KR102142010B1 - Marker for discriminating Hongro apple and its bud mutation cultivar and use thereof - Google Patents

Abstract

The present invention relates to a marker for discriminating a cultivar of a Hongro apple and a bud mutation thereof, and a use thereof. When using the marker of the present invention, it is possible to quickly and accurately discriminate the cultivar of the Hongro apple and the bud mutation thereof which do not show a large difference in the phenotype due to high genetic similarity. Thus, the present invention can contribute to the protection of intellectual property rights and promotion of branding of domestic cultivars of the Hongro apple and the bud mutation thereof and can also be used to secure competitiveness in related industries.

Description

Marker for discriminating Hongro apple and its bud mutation cultivar and use thereof}

The present invention relates to a Hongro apple and its marker for discrimination of azovaria and its use.

Apples ( Malus × domestica Borkh) are one of the main fruit crops, and compared to vegetable and grain crops, the breeding of apples takes longer, including the period from seed to flowering, and the genotypic composition is poor and not easy. In addition, because the cultivation area is also relatively large, the breeding period takes a long time, so the cultivation of apple varieties in Korea has been registered from azo mutations through grafting technology, and a number of varieties have been registered. The fruit varieties grown from the azo mutant are supplied in the state of seedlings, so it is almost impossible to discern the varieties. Classification of varieties by DNA markers can be easily and accurately identified varieties using extracted DNA without examining morphological traits such as branches, leaves, or fruits of trees, so that varieties can be prevented from distribution seedlings and protection of breeders' rights It is also very helpful. In addition, since the DNA marker essentially reflects the genetic characteristics of the cultivar, it is not affected by the environment and is highly useful in replacing and supplementing the phenotypic characteristics.

Single nucleotide polymorphism (SNP) between multiple individuals derived from genotyping by sequencing (GBS) technology is characterized by a variety of dynamics, such as genome-wide association studies, association mapping, and quantitative trait loci (QTL) mapping. · Used in genetic research of plant species. The development of plant genome analysis technologies such as NGS (Next Generation Sequencing) and GBS is contributing to the development of cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers through SNP search for sorting varieties. The Sequence Chracterized Amplified Region (SCAR) marker refers to a 15-30 bp primer pair produced by obtaining information on amplified sequencing from a random amplified polymorphic DNA (RAPD) marker. SCAR markers appear predominantly dominant for the target genetic region and can be easily detected by agarose gel electrophoresis.

On the other hand, Korean Registered Patent No. 1798644 discloses a composition for discriminating azo mutant varieties of Fuji apple, and Korean Registered Patent No. 1905458 discloses a'CAPS marker for discriminating apple varieties and uses thereof'. However, as for the'marker for discrimination of a red apple and its azovariate varieties and uses thereof', the present invention has not been disclosed.

The present invention was derived by the above-mentioned needs, and the present inventors searched for a polymorphic candidate region between Hongo apple and its sub-variant varieties magenta through GBS (Genotyping by sequencing) analysis, and produced and analyzed a primer set. It was confirmed that it is possible to discriminate between hongro and its azovariate varieties, and based on this, a result of producing and analyzing a magenta-specific primer, by confirming that it is possible to effectively discriminate between hongro and its azovariate varieties, the present invention Was completed.

In order to solve the above problems, the present invention is an erythroid apple and azo variation thereof comprising at least one primer set selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 1 and 2 and oligonucleotide primer sets of SEQ ID NOs: 3 and 4. A primer set for discriminating varieties is provided.

In addition, the present invention is the primer set; And a reagent for performing an amplification reaction.

In addition, the present invention comprises the steps of isolating genomic DNA from an apple sample; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set; And detecting a product of the amplification step.

The marker for discriminating a hongro apple and its azovariant varieties according to the present invention has a high genetic similarity, so that it is possible to quickly and accurately discriminate between the hongro apple and its azovarious varieties that do not show a significant difference in phenotype, and in the future the domestic iris apple and its azomut It can contribute to the protection of intellectual property rights and promotion of branding, and can be used to secure the competitiveness of related industries.

