KR102144673B1 - KASP primer set based on SNP for discriminating Korean melon cultivar and F1 hybrid purity checking and uses thereof - Google Patents

Abstract

The present invention relates to a primer set for SNP-based KASP and its use for distinguishing melon varieties and testing the purity of F ₁ seeds, and the 23 SNP markers of the present invention are distributed over all 12 chromosomes of melon, and quickly and accurately Since it can be distinguished, it will be able to be used for cultivation of melon varieties.

Description

SNP-based KASP primer set based on SNP for discriminating Korean melon cultivar and F1 hybrid purity checking and uses thereof for differentiation of melon varieties and F1 seed purity testing

The present invention relates to a primer set for SNP-based KASP and its use for distinguishing melon varieties and testing the purity of F ₁ seeds.

Melon ( cucumis melo var. makuwa ) is a vine plant belonging to the cucurbit family, and taxonomically, melon ( Cucumis melo ). The domestic seed market for gourd crops is about 32 billion won, accounting for about 20% of the total vegetable seed market, of which melon is the second largest seed market after watermelon among gourds, and the market size and cultivation area are somewhat larger every year. It is on an increasing trend.

Almost all of the melons currently in circulation are hybrid varieties produced through mating between parents. In one major hybrid (F ₁ ) cultivar, a purity test is performed on the produced seeds, which is a quality control that tests whether the intended parental crossing, that is, heterozygosity, is expressed as purity (%). Speak of an act. In this regard, there may be contamination of other pollen during fertilization, children of the parent, and simple mixing of other seeds. Among them, it is known that the frequency of occurrence of the parental child individuals is the highest. Therefore, all seed companies basically maintain their own test system for the purity test of F ₁ seeds. This is not only a quality control dimension that maintains the basic genetic characteristics of the hybrid variety, but also a technological means to secure high-purity seeds essential for the cultivation and production of high-quality agricultural products. It is a process that has a very important meaning, such as the effect of preventing civil complaints from occurring.

Melons are economically and commercially important crops in Korea, and the importance of fostering export varieties is also remarkably emerging. Since melon has a short generation and is well fixed compared to other crops, the time it takes to develop a line is very short, so for the protection of the developed variety and line, it is essential to develop a molecular marker for the protection of the variety.

On the other hand, Korean Patent No. 1516190 discloses'SSR primer sets for distinguishing melon strains or varieties and their uses', and Korean Patent No. 1447112 discloses'Dried persimmon melon varieties, which are Korean native species from cultivated melon varieties. A biomarker for screening and its use is disclosed, but there is no description of the primer set for SNP-based KASP and its use for the identification of melon varieties and F ₁ seed purity assay of the present invention.

The present invention was derived from the above requirements, and the inventors of the present invention refer to the Korean native species Dried persimmon melon as a reference genome, and various melon ( Cucumis melo var. makuwa ) After selecting 23 melon-specific single nucleotide polymorphism (SNP) markers that can distinguish melon cultivar from 12 chromosomes of melon through genome analysis, a primer set for KASP to amplify the marker was prepared and genotyped. By performing an analysis and confirming that 17 melon varieties can be distinguished, the present invention was completed.

In order to solve the above problems, the present invention comprises a set of 23 oligonucleotide primers, characterized in that three adjacent oligonucleotides starting from SEQ ID NO: 1 in the oligonucleotides represented by SEQ ID NOs: 1 to 69 are one primer set. , Melon ( Cucumis melo var. makuwa) provides a primer set for KASP (kompetiive allele specific PCR) for distinguishing breeds and F ₁ seed purity of black.

In addition, the present invention is the primer set; And it provides a kit for KASP for distinguishing melon varieties and assaying the purity of F ₁ seeds including reagents for performing an amplification reaction.

In addition, the present invention comprises the steps of separating genomic DNA from a melon sample; Using the isolated genomic DNA as a template and performing KASP using the primer set of the present invention; And analyzing the amplification product of the KASP; containing, it provides a method for distinguishing melon varieties and assaying the purity of F ₁ seeds.

The primer set for KASP of the present invention can distinguish various melon varieties and can test the purity of F ₁ seeds at the whole genome level of melon, so it will be usefully utilized as an efficient biomarker for the development of melon varieties, variety protection and seed production. I will be able to.

1 shows SNPs present on 12 chromosomes of melon, and the 23 SNPs finally selected are indicated in blue.

