KR20150101146A - Primer set for classifing balloon flower, Classification method for balloon flower using the same, and Classification kit for balloon flower using the same - Google Patents

Abstract

The present invention provides a primer set for classifying balloon flower, a method for classifying balloon flower using the same, and a classification kit for balloon flower using the same. The primer set for classifying the balloon flower comprises one or more primer set pairs selected from: a pair of primer including a primer of sequence number 1 and a primer of sequence number 2; a pair of primers including a primer of sequence number 3 and a primer of sequence number 4; a pair of primers including a primer of sequence number 5 and a primer of sequence number 6; a pair of primers including a primer of sequence number 7 and a primer of sequence number 8; a pair of primers including a primer of sequence number 9 and a primer of sequence number 10; and a pair of primers including a primer of sequence number 11 and a primer of sequence number 12. The present invention can classify balloon flower, specifically balloon flower having phylogenetic relationships, and thus can be used in breeding or the like.

Description

TECHNICAL FIELD The present invention relates to a primer set for platycodon classification, a platelet classification method using the same, and a platelet classification kit using the same.

The present invention relates to a primer set for bellflower classification, a bellflower classification method using the bellflower, and a bellflower classification kit using the same. More particularly, the present invention relates to a primer set for bellflower classification that can genetically classify bellflower- , And a bellflower sorting kit.

The bellflower is a plant belonging to the genus Chironobacterium, and after confirming the characterization and color fixation, it is registered as an organ of the National Institute of Agricultural Science and Technology.

In Korea, bellflower is consumed a lot, but there are not many studies to improve breeding and cultivation methods.

In order to improve breeding, breeding, breeding, and the like of such strains, it is necessary to develop a molecular genetic technique capable of classifying genetically stable strains.

However, existing studies related to the bellflower studies are known to be related to ISSR markers for genetic diversity and identification of Korean and Chinese bellflower, SCAR marker-related studies for distinguishing blue and white bellflower, and RAPD marker-related studies for distinguishing bellflower There are not many studies to classify strains that have genetically related relationships.

On the other hand, cleaved amplified polymorphic sequence (CAPS) analysis is a technique for genetic marker analysis, and PCR is used for faster analysis such as Restriction Fragment Length Polymorphism (RFLP). This method is based on the logic that genetic differences between individuals (breeds) make or eliminate restriction enzyme sites and that such differences can be known from the length of DNA fragments generated by digestion. That is, it can be said that it is a method of detecting DNA sequence difference by restriction enzyme treatment. In CAPS analysis, PCR amplification is carried out through restriction sites modified according to the individual (breed), and the amplification products are digested by restriction enzymes. When separated by agarose or acrylamide gel electrophoresis, the digested PCR products consistently exhibit characteristic band patterns, and by analyzing such patterns, it is possible to classify the varieties with large genetic linkages into the same group , It is possible to distinguish the breeds in contrast to the patterns of the standard varieties.

 S, Soonshik. 2009. ISSR Markers of Authentication for Korean and Chinese Platycodon grandiflorum. Korean J. Oriental Physiology & Pathology 23 (1): 214-218.  K, Ohseong., M, Kyuhuh. 2010. Genetic Diversity and Identification of Korean and Chinese Platycodon grandiflorum Using ISSR Markers. Department of Molecular Biology, Dongeui University  C, Geonpark, K, Hwan Bang et al. 2007. Development of SCAR Marker for Discriminating between Violet Flowered Lines and White Flowered Lines in Chinese Bellflower (Platycodon grandiflorum A.). Korean J. Medicinal Crop Sci. 15 (1): 1-5.

One problem to be solved by the present invention is to provide a primer set for bellflower classification capable of classifying strains having genetically related relationships.

In addition, another object to be solved by the present invention is to provide a method of classifying flower buds that can classify flower buds having genetically related relationships.

Another object to be solved by the present invention is to provide a brood classification kit capable of classifying broodstock having genetically related relationships.

The problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention also provides a primer pair comprising a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 2, a primer pair of a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4, a primer pair of SEQ ID NO: 5 and a primer of SEQ ID NO: A pair of primers consisting of the primer of SEQ ID NO: 7 and SEQ ID NO: 8, a pair of primers of SEQ ID NO: 9 and 10, and a primer pair of SEQ ID NO: 11 and SEQ ID NO: 12 And at least one primer pair selected.

The primer set may be for amplification of the fragment amplification polymorphism sequence (CAPS) of the bellflower.

The fragment amplification polymorphism sequence may be included in a CAPS marker having one base sequence selected from SEQ ID NOS: 31 to 36. [

The platelet-dividing primer set may be a primer set for platycodon classification for analysis of the platelet genetic mutual relation.

The platycode-dividing primer set may be a primer set for platycodon classification for identification of platycodon varieties.

The platycodon may be one or more selected from the twenty-one platycodon varieties described in Table 2.

The boll broth is selected from the group consisting of Guryeodoraji, Yongdong boll, Gosong broth, Pyeongchang boll, Danyang boll, Qingda boll, Sangchoon boll, Jinan boll, Geumsan boll, Nutritional boll, Mokpo boll, Ashen boll, , Yecheon bellflower, Jodong bellflower, Kimcheon bellflower, Jangseong bellflower, and Ansung bellflower.

The present invention also provides a bellflower classification method for classifying bellflower using the primer set of the present invention.

The method comprises: (A) a sample processing step in which a genomic DNA of a bell pepper sample is used as a template and an amplification product amplified using the primer set of the present invention is cleaved with a restriction enzyme; And (B) analyzing the pattern obtained by electrophoresis of the cleavage product.

The pattern analysis may be a comparative analysis with a pattern of a standard Doragi variety.

The above-mentioned standard dandelion variety may be at least one selected from the twenty-one dandelion varieties listed in Table 2. [

The pattern of the standard bellflower variety may be obtained by using genomic DNA of the standard bellflower variety as a template and digesting the amplification product PCR-amplified using the primer set with restriction enzymes and electrophoresis.

In addition, the present invention provides a flowering-sorting kit comprising a primer set of the present invention, a restriction enzyme, and a reagent for carrying out an amplification reaction.

The restriction enzyme may be one or more of the restriction enzymes listed in Table 1.

The reagent for carrying out the amplification reaction may include a DNA polymerase, dNTPs, and a buffer.

INDUSTRIAL APPLICABILITY The present invention has the effect that the bellflower can be classified, and particularly, the bellflower having a genetically related relationship can be classified and used for breeding. In addition, it has an effect that the variety of the bellflower can be distinguished.

1 is a view for explaining a bellflower classification method according to an embodiment of the present invention.
2 is a diagram showing a base sequence for explaining the primer pairs of SEQ ID NOS: 1 and 2.
3 is a diagram showing a nucleotide sequence for explaining the primer pairs of SEQ ID NOS: 3 and 4.
4 is a diagram showing a base sequence for explaining the primer pairs of SEQ ID NOS: 5 and 6.
5 is a diagram showing a nucleotide sequence for explaining the primer pairs of SEQ ID NOS: 7 and 8;
Fig. 6 is a diagram showing a nucleotide sequence for explaining the primer pairs of SEQ ID NOS: 9 and 10; Fig.
7 is a diagram showing a base sequence for illustrating the primer pairs of SEQ ID NOS: 11 and 12;
8 is a diagram showing the results of electrophoresis using the primer pairs of SEQ ID NOS: 1 and 2.
9 is a diagram showing the results of electrophoresis using the primer pairs of SEQ ID NOS: 3 and 4.
10 shows electrophoresis results using primer pairs of SEQ ID NOS: 5 and 6.
11 is a diagram showing the results of electrophoresis using the primer pairs of SEQ ID NOS: 7 and 8.
12 is a diagram showing the results of electrophoresis using the primer pairs of SEQ ID NOS: 9 and 10.
13 is a diagram showing the results of electrophoresis using the primer pairs of SEQ ID NOS: 11 and 12.
Fig. 14 is a view showing the tree number created in the embodiment B. Fig.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will be more apparent from the following detailed description taken in conjunction with the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. But is only provided to fully inform the owner of the scope of the invention, and the present invention is only defined by the scope of the claims.

