KR102248017B1 - SSR markers for discriminating Chrysanthemum morifolium cultivars and uses thereof - Google Patents

Abstract

The present invention relates to an SSR primer set for distinguishing chrysanthemum varieties, a kit for distinguishing chrysanthemum varieties including the SSR primer set, and a method of distinguishing chrysanthemum varieties using the SSR primer set.
Therefore, the SSR marker of the present invention can be a tool for evaluating the polymorphism of domestic chrysanthemum varieties, accurately separating and efficiently managing chrysanthemum varieties by using the nucleotide sequence of chrysanthemum that has not been previously utilized. It can be used in various fields such as building core groups, identifying varieties and analyzing family trees.

Description

{SSR markers for discriminating Chrysanthemum morifolium cultivars and uses thereof}

The present invention relates to an SSR primer set for distinguishing chrysanthemum varieties, a kit for distinguishing chrysanthemum varieties including the SSR primer set, and a method of distinguishing chrysanthemum varieties using the SSR primer set.

Chrysanthemum morifolium is a perennial herb belonging to the genus Chrysanthemum . It is mainly used for ornamental purposes. Drying flowers and putting them in a pillow is effective for headaches, and if you put them on a blanket, you can enjoy the delicate scent. In order to register a variety of chrysanthemums, the distinction from one or more control varieties must be clear (distinguishedness; distinctness), the irradiation characteristics must be uniformly expressed at the time (uniformity), and stably when grown in a general way. It must be maintained (stability).

Chrysanthemum is one of the major flower crops worldwide, and it is one of the three major crops in the domestic flower industry. In order to energize the chrysanthemum industry, there is an urgent need for a breeding technology for the development of innovative chrysanthemum varieties and a variety protection system that efficiently protects domestically grown varieties. However, chrysanthemums are an allele crop with a high genome size of 94 GB and a high degree of heterozygosity, and because of the large number of alleles as a polyploid crop, the interpretation is complicated during genotyping, which makes it difficult to systematically protect the chrysanthemum varieties.

Traditional plant research has been conducted on the basis of plant morphological or ecological differences, but it is difficult to identify due to the occurrence of plant mutations, low efficiency, and very limited among related species with close genetic characteristics, making it difficult to evaluate plant diversity. there was. Molecular biological analysis is being used to compensate for these limitations, and through this, analysis of genetic diversity or relationship of crops is efficiently performed. Molecular labeling markers used in the molecular biological assay include Restriction Fragment Length Polymorphism (RFLP), Random Amplification of Polymorphic DNA (RAPD), Amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), single nucleotide polymorphism (SNP), or CAPS (cleaved amplified polymorphic sequences).

Ideal markers should be highly polymorphic, evenly distributed throughout the genome, and genetic differences should be clear. And the cost of analysis should be low and reproducibility should be high. However, RFLP markers have high reproducibility, but polymorphism is difficult to identify, and RAPD markers and AFLP markers are easy to identify polymorphisms, but their reproducibility is low. For molecular breeding studies, SSR markers or SNP markers developed based on specific nucleotide sequences in the genome are mainly used. . Among them, SSR markers are distributed throughout the biological genome and are produced using differences in simple repetitive nucleotide sequences (1 to 4 bp) with large mutations, so it is easy to identify polymorphisms or relationships. Therefore, SSR markers must have co-dominant, and nucleotide sequence information is required at the time of development. SSR markers are commonly used in 27 crops such as pepper, melon, and lettuce based on the genetic analysis guidelines proposed by the National Seed Resources UPOV. However, as mentioned above, in the case of chrysanthemum, it is difficult to develop SSR markers for distinguishing chrysanthemum varieties due to the complex genome.

In Korean Patent Registration No. 10-1826732, a method and kit for identifying chrysanthemum varieties using a single nucleotide polymorphism (SNP) marker are provided. However, there is no disclosure of SSR markers for distinguishing varieties of chrysanthemum distributed in Korea while having high genetic polymorphism and reproducibility.

Accordingly, the present inventors made diligent efforts to use SSR markers suitable for analyzing the polymorphism or relationship of crops to distinguish chrysanthemum varieties, and as a result, based on RNA of standard chrysanthemums'Jungwoon' and'Seinoisei', which are domestically distributed varieties. As a result, SSR markers with high polymorphism were produced and selected, and when the SSR marker was used, it was confirmed that varieties of chrysanthemum can be distinguished with high accuracy, and the present invention was completed.

Accordingly, an object of the present invention is to provide an SSR primer set for distinguishing chrysanthemum varieties, a kit for distinguishing chrysanthemum varieties including the SSR primer set, and a method for distinguishing chrysanthemum varieties using the SSR primer set.

In order to achieve the above object, the present invention can provide an SSR primer set for distinguishing chrysanthemum varieties including any one or more SSR primer sets selected from the group consisting of 9 SSR primer sets.

According to a preferred embodiment of the present invention, the varieties of chrysanthemum are Yes Nuri, Yes Corus, Yellow Fang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Greenwich, Pink Pride, Purple Corn , Progi, Black Marble, Leopard, Argus, Purple, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange End, Bright End, SP13-154-01, Joy Pink, Pink Berry, Blue Hope, Golden Eye, Shinmyung, Ford, Delia Cream, January, Power End, Fire Pink, Dream Round, Dream Yellow, Field Green, Yellow Marble, Angel, Yesholic, Bubble Endy, Happy Endy, Sweet Candy, Yellow Kid, Select from the group consisting of Love Mine, Joy Cream, Magic Star, Loving Oil, Yes Luna, Yes Ruby, Green Angel, Peach Endy, Yes Song, Yes Lsa, Borami White, High Baek Mountain, Snow White, Baekseon, Shinma, Sumi and Baekma It may be any one or more varieties.

