KR102226016B1 - Marker for identification of chloroplast derived from Ilex integra and Primer set for identification of Ilex x wandoensis - Google Patents

Abstract

The present invention relates to a marker for identifying chloroplasts of a persimmon tree, a primer set for identifying a persimmon tree chloroplast, a primer set for identifying a wando holly, and a method for identifying a chloroplast derived from persimmon tree using the same. By using the marker composition for identifying chloroplasts of the persimmon tree of the present invention, the identification primer set, the identification method, the composition for identification, or the kit for identification, it is possible to quickly and effectively identify whether the origin of the chloroplast from the chloroplast DNA is the chloroplast derived from the persimmon tree. . In addition, if the primer set for identification of persimmon tree, holly or wando holly tree of the present invention, identification method, identification composition or identification kit is used, persimmon tree, holly or wando holly tree can be obtained from the DNA of the tree. Can be identified quickly and effectively. In addition, the primer set for identification of the chloroplast Wando holly tree derived from the persimmon tree, holly, or persimmon tree of the present invention, the identification method, the composition for identification, and the kit for identification From DNA, it is possible to quickly and effectively discriminate the chloroplast Wando holly or the chloroplast Wando holly derived from holly.

Description

Marker for identification of chloroplast derived from Ilex integra and Primer set for identification of Ilex x wandoensis}

The present invention relates to a marker for identifying chloroplasts of a persimmon tree, a primer set for identifying a persimmon tree chloroplast, a primer set for identifying a wando holly, and a method for identifying a chloroplast derived from persimmon tree using the same.

Wando holly (Ilex x wandoensis) is a natural hybrid of holly (Ilex cornuta) and Ilex integra native to the southwestern coastal region of Korea. The fruit, which ripens red in autumn, also hangs in winter, and has excellent ornamental value. The red fruits of the Ilex genus trees attract algae and are thus attracting attention as an environmental tree species.

Plant cells have genomes in the nucleus, chloroplasts, and mitochondria. Chloroplasts are the main organs responsible for photosynthesis, and are generally maternal inheritance. The size of the chloroplast genome is 120 to 217 kb, and about 130 genes are preserved and maintained with few mutations, whereas there are relatively many single nucleotide polymorphisms (SNPs) and indels between genes and genes (IGS, intergenic spacers). It has mutations such as (InDel), inversion, and translocation. Chloroplasts are circular in all plant cells, and there are hundreds of copies in one cell.

However, in the case of plants, hybrids between various species and genus have been frequently reported, and it is difficult to understand the maternal lineage and evolution for accurate identification of plant taxa due to the complex pattern of multiplexing. Therefore, the development of DNA capable of grasping the maternal lineage is desired.

Among these nuclear DNA markers, there are rDNA and intranuclear intergenic transcribed spacer (ITS) sections, but in the case of rRNA genes, differences between species and genus hardly appear, whereas ITS sections with a lot of mutations have thousands of copies per single genome. Because of this, it can provide a characteristic with useful information for systematic analysis.

The Wando holly tree has all ITS sequences inherited from its parents, such as holly (Ilex cornuta) and persimmon tree (Ilex integra), so cross-species hybrids can be identified.

Currently, there is a need to develop a genetic marker that can easily and accurately identify the origin of the chloroplasts of the Wando holly tree native to Korea from plants of the genus Persimmon tree having a lot of genetic identity. Genetic markers capable of identifying trees have not been developed at present, and development of genetic markers capable of identifying them is required.

Accordingly, the present inventors have tried to develop a marker for simply and accurately identifying the chloroplasts of the persimmon tree, and a marker that can identify the persimmon tree, holly, and wando holly. Therefore, it was analyzed a DNA site capable of discriminating the persimmon tree chloroplast in the persimmon tree chloroplast DNA, and in the present invention, it was confirmed that the ndhC-trnV IGS of the persimmon tree chloroplast DNA can be an important marker to identify the persimmon tree chloroplast. And, by identifying markers for identification of persimmon tree, holly tree, and wando holly tree targeting ITS in the DNA of the nucleus, and by constructing and verifying the tetra-primer amplification resistance mutant system-PCR as a means to detect the corresponding marker, The present invention has been completed.

Accordingly, an object of the present invention is to provide a marker composition for identification of chloroplasts of persimmon tree, a primer set for identification, an identification method, a composition for identification, or a kit for identification, and a primer set for identification of persimmon tree, holly or wando holly tree , Identification method, identification composition or kit for identification, and persimmon tree, holly, persimmon tree-derived chloroplasts Wando holly or hollywood-derived chloroplasts Wando hollywood identification primer set, identification method, for identification It is to provide a composition, a kit for identification.

In order to achieve the above object, the present invention provides a marker composition for chloroplast identification of persimmon tree (Ilex integra ) containing ndhC-trnV IGS (intergenic spacer) of persimmon tree chloroplast DNA.

In addition, the present invention provides a primer set for identification of chloroplasts of the persimmon tree comprising; a primer pair consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention (a) separating the DNA from the sample; (b) using the DNA as a template and amplifying a target sequence using the primer set; And (c) detecting the amplification product. It provides a method for identifying chloroplasts derived from persimmon tree comprising a.