Like reference numerals refer to like elements throughout the specification. Also, "and / or" include each and every combination of one or more of the components mentioned.

The terminology used herein is for the purpose of illustrating embodiments and is not intended to be limiting of the present invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. It is noted that the terms "comprises" and / or "comprising" used in the specification are intended to be inclusive in a manner similar to the components, steps, operations, and / Or additions.

A "primer set" means a plurality of primers. In addition, the primer set may further include a container for receiving the primer.

The primer set according to one embodiment of the present invention may include at least one selected from the primer pairs set forth in Table 1. Preferably, the primer set according to an embodiment of the present invention comprises a primer pair consisting of a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 2, a primer pair of a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: And a primer pair of SEQ ID NO: 6, a primer pair of a primer of SEQ ID NO: 7 and a primer of SEQ ID NO: 8, a pair of primers consisting of a primer of SEQ ID NO: 9 and a primer of SEQ ID NO: 10, A primer pair consisting of a primer and a primer of SEQ ID NO: 12.

[Table 1] Primer

In Table 1, the primer pair consists of a forward primer and a pair of reverse primers, F means forward, and R means reverse.

This primer set is for the amplification of the fragment amplification polymorphism sequence (CAPS) of the bellflower. The PCR amplified CAPS by the primer set is treated by the restriction enzyme, and the digested PCR product exhibits a characteristic band pattern. By analyzing such patterns, it is possible not only to classify the varieties having a large genetic relationship, but also to discriminate the varieties against the patterns of the standard varieties.

Therefore, one embodiment of the present invention may be for the bellflower sorting. In addition, one embodiment of the present invention can be used for the analysis of platycodon relation, and further, it can be used for identification of platycodon.

The platycodon varieties to be classified include, but are not limited to, one or more of the platycodon varieties described in Table 2, for example.

In Table 2, the registration number means the registration number of the bryophyte variety registered at the National Institute of Agricultural Science and Technology Center of the National Institute of Agricultural Science and Technology, the classification code is the abbreviation used in this specification, and the name refers to the breed name.

[Table 2] Bellflower

A primer is a base sequence having a short free 3 'hydroxyl group, which means a short base sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of a reagent for polymerization and four nucleoside triphosphates at the appropriate buffer solution and temperature.

The primer may be an oligonucleotide and the oligonucleotide may comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, intercalating agent.

The primer can be produced through a chemical synthesis method using a phosphoramidite method, a phosphodiester method, a diethylphosphoramidite method, or the like. It is to be understood that the primer sequence can be modified by means known in the art.

The base sequence to be amplified by the primer pair may be a CAPS marker, and the CAPS marker can be designed from the contig shown in Table 3 below. The CAPS marker may have CAPS, as shown in Figs. 2-7. That is, depending on the SNPs present in each strain, they may not be cleaved or cleaved by restriction enzymes. FIG. 2 is a diagram showing a base sequence for explaining the primer pairs of SEQ ID NOS: 1 and 2, FIG. 3 is a diagram showing a base sequence for explaining the primer pairs of SEQ ID NOS: 3 and 4, 5 and 6 show the nucleotide sequences for explaining the primer pairs of SEQ ID NOS: 7 and 8, and FIG. 6 shows the primer pairs of SEQ ID NOS: 9 and 10, respectively. FIG. 7 is a view showing a nucleotide sequence for explaining the primer pairs of SEQ ID NOs: 11 and 12. FIG. In the figure, the underlined part indicates that the restriction enzyme cut part is not cut or uncut according to the breed, and the box mark indicates the corresponding position of the primer sequence. Such a CAPS marker may have the nucleotide sequence shown in Table 4. < tb > < TABLE > The nucleotide sequence shown in Table 4 may be a cDNA sequence.

[Table 3] Contig

(Table 3 continued)

(Table 3 continued)

(Table 3 continued)

[Table 4] Marker sequence

In addition, it is possible to implement the platycodonization method of the present invention using a primer set which is an embodiment of the present invention.

Hereinafter, an exemplary embodiment of the present invention will be described in detail.

Specifically, the broodstock can be classified by a method including (A) the sample processing step shown in FIG. 1, and (B) the pattern analysis step.

(A) the sample processing step is a step of digesting an amplification product amplified by using a primer set, which is one embodiment of the present invention, with restriction enzymes using a genomic DNA of a bellows sample as a template, and (B) And analyzing the obtained pattern by electrophoresis.

Methods for isolating the genomic DNA of the bellows sample can be performed by a method known in the art. For example, a CTAB method, a commercially available wizard prep kit (Promega), and the like can be used. The target sequence can be amplified by performing amplification reaction using a separated genomic DNA as a template and using a primer set as an example of the present invention as a primer. At this time, the target sequence may be a CAPS marker. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with a Q [beta] replicase, or a method for amplifying a nucleic acid molecule known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are common in the art and commercially available kits can be used.

The restriction enzyme may be one or more of those listed in Table 1.

The pattern analysis may be a comparative analysis with a pattern of the standard bryophyte variety, and the standard bryophyte variety may be one or more selected from the 21 bryophyte varieties listed in Table 2. [

The pattern of the standard bellflower variety may be obtained by using the genomic DNA of the standard bellflower variety as a template, and digesting the amplification product amplified using the primer set with restriction enzymes and electrophoresis.

After cutting as described above, the pattern obtained by electrophoresis shows a characteristic pattern according to genetically related individuals (cultivars), so that the platelets can be classified based on the patterns.

That is, it is possible to classify the bellflower group or the bellflower group by analyzing the band pattern that appears when the fragments obtained after treatment with the restriction enzyme are separated using a gel electrophoresis apparatus.

In addition, the primer set which is one embodiment of the present invention may be included in the bellflower sorting kit.

Hereinafter, one embodiment of the platelet sorting kit according to the present invention will be described in detail.

Specifically, one embodiment of the strait classification kit according to the present invention may include a primer set, restriction enzymes, and a reagent for performing an amplification reaction, which are one embodiment of the present invention.

The restriction enzyme may be one or more of the restriction enzymes listed in Table 1.

In addition, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer, and the like.

In addition, the bellflower sorting kit may further include instructions describing optimal reaction performing conditions. The instructions may be a recording medium that describes how to use the kit, for example, PCR buffer preparation method, reaction conditions, and the like.

The matters referred to in each of the primer set for bellflower classification, the bellflower classification method and the bellflower classification kit, which are one embodiment of the present invention, are applied within the same range unless they are contradictory to each other.

Hereinafter, a primer set for bellflower classification, a bellflower classification kit, and a bellflower classification method, which are one embodiment of the present invention, will be described in detail with reference to specific examples.

The balloon used in the specific example is shown in Table 2. Unless otherwise noted, these platycodon were re-sold by the Horticultural Breeding and Molecular Breeding Laboratory of Andong University, which was sold by the National Institute of Agricultural Science and Technology.

<Specific Example A> A primer set for bell flower classification

A-1. RNA extraction

In order to maximize the genetic distance, RNA extraction was used to collect the strains collected from the most distant parts of the region. In order to achieve this purpose, Doraji 4 (Gangwon Province, Pyeongchang), Doraji 16 (Jeollanamdo, Sunchang) and Boraji 17 (Gyeongsangbuk-do, Yecheon) were used. One month old bellflower leaves are collected in 2 ml tubes with stainless beads and rapidly frozen with liquid nitrogen (LN2). Vortex, and RNeasy Plant Mini Kit (Cat No. 74904, QIAGEN, Valencia, CA, USA) was used. The experimental procedure was in accordance with the manufacturer's instructions. The RNA obtained is adjusted to a final concentration of 250 ng / L and a final volume of 50 L, and stored frozen at -20 ° C.

