KR101758610B1 - A method for identifying gerbera varieties using microsatellites markers - Google Patents
A method for identifying gerbera varieties using microsatellites markers Download PDFInfo
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- KR101758610B1 KR101758610B1 KR1020150056826A KR20150056826A KR101758610B1 KR 101758610 B1 KR101758610 B1 KR 101758610B1 KR 1020150056826 A KR1020150056826 A KR 1020150056826A KR 20150056826 A KR20150056826 A KR 20150056826A KR 101758610 B1 KR101758610 B1 KR 101758610B1
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- 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
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- 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
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/151—Modifications characterised by repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
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- 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
Abstract
The present invention relates to a method for identifying a Gerbera variety using a supersatellite primer set. More particularly, the present invention relates to a method for identifying Gerbera cultivars using a super-satellite primer set for identification of Gerbera variety which shows polymorphism in 30 varieties of Gerbera collected in Korea. Using the primer set according to the present invention as a super satellites marker for discriminating genotypes can greatly contribute to various fields such as identification of authenticity of gerbera varieties, arbitration of seed disputes and selection of control varieties for varietal protected varieties.
Description
The present invention relates to a breed identification method of Gerbera spp. Using a supersatellite marker. More particularly, the present invention relates to a super satellites primer set for identifying gerbera varieties, a kit including the primer set, and a method for identifying the Gerbera variety using the primer set.
DNA markers are specific nucleotide sequences that can be used to determine polymorphisms in the genome. These include marker-assisted selection (MAS), gene mapping, quantitative trait locus analysis, purity assay, genetic markers And is used in fields such as species diversity analysis and breed identification.
In general, the method to identify plant cultivars is divided into morphological characterization test through cultivation test and DNA analysis method. The DNA analysis method is advantageous in that it is more objective and reproducible because it is not influenced by cultivation environment and the growth stage of the crop compared with phenotype-based identification method. UPOV is in the process of discussing the use of DNA markers to investigate the characteristics of new varieties and to grant new crop protection rights. Although the simple sequence differences at the gene level have not yet been recognized as the distinction of the breeds, the possibility of using the molecular markers for breed identification is proposed. Specifically, SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) methods have been proposed to take into account the degree of polymorphism between varieties, the reproducibility of analysis, and the distribution of chromosomal patterns in the molecular markers.
As of December 1, 2004, Gerbera was designated as a cultivar protected crop, and it is urgent to cultivate new domestic varieties. It is one of the most important crop varieties to be protected and identification of registered varieties is very important. As of March 10, 2015, a new variety protection application was filed with the national seed resource, and the number of registered varieties is 153, and the number of gerbera varieties filed in Korea is increasing. In order to test the breed of Gerbera, it is necessary to carry out cultivation test. As the cultivation environment is influenced by the cultivation environment and it takes time to characterize it, the importance of breeding and breeding identification method using molecular beacon is increasing.
Therefore, in the present invention, we sought to select a supersatellite marker suitable for cultivar discrimination and to establish a breed identification system using it.
The present invention provides a primer set for identifying Gerbera variety.
The present invention provides a kit for identifying Gerbera varieties comprising the primer set.
The present invention provides a method for identifying a variety of gerbera using the primer set.