The present invention also, the SSR (Simple sequence repeat) primer set; And a reagent for carrying out an amplification reaction. It can provide a kit for distinguishing the chrysanthemum varieties, including.

According to a preferred embodiment of the present invention, reagents for performing the amplification reaction may be DNA polymerase, dNTPs, and buffers.

The present invention also comprises the steps of: i) separating genomic DNA from chrysanthemum;

ii) using the genomic DNA isolated in step i) as a template and performing an amplification reaction using the SSR primer set according to claim 1 or 2 to amplify the target sequence; And

iii) detecting the amplification product of step ii);

Including, it can provide a method of distinguishing the chrysanthemum varieties.

According to a preferred embodiment of the present invention, the genomic DNA of step i) may be isolated from a sprout or leaf of a chrysanthemum.

According to a preferred embodiment of the present invention, the detection of the amplification product of step iii) may be performed through gel electrophoresis, capillary electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement, or phosphorescence measurement.

According to a preferred embodiment of the present invention, the varieties of chrysanthemum are Yes Nuri, Yes Corus, Yellow Fang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Greenwich, Pink Pride, Purple Corn , Progi, Black Marble, Leopard, Argus, Purple, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange End, Bright End, SP13-154-01, Joy Pink, Pink Berry, Blue Hope, Golden Eye, Shinmyung, Ford, Delia Cream, January, Power End, Fire Pink, Dream Round, Dream Yellow, Field Green, Yellow Marble, Angel, Yesholic, Bubble Endy, Happy Endy, Sweet Candy, Yellow Kid, Select from the group consisting of Love Mine, Joy Cream, Magic Star, Loving Oil, Yes Luna, Yes Ruby, Green Angel, Peach Endy, Yes Song, Yes Lsa, Borami White, High Baek Mountain, Snow White, Baekseon, Shinma, Sumi and Baekma It may be any one or more varieties.

Therefore, the SSR marker of the present invention can be a tool for evaluating the polymorphism of domestic chrysanthemum varieties, accurately separating and efficiently managing chrysanthemum varieties by using the nucleotide sequence of chrysanthemum that has not been previously utilized, and management of chrysanthemum genetic resources in the future, It can be used in various fields such as building core groups, identifying varieties and analyzing family trees.

1 shows a unigene or contigue produced using short reads produced using standard chrysanthemum varieties'Jungwoon'and'Seinoisei'. The x-axis represents the length (base pair) of the contigs, and the y-axis represents the number.
2 shows the frequency of various repeat motifs present in the chrysanthemum SSR marker of the present invention. A is Di-nucleotide (27.5%), which means TG (10.1%) + AC (3.4%) + CA (6.7%) + TA (7.3%); B is Tri-nucleotide (61.2%), GAT(10.7%)+CAA(2.2%)+CCA(2.8%)+CGT(3.9)+GAG(2.2)+ACA(2.2)+TCA(5.6%) + TGA(5.1%)+ACC(2.8%)+ATA(4.5%)+AGT(2.8%)+CGC3.9%)+ATC(3.9%)+TTG(3.4%)+TGT(2.8%)+AAC( 2.2%); C is Tetra-nucleotide (11.3%), which means ATTT (3.5%) + TAAC (2.8%) + TGTT (2.8%) + TTAT (2.2%).
3 shows the frequency of various repeat motifs present in the chrysanthemum SSR marker of the present invention. Among the di-nucleotides, AT/TA type was found to be the most distributed among the tri-nucleotides, ATG/TGA/GAT.
4 shows polymorphic information content (PIC) of 31 selected markers.
5 shows gene information (Gene Diversity; GD) of 31 selected markers.
6 shows 8 chrysanthemum varieties (from left in turn, 1: Argus, 2: Borami, 3: Rainbow, 4: Yes Ruby, 5: Beksul, 6: Ringworm) using 9 markers selected for genetic analysis. , 7: Shinma, 8: Sumi).
Figure 7 shows 8 chrysanthemum varieties (from left in turn, 1: Argus, 2: Borami, 3: Rainbow, 4: Yes Ruby, 5: Snow White, 6: White Line) using 22 markers selected for breed identification. , 7: Shinma, 8: Sumi) shows the results of cultivar analysis.
Figure 8 is the nine markers selected for genetic analysis, 8 chrysanthemum varieties in [Table 4] and [Table 7] (a1 and b1: Argus, a2 and b2: Borami, a3 and b3: rainbow, a4 and b4: Yes Ruby, a5 and b5: Beksul, a6 and b6: Ringworm, a7 and b7: Shinma, a8 and b8: Sumi). It was confirmed that the nine markers have reproducibility.
9 is a schematic diagram showing the relationship between 56 of 64 chrysanthemum varieties with 8 markers selected for genetic analysis. The numbers mean genetic dissimilarity, the length of the tree is equal to the dissimilarity index value, and the scale bar is located at the bottom left.
10 shows the estimation of the polymorphism information content (PIC) of the chrysanthemum SSR marker of the present invention. (A) shows the correlation of PIC estimation between 8 chrysanthemum varieties and 56 chrysanthemum varieties among 8 KNOU_SSR markers. The data set fits a linear or polynomial regression model, and the equations and R-squared values of the linear and polynomial models are shown at the bottom and top of the plot, respectively. (B) shows the results of independently estimating the PIC of KNOU_CrySSR40 with 8, 24 and 40 varieties randomly selected from 56 varieties of chrysanthemum.
11 shows the results of cultivar analysis of 56 chrysanthemum varieties using markers selected for genetic analysis.

