Martins M., R. et al., Plant Cell Reports, 22:71-78, 2003; Williams, J. G. K. et al, Nucl. Acids Res., 18:6531-6535, 1990; Michelmore, R. W., et al., Proc. Natl. Acad. Sci. USA. 88:9828-9832, 1991; Martins et al, Plant Cell Rep., 22:71-79, 2003; Orita et al, Proc. Natl. Acad. Sci. USA 86:2766-2770, 1989). For the DAF analysis, a primer consisting of 5-8 consecutive
nucleotides can be designed and used. For example, a primer for the STS analysis can be designed so as to coincide with the terminal nucleotide sequence of an RFLP marker, and a primer for the SCAR analysis can be designed based on the terminal nucleotide sequence of an RAPD marker. Also, a primer for the CAPS analysis can be designed so as to contain a suitable restriction enzyme site. Preferably, the primer provided in the present invention may have the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10.
The inventive molecular marker may be very useful for the detection of TMV-resistant plants. Thus, the present invention provides a method for the detection of a TMV-resistant plant, which comprises analyzing the genomic DNA of a plant using the nucleic acid or the primer. This method may be performed using various DNA polymorphism analyses known in the art. Example of the DNA polymorphism analyses which can be used in the present invention include, but are not limited to, RFLP (restriction fragment length polymorphism), RAPD (randomly amplified polymorphic DNA), DAF (DNA amplification fingerprinting), AP-PCR (arbitrarily primed PCR), STS (sequence tagged site), EST (expressed sequence tag), SCAR (sequence characterized amplified regions), ISSR (inter-simple sequence repeat amplification), AFLP (amplified fragment length polymorphism), CAPS (cleaved amplified polymorphic sequence), PCR-SSCP (PCR- single strand conformation polymorphism) and the like (Jordan et al, Theor. Appl. Genet., 106:559-567, 2003; Martins M., R. et al, Plant Cell Reports, 22:71-78, 2003; Williams, J. G. K. et al, Nucl. Acids Res. 18:6531-6535, 1990; Michelmore, R. W., et al, Proc. Natl Acad. Sci. USA. 88:9828-9832, 1991; Martins et al, Plant Cell Rep., 22:71-79, 2003; Orita et al, Proc. Natl. Acad. Sci. USA 86:2766-2770, 1989). Preferably, the detection
method may be performed by the RFLP analysis.
Concretely, the detection method may be performed by the RAPD analysis comprising the steps of:
(a) performing PCR on each of genomic DNA templates obtained from TMV-resistant plants and TMV-susceptible plants, using a primer capable of amplifying the nucleotide sequence set forth in SEQ ID NO: 2;
(b) subjecting the PCR products to electrophoresis using agarose gel; and
(c) performing the comparison between the DNA band patterns of the electrophoresis finished gel. The primer in the step (a) may have all nucleotide sequences which can be designed by a person skilled in the art so as to amplify the nucleic acid set forth in SEQ ID NO: 2. Preferably, the primer may have a nucleotide sequence set forth in SEQ ID NO: 1.
Also, the inventive molecular marker may be used to determine the genotype of the TMV-resistant plant. Thus, the present invention provides a method for determining the genotype of a TMV-resistant plant, which comprises analyzing the genomic DNA of a plant using the inventive nucleic acid or primer. This method may be performed by various DNA polymorphism analyses known in the art. Concretely, this method is performed as described for the method for detecting the TMV-resistant plants.
The plant to which the inventive methods may be applied includes, but is not limited to, cucumber, watermelon, red pepper, melon, Chinese cabbage, tobacco, Petunia, cotton and rose.
Moreover, the present invention provides a kit for the detection of a TMV- resistant plant, which comprises either a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, or a series of nucleotide sequences selected from the nucleotide sequences of the above nucleic acids. The inventive kit may additionally comprise various reagents required not only in experiment procedures (PCR, Southern blot analysis, etc) to detect TMV-resistant plants using the above nucleic acid or primer but also in a procedure to examine the test results. For example, the reagents include PCR reaction mixture, restriction enzyme, agarose, buffer solution required for hybridization and/or electrophoresis, etc.
