KR20180046907A - Composition for determining bud mutation cultivar of Fuji apple - Google Patents

Abstract

The present invention relates to a composition for distinguishing bud mutant cultivar of Fuji apple. When the composition for distinguishing bud mutant cultivar of Fuji apple is used, the bud mutant cultivar of Fuji apple which does not show a large difference in phenotype due to high genetic similarity can be accurately distinguished. In addition, an enhanced coloring group, an early season group, a spur group, and a follicular group of the bud mutant cultivar of Fuji apple can be identified, so that the present invention can contribute to promotion of intellectual property right protection and branding of bud mutant cultivar of domestic Fuji apple, and secure competitiveness in related industries.

Description

[Technical Field] The present invention relates to a composition for discriminating azo varieties of Fuji apples {Composition for determining bud mutation cultivar of Fuji apple}

The present invention relates to a composition for discriminating an azo mutant of Fuji apple.

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. Among the apple varieties, Fuji apple is a hybrid of "Ralls Genet" and "Delicious" and is one of the most cultivated varieties in Korea. However, due to the genetic disadvantages of the above-mentioned Fuji apples, it has been difficult to produce high quality apples, and various varieties of Fuji apple azo mutants have been selected at home and abroad. Because of the high genetic similarity between Fuji apple and its azo mutant varieties, there is a great difficulty in identifying each of them because of the large differences in phenotype.

As a means of securing differentiation of varieties, mainly morphological traits of varieties have been compared, morphological traits are limited in number, and most traits are quantitative or continuous traits involving many genes, Since the expression of these traits is influenced by the external environmental condition, it is inevitable that the classification of cultivars using external morphological traits is limited. In order to solve the above problems, stable and non-environmentally sensitive DNA-based molecular markers have been used for the purpose of breed identification, stability test, and protection of domestic varieties of major crops, mainly in some European countries.

The molecular markers are widely used in molecular breeding systems for the search of useful traits, identification of species of organisms, identification of breed classification, and analysis of the relationship of group populations. First, RFLPs (Restriction Fragment Length Length Polymorphisms) method based on the difference in nucleotide sequence length caused by mutation of restriction enzyme recognition sites in chromosomes have been developed, but this method is troublesome to use radioactive isotopes. Thereafter, a randomly amplified polymorphic DNA (RAPD) method has been developed as a nucleic acid fingerprinting method using PCR (Polymerase Chain Reaction). In the PCR method, a small oligonucleotide (hereinafter, referred to as a primer) composed of 10 to 20 nucleotides is annealed with DNA or RNA of an organism, and then a heat-resistant DNA polymerase is added thereto, . This requires only a small amount of DNA (1-50 ng) compared to other methods, and has the advantage of being able to confirm the results easily and quickly. However, the RAPD method of the above-mentioned PCR analysis method has a disadvantage that the non-specific PCR product is amplified and thus the reproducibility is poor. The AFLP (Amplified Fragment Length Polymorphism) method is attracted by high DNA polymorphism detection, The emergence and analysis of bands is complex. The SSR (single sequence repeat) method is a method of analyzing a supersatellite in an individual by preparing a PCR primer based on nucleotide sequence information in a microsatellite region, which is a DNA repeat sequence. Because of the differences between individuals, when this part is amplified by PCR reaction, polymorphism occurs and this gene is actively used in the genetic studies of animals and plants. However, when the number of SSR markers is small, it is not sufficient for high density gene mapping. On the other hand, the single nucleotide polymorphism (SNP) method is a method of analyzing one or several nucleotide variations indicating the individual variation among the 3 billion nucleotide sequences of the chromosomes in the nucleus. Specifically, It is a method of analyzing DNA base sequences and reading hundreds of nucleotides and finding other bases at the same position. The SNPs have a higher resolution power than the SSR markers, making it possible to compensate for the shortcomings of SSR markers that are not sufficient for high density genetic mapping.

At present, domestic apple cultivars can be identified using SSR molecular markers (Ban et al. 2014), but identification of azo mutant varieties is difficult. In addition, we tried to discriminate the Azo varieties of Fuji using S-SAP (retrotransposon based) molecular markers (Zhao et al. 2010), but there was a disadvantage of low reproducibility and the accuracy and sensitivity for the discrimination of Azo varieties of Fuji A high selection method is required.