Hereinafter, the present invention will be described in more detail.

As described above, in the prior art, it was difficult to systematically protect chrysanthemum varieties due to the nature of the complex genome of chrysanthemums, and in the same context, it was difficult to apply SSR markers produced based on the genome information of crops to distinguish chrysanthemum varieties. By overcoming this, the SSR marker, which is easy to identify genetic polymorphisms or relationships of crops, has not been disclosed to effectively differentiate chrysanthemum varieties.

The SSR marker for distinguishing chrysanthemum varieties according to the present invention has a high numerical value such as polymorphic information content, so that varieties of chrysanthemum distributed in Korea can be distinguished with high efficiency or accuracy. It is effective as

Accordingly, the present invention provides a simple sequence repeat (SSR) primer set of SEQ ID NOs: 1 and 2; SSR primer set of SEQ ID NO: 3 and 4; SSR primer set of SEQ ID NO: 5 and 6; SSR primer set of SEQ ID NOs: 7 and 8; SSR primer set of SEQ ID NO: 9 and 10; SSR primer set of SEQ ID NO: 11 and 12; SSR primer set of SEQ ID NO: 13 and 14; SSR primer set of SEQ ID NO: 15 and 16; And SSR primer sets of SEQ ID NOs: 17 and 18; It provides an SSR primer set for distinguishing chrysanthemum varieties comprising any one or more SSR primer sets selected from the group consisting of.

The SSR primer set for distinguishing the chrysanthemum variety is one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, selected from the group consisting of the 9 SSR primer sets, It may contain 8 or more or 9 primer sets. Preferably, 6 or more primer sets are included, and more preferably, it is preferable that 8 or more primer sets are included. When the six or more primer sets are included, preferably, a simple sequence repeat (SSR) primer set of SEQ ID NOs: 1 and 2; SSR primer set of SEQ ID NO: 3 and 4; SSR primer set of SEQ ID NOs: 7 and 8; SSR primer set of SEQ ID NO: 9 and 10; SSR primer set of SEQ ID NO: 11 and 12; And SSR primer sets of SEQ ID NOs: 13 and 14; It is preferable that all are included. If 8 or more primer sets are included, preferably, a simple sequence repeat (SSR) primer set of SEQ ID NOs: 1 and 2; SSR primer set of SEQ ID NO: 3 and 4; SSR primer set of SEQ ID NOs: 7 and 8; SSR primer set of SEQ ID NO: 9 and 10; SSR primer set of SEQ ID NO: 11 and 12; SSR primer set of SEQ ID NO: 13 and 14; SSR primer set of SEQ ID NO: 15 and 16; And SSR primer sets of SEQ ID NOs: 17 and 18; It is preferable that all are included.

In addition, the SSR primer set for distinguishing the chrysanthemum variety is the SSR primer set of SEQ ID NO: 19 and 20; SSR primer set of SEQ ID NO: 21 and 22; SSR primer set of SEQ ID NO: 23 and 24; SSR primer set of SEQ ID NOs: 25 and 26; SSR primer set of SEQ ID NOs: 27 and 28; SSR primer set of SEQ ID NOs: 29 and 30; SSR primer set of SEQ ID NOs: 31 and 32; SSR primer set of SEQ ID NOs: 33 and 34; And an SSR primer set of SEQ ID NOs: 35 and 36; SSR primer set of SEQ ID NOs: 37 and 38; SSR primer set of SEQ ID NO: 39 and 40; SSR primer set of SEQ ID NOs: 41 and 42; SSR primer set of SEQ ID NOs: 43 and 44; SSR primer set of SEQ ID NOs: 45 and 46; SSR primer set of SEQ ID NOs: 47 and 48; SSR primer set of SEQ ID NO: 49 and 50; SSR primer set of SEQ ID NOs: 51 and 52; And SSR primer sets of SEQ ID NOs: 53 and 54; SSR primer set of SEQ ID NO: 55 and 56; SSR primer set of SEQ ID NO: 57 and 58; SSR primer set of SEQ ID NOs: 59 and 60; And SSR primer sets of SEQ ID NOs: 61 and 62; It may further include any one or more SSR primer sets selected from the group consisting of.

