KR101649589B1 - SSR primer derived from apple and use thereof - Google Patents

Abstract

The present invention relates to a primer set for SSR (simple sequence repeat) detection derived from apple, an apple variety discrimination method comprising the SSR detection primer set, and a composition and kit for apple apple variety discrimination. The SSR (Simple Sequence Repeat) primer set for discriminating apple cultivars according to the present invention can effectively detect the DNA polymorphism of the apple plant, and thereby the apple variety can be identified. In addition, it can be used in apple research institutes, agricultural product quality control centers, and distributors to solve problems related to the identification, production and distribution of varieties, protection of varieties, and marker-assisted backcross (MAB) Can be provided as basic information necessary for cultivating new varieties of apple.

Description

SSR primers derived from apples and their uses {SSR primer derived from apple and use thereof}

The present invention relates to a primer set for SSR (simple sequence repeat) detection derived from apple, an apple variety discrimination method comprising the SSR detection primer set, and a composition and kit for apple apple variety discrimination.

An apple ( Malus x domestica Borkh.) Is one of the main fruits. In Korea, Hongro ('Spur Earliblaze' × 'Spur Golden Delicious') varieties were cultivated in 1988 and 22 varieties were breed in Arisoo ('Yoko' × 'Senshu') varieties in 2010. In Korea, 18 varieties have been used only as breeders in planned breeding programs. However, the use of a limited number of breeders in the proposed breeding program has created a problem that limits the genetic diversity of available germplasm for crop improvement.

The morphological and physiological characteristics of apples have been used as tools for apple apple variety identification. However, the process is time-consuming, expensive and often disadvantageous by subjective decisions (Cooke R. J. (1995), Varietal identification of crop plants, p. 33-63). Because of the multigenic, quantitative and environmental factors affecting the expression of apple varietal characteristics, the biochemical and molecular biology of UPOV (International Union for Protection of New Varieties of Plants) The study group is requesting the use of molecular biologic primers to identify apple varieties.

Simple sequence repeat (SSR) (or microsatellites) is a short DNA fragment with high polymorphism due to changes in the number of eukaryotic genomes and repeat units. In addition, it has features of co-dominant inheritance. All SSR loci are defined by specific primer pairs and it is easy to share information between laboratories studying them (Gianfranceschi et al., (1998), Theor. Appl. Genet. 96: 1069-1076). Approximately 300 SSR primers are available in apples (Hokanson et al., (1998), Appl. Genet, 97, 671-683; Gianfranceschi et al., (1998), Appl. Genet. (2006), Tree Genetics and 205 Genomes. 2, 202-224). The SSR primers were used to isolate the genitalia (2006), Tree Genetics and 205 Genomes. 2, 202-224) and by breed identification (Liebhard et al., (2002), Molecular Breeding.10,217-241; Silfvergerg Dilworth et al. (2004), Int. J. Hort et al., (2004), Acta Hort. 663-639; (2003), Acta Hort. 839, 471-477) have been used for a variety of applications.

As a result of continuing the research for developing an SSR detection primer for discriminating domestic cultivated apple, the present inventors have found that a SSR detection primer set and a SSR detection primer set using a nucleotide sequence of a Golden Delicious apple apple It was confirmed that 22 kinds of apple cultivated in Korea can be identified quickly and accurately when using a primer set, and thus the present invention has been completed.

It is an object of the present invention to provide a primer set for SSR detection for discriminating apple cultivars comprising at least one primer pair selected from the group consisting of primer pairs represented by SEQ ID NOS: 1 to 44.

It is also an object of the present invention to provide a method for detecting a DNA fragment, comprising: (a) extracting DNA from a sample; (b) amplifying the extracted DNA using the primer set for SSR detection according to claim 1; And (c) detecting the amplification product.

It is also an object of the present invention to provide a composition for discriminating apple cultivars comprising the SSR detecting primer set.

It is also an object of the present invention to provide an apple cultivar discriminating kit comprising the SSR detecting primer set.

In order to solve the above problems, the present invention provides a primer set for SSR detection for discriminating apple cultivars, comprising at least one primer pair selected from the group consisting of the primer pairs shown in SEQ ID NOS: 1 to 44.

(A) extracting DNA from a sample; (b) amplifying the extracted DNA using the primer set for SSR detection according to claim 1; And (c) detecting the amplification product.

In addition, the present invention provides a composition for distinguishing apple cultivars comprising the SSR detecting primer set.

The present invention also provides a kit for discriminating apple cultivars comprising the SSR detecting primer set.

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

The present invention relates to a pharmaceutical composition comprising a compound of SEQ ID NO: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; SEQ ID NOS: 15 and 16; SEQ ID NOS: 17 and 18; SEQ ID NOS: 19 and 20; SEQ ID NOS: 21 and 22; SEQ ID NOS: 23 and 24; SEQ ID NOs: 25 and 26; SEQ ID NOS: 27 and 28; SEQ ID NOs: 29 and 30; SEQ ID NOS: 31 and 32; SEQ ID NOS: 33 and 34; SEQ ID NOS: 35 and 36; SEQ ID NOS: 37 and 38; SEQ ID NOS: 39 and 40; SEQ ID NOS: 41 and 42; And at least one primer pair selected from the group consisting of primers represented by SEQ ID NOs: 43 and 44. The primer set for SSR (Simple Sequence Repeat) detection for discriminating apple cultivars is provided.

