KR102617631B1 - Primer set for identifying species of scutellaria sp. and identification method using the same - Google Patents

Abstract

The present invention relates to a primer set for species identification of Scutellaria and a method for identifying species of Scutellaria using the same. Specifically, in Scutellaria, Scutellaria indica L. , Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria barbata ), and golden thimble flower ( Scutellaria baicalensis ). At least one species selected from the group consisting of Scutellaria baicalensis can be identified simply and with high accuracy. It relates to a primer set for plant species identification and a method for identifying species of Thimble genus using the same.

Description

Primer set for species identification of Thimble genus plants and method for species identification of Thimble genus plants using the same {PRIMER SET FOR IDENTIFYING SPECIES OF SCUTELLARIA SP. AND IDENTIFICATION METHOD USING THE SAME}

The present invention relates to a set of primers for identifying species of the genus Thimble genus and a method for identifying species of the genus genus using the same.

In the case of plants, hybridization between various species and genera is frequently reported, and due to the complex pattern of polyploidization, it is difficult to understand maternal lineage and evolution for accurate identification of plant taxa. Therefore, there is a demand for the development of DNA that can identify lineages or varieties.

With the recent development of molecular biology, the technology to search for polymorphisms in specific DNA base sequences and use them as molecular markers is not affected by the environment and has little variation between years, so it is used for genetic mapping, mapping of disease resistance genes, and cultivars. It can be used in various fields such as identification. In particular, the development of genetic molecular marker technology is increasing its usefulness in crop cultivation.

Plant species identification methods used to date include comparing morphological characteristics, qualitative and quantitative analysis of components, and comparing differences at the DNA level. Comparison of morphological characteristics can result in differences depending on the growth environment of the plant, making it difficult to identify plant species. In the case of differentiation through component analysis, taste sensors and nuclear magnetic resonance spectrometers (NMR spectrometers) have been used, but this method has the limitation that the components can be affected by the cultivation environment of the production area.

To solve the limitations of these conventional methods, molecular biological plant species identification using polymorphisms of DNA fragments, such as RAPD (Random Amplified Polymorphic DNA), SCAR (Sequence Characterized Amplified Regions), and AFLP (Amplified Fragment Length Polymorphism), is being attempted. Additionally, recently, the classification method using DNA barcodes proposed by the CBOL Plant Working Group has been recognized for its usefulness in plant species identification. In particular, the nrDNA-ITS (internal transcribed spacer) region of nuclear ribosomal DNA (nrDNA) present in the genome not only evolves faster than the coding region of other genes, but also has advantages such as generality, simplicity, and reproducibility. For this reason, it is known to be a base sequence widely used for phylogenetic classification and the search for genetic variation between species. In the case of rRNA genes, there is little difference between species and genera, whereas the ITS section, which has a lot of variation, has thousands of copies per single genome, so it can provide characteristics with useful information for phylogenetic analysis.

Currently, genetic markers that can accurately identify species of Scutellaria plants, which include many useful medicinal plants, have not been developed. Thimble genus, belonging to the Lamiaceae family, is the second largest genus within the family and includes approximately 470 taxa worldwide. Many plants of the thimble flower genus are mainly distributed in temperate regions and are used as medicinal plants or spices. Medicinal plants in the thimble genus include thimble flower, mountain thimble flower, cotyledon thimble flower, toothbrush thimble flower, and golden thimble flower. Among these, thimble flower and cotyledon thimble flower are difficult to classify based on their appearance, so species analysis method through genetic analysis is used. development is needed.

Scutellaria indica L. is a perennial plant with heart-shaped leaves and black spots on the flowers. In China, it is called Hansincho, and it is a medicinal plant that has been used in the folk world for its analgesic and hemostatic effects and to relieve boils, toothache, headache, vomiting blood, and hemoptysis.

Scutellaria pekinensis var. transitra is characterized by having opposite leaves, hairs on both sides, and serrated edges. Light purple flowers bloom from May to June, and young shoots and tender leaves are eaten as vegetables in spring.

