KR101912192B1 - Molecular marker and primer set for discriminating Platycodon grandiflorum cultivar and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker and a primer set for discriminating platycodon and a use thereof, and the chloromelic sequence-based InDel molecular marker and the primer set for the molecular marker amplification of the present invention can easily and quickly discriminate the species of the platycodon Therefore, it is considered that it is possible to effectively prevent mixed use among the blooming cultivars.

Description

Molecular markers and primer sets for distinguishing bollflower varieties and their uses {Molecular marker and primer set for discriminating Platycodon grandiflorum cultivar and uses thereof}

The present invention relates to a molecular marker and a primer set for the determination of the species of Doragi and a use thereof.

Recently, the rapid development of molecular biology has led to the development of nucleic acid fingerprinting methods and various DNA markers that enable the study of bio-diversity at the DNA level. This makes it possible to search for useful traits, identify species of organisms, classify and identify cultivars, and analyze the relationship of group populations. Fingerprinting using the polymerase chain reaction (PCR) developed so far includes randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphic DNA (AFLP), and simple sequence repeat (SSR) methods. Among the above methods, the RAPD method has a disadvantage in that the non-specific PCR product is amplified and thus the reproducibility is poor. The AFLP method is complicated in appearance and analysis of a band with low reproducibility, although it is spotlighted by high DNA polymorphism detection. The PCR primer is prepared based on the nucleotide sequence information in the microsatellite region, which is a repeated sequence, to analyze the supersatellite in the individual. Since the number of repetitions of the simple nucleotide sequence varies among cultivars or individuals, When amplified by the reaction, polymorphism may occur, but it is not sufficient for high density gene mapping.

On the other hand, the InDel (insertion / deletion) marker complements the disadvantage of the SSR marker which is insufficient for high-density gene mapping while maintaining the advantage of exhibiting polymorphism when amplified by PCR reaction. Analysis is used to construct a PCR primer based on insertion or deletion of a nucleotide sequence between cultivars.

Plants have three genomes: nuclear, mitochondria, and chloroplast genomes. The majority of plant genetic information is present in the nuclear genome, but the size of the genome is large and consists of many introns and exons. The nucleotide sequence of a specific gene is analyzed from genomic DNA, (ITS) region of nuclear ribosomal DNA (nrDNA), and specific intron regions of alcohol dehydrogenase are used. The plant mitochondrial genome is usually 400kb to 4000kb in size, unlike animal mitochondria, large in size, structurally very unstable, and very conservative in its nucleotide sequence, It is almost meaningless.

Plant chloroplasts, on the other hand, have become very important not only in the chloroplast itself, such as origin or evolution, but also in genetic research. The size of the chloroplast genome is about 160 Kb, structurally stable, and the non coding region with a large nucleotide sequence is useful for identification of endemic species, identification of subspecies and variants, and genetic variation have. Of the 120 genes present in the chloroplast genome, 84 genes are transcribed into proteins, which vary greatly depending on the gene. Some conserved genes can be used only in the taxa of higher taxa, but the sequence of the rapidly evolving genes can be used to discuss the interrelationships between consecutive or distinct species in the genus. Therefore, the nucleotide sequences of the chloroplast genome can be widely used to establish the validity, identification, and conservation strategies of the Korean genus.

Platycodon grandiflorum is a perennial herb that grows up to 20 years and belongs to Campanulaceae . In Korea, the species registered as a variety of platycodon is the domesticated platycodon and top platycodon obtained through the net analysis of native platycodon. In addition to these, there are also lines that have been cultivated from the white rosewood plants collected in Jeju, Miryang, and Asan. The Jangbaek Doraji system registered in the National Seed Supervisory Service is a domestic pure native species which was cultivated by Rural Development Administration in 2002 separated from native species collected in Miryang. The white flowers are white, the leaves are lanceolate, and the flowering period, rhizome, and hairy appearance are similar to those of native species, but their roots are long and thick.

As the consumer demand for the bellflower grows, the volume of imported agricultural products is increasing, and a validation method for distinguishing between domestic and foreign varieties is necessary due to the incorporation of various varieties. Therefore, it is necessary to develop molecular genetic techniques that can classify strains of genetically related strains for breeding, breeding protection, and breeding research.