The SSR detecting primer set of the present invention preferably comprises one or more, two or more, three or more, four or more, five or more, six or more, or more than one selected from the group consisting of the 22 SSR detecting primer sets. 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 More than 20, more than 21, and more than 22 SSR detecting primer sets can be used simultaneously, and most preferably, 22 SSR detecting primer sets can be used simultaneously.

In the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. 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 primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

SEQ ID NOS: 5 and 6; And SEQ ID NOs: 39 and 40 can discriminate apple cultivars, but it is preferably domestic cultivated apple cultivars, more preferably domestic apple cultivars excluding somatic mutants. But is not limited thereto.

In the present invention, "somatic mutation" is a gene mutation occurring during somatic cell division, meaning that economically important characteristics such as increase in color, size or taste of fruit are different in one or a few small regions .

The SSR detection primer set is derived from a plant of the genus Malus sp.

In the present invention, "apple plant" is a perennial plant, which takes a long period from seed to the time when it is bloomed and deleted, has not only a complicated gene composition but also self-incompatibility. Therefore, in order to increase breeding efficiency, it is important to understand the genetic mechanism of major traits and to collect and classify various genetic resources.

In the present invention, the above-mentioned apple can include all the plants belonging to it, preferably domestic cultivated apple varieties, more preferably Hongro, Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Seohong, Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Picnic, Yeohong, Danhong, Hwayoung, Greenball, Hwangok, Hwasa, Summer King and Arisoo varieties, But is not limited thereto.

The primer set for SSR detection provided in the present invention can be usefully used for detecting DNA polymorphism and analyzing genetic diversity in apple plants. By using the primer for detecting SSR according to the present invention, it is possible to efficiently evaluate and preserve genetic resources of apple plants. Accordingly, the present invention provides a method for detecting DNA polymorphism in a plant belonging to the genus Appleus, which comprises performing PCR using the SSR detection primer set.

The primer sets for detecting SSRs shown in SEQ ID NOS: 1 to 30 are derived from golden delicious varieties.

In the present invention, "Golden Delicious" is an apple variety found in 1914 as a coincidence in the United States. It is one of the widely cultivated varieties of the world, and has a shortage of sunshine, strong resistance to spotted leaves, and vulnerability to aphid damage.

In the present invention, the primer set for SSR detection represented by SEQ ID NOS: 1 to 30 is obtained by downloading chromosome sequence analysis information based on the Golden Delicious (NCBI GenBank Assembly ID: GCA_000148765.2) A primer set for SSR detection was constructed by searching repeated nucleotide sequences using a programming language perl which detects SSR primers.

The SSR detection primer set of SEQ ID NOS: 31 to 44 is derived from HiDRAS (High-Quality Disease Resistant Apples for a Sustainable Agriculture) database.

(A) extracting DNA from a sample; (b) amplifying the extracted DNA using the primer set for SSR detection according to claim 1; And (c) detecting the amplification product.

The extraction of DNA in step (a) can be carried out by a conventional phenol / chloroform extraction method, SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB separation method Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits.

The amplification of step (b) may be performed using a polymerase chain reaction, a multiplex polymerase chain reaction, a competitive polymerase chain reaction, a real-time polymerase chain reaction selected from the group consisting of Real-time Polymerase Chain Reaction, Real-time Quantitative Polymerase Chain Reaction, DNA chip, Loop-mediated isothermal amplification and combinations thereof Either method may be used.

In the present invention, the Polymerase Chain Reaction may be performed using a PCR reaction mixture containing various components known in the art and necessary for the PCR reaction. The PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water (dH ₂ O) in addition to the genomic DNA extracted from the apple plant to be analyzed and the primer set for detecting SSR provided in the present invention. The PCR buffer solution includes Tris-HCl, MgCl ₂ , KCl, and the like. At this time, MgCl ₂ concentration greatly affects amplification specificity and yield. Preferably in the range of 1.5-2.5 mM. In general, when Mg ²⁺ is excessive, nonspecific PCR amplification products are increased, and when Mg ²⁺ is insufficient, the yield of PCR products is decreased. The PCR buffer solution may further contain an appropriate amount of Triton X-100.

The detection of the amplification product in the step (c) may be performed by any one selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement, phosphorescence measurement, and combinations thereof.

In the present invention, the gel electrophoresis can be performed by agarose gel electrophoresis or acryl amide gel electrophoresis according to the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is performed, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement instrument such as a Geiger counter or a liquid island The radioactivity can be measured using a liquid scintillation counter.

In addition, the present invention provides a composition for distinguishing apple cultivars comprising the SSR detecting primer set.

The oligonucleotide used as a primer in the composition comprising the primer set also includes a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid Or may include an intercalating agent.

The present invention also provides a kit for discriminating apple cultivars comprising the SSR detecting primer set.

The kit of the present invention may contain a reagent for performing an amplification reaction, and may include a thermostable DNA polymerase, dNTPs, a buffer, and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

The SSR (Simple Sequence Repeat) primer set for discriminating apple cultivars according to the present invention can effectively discriminate apple cultivars by using DNA polymorphism of apple plant, , Can be used to solve problems related to the identification, distribution, and protection of varieties of distributed apples, and can be provided as basic information necessary for the cultivation of new varieties of apples through marker-assisted backcross (MAB). have.