A-2. mRNA purification and cDNA library creation

mRNA purification and cDNA library generation are described in Zhong et al. (2011) (Silin Zhong, Je-Gun Joung, Yi Zheng, Yun-ru Chen, Bao Liu, Ying Shao, Jenny Z. Xiang, (2011) High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation. Cold Spring Harb Protoc; doi: 10.1101 / pdb.prot5652). MRNA was isolated and purified from total RNA using Oligo (dT) 25 Dynabeads (Invitrogen 610-05). Prepare Oligo (dT) 25 Dynabeads (Invitrogen 610-05) for the amount of sample RNA and sample volume, then remove all storage solution on ice. Oligo (dT) 25 Dynabeads are washed with 200 ul 1x Binding buffer A (1% beta-ME) and magnet. Add 50ul total RNA to Oligo (dT) 25 Dynabeads treated with 50ul 2x Binding Buffer A (1% beta-ME) and mix well. Incubate at 65 ° C for 1 min and at room temperature for 10 min. Then, spin down the tube lightly to remove any solution. Wash with 150 μl wash buffer (1% beta-ME) per sample. Add 50 ul TE (1% beta-ME) to the tube and incubate for 1 minute at 70 ° C and place on ice. After adding 50 μl of 2x Binding buffer A (1% beta-ME) per sample, incubate at 65 ° C for 1 minute and at room temperature for 10 minutes. Lightly spin down and remove all solutions. Wash with 150ul Wash buffer (w / o 1% beta-ME) three times. For fragmentation of purified mRNA, beads were reproduced using 10ul Superscript Buffer mixture (0.5ul of 5x first strand buffer, 0.5ul of hexamer (1ug / ul), 0.5ul of oligo dT VN (100ng / ul) I will. After mRNA fragmentation at 94 ° C for 5 minutes, place it on ice. Lightly spin down and transfer the mRNA-containing solution to the new tube.

CDNA was prepared by using Superscript III RT kit (Invitrogen) for purified mRNA. Following the manufacturer's instructions, add a reverse transcription mix (TDN 5.88 ul, dNTP (10 mM) 1 ul, Actinomycin D 0.12 ul, DTT (100 mM) 2 ul, RNase Inhibitor 0.5 ul, Superscript III 0.5 ul) , At 50 ° C for 50 minutes. Immediately after the treatment, add 36ul RNA Clean XP beads (Agencourt A66514), mix well and incubate on ice for 15 minutes. Wash the magnet twice with 75% ethanol, dry for 2 minutes, and obtain RNA / cDNA hybrids with 10 ul of tertiary distilled water. Subsequently, a second strand reaction mixture (TDW 2.4 ul, dUTP (10 mM) + dATP (10 mM), dCTP (10 mM), dGTP (10 mM), 10 x Blue Buffer 1.5 ul, RNase H (5 U / , DNA pol I (10 U / ul) 0.5 ul) is prepared on ice. Add 5 ul of the mixture to an eluted RNA / cDNA hybrid and incubate at 16 ° C for 2 hours and 30 minutes. Add 1.8 volumes of AMPure XP beads (Agencourt A63881), incubate for 15 minutes, wash twice with 75 degrees Celsius ethanol, and then elute with tertiary distilled water at 10 degrees Celsius. End - repair Mix (Enzymatics, Y914 - LC - L) was used for the end - repair process of the prepared cDNA. Prepare dTTP 2.75 ul, dNTP mix (10 mM, 0.5 ul), 10x End-Repair Buffer 1.5ul and End-Repair Enzyme LC 0.25ul according to the manufacturer's instructions, add 5ul of each of the samples to each eluted dsDNA and mix well Incubate at 20 ° C for 30 minutes. Immediately after the end-repair process, washing is done using AMPure XP beads (Agencourt A63881). For the dA-Tailing process, Klenow 3'-5 'exo- (Enzymatics P701-LC-L) is used. Prepare dA-Tailing Mastermix (TDU 2.5ul, dATP mix (10mM) 0.5ul, 10x Blue Buffer 1.5ul, Klenow exo- 0.5ul) according to the manufacturer's instructions. Add 5ul dA-Tailing mastermix to the cDNA sample, mix well, and incubate at 37 ° C for 30 minutes. Immediately after the dA-Tailing process, again wash using AMPure XP beads (Agencourt A63881). Prepare ligation master mix (2 μl of 10 × ligation buffer, 0.5 μl of Truseq adapter (5 μM), 1 μl of T4 DNA Ligase, and 6.5 μl of TDW) using T4 DNAligaseEnzymatics L603-HC-L for barcode adapter ligation. Add 10 ul ligation mastermix to each cDNA and mix. Treated at 37 ° C for 30 minutes, then on ice for 15 minutes. For size selection and Uracil DNA Glycosylase (UDG) digestion, add 1.4 volumes of AMPure XP beads (Agencourt A63881) to each sample, incubate at room temperature for 10 minutes, and then wash twice with 75% ethanol. After elution with 10 ul TE (w / o 1% beta-ME), 4.5 ul TE and 0.5 ul UDG (Enzymatics G501L) are added to each sample and treated at 37 ° C for 15 minutes. Then add 1.4 volumes of AMPure XP beads (Agencourt A63881) to each sample and incubate at room temperature for 10 minutes. After washing twice with 75% ethanol, elution with 15ul TDW. For the PCR enrichment process, prepare PCR reaction (5 μl of UDG digested DNA, 1 μl of Index Primer (10 μM), 10 μl of 5x Buffer, 10 mM, 0.5 μl of dNTP, 33 μl of TDW, 0.5 μl of Phusion HF (New England Biolabs)). The PCR conditions were denaturation at 98 ° C for 2 minutes, denaturation at 98 ° C, annealing at 65 ° C for 30 seconds, extension at 72 ° C for 20 seconds, The reaction is carried out for 2 minutes. To confirm the preparation of the cDNA library, electrophoresis on 1.2% agarose gels at 4 μl per sample to confirm the presence of a band between 200-300 bp. Add 1.2 volumes of AMPure XP beads (Agencourt A63881), wash twice with 75% ethanol, and elute with 20ul TE. Then, quantify the library using the Qubit dsDNA HS assay kit (Invitrogen Q32851). The quantified library is mixed with 20 ug per library, then 1.4 volumes of AMPure XP beads (Agencourt A63881) are added and Elution is performed with 10ul TE.

A-3. Primer set design

The contigs of three species of bellflower were synthesized through the denovo assembly, and the SNPs were identified through alignment with each other using the RNA Seq information of bellflower 4, bellflower 16, and bellflower 17. All bioinformatics analyzes were performed using CLC Assembly Cell (CLC Bio, Denmark). The CAPS markers were designed using the CAPS design tool provided by the Sol Genomics Network (http://solgenomics.net) based on the identified SNPs, and the CAPS markers were designed based on the restriction sites and lengths Primer was designed.

The created contig is as shown in Table 3. The SNPs are shown by solid lines and dotted lines in Figs. 2 to 7, and the boxed portions in Figs. 2 to 7 correspond to the primers. The CAPS markers are as shown in Table 4 and Figs. 2 to 7.

Specifically, designed primers and restriction enzymes are as shown in Table 1. certain In Contig, the presence of specific restriction enzyme cleavage sites at the first SNP site was confirmed. Primer production was performed by designating primers at positions spaced by 100-200 bp in both 5 'and 3' directions at SNP positions. DNA oligomers used as primers were synthesized by Bioneer (Korea) and synthesized by applying 'phosphite triester' method, which connects phosphodiester bonds using cyanoethyl phosphoramidite developed by Koster. Since the CAPS marker was constructed based on the results of RNA seq, the subsequent experiments were conducted considering that PCR and restriction enzyme treatment using genomic DNA may be different from the prediction.

One or more pairs of the primers set forth in Table 1 prepared by such a method may be included to prepare a primer set for a bell flower sorting or a bellflower sorting kit.

