KR20200070935A - 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 for differentiation of a Korean melon cultivar and an F1 seed purity test, and a use thereof. 23 SNP markers of the present invention are distributed over 12 chromosomes of Korean melon, and are able to quickly and accurately differentiate the Korean melon cultivar, thereby being applied for the cultivation of the Korean melon cultivar.

Description

A primer set for SNP-based KASP for the identification of melon varieties and the purity of F1 seeds and its use{KASP primer set based on SNP for discriminating Korean melon cultivar and F1 hybrid purity checking and uses thereof}

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

melon(Cucumis melo var. makuwa) Is a vine plant belonging to the fruit family.Cucumis melo). The domestic seed market of gourds and crops is about 32 billion, accounting for about 20% of the total vegetable seed market. It is on an increasing trend.

Almost all melons in circulation are large-scale hybrids produced through mating. In one-hybrid (F ₁ ) varieties, a purity test is performed on the produced seeds. This is a quality control that tests whether the intended mating of the parents is established, that is, heterozygosity and displays the degree as purity (%). Refers to the act. In this regard, the reasons for poor purity may include contamination of other pollen during fertilization, offspring of the parent, and simple mixing of other seeds. Among them, it is known that the frequency of occurrence of child cases of the parent is the highest. Therefore, all seed companies basically maintain their own verification 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 species, but also a technical means to secure high-purity seeds essential for the cultivation and production of high-quality agricultural products. This is a process that has a very important meaning, such as the effect of fundamentally preventing the causes of complaints.

Melon is an economically and commercially important crop in Korea, and the importance of cultivating export varieties is also rising. Since the melon has a short generation and a good fixation time compared to other crops, it takes very short time to develop a lineage. Therefore, it is essential to develop a molecular label to protect the developed variety and lineage.

On the other hand, Korean Registered Patent No. 1516190 discloses'SSR primer sets for differentiation of melon strains or varieties and their use.' Biomarkers for screening and uses thereof' have been disclosed, but there has been no description of the primer set for SNP-based KASP and its use for discrimination of melon varieties and F ₁ seed purity of the present invention.

The present invention was derived by the above-described needs, and the present inventors used a variety of melons ( Cuccumis) by using Korean native species of dried persimmon melon as a reference dielectric. melo var. makuwa ) After selecting 23 melon-specific SNP (single nucleotide polymorphism) markers capable of distinguishing melon varieties from 12 chromosomes through genome analysis of varieties, a primer set for KASP to amplify the markers was prepared and genotyped By performing the 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 includes 23 oligonucleotide primer sets characterized in that three adjacent oligonucleotides are one primer set starting from SEQ ID NO: 1 in the oligonucleotides represented by SEQ ID NOs: 1 to 69. , 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 the melon variety discrimination and F ₁ seed purity test comprising a reagent for performing an amplification reaction.

In addition, the present invention comprises the steps of isolating 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 products of the KASP; provides a method for discriminating melon varieties and determining the purity of F ₁ seeds.

The primer set for KASP of the present invention can distinguish various melon varieties, and since F ₁ seed purity can be tested at the whole genome level of melon, it can be usefully utilized as an effective biomarker for varieties development, varietal protection, and seed production. Will be able to.

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

In order to achieve the object of the present invention, the present invention provides 23 oligonucleotide primer sets characterized in that three adjacent oligonucleotides are one primer set starting from SEQ ID NO: 1 in the oligonucleotides shown in SEQ ID NOS: 1 to 69. 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 bokbok honey melon, bokbok honey plus melon, oyster honey melon, honey love melon, native honey melon, rich honey melon, wangju honey melon, gold ladle melon, jackpot honey melon, gold melon, manata Honey melon, Dae Jang Geum Plus melon, Bokbok honey melon, Yellow honey melon, Golden axe melon, melon melon, or super gold melon, but are not limited thereto.

The term "KASP (kompetitive allele specific PCR)" of the present invention is one of PCR (polymerase chain reaction) based analysis methods, and is a homogenous and fluorescence based genotyping 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 specifically differentiated from a melon locus.

In the primer set of the present invention, consecutive oligonucleotides of SEQ ID NO: 3 form one primer set, and are composed 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 bound to the 3'end, and the reverse primer is composed of the same base pair in 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 simultaneously, melon varieties can be more efficiently distinguished. .

The primer of the present invention is 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 according to the sequence length of each primer Oligonucleotides consisting of segments of at least 25, at least 26, consecutive nucleotides. For example, 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 a fragment of 26 or more, 27 or more nucleotides. In addition, the primer may also include addition, deletion or substituted sequences 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 the 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 forward primers to which HEX is attached, 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.