Cotyledon thimble flower ( Scutellaria indica var. tsusimensis ) is very similar in appearance to thimble flower, and its leaves are slightly thicker and hairier than thimble flower. The young leaves are eaten as a vegetable and used as an herbal medicine with effects such as detoxification and pain relief.

Toothbrush thimble flower ( Scutellaria barbata ) is also called semi-lily, and the above-ground part is mainly used as medicine. It has the effect of reducing fever and swelling, and its anti-cancer effect has also been studied.

Scutellaria baicalensis is a naturalized plant native to China and is widely cultivated as a medicinal plant. The roots are mainly used, and it is a valuable biological resource as a medicinal and ornamental plant. Pharmacological effects include reducing heart and lung fever or stopping coughing and bleeding, and have anti-allergic, antioxidant, anti-inflammatory, and antibacterial effects on the skin, such as atopic dermatitis, bacterial dermatitis such as acne, and skin whitening. It is used as an active ingredient in cosmetics.

KR 101804120 B1

The present invention relates to plants belonging to the genus Scutellaria , including thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria barbata ), and The purpose is to provide a set of primers that can be used to identify or distinguish at least one species selected from the group consisting of Scutellaria baicalensis .

In addition, another purpose is to provide a method for identifying species of Thimble genus using the primer set.

In addition, another object is to provide a kit for species identification of Thimbium genus including the primer set.

According to one aspect of the present invention, it includes a first primer set including the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 2, and the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4. It further includes at least one primer set selected from the group consisting of a second primer set containing the base sequence shown in SEQ ID NO: 5 and a third primer set containing the base sequence shown in SEQ ID NO: 6. However, a set of primers for species identification of Scutellaria plants that can identify or distinguish Scutellaria indica L. and cotyledonous Scutellaria indica var. tsusimensis may be provided.

In addition, the first primer set is capable of amplifying a sequence containing a species-specific base sequence in the CYC2B gene sequence, and the second primer set is capable of amplifying a sequence containing a species-specific base sequence in the AtpI gene sequence. The third primer set may be capable of amplifying a sequence containing a species-specific base sequence within the rbcL_3 gene sequence.

According to another aspect of the present invention, a second primer set comprising the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4, and the base sequence shown in SEQ ID NO: 5 and the base shown in SEQ ID NO: 6 A species of Scutellaria indica var. tsusimensis , comprising at least one primer set selected from the group consisting of a third primer set comprising a sequence, capable of identifying Scutellaria indica var. tsusimensis from Scutellaria. A set of primers for identification may be provided.

According to another aspect of the present invention, a first step of isolating nucleic acid from an individual of the genus Thimble flower; A second step of performing an amplification reaction using the primer set using the isolated nucleic acid as a template; And a third step of detecting the product of the amplification reaction; a plant of the genus thimble, which includes, and can identify or distinguish between thimble flower ( Scutellaria indica L. ) and cotyledon thimble flower ( Scutellaria indica var. tsusimensis ) in the thimble flower genus. Species identification methods may be provided.

In addition, when the product of the amplification reaction using the first primer set is detected and the product of the amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is not detected It is identified as a thimble flower, the product of the amplification reaction using the first primer set is detected, and the product of the amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is If detected, it can be identified as cotyledon flower.

In addition, in the second step, a fourth primer set including the base sequence shown in SEQ ID NO: 7 and the base sequence shown in SEQ ID NO: 8; And, a fifth primer set comprising the base sequence shown in SEQ ID NO: 9 and the base sequence shown in SEQ ID NO: 10; wherein the product of the amplification reaction using the first primer set is detected and the second primer When the product of the amplification reaction using at least one primer set selected from the group consisting of the set and the third primer set is not detected and the product of the amplification reaction using the fourth primer set and the fifth primer set is not detected Identified as thimble flower, the product of the amplification reaction using the first primer set is detected, and the product of the amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is detected And if the product of the amplification reaction using the fourth primer set and the fifth primer set is not detected, it can be identified as a cotyledon flower.