Therefore, in order to improve the accuracy and simplicity of discrimination of Azo mutation of Fuji apple, the present inventors have confirmed the relationship between the specific single nucleotide polymorphism site and the azo mutant variety of Fuji apple, and can identify the azo mutant of Fuji apple The present invention has been completed by developing a composition.

Accordingly, an object of the present invention is to provide a composition for distinguishing azo varieties of Fuji apple.

Another object of the present invention is to provide an azo mutant variety kit of Fuji apple.

It is another object of the present invention to provide a method for discriminating azo varieties of Fuji apple.

In order to accomplish the above object, the present invention provides a chromosome of chromosome 3 (NC_024241.1) at position 10473276; 25021180 th position of chromosome 3 of apple (NC_024241.1); 18260085 th position of chromosome 4 of apple (NC_024242.1); Position 17139584 of the chromosome 6 of apple (NC_024244.1); Position 16892600 of the chromosome 12 of the apple (NC_024250.1); And position 9155427 of the chromosome 15 of apple (NC_024253.1); (SNP) marker of one or more loci selected from the group consisting of the nucleotide polymorphisms (SNPs) of the present invention.

The present invention also provides an azo mutant variety kit of Fuji apple comprising the above composition.

Further, the present invention provides a method for producing a fungus, comprising the steps of: (a) extracting DNA of Fuji apple; (b) performing a PCR using the DNA of step (a) as a template and using the composition for distinguishing an azo variant of fuzzy apple; And (c) analyzing the polymerase chain reaction products to determine the variety. The method for distinguishing Azo varieties of Fuji apple is provided.

When the composition for discriminating the azo mutant of the fuzi apple of the present invention is used, it is possible to accurately discriminate the azo mutant of the fuzi apple which does not show a large difference in the phenotype due to high genetic similarity. In addition, it is possible to identify the pigment system, azo system, phylogenetic system, phylogenetic system and phylogenetic system of azo mutant of Fuji apple, and contribute to promotion of intellectual property right protection and branding of azo mutant varieties of domestic Fuji apple, . ≪ / RTI >

FIG. 1 is a diagram showing a process for selecting SNP markers for discriminating azo mutants of Fuji apple according to the present invention.
FIG. 2 is a graph showing the effect of the calling mutant Fig.
FIG. 3 is a graph showing SNP comparison results of selected four fuji apples and azo mutants according to the present invention.
FIG. 4 is a diagram showing results of performing allele-specific PCR for discriminating the Hong Kong variety according to the present invention.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention relates to a chromosome of chromosome 3 (NC_024241.1) at position 10473276; 25021180 th position of chromosome 3 of apple (NC_024241.1); 18260085 th position of chromosome 4 of apple (NC_024242.1); Position 17139584 of the chromosome 6 of apple (NC_024244.1); Position 16892600 of the chromosome 12 of the apple (NC_024250.1); And position 9155427 of the chromosome 15 of apple (NC_024253.1); (SNP) marker of one or more loci selected from the group consisting of the nucleotide polymorphisms (SNPs) of the present invention.

In the present invention, " single nucleotide polymorphism (SNP) " means a genetic change or mutation showing a difference in one nucleotide sequence (A, T, G, C) It is the most common form of polymorphism.

In the present invention, "polymorphism" refers to a case in which two or more alleles exist in one locus, and among the polymorphic sites, only a single base is different according to a person and a single base polymorphism (single nucleotide polymorphism, SNP). Preferred polymorphic markers have two or more alleles exhibiting an incidence of 1% or more, more preferably 10% or 20% or more, in the selected population.

In the present invention, the term "allele" refers to various types of a gene existing on the same gene locus of a homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two kinds of bialles.

In the present invention, the term "marker" means a nucleotide sequence used as a reference point when identifying a genetically locus unrelated to genetics, and the position of the marker on the gene map can be represented by the locus have.