&Lt; Specific Example B >

B-1. Genomic DNA extraction

The platelet DNA extraction described in Table 2 is carried out by rapid-freezing with liquid nitrogen (LN2) after collecting young leaves in a 2 ml tube containing Stainless bead at 4-5 leaf stage during growth. Finely crush with Vortex, add 300ul of CTAB Buffer containing 0.2% sodium bisulfite, and mix well. After processing for 30 minutes in a water bath at 65 ° C, add 150ul of Chloroform and mix well. Then centrifuge at 4 ° C for 15 minutes to induce layer separation. Transfer 200 ul of the layered supernatant to a new tube, add 200 ul of pre-chilled 100% Isopropanol, and slowly invert. Centrifuge for 30 seconds to leave only the DNA pellet and remove any liquid from the tube. After adding 750 ul of 70% Ethanol, centrifuge for 30 seconds after inverting as before. After completely removing the liquid from the tube, dissolve in 100 μl of DNase-free water in a tube containing only the DNA pellet, and store at -20 ° C.

B-2. Amplification (PCR)

PCR was carried out using 1 μl of gDNA, 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl, 2 mM MgSO ₄ , 0.1% 0.2 mM each dNTP, 0.4 mM forward and reverse primers, 5 units Taq polymerase (TaKaRa, Otsu, Shiga, Janpan), a total of 25ul. PCR was performed using a T-100 Thermal Cycler (BIO-RAD, Hercules, CA, USA) for PCR reaction. The PCR conditions were denaturation at 94 ° C for 10 min and denaturation at 94 ° C for 1 min. Annealing at annealing temperature, extension at 72 ° C for 1 min was repeated 35 times and reaction was carried out at 72 ° C for 10 minutes.

B-3. Restriction enzyme treatment

PCR product 10ul of the total volume of 25ul was electrophoresed to confirm whether the PCR amplification was carried out. 2ul of the restriction enzyme shown in Table 1, 2ul of buffer and 2ul of tertiary distilled water were added to 15ul PCR product, For 1 hour.

B-4. Electrophoresis

For genomic DNA, agarose gel was prepared at the concentrations listed in Table 1, taking into account the expected fragment length. Gel was stained with EtBr after electrophoresis and PCR amplification was performed using Gel Doc 2000 (BIO-RAD, Hercules, Genotyping was performed. 8 to 13 show electrophoresis results.

B-5. Pattern analysis

The electrophoresis results (FIG. 8 to FIG. 13) obtained according to the primer pairs were analyzed, and platycodon species (lines) showing the same pattern were classified and the phylogenetic tree was created using the results.

FIG. 8 shows the results of electrophoresis using the primer pairs of SEQ ID NOS: 1 and 2, FIG. 9 shows the results of electrophoresis using the primer pairs of SEQ ID NOS: 3 and 4, Fig. 11 shows the results of electrophoresis using the primer pairs of SEQ ID NOs: 7 and 8, Fig. 12 shows the results of electrophoresis using the primer pairs of SEQ ID NOs: 9 and 10, Pair electrophoresis results.

In FIGS. 8 to 13, numerals 01 to 21, which are integers, denote a serial number denoting the platelets of Table 2, and M denotes a 1 Kb DNA ladder, and one uppercase letter denotes a group classified by each primer pair. WD indicates that the PCR showed poor results.

As shown in FIG. 8, by the analysis of PS03 CAPS marker, 21 kinds of bellflower were classified into two groups of genotyping, and 15 kinds and 6 kinds belong to each group.

Also, as shown in FIG. 9, by the analysis of PS08 CAPS markers, 21 kinds of balloons were classified into three groups of genotyping, and 7 kinds, 3 kinds, and 11 kinds belong to each group.

As shown in FIG. 10, genotyping was classified into 6 groups according to the analysis of PS10 CAPS marker, and 21 strains were classified into 6 groups, and 5 strains, 1 strain, 5 strains, 8 strains, 1 strain, 1 strain Can be found.

Also, as shown in FIG. 11, by the analysis of the PS13 CAPS marker, 21 kinds of broodstock were classified into three groups of genotyping, and 5 kinds, 5 kinds, and 11 kinds belong to each group.

As shown in FIG. 12, the genotyping was classified into 7 groups according to the analysis of the PS23 CAPS marker, and 21 strains were classified into 7 groups. Three strains, one strain, four strains, nine strains, one strain, one strain , And one variety belongs.

Also, as shown in FIG. 13, by the analysis of the PS24 CAPS markers, 21 types of broodstock were classified into two groups of genotyping, and 13 cultivars and 8 cultivars belong to each group.

This pattern was used as a pattern of a standard Dorago cultivar, and B-1 of Example B was applied to an unknown platelet sample. To B-4. By comparing the obtained patterns, it can be seen that the bellows samples can be classified. It is also possible to classify the cultivars. This can be confirmed by phylogenetic tree analysis.

[Analysis of phylogenetic tree]

Based on the results of the pattern analysis of B-5., Similarity to the genotype of the bellflower was evaluated using the DARwin 5.0 version (CIRAD, French) to calculate the arithmetic mean UPGMA (unweighted pair-group method with arithmetic average) Based method. A pattern of the PCR amplification product of a specific size was expressed as a natural number, and a phylogenetic tree was prepared. The results are shown in Fig.

Figure 14 shows the results of the phylogenetic analysis. The natural number of the tree species means the platycodon of Table 2.

As can be seen from FIG. 14, the results of the comprehensive flexible relationship analysis on the six markers show that all the blooming varieties (21 species) to be analyzed are classified into the respective groups. Thus, it can be seen that not only the bellflower classification but also the bellflower variety to be analyzed can be discriminated by using the six markers.

From these results, it can be seen that not only the broodstock can be classified by the primer set of the present invention, but also the variety can be further classified. For the unknown bellflower sample, B-1 of Example B was used. To B-4. By comparing the patterns obtained in the same manner with the patterns of the standard Doragi cultivars, it is possible to identify cultivars by checking whether they belong to each of the 21 cultivars. Platycodon, which does not correspond to this species, can be classified as a separate species.