FIG. 1 is a diagram showing genetic similarity of 38 domestic varieties of apple cultivars based on genetic distance by CS Chord 1967.
FIGS. 2A, 2B, and 2C are diagrams showing the results of discriminating 22 domestic cultivated apple cultivars using 22 pairs of primer sets for SSR detection. FIG.
FIG. 3A and FIG. 3B are diagrams showing the results of lineage analysis of 22 domestic cultivated apple cultivars using 22 pairs of SSR detecting primer sets.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1 DNA Extraction and Amplification

Example 1-1. DNA extraction of apple samples

In order to extract the apple sample DNA of the present invention and to check its DNA amount and purity, the following experiment was conducted.

More specifically, in order to discriminate 22 domestic cultivated apple varieties, 38 leaves of 38 varieties were collected from the above-mentioned 22 varieties and 16 varieties crossed with each other by shoot type at the Horticultural Research Institute of Rural Development Administration. This is shown in Table 1 below. DNA extraction of the collected young leaves was performed by Lodhi et al. of CTAB (cetyl-tri-methyl ammonium bromide) method was used. (100 mM Tris-HCl (pH 8.0), 500 mM NaCl and 50 mM EDTA), 400 μl and 2 × CTAB buffer (2% CTAB) were added to each well of a mortar, 400 μl of a 10 mM mixed solution of 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA (pH 8.0) and 1% PVP-40), β-mercaptoethanol and 10 μl of 10% And mixed. The sample was transferred to a 1.5-ml tube, reacted at 65 ° C for 20 minutes, 200 μl of 5M KOAc (potassium acetate) was added, and the mixture was treated with ice for 20 minutes. 700 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1 by volume) was added and centrifuged at 14,000 rpm for 10 minutes. 700 μl of the supernatant was transferred to a new tube. 700 μl of chloroform: isoamyl alcohol (24: 1 by volume) was added to the tube so that the volume ratio with the supernatant was 1: 1. The supernatant was centrifuged at 14,000 rpm for 10 minutes, and the supernatant was transferred to a new tube. Cold isopropanol was added to the tube, and the mixture was treated at -70 ° C for 10 minutes, centrifuged at 14,000 rpm for 10 minutes, discarded, and centrifuged by adding 1 ml of 70% EtOH. The remaining pellet was discarded from the centrifuged tube, dried, and dissolved in sterilized water (50 μl). 2 μl of RNase (10 mg / ml) was added to the sample, treated at 37 ° C for at least 30 minutes, and stored at -20 ° C.

The amount and purity of the extracted DNA were measured using a spectrophotometer (smartspec plus spectrophotometer 273 BR 02930).

Cultivar Reported Parentage Year Released Hongro Spur Earliblaze × Spur Golden Delicious 1988 Seokwang Mollie's Delicious × Gala 1995 Chukwnng Fuji × Mollie's Delicious 1992 Saenara Spur Earliblaze × Spur Golden Delicious 1997 Gamhong Spur Earliblaze × Spur Golden Delicous 1992 Hwahong Fuji × Sekaiichi 1992 Sunhong Hongro × Chukwang 2000 Seohong Tsugaru × Chukwang 2004 Summer Dream Tsugaru × Natsumidori 2005 Honggeum Senshu × Hongro 2004 Manbok Earliblaze × Fuji 2006 Hongso Yoko × Hongro 2006 Hongan Fuji × Jonathan 2006 Picnic Fuji × Sansa 2008 Yeohong Jonathan × Fuji 2007 Danhong Sport of Fuji 2008 Hwayoung Sport of Fuji 2008 Greenball Golden Delicious × Fuji 2008 Hwangok Kougetsu × Yataka Fuji 2009 Hwasa Mollie's Delicious × Fuji 2009 Summer King Fuji × Golden Delicious 2010 Arisoo Yoko × Senshu 2010

Examples 1-2. SSR (Simple Sequence Repeat) primer selection

In order to identify the 22 apple cultivars cultivated in Korea, SSR detection primer sets were selected by the following two methods in order to analyze the genetic diversity of 38 cultivars including 22 cultivars and 16 transplanted cultivars .

More specifically, The first method was selected from 300 SSR primers reported in HiDRAS (High-quality Disease Resistant Apples for a Sustainable Agriculture).

As a second method, SSR primers were prepared based on the full-length sequence of Golden Delicious apple varieties (NCBI GenBank Assembly ID: GCA_000148765.2). More specifically, the program language perl, which detects SSR primers by searching for malaria domestica in the NCBI taxonomy, selecting projects related to genomic DNA, and downloading chromosome sequencing information, Were used to find repeated nucleotide sequences. Over 90% of the detected SSR primer information was located in similar regions of the same chromosome or other chromosomes but similar regions. The final SSR primer region was derived and 6,442 SSR primers were developed, and 227 overlapping with other primers were removed, and a total of 6,165 SSR primers were selected for 17 chromosomes (Table 2). Of these, 15 SSR primer sets were selected based on the polymorphism test between varieties. The size range (mer) of the primer is 18-22, the temperature range is 58-64 ° C, the GC content range is 40-60, and the PCR product size is 100-250bp. Table 3 shows SSR primers based on Golden Delicious varieties.