In addition, in the second step, a sixth primer set comprising the base sequence shown in SEQ ID NO: 11 and the base sequence shown in SEQ ID NO: 12; And, a seventh primer set comprising the base sequence shown in SEQ ID NO: 13 and the base sequence shown in SEQ ID NO: 14; further used, wherein the first primer set, the sixth primer set, and the seventh primer set are used. If the product of the amplification reaction using the amplification reaction is detected and the product of the amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is not detected, it is identified as thimble flower, and the first primer set A product of an amplification reaction using a primer set, the sixth primer set, and the seventh primer set is detected, and an amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set. If the product is detected, it can be identified as cotyledon flower.

According to another aspect of the present invention, a first step of isolating nucleic acid from an individual of the genus Thimble flower; A second step of performing an amplification reaction using the primer set using the isolated nucleic acid as a template; and a third step of detecting the product of the amplification reaction. A method for identifying the species of a plant of the genus Scutellaria can be provided, which includes and can identify Scutellaria indica var. tsusimensis from the genus.

According to another aspect of the present invention, a thimble comprising the primer set and capable of identifying or distinguishing thimble flowers ( Scutellaria indica L. ) and cotyledon thimble flowers ( Scutellaria indica var. tsusimensis ) in the genus Scutellaria. Kits for species identification of flowering plants may be provided.

According to another aspect of the present invention, a kit for species identification of plants of the genus Scutellaria , including the primer set and capable of identifying Scutellaria indica var. tsusimensis from Scutellaria, may be provided. there is.

Using the primer set according to an aspect of the present invention or an identification kit containing the same, PCR is performed to identify specific species of thimble genus plants, specifically thimble flower, mountain thimble flower, cotyledon thimble flower, toothbrush thimble flower, and golden thimble flower. At least one species selected from the group can be identified simply and with high accuracy. Through this, it can be useful in preventing mixing between varieties. In addition, it can be applied to a simple, yet highly accurate information provision method for identifying plant species of the genus genus.

The identification method according to another aspect of the present invention uses species-specific primers of the genus plant, thereby identifying plant species of the genus genus more quickly, conveniently, and with high accuracy compared to the appearance comparison performed to identify conventional plant species of the genus genus. can do. Through this, the time and effort required to identify thimble genus plant species can be reduced.

Figure 1 shows thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria barbata ) and gold ( Scutellaria baicalensis) among plants of thimble flower genus . Comparison of leaf phenotypes of species;
Figure 2 shows a method for producing species-specific primers using atpF registered in GeneBank;
Figure 3 shows PCR results of species-specific primers using atpF registered in GeneBank;
Figure 4 shows the nucleotide sequence and BLAST results of the entire gene of Cotyledon thimble and multiple species of Thimble genus registered in GeneBank;
Figure 5 shows the AtpI_0 base sequence and the position amplified by primers within the base sequence;
Figure 6 shows the rbcL_3 base sequence and the position amplified by primers within the base sequence;
Figures 7 and 8 show PCR results using the prepared species-specific primers.

The present invention relates to a primer set for identifying or distinguishing species of Scutellaria plants, an identification composition using the same, and a method for providing information.

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

Technical and scientific terms used in this specification can be interpreted as meanings commonly understood by those skilled in the art in the technical field to which this invention pertains.

The term DNA marker used in the present invention refers to a specific marker on the DNA of each plant species of the genus Thimmelum that can distinguish between the species of plants of the genus Thimmophyllum. It can be used interchangeably with species-specific base sequences.

The term single nucleotide polymorphism (SNP) used in the present invention refers to any position accompanied by a base sequence containing one or more modified bases. Single nucleotide polymorphisms are generally defined as differences from a constructed reference DNA sequence, or as differences found among subpopulations of individuals drawn from the general population.