1 is an electrophoresis photograph of a PCR amplification product using a CP-GBS-JAHONG primer set. 1: magenta (repeat 1), 2: magenta (repeat 2), 3: magenta (repeat 3), 4: red, 5: control (water + primer), M: 100 bp ladder.
2 is an electrophoresis photograph of a PCR amplification product using a CP-SCAR-JAHONG primer set. 1: Hongro, 2: Magenta (repeat 1), 3: Magenta (repeat 2), 4: Magenta (repeat 3), 5: Control (water + primer), M: 100bp ladder.

In order to achieve the object of the present invention, the present invention is an erythroid apple containing at least one primer set selected from the group consisting of the oligonucleotide primer set of SEQ ID NO: 1 and 2 and the oligonucleotide primer set of SEQ ID NO: 3 and 4, and its Provides a primer set for discriminating azomorphic varieties.

In the present invention, "Hongro apple" is a variety that was nurtured by the National Academy of Horticultural Sciences, and was selected in 1988 by crossing'Spey Golden Delicacies' in'Spare Blaze' in 1980.

In the present invention, "bud mutation" means that a gene mutation occurs at the growth point of a branch and a stem during growth, resulting in a branch or stem having two or three different traits, also referred to as a branch variant. When only the mutated part is reproduced by grafting or folding, it is possible to obtain an individual whose trait is different from the parent strain, and the obtained individual is called a cultivar or system.

The primer set of the present invention is a oligonucleotide primer set of SEQ ID NOs: 1 and 2 or an oligonucleotide of SEQ ID NOs: 3 and 4, capable of discovering and amplifying regions showing genetic polymorphism between the red and green apples and its azo variant varieties. As a primer set, the oligonucleotide primer set of SEQ ID NOs: 1 and 2 is a primer set for CP-GSB-JAHONG marker amplification based on the SNP polymorphic position base between red and magenta, and the oligonucleotide primer set of SEQ ID NOs: 3 and 4 is in magenta As a specific primer set, it is a primer set for CP-SCAR-JAHONG marker amplification produced based on Examples 1 and 2. SEQ ID NOs: 1 and 3 are forward primers, and SEQ ID NOs: 2 and 4 are reverse primers.

When both the oligonucleotide primer sets of SEQ ID NO: 1 and 2 and the oligonucleotide primer sets of SEQ ID NO: 3 and 4 of the present invention are used, red and magenta can be more accurately determined.

According to the sequence length of each primer, 15 or more, 17 or more, 18 or more, 19 or more, 20 in the oligonucleotide primer sequences of SEQ ID NOs: 1 and 2 and the oligonucleotide primer sequences of SEQ ID NOs: 3 and 4 Oligonucleotides consisting of segments of two or more consecutive nucleotides. In addition, the primers are oligonucleotide primer sequences of SEQ ID NOs: 1 and 2; And addition, deletion or substituted sequences of base sequences in the oligonucleotide primer sequences of SEQ ID NOs: 3 and 4.

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

As used herein, oligonucleotides used as primers may also include nucleotide analogs, such as phosphorothioates, alkylphosphorothioates or peptide nucleic acids, or It may include an intercalating agent.

In the primer set for discriminating a hongro apple and its azovarious varieties of the present invention, the azovarious varieties of the hongro apple may be magenta, but are not limited thereto.

In addition, the present invention is the primer set; And a reagent for performing an amplification reaction.

In the kit of the present invention, reagents for performing the amplification reaction may include DNA polymerase, dNTPs, buffers, and the like. In addition, the kit of the present invention may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout that explains how to use the kit, for example, how to make PCR buffers, and suggested reaction conditions. The guide includes brochures in the form of brochures or flyers, labels attached to the kit, and descriptions on the surface of the package containing the kit. In addition, the handbook includes information that is disclosed or provided through electrical media, such as the Internet.

In addition, the present invention

Isolating genomic DNA from the apple sample;

Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set; And

It provides a method for determining the red roe apple and its azo variant varieties comprising the step of detecting the product of the amplification step.

The method of the present invention includes isolating genomic DNA from an apple sample. As the method for isolating genomic DNA, a method known in the art may be used, for example, a CTAB method may be used, or a wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, the target sequence can be amplified by performing an amplification reaction using the oligonucleotide primer set according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. amplification system, strand displacement amplification or any other suitable method for amplifying via Qβ replicates or amplifying nucleic acid molecules known in the art. Among these, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In one embodiment of the invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling material may be a material emitting fluorescence, phosphorescence or radioactivity, but is not limited thereto. Preferably, the labeling material is Cy-5 or Cy-3. When PCR is performed by labeling Cy-5 or Cy-3 on 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, a label using a radioactive material, when a radioactive isotope, such as ³² P or ³⁵ S, is added to the PCR reaction solution during PCR, the amplification product is synthesized and radioactive is incorporated into the amplification product, so that the amplification product can be radioactively labeled. The oligonucleotide primer set used to amplify the target sequence is as described above.