Furthermore, the present invention provides a TMV-resistant plant which comprises a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO:4, the nucleic acid being a molecular marker having a close association with the TMV resistant trait of plants. As used herein, the term "a TMV- resistant plant" refers to the plant showing a resistance trait to TMV. The plant includes, but is not limited to, cucumber, watermelon, red pepper, melon, Chinese cabbage, tobacco, Petunia, cotton and rose. The red pepper is preferable. Also, the TMV-resistant plant provided in the present invention includes the organ, tissue, cell, seed and callus of plants. Experiments on whether the TMV resistance of the inventive TMV-resistant plants is hereditary were performed, and the experiment results proved that the TMV resistance is determined by a dominant single gene. The inventive TMV-resistant plant can reproduce asexually by a general tissue culture method known in the art. For example, the inventive plant can reproduce asexually by fine reproduction by organ generation (e.g., a method for culturing tissue, such as leaves having no organ formed therein, leaves, petioles, stem nodes, cotyledons,
cotyledon axes or the like, to induce fresh shoots on the surface of the tissue) or regeneration by callus induction, and the like. In addition, the present invention provides a seed which is obtained from the TMV-resistant plant and comprises the nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO:4.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photograph showing the results of RAPD analysis performed using a TMV-resistant DNA pool and a TMV-susceptible DNA pool as templates, and using an operon primer (OPI-20).
FIG. 2 is a photograph showing the results of RAPD analysis performed for the F2 population TMVpop 1 using an OPI-20 primer (SEQ ID NO: 1).
FIG. 3 is a photograph showing the results of RAPD analysis performed for the F2 population TMVpop 2 using an OPI-20 primer (SEQ ID NO: 1). FIGs 4 and 5 show the deletion site, primer site and Hind III site through the comparison of the nucleotide sequence of SEQ ID NO:3, the nucleotide sequence of SEQ ID NO:4 and the nucleotide sequence of TMV-susceptible plants.
FIG. 6 is a photograph showing the results of PCR analysis performed using DNAs extracted from TMV-resistant plants, TMV-susceptible plants and F2 population as templates, and using primers of SEQ ID NO: 6 and SEQ ID NO: 8. M: Molecular weight marker(lkb ladder marker)
FIG. 7 is a photograph showing the results of PCR analysis performed using DNAs extracted from TMV-resistant plants, TMV-susceptible plants and F2 population as templates, and using primers of SEQ ID NO:9 and SEQ ID NO: 10. M: Molecular weight marker(lkb ladder marker);
Lane 1 : TMV-resistant plants; Lane 2: TMV-susceptible plants; Lanes 3-22: F2 plants.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in further detail by the following examples. It is to be understood, however, that these examples are given for illustrative purpose only and are not intended to limit the scope of the present invention.
Example 1: Construction of crossed population for molecular marker development of TMV-resistant red pepper plants and examination of hereditary pattern of TMV resistance
TMV was inoculated into a variety of red pepper plants so as to screen TMV- resistant plants. The results showed that one plant, PI260429, of the Capsicum chacoense lines was TMV-resistant. The screened plant PI260429 was cross-bred with TMV-susceptible plants Capsicum annuum FP21 and FP22 to produce Fl seeds. In order to anticipate the genotype of TMV-resistance in the F2 populations, the Fl seeds were self-pollinated to produce the F2 populations(TMVpopl and TMVpop2), and each of the F2 populations was self-pollinated to produce the F3 plants. The F2 and F3 populations were tested for TMV resistance, and the results showed that the TMV resistance was hereditary at a ratio of 3 :1 (resistance: susceptibility) in the phenotype of the F2 populations, and at a ratio of 1 :2: 1 (resistance homo: resistance hetero: susceptibility homo) in the genotype of the F3 populations. This suggests that the TMV resistance is determined by a dominant single gene.