In addition, the present invention provides a composition for identification of persimmon tree chloroplasts comprising the primer set.

In addition, the present invention provides a kit for identification of persimmon tree chloroplasts comprising the composition for identification of persimmon tree chloroplasts.

In addition, the present invention is composed of a primer represented by the nucleotide sequence of SEQ ID NO: 3, a primer represented by the nucleotide sequence of SEQ ID NO: 4, a primer represented by the nucleotide sequence of SEQ ID NO: 5, and a primer represented by the nucleotide sequence of SEQ ID NO: 6 Primer set; It provides a primer set for identification including persimmon tree, holly or wando holly.

In addition, the present invention (a) separating the DNA from the sample; (b) using the DNA as a template, and using the primer set for identification of persimmon tree, holly or wando holly, tetra-primer amplification resistance mutation system-PCR (tetra-primer amplification refractory mutation system-PCR, ARMS- PCR) to amplify the target sequence; And (c) detecting the amplification product; it provides a method for identifying a persimmon tree, a holly or a wando holly tree.

In addition, the present invention provides a composition for identifying a persimmon tree, a holly or a wando holly tree comprising a primer set for identifying the persimmon tree, holly or wando holly.

In addition, the present invention provides a kit for identifying a persimmon tree, a holly or a wando holly tree comprising the composition for identifying the persimmon tree, holly or wando holly.

In addition, the present invention is a primer pair consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2; And a primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 3, a primer represented by the nucleotide sequence of SEQ ID NO: 4, a primer represented by the nucleotide sequence of SEQ ID NO: 5, and a primer represented by the nucleotide sequence of SEQ ID NO: 6. It provides a primer set for identification of chloroplasts derived from persimmon tree, holly, persimmon tree, or chloroplast derived from holly or holly.

In addition, the present invention comprises the steps of: (a) amplifying the DNA of the sample and detecting the amplification product using the primer set for identifying the chloroplasts of the persimmon tree; And (b) amplifying the DNA of the sample and detecting the amplification product using a primer set for identifying the persimmon tree, holly or Wando holly tree; containing persimmon tree, holly, persimmon tree-derived chloroplast Provides a method of identifying Wando holly or Wando holly chloroplast derived from holly.

In addition, the present invention is a persimmon tree, holly, persimmon tree-derived chloroplast wando holly or hollywood-derived chloroplast wand holly, including a primer set for identification of persimmon tree, holly, persimmon tree-derived chloroplast wand It provides a composition for identification of holly or holly chloroplasts Wando holly tree derived from holly.

In addition, the present invention is a persimmon tree, holly, persimmon tree-derived chloroplast Wando holly tree or hollywood-derived chloroplast Wando holly, including a composition for identification of the persimmon tree, holly, persimmon tree-derived chloroplast Wando holly Provides a kit for identification of chloroplasts derived from thorns or holly.

If the marker composition for identification of chloroplasts of the persimmon tree of the present invention, the identification primer set, the identification method, the composition for identification, or the kit for identification is used, it is possible to quickly and effectively identify the origin of the chloroplast from the chloroplast DNA. .

In addition, if the primer set for identification of persimmon tree, holly or wando holly tree of the present invention, identification method, identification composition or identification kit is used, persimmon tree, holly or wando holly tree can be obtained from the DNA of the tree. Can be identified quickly and effectively.

In addition, the primer set for identification of the chloroplast Wando holly tree derived from the persimmon tree, holly, or persimmon tree of the present invention, the identification method, the composition for identification, and the kit for identification From DNA, it is possible to quickly and effectively discriminate the chloroplasts derived from persimmon trees, holly, persimmon trees, and chloroplasts from Wando holly or holly, and preferably persimmon, holly, and Wando holly. It is possible to quickly and effectively discriminate the chloroplast Wando holly or holly-derived chloroplast Wando holly from any DNA sample selected from among them.

1 is a diagram schematically showing the location of ndhC-trnV IGS in chloroplast DNA.
Fig. 2 is a diagram schematically showing the principle of a chloroplast DNA-specific gene marker and an amplification product analysis method.
Figure 3 is a diagram confirming the results of amplification specific to the chloroplasts of the chloroplasts of a plurality of primer pairs prepared to select the chloroplast markers of the stalks trees.
Figure 4 is a diagram confirming the ability to detect chloroplasts derived from the persimmon tree of a pair of primers for identification of persimmon tree chloroplasts of SEQ ID NO: 1 and SEQ ID NO: 2.
5 is a diagram schematically showing the structure of ITS DNA in the nucleus.
6 is a diagram schematically showing the manufacturing process of the gene marker for confirming the hybrid of Wando holly.
7 is a diagram showing a method of analyzing a gene amplification product confirming a hybrid of Wando holly.
8 is a diagram comparing the effect of PCR amplification when the annealing conditions are changed when performing PCR using the primer set for identifying Wando holly tree of SEQ ID NOs: 3 to 6.
9 is a diagram confirming the ability to identify Wando holly thorn hybrids of the Wando holly thorn identification primer set of SEQ ID NOs: 3 to 6.
FIG. 10 is from a DNA sample of a persimmon tree, a holly tree, and a wando holly tree using a primer set of SEQ ID NOs: 1 to 2 and a primer set of SEQ ID NOs: 3 to 6, persimmon tree, holly tree, persimmon tree It is a diagram showing the process of identifying the chloroplast Wando holly tree derived from the chloroplast Wando holly tree or the holly holly tree derived chloroplast Wando holly tree.