According to a preferred embodiment of the present invention, the single nucleotide polymorphism marker of the present invention is a single nucleotide polymorphism marker in which the 10473276 base of the apple chromosome 3 (NC_024241.1) is T; A single nucleotide polymorphism marker in which the 25021180 base of the apple chromosome 3 (NC_024241.1) is A; A single nucleotide polymorphism marker with the nucleotide 18260085 of the apple chromosome 4 (NC_024242.1); A single nucleotide polymorphism marker of nucleotide 17139584 of the chromosome 6 of apple (NC_024244.1); A single nucleotide polymorphism marker in which the 16892600 base of the apple chromosome 12 (NC_024250.1) is A; And a single base polymorphism marker in which base 9155427 of the chromosome 15 of apple (NC_024253.1) is C is preferable.

According to a preferred embodiment of the present invention, the agent capable of detecting the single base polymorphic marker of the present invention comprises a polynucleotide consisting of 10 to 100 consecutive DNA sequences or a complementary poly It is preferable that the polynucleotide is a polynucleotide that specifically hybridizes with a nucleotide. The polynucleotide is preferably at least one selected from the group consisting of primers, probes, and combinations thereof capable of detecting a single nucleotide polymorphism (SNP) marker, but is not limited thereto. Also, the primer can be complementarily bound to a single base polymorphic site at the 3 'last base.

In the present invention, "Fuji apple" is a hybrid of Ralls Genet and Delicious. It is one of the most cultivated varieties in Korea since it was cultivated in Japan in 1958. The above-mentioned Fuji apple has a disadvantage that it is difficult to produce high quality fruit due to its genetic characteristics. To overcome the genetic disadvantage, many azo varieties were selected at home and abroad. However, the azo mutant varieties have a high genetic similarity, so that they do not show a large difference in phenotype and are in a difficult state to discriminate.

In the present invention, the term " bud mutation "refers to a mutation in a branch of a stem and a branch of a stem during growth, resulting in two or three different traits or trunks. When only the mutated part is propagated by grafting or folding, it is possible to obtain an individual having a different trait from that of the parent strain. The obtained individual is called a variety or system. The azo mutant varieties can be classified into a coloring system, a compost system, a stage system, and a system system.

In the present invention, the term "coloring system" refers to an improvement in coloration of the Fujiazo system, which has improved coloration compared to ordinary Fuji. "Choyo" is characterized by faster maturity than ordinary Fuji. Compared with ordinary Fuji, the "sweet potato" is advantageous in that the sweet pot storage area is short and the flower pot is easy to be pollinated. "Daigaku" is characterized by a larger volume of fruit than ordinary Fuji.

The coloring system is represented by Danhong, Hangawi, Akifu-1, Aofu-5, Iwafu-10, Morihofu 3A Kiku 8, Nagafu 2, Nagafu 6, Nagafu 12, Sun Fuji, and Sairai Fuji, all of which will be on display at the same time. Miyama Fuji, Champion Fuji, Akifu-47, Ryoka no kisetsu, Misima Fuji, Royal Fuji, Minemuragei, Fuji, and Myra Red, but is not limited thereto.

The premixing system may be at least one selected from the group consisting of Benishogun, Yataka, Hirosaki, Ryoka no kisetsu, Mimaki Spur Fuji, But is not limited thereto.

The stage and the background are not limited to one or more kinds selected from the group consisting of Spur Fuji, Jubilee Fuji and Gallery.

The above-mentioned pedigree system is a natural variety, but is not limited thereto.

In the present invention, "discrimination" means discriminating the azo mutant variety of Fuji apple in the biological sample to be analyzed.

In a preferred embodiment of the present invention, the azo mutant of the fuji apple of the present invention preferably discriminates one or more strains selected from the group consisting of a coloring system, a densitometer, a stage system and a ferment system. Also, the coloring system is at least one selected from the group consisting of sweet potato and mackerel, and the atmospheric meter is preferably at least one selected from the group consisting of Hong genera and yata taka, but is not limited thereto.

According to another aspect of the present invention, there is provided an azo mutant variety kit for Fuji apple comprising a composition for discriminating azo mutants of fuji apple.

In the present invention, the " discrimination kit " can discriminate a variety of azo mutants of Fuji apple by detecting a single nucleotide polymorphism marker. The azo mutant breeding kit of Fuji apple may include a polynucleotide, a primer, a probe and the like for detecting a single nucleotide polymorphism marker, and may include one or more other components, solutions or devices suitable for the assay method .