The present invention relates to a Ger-1 primer set comprising oligonucleotides of SEQ ID NOS: 1 and 2; A Ger-2 primer set consisting of the oligonucleotides of SEQ ID NOS: 3 and 4; A Ger-3 primer set consisting of the oligonucleotides of SEQ ID NOS: 5 and 6; A Ger-4 primer set consisting of the oligonucleotides of SEQ ID NOS: 7 and 8; A Ger-5 primer set consisting of the oligonucleotides of SEQ ID NOS: 9 and 10; A Ger-6 primer set consisting of the oligonucleotides of SEQ ID NOS: 11 and 12; A Ger-7 primer set consisting of the oligonucleotides of SEQ ID NOS: 13 and 14; A Ger-8 primer set consisting of the oligonucleotides of SEQ ID NOS: 15 and 16; A Ger-9 primer set consisting of the oligonucleotides of SEQ ID NOS: 17 and 18; A Ger-10 primer set consisting of the oligonucleotides of SEQ ID NOS: 19 and 20; A Ger-11 primer set consisting of the oligonucleotides of SEQ ID NOS: 21 and 22; A Ger-12 primer set consisting of the oligonucleotides of SEQ ID NOS: 23 and 24; A Ger-13 primer set consisting of the oligonucleotides of SEQ ID NOS: 25 and 26; A Ger-14 primer set consisting of the oligonucleotides of SEQ ID NOS: 27 and 28; A Ger-15 primer set consisting of the oligonucleotides of SEQ ID NOS: 29 and 30; A Ger-16 primer set consisting of the oligonucleotides of SEQ ID NOS: 31 and 32; A Ger-17 primer set consisting of the oligonucleotides of SEQ ID NOS: 33 and 34; A Ger-18 primer set consisting of the oligonucleotides of SEQ ID NOS: 35 and 36; At least one primer set selected from the group consisting of a Ger-19 primer set consisting of the oligonucleotides of SEQ ID NOs: 37 and 38 and a Ger-20 primer set consisting of the oligonucleotides of SEQ ID NOs: 39 and 40; (Hereinafter also referred to as at least one primer set of Ger-1 to Ger-20 primer sets).
The primer set may exhibit high polymorphism for the domestic varieties of gerbera. Thus, the primer set can be used to identify the Gerbera variety. The primer set may be used, for example, to identify the Gerbera variety described in Table 2 below.
Wherein the primer set comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least two of the Ger-1 to Ger- At least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 primer sets. The primer set may include all of the Ger-1 to Ger-20 primer sets. 20 pairs of primer sets containing oligonucleotides of SEQ ID NOS: 1 to 40 of the present invention are shown in Table 1 below. As used herein, 'marker' may refer to an amplification product obtained through each primer set or each primer set itself.
The oligonucleotide constituting the primer set may include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, a peptide nucleic acid or an intercalating agent. The oligonucleotide may further comprise fluorescent, phosphorescent or radioactive labeling substance. The fluorescent labeling substance may be VIC, NED, FAM, PET, or a combination thereof. The labeling substance may be labeled at the 5 ' end of the oligonucleotide. In addition, the radioactive labeling substance can be incorporated into the amplification product through PCR reaction using a PCR reaction solution in which a radioisotope such as 32 P or 35 S is added.
In addition, the present invention provides a kit for identifying Gerbera varieties, comprising at least one primer set of Ger-1 to Ger-20 primer sets. Wherein said kit comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 of Ger-1 to Ger-20 primer sets At least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 primer sets. The kit may include all of the Ger-1 to Ger-20 primer sets.
The kit may further comprise DNA polymerase, dNTP and PCR buffer solution. In addition, components necessary for PCR reaction or confirmation of an amplification product may be further included in the kit. The PCR buffer may contain KCl, Tris-HCl and MgCl 2. The kit may include a brochure. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.
Further, the present invention provides a method for amplifying a nucleic acid comprising the steps of: amplifying a nucleic acid using genomic DNA derived from gerbera as a template and using at least one primer set of Ger-1 to Ger-20 primer sets as primers; And determining the varieties of the gerbera from the resulting amplification products.
The genomic DNA may be derived from leaves, stems, petals, or combinations thereof of the gerbera. The genomic DNA can be obtained using a conventional method for obtaining DNA from a plant. The genomic DNA can be obtained, for example, using a phenol extraction method.
In this method, amplification of the nucleic acid can be performed using PCR. The amplification may be performed using a minimum set of primers necessary for identification of the Gerbera variety among Ger-1 to Ger-20 primer sets. The primers used for the amplification include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 of the Ger-1 to Ger-20 primer sets Or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more or 19 or more. The amplification may be performed using all of the Ger-1 to Ger-20 primer sets. PCR methods are well known in the art, and commercially available kits can be used. The PCR reaction can be performed using a PCR reaction solution containing various components known to be required for PCR reaction in the art. The PCR reaction solution may include, for example, genomic DNA derived from the genome of Gerbera to be analyzed, a primer set of the present invention, a DNA polymerase (for example, Taq polymerase), a dNTP mixture, a PCR buffer solution and water have.