The present invention provides a marker composition for identification of chloroplasts of persimmon tree (Ilex integra ) containing ndhC-trnV IGS (intergenic spacer) of persimmon tree chloroplast DNA.

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

The'Ilex integra ' of the present invention is an evergreen broad-leaved small arboreous tree native to Asia and distributed in Japan, Taiwan, and China. It is often grown on the coasts of Jeju Island and Ulleungdo and southern regions. It reaches 10m in height, and the bark is gray or grayish-white, flat, but the bark is rough.

The term'IGS (intergenic spacer)' in the present invention is a non-coding DNA region in a gene.

The ndhC-trnV IGS of the present invention is an IGS located in chloroplast DNA, and the ndhC-trnV IGS sequence obtained in the present invention was registered in NCBI Accession numbers MF324931 to MF324938 and MH424109 to MH424118.

The'marker for identification of chloroplasts of Ilex integra ' of the present invention refers to a material capable of distinguishing whether chloroplasts are derived from chloroplasts in a biological sample. In a specific embodiment of the present invention, a marker for identifying the chloroplasts of the persimmon tree is detected in a Wando holly tree sample having a persimmon tree or a persimmon tree-derived chloroplast, and can be used as a marker for identifying the chloroplasts of the persimmon tree.

In addition, the present invention provides a primer set for identification of chloroplasts of the persimmon tree comprising; a primer pair consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2.

In the present invention, the'primer' is a template-directed DNA synthesis under appropriate conditions (e.g., 4 different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer solution. It refers to a single-stranded oligonucleotide that can act as the starting point of. The appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 nucleotides, preferably 15 to 25 nucleotides, but is not limited thereto.

Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but must be sufficiently complementary to hybridize to the template. For example, the primer of the present invention can be chemically synthesized using a method known in the art, such as a phosphoramidite solid support method. In addition, it can be modified by methylation, capping, or the like by a known method. In addition, the primers of the present invention may contain a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (e.g. horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g., 32P), fluorescent molecules, chemical groups (e.g., biotin), etc. have.

In the present invention, variants of the nucleotide sequences represented by SEQ ID NOs: 1 and 2 are also included within the scope of the present invention. The nucleic acid molecule of the primer set of the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, a partial nucleotide sequence of one of the primer sets for identification of the chloroplast of the persimmon tree is deleted, substituted, or inserted. It is a concept that includes variants that have been transformed by (insertion), but that can function functionally with the effect of identifying the chloroplasts of the persimmon tree. Specifically, the primer set for identifying the chloroplast of the persimmon tree is 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably, each of the base sequence of any one of SEQ ID NOs: 1 and 2 Preferably, it may include a base sequence having 95% or more sequence homology. The'% of sequence homology' for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

In the present invention, the primer set can identify a tree from which the chloroplast is derived from a persimmon tree, and preferably, the chloroplast is from a persimmon tree, and can identify a wando holly (Ilex x wandoensis).

The term'single nucleotide polymorphism (SNP)' in the present invention is a single nucleotide polymorphism, which is a general mutation that appears in one of several DNA bases at a single site of a chromosome.

In the present invention, the primer represented by the nucleotide sequence of SEQ ID NO: 2 is upstream from the SNP position (323th base, holly: A, persimmon tree: C) of the ndhC-trnV IGS target DNA strand (GenBank MF324933). It is characterized by increasing the specificity of PCR amplification by giving an artificial mismatch to the second or third base position.

By using the primer set for identifying the chloroplasts of the persimmon tree of the present invention, it is possible to quickly and accurately identify whether or not the origin of the persimmon tree is a persimmon tree.

In addition, the present invention (a) separating the DNA from the sample; (b) using the DNA as a template and amplifying a target sequence using the primer set; And (c) detecting the amplification product. It provides a method for identifying chloroplasts derived from persimmon tree comprising a.

In the present invention, the sample in step (a) refers to an unknown substance for extracting nucleic acids, in particular DNA, required to determine whether or not chloroplasts of persimmon tree, and includes, for example, wood, food, biological samples, etc. It is not limited to this.

In the present invention, the sample in step (a) may be a mixed sample, and the mixed sample may be a sample mixed with a mixture such as an environmental sample, not a pure culture solution in which plant DNA separation has been completed.

In the present invention, the amplification of step (b) is polymerase chain reaction (PCR), ligase chain reaction (LCR), Gap-LCR, repair chain reaction, transcription-mediated amplification (TMA), Self sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence priming polymerase chain reaction (CP-PCR), random priming polymerase chain reaction (AP) -PCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, ring-mediated constant temperature amplification (LAMP), multiplex PCR, nested- PCR), single tube nested-PCR, reverse transcription-polymerase chain reaction (RT-PCR), inverse PCR, real-time polymerase chain reaction (RT-PCR) , And real-time polymerase chain reaction quantitative test (RQ-PCR) can be performed by using one or more methods selected from the group consisting of, preferably by using a polymerase chain reaction (PCR) method, It is not limited thereto.

