KR101804120B1 - Complete sequencing of chloroplast genome and nrDNA of Aloe vera, Aloe saponaria and Aloe Saengjang-derived barcoding marker, DNA primer set for discrimination of origin and species and uses thereof - Google Patents

Abstract

The present invention relates to an origin species identification marker and primer set based on complete translation of chloroplast genome and nuclear ribosome DNA sequence of Aloe vera, Aloe saponaria and Saeng Jang, and uses thereof. The molecular marker and primer set of the present invention can identify Aloe vera, Aloe saponaria and Saeng Jang at low costs and with high efficiency, can distinguish raw materials even in Saeng Jang processed foods and can be used for maintaining purity of Saeng Jang, protecting a variety and solving the seed war.

Description

Complete sequencing of chloroplast genomes and nrDNA of aloe vera, aloe saponaria and aloe growth, and complete decode-based species identification markers and primer sets of nucleosome ribosome DNA sequences and their use barcoding marker, DNA primer set for discrimination of origin and species and uses thereof,

The present invention relates to a complete detoxification-based species identification marker and primer set of chloroplast genomic and nuclear ribosomal DNA sequences of Aloe vera, aloe saponaria and aloe, and uses thereof.

Aloe is a succulent plant of the genus Lilium native to Africa. The origin of the plant belonging to the genus Aloe is mainly South Africa, Madagascar and Arabia, mainly in the African continent and neighboring archipelago. Today, it is cultivated extensively in tropical and temperate regions. Leaves are thick-knife-shaped, with prickles on the edges and orange or pink flowers. Aloe, and saponin, it has been developed and used as medicines, health supplements, and cosmetics since ancient times, and it is popular for ornamental use because it can appreciate green leaf stalks all year round. More than 600 kinds of aloe are known, of which Aloe vera , Aloe arborescens and Aloe saponaria are currently available.

In recent years, as the molecular biology has developed, techniques for searching for polymorphisms of specific DNA sequences and using them as molecular markers have not been affected by the environment and have little annual variation. Therefore, mapping of genes, mapping of disease- It can be used in various fields such as breed identification. In particular, the development of genetic molecular marker technology has increased its value in cultivating crops.

There are morphological characteristics, qualitative and quantitative analysis of components, and comparison of differences at DNA level. Morphological characteristics may be different depending on the growth environment of the plant, which makes it difficult to identify the plant species. In addition, a taste sensor, a nuclear magnetic resonance spectrometer (NMR spectrometer) and the like have been used for distinguishing by the component analysis. However, this method has a limitation that components may be affected depending on the cultivation environment of the production site. In order to solve these problems, attempts have been made to identify molecular biological plant species using polymorphisms of DNA fragments such as RAPD (Random Amplified Polymorphic DNA), SCAR (Sequence Characterized Amplified Regions) and AFLP (Amplified Fragment Length Polymorphism). Recently, the classification method using the DNA barcode proposed by the CBOL Plant Working Group in the plant species discrimination has been recognized as useful. In particular, the nrDNA-ITS (internal transcribed spacer) region of the nuclear ribosomal DNA (nrDNA) existing in the genome not only evolves faster than the coding region of other genes, but also has advantages such as generality, simplicity and reproducibility, It is known to be a widely used base sequence for classification and interspecies genetic mutation searches.

Accordingly, the present inventors analyzed the entire sequence of the chloroplast genome of aloe vera, aloe saponaria and aloe, and 45S nrDNA, and compared them with each other to determine the sequence of eight (8) genes capable of efficiently discriminating aloe vera, aloe saponaria and aloe Molecular markers were developed.

Korean Patent No. 1402607 discloses 'metabolite profiling based on mass spectrometry of various sizes of aloe vera and its biomarker', Korean Patent No. 1508744 discloses 'chloroplast DNA marker for discrimination of Chinese cabbage and crop species' And a discrimination method using the same. However, the complete decode-based genomic identification marker and primer set of chloroplast genomic and nuclear ribosomal DNA sequences of aloe vera, aloe saponaria and aloe growth of the present invention and their uses have been disclosed There is no bar.

The present invention has been completed by analyzing the entire sequence of the chloroplast genome and the nucleotide ribosomal DNA (nrDNA) of aloe vera, aloe saponaria and aloe, and the sequence of the completed nucleotide sequence A total of eight kinds of markers were prepared by comparing the information and the DNA samples were prepared by preparing a primer set capable of amplifying the molecular markers. As a result, it was found that the molecular markers of the present invention were aloe vera, aloe saponaria and aloe Can be clearly distinguished from each other, thereby completing the present invention.