<110> Andong National University Industry-Academic Cooperation Foundation <120> Primer set for classifing balloon flower, Classification method          for balloon flower using thd same, and Classification kit for          balloon flower using the same <130> GP14005 <160> 36 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PS03 <400> 1 atgccatact ctatcgtt 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PS03 <400> 2 tctctaggag ctcgaaga 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PS08 <400> 3 cgcgtcccgt agcgcatt 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PS08 <400> 4 accgaaggtt gcgattgg 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PS10 <400> 5 ccacttgtgc aattgcgc 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PS10 <400> 6 gagattttga tccacaat 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PS13 <400> 7 taacgacggc cgcgccca 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PS13 <400> 8 tctcgtgctg ccatctac 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PS23 <400> 9 gacggaggag gcgatgacct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PS23 <400> 10 acccaatgac ttcaatctcg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PS24 <400> 11 ttttccacgt gggcttcgat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PS24 <400> 12 ctggaggtga agtggtcgtc 20 <210> 13 <211> 602 <212> DNA <213> Platycodon grandiflorum <400> 13 atgccatact ctatcgttcc tgttccacaa caccctattg aagtctctga aaagccatat 60 tttttgggat ttttgattgc gcttagcatg agattgtagg cgtcgacgta gatagttctc 120 atccctagtg tggttttcag ggtaaccaat gtttggttaa tcttggaatt gaacgagtga 180 gccactgcgt tataggtagc gtcacatttg gtttgaccct ttaatgtctt gactagagga 240 acgcatccga acggtagaac cccaacaact cccaatcttc gagctcctag agaatgcatc 300 gtcttaacat aagaaaccaa gcaagagatg agataatctt ggtattgttc cacagtgaaa 360 tttgcagggc gattaggttc aacatagtaa ttttggagaa aatcatttgt tcccaaactc 420 ataacaaatg caccgttttg gatgattttt tcagcctgtt tcttccccac gattttcgct 480 agatggatct tgtaatgctt gaaatatact agttgttggg acatagataa cacacccgag 540 agttcagcgg ttagattatc ataaccagaa ccagctgaag caaaactaac accatgtaaa 600 ag 602 <210> 14 <211> 602 <212> DNA <213> Platycodon grandiflorum <400> 14 atgccatact ctatcgttcc tgttccacaa caccctattg aagtctccga aaagccatat 60 tttttgggat tttcaattgc gcttagcatg agattgtagg cgtcgacgta gatagttctc 120 atccctagtg tggttttcag ggtaaccaat gtttggttaa tcttggaatt gaacgagtga 180 gccactgcgt tataggtagc gtcacatttg gtttgaccct ttaatgtctt gactagagga 240 atgcatccga acggtagaac cccaacaact cccaatcttc gagctcctag agaatgcatc 300 gtcttaacat aagaaaccaa gcaagagatg agataatctt ggtattgttc cacagtgaaa 360 tttgcagggc gattaggttc aacatagtaa ttttggagaa agtcatttgt tcccaaactc 420 ataacaaatg caccgttttg gatgattttt tcagcctgtt tcttccccac gattttcgct 480 agatggatct tgtaatgctt gaaatatact agttgttggg acatagataa cacacccgag 540 agttcagcgg ttagattatc ataaccagaa ccagctgaag caaaactaac accatgtaaa 600 ag 602 <210> 15 <211> 541 <212> DNA <213> Platycodon grandiflorum <400> 15 ttttttggga ttttcaattg cgcttagcat gagattgtag gcgtcgacgt agatagttct 60 catccctagt gtggttttca gggtaaccaa tgtttggtta atcttggaat tgaacgagtg 120 agccactgcg ttataggtag cgtcacattt ggtttgaccc tttaatgtct tgactagagg 180 aatgcatccg aacggtagaa ccccaacaac tcccaatctt cgagctccta gagaatgcat 240 cgtcttaaca taagaaacca agcaagagat gagataatct tggtattgtt ccacagtgaa 300 atttgcaggg cgattaggtt caacatagta attttggaga aagtcatttg ttcccaaact 360 cataacaaat gcaccgtttt ggatgatttt ttcagcctgt ttcttcccca cgattttcgc 420 tagatggatc ttgtaatgct tgaaatatac tagttgttgg gacatagata acacacccga 480 gagttcagcg gttagattat cataaccaga accagctgaa gcaaaactaa caccatgtaa 540 a 541 <210> 16 <211> 858 <212> DNA <213> Platycodon grandiflorum <400> 16 ctgacggcag gttctgagtt cgcttaacaa acgcgtcccg tagcgcattg tacgcctgag 60 tttgcaaccg cgtcaccgcg gttcccgagt caactatgat cccgcctcgg ccactttcgt 120 cgactgcgaa taccgacggc gagatcgaca gcggctcccc accgacgctg attccggtta 180 gtccaacgta gaagaaggta cgcactctgg agttgcgaag caagggcgcg acaaccgagt 240 tgcctggacg agccgagttg aactcgagcg tcgacgacga ggtggcgtca cggctcacga 300 ggcagtacga gaacgacgag gccttgaact gagacggaag cgagagagag ccgccgccga 360 gccccataag cccagccgag ccgacaaaca gtccttcgtt gtcgtgaccg cacccaatcg 420 caaccttcgg tatcgagcct gagttggtaa acgacagcgt ttcggtgacg aattccccca 480 cggtgaaaga tccgtcaccg aaggaaacct ggtacagaca cgtgtcactc ctgcaggcgg 540 agatgtcgag agcggagcac tgttgtgaac cgcaggataa gggactgtaa gtggaggaag 600 cggacgggtt aaaaaccggg tcggactgtt ggtaacagtc tatacatggc tcgcattgga 660 tccaagtgac gtcacttcca gtgtcaatgg ccatgtaaaa attcttaacg ggttgaccca 720 ctccaagtcg ggcaaagtac tcaccgctgc cttgagaaaa cccggaggta ataggagtcg 780 aaagttcctc cggttggacg acagtctcaa caggtttcag gtcagacctg ttgaaaccag 840 tgagtgcgaa ctcgagtt 858 <210> 17 <211> 858 <212> DNA <213> Platycodon grandiflorum <400> 17 ctgacggcag gttctgagtt cgcttaacaa acgcgtcccg tagcgcattg tacgcctgag 60 tttgcaaccg cgtcacggcg gttcccgagt caactatgat cccgcctcgg ccactttcgt 120 cgactgcgaa taccgacggc gagatcgaca gcggctcccc accgacgctg attccggtta 180 gtccaacgta gaagaaggta cgcactctgg agttacgaag caagggcgcg acaaccgagt 240 tgcctggacg agccgagttg aactcgagcg tcgacgacga ggtggcgtca cggctcacga 300 ggcagtacga gaacgacgag gccttgaact gagacggaag cgagagagag ccgccgccga 360 gccccataag cccagccgag ccgacaaaca gtccttcgtt gtcgtgaccg cacccaatcg 420 caaccttcgg tatcgagcct gagttggtaa acgacagcgt ttcggtgacg aattccccca 480 cggtgaaaga tccgtcaccg taggaaacct ggtacagaca cgtgtcgctc ctgcaggcgg 540 agatgtcgag agcggagcac tgttgtgaac cgcaggataa gggactgtaa gtggaggaag 600 cggacgggtt aaaaaccggg tcggactgtt ggtaacagtc tatacatggc tcgcattgga 660 tccaagtgac gtcacttcca gtgtcaatgg ccatgtaaaa attcttaacg ggttgaccca 720 ctccaagtcg ggcaaagtac tcaccgctgc cttgagaaaa cccggaggta ataggagtcg 780 caagttcctc cggttggacg accgtctcaa caggtttcag gtcagacctg ttgaaaccag 840 tgagtgcgaa ctcgagtt 858 <210> 18 <211> 681 <212> DNA <213> Platycodon grandiflorum <400> 18 tgtcaaccat gcaaccccat gcctgttgtc gtttacgtga acatttgatg tatacataac 60 ggttacattt taatacaccc ttactctttc acttatttgc attacatgcc acttgatgga 120 aatactaaag gaatcaaaca tactacctac cacggtacca ccctcaattg tgggcattcg 180 cacttaaatc acttattaca gttggatccc acctagacgg gcaaccaccc tagcagggcc 240 ccacctctta ataacaccaa taaatatttt cacctgtccc ccatctttac accagtattt 300 atcaaaatac aaataatatc tccttctccg ttcagggatg aaaaagtaat ttcacaaact 360 atattgattg gttcaaattt tcgttttaac agttattggg cgtaaagccg acaagggaat 420 tggctatatc ataactaaca cgtgtcccct gctgctgtat gttgcctatg atggacagcg 480 acccttgcgt cgccgcaaac gctaggcaga atttgccact tgaatctact ggtaccaggt 540 aattcttcgg ctgcaaaaac agcgttttcc ctcctgagaa ctgaaacgac accgtcggca 600 cggaagccgt ggtcattgac gacaggtcgt aacaagtatc gaacaacgaa aaactgccac 660 ctgacggcag gttctgagtt c 681 <210> 19 <211> 765 <212> DNA <213> Platycodon grandiflorum <400> 19 caacaacggg accacaaagt gaagcgaaat catcgctcct catagtgtaa aatgtgctaa 60 ggaacataat cctggttcta gtagtagtag ccacaaaccg gagaattgcc cgtttgtatc 120 cacctttgcc cttagattca taaggcacct taaccgtgtc tttaccggca aacgcctcca 180 ctatcattga cccttcacaa gagttgctag catctcccac agcaaacgag agctggtagg 240 ttgtcccaac cttggttcgg gccacttgtg caattgcgct ttctttacca gcaacaagct 300 caacggctcg ttttccttgt ggaactgaga agtgatcgga atctatgtac ttgacggctt 360 tgagggattc gatcatccat gccgggaggg gagagtgatc atcttcaatg tttgggggta 420 ctaggactcc ccaagatgta ttggggaaaa catatggacc ttcttcaaag tcaccatttt 480 tcaataagtt ctgattagta gcttttggag ggtacagagc tttaatggca atggaatcga 540 tgagcggccc acaagcagga tcctcctcaa ctcctggatt gtggcaaa atctccacca 600 cattatacat tgcctgaaat gcccacgcgt acgagtccca tccattacta