The method may include detecting the resulting amplification product by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement. When detected by gel electrophoresis, agarose gel electrophoresis or acrylamide gel electrophoresis can be used depending on the amplification product size. In the case of detection through fluorescence measurement, fluorescence can be measured using a fluorescence meter after carrying out PCR using the primer set of the present invention labeled with a fluorescent substance. When detecting by radioactivity, the radioactive material is added to the PCR reaction solution to label the amplified product, and radioactivity is measured using a radioactivity meter such as a Geiger counter or a liquid scintillation counter .
In the above method, the step of determining the breed of Gerbera from the obtained amplification products may comprise the step of amplifying a nucleic acid of a known Gerbera variety using one or more primer sets of Ger-1 to Ger-20 primer sets as primers, Or by comparing the products.
Specifically, the step of determining the Gerbera variety from the amplified product may be performed by confirming the size of the amplified product (peak) using an automatic nucleotide sequencer by a computer program. For example, the presence of the amplified product (corresponding to each allele) through the primer set of the present invention for known gerbera cultivars is summarized in one table in advance. Specifically, "1" is indicated when an allele is present, and "0" is indicated when an allele is not present. Genomic DNA was isolated from a genome of the genus which was to be identified, amplified using the primer set of the present invention, analyzed for the size of the amplified product, and the result of the amplification was used to amplify the nucleic acid of the known gerbera variety The varieties of gerbera can be determined by comparison using statistical programs.
The primer set for identifying Gerbera variety according to the present invention is excellent in discrimination of various Gerbera varieties and can be used in various fields such as identification of authenticity of Gerbera varieties, examination of the seeds to be tested against the seeds, and selection of the control varieties of varieties for the protection varieties.
FIGS. 1A and 1B show the results of encoding the existence of alleles identified by using the genome sequence-identifying primer set according to the present invention in 30 varieties of Gerbera.
FIG. 2 shows the genetic similarity of each of the Gerbera cultivars analyzed by a primer set for identifying Gerbera variety according to the present invention.
The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.
Example One: Gerbera For breed identification Marker evaluation
(One) Gerbera sample
In the present invention, 30 varieties of Gerbera described in Table 2 were used for the discrimination test of Gerbera variety.
The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.
Example One: Gerbera For breed identification Marker evaluation
(One) Gerbera sample
In the present invention, 30 varieties of Gerbera described in Table 2 were used for the discrimination test of Gerbera variety.
(2) Gerbera Genome DNA Separation of
The leaves of the Gerbera cultivars described in Table 2 were collected, two tungsten beads were placed in a 2 ml Eppendorf tube, and the seeds were evenly ground by a grinder (FRITSCH, Germany). Genomic DNA was then isolated using a NucleoSpin ® Plant II (Macherey-Nagel Cat. 740 770.250) kit. The extracted genomic DNA was electrophoresed on 2% agarose gel to confirm the DNA concentration and quantified and stored in a freezer (-20 ° C) for PCR analysis.
(3) primer Production of sets
DNAs isolated from Weinou, Sweet Yellow, Lingo, Dune, Pinkver, Etta-basic, Flare, and Hymen were amplified using 99 primer sets , And 22 markers with high polymorphism were selected. For these 22 molecular beacons, fluorescently labeled oligonucleotides were prepared with one of the fluorescent materials VIC, NED, FAM and PET and used as forward primers. As a result of the analysis through the automatic genome analyzer (Genetic Analyzer 3130XL, Applied Biosystem), 20 markers having high reproducibility and high polymorphism of the band pattern were selected for the 30 Gerbera varieties. The selected 20 sets of primer sets are shown in Table 1 above.
(4) PCR Amplification and electrophoresis
PCR amplification and electrophoresis were performed using genomic DNA isolated from 30 Gerbera cultivars and 20 pairs of primer sets.