The SSR primers are SSR (Simple sequence repeat) primer sets of SEQ ID NOs: 1 and 2 according to the sequence length of each SSR primer; SSR primer set of SEQ ID NO: 3 and 4; SSR primer set of SEQ ID NO: 5 and 6; SSR primer set of SEQ ID NOs: 7 and 8; SSR primer set of SEQ ID NO: 9 and 10; SSR primer set of SEQ ID NO: 11 and 12; SSR primer set of SEQ ID NO: 13 and 14; SSR primer set of SEQ ID NO: 15 and 16; And an oligonucleotide consisting of segments of 18 or more, 19 or more, 20 or more, or 21 or more contiguous nucleotides in the SSR primer set of SEQ ID NOs: 17 and 18. For example, the SSR primer of SEQ ID NO: 1 (22 oligonucleotides) may comprise an oligonucleotide consisting of segments of 18 or more, 19 or more, 20 or more, or 21 or more contiguous nucleotides in the sequence of SEQ ID NO: 1. have. In addition, the SSR primers are SSR (Simple sequence repeat) primers of SEQ ID NOs: 1 and 2; SSR primers of SEQ ID NOs: 3 and 4; SSR primers of SEQ ID NOs: 5 and 6; SSR primers of SEQ ID NOs: 7 and 8; SSR primers of SEQ ID NOs: 9 and 10; SSR primers of SEQ ID NOs: 11 and 12; SSR primers of SEQ ID NOs: 13 and 14; SSR primers of SEQ ID NOs: 15 and 16; And the addition, deletion or substitution of the SSR primer nucleotide sequences of SEQ ID NOs: 17 and 18 may also be included.

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 the synthesis of the extension product to begin. 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 required DNA or RNA target.

In the present specification, the oligonucleotide used as a primer may also comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or It may contain an intercalating agent.

The varieties of the chrysanthemum of the present invention are Yes Nuri, Yes Corus, Yellow Fan Pang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Green Witch, Pink Pride, Purple Corn, Progi, Black Marble, Leopard , Argus, Borami, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange Endy, Bright Endy, SP13-154-01, Joy Pink, Pink Berry, Blue Hope, Golden Eye, Shinmyung , Ford, Delia Cream, January, Power End, Fire Pink, Dream Round, Dream Yellow, Field Green, Yellow Marble, Angel, Yes Holic, Bubble End, Happy End, Sweet Candy, Yellow Kid, Love Mine, Joy Cream, Magic Star, Loving Oil, Yes Luna, Yes Ruby, Green Angel, Peach & D, Yes Song, Yes Elsa, Borami White, High Baek Mountain, Snow White, Baekseon, Shinma, Sumi and Baekma. It is preferable to be a variety, more preferably Yes Nuri, Yes Corus, Yellow Fan Pang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Green Witch, Pink Pride, Purple Corn, Progi, Black Marble , Leopard, Argus, Purple, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange Endy, Bright Endy, SP13-154-01, Joy Pink, Blue Hope, Golden Eye, Shinmyung, Delia Cream, January, Power End, Fire Pink, Dream Round, Field Green, Yellow Marble, Angel, Yes Holic, Bubble End, Sweet Candy, Yellow Kid, Love Mine, Joy Cream, Magic Star, Loving Oil, Yes Luna, Yes It is preferable to be one or more varieties selected from the group consisting of 56 varieties of Ruby, Peach Andy, Yessong, YesLsa, Beksul, Baekseon, Shinma, and Sumi, most preferably Yesnuri, Yessorus, Yellow Pang, and Dream. Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Greenwich, Pink Pride, Purple Corn, Progi, Black Marble, Argus, Purple, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blu Rutsum, Secret Pink, Orange Andy, Brighten D, SP13-154-01, Joy Pink, Blue Hope, Golden Eye, Shinmyung, Delia Cream, January, Power End, Fire Pink, Dream Round, Field Green, Yellow Marble, Angel, Yesholic, Bubble Endy, Sweet Candy, It is preferable to be any one or more varieties selected from the group consisting of 54 varieties of Yellow Kid, Lovemine, Joy Cream, Magic Star, Yes Luna, Yes Ruby, Peach Andy, Yes Song, Yes Lsa, Beksul, Baekseon, Shinma and Sumi. Do. The group consisting of Borami White, Happy Endy, Hibaeksan, and Pink Berry, the group consisting of White Horse and Dream Yellow, the group consisting of Ford and Green Angel, and the group consisting of Leopard and Loving Oil can be distinguished from each of the 54 kinds of chrysanthemum varieties. , Each can be separated as a group (Fig. 9).

The present invention also, the SSR (Simple sequence repeat) primer set; And a reagent for carrying out an amplification reaction. It provides a kit for distinguishing chrysanthemum varieties, including.

Reagents for performing the amplification reaction of the present invention are preferably DNA polymerase, dNTPs, and buffers, but are not limited thereto. In addition, the kit of the present invention may further include a user's guide describing the optimum reaction performance conditions. The guide is a printout explaining how to use the kit, e.g., how to prepare PCR buffer, suggested reaction conditions, and so on. The guide includes a brochure in the form of a pamphlet or leaflet, 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 comprises the steps of: i) separating genomic DNA from chrysanthemum;

ii) using the genomic DNA isolated in step i) as a template and performing an amplification reaction using the SSR primer set according to claim 1 or 2 to amplify the target sequence; And

iii) detecting the amplification product of step ii);

It provides a method for distinguishing the chrysanthemum varieties, including.

The genomic DNA of step i) of the present invention is preferably isolated from the sprouts or leaves of chrysanthemums, but is not limited thereto. As a method for separating genomic DNA from the sample, a method known in the art may be used, for example, a kit for separating DNA may be used. Using the isolated genomic DNA as a template, a target sequence may be amplified by performing an amplification reaction using the SSR primer set of the present invention as a primer. Methods of amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, There are 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 target 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 is 6-FAM (6-Carboxyfluorescein), NED, VIC, PET or ROX. When performing PCR by labeling 6-FAM, NED, VIC, PET or ROX at the 5'-end of the primer when amplifying the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. In addition, the labeling material may include Cy-5 or Cy-3. In addition, when a ^{radioactive isotope such as 32} P or ³⁵ S is added to the PCR reaction solution when performing PCR, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product may be radiolabeled. The SSR primer set used to amplify the target sequence is as described above.