In the present invention, the step of detecting the amplification product of step (c) may use one or more methods selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement. , But is not limited thereto.

In addition, the present invention provides a composition for identification of persimmon tree chloroplasts comprising the primer set.

In the present invention, the composition may include one or more other component compositions, solutions, or devices suitable for the analysis method. It may include conventional constituents included in the composition for genetic diagnosis together with the composition for identifying the persimmon tree chloroplast.

In addition, the present invention provides a kit for identification of persimmon tree chloroplasts comprising the composition for identification of persimmon tree chloroplasts.

In the present invention, the kit is a polymerase chain reaction (PCR) kit, a ligase chain reaction (LCR) kit, Gap-LCR, a repair chain reaction kit, a transcription-mediated amplification (TMA) kit, and Self sustained sequence replication kit, selective amplification of target polynucleotide sequences kit, consensus sequence priming polymerase chain reaction (CP-PCR) kit, random priming polymerase chain reaction (AP-PCR) kit, nucleic acid sequence-based amplification (NASBA) kit, strand displacement amplification kit, ring-mediated constant temperature amplification (LAMP) kit, multiplex PCR kit, dual Polymerase chain reaction (nested-PCR) kit, single tube nested-PCR kit, reverse transcription-polymerase chain reaction (RT-PCR) kit, inverse PCR kit , Real-time polymerase chain reaction (RT-PCR) kit, and real-time polymerase chain reaction quantitative test (RQ-PCR) may be one or more selected from the group consisting of kits, preferably the polymerase chain reaction kit. However, the present invention is not limited thereto.

In the present invention, the kit may also include one or more other constituent compositions, solutions, or devices suitable for the analysis method, and the conventional genetic diagnostic kit included in the genetic diagnostic kit together with the primer set for identifying the chloroplast of the persimmon tree Constituents may be included. The constituents may include a reaction buffer solution, DNA polymerase, dNTP and MgCl ₂ , but are not limited thereto.

Furthermore, in the present invention, the kit may include at least one container including a container containing a compartmentalized carrier means for containing a sample, a container, a PCR reaction buffer, and RNA polymerase, in addition to the primer set, and the carrier The means are suitable for containing one or more containers such as bottles, tubes, each container comprising independent components used in the method of the present invention, and those skilled in the art can easily dispense the necessary formulations in the container. can do. In addition, the kit of the present invention may further include a user's guide describing the optimum reaction performance conditions. The guide is a printout explaining how to use the kit, e.g., how to prepare PCR buffer, suggested reaction conditions, and so on. The guide includes a brochure in the form of a pamphlet or flyer, a label affixed to the kit, and a description on the surface of the package containing the kit. In addition, the guide contains information disclosed or provided through electronic media such as the Internet.

In addition, the present invention is composed of a primer represented by the nucleotide sequence of SEQ ID NO: 3, a primer represented by the nucleotide sequence of SEQ ID NO: 4, a primer represented by the nucleotide sequence of SEQ ID NO: 5, and a primer represented by the nucleotide sequence of SEQ ID NO: 6 Primer set; It provides a primer set for identification including persimmon tree, holly or wando holly.

The term internal transcribed spacer (ITS) of the present invention refers to a DNA region located between small-subunit ribosomal RNA (SSU rRNA) and large-subunit rRNA (LSU rRNA) genes of a chromosome.

ITS sequences obtained in the present invention were registered in NCBI Accession numbers KY682211 to KY682256 and MK240391 to MK240428.

The primer set for identifying the persimmon tree, holly or Wando holly tree of the present invention includes the ITS nucleotide sequence of the Wando holly tree and the ITS nucleotide sequence of the holly tree and the persimmon tree, holly and persimmon tree The difference between the ITS sequence SNP (Single Nucleotide polymorphism) in the nucleus between holly and persimmon tree and containing the recombined ITS sequence of (GenBank AC# KY682252, 187th nucleotide in ITS1 G: holly, T: persimmon tree) It may be designed based on ).

When amplifying the tetra-primer amplification refractory mutation system-PCR (ARMS-PCR) using the primer set for identifying the persimmon tree, holly or wando holly of the present invention, holly The product of 175, 262 bp of tree DNA is amplified, the product of 142, 262 bp of persimmon tree is amplified, and the product of 142, 175, 262 bp of Wando holly is amplified. Holly can be identified.

In the present invention, variants of the nucleotide sequences represented by SEQ ID NOs: 3 to 6 are also included within the scope of the present invention. The nucleic acid molecule of the primer set of the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, a partial nucleotide sequence of one of the primer sets for identification of persimmon tree, holly or walnut holly tree is deleted. , It is a concept that includes variants that have been transformed by substitution or insertion, but that can function functionally with the effect of identifying persimmon, holly, or wando holly. . Specifically, the primer set for identification of the persimmon tree, holly or Wando holly tree is 70% or more, more preferably 80% or more, and even more preferably, the base sequence of any one of SEQ ID NOs: 3 to 6, respectively. May comprise a base sequence having 90% or more, most preferably 95% or more sequence homology. The'% of sequence homology' for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

If the primer set for identifying the persimmon tree, holly or Wando holly tree of the present invention is used, whether it is persimmon tree, holly or Wando holly tree can be quickly and accurately identified with only one inspection.