The term primer used in the present invention is a base sequence with a short free 3' hydroxyl group, which can form a base pair with a complementary template sequence and can be used to copy the template strand. refers to a short sequence that serves as a starting point for . To amplify a base sequence through template copying, a set of forward and reverse primers is generally used.

According to one aspect of the present invention, among plants of the genus Scutellaria , thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria) A primer set for identifying or distinguishing at least one species selected from the group consisting of Scutellaria baicalensis and Scutellaria baicalensis may be provided.

The primer set includes a first primer set including the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 2; A second primer set comprising the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4; A third primer set comprising the base sequence shown in SEQ ID NO: 5 and the base sequence shown in SEQ ID NO: 6; A fourth primer set comprising the base sequence shown in SEQ ID NO: 7 and the base sequence shown in SEQ ID NO: 8; A fifth primer set comprising the base sequence shown in SEQ ID NO: 9 and the base sequence shown in SEQ ID NO: 10; A sixth primer set comprising the base sequence shown in SEQ ID NO: 11 and the base sequence shown in SEQ ID NO: 12; and a seventh primer set including the base sequence shown in SEQ ID NO: 13 and the base sequence shown in SEQ ID NO: 14.

The primer set may be for amplifying species-specific base sequences that are specifically different between species. The species-specific base sequence may include, but is not limited to, at least one position selected from the group consisting of single nucleotide polymorphism (SNP), base insertion, and base deletion.

Primers included in the primer set may be modified as needed, for example, methylation, capping, nucleotide substitution or modification between nucleotides, for example, uncharged linkages (e.g., methyl phosphonate, phosphotri) There may be modifications to esters, phosphoroamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The species-specific base sequences include CYC2B gene, AtpI (ATP synthase subunit a, chloroplastic) gene, rbcL_3 ( ribulose bisphosphate carboxylase large chain ) gene, BOP028266 chloroplast genome (MF521633.1) sequence, trnH-PsbA gene, and Scutellaria insignis. It may be located within at least one sequence selected from the group consisting of a chloroplast genome (KT750009.1) sequence and a psbK-psbI intergenic spacer (KX059898.1) sequence. The detailed sequence information of the above-mentioned genes and genomes can be found in GeneBank (https://www.ncbi.nlm.nih.gov/genbank/). The AtpI gene may be represented by AtpI_0 and may include the base sequence represented by SEQ ID NO: 15. The rbcL_3 gene may include the base sequence represented by SEQ ID NO: 16.

In a preferred embodiment according to the present invention, a primer set capable of amplifying a sequence containing a species-specific base sequence in the CYC2B gene sequence is used as a first primer set, and a sequence containing a species-specific base sequence in the AtpI gene sequence is used as the first primer set. A primer set capable of amplifying is the second primer set, and a primer set capable of amplifying a sequence containing a species-specific base sequence in the rbcL_3 gene sequence is the third primer set, BOP028266 chloroplast genome (MF521633.1 ) A primer set capable of amplifying a sequence containing a species-specific base sequence within the sequence is provided as the fourth primer set, and a primer set capable of amplifying a sequence containing a species-specific base sequence within the trnH-PsbA gene sequence is provided. The 5 primer set is a primer set that can amplify a sequence containing a species-specific base sequence in the Scutellaria insignis chloroplast genome (KT750009.1) sequence, and the 6th primer set is the psbK-psbI intergenic spacer (KX059898. 1) The primer set that can amplify the sequence containing the species-specific base sequence in the sequence is the seventh primer set, and each primer set is used alone or in combination for cross-validation to perform PCR (polymerase chain reaction). By doing so, it was used to determine the species of the thimble flower genus. In detail, thimble flowers can be identified using at least one primer set selected from the group consisting of the second primer set and the third primer set, and the first primer set, and the second primer set and Cotyledon thimble can be identified using at least one primer set selected from the group consisting of the third primer set, gold can be identified using the fourth primer set, and using the fifth primer set Thus, it was confirmed that the mountain thimble flower could be identified and that the toothbrush thimble flower could be identified using the sixth and seventh primer sets.