In one embodiment of the present invention, the method for discriminating the hongro apple and its azomutated varieties includes detecting a product of the amplification step, wherein the detection of the amplification product is capillary electrophoresis, DNA chip, gel Electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement may be performed, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis may be performed, and gel electrophoresis may use acrylamide gel electrophoresis or agarose gel electrophoresis depending on the size of the amplification product. In addition, capillary electrophoresis can be performed. For capillary electrophoresis, for example, an ABi Sequencer can be used. In addition, in the fluorescence measurement method, when PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer, the target sequence is labeled with a detectable fluorescent labeling material, and the fluorescence thus labeled is measured using a fluorescence meter. can do. In addition, in the radioactive measurement method, when performing PCR, a radioactive isotope such as ³² P is added to the PCR reaction solution to label the amplified product, and then a radioactive measuring instrument, for example, a Geiger counter or a liquid scintillation counter (liquid). Radioactivity can be measured using a scintillation counter.

Hereinafter, it will be described in detail by examples of the present invention. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Materials and methods

DNA extraction and quantification

The leaves were collected from the tip of the red apple and its azovarious varieties, magenta, washed with distilled water, packaged individually and stored at -80°C. For the DNA extraction of the collected leaves, CTAB (cetyl trimethyl ammonium bromide) method was used. The young leaves were ground in a mortar with liquid nitrogen and then transferred to a sample tube. 500 µl of CTAB buffer was added, and 2 µl of 2-mercaptoethanol was added, followed by reaction in a constant temperature water bath at 65° C. for 30 minutes. 200 μl of 5M potassium acetate was added and treated on ice for 30 minutes. Subsequently, 600 µl of a solution of phenol, chloroform and isoamyl alcohol mixed at 25:24:1 was added and centrifuged at 13,000 rpm at 4°C for 10 minutes, the supernatant was transferred to a new tube, and 600 µl of chloroform and isoamyl After adding the mixed solution of alcohol 24:1, it was centrifuged at 13,000rpm at 4℃ for 10 minutes. The supernatant was transferred back to a new tube, 600 μl of cold isopropanol was added, and a dehydration reaction was conducted at -20°C for 30 minutes, followed by centrifugation at 13,000 rpm for 5 minutes at 4°C and sedimentation was confirmed. The isopropanol was discarded, and 500 µl of cold 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 5 minutes at 4°C. After ethanol was discarded and dried, 30 µL of TE (Tris-EDTA) buffer was added. Thereafter, RNase A was added, treated at 37°C for 1 hour, and stored at -80°C. The extracted genomic DNA was quantified at 24 ng/μl.

GBS (Genotyping by sequencing) analysis

For sequencing and genotyping, a library for GBS was constructed against DNA of 95 apple genetic resources, including Hongro apple and its sub-variant varieties, Maghong (Elshire et al., 2011, PloS one, 6( 5):e19379.), the library was sequenced according to the standard protocol of the Illumina Hiseq2000 sequencer. Since the generated fragment sequences were used for bioinformation analysis, the reference genome of the total genomic sequence of'Golden delicious' (https://www.ncbi.nlm.nih.gov/assembly/GCF_000148765.1) for bioinformation analysis It was used as. Through the DNA sequence data analysis of the Hongro apple and its sub-variant varieties Magenta, the regions showing the differences between the DNA sequences in 349 regions were identified. 338 were single nucleotide polymorphisms (SNPs), which represent DNA mutations caused by one base sequence being substituted with another, and 11 were InDel (Insertion/Deletion), which means relative insertion or deletion. Of the 338 candidate SNPs, 7 primer sets for PCR assays were prepared for 7 candidate SNPs considering the read depth. Based on the coordinate values of the apple reference genome of the candidate SNP, after extracting a total length of 1,000 bp by 500 bp back and forth, primers were prepared, and one primer set (CP-GBS-JAHONG) was selected.