As shown in Table 3, among the 300 SSR primers reported in HiDRAS, CH05g08 (SEQ ID NO: 31 and 32), Hi07d08 (SEQ ID NO: 33 and 34), Hi04e04 (SEQ ID NO: 35 and 36), CH04e03 A total of 7 SSR primer sets were selected: CHO3d07 (SEQ ID NOs: 39 and 40), Hi05b09 (SEQ ID NOs: 41 and 42), and CHO3d08 (SEQ ID NOs: 43 and 44)

SSR primers based on Golden Delicious apple cultivars were KFBG-2 (Sequence 1 and 2), KFBG-4 (Sequence 3 and 4), KFBG-7 (Sequence 5 and 6), KFBG-9 (SEQ ID NOS: 7 and 8), KFBG-11 (SEQ ID NOS 9 and 10), KFBG-12 (SEQ ID NOS 11 and 12), KFBG-13 (SEQ ID NOS 13 and 14, KFBG- ), KFBG-20 (SEQ ID NOs: 17 and 18), KFBG-24 (SEQ ID NOs: 19 and 20), KFBG-26 (SEQ ID NO: 21 and 22), KFBG- A total of 15 sets of SSR primers were selected: KFBG-43 (SEQ ID NOs: 27 and 28) and KFBG-45 (SEQ ID NOs: 29 and 30).

Therefore, a total of 22 pairs of SSR primers were selected to identify 22 domestic cultivars

Chromosome
Detected SSR markers After remove overlap

SSR markers Percentage of overlap

(%) Chr1 335 329 1.8 Chr2 436 417 4.6 Chr3 393 381 3.1 Chr4 273 262 4.2 Chr5 406 389 4.4 Chr6 296 285 3.9 Chr7 319 309 3.2 Chr8 381 364 4.7 Chr9 401 384 4.4 Chr10 409 390 4.9 Chr11 454 442 2.7 Chr12 387 368 5.2 Chr13 443 422 5.0 Chr14 373 344 8.4 Chr15 551 517 6.6 Chr16 267 258 3.5 Chr17 318 304 4.6 Total 6,442 6,165 4.5 Mean 378.9 362.6 4.5

Marker SEQ ID NO: F / R Sequence
5'-3 ' KFBG-2 One F CTG GAG AAA GGA TCT TGA GAA
GTT GTG GCT TAG ACA TTT GCA 2 R KFBG-4 3 F CTA GCC GAC ATT CAC CTC TC
GTT CTT CAG CGC ACT GGA GTT 4 R KFBG-7 5 F CTG AAT ATA CTC CCT GCC AAC
GTT GGT GCA GCA CGC TGA CAA 6 R KFBG-9 7 F GAT GCA GGC TCG GAT TGT AA
GTT TGA GTT GTT CTG CTA AGC 8 R KFBG-11 9 F GAG AAT AGT CCT AAG CAG CAG
GTG TTA GTC CTC CCA TTA AGA 10 R KFBG-12 11 F TTA GTT TGC GGA TTG ACA GGGA
GTG AAT CAG TGT CTT CCA GGT T 12 R KFBG-13 13 F GTA GAC GGC TTG ACA AAT CAC
GTC TTC CAG TTC CGA GCT TAC 14 R KFBG-16 15 F TTC TTG GGT GTG GCC GTA CAA
GTG AAA TGA CGA TGC TGC CCT 16 R KFBG-20 17 F GGA GCC AGT ATC GTC CTC TC
GTG CTG CCA TGC CTA GTG AAC 18 R KFBG-24 19 F TGA GAA CGT TTT GTA GGG AAG
GTT TAA TCT CAC CTG ATG AGGA 20 R KFBG-26 21 F TAC TTT ATG CTG AGC TGG CAC
GTG TGT TCC TAG GCT CTA ATC 22 R KFBG-32 23 F GTG GTG CTC ATT CGT CAA AGA
GTG CGC ATA TCC GAC TTC AC 24 R KFBG-36 25 F GTC AAT TAG GCA CCA AGT TTA G
GTC AGG GAG GCC ATA GTT TCA 26 R KFBG-43 27 F TCA CGA TTA AGT CAT GTC AGC
GTA ATT TGA CCA TCG TGG GCA 28 R KFBG-45 29 F ATG TTA TCG AGC GAG TAG TCA
GTT CAG GAG ATT GAG GGA TGA 30 R CH05g08 31 F CCA AGA CCA AGG CAA CAT TT
CCC TTC ACC TCA TTC TCA CC 32 R Hi07d08 33 F TGA CAT GCT TTT AGA GGT GGA C
GTT TGA GGG GTG TCC GTA CAA G 34 R Hi04e04 35 F GA CCA CGA AGC GCT GTT AAG
GTT TCG GTA ATT CCT TCC ATC TTG 36 R CHO4e03 37 F TTG AAG ATG TTT GGC TGT GC
TGC ATG TCT GTC TCC TCC AT 38 R CH03d07 39 F CAA ATC AAT GCA AAA CTG TCA
GGC TTC TGG CCA TGA TTT TA 40 R Hi05b09 41 F AAA CCC AAC CCA AAG AGT GG
GTT TCT AAC GTG CGC CTA ACG TG 42 R CH03d08 43 F CAT CAG TCT CTT GCA CTG GAAA
TAG GGC TAG GGA GAG ATG ATGE 44 R

Example 2. SSR analysis

In order to perform SSR analysis using the DNA of the apple sample extracted in Example 1-1 and the SSR primer set selected in Example 1-2, the following experiment was conducted.