The PCR reaction mixture contained 20 ng of the genomic DNA of gerbera, a forward and reverse primer set of 10 pmol, a mixture of 2.0 μl dNTP mixture (2.5 mM), 1 μl of Taq DNA polymerase (GeNet bio, Korea), 2.5 μl of 10 × PCR buffer , 20 mM Tris-HCl, pH 8.0, 2.0 mM MgCl 2 ), and the total volume was adjusted to 25 μl. PCR (Biometra, Germany) amplification was performed by denaturing the gerbera genomic DNA at 94 ° C for 4 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 45 seconds, For final elongation, extension was carried out at 72 ° C for 10 minutes. After completion of the PCR, amplification was confirmed by electrophoresis on 2% agarose gel. The amplified samples were stored in a refrigerator at 4 ° C for the next experiment. The amplified sample was diluted to a suitable concentration in 220 μl of sterilized distilled water, and 1 to 3 μl of the diluted PCR product was mixed well with 10 μl of deionized formamide and 0.25 μl of a size marker (LIZ 500 size standard) And denatured at 94 ° C for 2 minutes. The denatured amplification product was electrophoresed through an automated sequencer. The exact size of each allelic marker was determined using a GeneMapper computer program (Applied Biosystems).
(5) According to the present invention Marker Gerbera Test breed identification
The possibility of identifying the Gerbera variety of the marker according to the present invention was investigated. Polymorphism information content (PIC) values were calculated using the following equation (1). K is the total number of alleles of each allele, and P i is the frequency of the i-th common band pattern among the bands of the marker (Anderson et al. 1993).
<
The annealing temperature (° C) by marker, the size (bp) of the amplified product, the number of alleles and the PIC value are shown in Table 3 below.
1 and 2 (marker name: Ger-1), SEQ ID NOS: 3 and 4 (marker name: Ger-2), SEQ ID NO: 5 9 and 10 (Marker name: Ger-5), SEQ ID NO: 11 and 12 (Marker name: Ger- SEQ ID NOS: 13 and 14 (marker name: Ger-7), SEQ ID NOS: 15 and 16 (marker name: Ger-8), SEQ ID NOS: 17 and 18 SEQ ID NOS: 21 and 22 (marker name: Ger-11), SEQ ID NOS: 23 and 24 (marker name: Ger-12), SEQ ID NOS: 25 and 26 (marker name: Ger- , SEQ ID NOS: 27 and 28 (marker name: Ger-14),
In addition, the presence or absence of alleles (bands) in the marker according to the present invention is judged. When there is a band, the score is coded as "1", and when there is no band, it is coded as "0".
FIGS. 1A and 1B show the results of encoding the existence of alleles identified by using the genome sequence-identifying primer set according to the present invention in 30 varieties of Gerbera. For example, the genome DNA can be isolated from the genome of the genome to be identified, amplified using the primer set of the present invention, and the amplified product can be compared with the result of Fig. 1 to identify the genus Gerbera.
FIG. 2 shows the genetic similarity of each of the Gerbera cultivars analyzed by a set of primers for identifying Gerbera variety according to the present invention. The presence of alleles was entered into the computer program of NTSYS-pc (version 2.21m) (Rohlf, 2000) and the genetic similarity value was calculated according to Jaccard's method (Sneath and Sokal, 1973). Using the calculated genetic similarity, an unweighted pair-group method with an arithmetical average (Sneath and Sokal, 1973) was used to analyze the cluster and a dendrogram was created. The results are shown in FIG.
Thus, it was confirmed that all the cultivars can be distinguished by using the primer set of the present invention.
