KR102335806B1 - Molecular marker based on chloroplast genome sequence for discriminating Zizyphus jujuba 'SanJo' cultivar and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker based on chloroplast genome sequence for distinguishing 'Sanjo', which is a variety of Ziziphus jujuba Mill. and a use thereof. A molecular marker of the present invention can rapidly and accurately discriminate various varieties of Ziziphus jujuba Mill. Therefore, the present invention is expected to be effectively used in preventing the mixing of varieties of Ziziphus jujuba Mill. distributed in the domestic market as well as be utilized for quality control purposes.

Description

Molecular marker based on chloroplast genome sequence for discriminating Zizyphus jujuba 'SanJo' cultivar and uses thereof}

The present invention relates to a molecular marker based on a chloroplast genome sequence for differentiating jujube sanjo varieties and a use thereof.

Jujube ( Zizyphus jujuba ) is a plant belonging to the genus Zizyphus of the Rhamnaceae family, and its origin is China, and is cultivated in about 30 countries including China, Korea and Japan, Asia, Europe, the United States, Canada, Australia, etc. It is a crop. Jujube, a stone fruit, is particularly rich in carbohydrates, vitamin C, amino acids, and phenols. The seeds contain unsaturated fatty acids such as oleic acid. It is widely used as a food supplement. Vitamin C is about 4 times higher than that of kiwi, and sugar is about twice that of sugar beet. Since it is a crop with such a high utility value, various varieties of jujube have been developed, but a method for accurately classifying varieties is still lacking. In particular, jujubes are mainly vegetatively propagated, so it is difficult to distinguish varieties with the naked eye except for some species, and it is very difficult to distinguish them because they are genetically similar. However, with the radical development in the field of molecular biology, molecular biological methods are being used in a variety of ways to classify plant genus, interspecies, or within species. have. Differentiation at the molecular level by DNA analysis has the advantage of being able to grasp a number of characteristics as well as the quantitative level and excluding the influence of the environment.

By comparing DNA information analyzed through next generation sequencing (NGS) technology, and finding differences, the development of molecular markers using this technology enables accurate differentiation of jujube varieties. Among the various types of molecular markers based on genomic information, the InDel (Insertion/Deletion) marker is a method for detecting polymorphisms using PCR (polymerase chain reaction) technology. The InDel marker uses the difference in the nucleotide sequence inserted or deleted from the nucleotide sequence, and it is a molecular marker suitable for cultivar classification because it exists in various genotypes not only between species but also within species.

On the other hand, Korean Patent No. 009712 discloses 'a primer set for discriminating jujube demodulation varieties and their use', and Korean Patent No. 1860232 discloses 'Jujube varieties using SSR markers that can discriminate jujube varieties Boeun and Chuseok'. ' is disclosed, but there is no description of 'a molecular marker based on the chloroplast genome sequence and its use for differentiating jujube sanjo varieties' of the present invention based on the InDel polymorphism.

The present invention was derived in response to the above needs, and the present inventors compared the chloroplast genome information of the main domestic jujube varieties Sanjo, Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Venus and Wolchul to compare the InDel (insertion/deletion) polymorphism. After preparing a primer set for amplifying a nucleotide sequence showing By confirming that only varieties can be specifically distinguished, the present invention has been completed.

In order to solve the above problems, the present invention provides a primer set for distinguishing Sanjo varieties from Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties, comprising an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 do.

In addition, the present invention is the primer set; And it provides a kit for distinguishing sanjo cultivars from jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties, including reagents for performing an amplification reaction.

In addition, the present invention comprises the steps of isolating genomic DNA from a jujube sample; amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; and detecting the product of the amplification step; provides a method for distinguishing Sanjo varieties from Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties, including.

The InDel molecular marker of the present invention clearly distinguishes the jujube varieties distributed in the domestic fruit tree market and the oriental medicine market, and can be usefully used as an index for preventing raw material mixing of jujube varieties distributed in the domestic market and controlling the quality of raw materials.