In order to achieve the object of the present invention, the present invention provides a set of 23 oligonucleotide primers, characterized in that three adjacent oligonucleotides starting from SEQ ID NO: 1 in the oligonucleotides represented by SEQ ID NOs: 1 to 69 are one primer set. Containing, melon ( Cucumis melo var. makuwa) provides a primer set for KASP (kompetiive allele specific PCR) for distinguishing breeds and F ₁ seed purity of black.

The melon varieties of the present invention are five bok honey melon, five bok honey plus melon, champignon honey melon, honey love melon, native honey melon, rich honey melon, king honey melon, new gold melon, daebak honey melon, gold melon, manata It may be honey melon, Daejanggeum plus melon, eight bok honey melon, yellow honey melon, gold ax melon, chimney melon or super gold lump melon, but is not limited thereto.

The term "kompetitive allele specific PCR (KASP)" of the present invention is one of a polymerase chain reaction (PCR)-based analysis method, and is a homogenous and fluorescence-based genotyping analysis technique. KASP is a technology based on fluorescence resonance energy transfer for allele-specific oligo extension and signal generation.

The primer set of the present invention is a primer set that detects the base type of a single nucleotide polymorphism (SNP) marker that is specifically differentiated in a melon locus.

In the primer set of the present invention, three consecutive oligonucleotides of SEQ ID NOs form one primer set, and consist of two forward primers and one reverse primer. Specifically, a FAM or HEX fluorescent substance is attached to the 5'end of the forward primer, and a base representing SNP is bonded to the 3'end, and the reverse primer consists of the same base pair to the allele of the SNP portion of the forward primer. (Table 3).

The primer set of the present invention may preferably include one or more primer sets selected from the group consisting of the 23 primer sets, but when the 23 primer sets are used at the same time, melon varieties can be more efficiently distinguished. .

The primers of the present invention are 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, depending on the sequence length of each primer. It may include an oligonucleotide consisting of segments of at least two, at least 25, or at least 26 contiguous nucleotides. For example, the primers of SEQ ID NO: 1 (28 oligonucleotides) are 15 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more in the sequence of SEQ ID NO: 1, It may include an oligonucleotide consisting of fragments of 26 or more and 27 or more nucleotides. In addition, the primer may include an addition, deletion or substitution of the nucleotide sequences of SEQ ID NOs: 1 to 69. Oligonucleotides of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64 and 67 The primer is a forward primer to which a fluorescent substance FAM is attached, and SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, The oligonucleotide primers of 56, 59, 62, 65 and 68 are HEX-attached forward primers, and SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, Oligonucleotide primers of 42, 45, 48, 51, 54, 57, 60, 63, 66 and 69 are reverse primers.

In the primer set according to an embodiment of the present invention, the aforementioned 17 melon varieties may be distinguished by combining 23 primer sets represented by SEQ ID NOs: 1 to 69. For example, the ohbok honey melon (1) of Table 5 below is SEQ ID NO: 13, 14 and 15; SEQ ID NOs: 16, 17 and 18; SEQ ID NOs: 19, 20 and 21; SEQ ID NOs: 22, 23 and 24; SEQ ID NOs: 25, 26 and 27; SEQ ID NOs: 28, 29 and 30; SEQ ID NOs: 31, 32 and 33; SEQ ID NOs: 34, 35 and 36; SEQ ID NOs: 37, 38 and 39; SEQ ID NOs: 49, 50 and 51; SEQ ID NOs: 52, 53 and 54; SEQ ID NOs: 55, 56 and 57; SEQ ID NOs: 58, 59 and 60; SEQ ID NOs: 61, 62 and 63; And it can be distinguished by using the primer sets of SEQ ID NOs: 64, 65 and 66. In addition, in addition to Obok Honey Melon, Obok Honey Plus Melon, Chamsiso Honey Melon, Honey Love Melon, Native Honey Melon, Rich Honey Melon, King Honey Melon, Shingeum Salagi Melon, Daebak Honey Melon, Gold Melon, Manata Honey Melon, Daejanggeum Plus melon, eight bok honey melon, yellow honey melon, golden ax melon, melon or super gold melon can also be distinguished by combining 23 primer sets.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence that is complementary to a nucleic acid strand to be copied, and can serve as an initiating point for the synthesis of a primer extension product. The length and sequence of the primers should allow starting the synthesis of the extension product. The specific length and sequence of the primers will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required.