ctatacaacg 660 tctgcatcgg caacacgcca gagtccggtg caacggacac gttcagctgt tcctcttggg 720 cacaagtacg ggcagcgcta aacgtgatgg aatagtacat tcctt 765 <210> 20 <211> 765 <212> DNA <213> Platycodon grandiflorum <400> 20 caacaacggg accacaaagt gaagcgaaat catcgctcct catagtgtaa aatgtgctaa 60 ggaacataat cctggtccta gtagtagtag ccacaaaccg gagaattgcc cgtttgtatc 120 cacctttgcc cttagattca taaggcacct taaccgtgtc tttaccggca aacgcctcca 180 ctatcattga cccttcacaa gagttgctag catctcccac agcaaacgag agctggtagg 240 ttgtcccaac cttggttcgg gccacttgtg caattgcgct ttctttaccg gcaacaagct 300 caacggctcg ttttccttgt ggaactgaga agtgatcgga atctatgtac ttgacggctt 360 tgagggattc gatcatccat gccgggaggg gagagtgatc atcttcaatg tttgggggta 420 ctaggactcc ccatgatgta ttggggaaaa catatggacc ttcttcaaag tcaccatttt 480 tcaataagtt ctgattagtg gcttttggag ggtacagagc tttaatggct atggaatcga 540 tgagcggtcc acaagcagga tcctcctcaa ctcctggatt gtggcaaa atctccacca 600 cattatacat tgcctgaaat gcccacgcgt acgagtccca tccattacta ctatacaacg 660 tctgcatcgg caacacgcca gagtccggtg caacggacac gttcagctgt tcctcttggg 720 cacaagtacg ggcagcgcta aacgtgatgg aatagtacat tcctt 765 <210> 21 <211> 501 <212> DNA <213> Platycodon grandiflorum <400> 21 gtcaacaacg ggaccacaaa gtgaagcgaa atcatcgctc ctcatagtgt aaaatgtgct 60 aaggaacata atcctggtcc tagtagtagt agccacaaac cggagaattg cccgtttgta 120 tccacctttg cccttagatt cataaggcac cttaaccgtg tctttaccgg caaacgcctc 180 cactatcatt gacccttcac aagagttgct agcatctccc acagcaaacg agagctggta 240 ggttgtccca accttggttc gggccacttg tgcaattgcg ctttctttac cggcaacaag 300 ctcaacggct cgttttcctt gtggaactga gaagtgatcg gaatctatgt acttgacggc 360 tttgagggat tcgatcatcc atgccgggag gggagagtga tcatcttcaa tgtttggggg 420 tactaggact ccccatgatg tattggggaa aacatatgga ccttcttcaa agtcaccatt 480 tttcataag ttctgattag t 501 <210> 22 <211> 909 <212> DNA <213> Platycodon grandiflorum <400> 22 cgagaaggtg ttttacacaa gagaatggtt atggttgaat ccaaaggaaa agcccaataa 60 aggtctcgac ggaagctcta tcttttagct gaggggttcc cgtctgtaca taagtgacca 120 ttagtttggg ccggcaatag ccttcaaaac gttaaagaag tcatgtttgt atccactgga 180 atggaaatgt tgacgcggga tgaccttcgt gtacaaatag aagacgatgc aagcaattgt 240 aggcccaacc caaaaaaccc agtgaccatt ccataggtga cctcctctaa cgacggccgc 300 gcccaaacac cgagcagggt tcattccagc accagcataa cccttctttg cagtaacagt 360 tgtcgatata aacaccagta ggcccaaaat gattccgatg actgagcaaa caatcacacg 420 gcccaactcc tttgcttgac gatgatcgta ggccatccaa atcgaagcaa aaagaaaaat 480 gaatgtacaa attatctcga gccaaagagc ctggctagtc tcaaggccca ctacaacagg 540 gccatttggg cctggtgcaa ttactgtgag ggtacagcct ccaagtgaaa aggtttttgc 600 aatagtgctg ctcaccacgg ctttgagtgc tagtgcacct aggacagaac caagacattg 660 tgcaacaatg tagatggcag cacgagagag cgaaataaga ccaacgaggg cggcggagaa 720 ggagatcaca ggattcatgt ggccgccgga aattggcaag acggcgagaa gcagaattgt 780 gatagtaata gctacaagga tcgatattaa caagtttggc atcttggttt ctgtctcaaa 840 cgaagagatg actatagtgt caagcgcaaa gacaagtaca gctgagccga acagctcccc 900 tatcgatgc 909 <210> 23 <211> 909 <212> DNA <213> Platycodon grandiflorum <400> 23 cgagaaggtg ttttacacaa gagaatggtt atggttgaat ccaaaggaaa agcccaataa 60 aggtctcgac ggaagctcta tcttttagct gaggggttcc cgtctgtaca taagtgacca 120 ttagtttggg ccggcaatag ccttcaaaac gttaaagaag tcatgtttgt atccactgga 180 atggaaatgt tgacgcggga tgacctttgt gtataaatag aagacgatgc aagcaattgt 240 aggcccaacc caaaaaaccc agtgaccatt ccataggtga cctcctctaa cgacggccgc 300 gcccaaacac cgagcagggt tcattccagc accagcataa cccttctttg cagtaacagt 360 tgtcgatata aacaccagta ggcccaaaat gattccgatg actgagcaaa caatcacacg 420 gcccaactcc tttgcttgac gatgatcgta ggccatccaa atcgaagcaa aaagaaaaat 480 gaatgtacaa attatctcga gccaaagggc ctggctcgtc tcgaggccca ctacaacagg 540 gccatttggg cctggtgcaa taactgtgag ggtacagcct ccaagtgaaa aggtttttgc 600 aatagtgctg ctcaccacgg ctttaagtgc tagtgcacct agtacagaac caagacattg 660 tgcaacaatg tagatggcag cacgagagag agaaataaga ccaacgagcg cggcggagaa 720 ggagatcaca ggattcatgt ggccgccgga aattggcaag acggcgagaa gcagaattgt 780 gatagtaata gctacaagga tggatattaa caggtttggc atcttggttt ctgtctcaaa 840 cgaagagatg actatagtgt caagcgcaaa gacaagtaca gctgagccga acagctcccc 900 tatcgatgc 909 <210> 24 <211> 504 <212> DNA <213> Platycodon grandiflorum <400> 24 gttttacacaga agagaatggt tatggttgaa tccaaaggaa aagcccaata aaggtctcga 60 ccgaagctct atcttttagc tgaggggttc ccgtctctac attagtgacc attagtttgg 120 gccggcaata gccttcaaaa cgttaatgaa gtcatgtttg tatccactgg aatggaaatg 180 ttgacgcggg atgaccttcg tgtacaaata gaagacgatg caagcaattg taggcccaac 240 ccaaaaaacc cagtgaccat tccataggtg acctcctcta acgacggccg cgcccaaaca 300 ccgagcaggg ttcattccag caccagcata acccttcttt gcagtaacag ttgtcgatat 360 aaacaccagt aggcccaaaa tgattccgat gactgagcaa acaatcacac ggcccaactc 420 ctttgcttga cgatgatcgt aggccatcca aatcgaagca aaaagaaaaa tgaatgtaca 480 aattatctcg agccaaagag cctg 504 <210> 25 <211> 641 <212> DNA <213> Platycodon grandiflorum <400> 25 gccaaccacg gacgggaagg tgaagtagtg gaacccgcac actgcccggc agaaatcctg 60 tacggtgacg tctacggaag ttaggacgag gtagatccct tttttgtggt ctactgggaa 120 acgggcggtc ttgacggagg aggcgatgac ctcttggata gacaaacggg tgaggtgagt 180 accgtgagag tagaggtggt ctgtgtattc tccggcgacg aggacggaac gggagatgtt 240 ggcgccggtt tggtcggtgt agagggagat ggtttgccac cagtcggaga cggaggggaa 300 aggggcggcg cggcggttgg tggtggagat ggagagaagg aagtccttga tgaggagctt 360 ctggggaggg gaccacttgc cgtaccagat gatgtaaatg ttgatgggtg aggagagaac 420 gggacccatg tggtaacgga ggttgacgag ctccgatgag ccctcgaatt tcttggagga 480 tgttagggtt ctgggtggga gttgggggtt gacgagattg aagtcattgg gttgttttac 540 aatattgagg gtttggacta gagagtggga gtgaatggag tagaagaaga aggagaagga 600 ggaggagagg aaaaggaggg tactgaggaa aaaagacatg a 641 <210> 26 <211> 641 <212> DNA <213> Platycodon grandiflorum <400> 26 gccaaccacg gacgggaagg tgaagtagtg gaacccgcac actgcccggc agaaatcctg 60 tacggtgacg tctacggaag ttaggacgag gtagatccct tttttgtggt ctactgggaa 120 acgggcggtc ttgacggagg aggcgatgac ctcttggata gacaaacggg tgaggtgagt 180 accgtgggag tagaggtggt ctgtgtattc tccggcgacg aggacggaac gtgagatgtt 240 ggcgccggtt tggtcggtgt agagggagat ggtttgccac cagtcggaga cggaggggaa 300 aggggcggcg cggcggttgg tggtggagat ggagagaagg aagtccttga tgaggagctt 360 ctggggaggg gaccacttgc cgtaccagat gatgtaaatg ttgatgggtg aggagagaac 420 gggacccatg tggtaacgga ggttgacgag ctccgatgag ccctcgaatt tcttggagga 480 tgttagggtt ctgggtggga gttgggggtt gacgagattg aagtcattgg gttgttttac 540 aatattgagg gtttggacta gagagtggga gtgaatggag tagaagaaga aggagaagga 600 ggaggagagg aaaaggaggg tactgaggaa aaaagacatg a 641 <210> 27 <211> 683 <212> DNA <213> Platycodon grandiflorum <400> 27 ctgtttaccc gagtttccca cccaagcgta gggcaaagtg tagccaacca cggacgggaa 60 ggtgaagtag tggaacccgc acactgcccg gcagaaatcc tgtacggtga cgtctacgga 120 agttaggacg aggtagatcc cttttttgtg gtctactggg aaacgggcgg tcttgacgga 180 ggaggcgatg acctcttgga tagacaaacg ggtgaggtga gtaccgtggg agtagaggtg 240 gtctgtgtat tctccggcga cgaggacgga acgggagatg ttggcgccgg tttggtcggt 300 gtagagggag atggtttgcc accagtcgga gacggagggg aaaggggcgg cgcggcggtt 360 ggtggtggag atggagagaa ggaagtcctt gatgaggagc ttctggggag gggaccactt 420 gccgtaccag atgatgtaaa tgttgatggg tgaggagaga acgggaccca tgtggtaacg 480 gaggttgacg agctccgatg agccctcgaa tttcttggag gatgttaggg ttctgggtgg 540 gagttggggg ttgacgagat tgaagtcatt gggttgtttt acaatattga gggtttggac 600 tagagagtgg gagtgaatgg agtagaagaa gaaggagaag gaggaggaga ggaaaaggag 660 ggtactgagg aaaaaagaca tga 683 <210> 28 <211> 1192 <212> DNA <213> Platycodon grandiflorum <400> 28 acaactagta aggctttaat aaatctagcg tgtcctacaa tttacagttt ctgcagaatc 60 tctatacatt tgagatcacg tggcagggaa catttctttc aaggactcca aagttttctt 120 tctttatgac