The composition of the PCR mixture is 40 μl in total by adding 2 μl (20 ng) of extracted red and magenta gDNA, 6 μl of 2×Hot Taq polymerase (GeNetbio, Korea) and 2 μl of primer set (1 μl each of forward and reverse primers). PCR reaction mixtures were prepared respectively. The PCR process was repeated 35 times at 94°C for 30 seconds, annealing at a melting temperature (Tm) temperature described in Table 1 below for 45 seconds, and 72°C for 30 seconds, and finally stretching at 72°C for 7 minutes ( extension). The amplified PCR products were electrophoresed on a 1.5% agarose gel to confirm polymorphism.

Primers used in the present invention primer Base sequence (SEQ ID NO) Expected size (bp) Tm(℃) CP-GBS-JAHONG F: TCC CAC CTT TGG CCA G (1) 750 63.7 R: TCT GAA CCT TTT CTT CTC TGT (2)

SCAR marker development

In the CP-GBS-JAHONG primer set and PCR amplification products of'magenta', an agarose electrophoresis fragment for a PCR amplification product having the same size as expected on the reference genomic data was cut out, followed by Sanger sequencing. One primer set (CP-SCAR-JAHONG) was re-produced using primer 3 software from the base sequence obtained through sequencing of Sanger of'magenta' (Table 2).

Primers used in the present invention primer Base sequence (SEQ ID NO) Expected size (bp) Tm(℃) CP-SCAR-JAHONG
F: CCT TTC AGA TCT GCC ACA CA (3) 640 61.6 R: AGA AAC TTT CGC ATC CAC TGA (4)

Example 1. Discrimination of Hongro apple and its sub-variant magenta using CP-GBS-JAHONG primer set

PCR and agarose gel electrophoresis were performed on the red rose apple and its azo variant varieties, magenta. As a result of confirming the PCR reaction product using the CP-GBS-JAHONG primer set of the present invention, as shown in FIG. 1, it was confirmed that the band pattern was different between 3 magenta, 1 hongro and control (water+primer). . In particular, the CP-GBS-JAHONG primer set contained a sequence (yellow arrow portion) other than 750 bp of the expected PCR amplification product size based on the reference genomic sequence data in magenta. Through this, it was confirmed that the CP-GBS-JAHONG primer set of the present invention can be used for discrimination between Hongro apple and its sub-variant varieties Magenta.

Example 2. Discrimination of Hongro apple and its sub-variety magenta using CP-SCAR-JAHONG primer set

As disclosed in Example 1, the CP-GBS-JAHONG primer set produced a PCR amplification product other than the size expected in the PCR reaction between Hongro apple and its sub-variant, magenta. In order to discriminate two more stable varieties, the PCR amplification fragment of 750bp, which is specifically amplified in magenta and has the same size as the primer designed in the reference genome, was cut out and the base sequence was read again through Sanger sequencing. As a result of performing PCR by applying the re-produced CP-SCAR-JAHONG primer set to 1 of Hongro, 3 of Jahong and control (water+primer), agarose electrophoresis was performed, resulting in CP-SCAR-JAHONG primer set. It was confirmed that only magenta was stable and specifically amplified (FIG. 2 ).

Through this, it was found that the CP-GBS-JAHONG primer set and CP-SCAR-JAHONG primer set of the present invention can be used for discrimination between Hongro apple and its sub-variant varieties Magenta.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Marker for discriminating Hongro apple and its bud mutation cultivar and use thereof <130> PN19180 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tcccaccttt ggccag 16 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tctgaacctt ttcttctctg t 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cctttcagat ctgccacaca 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agaaactttc gcatccactg a 21

Claims (6)

A primer set for discriminating a crimson apple and its azovarious varieties, magenta, comprising at least one primer set selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 1 and 2 and oligonucleotide primer sets of SEQ ID NOs: 3 and 4. delete The primer set according to claim 1; And a reagent for performing an amplification reaction. A kit for determining magenta, which is a red berry apple and its azovariate varieties. [4] The kit of claim 3, wherein the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs, and buffer. Isolating genomic DNA from the apple sample;
Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to claim 1; And
A method of determining the erythroid apple and its azo variant varieties magenta, comprising the step of detecting the product of the amplification step. The method according to claim 5, wherein the detection of the amplification product is performed by gel electrophoresis, capillary electrophoresis, DNA chip, radiometric measurement, fluorescence measurement, or phosphorescence measurement. How to.

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