1 is a PCR amplification product of jujube Sanjo, Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Venus and Wolchul cultivar resources using the Zj-cp-InDel-21 primer set prepared based on the InDel polymorphism between jujube varieties of the present invention. is the result of gel electrophoresis.

In order to achieve the object of the present invention, the present invention provides a primer set for distinguishing Sanjo varieties from Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties, including the oligonucleotide primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 provides

In the primer set of the present invention, the jujube sanjo variety refers to resource number 10498 possessed by the National Forest Varieties Management Center of the Korea Forest Service.

The primer is an oligonucleotide consisting of a fragment of 13 or more, 14 or more, 15 or more, 16 or more, 17 or more consecutive nucleotides in the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 according to the sequence length of each primer. may include For example, the primer of SEQ ID NO: 1 (20 oligonucleotides) may include an oligonucleotide consisting of a fragment of 17 or more, 18 or more, 19 or more consecutive nucleotides in the sequence of SEQ ID NO: 1. In addition, the primer may also include an addition, deletion or substitution of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. Odd-numbered oligonucleotide primers in the SEQ ID NO: are forward primers, and even-numbered oligonucleotide primers are reverse primers.

The oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention is a primer set capable of amplifying the InDel marker located in the intergenic region of rps8 and rpl14 of the date chloroplast genome, Amplification products of different sizes were identified by insertion of a 20 bp base in the chloroplast genome sequence of the jujube 'Sanjo' cultivar compared to the 'Mudeung, Chuseok, Venus and Wolchul' varieties. And only the Sanjo variety can be specifically discriminated from the Wolchul variety.

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

As used herein, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.

The present invention also includes the primer set; And it provides a kit for distinguishing sanjo cultivars from jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties, including reagents for performing an amplification reaction.

In one embodiment of the present invention, the reagent for performing the amplification reaction may include, but is not limited to, DNA polymerase, dNTPs, and a buffer.

In one embodiment of the present invention, the kit may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and PCR buffer, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.

The present invention also

isolating genomic DNA from the jujube sample;

amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; and

It provides a method for distinguishing Sanjo varieties from Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties, including the step of detecting the product of the amplification step.

In the method according to an embodiment of the present invention, the jujube sanjo variety refers to resource number 10498 possessed by the National Forest Varieties Management Center of the Korea Forest Service, and the primer set is the same as described above.

The method of the present invention includes isolating genomic DNA from a jujube sample. A method for isolating the genomic DNA may use a method known in the art, for example, the CTAB method may be used, or a Wizard prep kit (Promega) may be used. The target sequence may be amplified by performing an amplification reaction using the isolated genomic DNA as a template and 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 systems. amplification system), strand displacement amplification or amplification via Qβ replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a 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 present 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 substance is Cy-5 or Cy-3. When PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer during amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. ^{In addition, when a radioactive isotope such as 32} P or ³⁵ S is added to a PCR reaction solution for labeling using a radioactive material, the amplification product is synthesized and radioactivity 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 the same as described above.

In one embodiment of the present invention, the method for distinguishing a Sanjo variety from the Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties comprises the step of detecting the product of the amplification step, Detection of the product may be performed through, but not limited to, DNA chip, gel electrophoresis, capillary electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement. As one of the methods for detecting the amplification product, capillary electrophoresis may be performed. Capillary electrophoresis may use, for example, an ABi Sequencer. In addition, gel electrophoresis can be performed, and agarose gel electrophoresis or acrylamide gel electrophoresis can be used for gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, when PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorescence meter. can do. In addition, the radioactive measurement method is a ^{radioactive isotope such as 32} P or ³⁵ S added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter (Geiger counter) or liquid scintillation The radioactivity can be measured using a liquid scintillation counter.