More specifically, in order to perform polymerase chain reaction (PCR) amplification, the reaction mixture contained 20 ng template DNA (template DNA), SSR primer shown in Table 2 selected in Example 1-2 from each 10 μM, 5 2 μl of Hot Taq Master Mix (PKT) was used and the final volume was adjusted to 10 μl. PCR was performed at 94 ° C for 5 minutes in pre-denaturation step, at 94 ° C for 30 seconds at 35 cycles, at 60 ° C for 30 seconds, at 72 ° C for 1 minute; And 72 < 0 > C for 7 min. In order to confirm the amplified PCR products were analyzed using an automated capillary gel electrophoresis device ^{(Fragment Analyzer TM, Advanced Analytical Technologies} , Inc, USA).

Example 3. Screening of polymorphism among domestic cultivated apple varieties through SSR analysis

The number of alleles of 38 cultivars, including 16 cultivated cultivars, and the size range of molecular weights of the alleles (number of alleles Genetic diversity and polymorphic information content (PIC) values were analyzed using the following analysis.

More specifically, the SSR fragment product was converted to an appropriate format for data application using PRO Size 2.0 software (Advanced Analytical Technologies Inc., USA) to measure the allele molecular weight size range, and an automatic capillary gel analysis device (Fragment Analysis) The size of the allele was used as a ± 2-bp error range in the sample analysis.

In addition, the polymorphism information content (PIC) was calculated from the polymorphic bands of the 22 SSR primer sets. The PIC value represents a measure of allelic variability in the locus. The PCR-based primer was calculated by the following equation (1), and Pij represents the frequency of the PCR pattern of j. The results are shown in Table 4 below.

Marker No. of alleles Range of allele size (bp) Genetic diversity PIC KFBG-2 17 242 - 273 0.8923 0.8833 KFBG-4 13 172 - 236 0.8608 0.8453 KFBG-7 25 212 - 260 0.9467 0.9441 KFBG-9 17 211 - 235 0.9048 0.8970 KFBG-11 13 189 - 212 0.8767 0.8643 KFBG-12 6 144 - 152 0.6794 0.6222 KFBG-13 11 154 - 181 0.8310 0.8166 KFBG-16 22 154 - 268 0.9172 0.9115 KFBG-20 13 189 - 211 0.8165 0.7956 KFBG-24 12 235 - 280 0.8051 0.7813 KFBG-26 11 183 - 241 0.8310 0.8112 KFBG-32 9 170 - 235 0.8134 0.7882 KFBG-36 19 217 - 248 0.8954 0.8870 KFBG-43 20 226 - 255 0.9103 0.9039 KFBG-45 24 156 - 182 0.9301 0.9259 CH05g08 25 165 - 198 0.9335 0.9297 Hi07d08 13 176 - 190 0.8729 0.8603 Hi04e04 24 174 - 261 0.9356 0.9319 CHO4e03 26 173 - 228 0.9387 0.9354 CH03d07 28 171 - 221 0.9404 0.9374 Hi05b09 14 122 - 140 0.8681 0.8547 CH03d08 16 118 - 139 0.8847 0.8745 Mean 17 118 - 280 0.8766 0.8637

As shown in Table 4, a total of 378 polymorphic alleles were amplified, and the number of alleles per each SSR primer set was 6 to 28, with an average of 17. The PIC value per locus was found to be 0.6222 in the KFBG-12 (SEQ ID NOs: 11 and 12) primer set and from this to the CHO3d07 (SEQ ID NOs: 39 and 40) primer set to 0.9374. Also, the average value of PIC was 0.8637.

Example 4. Analysis of Genetic Relationship between Domestic Cultivated Apple Cultivars by SSR Analysis

For the identification of 22 apple cultivars cultivated domestically, 22 genotypes were analyzed by using 22 pairs of SSR primer sets selected as follows. A total of 38 cultivars including 22 cultivars and 16 transplanted cultivars were analyzed as follows.

The primary genetic relationship diagram is the C.S. group of the neighbor-joining method. Chord (1967) was drawn using genetic distances obtained by a distance function. The final diagrams were drawn using PowerMarker V3.25 (Bioinformatics program in North Carolina State University) and TreeView. 38 varieties were classified into 6 groups based on the genetic mutual relation between varieties. The results are shown in Fig.

As shown in Figure 1, Group I includes Gala (from New Zealand); Molly's Delicious, originating in the United States; Arisoo (Yoko x Senshu); Tsugaru (origin in Japan); Hwasa (Mollie's Delicious × Fuji); Summer King (Fuji × Golden Delicious); Kougetsu (origin in Japan); And Hwangok (Kougetsu × Yataka Fuji) varieties were included.

Group II is Yeohong (Jonathan × Fuji); Seohong, Tsugaru × Chukwang; Manbok, Earliblaze × Fuji; Early Blaze (Earliblaze, origin in the United States); Spur Earliblaze, originating in the United States; Hongso (Yoko × Hongro); Yoko (origin in America); And Natsumidori (originated from Japan) varieties.

Group III is Golden Delicious (originating in the United States); Sunhong (Hongro × Chukwang); Gamhong (Spur Earliblaze × Spur Golden Delicous); Spur Golden Delicious, originating in the United States; And Chukwang (Fuji x Gollie's Delicious) varieties.