Accordingly, the inventors of the present invention have developed an InDel molecular marker based on a chloroplast sequence for the breeding of bellflower, and confirmed that the bellflower variety can be identified by preparing a primer capable of amplifying the molecular marker.

Korean Patent Laid-Open Publication No. 2017-0055613 discloses a method for judging whether or not a ginseng sample, a bellflower, or a mixture thereof is incorporated. In Korean Patent Publication No. 2012-0051937, Discloses a method for determining the number of years of cultivation. However, there is no description of a molecular marker, a primer set, and a use thereof for the determination of the species of the present invention.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and the inventors of the present invention compared the chloroplast genome sequences of 11 varieties of Platycodon grandiflorum to determine the nucleotide sequence of InDel (insertion / deletion) polymorphism at the trnQ- , And a primer set capable of amplifying the InDel molecular marker was prepared and PCR was performed on the bell sample. As a result, it was confirmed that the InDel molecular marker could discriminate the bellflower MARIES 2, Thereby completing the invention.

In order to solve the above-described problems, the present invention provides a primer set for determining the species of the blooming MARIES 2 comprising the oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention provides a kit for distinguishing a blooming MARIES2 cultivar comprising the primer set.

In addition, the present invention provides a method for distinguishing the blooming maries 2 cultivars using the primer set.

The InDel molecular marker of the present invention is based on a nucleotide sequence exhibiting polymorphism due to insertion or deletion in the chloroplast genome of Platycodon. Using the InDel molecular marker of the present invention, it is possible to easily and quickly discriminate platycodon species, And it can be usefully used as an index for prevention of mixing of raw materials and quality control of raw materials.

FIG. 1 is a schematic diagram showing a region showing an InDel polymorphism (green solid line) in the trnQ-UUG gene among the platelet chloroplast genome nucleotide sequences.
FIG. 2 shows PCR results of a total of 11 cultivars using a CPPlg1 primer set for amplifying InDel molecular markers.

In order to accomplish the object of the present invention, the present invention provides a primer set for determining the species of the Bloom maries 2 comprising the oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2.

The primers may include oligonucleotides consisting of fragments of at least 15, at least 16, at least 17, at least 18, at least 19 contiguous nucleotides in the sequence of SEQ ID NOs: 1 and 2, depending on the sequence length of each primer have. For example, the primer (18 oligonucleotides) of SEQ ID NO: 1 may comprise oligonucleotides consisting of fragments of 14 or more, 15 or more, 16 or more, 17 or more consecutive nucleotides in the sequence of SEQ ID NO: 1 . The primers may also include additional, deleted, or substituted sequences of the nucleotide sequences of SEQ ID NOS: 1 and 2.

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.

As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.

The present invention also relates to oligonucleotide primer sets according to the present invention; And a reagent for carrying out an amplification reaction.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, 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 manual includes instructions on the surface of the package including a 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.

In addition,

Isolating the genomic DNA from the bellflower sample;

Amplifying the target sequence by using the separated genomic DNA as a template and carrying out an amplification reaction using the oligonucleotide primer set; And

And detecting the product of the amplification step.

The method of the present invention comprises isolating genomic DNA from a bellflower sample. As a method for separating the genomic DNA from the bellflower sample, a method known in the art can be used. For example, a CTAB method may be used, or a Wizard prep kit (Promega) may be used. Using the separated genomic DNA as a template, an amplification reaction may be performed using a primer set according to an embodiment of the present invention to amplify the target sequence.

In one embodiment of the invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The oligonucleotide primer combinations used to amplify the target sequence are as described above.