In addition, the present invention (a) separating the DNA from the sample; (b) using the DNA as a template, and using the primer set for identification of persimmon tree, holly or wando holly, tetra-primer amplification resistance mutation system-PCR (tetra-primer amplification refractory mutation system-PCR, ARMS- PCR) to amplify the target sequence; And (c) detecting the amplification product; it provides a method for identifying a persimmon tree, a holly or a wando holly tree.

The tetra-primer amplification refractory mutation system-PCR (ARMS-PCR) of the present invention is a simple gene typing method for distinguishing polymorphism/mutation through one PCR. In the tetra primer amplification resistance mutant system-PCR, a common outer primer pair produces a non-trait specific amplification product, and an inner primer pair produces two trait-specific amplification products according to the combination.

Using the method of identifying persimmon tree, holly or wando holly tree of the present invention, a persimmon tree, holly or wando holly tree can be identified simply and quickly through only one amplification.

In addition, the present invention provides a composition for identifying a persimmon tree, a holly or a wando holly tree comprising a primer set for identifying the persimmon tree, holly or wando holly.

In addition, the present invention provides a kit for identifying a persimmon tree, a holly or a wando holly tree comprising the composition for identifying the persimmon tree, holly or wando holly.

In addition, the present invention is a primer pair consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2; And a primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 3, a primer represented by the nucleotide sequence of SEQ ID NO: 4, a primer represented by the nucleotide sequence of SEQ ID NO: 5, and a primer represented by the nucleotide sequence of SEQ ID NO: 6. It provides a primer set for identification of chloroplasts derived from persimmon tree, holly, persimmon tree, or chloroplast derived from holly or holly.

Persimmon tree, holly, persimmon tree-derived chloroplasts Wando holly or hollywood-derived chloroplasts Wando hollywood identification primer set of the present invention, in a specific embodiment, represented by the nucleotide sequence of SEQ ID NO: 1 Using a primer pair consisting of a primer and a primer represented by the nucleotide sequence of SEQ ID NO: 2, identifying whether a chloroplast derived from a persimmon tree or a chloroplast derived from a non- persimmon tree from the chloroplast DNA in the sample; And a primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 3, a primer represented by the nucleotide sequence of SEQ ID NO: 4, a primer represented by the nucleotide sequence of SEQ ID NO: 5, and a primer represented by the nucleotide sequence of SEQ ID NO: 6. Using the step of identifying whether or not persimmon tree, holly or Wando holly tree from the DNA in the sample; chloroplast derived from persimmon tree, holly, persimmon tree from the DNA in the sample, Wando holly or holly tree derived It can be used to identify whether the chloroplast Wando holly is present.

Using the primer set for identification of persimmon tree, holly, persimmon tree-derived chloroplast Wando holly or holly chloroplast Wando holly tree containing the above of the present invention, persimmon tree, holly, Chloroplasts derived from persimmon tree Wando holly tree or chloroplast Wando holly tree derived from holly can be identified.

In addition, the present invention comprises the steps of: (a) amplifying the DNA of the sample and detecting the amplification product using the primer set for identifying the chloroplasts of the persimmon tree; And (b) amplifying the DNA of the sample and detecting the amplification product using a primer set for identifying the persimmon tree, holly or Wando holly tree; containing persimmon tree, holly, persimmon tree-derived chloroplast Provides a method of identifying Wando holly or Wando holly chloroplast derived from holly.

Using the method of identifying persimmon tree, holly, persimmon tree-derived chloroplast Wando holly or holly chloroplast Wando holly tree containing the above of the present invention, persimmon tree, holly, persimmon tree Derived chloroplasts Wando holly tree or chloroplasts derived from holly tree Wando holly can be identified.

In addition, the present invention is a persimmon tree, holly, persimmon tree-derived chloroplast wando holly or hollywood-derived chloroplast wand holly, including a primer set for identification of persimmon tree, holly, persimmon tree-derived chloroplast wand It provides a composition for identification of holly or holly chloroplasts Wando holly tree derived from holly.

In addition, the present invention is a persimmon tree, holly, persimmon tree-derived chloroplast Wando holly tree or hollywood-derived chloroplast Wando holly, including a composition for identification of the persimmon tree, holly, persimmon tree-derived chloroplast Wando holly Provides a kit for identification of chloroplasts derived from thorns or holly.

Redundant content is omitted in consideration of the complexity of the present specification, and terms that are not otherwise defined herein have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Securing wood samples and extracting DNA

Tree samples to be used in this example were collected from Wando Arboretum and Bulgok branch of elementary schools outside Wando-gun, and DNA was extracted from the leaves of the tree samples using the CTAB method (Doyle, 1987) and the QIAGEN DNeasy Plant Mini kit, and agarose gel electrophoresis. The amount of DNA was quantified through electrophoresis. Information on the wood sample is shown in Table 1.