The oligonucleotide primer set of SEQ ID NOs: 3 and 4, the oligonucleotide primer set of SEQ ID NOs: 5 and 6, the oligo oligonucleotide primer set of SEQ ID NOs: 7 and 8, Oligonucleotide primer sets of SEQ ID NOs: 9 and 10, oligonucleotide primer sets of SEQ ID NOs: 11 and 12, oligonucleotide primer sets of SEQ ID NOs: 13 and 14, and oligonucleotide primer sets of SEQ ID NOs: 15 and 16 And a set of primers for distinguishing aloe vera, aloe saponaria and aloe growth, comprising at least one primer set selected from the group consisting of:

In addition, the present invention provides a kit for distinguishing aloe vera, aloe saponaria and aloe growth, comprising the primer set and a reagent for carrying out an amplification reaction.

In addition, the present invention provides a method for distinguishing aloe vera, aloe saponaria and aloe growth using the primer set.

The molecular markers of the present invention have been found in a region where hundreds to thousands of copies are present in the cell, but the mutation is very rare in the species. Therefore, it is possible to carry out a very stable verification, Saponaria and aloe growth can be identified, which is highly utilizable. In addition, since it is possible to discriminate the raw materials in aloe-grown processed products, it can improve the value of aloe-grown processed products, maintain the purity of aloe, protect the breed, and resolve seed conflicts.

Fig. 1 is photographs of aloe vera, aloe saponaria and aloe. Aloe vera , aloe vera; Aloe saponaria , aloe saponaria ; Saeng Jang, Aloe growth.
Figure 2 is a complete chloroplast genome map of Aloe vera, aloe saponaria and aloe. Aloe vera , aloe vera; Aloe saponaria , Aloe saponaria ; Saeng Jang, Aloe growth.
Fig. 3 shows the result of excavation of the interspecies mutation region through comparative analysis of 45s nrDNA sequences of aloe vera, aloe saponaria and aloe.
Fig. 4 shows the results of the phylogenetic analysis of three chlorophyll-based aloe species. Iris gate when the child (Iris gatesii) was used out of the group (outgroup).
Figure 5 shows the results of phylogenetic analysis of three species of aloes based on the 45s nrDNA sequence.
Fig. 6 shows the results of 45s nrDNA sequence analysis of aloe vera, growth aloe and aloe saponaria.
Fig. 7 shows the result of distinguishing aloe saponaria through the InDel marker of the present invention.
Fig. 8 shows the results of identification of aloe vera, aloe saponaria and aloe growth using the dCAPs marker of the present invention. Aloe_d_13: Aloe vera specific marker; Aloe_d_5,17: Growth aloe specific marker.
FIG. 9 shows the results of identification of aloe vera, aloe saponaria and aloe growth using the HRM markers of the present invention. Aloe_h_1: Aloe saponaria specific marker; Aloe_h_10: Aloe vera specific marker.

In order to achieve the object of the present invention, the present invention provides a primer set for distinguishing aloe vera, aloe saponaria and aloe growth.

Aloe Vera (Aloe vera) belongs to the fastest growing section during 1-2 years after growing only deodiji two years, the flowers come up with a long spine, regardless of the season of the year smokes a yellow or reddish orange flowers. It is bitter taste when it eats peel. When it peels and eats it, it is often used for peeled off because it is a variety with no taste (odorless) and no jelly quality.

Aloe saponaria ( Aloe saponaria ) belongs to a small species of aloe, grows attached to the ground, and has leaves with white spots. The peduncle comes up long and blossoms in red. There is no bitter taste in the skin.

Aloe vera and Aloe saponaria are cultivated by mixing Aloe vera and aloe saponaria. It is a new varieties cultivated through system breeding by inducing natural hybrids and obtaining actual seedlings. It has low plant height, high number of leaves, has white spots within the pale green leaf, thin skin of leaves, and soft spines, so it can be used to peel aloe leaves without removing the skin. Especially in the case of new varieties of aloe, it may be difficult to distinguish them from aloe vera or aloe saponaria in the eyes of younger birds or in processing distribution.

Accordingly, the present inventors have developed eight kinds of molecular markers capable of efficiently discriminating aloe vera, aloe saponaria and aloe growth. The marker consists of three Indel (insertion-deletion), three dCAPs (derived cleaved amplified polymorphic sequence) and two HRM (high resolution melting) marker types (see Table 6).

The primer set of the present invention specifically comprises an oligonucleotide primer set of SEQ ID NOs: 1 and 2, an oligonucleotide primer set of SEQ ID NOs: 3 and 4, an oligonucleotide primer set of SEQ ID NOs: 5 and 6, an oligonucleotide primer of SEQ ID NOs: A set of oligonucleotide primers of SEQ ID NOs: 9 and 10, an oligonucleotide primer set of SEQ ID NOs: 11 and 12, an oligonucleotide primer set of SEQ ID NOs: 13 and 14, and an oligonucleotide primer set of SEQ ID NOs: 15 and 16 Lt; / RTI > primer set.