The primer set according to an embodiment of the present invention can distinguish the 17 melon varieties described above by combining the 23 primer sets represented by SEQ ID NOs: 1 to 69. For example, the five melon honey melon (1) in Table 5 below are SEQ ID NOs: 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 the primer sets of SEQ ID NOs: 64, 65 and 66. In addition, in addition to Obok Honey Melon, Obok Honey Plus Melon, Omi Honey Melon, Honey Love Melon, Native Honey Melon, Rich Honey Melon, King Honey Melon, Shingeumlagi Melon, Jackpot Honey Melon, Gold Melon, Mana Honey Honey Melon, Dae Jang Geum Plus melon, bokbok honey melon, yellow honey melon, golden ax melon, melon melon or super gold melon can be distinguished by combining 23 primer sets.

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

In the present invention, oligonucleotides used as primers may also include nucleotide analogs, such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or And an intercalating agent. In addition, primers can incorporate additional features that do not change the basic properties of the primer, which serves as the starting point for DNA synthesis. The primer nucleic acid sequence of the present invention can include a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (e.g. horse radish peroxidase (HRP), alkaline phosphatase), radioactive isotopes (e.g. ³² P), fluorescent molecules, chemical groups (e.g. biotin), etc. have.

The suitable length of the primer is determined by the properties of the primer to be used, but is usually used in a length of 15 to 30 bp. The primer need not 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 provides the primer set; And it provides a kit for KASP for the melon variety discrimination and F ₁ seed purity test comprising a reagent 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 DNA polymerase can be used as a heat-resistant DNA polymerase, commercially available polymerases such as Taq DNA polymerase and Tth DNA polymerase. In addition, the kit of the present invention may further include a user manual describing optimal conditions for performing the reaction. The guide is a printout that explains how to use the kit, for example, how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented. The guide includes brochures in the form of brochures or flyers, labels attached to the kit, and descriptions on the surface of the package containing the kit. In addition, the handbook includes information that is disclosed or provided through electronic media, such as the Internet.

The present invention also

Isolating 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 products of the KASP; provides a method for discriminating melon varieties and F ₁ seed purity.

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

The method of the present invention includes isolating genomic DNA from a melon sample. As the method for separating the genomic DNA, a method known in the art may be used, for example, a CTAB method may be used, or a Wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, an amplification reaction may be performed using a primer set according to an embodiment of the present invention as a primer to amplify a target sequence. Methods for amplifying the target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. amplification system, strand displacement amplification, or any other suitable method for amplifying via Qβ replication enzymes or amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the 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 can be labeled with a detectable labeling substance. In one embodiment, the labeling material may be a material emitting fluorescence, phosphorescence or radioactivity, but is not limited thereto. Preferably, the labeling material may be FAM, HEX, VIC, JOE, ROX, TAMRA, Cy3 or Cy5, and the like. Upon amplification of 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 oligonucleotide primer set used to amplify the target sequence is as described in Table 3 below.

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

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely 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 prepare the reference genome, genomic information was obtained by Next Generation Sequencing (NGS) and Hi-C method for persimmon melon, and melon genome information composed of 12 chromosomes was constructed through various bioinformation analysis methods. (Laboratory). NGS reads for genomic analysis of 36 different native melons and melon strains, including hot bone melon, Euncheon melon, Anjong melon, Gangseo melon, Chosun melon, Yellow melon, Ganchi melon, Wild species melon, and wild species melon Was produced, and after removing the low-quality sequence, the produced lead was mapped to the reference sequence of persimmon melon using BWA (Burrows-Wheeler Alignment tool). The mapped leads are double masked, localized using the genomic analysis tool GATK (http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit) to increase the accuracy of SNP calling. After performing the local realignment process, UnifiedGenotyper was used to predict SNP (single nucleotide polymorphism) and filtering process to obtain 526,271 SNPs (Table 1). It was summarized (Table 2 and FIG. 1), and 4 SNPs per chromosome were analyzed so that SNPs were evenly distributed by 12 chromosomes, and it was confirmed that 34 SNPs responded properly.

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 marker selected by 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 prepared based on the selected 34 SNPs is composed of two forward primers and one reverse primer. The forward primer was designed so that the FAM or HEX fluorescent material is attached to the 5'end and the difference in base sequence such as SNP is distinguished at the 3'end. And the reverse primer was designed to work with the two forward primers so that the PCR reaction can proceed (Table 3). When PCR was performed, amplification was carried out with different fluorescent substances attached to the 5'ends of the two forward primers, so genotyping was analyzed according to the wavelength difference of the fluorescent substances. If the genotype is homozygous, then only one fluorescence of the two fluorescences (FAM or HEX) is detected, and if FAM is detected, it can be determined as genotype'A', and when HEX is detected, genotype'B'. The SNP present in the reference genome (persimmon melon) was designed to be detected by the FAM-attached forward primer.

4. SNP Marker For detecting 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 are KASP 2X master mix and KASP Assay mix (LGC genomics). KASP reaction conditions are shown in Table 4 below.