Example 2. Sequence analysis of chloroplast DNA

Universal marker (trnH-psbA IGS) and directly produced ycf3-rps4 IGS, rpl16-rps3 IGS, ndhA, ndhC-trnV IGS, matK-rps16 IGS, rpl32-ccsA IGS chloroplasts from DNA extracted in Example 1 with six markers DNA sequence was obtained. To design the primer, the Primer-BLAST program was used on the NCBI website, and conditions such as the length and temperature of the marker were added, and the chloroplast gene (NCBI accession number: KX426465) of the broad leaf holly (Ilex latifolia) was used. Based on this, forward and reverse primers were prepared. Universal markers (ITS5 and ITS4) were used to obtain an internal transcribes spacer (ITS) base sequence in the nucleus. The obtained chloroplast DNA nucleotide sequence and ITS nucleotide sequence were registered in NCBI GenBank, and information on the primers used in Example 2 and the identified nucleotide sequence are shown in Table 2.

Example 3. Preparation of chloroplast-specific gene markers of persimmon tree

3.1 Persimmon tree chloroplast DNA-specific genetic marker screening

For the analysis of single nucleotide polymorphism (SNP) based on the nucleotide sequence analyzed in Example 2, an allele specific-polymerase chain reaction (AS-PCR) technique was used. To prepare the forward primer cp-ndhC-F (hereinafter, F), and to increase the specificity of PCR amplification, the SNP position (base 323, holly: A, walnut tree) of the ndhC-trnV IGS target DNA strand (GenBank MF324933) : Reverse primers cp-ndhC-R, cp-ndhC-3G-R, cp-ndhC-3A-R, cp-ndhC-3C- that gave artificial mismatch at the second or third base position upstream from C) R, cp-ndhC-4G-R, cp-ndhC-3A-R and cp-ndhC-3C-R were prepared, respectively. Apply constant PCR amplification conditions to 8 samples (ICM, ICF, IIM, IIF, IWM, IWF, IRM, IRF) among the DNA samples extracted in Example 1, and check the presence or absence of amplification products on a 2% agarose gel. The presence or absence of was observed and grasped. The PCR amplification conditions and the primer pairs that gave the artificial mismatch are shown in Table 3 by underlined in the mismatch portion, and the composition of the PCR reaction buffer is shown in Table 4. The position of ndhC-trnV IGS in the chloroplast DNA is schematically shown in FIG. 1, and the principle of the chloroplast DNA-specific gene marker and the amplification product analysis method are schematically shown in FIG. 2, and the PCR amplification result is shown in FIG. I got it.

As shown in Figure 3, when the reverse primer is cp-ndhC-3G-R (hereinafter, 3G-R), that is, when the second nucleotide in the SNP is G, IIM, IIF, which are samples with chloroplast DNA, In the IWM and IWF samples, ndhC-trnV IGS was amplified in the chloroplast of the persimmon tree and a band was formed at the position of 208 bp, and the band was not formed in ICM, ICF, and IRF and IRM used as outgroups and samples with holly chloroplast DNA. It was confirmed that ndhC-trnV IGS in holly chloroplast was not amplified. Accordingly, forward primer F and reverse primer 3G-R were completed as a pair of primers to detect a persimmon tree chloroplast DNA-specific gene marker, and the forward primer F is represented by SEQ ID NO: 1, and the reverse primer 3G-R is represented by SEQ ID NO: 2. I got it.

3.2 Verification of DNA-specific genetic markers of persimmon tree chloroplast

3.2.1 Confirmation of the chloroplast gene origin of the sample of the experimental group

An experiment was conducted to additionally verify the effect of amplification of persimmon tree chloroplast DNA-specific amplification effect of persimmon tree chloroplast marker primers F and 3G-R identified in Example 3.1. Specifically, the chloroplast nucleotide sequences of samples BM1 to BM7 and WA2 described in Table 1 were described in Table 2 as ycf3-rps4 IGS, rpl16-rps3 IGS, ndhA, ndhC-trnV IGS, matK-rps16 IGS, rpl32-ccsA IGS and trnH- Using the psbA IGS primer, all the nucleotide sequences of each sample were analyzed, and if it is the same as the chloroplast nucleotide sequence of the persimmon tree, it is marked as II, and if it is the same as the chloroplast nucleotide sequence of holly, the chloroplast analysis result is expressed as IC. It is shown in Table 5.

As shown in Table 5, as a result of analyzing the chloroplast nucleotide sequence of W. holly BM1 to BM7, WA2, it was confirmed that the chloroplasts of BM4, BM6, BM7 and WA2 were the same as the chloroplast nucleotide sequence of the cocoon tree. It was confirmed that the chloroplasts of BM1, BM2, BM3 and BM5 were the same as the chloroplast nucleotide sequence of the holly tree, and it was confirmed that the chloroplasts were derived from holly.

3.2.2 Verification of DNA-specific genetic markers of the chloroplast DNA of the persimmon tree

As an experimental group, Wando holly samples BM1 to BM7, WA2 and holly samples WA1 and BM8, which confirmed the chloroplast origin in Example 3.2.1, were set. IWF was set as a positive control and distilled water was set as a negative control.