Preferably, the primer set of the present invention comprises (a) an oligonucleotide primer set of SEQ ID NOs: 1 and 2, an oligonucleotide primer set of SEQ ID NOs: 3 and 4, an oligonucleotide primer set of SEQ ID NOs: 5 and 6, At least one primer set selected from the group consisting of a set of oligonucleotide primers; (b) at least one primer set selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 9 and 10 and oligonucleotide primer sets of SEQ ID NOs: 11 and 12; And (c) at least one primer set selected from the group consisting of an oligonucleotide primer set of SEQ ID NOs: 7 and 8 and a set of oligonucleotide primers set forth in SEQ ID NOs: 15 and 16, in combination with aloe vera, aloe saponaria and aloe May be a primer set for distinguishing.

According to one embodiment of the present invention, oligonucleotide primer sets of SEQ ID NOs: 1 and 2, oligonucleotide primer sets of SEQ ID NOs: 3 and 4, oligonucleotide primer sets of SEQ ID NOs: 5 and 6, and oligonucleotides of SEQ ID NOs: A set of primers set forth in SEQ ID NOs: 7 and 8, and a set of primers set forth in SEQ ID NOS: 15 and 16, wherein the set of primers set forth in SEQ ID NOs: 7 and 8 is a set of primers for distinguishing aloe vera from aloe vera and aloe growth comprising at least one primer set selected from the group consisting of An oligodeoxynucleotide primer set of SEQ ID NOs: 9 and 10, which comprises at least one primer set selected from the group consisting of an oligonucleotide primer set And a primer set to distinguish growth from the aloe and aloe vera Aloe sand paper or Ria comprising at least one primer set of oligonucleotide SEQ ID NO: 11 and 12 selected from the group consisting of a nucleotide primer set.

The primer set of the present invention is a set of oligonucleotide primers capable of amplifying a total of eight markers developed based on the complete chloroplast genome sequencing of aloe vera, aloe saponaria and aloe growth, 5, 7, 9, 11, 13 and 15 are forward primers and SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16 are reverse primers.

According to the sequence length of each primer, the primers include SEQ ID NOS: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; At least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 within SEQ ID NO: At least 26, at least 27, at least 28, at least 29 contiguous nucleotides. For example, a primer (25 oligonucleotides) of SEQ ID NO: 1 may comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, An oligonucleotide consisting of at least 22, at least 23, and at least 24 consecutive nucleotide fragments. In addition, the primers include SEQ ID NOs: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; Addition, deletion or substitution of the nucleotide sequences of SEQ ID NOS: 15 and 16.

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.

In order to achieve still another object of the present invention,

There is provided a kit for distinguishing aloe vera, aloe saponaria and aloe growth, comprising a primer set 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 order to achieve still another object of the present invention,

Isolating genomic DNA from aloe vera, aloe saponaria and aloe growth suspected specimens;

Amplifying the polymorphic nucleotide by performing an amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set of the present invention; And

And detecting the amplification product. The method comprises the steps of: detecting Aloe vera, Aloe saponaria, and Aloe.

The method of the present invention comprises isolating the genomic DNA from aloe vera, aloe saffronaria and aloe growth suspected samples. A method known in the art may be used for separating the genomic DNA from the sample. For example, a CTAB method may be used, or a Wizard prep kit (Promega, USA) may be used. The target sequence can be amplified by performing amplification reaction using the separated genomic DNA as a template and using a 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, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] 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 pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. 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 primer set used to amplify the target sequence is as described above.

The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As a method of detecting the amplification product, gel electrophoresis can be performed, and gel electrophoresis can be performed using acrylamide gel electrophoresis or agarose gel electrophoresis according to the size of the amplification product. In addition, capillary electrophoresis can be performed. Capillary electrophoresis can be performed, for example, using the ABI Genetic Analyzer. In the fluorescence measurement method, when a fluorescent dye is labeled at the 5'-end of the primer and PCR is performed, the target sequence is labeled with a fluorescent label capable of detecting the fluorescence. The fluorescence thus labeled can be measured using a fluorescence analyzer. 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 with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1. NGS analysis of aloe vera, aloe saponaria and aloe growth

The chloroplast genome and nucleus 45S nrDNA (nuclear ribosomal DNA) sequences of aloe vera, aloe saponaria and aloe were assembled and analyzed using NGS (Next Generation Sequences) method. The size of the completely-cleaved chloroplast genome was 152,875 bp for aloe vera, 153,175 bp for aloe saponaria, and 152,879 bp for aloe growth (Table 1). The nucleus 45S nrDNA was identified as three types in aloe vera, two types in saponaria, and four types in aloe growth. Table 1 specifies representative 45s nrDNA type information.