In the present invention, the oligonucleotide used as a primer may also comprise a nucleotide analogue, for example, phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or It may include an intercalating agent. In addition, the primers can incorporate additional features that do not change the basic properties of the primers serving as the starting point for DNA synthesis. If necessary, the primer nucleic acid sequence of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg, horse radish peroxidase (HRP), alkaline phosphatase), radioactive isotopes (eg, ³² P), fluorescent molecules, chemical groups (eg, biotin), and the like. have.

The suitable length of the primer is determined by the characteristics of the primer to be used, but is usually used in a length of 15 to 30 bp. The primer does not have to be exactly complementary to the sequence of the template, but must be complementary enough to form a hybrid-complex with the template.

The present invention also, the primer set; And it provides a kit for KASP for distinguishing melon varieties and assaying the purity of F ₁ seeds including reagents for performing an amplification reaction. Reagents for performing the amplification reaction may include DNA polymerase, dNTPs, and buffers, but are not limited thereto. The dNTPs include dATP, dCTP, dGTP, and dTTP, and the DNA polymerase may be a commercially available polymerase such as Taq DNA polymerase or Tth DNA polymerase as a heat-resistant DNA polymerase. In addition, the kit of the present invention may further include a user's manual describing the optimum reaction performance conditions. The guide is a handout explaining how to use the kit, e.g., how to prepare reverse transcription and PCR buffers, and the reaction conditions presented. The guide includes a brochure in the form of a brochure or flyer, a label affixed to the kit, and a description on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.

The present invention also,

Separating genomic DNA from the melon sample;

Using the isolated genomic DNA as a template and performing KASP using the primer set for KASP of the present invention; And

Analyzing the amplification product of the KASP; containing, it provides a method for distinguishing melon varieties and assaying the purity of F ₁ seeds.

In the method for distinguishing melon varieties of the present invention, the primer set for KASP is the same as described above.

The method of the present invention includes the step of isolating genomic DNA from a melon sample. As a method of separating the genomic DNA, a method known in the art may be used. For example, the CTAB method may be used, or the Wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template and using the primer set according to an embodiment of the present invention as a primer, an amplification reaction may be performed to amplify a target sequence. Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. amplification system), strand displacement amplification, or amplification via Qβ replicase or any other suitable method for amplifying nucleic acid molecules 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 well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified sequence may be labeled with a detectable labeling material. In one embodiment, the labeling material may be a material that emits fluorescence, phosphorescence, or radioactivity, but is not limited thereto. Preferably, the labeling material may be FAM, HEX, VIC, JOE, ROX, TAMRA, Cy3 or Cy5. When amplifying the target sequence, PCR is performed by labeling the labeling material at the 5'-end of the primer, so that the target sequence can be labeled with a detectable fluorescent labeling material. The set of oligonucleotide primers used to amplify the target sequence is as described in Table 3 below.

In addition, in the method according to an embodiment of the present invention, the step of analyzing the amplification product of KASP may be performed through genotyping. Specifically, the genotype can be distinguished through the fluorescent substance FAM or HEX attached to the 5'end of the forward primer and the SNP bonded to the 3'end to distinguish the melon variety. If the genotype is homozygous, only one of the two fluorescence (FAM or HEX) is detected. If FAM is detected, it can be determined as genotype'A', and if HEX is detected, it can be determined as genotype'B'.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

1.SNP Marker Information

To create a reference genome, genome information was obtained from dried persimmon melon by Next Generation Sequencing (NGS) and Hi-C method, and information on the melon genome consisting of 12 chromosomes was constructed through various bioinformation analysis methods. (Laboratory). NGS read for genome analysis of 36 different native melon and melon strains, including Yeolgol melon, Euncheon melon, Anjong melon, Gangseo melon, Chosun melon, Mook melon, Yellow melon, Ganchi melon, Gaeruri melon, and wild melon. Was produced, and the produced reads were mapped to the dried persimmon melon reference sequence using the BWA (Burrows-Wheeler Alignment tool) after removing the sequence of low quality. The mapped reads are double masked and localized using GATK (http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit), a genome analysis tool, to increase the accuracy of SNP calling. After performing the local realignment process, SNP (single nucleotide polymorphism) was predicted using the Unified Genotyper, and 526,271 SNPs were obtained through the filtering process (Table 1). Among them, 519 SNPs that can be analyzed were selected for each chromosome. In addition, 4 SNPs per chromosome were analyzed for even distribution of SNPs by 12 chromosomes (Table 2 and FIG. 1), and it was confirmed that 34 SNPs were properly reacted.