PCR was performed as in Example 3.1 using F and 3G-R primers for the experimental group and the control group set above, and the PCR results are shown in FIG. 4.

As shown in Fig. 4, it was confirmed that specific PCR bands of 208 bp appeared on the agarose gel in BM4, BM6, BM7, WA2, and IWF. The result of the presence or absence of this PCR band is consistent with the confirmation that the chloroplasts of BM4, BM6, BM7, and WA2 are derived from persimmon tree in Example 3.2.1. Figure 4 The band located at the bottom of the agarose gel is a non-specific primer (ITS2C-S: 5'-ATGCCCAGCCGGATATTG-3', ITS-4: 5'-TCCTCCGCTTATTGATATGC) for Wando holly and holly when the same sample is targeted. As a result of performing ITS PCR with -3'), the amplification product was observed from all samples.

Example 4. Preparation and verification of genetic markers for confirming Wando hollyhock hybrids

4.1 Preparation of genetic markers to identify Wando hollyhock hybrids

An experiment was conducted to create a Wando holly hybrid identification genetic marker to confirm whether the unknown species of the tree belongs to any one of holly, persimmon and Wando holly. Specifically, ITS nucleotide sequences of holly, persimmon tree, and monk tree were obtained by direct sequencing method as in Example 2, cloned into pGEM-T vector and transformed into JM109 bacteria, and then primer T7 in the vector. Using the promoter primer and Sp6 promoter primer, the ITS nucleotide sequence of Wando holly tree was secured. As a result of nucleotide sequence analysis, it was confirmed that the ITS sequence of the Wando holly tree contained the ITS sequence of the holly and the ITS tree, and the ITS sequence that was homologous recombination between the two tree ITS was also included. I did. Wando holly (a hybrid between two trees) based on the above-identified ITS sequence difference in the nucleus between holly and persimmon trees (GenBank AC# KY682252, 187th nucleotide in ITS1 G: holly, T: persimmon tree) In the case of 142, 175, 262 bp, three amplification products are made, in the case of holly, two amplification products of 175, 262 bp, and 142, 262 bp in the case of a holly tree, a tetra-primer amplification resistance mutant system-PCR ( The tetra-primer amplification refractory mutation system-PCR, ARMS-PCR) primers were prepared through WASP (http://bioinfo.biotec.or.th/WASP). The prepared primers represented ITS-F-outer as SEQ ID NO: 3, ITS-R-outer as SEQ ID NO: 4, ITS-F-inner as SEQ ID NO: 5, and ITS-R-inner as sequence It is represented by number 6. Among the produced tetraprimer amplification-resistant mutant system-PCR primers, ITS-F-inner primer artificially mismatches the second nucleotide from SNP upstream for PCR specificity, and ITS-R-inner primer is from SNP to downstream for PCR specificity. It gave an artificial mismatch to the second nucleotide. The nucleotide sequence and PCR reaction conditions of the tetraprimer amplification resistance mutant system-PCR primer are shown in Table 6, and the buffer composition for the PCR reaction is shown in Table 7. The structure of the ITS DNA in the nucleus is schematically shown in Fig. 5, and the manufacturing process of the gene marker for confirming the Wando holly thorn hybrid is shown in Fig. 6, and the method of analyzing the gene amplification product confirming the Wando holly thorn hybrid is shown in Fig. 7 Shown in.

4.2. Verification of genetic markers for confirming Wando holly hybrids

4.2.1 Setting the PCR annealing temperature of the gene marker for confirming the Wando hollyhock hybrid

An experiment for setting an annealing temperature with high PCR efficiency of the tetraprimer amplification resistance mutant system-PCR primer prepared in 4.1 above was performed. Specifically, PCR was performed using the primers and PCR conditions shown in Table 6 and the PCR buffer composition shown in Table 7. When the annealing conditions were different, in order to compare the PCR amplification effect, the primer and annealing temperature were 72. The experiment was conducted by dividing the case at ℃ and 70 ℃, and the amplification product was electrophoresed for 20 to 30 minutes at 135V in 2% agarose gel. IWM, IWF, IIM, IIF, ICM and ICF of Table 1 were used as samples, and the results are shown in FIG. 8.

As shown in FIG. 8, it was confirmed that bands of 142 bp, 175 bp and 262 bp were formed in the Wando holly tree sample in both the case of the annealing temperature of 70° C. and 72° C. among the PCR conditions, and It was confirmed that a band of 142 bp was formed, and bands of 175 bp and 262 bp were formed in the sample of holly. In addition, when electrophoresis was performed for more than 30 minutes, a large single band was broken, and it was confirmed that performing the electrophoresis condition at 135V for about 20 to 30 minutes is most effective in reading the results. Accordingly, in the PCR test for identifying the Wando hollyhock, which is a hybrid tree of the Wando hollyhock and the Wando holly, a hybrid of the Wando hollyhock hybrid identification gene marker, the optimal conditions for PCR and electrophoresis were confirmed.