Example 2: DNA isolation from plants
DNA extraction was performed by- a modification to the method of Prince, J. P., et al.( Prince, J. P., et al, HortScience 32:937-939, 1997). Red pepper leaves stored at -80 °C were crushed with liquid nitrogen, and then well mixed with 25 ml of DNA extraction buffer containing 7M urea, 0.35M sodium chloride, 0.05M Tris-HCl pH 8.0, 0.02M EDTA, 0.25% sarkosyl, 5% phenol and 0.2% sodium bisulfate. Then, 0.75 ml of 20% SDS was added into the mixture, and the mixture was incubated at 65 °C for 30 minutes while shaking at intervals of 10 minutes. A solution containing chloroform and isoamyl alcohol in a volume ratio of
24:1 was filled to the end of a 50-ml tube and well mixed for 15 minutes. The mixture solution was centrifuged at 5,000 rpm for 15 minutes, and the obtained supernatant was transferred into a fresh 50-ml tube using cheesecloth. Next, the same volume of isopropanol was added to the tube and mixed, and stored for 1 hour to allow of the precipitation of DNA. The precipitated DNA was collected using a U- shaped Pasteur pipette and placed in a 1.5-ml micro-tube to which 70% ethanol was then added to reprecipitate the DNA. The resulting DNA was dried at room temperature for 30-40 minutes. 600-700 μl of TE buffer (10 mM Tris-Cl, 1 mM EDTA) was added to the dried DNA pellet, and the DNA solution was incubated at 65 0C for 1 hour. The culture solution was centrifuged three times. Then, the resulting DNA was transferred into a 1.5-ml tube and added with 100 μg/ml of RNase so as to remove RNA. The final concentration of the purified DNA was measured with a fluorometer.
Example 3: Selection of TMV resistance-associated RAPD primer
In order to select a DNA molecular marker associated with a TMV -resistant trait, RAPD(Random Amplified Polymorphic DNAs) (Williams, J. G. K. et al, Nucl. Acids Res. 18, 6531-6535, 1990) and BSA (Bulked Segregant Analysis) (Michelmore, R. W. et al, Proc. Nat. Acad. Sci., 88:9828-9832, 1991) were used. Based on the TMV resistance assay results from the F2 population of Example 1, a DNA pool of 20 resistant F2 plants and a DNA pool of 20 susceptible F2 plants were prepared, respectively. The each DNA pool was controlled to a DNA concentration of 50 ng/μl. Then, the PCR was performed using the DNA as a template. In this case, for RAPD primers, about 340 primers of Operon RAPD 10-mer kits series A to series Q (Operon, Alameda, CA, USA) were used to screen bands showing a specific difference in amplification between the resistant DNA pool and the susceptible DNA pool.
The PCR reaction mixture consisted of a 25 μl total volume of 1 X PCR buffer, 100 ng of the template DNA, 0.2 rnM dNTP, 0.4 μM primer, 3.5 mM MgCl2, and 1 Unit Taq DNA polymerase (Takara, Japan). The PCR amplification consisted of the following: denaturation of template DNA of 4 minutes at 94 0C; 45 cycles each consisting of 1 minute at 94 0C, 1 minute at 35 0C, and 1 minute and 30 seconds at 72 °C; and final extension of 7 minutes at 72 0C. As a result, as shown in FIG. 1, a molecular marker which had shown specificity to only one resistant pool in the PCR reaction using an OPI-20 primer (SEQ ID NO: 1) was selected.
In order to examine whether the molecular marker is associated with a TMV- resistant trait, RAPD analysis was further performed on the TMVpopl and TMVpop2 as F2 plants using the OPI-20 primer (SEQ ID NO:1) under the same reaction conditions as the above RAPD analysis. As shown in FIGS. 2 and 3, the analysis results showed that the molecular marker was 99% co-segregated.
Example 4: Cloning and nucleotide sequence determination of RAPD molecular marker
The DNA amplified with the OPI-20 primer having the nucleotide sequence set forth in SEQ ID NO:1 was isolated and purified from gel, and cloned into a pGEM-T vector (Promega). The cloned plasmid was introduced into E. coli DH 1OB by electroporation, and a transformant was selected in an antibiotic-containing medium. The recombinant plasmid was isolated from the selected transformant, and the nucleotide sequence of DNA contained in the plasmid was analyzed by Zenotech Co. Ltd. The determined nucleotide sequence is set forth in SEQ ID NO:2.
Example 5: Determination of nucleotide sequence of DNA close to TMV- resistant gene
The parts of the nucleotide sequence of SEQ ID NO:2 analyzed in Example 4 were used to construct nested primers. The nucleotide sequence of each of the constructed primers is given in Table 1 below.