Group IV is Summer Dream (Tsugaru × Natsumidori); Seokwang (Mollie's Delicious × Gala); Hwahong (Fuji × Sekaiichi); Hongan (Fuji × Jonathan); Sansa (origin in Japan); Jonathan (origin in America); Picnic (Picnic, Fuji × Sansa); Greenball (Golden Delicious × Fuji); Hwayoung, Sport of Fuji; And Danhong (Sport of Fuji) varieties.

Group V is the Hongro (Sprout Earliblaze × Spur Golden Delicious); Senshu (origin in Japan); And Sekaiichi (originated in Japan) varieties.

The group Ⅵ is Saenara (Spur Earliblaze × Spur Golden Delicious); Honggeum (Senshu × Hongro); Yadaka Fuji (origin in Japan); And Fuji (originated in Japan) varieties.

As a result, it was confirmed that most of the 16 varieties cultivated in Korea and their crosses were divided into the same group. Therefore, it was confirmed that the genotypic relationship between the domestic cultivated apple cultivars was basically consistent with the known pedigree.

Example 5. Identification of domestic cultivated apple varieties using SSR primer set

5-1. Identification of domestic cultivated apple varieties using SSR primer set

In order to discriminate 22 domestic cultivated apple varieties using 22 pairs of selected SSR primer sets, alleles of 38 varieties including 22 cultivars and 16 transplanted cultivars were identified as follows.

More specifically, a total of 22 primer sets selected from the above Example 1-2 were used for PCR of 22 domestic cultivated apple varieties and 16 hybrid varieties thereof in the same manner as in Example 2, followed by automatic capillary gel electrophoresis (Fragment Analyzer ^™ , Advanced Analytical Technologies, Inc., USA) and analyzed using Pro size 2.0. The size of alleles identified for each type of each primer set (base pair, bp ). In order to discriminate the cultivars, if the genotype results of bp identified by the primer set are discriminated from each other and if it can not be discriminated by a single primer, two or more combinations are discriminated to identify bp (Figs. 2A, 2B and 2C). In order to confirm the accuracy of the SSR data of domesticated apple cultivars identified using the 22 primer sets shown in FIGS. 2A, 2B, and 2C, the alleles estimated in the present invention and the known genotypes were compared. The results are shown in Figs. 2A, 2B, 2C, 3A and 3B.

As shown in FIG. 2A, FIG. 2B, FIG. 2C, FIG. 3A and FIG. 3B, it was confirmed that 76% (337/440) of the strains were identical between 22 domestic cultivars and 22 SSR primer sets.

5-2. Identification of domestic cultivated apple varieties using minimum SSR primer set

The following experiment was conducted to confirm that 22 apple cultivars cultivated domestically can be discriminated with minimum primer sets.

More specifically, Using a total of 22 primer sets selected in Example 1-2, 22 domestic apple cultivars and 16 hybrid cultivars were PCR-amplified in the same manner as in Example 2, and then subjected to automatic capillary gel electrophoresis Analyzer ^TM , Advanced Analytical Technologies, Inc, USA) and analyzed using Pro size 2.0. The results are shown in Table 5.

Marker combination Distinguishable pairs (no.) Observed not observed CH03d07 24 14 CH03d07 + KFBG-7 14 0

As shown in Table 5, the successful differentiation of domestic cultivated apple cultivars other than somatic mutants was determined using CH03d07 (SEQ ID No. 39 and 40) and KFBG-7 (SEQ ID No. 5 and 6) SSR primer set .

Example 6. Parent-offspring relationship of three varieties

In order to find the bloodlines of Gamhong, Hongan and Green Ball, whose bloodlines are not yet known, the following experiment was conducted.

More specifically, using the same method as in Example 5-1, The candidate varieties were selected based on the S-genotype (Heo et al., (2011), Environ. Biotechnol. 52 (2): 158-162). Calomel (S ₁ S ₉₎ cultivar Yoko _{(Yoko) (S 3 S 9} ), Fuji _{(Fuji) (S 1 S 9} ), sekayi value _{(Sekaiichi) (S 3 S 9} ), chugwang (Chukwang) (S ₃ S ₉ ) and Jonathan (S ₇ S ₉ ) were selected as the candidate strain.

In addition, Hongan (S ₁ S ₃ ) varieties were identified as Spur Golden Delicious (S1S3), Hongro (S ₁ S ₃ ), Tsugaru (S ₃ S ₇ ) and Natsumidori ) (S ₃ S ₉ ) were selected as the candidate strain.

In addition, Green Ball (S ₃ S ₇ ) varieties were classified as Molly Delicious (S ₃ S ₇ ), Tsugaru (S ₃ S ₇ ), Jonathan (S ₇ S ₉ ) and Sans (S ₅ S ₇ ) were selected as the candidate strain. In order to analyze the lineage of the three cultivars, SSR primers selected in the same manner as in Example 5-1 were analyzed. The results are shown in Table 6 below.

Cultivar Reported parents Our results Gamhong (S ₁ S ₉₎ Spur Earliblaze (S ₁ S ₁₉ ) × Spur Golden Delicious (S ₂ S ₃ ) Spur Earliblaze (S ₁ S ₁₉ ) × Fuji (S ₁ S ₉ ) Hongan (S ₁ S ₃₎ Fuji (S ₁ S ₉ ) × Jonathan (S ₇ S ₉ ) Fuji (S ₁ S ₉ ) × Hongro (S ₁ S ₃ ) or Natumidori (S ₃ S ₉ ) Green Ball (S ₃ S ₇ ) Golden Delicious (S ₂ S ₃ ) × Fuji (S ₁ S ₉ ) Golden Delicious (S ₂ S ₃ ) × Jonathan (S ₇ S ₉ )

As shown in Table 6, 22 SSR primer analysis results confirmed that one allele for each locus of the psyllium varieties was derived from the fuji variety except CH03d08 (SEQ ID NOs: 43 and 44) SSR primers.