4.2.2 Verification of genetic markers for confirming Wando hollyhock hybrids

An experiment was conducted to additionally verify the effect of the tetraprimer amplification resistance mutant system-PCR primers prepared in 4.1 above for confirming the Wando holly hybrids. Specifically, PCR was performed using the primers and PCR conditions shown in Table 6 and the PCR buffer composition shown in Table 7, and the amplification products were electrophoresed on a 2% agarose gel. BM1 to BM6, WA1, WA2, IWF, ICF and IIF of Table 1 were used as samples, and the results are shown in FIG. 9.

As shown in Figure 9, it was confirmed that bands of 142 bp, 175 bp and 262 bp were formed in Wando holly samples BM1 to BM6, WA2, and IWF, and 175 bp and 262 bp bands were formed in WA1 and ICF holly samples. Formation was confirmed, and it was confirmed that bands of 142 bp and 262 bp were formed in IIF, a sample of persimmon tree. Accordingly, the effect of confirming Wando hollywood hybrids using the present invention tetraprimer was once again confirmed.

Based on the results confirmed in Examples 1 to 4, the persimmon tree chloroplast DNA-specific gene marker of Example 3 and the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2 for detection thereof, and the Wando holly hybrid of Example 4 It is possible to confirm the origin of the species and chloroplasts of a sample of persimmon tree, holly or walnut tree whose species are not identified by using a confirmed genetic marker and a pair of primers of SEQ ID NOs: 3 to 6 for detection thereof. have. This process is schematically shown in FIG. 10.

<110> Republic of Korea (National Forensic Service Director Ministry of Interior and Safety) <120> Marker for identification of chloroplast derived from Ilex integra and Primer set for identification of Ilex x wandoensis <130> Scientific Investigation1-10P <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for identification of chloroplast derived from Ilex integra <400> 1 tcatctcacc taggattctc tct 23 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for identification of chloroplast derived from Ilex integra <400> 2 caaacagagc tcccgatgcg 20 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer 1 for identification of Ilex x wandoensis <400> 3 tgcggaagga tcattgtcga tgcctgcaa 29 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer 1 for identification of Ilex x wandoensis <400> 4 aaagatgcaa atgcctcccg tgcacaccg 29 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer 2 for identification of Ilex x wandoensis <400> 5 tttggcttgc gttcccctag cgggggct 28 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer 2 for identification of Ilex x wandoensis <400> 6 ggggttcgtt gtcgggagct tggctgc 27

Claims (15)

Primer pair consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2; a primer set for identification of chloroplasts of the persimmon tree.
The primer set according to claim 1, wherein the primer set is capable of discriminating the wando holly tree (Ilex x wandoensis) from which the chloroplast is derived from the persimmon tree.
The method of claim 1, wherein the primer represented by the nucleotide sequence of SEQ ID NO: 2 is artificial at the second nucleotide position upstream from the 323th base of the target DNA, ndhC-trnV IGS (intergenic spacer) DNA strand (GenBank MF324933). A primer set, characterized in that it is a reverse primer given a mismatch.
(a) separating DNA from the sample;
(b) using the DNA as a template and amplifying a target sequence using the primer set of claim 1; And
(c) detecting the amplification product; Persimmon tree-derived chloroplast identification method comprising a.
Persimmon tree chloroplast identification composition comprising the primer set according to claim 1.
Persimmon tree chloroplast identification kit comprising the composition for identification of persimmon tree chloroplast according to claim 5.
Including a primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 3, a primer represented by the nucleotide sequence of SEQ ID NO: 4, a primer represented by the nucleotide sequence of SEQ ID NO: 5, and a primer represented by the nucleotide sequence of SEQ ID NO: 6; Primer set for identification of persimmon tree, holly or wando holly.
(a) separating DNA from the sample;
(b) using the DNA as a template and performing a tetra-primer amplification refractory mutation system-PCR (ARMS-PCR) using the primer set of claim 7 to amplify the target sequence ; And
(c) detecting the amplification product; persimmon tree, holly or wando holly tree identification method comprising.
Persimmon tree, holly or wando holly tree identification composition comprising the primer set of claim 7.
Persimmon tree, holly or wando holly tree identification kit comprising the composition of claim 9.
A primer pair consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2; And a primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 3, a primer represented by the nucleotide sequence of SEQ ID NO: 4, a primer represented by the nucleotide sequence of SEQ ID NO: 5, and a primer represented by the nucleotide sequence of SEQ ID NO: 6. Primer set for identifying Wando holly with chloroplasts derived from persimmon tree, holly, persimmon tree or Wando holly with chloroplasts derived from holly.
(a) amplifying the DNA of the sample using the primer set of claim 1 and detecting the amplification product; And
(b) amplifying the DNA of the sample using the primer set of clause 7 and detecting the amplification product; including chloroplasts derived from persimmon tree, holly, persimmon tree and chloroplast derived from persimmon tree or holly tree Wando holly tree identification method with.
Persimmon tree, holly, persimmon tree-derived chloroplasts containing the primer set of claim 11 Wando hollywood or hollywood-derived chloroplasts with a chloroplast Wando hollywood identification composition.
Persimmon tree, holly, persimmon tree-derived chloroplasts including the composition of claim 13 Wando holly or Wando holly with chloroplasts derived from hollywood identification kit.
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