Table 1 : Primers used in close DNA nucleotide sequence working
The experiment was performed on resistant plants using the primers set forth
in Table 1 above and a DNA working kit (Seegene; K-1502) kwon in the art. The first PCR was performed using the nucleotide sequence of SEQ ID NO:5. The PCR reaction composition was prepared by mixing lOOng of a template DNA, 4 μl of 2.5 μM DW-ACP, 10 μM primer, 25 μl of 2 X SeeAMP™ ACP™, and 18 μl of triple distilled water, and its total volume was regulated to 50 μl. The first PCR amplification consisted of the following: denaturation of template DNA of 5 minutes at 94 °C, reaction of 1 minute at 42 0C and 2 minutes at 72 °C, 30 cycles each consisting of 40 seconds at 94 0C, 1 minute at 42 0C and 1 minute at 72 °C; and final extension of 7 minutes at 72 0C. The second PCR was performed with the first PCR products as a template using a primer of SEQ ID NO:6. The second PCR reaction composition was prepared by mixing lOOng of a template DNA, 1 μl of 10 μM DW- ACP, 10 μM primer, 10 μl of 2 X SeeAMP™ ACP™, and 6 μl of triple distilled water, and its total volume was regulated to 20 μl. The second PCR amplification consisted of the following: denaturation of template DNA of 5 minutes at 94 0C, 35 cycles each consisting of 40 seconds at 94 0C, 40 seconds at 55 0C and 1 minute at 72 0C; and final extension of 7 minutes at 72 0C. The third PCR was performed with the second PCR products as a template using a primer of SEQ ID NO:7. The third PCR reaction composition was prepared by mixing 50ng of a template DNA, 10 μM universal primer, 10 μM primer, 10 μl of 2 X SeeAMP™ ACP™, and 7 μl of triple distilled water, and its total volume was regulated to 20 μl. The third PCR amplification consisted of the following: denaturation of template DNA of 5 minutes at 94 °C, 30 cycles each consisting of 40 seconds at 94 0C, 40 seconds at 55 °C and 1 minute at 72 0C; and final extension of 7 minutes at 72 0C. The nucleotide sequences of the third PCR products obtained by the method above were analyzed by Zenotech Co., Ltd.
As a result, the DNA nucleotide sequences set forth in SEQ ID NO: 3 and
SEQ ID NO:4 were determined. The nucleotide sequences of SEQ ID NO:3 and
SEQ ID NO:4 can be distinguished by a restriction enzyme Hind III, i.e., the nucleotide sequence of SEQ ID NO: 3 had no site of Hind III, and the nucleotide sequence of SEQ ID NO:4 had the site of Hind III (see FIGs. 4 and 5.)
Example 6: Conversion into co-dominant molecular marker After finding out deletion of specific parts of the resistant nucleotide sequences by comparing the nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:4 with the susceptible nucleotide sequence, primers capable of amplifying DNA fragments showing the polymorphism between genomic DNAs of resistant plants and susceptible plants were constructed from the specific parts before and behind the deletion sites of the genomic DNAs of resistant plants and susceptible plants (see FIGs 4 and 5). That is, 4 primers each consisting of a series of nucleotide sequences selected from the nucleotide sequences set forth in SEQ ID NO:3 and SEQ ID NO:4 were constructed (see Table 2 below).
Table 2: Primers for development of co-dominant molecular marker for examination of TMV resistance
The genomic DNAs were extracted from a resistant plant, a susceptible plant and their F2 population, respectively, and used as a template for PCR. The PCR was performed with the DNA template and a primer set selected from a combination of SEQ ID NO:6 and SEQ ID NO:8, and SEQ ID NO:9 and SEQ ID NO:10. The PCR amplification consisted of the following: denaturation of template DNA of 4 minute at 94 0C; 35 cycles each consisting of 1 minute at 94 0C, 1 minute at 54 °C, and 1 minute and 30 seconds at 72 0C; and final extension of 7 minutes at 72 0C. Thereafter, each of the amplified DNAs was subjected to electrophoresis using agarose gel so as to examine if the amplified DNAs were divided into genotypes, i.e., homo (RR) and hetero (Rr).