For example, the azo mutation discrimination kit of Fuji apple of the present invention may be a kit containing essential elements for performing allele specific PCR. Allele-specific PCR kits can be used in conjunction with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentrations), deoxynucleotides (dNTPs), and the like, as well as specific polynucleotides, primers or probes for the single base polymorphic markers of the invention. Enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water and sterile water.

According to still another aspect of the present invention, there is provided a method for producing a fungus, comprising: (a) extracting DNA of a fungus apple; (b) performing a PCR using the DNA of step (a) as a template and using the composition for distinguishing an azo variant of fuzzy apple; And (c) analyzing the polymerase chain reaction products to determine the variety. The method for distinguishing Azo varieties of Fuji apple is provided.

In the present invention, the term " polymerase chain reaction " means an experimental method in which only a desired portion of a DNA chain is amplified in a large amount. Examples thereof include polymerase chain reaction, allele specific Polymerase Chain Reaction, Nested-Polymerase Chain Reaction, Multiplex Polymerase Chain Reaction, Competitive Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, One or more selected from the group consisting of real-time polymerase chain reaction, real-time quantitative polymerase chain reaction, DNA chip, and loop-mediated isothermal amplification Method, preferably allele-specific polymerase chain reaction, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One. Fuji apple  And objection Azo  DNA extraction of variant varieties

There are various azo varieties of Fuji apples in Korea. The varieties of apples used in this Example are shown in Table 1.

As shown in Table 1, the genomic DNA of Fuji apple and its four azo varieties (Danhong, Hangawi, Benishogun and Yataka) was extracted Respectively. More specifically, the apples used in this example were obtained from the National Institute of Horticultural Science, Apple Research Center, and stored at -80 ° C until used in the experiment. The DNA extraction was performed using DNeasy Plant Mini Kit (Qiagen). The experiment was carried out according to the manufacturer's manual.

The SNP markers for the determination of azo mutants of Fuji apple were selected using the genomic DNA of four cultivars of the extracted Fuji apple and its azo mutant. The SNP marker selection process is shown in Fig.

Example  2. DNA library production and Fuji apple Azo  Dielectric for determination of mutation A recombinant sequence Re-sequencing analysis

In order to select SNP markers for discrimination of fuzzy apple azo mutants, the following experiment was conducted. First, DNA library was constructed for genome re-sequencing analysis for analyzing mutations of specific individuals in Fuji apple cultivars with known genome, Was used.

Specifically, in order to analyze the gene sequences of four varieties of Fuji and its azo mutants, next-generation sequencing was performed. For this procedure, a DNA library for Illumina Hiseq 2000 was prepared using the genomic DNA of each apple sample extracted in Example 1 above. Thereafter, the constructed DNA library was subjected to a rearrangement sequence analysis using Illumina Highseq 2000, and then a dielectric was assembled to prepare a contig sequence. The assembled assembly sequence was predicted using the fugi apple reported in the gene bank as a reference, and the structure of the gene was determined, and the recombinant sequence analysis data was constructed.

Example  3. Fuji apple Azo  For mutation discrimination Trimming (trimming) and alignment

Trimming and alignment were performed to select SNP markers for discrimination of apple azo mutants.

More specifically, for data preprocessing, the assembly sequence (contig) obtained in Example 2 above was trimming low sequences that did not match using the Solexa QA package software. The data trimming results are shown in Table 2.

According to the result of the trimming of the sequenced shot read shown in Table 2, it can be seen that the range of the sequencing depth of the four varieties of Fuji apple and its azo varieties is 12.90 to 19.31 X. [

Thereafter, the Burrows-Wheeler Alignment tool was used to align the readings to the existing sequences and perform alignment. The data alignment results are shown in Table 3.

As shown in Table 3, about 70% of the total read was used for mapping, and both the Fuji apple and its azo mutant varieties covered about 70% of the reference genome sequence .

According to the results of trimming and sorting, the Danhong, Hanqiao, Hong generations and Yataka varieties have different varieties than the conventional varieties.