aagttgagga tctttctttc caggtaattg aacctctgga tgtcctcaaa 180 ttctgtcgga cgcactgaca taaaacattt caaggtaacc cgagatcgat tccccccaaa 240 acgagctagg atccattctg gttagctttt gacagcaatt atttgtatag caggaatggc 300 ttcaagggtg tggcgtagct gtattgtagt attctatctt atgcagactg caacgtcacc 360 tggcccgcag ttgtttgctc aaactccgca caagcctctg gggtgtcttc ttcactaggt 420 ggaaccagtt gccagaacaa tggcagatat ttgtcccact cgttcaagat agccaagcct 480 ttgctgcttc cagttttttc cacgtgggct tcgataaggc tcttcagctg catctgaccc 540 acaggtgcaa ccactctctg gatcttcacg atttccttgt ttaccttggg aataagagta 600 tcatcttcat caagaatgta tgccaagcct ccagtcatac cagctgctac atttcgacca 660 acttttccaa gcactacaac acaacctcca gtcatgtact cgcaactatg atctccggtg 720 ccttccacta ctgcttgagc aagtgaattt ctaacagcaa aacgctcccc agctttgcct 780 ctaacaaata gttgaccacc tgttgctcca tataagcagg tgttccctat tatggtggcc 840 tcctcggggc aaaatccagt cttctcaaca ggagtgacga ccacttcacc tccagccata 900 cccttcccca catagtcgtt agcttctcct actaatcgaa tgttcattcc aggagtcaaa 960 aaacaagcaa atgattggcc agcactccct gtaaatgtta tattcaactg cccagcaaaa 1020 ccagtgtcac catacttctt tgcaactaca cctgctaggc gcccacaaac agcacggtcc 1080 acattgtata tctgtatggt cttatttacc actttttcat tctcaatggc ttctgctatc 1140 tcaacatctg aaagcaaagt atcatccaga acaggaccat tactgtgagc at 1192 <210> 29 <211> 1193 <212> DNA <213> Platycodon grandiflorum <400> 29 acaactagta aggctttaat aaatctagcg tgtcctacaa tttacagttt ctgcagaatc 60 tctatacatt tgagatcacg tggcagggaa catttctttc aaggactcca aagttttctt 120 tctttatgac aagttgagga tatttctttc caggtaattg aacctctgga tgtcctcaaa 180 ttctgtcgga cgcactgaca taaaacattt caaggtaacc cgagatcgat tccccccaaa 240 acgagctagg atccattctg gttagctttt gacagcaatt atttgtatag caggaatggc 300 ttcaagggtg tggcgtagct gtattgtagt attagaatct tatgcagact gcaacgtcac 360 gt; tggaaccagt tgccagaaca atggcagata tttgtcccac tcgttcaaga tagccaagcc 480 tttgctgctt ccagtttttt ccacgtgggc ttcgataagg ctcttcagct gcatctgacc 540 cacaggtgca accactctct ggatcttcac gatttccttg tttaccttgg gaataagagt 600 atcatcttca tcaagaatgt atgccaagcc tccagtcata ccagctgcta catttcgacc 660 aacttttcca agcactacaa cacaacctcc agtcatgtac tcgcaactat gatctccggt 720 gccttccact actgcttgag caagtgaatt tctaacagca aaacgctccc cagctttgcc 780 tctaacaaat agttgaccac ctgttgctcc atataagcag gtgttcccta ttatggtggc 840 ctcctcgggg caaaatccaa tcttctcaac aggagtgacg accacttcac ctccagccat 900 acccttcccc acatagtcgt tagcttctcc tactaatcga atgttcattc caggagtcaa 960 aaaacaagca aatgattggc cagcactccc tgtaaatgtt atattcaact gcccagcaaa 1020 accagtgtca ccatacttct ttgcaactac acctgctagg cgcccacaaa cagcacggtc 1080 cacattgtat atctgtatgg tcttatttac cactttttca ttctcaatgg cttctgctat 1140 ctcaacatct gaaagcaaag tatcatccag aacaggacca ttactgtgag cat 1193 <210> 30 <211> 1434 <212> DNA <213> Platycodon grandiflorum <400> 30 ttttttttta acaaaggaga gcttttcgga tatagctctg atagaaagga aactttaaag 60 acagaacgtc actttcagaa tacaacagta cttgaataaa tgatagtgtt cgagatcatt 120 tctagccaat gcagaaattt ggataacgga tttagaaaac ttgaagaagc actgatgaac 180 ctattatctt tcaatatgct ttttcttaat tacgtgaagg aagttaccag tagtaacaac 240 aagccatcac aactagtaag gctttaataa atctagtatg tcctacaatt tacagtttct 300 gcagaatctc tatacatttg agatcacgtg gcagggaaca tttctttcaa ggactccgaa 360 gttttctttc tttatgacaa gttgaggatc tttctttcca ggtaattgaa cctctggatg 420 tcctcaaatt ctgtcggacg cactgacata aaacatttca aggtaacccg agatcgattc 480 cccccaaaac gagctaggat ccattctggt tagcttttga cagcaattat ttgtatagca 540 ggaatggctt caagggtgtg gcgtagctgt attgtagtat tagaatctta tgcagactgc 600 aacgtcacct ggcccgcagt tgtttgctca aactccgcac aagcctctgg ggtgtcttct 660 tcactaggtg gaaccagttg ccagaacaat ggcagatatt tgtcccactc gttcaagata 720 gccaagcctt tgctgcttcc agttttttcc acgtgggctt cgataaggct cttcagctgc 780 atctgaccca caggtgcaac cactctctgg atcttcacga tttccttgtt taccttggga 840 ataagagtat catcttcatc aagaatgtat gccaagcctc cagtcatacc agctgctaca 900 tttcgaccaa cttttccaag cactacaaca caacctccag tcatgtactc gcaactatga 960 tctccggtgc cttccactac tgcttgagca agtgaatttc taacagcaaa acgctcccca 1020 gctttgcctc taacaaatag ttgaccacct gttgctccat ataagcaggt gttccctatt 1080 atggtggcct cctcagggca aaatccagtc ttctcaacag gagtgacgac cacttcacct 1140 ccagccatac ccttccccac atagtcgtta gcttctccta ctaatcgaat gttcattcca 1200 ggagtcaaaa aacaagcaaa tgattggcca gcactccctg taaatgttat attcaactgc 1260 ccagcaaaac cagtgtcacc atacttcttt gcaactacac ctgctaggcg cccacaaaca 1320 gcacggtcca cattgtatat ctgtatggtc ttatttacca ctttttcatt ctcaatggct 1380 tctgctatct caacatctga aagcaaagta tcatccagaa caggaccatt actg 1434 <210> 31 <211> 293 <212> DNA <213> Platycodon grandiflorum <400> 31 atgccatact ctatcgttcc tgttccacaa caccctattg aagtctccga aaagccatat 60 tttttgggat tttcaattgc gcttagcatg agattgtagg cgtcgacgta gatagttctc 120 atccctagtg tggttttcag ggtaaccaat gtttggttaa tcttggaatt gaacgagtga 180 gccactgcgt tataggtagc gtcacatttg gtttgaccct ttaatgtctt gactagagga 240 atgcatccga acggtagaac cccaacaact cccaatcttc gagctcctag aga 293 <210> 32 <211> 400 <212> DNA <213> Platycodon grandiflorum <400> 32 cgcgtcccgt agcgcattgt acgcctgagt ttgcaaccgc gtcaccgcgg ttcccgagtc 60 aactatgatc ccgcctcggc cactttcgtc gactgcgaat accgacggcg agatcgacag 120 cggctcccca ccgacgctga ttccggttag tccaacgtag aagaaggtac gcactctgga 180 gttgcgaagc aagggcgcga caaccgagtt gcctggacga gccgagttga actcgagcgt 240 cgacgacgag gtggcgtcac ggctcacgag gcagtacgag aacgacgagg ccttgaactg 300 agacggaagc gagagagagc cgccgccgag ccccataagc ccagccgagc cgacaaacag 360 tccttcgttg tcgtgaccgc acccaatcgc aaccttcggt 400 <210> 33 <211> 334 <212> DNA <213> Platycodon grandiflorum <400> 33 ccacttgtgc aattgcgctt tctttaccag caacaagctc aacggctcgt tttccttgtg 60 gaactgagaa gtgatcggaa tctatgtact tgacggcttt gagggattcg atcatccatg 120 ccgggagggg agagtgatca tcttcaatgt ttgggggtac taggactccc caagatgtat 180 tggggaaaac atatggacct tcttcaaagt caccattttt caataagttc tgattagtag 240 cttttggagg gtacagagct ttaatggcaa tggaatcgat gagcggccca caagcaggat 300 cctcctcaac tcctggattg tggatcaaaa tctc 334 <210> 34 <211> 400 <212> DNA <213> Platycodon grandiflorum <400> 34 taacgacggc cgcgcccaaa caccgagcag ggttcattcc agcaccagca taacccttct 60 ttgcagtaac agttgtcgat ataaacacca gtaggcccaa aatgattccg atgactgagc 120 aaacaatcac acggcccaac tcctttgctt gacgatgatc gtaggccatc caaatcgaag 180 caaaaagaaa aatgaatgta caaattatct cgagccaaag agcctggcta gtctcaaggc 240 ccactacaac agggccattt gggcctggtg caattactgt gagggtacag cctccaagtg 300 aaaaggtttt tgcaatagtg ctgctcacca cggctttgag tgctagtgca cctaggacag 360 aaccaagaca ttgtgcaaca atgtagatgg cagcacgaga 400 <210> 35 <211> 400 <212> DNA <213> Platycodon grandiflorum <400> 35 gacggaggag gcgatgacct cttggataga caaacgggtg aggtgagtac cgtgggagta 60 gaggtggtct gtgtattctc cggcgacgag gacggaacgt gagatgttgg cgccggtttg 120 gtcggtgtag agggagatgg tttgccacca gtcggagacg gaggggaaag gggcggcgcg 180 gcggttggtg gtggagatgg agagaaggaa gtccttgatg aggagcttct ggggagggga 240 ccacttgccg taccagatga tgtaaatgtt gatgggtgag gagagaacgg gacccatgtg 300 gtaacggagg ttgacgagct ccgatgagcc ctcgaatttc ttggaggatg ttagggttct 360 gggtgggagt tgggggttga cgagattgaa gtcattgggt 400 <210> 36 <211> 400 <212> DNA <213> Platycodon grandiflorum <400> 36 ttttccacgt gggcttcgat aaggctcttc agctgcatct gacccacagg tgcaaccact 60 ctctggatct tcacgatttc cttgtttacc ttgggaataa gagtatcatc ttcatcaaga 120 atgtatgcca agcctccagt cataccagct gctacatttc gaccaacttt tccaagcact 180 acaacacaac ctccagtcat gtactcgcaa ctatgatctc cggtgccttc cactactgct 240 tgagcaagtg aatttctaac agcaaaacgc tccccagctt tgcctctaac aaatagttga 300 ccacctgttg ctccatataa gcaggtgttc cctattatgg tggcctcctc ggggcaaaat 360 ccagtcttct caacaggagt gacgaccact tcacctccag 400