As a result, the amplified resistant DNA fragment obtained by performing the PCR amplification with the primer combination of SEQ ID NO: 6 and SEQ ID NO: 8 was 466 bp in size. It could be found that these band patterns were co-segregated with the genotype of the F2 plants, which had been expected by testing the disease resistance of F3 in Example 1 , and the band patterns of the susceptible and resistant plants were different from each other (see FIG. 6).
Additionally, the amplified resistant DNA fragment obtained by performing the PCR amplification with the primer combination of SEQ ID NO:9 and SEQ ID NO: 10 was 505 bp in size. It could be found that the band patterns of the susceptible and resistant plants were different from each other, and these band patterns were co- segregated with the disease resistance results shown in Example 1 (see FIG. 7).
The above results show that the primers constructed from a series of any nucleotide sequences selected from the nucleotide sequences set forth in SEQ ID NO: 3 and SEQ ID NO:4 are used to perform PCR, thereby determining the presence or absence of a TMV -resistant gene and its genotype (i.e., homo or hetero). Namely, since the nucleotide sequences set forth in SEQ ID NO:3 and SEQ ID NO:4 significantly approach to TMV -resistant genes, the use of any polymorphism shown by the nucleotide sequences allows to determine the presence or absence of TMV- resistant genes.
This application claims priority to Korean Patent Application No 10-2006-
0018325, filed on February 24, 2006, the contents of which are hereby incorporated by reference.
INDUSTRIAL APPLICABILITY As discussed above, the present invention provides the molecular marker comprising the nucleotide sequence highly linked to TMV (Tobacco mosaic virus)- resistance gene, such that TMV-resistant plants can be diagnosed effectively. Moreover, the use of the inventive molecular marker allows detecting TMV-resistant plants in a rapid and precise manner without inoculating TMV directly into plants, and also allows determining the genotype of TMV-resistant plants.
Claims
1. An isolated nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.
2. A primer for the detection of tobacco mosaic virus (TMV)-resistant plants, which comprises consecutive nucleotide sequences selected from the nucleotide sequences set forth in SEQ ID NO:3 and SEQ ID NO:4.
3. The primer of Claim 2, wherein the primer has the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10.
4. A kit for the detection of TMV-resistant plants, which comprises the nucleic acid of Claim 1 or the primer of Claim 2.
5. A method for the detection of a TMV-resistant plant, comprising analyzing the genomic DNA of a plant in the presence of the nucleic acid of Claim 1 or the primer of Claim 2.
6. The method of Claim 5, wherein the analysis is performed by any one method selected from the group consisting of RFLP (restriction fragment length polymorphism), RAPD (randomly amplified polymorphic DNA), DAF (DNA amplification fingerprinting), AP-PCR (arbitrarily primed PCR), STS (sequence tagged site), EST (expressed sequence tag), SCAR (sequence characterized amplified regions), ISSR (inter-simple sequence repeat amplification), AFLP (amplified fragment length polymorphism), CAPS (cleaved amplified polymorphic sequence) and PCR-SSCP (PCR-single strand conformation polymorphism).
7. The method of Claim 5, wherein the plant is selected from the group consisting of cucumber, watermelon, red pepper, melon, Chinese cabbage, tobacco, Petunia, cotton and rose.
8. A method for determining the genotype of a TMV -resistant plant, comprising analyzing the genomic DNA of a plant in the presence of the nucleic acid of Claim 1 or the primer of Claim 2.
9. The method of Claim 8, wherein the analysis is performed by any one method selected from the group consisting of RFLP (restriction fragment length polymorphism), RAPD (randomly amplified polymorphic DNA), DAF (DNA amplification fingerprinting), AP-PCR (arbitrarily primed PCR), STS (sequence tagged site), EST (expressed sequence tag), SCAR (sequence characterized amplified regions), ISSR (inter-simple sequence repeat amplification), AFLP (amplified fragment length polymorphism), CAPS (cleaved amplified polymorphic sequence), and PCR-SSCP (PCR-single strand conformation polymorphism).
10. The method of Claim 8, wherein the plant is selected from the group consisting of cucumber, watermelon, red pepper, melon, Chinese cabbage, tobacco, Petunia, cotton and rose.
11. A TMV-resistant plant which is reproduced asexually by tissue culture and comprises a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO.4.