Example  4. Fuji apple Azo  For mutation discrimination Calling Mutant calling variation analysis, Filtering (Filtering) and SNP Marker  Selection

Calling variants of four varieties of Fuji apple and its azo mutants were analyzed. The results of the calling mutant analysis are shown in Table 4.

Filtering was performed using SAM tools to isolate genetically meaningful variants of the calling variants of Table 4. The results of the filtering are shown in Fig.

As shown in Fig. 2, it can be seen that the number of heterozygous SNPs (heterozygous SNPs) is larger than that of homozygous SNPs in all four azo mutants. Also, the YATAKA variety has the lowest sequencing depth, but it has the greatest variation.

Based on the above filtering results, SNPs of four varieties of Fuji apple and its azo mutant were selected. SEEDERS in-house script was used for the SNP selection. We compared the SNPs of selected four varieties of Fuji apple and its azo mutant. The results of the comparison of the selected SNPs of Fuji apple and its azo varieties are shown in Fig. 3, and the results of the addition of the selected SNPs of Hong group, Fuji apple, Yamataka and Alps autosomal (Malus domestica) are shown in Table 5.

As shown in Fig. 3, SNPs selected for the breed were Chr6.139584 for Fuji apple, Chr3.10473276 for Chongqing, Chr3.25021180 for Chongqing, Chr15.9155427 for Chongqing, Chr12.16892600 for Chongqing .

Ref. chr
(MalDomGD1.0_Primary_Assembly.fa) 4 Position 18260085 Ref C Fuji C Benishogun T Lie C Genic LOC103433818 Transcript XM_008372106.2 Gene.feature exon Gene.desc rhicadhesin receptor-like

In addition, as shown in Table 5, the selected Hong group SNP is Chr 4.18260085, and its base is different from Fuji apple, Yataka and Alps Autome.

From these results, it can be discriminated as a cultivar of reddish when the 10473276th base of Apples 3 (NC_024241.1) is T. Also, when the 25021180th base of apple chromosome 3 (NC_024241.1) is A; If the 18260085th base of apple chromosome 4 (NC_024242.1) is T, it is a Hong generic variety; If the 17139584th base of the chromosome 6 of the apple (NC_024244.1) is A, use Fuji apple; If the 16892600th base of the apple chromosome 12 (NC_024250.1) is A, it is a Yataka variety; Chromosome 15 of the apple (NC_024253.1) has the base C of 9155427; Respectively.

In addition, the selected SNP site can be used as a SNP marker for discriminating Fuji apple and its azo varieties, and it is possible to more easily identify the azo mutant by preparing a primer for amplifying the SNP marker.

Example  5. Fuji apple Azo  For mutant variety identification primer  Production and identification of varieties using them

On the basis of Table 5 of Example 4, a primer SFY4A for the identification of the genus Hong, which is a variant of azo mutant of Fuji apple, was prepared. The primer SFY4A was constructed so that the last base of the primer 3 'was complementarily bound to the 18260085th base of the chromosome 4 of FUJI apple, SNP site. Information on the primer SFY4A is shown in Table 6.

Primer SFY4A length 20 nt Tm Fw 61.4 Tm Rv 61.4 GC Fw (%) 60 GC Rv (%) 60

Allele specific polymerase chain reaction was performed using 20 nt long primer SFY4A prepared as shown in Table 6 above. The above-mentioned allele-specific PCR does not amplify the DNA unless the 3 ' final base sequence of one of the two primers used in the PCR is complementary to the template DNA. Therefore, if PCR is performed so that the base pair corresponding to the SNP in the allele in which the SNP is present becomes the last base pair of the primer, the allele complementary to the whole sequence of the primer is amplified and complementary to the primer 3 ' Not alleles are not amplified. Therefore, SNP can be confirmed by the presence or absence of PCR amplification products.