<110> Korea seed and variety service <120> A method for identifying gerbera varieties using microsatellites markers <130> PN109509 <160> 40 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-1 F <400> 1 gcgaatatga gaatgtgaat 20 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Ger-1 R <400> 2 aagtgtagta tgatctttag aatcc 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-2 F <400> 3 caccctcatg ggtatcaagg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-2 R <400> 4 gaacaaccgg aaccgtaatg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-3 F <400> 5 catatgtaca aaacaacatt 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ger-3 R <400> 6 gaaaagtaat tacacattca t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-4 F <400> 7 aaggcagatg acccttctct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-4R <400> 8 ttgcacctta aatcacacca 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Ger-5 F <400> 9 gcacacaaca actaactggt aatc 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-5 R <400> 10 gtcgggagga tcctaaatgt 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ger-6 F <400> 11 cctacaacca aaccaaacca t 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-6 R <400> 12 ggtcctcagg aacgtttttc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-7 F <400> 13 cctcttgaaa aaccaaaacg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-7 R <400> 14 aggaagagga agaggagtgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-8 F <400> 15 tgaaacacaa caacacagat 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ger-8 R <400> 16 tttgttgttt tgacaccat 19 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Ger-9 F <400> 17 tttgtattga tatgacaaaa aggtg 25 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-9 R <400> 18 agacatgccg agaaagaaag 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-10 F <400> 19 cgggtgtttt tgattttgat 20 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ger-10 R <400> 20 tcggatcttc gattattaac ttg 23 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-11 F <400> 21 tctgctaggg tggttgtgat 20 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ger-11 R <400> 22 tgacataatt attgccaaca aca 23 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ger-12 F <400> 23 gcctaaatgt agtgtgccat t 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ger-12 R <400> 24 acaacgagca ttttgaagtt t 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ger-13 F <400> 25 cagccttggt attctcctcc t 21 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-13 R <400> 26 gcttcaaggc cgtacttctg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-14 F <400> 27 ggaagaagaa ggcgacattg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-14 R <400> 28 cccagtcgtt ggttatgctt 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-15 F <400> 29 gacgagtggt gttgaagagg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-15 R <400> 30 cgcatggttt ctttgacact 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-16 F <400> 31 cacacctccc cactcttcat 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-16 R <400> 32 cagcactgac accaaaatcg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-17 F <400> 33 tggtcgcaca gttggtttag 20 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Ger-17 R <400> 34 aaaacgcgat acaaaagtac aaca 24 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ger-18 F <400> 35 ggtgtgaaag aacatggtgg t 21 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Ger-18 R <400> 36 tgcagtttat tagcacagaa tcaag 25 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-19 F <400> 37 gagcccgatg atgatgttgt 20 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Ger-19 R <400> 38 tcgtcctcgt caccatcc 18 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ger-20 F <400> 39 ggttgtgtta gccagggaga g 21 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ger-20 R <400> 40 catttgttgc acgggagaac 20
Claims (7)
A Ger-2 primer set consisting of the oligonucleotides of SEQ ID NOS: 3 and 4;
A Ger-3 primer set consisting of the oligonucleotides of SEQ ID NOS: 5 and 6;
A Ger-4 primer set consisting of the oligonucleotides of SEQ ID NOS: 7 and 8;
A Ger-5 primer set consisting of the oligonucleotides of SEQ ID NOS: 9 and 10;
A Ger-6 primer set consisting of the oligonucleotides of SEQ ID NOS: 11 and 12;
A Ger-7 primer set consisting of the oligonucleotides of SEQ ID NOS: 13 and 14;
A Ger-8 primer set consisting of the oligonucleotides of SEQ ID NOS: 15 and 16;
A Ger-9 primer set consisting of the oligonucleotides of SEQ ID NOS: 17 and 18;
A Ger-10 primer set consisting of the oligonucleotides of SEQ ID NOS: 19 and 20;
A Ger-11 primer set consisting of the oligonucleotides of SEQ ID NOS: 21 and 22;
A Ger-12 primer set consisting of the oligonucleotides of SEQ ID NOS: 23 and 24;
A Ger-13 primer set consisting of the oligonucleotides of SEQ ID NOS: 25 and 26;
A Ger-14 primer set consisting of the oligonucleotides of SEQ ID NOS: 27 and 28;
A Ger-15 primer set consisting of the oligonucleotides of SEQ ID NOS: 29 and 30;
A Ger-16 primer set consisting of the oligonucleotides of SEQ ID NOS: 31 and 32;
A Ger-17 primer set consisting of the oligonucleotides of SEQ ID NOS: 33 and 34;
A Ger-18 primer set consisting of the oligonucleotides of SEQ ID NOS: 35 and 36;
A Ger-19 primer set consisting of the oligonucleotides of SEQ ID NOS: 37 and 38; And
A primer set comprising a set of Ger-20 primers consisting of oligonucleotides of SEQ ID NOs: 39 and 40 and identifying one or more of the Gerbera varieties listed in the following table
[table]
[table]
From the obtained amplification products, a method for identifying the Gerbera variety, comprising the step of determining the genus of the Gerbera among the Gerbera varieties listed in the following table
[table]
[table]
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Non-Patent Citations (3)
| Title |
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| AJCS 8(5):722-729 (2014) |
| Mol Breeding (2010) 26:125-132 |
| POJ 5(2):159-166 (2012) |
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