Claims (8)

A primer pair consisting of a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 2,
A primer pair consisting of a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4,
A primer pair consisting of a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 6,
A primer pair consisting of a primer of SEQ ID NO: 7 and a primer of SEQ ID NO: 8,
A primer pair consisting of a primer of SEQ ID NO: 9 and a primer of SEQ ID NO: 10, and
A primer set of SEQ ID NO: 11 and at least one primer pair selected from the primer pairs consisting of the primers of SEQ ID NO: 12. 2. The primer set of claim 1, wherein the primer set is for amplification of fragment amplification polymorphism sequence (CAPS) of bellflower. The primer set according to claim 1, wherein the platelet-dividing primer set is a primer set for platycodon classification for analysis of platycodon genetic affinity. 2. The primer set according to claim 1, wherein the primer set for platycodon classification is a primer set for platycodon classification. A method for classifying a bellflower using the primer set of claim 1. 6. The method of claim 5,
The bellflower classification method
(A) a sample processing step of using a genomic DNA of a bellows sample as a template and digesting the amplified product amplified with the primer set of claim 1 with a restriction enzyme; And
(B) a pattern analysis step of analyzing a pattern obtained by electrophoresis of the cleavage product. A straining kit comprising the primer set of claim 1, a restriction enzyme, and an amplification reaction performing reagent. [Claim 7] The flowering-sorting kit according to claim 7, wherein the restriction enzyme is at least one of the restriction enzymes listed in Table 1, and the amplification reaction-performing reagent comprises a DNA polymerase, dNTPs, and a buffer.

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