The detection of the amplification product of step iii) of the present invention is preferably performed through gel electrophoresis, capillary electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto.

The gel electrophoresis may use acrylamide gel electrophoresis or agarose gel electrophoresis depending on the size of the amplification product. For the capillary electrophoresis, for example, an ABI Genetic Analyzer may be used. In the fluorescence measurement method, when PCR is performed by labeling a fluorescent dye at the 5'-end of the primer, the target sequence is labeled with a detectable fluorescent labeling material, and thus labeled fluorescence can be measured using a fluorescence measuring device. The radioactivity measurement method includes ^{labeling the amplified product by adding a radioactive isotope such as 32} P or ³⁵ S to the PCR reaction solution when performing PCR, and then using a radioactivity measuring instrument, for example, a Geiger counter or a liquid scintillation counter. (liquid scintillation counter) can be used to measure radioactivity.

The varieties of the chrysanthemum of the present invention are Yes Nuri, Yes Corus, Yellow Fan Pang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Green Witch, Pink Pride, Purple Corn, Progi, Black Marble, Leopard , Argus, Borami, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange Endy, Bright Endy, SP13-154-01, Joy Pink, Pink Berry, Blue Hope, Golden Eye, Shinmyung , Ford, Delia Cream, January, Power End, Fire Pink, Dream Round, Dream Yellow, Field Green, Yellow Marble, Angel, Yes Holic, Bubble End, Happy End, Sweet Candy, Yellow Kid, Love Mine, Joy Cream, Magic Star, Loving Oil, Yes Luna, Yes Ruby, Green Angel, Peach Andy, Yes Song, Yes Elsa, Borami White, Hibaeksan, Snow White, Baekseon, Shinma, Sumi and Baekma. desirable. Since the chrysanthemum variety is the same as the chrysanthemum variety in the SSR primer set for distinguishing the chrysanthemum variety, the description will be substituted for the description.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Chrysanthemum SSR marker production

<1-1> Chrysanthemum SSR mass extraction

The publicly available materials are collected from two varieties of standard chrysanthemums'Jungwoon' and'Seinoisei', which are domestically distributed varieties at Yesan Flower Research Institute, and extracted 1 sprout and 1 young leaf, respectively, and RNA was extracted with Total RNA Isolation System (238,635,903). basepair (bp)), and after sequencing with Hiseq2500, respectively, QC and cDNA were synthesized to produce a library and a short read based on NGS. As a result, 280,434 unigenes were produced, and the average unigene length was 851 bp (Fig. 1). And de novo assembly and read mapping were performed. In the sample pooling, SSR sequence filtering was first performed, and then a large amount of SSR was extracted through polymorphic SSR sequence filtering.

<1-2> Primer design

Based on the unigene sequence produced in Example <1-1>, 31,121 simple sequence repeats (SSRs) were selected, and De novo assembly was performed based on each RNAseq information to find the polymorphic SSR between Jungwoon and Seinoisei. Performed. Then, after pairwise blasting the SSR sequences selected for each de novo assembly result, the difference between each SSR loci repeat number was investigated, and as a result, 17,551 SSR loci (about 56%) were found to be polymorphic. And among them, 207 SSR loci were selected to design primers with high polymorphism and amplification product size in the range of 200 to 300 bp, and 50 of them were selected, and primers were designed using them.

<1-3> PCR amplification and primer selection

Using the 50 primers designed in Example <1-2>, DNA polymerase chain reaction (PCR) was performed using Jungwoon and Seinoisei DNA as templates. After that, DNA was extracted using the DNeasy Plant Mini kit (Qiagen, Valencia, CA, USA), and only primers amplified through PCR were selected. As a result, only 31 primers out of 50 primers were amplified as shown in Table 1 below, and finally 31 SSR markers were selected.

Reviewed
Number of markers SSR marker type Polymorphic
Number of markers Amplified SSR Marker Polymorphism SSR marker source 50 Chrysanthemum's genomic SSR 31 31/50 Made in the present invention

SSR for genetic analysis Marker And SSR for variety identification Marker Classification

<2-1> Materials to be disclosed

Disclosure materials for classifying the markers selected in [Example 1] are spray chrysanthemum Argus, Borami, Mujigae, and Yes Ruby, which are generally well known among 64 varieties of chrysanthemums distributed on the market. Ruby) 4 varieties and 4 varieties of standard chrysanthemum Baekseol, Baekson, Zinba and Sumi were selected as representative varieties (Table 2) .

NO. Cultivar name Type classification NO. Cultivar name Type classification One Argus spray 5 Snow white standard 2 Borami spray 6 ringworm standard 3 Rainbow spray 7 Shinma standard 4 Yes Ruby spray 8 Sumi standard

<2-2> DNA extraction and PCR Amplification confirmation

The young leaves of 8 varieties of chrysanthemums of the above [Table 2] were collected from the Flower Research Institute of Chungcheongnam-do Agricultural Technology Institute, placed in a tube, and stored in a -80°C defroster. The chrysanthemum sample to be used in the experiment was put in liquid nitrogen in a square container, crushed in a crusher (tissue riser), and the tube was immersed in liquid nitrogen.