Specifically, for the allele-specific PCR for the identification of the Hong Kong species, 20 ng of the genomic DNA of the red blood group and 0.5 μM of the concentration SFY4A primer set (Fw and Rv), 1 × Taq reaction buffer , 0.2 mM dNTP mix 1.25 unit Taq DNA polymerase and sterilized water were mixed to prepare a reaction solution. The reaction solution was denatured at 94 DEG C for 30 seconds, annealed at 58 DEG C for 30 seconds, and then extended at 72 DEG C for 60 seconds. The PCR product was electrophoresed in 1.5% agarose gel at 100 V for 40 minutes and amplified by UV irradiation. Allele specific PCR was performed in the same manner as for Fuji apple and Yata taka. The result of allele-specific PCR for the identification of the Hong generations is shown in FIG.

As shown in Fig. 4, it can be seen that the DNA was amplified by complementary binding of the primers to Fuji apple and yata taka. On the other hand, the primate was not complementarily binding due to SNP, and DNA was not amplified. These results indicate that allele - specific PCR is performed using the SFY4A primer set, so that it is possible to discriminate the genus Hongo, the azo mutant of Fuji apple.

In general, when the selected SNP markers of the present invention and a primer set for amplifying the SNP markers of the present invention are used, it is possible to accurately discriminate the azo mutant variety of Fuji apple which does not show a large difference in phenotype due to high genetic similarity. In addition, we can confirm the color system, azo system, phylogenetic system, phylogenetic system and phylogenetic system of Azo mutant of Fuji apple. In future, we can contribute to promotion of intellectual property protection and branding of azo mutant varieties of domestic Fuji apple, It can be used to secure competitiveness.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

10473276 th position of chromosome 3 of apple (NC_024241.1);
25021180 th position of chromosome 3 of apple (NC_024241.1);
18260085 th position of chromosome 4 of apple (NC_024242.1);
Position 17139584 of the chromosome 6 of apple (NC_024244.1);
Position 16892600 of the chromosome 12 of the apple (NC_024250.1); And
Position 9155427 of chromosome 15 of apple (NC_024253.1); (SNP) marker of at least one locus selected from the group consisting of the nucleotide sequence of SEQ ID NO:
The method according to claim 1,
Wherein the single nucleotide polymorphism marker comprises:
A single nucleotide polymorphism marker with T at base 10473276 of the chromosome 3 (NC_024241.1) of apple;
A single nucleotide polymorphism marker in which the 25021180 base of the apple chromosome 3 (NC_024241.1) is A;
A single nucleotide polymorphism marker with the nucleotide 18260085 of the apple chromosome 4 (NC_024242.1);
A single nucleotide polymorphism marker of nucleotide 17139584 of the chromosome 6 of apple (NC_024244.1);
A single nucleotide polymorphism marker in which the 16892600 base of the apple chromosome 12 (NC_024250.1) is A; And
Wherein the 9155427 base of the chromosome 15 of the apple (NC_024253.1) is a single nucleotide polymorphism marker of C. The composition for discriminating the azo mutant of fuji apple.
The method of claim 1, wherein
Wherein the agent is a polynucleotide that specifically hybridizes with a polynucleotide consisting of 10 to 100 consecutive DNA sequences or a complementary polynucleotide thereof, including a single base polymorphic marker site. Compositions for discriminating azo mutants.
The method of claim 3,
Wherein the polynucleotide is at least one selected from the group consisting of primers, probes, and combinations thereof capable of detecting single nucleotide polymorphism (SNP) markers.
5. The method of claim 4,
Wherein the primer is complementarily bound to a single base polymorphic site at the 3 'last base.
The method according to claim 1,
The azo varieties of Fuji apples are distinguished by at least one system selected from the group consisting of a coloring system, a precocious system, a monoculture system, and a phylogeny system.
The method according to claim 6,
Wherein the coloring system is at least one selected from the group consisting of Danhong and Kyonggi.
The method according to claim 6,
Wherein the premixing system comprises at least one member selected from the group consisting of Hong genera and Yata taka.
9. An azo mutant strain of the Fuji apple comprising the composition according to any one of claims 1 to 8.
(a) extracting DNA of Fuji apple;
(b) performing a polymerase chain reaction using the DNA of step (a) as a template and using the composition according to any one of claims 1 to 8; And
(c) analyzing the PCR product to discriminate the variety; and determining the azo mutant strain of Fuji apple.
11. The method of claim 10,
Wherein the polymerase chain reaction of step (b) is an allele-specific polymerase chain reaction.

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