Number of SNPs per chromosome chromosome SNP number chr01 30,722 chr02 56,304 chr03 46,341 chr04 39,895 chr05 62,051 chr06 101,010 chr07 67,189 chr08 66,693 chr09 17,183 chr10 11,224 chr11 16,472 chr12 11,187 Sum 526,271

SNP markers selected for each chromosome chromosome Length (Kb) SNP number Average number of SNPs per 500 Kb chr01 30,382.071 49 0.81 chr02 22,746.494 39 0.86 chr03 21,121.645 36 0.85 chr04 29,198.730 50 0.86 chr05 27,871.970 39 0.70 chr06 34,894.544 55 0.79 chr07 23,387.212 42 0.90 chr08 32,358.245 55 0.85 chr09 19,018.732 34 0.89 chr10 19,826.103 36 0.91 chr11 26,513.796 48 0.91 chr12 20,136.143 36 0.89 Sum 307,455.685 519 0.84

3. SNP Marker Selection and SNP based KASP primer making

The KASP (Kompetitive allele specific PCR) primer set constructed based on the selected 34 SNPs consists of two forward primers and one reverse primer. The forward primer was designed so that a FAM or HEX fluorescent substance was attached to the 5'end and the difference in base sequence such as SNP was distinguished at the 3'end. And the reverse primer was designed to work with the two forward primers to allow the PCR reaction to proceed (Table 3). When PCR was performed, amplification was performed with different fluorescent substances attached to the 5'ends of the two forward primers. Therefore, genotype was analyzed according to the difference in wavelength of the fluorescent substances. If the genotype is homozygous, only one of the two fluorescence (FAM or HEX) is detected. If FAM is detected, it can be determined as genotype'A', and if HEX is detected, it can be determined as genotype'B'. SNPs present in the reference genome (dried persimmon melon) were designed to be detected by the FAM-attached forward primer.

4. SNP Marker For detection KASP Reaction conditions

It was carried out by the method recommended by LGC genomics (Laboratory of the Government Chemist Genomics), and the reagents used in the experiment were KASP 2X master mix and KASP Assay mix (LGC genomics). KASP reaction conditions are shown in Table 4 below.

KASP conditions step Temperature(℃) time Number of repetitions Hot-start activation 94 15 seconds One Denature 94 20 seconds 10 Annealing/Elongation 61~55
(Decrease by 0.6℃ per cycle) 1 min Denature 94 20 seconds 26
Annealing/Elongation 57 1 min

Example One. KASP for primer Distinguish melon varieties using a set

Genotyping was performed on 17 melon varieties using the 34 KASP primer sets. The 17 melon varieties are as follows; 1. Obok Honey Melon (Nongwoo Bio); 2. Obok Honey Plus Melon (Nongwoo Bio); 3. True smile honey melon (Nongwoo Bio); 4. Honey love melon (Nongwoo Bio); 5. Native honey melon (Pam Hannong); 6. Rich honey melon (Pam Hannong); 7. King Honey Melon (Pam Hannong); 8. Shingeum Salagi Melon (Pam Hannong); 9. Daebak Honey Melon (Palm Hannong); 10. Golden melon (Asian seedlings); 11. Manata honey melon (Asian seedlings); 12. Daejanggeum Plus melon (Asian seedlings); 13. The Beatitude Honey Melon (Hyundai Jongmyo); 14. Hwangkal Honey Melon (Hyundai Jongmyo); 15. Golden Ax Melon (Hyundai Jongmyo); 16. Chamkal melon (Koregon); And 17. Super gold chunk melon (Cheiljongmyo). SNPs present in the reference genome can be detected by a forward primer attached with a FAM fluorescent substance, and are represented by genotype A.