In one embodiment of the present invention, the method for distinguishing the strains of BLOODY MARIES includes detecting the amplification product, wherein the amplification product is detected by a DNA chip, gel electrophoresis, capillary electrophoresis, Measurement, or phosphorescence measurement. As one method of detecting the amplification product, capillary electrophoresis can be performed. Capillary electrophoresis can, for example, use an ABI Sequencer. In addition, gel electrophoresis can be performed, and gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis depending on 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 carried out, 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 device such as a Geiger counter or liquid scintillation counter 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 are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. DNA extraction of bellflower

A variety of cultivars such as Platycodon grandiflorum DC. Jangback, MARIES2, HAKONE double white, HAKONE double blue, FUJI white, FUJI pink, FUJI blue, ASTRA white, ASTRA blue, ASTRA blue -double blue leaves were used. Reproducibility was checked by repeating three times for each individual. The collected bloom of the blooming blooms was immersed in liquid nitrogen, frozen and then crushed into a mortar, and the genomic DNA was extracted from the crushed bloom of the bloom by CTAB (Cetyl Trimethyl Ammonium Bromide) method. The extracted DNA was dissolved in TE buffer and stored.

2. Polymerase chain reaction (PCR)

10 ng of DNA, 10 pmoles of each primer, and other PCR reaction reagents were mixed to prepare a final PCR reaction mixture of 20 μl, followed by PCR. The conditions are shown in Table 1 below. The PCR amplification product was confirmed by electrophoresis on 2% agarose gel.

PCR reaction conditions step Temperature (℃) time Pre-denaturation 95 3 minutes denaturalization 95 1 minute Combination 56 1 minute kidney 72 1 minute Go back to denaturation stage and repeat 34 times Final height 72 5 minutes keep 4 ∞

Example 1. Analysis of mutation regions by comparison of nucleotide sequences of chloroplasts according to the species of dorp

Genomic DNA of 11 varieties of Drosophila melanogaster extracted from the above was sequenced and submitted to Terajen Eitex (Korea), and assembled using CLC Genomics Workbench 7.5 program. Among the sequencing results of the bellflower, the chloroplast genome contours were aligned and compared, and insertion or deletion base sequences were analyzed.

As a result, it was confirmed that a 90-bp base sequence was inserted or deleted in the trnQ-UUG gene region on the chloroplast genome of one bellflower (Fig. 1).

Example 2. Determination of Platycodon sp. Varieties using molecular markers

A primer set CPPlg1 capable of amplifying the InDel nucleotide sequence in the trnQ-UUG gene selected in Example 1 was prepared.

Primer for the determination of the bellflower variety Name of the primer The base sequence (5 '- > 3') Tm (占 폚) location CPPlg1 F AAGGCGTGATAGCGGTAA
(SEQ ID NO: 1) 54.5
trnQ-UUG CPPlg1 R TGGGACGAAAGGGATCGAA
(SEQ ID NO: 2) 57.1

PCR analysis was carried out using a total of 11 different strains of Streptococcus thermophilus as a template in order to confirm whether the seedlings were distinguishable by using the primer set CPPlg1.

As a result, it was found that the blooming maries2 and other 10 blooming varieties (Jangbaek, HAKONE double white, HAKONE double blue, FUJI white, FUJI pink, FUJI blue, ASTRA white, ASTRA pink, ASTRA blue, ASTRA semi-double blue), indicating that the MARIES 2 cultivars were correctly distinguished (FIG. 2).

Therefore, it was confirmed from the above results that the molecular marker of the present invention based on the nucleotide sequence showing InDel polymorphisms in the platelet chloroplast genome sequence and the primer set CPPlg1 for amplifying the same can be utilized for effectively discriminating the cultivar MARIAIS2.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Molecular marker and primer set for discriminating Platycodon          grandiflorum cultivar and uses thereof <130> PN17383 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 aaggcgtgat agcggtaa 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgggacgaaa gggatcgaa 19

Claims (5)

A primer set for determining the species of the blooming MARIES 2 comprising an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2. An oligonucleotide primer set according to claim 1; And a reagent for carrying out the amplification reaction.  3. The kit according to claim 2, wherein the reagent for carrying out the amplification reaction comprises a DNA polymerase, dNTPs and a buffer. Isolating the genomic DNA from the bellflower sample;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set according to claim 1; And
And detecting the product of said amplification step. 5. The method of claim 4, wherein the detection of the product of the amplification step is performed by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.

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