12. The TMV-resistant plant of Claim 11, wherein the plant is selected from the group consisting of cucumber, watermelon, red pepper, melon, Chinese cabbage, tobacco, Petunia, cotton and rose.
13. A seed which is obtained from the TMV-resistant plant of Claim 11 or claim 12 and comprises a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020060018325A KR20070088123A (en) | 2006-02-24 | 2006-02-24 | Molecular marker associated with tmv resistance and use thereof |
| KR10-2006-0018325 | 2006-02-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2007097574A1 true WO2007097574A1 (en) | 2007-08-30 |
| WO2007097574A8 WO2007097574A8 (en) | 2009-11-05 |
Family
ID=38437577
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2007/000920 WO2007097574A1 (en) | 2006-02-24 | 2007-02-22 | Molecular marker associated with tmv resistance and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20070088123A (en) |
| WO (1) | WO2007097574A1 (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899523A (en) * | 2010-08-13 | 2010-12-01 | 中国农业科学院蔬菜花卉研究所 | Primer sequence for detecting Bt-resistant plutella xylostella and application thereof |
| CN102586239A (en) * | 2012-02-23 | 2012-07-18 | 山东省农业科学院蔬菜研究所 | Co-segregation SSR (Simple Sequence Repeat) codominant molecular marker of onion male sterility gene Ms site and application thereof |
| CN102605047A (en) * | 2011-12-09 | 2012-07-25 | 郑州轻工业学院 | SCAR (Sequence-characterized Amplified Regions) primers and method for identifying tobacco varieties |
| CN102703598A (en) * | 2012-07-04 | 2012-10-03 | 中国热带农业科学院香料饮料研究所 | Method for identifying pepper germplasm resources |
| CN103865925A (en) * | 2014-03-09 | 2014-06-18 | 中国水稻研究所 | Molecular marker of paddy rice flag leaf width controlling gene NAL1 and application thereof |
| CN109355419A (en) * | 2018-11-20 | 2019-02-19 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the downstream tobacco TMV resistant gene N (3 ' end) Linkage drag and its application |
| CN109439789A (en) * | 2018-11-20 | 2019-03-08 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the upstream tobacco TMV resistant gene N (5 ' end) Linkage drag and its application |
| WO2019174721A1 (en) | 2018-03-13 | 2019-09-19 | Dümmen Group B.V. | Tobamovirus resistant solanaceae plant |
| CN114381539A (en) * | 2020-10-21 | 2022-04-22 | 北京市农林科学院 | Cucurbita pepo ZYMV virus disease molecular marker and method for identifying resistance of Cucurbita pepo ZYMV virus disease |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101522600B1 (en) * | 2013-04-01 | 2015-05-26 | 대한민국 | SNP molecular markers associated with the clubroot and mosaic virus disease resistance and leaf traits of Chinese cabbages and uses thereof |
| CN104450697B (en) * | 2014-12-02 | 2017-01-11 | 中国科学院海洋研究所 | SNP marker associated with oyster antiviral properties and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5596132A (en) * | 1990-03-12 | 1997-01-21 | Cornell Research Foundation, Inc. | Induction of resistance to virus diseases by transformation of plants with a portion of a plant virus genome involving a read-through replicase gene |
| KR970025376A (en) * | 1995-11-27 | 1997-06-24 | 이세영 | Development of Virus-Resistant Plants Using Domain 1 Sequences in the Replication Enzyme of Tobacco Mosaic Virus |
| KR970074936A (en) * | 1996-05-31 | 1997-12-10 | 박명규 | Method for preparing transformed tobacco plants having resistance to tobacco mosaic virus |
| KR20000005741A (en) * | 1998-06-16 | 2000-01-25 | 클라크 3세 존 엠. | Scan flip-flop that simultaneously holds logic values from a serial load and a subsequent parallel load |
-
2006
- 2006-02-24 KR KR1020060018325A patent/KR20070088123A/en not_active Application Discontinuation
-
2007