Into the tube, 30 ml of an extraction buffer mix and 30 ml of a reducing agent β-mercaptoethanol were added, vortexed, and then bathed in a heating machine for 2 hours. Thereafter, 700 µl of the PCI solution stored at 4°C was added and mixed using a machine for 15 minutes, followed by centrifugation at 12,000, 15 minutes, and 4°C conditions. 500 µl of the supernatant was obtained with a pipette, 500 µl of PCI was added, mixed using a machine for 15 minutes, and then centrifuged at 12,000, 15 minutes, and 4°C, and 400 µl of the supernatant was obtained. In order to precipitate the DNA, 400 µl of isopropanol was added, and 2 µl of RNAse-A was added to remove the remaining RNA and left on ice for 30 minutes. After vortexing, centrifugation was performed under conditions of 12,000, 15 minutes, and 4°C. After discarding the supernatant, 700 µl of 70% primary ethanol was added and centrifuged for 15 minutes. The supernatant was discarded, 700 µl of 70% secondary ethanol was added, and centrifuged for 5 minutes. After discarding the ethanol and centrifuging for 3 minutes, the remaining ethanol was removed with a pipette. After evaporating ethanol for 15 minutes at room temperature, 100 µl of distilled water was added to dissolve the pellet. DNA was extracted and PCR was carried out in the following cycle using the PCR Mixture reaction solution shown in Table 3 below; After inital denturation at 95°C for 3 minutes, denturation at 95°C for 1 minute, Annealing at 55°C for 45 seconds, and Extension at 72°C for 1 minute, these three steps were repeated 35 times. And final extension was performed at 72℃ for 10 minutes. The amplified DNA was subjected to electrophoresis, and the amplification result was confirmed with a UV photo of a gel. DNA was quantified in nanodrums.

The results are shown in [Table 4] to [Table 7].

<2-3> Calculation of polymorphism index and analysis of marker photos

It was attempted to investigate the degree of polymorphism using the 31 primers selected in <Example 1>.

Specifically, polymorphism was analyzed using a capillary electrophoresis ZAG (Zero Agarose Gel) at the Suwon Practicalization Foundation, and the peak point data were organized using PROSize 2.0. Using the PROSize 2.0 program, capillary electrophoresis data was calculated using the PowerMaker V3.25 program to calculate PIC (polymorphism information contents) values. Then, by analyzing the derived PIC values and marker pictures, markers for genetic analysis and markers for breed identification were selected.

As a result, as shown in the following [Table 8] and [Table 9], the PIC (Polymorphism information content) value indicating the degree of polymorphism was distributed in the range of 0.051 to 0.908, and the average value was high as 0.779, and the genetic diversity value Phosphorus GD (Gene diversity) also showed a high average of 0.805, showing similar patterns ([Fig. 4] and [Fig. 5]). Heterozygosity H (CrySSR_16, CrySSR_17, CrySSR_18, CrySSR_40, CrySSR_49, CrySSR_50) showed a single band pattern and an average of 0.466. Knou CrySSR_4, Knou CrySSR_7, knou CrySSR_9, Knou CrySSR_16, knou CrySSR_31, knou CrySSR_40, Knou CrySSR_42, knou CrySSR_45, and Knou CrySSR_50 were selected 9 for genetic analysis (also selected for genetic analysis). ), and the rest were selected for cultivar identification only capable of cultivar identification (FIG. 7, Table 11).

Confirmation of reproducibility of selected markers

It was attempted to investigate the reproducibility of the nine genetic analysis markers selected in <Example 2>. Specifically, among the first 8 varieties (Table 2) and the second 64 varieties (Table 15), the same 8 varieties were analyzed with 9 markers for genetic analysis, and the peak points corresponding to each of the varieties were recorded and compared.

As a result, it was confirmed that the positions of a and b had the same pattern as shown in the following [Table 12] to [Table 14] and [FIG. 8]. The differences in the location patterns of a and b were considered to be errors in the analysis process. Therefore, it was confirmed that the marker for genetic analysis of the present invention has reproducibility.

Selected With a marker Chrysanthemum relationship analysis

<4-1> Materials to be disclosed

The publicly available materials for the analysis of the relationship are sprays or standard chrysanthemums distributed in Korea among varieties grown at the Yesan Flower Research Institute of Chungcheongnam-do Agricultural Research and Development Institute, and a total of 56 varieties (Table 15) to Borami White, Happy Endy, Hibaeksan, Pink Berry, Baekma, Dream Yellow, Ford and Green Angel were all used. This included 8 varieties (Table 2) used in the first experiment for the connectivity of the experiment. In addition, correlation analysis of 64 kinds of publicly available materials was performed using 8 markers for genetic analysis among 9 markers for genetic analysis selected in Example 2 above.

<4-2> DNA extraction and PCR Amplification confirmation

DNA extraction and PCR amplification confirmation were performed in the same manner as in Example <2-2>. The results are as shown in [Table 16] and [Table 17] below, and it was confirmed that more than 55 varieties of chrysanthemum can be sufficiently genetically distinguished with only the primer sets of KnouCry_4, KnouCry_7, KnouCry_16, KnouCry_31, KnouCry_40 and KnouCry_42.