In addition, two varieties of red blood cultures were identified. One is that one allele for each locus of the Hongyan variety was derived from the Hongo variety except for KFBG-7 (SEQ ID NOs: 5 and 6) and KFBG-24 (SEQ ID NOs: 19 and 20) SSR primer sets, And confirmed as a result of primer analysis. Another was that the SSR primer analysis was performed to derive one allele for each locus of the red sea rum varieties from the Natsumidori variety, except for KFBG-45 (SEQ ID NOs: 29 and 30) and CH04e03 (SEQ ID NOs: 35 and 36) SSR primers As a result.

As a result of the SSR primer analysis, it was confirmed that one allele for each locus of the green ball variety originated from the Jonathan variety, and that the Green ball originated from Golden Delicious and Jonathan varieties.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> SSR primer derived from apple and use thereof <130> KNU1.187 <160> 44 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-2 forward primer <400> 1 ctggagaaag gatcttgaga a 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-2 reverse primer <400> 2 gttgtggctt agacatttgc a 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KFBG-4 forward primer <400> 3 ctagccgaca ttcacctctc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-4 reverse primer <400> 4 gttcttcagc gcactggagt t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-7 forward primer <400> 5 ctgaatatac tccctgccaa c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-7 reverse primer <400> 6 gttggtgcag cacgctgaca a 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KFBG-9 forward primer <400> 7 gatgcaggct cggattgtaa 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-9 reverse primer <400> 8 gtttgagttg ttctgctaag c 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-11 forward primer <400> 9 gagaatagtc ctaagcagca g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-11 reverse primer <400> 10 gtgttagtcc tcccattaag a 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KFBG-12 forward primer <400> 11 ttagtttgcg gattgacagg ga 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KFBG-12 reverse primer <400> 12 gtgaatcagt gtcttccagg tt 22 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-13 forward primer <400> 13 gtagacggct tgacaaatca c 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-13 reverse primer <400> 14 gtcttccagt tccgagctta c 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-16 forward primer <400> 15 ttcttgggtg tggccgtaca a 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-16 reverse primer <400> 16 gtgaaatgac gatgctgccc t 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KFBG-20 forward primer <400> 17 ggagccagta tcgtcctctc 20 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-20 reverse primer <400> 18 tactttatgc tgagctggca c 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-24 forward primer <400> 19 tgagaacgtt ttgtagggaa g 21 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KFBG-24 reverse primer <400> 20 gtttaatctc acctgatgag ga 22 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-26 forward primer <400> 21 tactttatgc tgagctggca c 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-26 reverse primer <400> 22 gtgtgttcct aggctctaat c 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-32 forward primer <400> 23 gtggtgctca ttcgtcaaag a 21 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KFBG-32 reverse primer <400> 24 gtgcgcatat ccgacttcac 20 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KFBG-36 forward primer <400> 25 gtcaattagg caccaagttt ag 22 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-36 reverse primer <400> 26 gtcagggagg ccatagtttc a 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-43 forward primer <400> 27 tcacgattaa gtcatgtcag c 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-43 reverse primer <400> 28 gtaatttgac catcgtgggc a 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-45 forward primer <400> 29 atgttatcga gcgagtagtc a 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KFBG-45 reverse primer <400> 30 gttcaggaga ttgagggatg a 21 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CH05g08 forward primer <400> 31 ccaagaccaa ggcaacattt 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CH05g08 reverse primer <400> 32 cccttcacct cattctcacc 20 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Hi07d08 forward primer <400> 33 tgacatgctt ttagaggtgg ac 22 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Hi07d08 reverse primer <400> 34 gtttgagggg tgtccgtaca ag 22 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hi04e04 forward primer <400> 35 gaccacgaag cgctgttaag 20 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Hi04e04 reverse primer <400> 36 gtttcggtaa ttccttccat cttg 24 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CH04e03 forward primer <400> 37 ttgaagatgt ttggctgtgc 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO4e03 reverse primer <400> 38 tgcatgtctg tctcctccat 20 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CH03d07 forward primer <400> 39 caaatcaatg caaaactgtc a 21 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CH03d07 reverse primer <400> 40 ggcttctggc catgatttta 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hi05b09 forward primer <400> 41 aaacccaacc caaagagtgg 20 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Hi05b09 reverse primer <400> 42 gtttctaacg tgcgcctaac gtg 23 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CH03d08 forward primer <400> 43 catcagtctc ttgcactgga aa 22 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CH03d08 reverse primer <400> 44 tagggctagg gagagatgat ga 22

Claims (9)