- 2007-02-22 WO PCT/KR2007/000920 patent/WO2007097574A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5596132A (en) * | 1990-03-12 | 1997-01-21 | Cornell Research Foundation, Inc. | Induction of resistance to virus diseases by transformation of plants with a portion of a plant virus genome involving a read-through replicase gene |
| KR970025376A (en) * | 1995-11-27 | 1997-06-24 | 이세영 | Development of Virus-Resistant Plants Using Domain 1 Sequences in the Replication Enzyme of Tobacco Mosaic Virus |
| KR970074936A (en) * | 1996-05-31 | 1997-12-10 | 박명규 | Method for preparing transformed tobacco plants having resistance to tobacco mosaic virus |
| KR20000005741A (en) * | 1998-06-16 | 2000-01-25 | 클라크 3세 존 엠. | Scan flip-flop that simultaneously holds logic values from a serial load and a subsequent parallel load |
Non-Patent Citations (5)
| Title |
|---|
| DATABASE GENBANK [online] 24 November 2005 (2005-11-24), ANTONELLIS A. ET AL., accession no. NCBI Database accession no. (AC139112) * |
| LEE G.J. ET AL.: "Induction of a pepper cDNA encoding SAR8.2 protein during the resistance response to tobacco mosaic virus", MOL. CELLS, vol. 12, no. 2, 31 October 2001 (2001-10-31), pages 250 - 256 * |
| PARK C.J. ET AL.: "A hot pepper cDNA encoding a pathogenesis-related protein 4 is induced during the resistance response to tobacco mosaic virus", MOL. CELLS, vol. 11, no. 1, 28 February 2001 (2001-02-28), pages 122 - 127 * |
| SHIGEHARU TAKEUCHI ET AL.: "Discrimination between tobamoviruses and their pathotypes for L-gene-mediated resistance in green pepper (Capsicum annuum L.) by reverse transcription-polymerase chain reaction", J. GEN. PLANT PATHOL., vol. 71, 2005, pages 60 - 67 * |
| TSUDA S. ET AL.: "Characterization of a pepper mild mottle tobamovirus strain capable of overcoming the L3 gene-mediated resistance, distinct from the resistance-breaking Italian isolate", MOL. PLANT MICROBE INTERACT., vol. 11, no. 4, April 1998 (1998-04-01), pages 327 - 331 * |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899523B (en) * | 2010-08-13 | 2013-08-07 | 中国农业科学院蔬菜花卉研究所 | Primer sequence for detecting Bt-resistant plutella xylostella and application thereof |
| CN101899523A (en) * | 2010-08-13 | 2010-12-01 | 中国农业科学院蔬菜花卉研究所 | Primer sequence for detecting Bt-resistant plutella xylostella and application thereof |
| CN102605047A (en) * | 2011-12-09 | 2012-07-25 | 郑州轻工业学院 | SCAR (Sequence-characterized Amplified Regions) primers and method for identifying tobacco varieties |
| CN102586239A (en) * | 2012-02-23 | 2012-07-18 | 山东省农业科学院蔬菜研究所 | Co-segregation SSR (Simple Sequence Repeat) codominant molecular marker of onion male sterility gene Ms site and application thereof |
| CN102586239B (en) * | 2012-02-23 | 2013-09-04 | 山东省农业科学院蔬菜研究所 | Co-segregation SSR (Simple Sequence Repeat) codominant molecular marker of onion male sterility gene Ms site and application thereof |
| CN102703598A (en) * | 2012-07-04 | 2012-10-03 | 中国热带农业科学院香料饮料研究所 | Method for identifying pepper germplasm resources |
| CN103865925A (en) * | 2014-03-09 | 2014-06-18 | 中国水稻研究所 | Molecular marker of paddy rice flag leaf width controlling gene NAL1 and application thereof |
| CN103865925B (en) * | 2014-03-09 | 2016-01-20 | 中国水稻研究所 | Flag leaf width of rice regulatory gene NAL1 molecule marker and application thereof |
| JP2021518752A (en) * | 2018-03-13 | 2021-08-05 | デュメン・グループ・ベー・フェーＤｕｍｍｅｎ Ｇｒｏｕｐ Ｂ．Ｖ． | Tobamovirus resistant Solanaceae |
| US11659806B2 (en) | 2018-03-13 | 2023-05-30 | Dümmen Group B.V. | Tobamovirus resistant Solanaceae plant |
| WO2019174721A1 (en) | 2018-03-13 | 2019-09-19 | Dümmen Group B.V. | Tobamovirus resistant solanaceae plant |
| CN112312763A (en) * | 2018-03-13 | 2021-02-02 | 多盟集团公司 | Tobacco mosaic virus resistant solanaceae plants |
| CN109355419A (en) * | 2018-11-20 | 2019-02-19 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the downstream tobacco TMV resistant gene N (3 ' end) Linkage drag and its application |