As a result, for example,'Obok Honey Melon' is genotype A, bgs00659, bgs04958 in genotyping analysis results using primer sets for amplifying markers C01-sp010886, bgs00289, C09-sp000673, C08-sp000276, C03-sp004954 and bgs01444. , C09-sp007875, bgs04175, bgs04286, bgs05223, bgs00068, bgs52645 and bgs02242 In the result of genotyping using primer sets for marker amplification, genotype B was shown, and the combination of such genotyping results could be distinguished from other melon varieties; 'Obok Honey Plus Melon' is genotype A, bgs00659, bgs04958, C09-sp007875, bgs04286, bgs05223, bgs00068 in the results of genotyping analysis using primer sets for amplifying markers C01-sp010886, bgs00289, C09-sp000673, C08-sp000276 and bgs01444. , bgs52645 and bgs02242 In the result of genotyping using a primer set for amplifying markers, genotype B was indicated, and it could be distinguished from other melon varieties through the combination of the results of the gene expression analysis. However, among the 34 primer sets, the genotyping result of 11 primer sets is not clearly identified as isotype (A or B) or heterotype (H), and results indicated by A/H, B/H genotype, or'-' Was confirmed, and was excluded from the primer set for finally distinguishing the melon variety (primer set capable of amplifying the marker indicated in blue in Table 5 below).

3-F; A copy of the true smile honey melon.

3-M; A model of true smile honey melon.

4-F; A copy of honey love melon.

4-M; An example of honey love melon.

Example 2. KASP for primer Melon F with set _One Seed purity test

Genotyping analysis was performed on the copies and mothers of the true smile honey melon or honey love melon using the above primer set for KASP. As a result, as shown in Table 5, it was detected as homozygous in both the parent and the counterpart of the true miso honey melon or honey love melon. This means that the locus of the developed marker is a well-fixed position in the fixed species (original or parent) used for breed development, so it is a useful position for purity testing in F ₁ . In addition, primer sets of bgs01227, bgs14324, bgs03305, bgs21920, C01-sp011515, bgs00074, bgs00659, bgs04958, bgs05223, bgs05705, bgs07516, bgs08106, bgs02460 or bgs02242 can be used for F ₁ purity testing because they exhibit polymorphism between the master and the counterpart. And it was found.

The primer set for KASP of the present invention was developed based on the SNPs that exist for each chromosome of melon, and it was thought that the genotype could be analyzed at the level of the whole genome, so that the purity test could be performed more accurately and efficiently.