<4-3> Calculation of polymorphism index and creation of tree diagram

Polymorphism The Suwon Commercialization Foundation analyzed the data with a capillary electrophoresis ZAG (Zero Agarose Gel) and organized the data with PROSize 2.0. Capillary electrophoresis data using the PROSize 2.0 program were transferred using the PowerMaker V3.25 program, allele frequency (MAF: Major. Allele. Frquency), allele number (NA: No. of alleles), genetic polymorphism information content ( PIC: Polymorphic Information Conent) and Heterozyosity (H: Heterozyosity) were measured, and a phylogenetic tree was created using the UPGMA (unweighted pair-group method with arithmetical average) method.

As a result, the [Table 18] shows the sequence information for the marker for genetic analysis and the polymorphism index of the 56 varieties (Table 15) out of 8 varieties (Table 2) to 64 varieties using the same. At this time, as shown in [Fig. 10], only about 58% of the data sets based on PIC are described as linear regression, whereas when the curve regression set as a quadratic function is applied, about 81% of the data sets are described as related regression analysis I could see this. In particular, SSR loci groups such as KNOU_CrySSR4, which have high polymorphism, do not significantly change the degree of polymorphism as the number of samples increases. There was a distinct increase. In the case of loci with a PIC value of 0.75 or less, such as KNOU_CrySSR40, when the size of the group is only 8 varieties, it was confirmed that the polymorphism prediction of the relevant marker could be devalued. As a result of expanding the sample size for KNOU_CrySSR40 to 8, 24, and 40 varieties, the error range for PIC prediction gradually decreased, and it was observed that the devaluation effect was mitigated especially when the group size was more than 24 varieties. Therefore, it was determined that at least 24 varieties or resources should be used to secure accuracy when predicting the Chrysanthemum SSR marker polymorphism of the present invention.

In addition, it was possible to create a schematic diagram of [Fig. 9]. The markers for genetic analysis of the present invention are Yes Nuri, Yes Corus, Yellow Fan Pang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Green Witch, Pink Pride, Purple Corn, Progi, Black Marble, Ar Gus, Borami, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange Endy, Bright Endy, SP13-154-01, Joy Pink, Blue Hope, Golden Eye, Shinmyung, Delia Cream, January , Power End, Fire Pink, Dream Round, Field Green, Yellow Marble, Angel, Yes Holic, Bubble End, Sweet Candy, Yellow Kid, Love Mine, Joy Cream, Magic Star, Yes Luna, Yes Ruby, Peach End, Yes Song , Yeselsa, Beksul, Baekseon, Shinma, or Sumi were all individually isolated. In addition, Borami White, Happy Endy, Hibaeksan and Pinkberry into one group, Baekma and Dream Yellow into one group, Pod and Green Angel into one group, and Leopard and Loving Oil into one group. Could.

Therefore, it was confirmed that the nine markers for genetic analysis of the present invention can classify all 64 varieties.

<4-4> Distinguishing Chrysanthemum Varieties Using Selected Markers

Chrysanthemum varieties were genetically analyzed in the same manner as in Example <2-3>.

As a result, as shown in [Fig. 11], it was confirmed that the marker for genetic analysis of the present invention can clearly distinguish 56 varieties of chrysanthemum.

<110> Korea National Open University Industry-Academic Cooperation Foundation <120> SSR markers for discriminating Chrysanthemum morifolium cultivars and uses thereof <130> 1066126 <160> 62 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_4_F <400> 1 acagtacaca cacagccaac ac 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_4_R <400> 2 gtaatgcttc cgtctgcata gc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_7_F <400> 3 aggcccaact ttattcaccc 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_7_R <400> 4 ccaaatccaa tctccggc 18 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_9_F <400> 5 cattcacact cacctacaca cc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_9_R <400> 6 cctccctctc ttacaatgtc ac 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_16_F <400> 7 acaagtagta ggaggaggag ga 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_16_R <400> 8 cagtgtagcc ggtacagaag a 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_31_F <400> 9 ggaagcaagt gtgtggtttc 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_31_R <400> 10 acctccccat agaatcttga gc 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_40_F <400> 11 gacggatttt gagcttggag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_40_R <400> 12 gaaccaataa tcccgacacc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_42_F <400> 13 caaagtacta ccaaacgcgg 20 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_42_R <400> 14 gtaacattga gggtgtagca gc 22 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_45_F <400> 15 aaacagcctg acccaatctc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_45_R <400> 16 gtcatcatcc aaccaccaac 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_50_F <400> 17 gatggtgaga cattgcgtct 20 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_50_R <400> 18 gctcaaggat tatggacact gg 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_1_F <400> 19 gagttcttcg gagtaagatc cg 22 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_1_R <400> 20 tggcgtgtac aagtttcg 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_5_F <400> 21 accataacca ccaccactgt 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_5_R <400> 22 gaggtaggtt tggtggtcg 19 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_6_F <400> 23 ggaggctcgg aatttgtt 18 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_6_R <400> 24 ccatcgacga aactaacgag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_8_F <400> 25 ctcgacattc gtcaaagtcc 20 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_8_R <400> 26 gaatcccatt ctgcttcg 18 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_10_F <400> 27 gaacatgcaa tggatggg 18 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_10_R <400> 28 ccaccacact ccgtcattat 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_11_F <400> 29 cccatctcaa tccttcactc 20 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_11_R <400> 30 gtaacaccaa ccctaccttc ct 22 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_14_F <400> 31 gaagggaagg gtatggaaga 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_14_R <400> 32 tagcataagc ttgggctctc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_15_F <400> 33 gacttcaaac cctgacaacg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_15_R <400> 34 gggacatgtt tttgtgacgg 20 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_17_F <400> 35 gttcggatcg cttcatca 18 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_17_R <400> 36 taaccgcggt gatagttcg 19 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_18_F <400> 37 cgcgtatgct aatgaaggac 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_18_R <400> 38 ccttcaaacc caaaccctac 20 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_20_F <400> 39 ggctctgatc acccatttct ac 22 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_20_R <400> 40 accaaacact accaccgtga 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_22_F <400> 41 gatccatagc ccaaatggtg 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_22_R <400> 42 gccgagagct aacaagatca 20 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_23_F <400> 43 gtgataagga gcaagtccga g 21 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_23_R <400> 44 cggcattaca actctcccta gt 22 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_25_F <400> 45 gttcgaatac ctcgtttggc 20 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_25_R <400> 46 gaaacacctc tagttcacgc tc 22 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_27_F <400> 47 cgtacactcc aacaccactt c 21 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_27_R <400> 48 caacaagacc cgaatcctc 19 <210> 49 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_29_F <400> 49 ctctctcaag tgaagttgtg gc 22 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_29_R <400> 50 agaaagggtc ggcttgat 18 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_30_F <400> 51 caactacctg atcaacctcc tc 22 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_30_R <400> 52 ggagtcgtga aagtggtcat 20 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_35_F <400> 53 gtgaggagac cttttacagc ag 22 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_35_R <400> 54 atccagtggt gggaggtaac 20 <210> 55 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_37_F <400> 55 cacttctcgt tatcctcaca cc 22 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_37_R <400> 56 ctgtgtctac gattggcatc 20 <210> 57 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_38_F <400> 57 tattgccaca tgcacacc 18 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_38_R <400> 58 gtatatcact aacccccatc cc 22 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_48_F <400> 59 ctccatccat ccatgtgagt 20 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_48_R <400> 60 accacagcac tggcacat 18 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_49_F <400> 61 ctttgctagt gctgatactg gc 22 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Knou CrySSR_49_R <400> 62 ataatccgcc agctagggt 19