| CN109355419B (en) * | 2018-11-20 | 2021-05-18 | 云南省烟草农业科学研究院 | Group of molecular markers for breaking N downstream (3' end) linkage drag of tobacco TMV resistance gene and application thereof |
| CN109439789A (en) * | 2018-11-20 | 2019-03-08 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the upstream tobacco TMV resistant gene N (5 ' end) Linkage drag and its application |
| CN114381539A (en) * | 2020-10-21 | 2022-04-22 | 北京市农林科学院 | Cucurbita pepo ZYMV virus disease molecular marker and method for identifying resistance of Cucurbita pepo ZYMV virus disease |
| CN114381539B (en) * | 2020-10-21 | 2023-11-24 | 北京市农林科学院 | Molecular marker of zucchini ZYMV virus and method for identifying zucchini ZYMV virus resistance |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20070088123A (en) | 2007-08-29 |
| WO2007097574A8 (en) | 2009-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2007097574A1 (en) | Molecular marker associated with tmv resistance and use thereof | |
| Vilanova et al. | An apricot (Prunus armeniaca L.) F2 progeny linkage map based on SSR and AFLP markers, mapping plum pox virus resistance and self-incompatibility traits | |
| Lu et al. | Development of SCAR markers linked to the gene Or5 conferring resistance to broomrape (Orobanche cumana Wallr.) in sunflower | |
| AU2020202265B2 (en) | Genetic markers for myb28 | |
| Ohmori et al. | Identification of RAPD markers linked to the Tm-2 locus in tomato | |
| Ikeda et al. | Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice | |
| JP5908834B2 (en) | Pathogen resistant plant | |
| KR100984736B1 (en) | Marker for selecting brown planthopper-resistant rice cultivar | |
| US8026414B2 (en) | Molecular marker associated with CMV resistance and use thereof | |
| KR100781206B1 (en) | SSR primer isolated from Sesamum sp. and use thereof | |
| JP2004512828A (en) | Map-based genome collection method for identifying regulatory loci that control the amount of gene transcripts or products | |
| BR112021012597A2 (en) | METHODS AND COMPOSITIONS TO SELECT AND/OR PREDICT COTTON PLANTS RESISTANT TO FUSERIUM RACE-4 RESISTANCE IN COTTON | |
| JP5288608B2 (en) | Genes that increase grain seeds and their use | |
| EP2102364B1 (en) | Genetic markers for orobanche resistance in sunflower | |
| KR101242434B1 (en) | DNA marker to genic male sterile in tomato and use thereof | |
| EP3193586B1 (en) | New phytophthora resistance gene | |
| KR20140040374A (en) | Primer set, method and kit for selecting tswv-resistant pepper cultivar | |
| KR101401980B1 (en) | Specific primer for selecting Phytophthora blight resistant pepper and uses thereof | |
| Wu et al. | Cytogenetic and genomic characterization of a novel wheat-tetraploid Thinopyrum elongatum 1BS· 1EL translocation line with stripe rust resistance | |
| JP3047022B1 (en) | Primer set for identifying the presence or absence of the Ryadg gene involved in potato potato virus Y resistance, method for identifying the presence or absence of the Ryadg gene, polymerase linkage usable as SCAR marker of the Ryadg gene Reaction product | |
| Yang et al. | Identification of inconsistent S-genotypes in 15 Chinese pear (Pyrus L.) cultivars | |
| Liu | Genetic mapping and quantitative trait locus (QTL) analysis of root-knot nematode resistance in pearl millet | |
| Clark | Mapping molecular markers linked with two race specific loci conferring Colletotrichum trifolii resistance in Medicago sativa | |
| Gaafar | Fine mapping of the bolting gene of sugar beet (Beta vulgaris L.) using BAC-derived sequences | |
| KR20100087892A (en) | Dna diversity dectection method of azuki-bean and identification method of azuki-bean |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: RULE 160 1 EPC |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 07715358 Country of ref document: EP Kind code of ref document: A1 |