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

Materials and Methods

1. Jujube resource collection and genomic DNA extraction

The varieties of jujubes 'Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul' used in the experiment were provided by the Chungcheongbuk-do Agricultural Research and Extension Services, and the 'Sanjo' variety was provided by the National Forest Breeding Center (Resource No.: 10498). After collecting young leaf samples from each resource, DNA for NGS analysis was extracted using Qiagen DNeasy Plant Mini Kit, and large-capacity DNA for PCR was extracted using CTAB (Cetyl Trimethyl Ammonium Bromide) method.

2. Development of Chloroplast-based InDel Marker to Discriminate Jujube Varieties

After obtaining the DNA sequence information of the jujube varieties by performing NGS (Next Generation Sequencing) analysis on the genomic DNA of the extracted jujube varieties 'Sanjo, Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul', By comparing and analyzing the nucleotide sequences of the jujube varieties using the CLC Main Workbench program (version 8.0.1), the InDel polymorphic region showing the difference in insertion or deletion between the chloroplast nucleotide sequences of the eight jujube varieties was identified. A primer set was prepared for amplifying the InDel polymorphic region (Table 1).

The InDel marker of the present invention was constructed based on a polymorphic region in which a 20 bp base was inserted from the chloroplast genome sequence of the Sanjo variety in the intergenic region of rps8 and rpl14 of the date chloroplast genome.

InDel marker-based primer set information of the present invention Primer name Sequence (5'→3') (SEQ ID NO:) gene location Zj-cp-InDel-21F GACACAACCAATCTACTACT (1) rps8 to rpl14 Zj-cp-InDel-21 R GAAGGAAATCCAAAAGGAAC (2)

3. DNA amplification using polymerase chain reaction (PCR)

Dilute the DNA of jujube extracted using the CTAB method to 10 ng/μl, respectively, and then 2 μl (10 ng) of DNA, 2 μl of 2 μM forward primer, 2 μl of 2 μM reverse primer, 10 μl of Taq mixture, 4 μl of distilled water were mixed to make 20 μl of a PCR reaction mixture. The PCR process was pre-denatured at 94°C for 3 minutes; A total of 35 cycles of denaturation at 94°C for 30 seconds, annealing at 51°C for 30 seconds, and extension at 72°C for 1 minute; It was carried out under the condition of final elongation 72°C for 5 minutes. PCR amplification products were confirmed by electrophoresis on a 3% agarose gel.

Example 1. Identification of jujube varieties using InDel molecular markers

Using the Zj-cp-InDel-21 primer set prepared based on the InDel polymorphism between jujube cultivars, PCR was performed on DNA samples of various jujube cultivars as templates, and then electrophoresed on an agarose gel to Zj-cp-InDel- Polymorphic pattern analysis of 21 primer sets was performed.

As a result, a 237 bp band was confirmed in the 'Sanjo' variety, and a 217 bp band was confirmed in the 'Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul' varieties (Fig. 1), , Mudeung, Chuseok, Geumseong, and Wolchul were confirmed that it is possible to specifically distinguish the Sanjo variety.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Molecular marker based on chloroplast genome sequence for discriminating Zizyphus jujuba 'SanJo' cultivar and uses thereof <130> PN20288 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gacacaacca atctactact 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gaaggaaatc caaaaggaac 20

Claims (5)

A primer set for distinguishing Sanjo varieties from Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Venus and Wolchul varieties, comprising the oligonucleotide primer sets of SEQ ID NO: 1 and SEQ ID NO: 2. The oligonucleotide primer set of claim 1; And a kit for distinguishing Sanjo varieties from Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties comprising reagents for performing an amplification reaction. The kit according to claim 2, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs and a buffer. isolating genomic DNA from the jujube sample;
amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the oligonucleotide primer set of claim 1; and
A method of distinguishing Sanjo varieties from Jujube Boeun, Bokjo, Cheonsang, Mudeung, Chuseok, Geumseong and Wolchul varieties, comprising the step of detecting the product of the amplification step. The method according to claim 4, wherein the detection of the product of the amplification step is performed through a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement.

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