Claims (8)

Simple sequence repeat (SSR) primer set of SEQ ID NOs: 1 and 2; SSR primer set of SEQ ID NO: 3 and 4; SSR primer set of SEQ ID NOs: 7 and 8; SSR primer set of SEQ ID NO: 9 and 10; SSR primer set of SEQ ID NO: 11 and 12; And SSR primer sets of SEQ ID NOs: 13 and 14; SSR primer set for distinguishing chrysanthemum varieties containing all.
The method of claim 1, wherein the chrysanthemum varieties are Yes Nuri, Yes Corus, Yellow Fang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Greenwich, Pink Pride, Purple Corn, Progi, Black Marvel, Leopard, Argus, Purple, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange Endy, Bright Endy, SP13-154-01, Joy Pink, Pink Berry, Blue Hope, Golden Eye, Shinmyung, Ford, Delia Cream, January, Power End, Fire Pink, Dream Round, Dream Yellow, Field Green, Yellow Marble, Angel, Yesholic, Bubble End, Happy End, Sweet Candy, Yellow Kid, Love Mine, Joy Cream, Magic Star, Loving Oil, Yes Luna, Yes Ruby, Green Angel, Peach Endy, Yes Song, Yes Elsa, Borami White, Hibaeksan, Snow White, Baekseon, Shinma, Sumi, and Baekma. SSR primer set for distinguishing chrysanthemum varieties, characterized in that the variety.
The simple sequence repeat (SSR) primer set of claim 1; And a reagent for performing an amplification reaction; kit for distinguishing chrysanthemum varieties comprising.
The kit for distinguishing chrysanthemum varieties according to claim 3, wherein the reagents for performing the amplification reaction are DNA polymerase, dNTPs, and buffers.
i) separating genomic DNA from chrysanthemum;
ii) using the genomic DNA isolated in step i) as a template and performing an amplification reaction using the SSR primer set according to claim 1 or 2 to amplify the target sequence; And
iii) detecting the amplification product of step ii);
Containing, how to distinguish chrysanthemum varieties.
The method of claim 5, wherein the genomic DNA of step i) is isolated from the sprouts or leaves of chrysanthemums.
The method of claim 5, wherein the detection of the amplification product in step iii) is performed through gel electrophoresis, capillary electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement, or phosphorescence measurement. .
The method of claim 5, wherein the chrysanthemum varieties are Yes Nuri, Yes Corus, Yellow Fang, Dream Prince, Castelli, Mantree, Yes Together, Yes Now, Yes Star, Greenwich, Pink Pride, Purple Corn, Progi, Black Marvel, Leopard, Argus, Purple, Rainbow, Whistle, Yes Morning, Magic, Yellow Cap, Cherry Blossom, Secret Pink, Orange Endy, Bright Endy, SP13-154-01, Joy Pink, Pink Berry, Blue Hope, Golden Eye, Shinmyung, Ford, Delia Cream, January, Power End, Fire Pink, Dream Round, Dream Yellow, Field Green, Yellow Marble, Angel, Yesholic, Bubble End, Happy End, Sweet Candy, Yellow Kid, Love Mine, Joy Cream, Magic Star, Loving Oil, Yes Luna, Yes Ruby, Green Angel, Peach Endy, Yes Song, Yes Elsa, Borami White, Hibaeksan, Snow White, Baekseon, Shinma, Sumi, and Baekma. A method of distinguishing chrysanthemum varieties, characterized in that they are varieties.

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