SEQ ID NOS: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; SEQ ID NOS: 15 and 16; SEQ ID NOS: 17 and 18; SEQ ID NOS: 19 and 20; SEQ ID NOS: 21 and 22; SEQ ID NOS: 23 and 24; SEQ ID NOs: 25 and 26; SEQ ID NOS: 27 and 28; SEQ ID NOs: 29 and 30; SEQ ID NOS: 31 and 32; SEQ ID NOS: 33 and 34; SEQ ID NOS: 35 and 36; SEQ ID NOS: 37 and 38; SEQ ID NOS: 39 and 40; SEQ ID NOS: 41 and 42; And Seqwang, Chukwang, Saenara, Gamhong, Hwahong, and Sunhong, including a set of primers consisting of the nucleotide sequences shown in SEQ ID NOs: Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Yeohong, Danhong, and others. In addition, Hwayoung, Greenball, Hwangok, Hwasa, Summer King, Arisoo, Gala, Molie's Delicious, Tsugaru Tsugaru, Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan, Yoko, Senshu, One species selected from the group consisting of Sekaiichi, Yadaka Fuji, Fuji, Golden Delicious and Spur Golden Delicious varieties Of the SSR (simple sequence repeat) for detecting primer set for determining the apple varieties. delete The method according to claim 1,
The primer set is derived from plants of the genus Malus sp., Such as Hongro, Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Seohong, Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Yeohong, Danhong, Hwayoung, Greenball, Hwangok, Hwasa, Summer King, Arisoo, Gala, Molie's Delicious, Tsugaru, ), Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan, Yoko, Senshu, (Sekaiichi). SSR (simple sequence repeat) detection for discriminating one or more apple cultivars selected from the group consisting of Yadaka Fuji, Fuji, Golden Delicious and Spur Golden Delicious varieties Primer set. The method according to claim 1,
The primer set consisting of the nucleotide sequences shown in SEQ ID NOS: 5 and 6 is a primer set derived from Hongro, Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Seohong, Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Hwangok, Summer King, Arisoo, Gala, Hwangok, Greenball, Hwangok, Yeongong, Danhong, Hwayoung, Molly's Delicious, Tsugaru, Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan, Yoko, Senshu, and Sekaiichi. SSR (simple sequence repeat) detection for discriminating one or more apple cultivars selected from the group consisting of Yadaka Fuji, Fuji, Golden Delicious and Spur Golden Delicious varieties Primer set. (a) extracting DNA from a sample;
(b) amplifying the extracted DNA using the SSR primer set of claim 1; And
(c) detecting the amplification product, wherein the amplified products are detected by the method of Hongro, Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Yeohong, Danhong, Hwayoung, Seonghong, Greenball, Hwangok, Hwasa, Summer King, Arisoo, Gala, Molie's Delicious, Tsugaru, Kogetsu, Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan, Yoko, Senshu, and Sekaiichi. A method for distinguishing one or more apple cultivars selected from the group consisting of Yadaka Fuji, Fuji, Golden Delicious, and Spur Golden Delicious varieties. 6. The method of claim 5,
The amplification of step (b) may be performed using a polymerase chain reaction, a multiplex polymerase chain reaction, a competitive polymerase chain reaction, a real-time polymerase chain reaction a real time polymerase chain reaction (PCR), a real time polymerase chain reaction (PCR), a DNA chip, an isothermal amplification (Loopmediated isothermal amplification), and a combination thereof. (Summer Dream), which is the use of the Korean Wave (Hongro), Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Seohong, Honggeum, Manbok, Hongso, Hongan, Picnic, Yeohong, Danhong, Hwayoung, Greenball, Hwangok, ), Hwasa, Summer King, Arisoo, Gala Gala, Molie's Delicious, Tsugaru, Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan Jonathan, Yoko, Senshu, and Sekaiichi. A method for distinguishing one or more apple cultivars selected from the group consisting of Yadaka Fuji, Fuji, Golden Delicious, and Spur Golden Delicious varieties. 6. The method of claim 5,
The detection of the amplification product of step (c) may be performed using any one of the methods selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Seohong, Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Yeohong, Danhong, Hwayoung, Greenball, Hwangok, Hwasa, Summer King, Arisoo, Gala, Molie's Delicious, Tsugaru, Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan, Yoko, Senshu, and Sekaiichi. In addition, there are many places where you can enjoy the view. A method for distinguishing one or more apple cultivars selected from the group consisting of Yadaka Fuji, Fuji, Golden Delicious, and Spur Golden Delicious varieties. A method of screening for SSR comprising the primer set for SSR according to any one of claims 1 to 3, wherein the primers are selected from the group consisting of Hongro, Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Seohong, Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Yeohong, Danhong, Hwayoung, Green Ball, Greenball, Hwangok, Hwasa, Summer King, Arisoo, Gala, Molie's Delicious, Tsugaru, Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan, Yoko, Senshu, and Sekaiichi. It is also known as the " A composition for discriminating at least one apple cultivar selected from the group consisting of Yadaka Fuji, Fuji, Golden Delicious and Spur Golden Delicious. A method of screening for SSR comprising the primer set for SSR according to any one of claims 1 to 3, wherein the primers are selected from the group consisting of Hongro, Seokwang, Chukwang, Saenara, Gamhong, Hwahong, Sunhong, Seohong, Summer Dream, Honggeum, Manbok, Hongso, Hongan, Picnic, Yeohong, Danhong, Hwayoung, Green Ball, Greenball, Hwangok, Hwasa, Summer King, Arisoo, Gala, Molie's Delicious, Tsugaru, Kougetsu, Earliblaze, Spur Earliblaze, Natsumidori, Sansa, Jonathan, Yoko, Senshu, and Sekaiichi. It is also known as the " At least one apple cultivar discriminating kit selected from the group consisting of Yadaka Fuji, Fuji, Golden Delicious and Spur Golden Delicious varieties.

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