Example 2: The chloroplast and nrDNA sequences of aloe vera, aloe saponaria and aloe growth were completed.

In the present invention, the chloroplast base sequence of aloe vera, aloe saponaria, and growth aloe was completed. The basic information and structure of the chloroplast completed in the three plants are shown in FIG. 2 and Table 2.

Aloe vera, aloe saponaria, and growth aloe had chloroplast lengths of 152,875-153,175bp. The lengths of chloroplast sequences of these two species and saponaria were found to differ between the aloe vera and the growth aloe (Aloe vera: 152,875 bp, Growth aloe: 152,879 bp) Showed a difference in sequence length.

In addition, the 45s nrDNA sequence was completed in Aloe vera, Aloe saponaria, and Growl allosus using the NGS sequence (Table 3, Fig. 3). It was confirmed that there are two or more 45s nr DNAs in all three aloe species (3 types, aloe vera: 2 types, 4 types of aloe).

Example 3. Comparative analysis of aloe vera, aloe saponaria, growth aloe chloroplast and nrDNA sequences

Aloe vera, aloe saponaria, and growth aloe were compared with each other to identify mutations in three chloroplast sequences. There were 62 SNPs (0.04% of the total sequence) and 65 InDel (0.04% of the entire sequence) between the aloe vera and the growth aloe, and between the aloe saponaria and the growth aloe were 875 SNP ) And 138 InDel (0.09% of the total sequence) (Table 4 and 5).

Aloe vera, aloe saponaria, and grown in aloe, based on the chloroplast sequence was confirmed the relationship between the three species of aloe. Aloe vera and growth aloe formed the same family group, and Aloe saponaria formed a different family group than the two aloe species. Although there were some differences in sequence between the chloroplast sequences of the aloe aloe and aloe vera, it was confirmed by the sequence-based phylogenetic analysis of the chloroplast bearing the mother lineage (Fig. 4).

45s nrDNA sequences were compared and analyzed between three kinds of aloes (aloe vera, aloe saponaria, and growth aloe). The 45s nrDNA sequence is derived from the nucleus. The 45s nr DNA sequence of the offspring generation is mixed with the 45s nrDNA sequence of the sample and the subunit. Comparing 45s nrDNA sequences from three species of aloe, the 45s nrDNA sequences of the growth aloe (4 types of growth aloe) and alovevera (3 types of aloe vera) are quite similar, while the aloe saponaria sequence Saponaria: 2 types) differed from the nrDNA sequences of these two species (Figures 5 and 6).

Example 4. Development of marker for distinguishing species from aloe vera, aloe saponaria, aloe chloroplast and nrDNA sequence

A total of eight markers were constructed by selecting regions that can distinguish Aloe vera, Aloe saponaria, and growth aloe among the mutation regions obtained by comparing the chloroplasts of the three aloe species and the 45s nrDNA sequence. 2 markers capable of distinguishing growth aloe from 2, and 4 markers capable of distinguishing aloe saponaria were developed. Marker development area and primer information are shown in Table 6.

The above-mentioned primer set was used to verify the molecular markers of the present invention. PCR analysis was performed using DNA samples of aloe vera, aloe saponaria and aloe. As a result, it was possible to clearly distinguish three kinds of aloe from chloroplasts and 45s nrDNA sequence-derived markers, It will be useful for distinguishing aloe (Figs. 7 to 9).

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Claims (7)

(a) an oligonucleotide primer set of SEQ ID NOs: 1 and 2, an oligonucleotide primer set of SEQ ID NOs: 3 and 4, an oligonucleotide primer set of SEQ ID NOs: 5 and 6, and an oligonucleotide primer set of SEQ ID NOs: 13 and 14 At least one primer set selected;
(b) at least one primer set selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 9 and 10 and oligonucleotide primer sets of SEQ ID NOs: 11 and 12; And
(c) at least one primer set selected from the group consisting of an oligonucleotide primer set of SEQ ID NOs: 7 and 8 and an oligonucleotide primer set of SEQ ID NOs: 15 and 16; and a kit for distinguishing aloe vera, aloe saponaria and aloe Primer set. delete delete delete delete A kit for distinguishing aloe vera, aloe saponaria and aloe growth, comprising the primer set of claim 1 and a reagent for carrying out an amplification reaction. Isolating genomic DNA from aloe vera, aloe saponaria and aloe growth suspected specimens;
Amplifying the polymorphic nucleotide by performing an amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set of claim 1; And
And detecting said amplification product. &Lt; Desc / Clms Page number 18 &gt; 21. A method for distinguishing aloe vera, aloe saponaria and aloe growth.

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