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cggctcctat ttgacacttt atacatgta 29 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gccacgcttg ttggagatga c 21 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 ggccacgctt gttggagatg at 22 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 tctccacttc ctccgaaaat aactagttt 29 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 ctcgatgtgt tattccactt gc 22 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 cctctcgatg tgttattcca cttgt 25 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 tggtatgaca gggcagccag ca 22 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 cagcacaatt aggattttga tggcc 25 <210> 41 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 cagcacaatt aggattttga tggct 25 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 cgacttggca aactaagtcg ccatt 25 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 aagaacctca aaacttttgt atactcaaac 30 <210> 44 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 gaaagaacct caaaactttt gtatactcaa at 32 <210> 45 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 cgcttatcaa agcttgcctc gacta 25 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 cttatgattg ccaacaatat tcctgt 26 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 cttatgattg ccaacaatat tcctgc 26 <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 caaaagctta aattagtggg tgaaggcaa 29 <210> 49 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 gttagttaat cttaaatcgt tggagctca 29 <210> 50 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 agttaatctt aaatcgttgg agctcg 26 <210> 51 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 tagtgtggga acctaatgga ccaataaat 29 <210> 52 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 aatgggttat cttcaacaac tattaaaagc 30 <210> 53 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 aaaatgggtt atcttcaaca actattaaaa gt 32 <210> 54 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 gagtacatgc caaacactac aggaaataa 29 <210> 55 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 tccaactcac tcaattgtga cttaca 26 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 ccaactcact caattgtgac ttacc 25 <210> 57 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 57 gcctccaagt gttgagacgt acaaa 25 <210> 58 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 aattgatgaa acttataagt gcaagtgg 28 <210> 59 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 gtaattgatg aaacttataa gtgcaagtga 30 <210> 60 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 cgttgctcta atctccattc acaaatcat 29 <210> 61 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 aattggttta aggtccaagt tataaactg 29 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 caattggttt aaggtccaag ttataaacta 30 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 caccgtctca ttagctcgag agaaa 25 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 64 gtccaggtta tttgtaagcc tttgg 25 <210> 65 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 gtccaggtta tttgtaagcc tttgc 25 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 tgggtattca taaaacttca cgaaaaccta 30 <210> 67 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 acattcaaat agcaaatgac taacagattc 30 <210> 68 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 aatacattca aatagcaaat gactaacaga ttt 33 <210> 69 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 cctgtcattg ttgatgatgt ctttggtaa 29 <210> 70 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 gttcaaattc ggttcctaca acttattaaa 30 <210> 71 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 71 caaattcggt tcctacaact tattaag 27 <210> 72 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 ggatgtcgtt aaggtaattg gtcatgttt 29 <210> 73 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 gagcacatgt caaagcagta gtatct 26 <210> 74 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 74 agcacatgtc aaagcagtag tatcc 25 <210> 75 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 cgtttaagag tctttgttgg cttagagat 29 <210> 76 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 76 ccccgattat aacaggttgc acc 23 <210> 77 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 77 accccgatta taacaggttg caca 24 <210> 78 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 78 gtcggtggtt attatagttt gtcaggtat 29 <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 79 gatgtctcgt aggaagactc gg 22 <210> 80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 80 agatgtctcg taggaagact cga 23 <210> 81 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 tgtgggattt catacagatg cctaatgat 29 <210> 82 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 82 gatgtgtatg ttcatatagg aattgtcg 28 <210> 83 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 83 atgatgtgta tgttcatata ggaattgtca 30 <210> 84 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 84 tttccataaa gttgctcgat tgcaaggat 29 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 85 cacacgcgtc atcatcacgt tg 22 <210> 86 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 86 aatcacacgc gtcatcatca cgttt 25 <210> 87 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 87 cacatgcaag aaataagtga attccatgta 30 <210> 88 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 88 cataccatct agttggtgca tctg 24 <210> 89 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 89 gcataccatc tagttggtgc atcta 25 <210> 90 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 90 cagaaaacaa aggcctacag ctattgaaa 29 <210> 91 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 91 aataattttg aaaacggaaa aagcctactg 30 <210> 92 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 92 aaaataattt tgaaaacgga aaaagcctac ta 32 <210> 93 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 93 cgcgtattga ttgggacagt ttttgtatt 29 <210> 94 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 94 acacgattgt ttagatttgg ttaccc 26 <210> 95 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 95 acacgattgt ttagatttgg ttacct 26 <210> 96 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 96 tggggtaacg atcatttagt caaatctaaa 30 <210> 97 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 97 aatcttcata tgtaggctaa acgatcc 27 <210> 98 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 98 ataaatcttc atatgtaggc taaacgatct 30 <210> 99 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 99 gtcgaagaac ctgcatgagt tgagat 26 <210> 100 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 100 gcatattaat ttgtatcttg aatttgctcg 30 <210> 101 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 101 gcatattaat ttgtatcttg aatttgctcc 30 <210> 102 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 102 tacaaattac agcacgcttg gaggaattt 29

Claims (5)

In the oligonucleotides represented by SEQ ID NOs: 1 to 69, the oligonucleotides of SEQ ID NO: 3n, SEQ ID NO: 3n-1, and SEQ ID NO: 3n-2 in which n has the same value are one primer set (n is a natural number of 1 to 23) Including a set of 23 oligonucleotide primers, characterized in that in, melon ( Cucumis melo var. makuwa ) cultivar distinction or KASP (kompetiive allele specific PCR) primer set for F ₁ seed purity assay. The method of claim 1, wherein the melon varieties are Obok honey melon, Obok honey plus melon, Jummi honey melon, honey love melon, native honey melon, rich honey melon, king honey melon, Shingeumsalagi melon, Daebak honey melon, gold melon , Manata honey melon, Daejanggeum plus melon, eight bok honey melon, yellow honey melon, golden ax melon, melon variety distinction or F ₁ seed purity test for the KASP primer set. The primer set of claim 1; And KASP kit for distinguishing melon varieties or F ₁ seed purity assay comprising a reagent for performing an amplification reaction. According to claim 3, the reagents for performing the amplification reaction is a DNA polymerase, dNTPs, and melons cultivars or distinguish F ₁ seed kit for KASP for pure black comprising the buffer to. Separating genomic DNA from the melon sample;
Using the isolated genomic DNA as a template, performing KASP using the primer set of claim 1; And
Analyzing the amplification product of the KASP; containing, melon variety distinction or F ₁ seed purity assay method.

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