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

Example One. KASP for primer Differentiation of melon varieties using sets

Genotyping was performed on the 17 melon varieties using the 34 sets of KASP primers. The 17 melon varieties are; 1. Obok honey melon (Nongwoo Bio); 2. Obok Honey Plus Melon (Nongwoo Bio); 3. Chammi honey melon (Nongwoo Bio); 4. Honey Love Melon (Nongwoo Bio); 5. Native honey melon (Palm Hannong); 6. Rich Honey Melon (Palm Hannong); 7. King Honey Melon (Palm Hannong); 8. Shingeumlagi melon (Palm Hannong); 9. Jackpot honey melon (Palm Hannong); 10. Golden Melon (Asian Seedling); 11. Manata honey melon (Asian seedling); 12. Dae Jang Geum Plus Melon (Asian Seedling); 13. Falbee honey melon (modern seedling); 14. Yellow honey melon (modern seedling); 15. Golden ax melon (modern seedling); 16. Chamkal Melon (Koregon); And 17. Super Gold Loaf Melon (First Seedling). The SNP present in the reference genome can be detected by the FAM fluorescent material-attached forward primer and is designated as genotype A.

As a result, for example,'Okin honey melon' is genotype A in the genotyping results using primer sets for C01-sp010886, bgs00289, C09-sp000673, C08-sp000276, C03-sp004954 and bgs01444 markers, bgs00659, bgs04958 , C09-sp007875, bgs04175, bgs04286, bgs05223, bgs00068, bgs52645 and bgs02242 The genotyping results using primer sets for marker amplification showed genotype B and could be distinguished from other melon varieties through a combination of these genotyping results; 'Okin Honey Plus Melon' is genotype A from the genotyping results using primer sets for C01-sp010886, bgs00289, C09-sp000673, C08-sp000276 and bgs01444 marker amplification, bgs00659, bgs04958, C09-sp007875, bgs04286, bgs05223, bgs00068 , bgs52645 and bgs02242 genotyping results using the primer set for marker amplification showed genotype B, and thus it could be distinguished from other melon varieties through a combination of these genotyping results. However, among the 34 primer sets, 11 primer sets are not clearly identified as homogeneous (A or B) or heterozygous (H), and A/H, B/H genotype, or'-' Was confirmed, and was excluded from the primer set for the final melon variety discrimination (a primer set capable of amplifying the blue marker in Table 5 below).

3-F; It is a copy of the melon and melon.

3-M; The model of the tuna, honey and melon.

4-F; Honey Love Melon's copy.

4-M; Example of honey love melon.

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

Using the primer set for KASP, genotyping was performed on the copy and parent of the honey melon or honey love melon. As a result, as shown in Table 5, it was detected homozygous (homozygous) in both the parent and the parent of the honey melon or honey love melon. This means that the genetic position of the developed marker is a well-fixed position in a fixed species (a native species or a parent) used for breed development, which means it is a useful site 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 exhibit polymorphism between the parent and copy F ₁ can be used for purity testing And it was found.

The primer set for KASP of the present invention was developed based on SNPs present in each chromosome of a melon, and it was thought that the genotype can be analyzed at the level of the entire genome, thereby making it possible to more accurately and efficiently perform purity testing.

<110> Korea Research Institute of Bioscience and Biotechnology <120> KASP primer set based on SNP for discriminating Korean melon cultivar and F1 hybrid purity checking and uses thereof <130> PN18449 <160> 102 <170> KoPatentIn 3.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cctttcattt tctatggtct ttgacttc 28 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atcctttcat tttctatggt ctttgacttt 30 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 taatgtgatg tcgacacaca cattccaat 29 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aataagaaat gggagtgaga gtaagg 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 caataagaaa tgggagtgag agtaaga 27 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cttcctccat ggtttatttt cgtcacatt 29 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 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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)

Melon ( Cucumis) containing 23 oligonucleotide primer sets characterized in that three adjacent oligonucleotides are one primer set starting from SEQ ID NO: 1 in the oligonucleotides shown in SEQ ID NOS: 1 to 69 melo var. Primer set for makuwa) varieties distinguished and F ₁ seeds KASP (kompetiive allele specific PCR) for pure black. The method of claim 1, wherein the melon varieties are bokbok honey melon, bokbok honey plus melon, oyster honey melon, honey love melon, native honey melon, rich honey melon, wangju honey melon, gold ladle melon, jackpot honey melon, gold melon A primer set for KASP for differentiation of melon varieties and F ₁ seed purity testing, characterized by being mana honey melon, Dae Jang Geum plus melon, Bokbok honey melon, Hwangkyeol honey melon, Golden axe melon, melon melon or super gold lump melon. The primer set of claim 1; And a reagent for performing an amplification reaction, a kit for KASP for melon variety discrimination and F ₁ seed purity assay. The kit of claim 3, wherein the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs, and a buffer, and the KASP kit for melon variety discrimination and F ₁ seed purity assay. Isolating genomic DNA from the melon sample;
Using the isolated genomic DNA as a template and performing KASP using the primer set of claim 1; And
Analyzing the amplification products of the KASP; containing, melon varieties and F ₁ seed purity test method.

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