As described above, at least one primer set selected from the 1st to 7th primer sets can show specific amplification results depending on the species of the genus plants, and when using it, the species within the genus can be identified more easily and with greater accuracy. There is an advantage in that identification can be performed.

According to another aspect of the present invention, a method for species identification of Thimble genus plants using the primer set according to one aspect of the present invention described above can be provided.

In detail, after isolating the nucleic acid from an individual of the genus of Thimbrella whose species is to be identified, PCR is performed using the isolated nucleic acid as a template and using at least one primer set selected from the group consisting of the first to seventh primer sets. After performing, species identification can be performed by checking the PCR reaction products.

Confirmation of the PCR reaction product can be done by detecting the DNA amplification product to determine the presence or absence of a species-specific amplification reaction or by determining the size of the product. Detection of the product may be performed using a DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto.

According to a preferred embodiment of the present invention, a product of an amplification reaction using the first primer set is detected, and amplification is performed using at least one primer set selected from the group consisting of the second primer set and the third primer set. If the product of the reaction is not detected, it is identified as a thimble flower, and if the product of an amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is detected, it is identified as a thimble flower. is identified as gold when the product of the amplification reaction using the fourth primer set is detected, and is identified as sorghum flower when the product of the amplification reaction using the fifth primer set is detected, and the seventh primer set is identified as If the product of the amplification reaction using the sixth primer set is detected and the product of the amplification reaction using the sixth primer set is not detected, it can be identified as toothbrush thimble flower.

According to another aspect of the present invention, a composition for species identification of a thimble genus plant comprising a primer set according to an aspect of the present invention may be provided.

Preferably, the composition for species identification may be provided in the form of a kit. The kit may be a PCR kit or a DNA analysis kit, but is not limited thereto. For example, the PCR kit may be a kit that includes essential elements necessary to perform at least one PCR selected from the group consisting of single PCR, real-time PCR, and multiplex PCR. The essential elements include, for example, test tubes, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase, DNase, RNAse inhibitors, DEPC-water, and sterilized water. There may be at least one, but it is not limited to this. The DNA analysis kit may be, for example, a DNA chip.

The kit can be used to distinguish specific species quickly and with excellent accuracy by amplifying and confirming the specific base sequence for each species of Thimble genus plants using the primer set according to one aspect of the present invention.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but these examples are only illustrative of the present invention and do not limit the scope of the appended claims, and are examples within the scope and technical idea of the present invention. It is obvious to those skilled in the art that various changes and modifications are possible, and it is natural that such changes and modifications fall within the scope of the appended patent claims.

Example

<Extraction of nucleic acid from target>

Among thimble genus plants, leaf samples were prepared and used for each species of thimble flower, mountain thimble flower, cotyledon thimble flower, toothbrush thimble flower, and golden thimble flower. The phenotype of each leaf is shown in Figure 1. In Figure 1, a is a thimble flower, b is a thimble flower, c is a cotyledon thimble flower, d is a toothbrush thimble flower, and e is a golden leaf.

Approximately 100 mg of fresh or frozen leaf samples for each plant species were placed in a mortar and crushed to a fine powder using liquid nitrogen. The powdered sample was transferred to a 1.5 ml tube. Genomic DNA was extracted according to the manual using Qiagen's DNeasy Plant Mini Kit, and RNA was extracted using Bioneer's Universal RNA Extraction kit. The extracted nucleic acid was used as a template for PCR reaction. If necessary, cDNA was synthesized and used as a template.

<Setting PCR conditions>

The reaction product composition for PCR amplification using the barcode primer used in the present invention is 0.5㎕ of Taq polymerase (5 units/㎕) (nTaq Mg ²⁺ , enzynomics, Republic of Korea), 2㎕ of 10X nTaq buffer, and 2㎕ of Mix dNTP Mixture (2.5mM), 1㎕ of forward primer (10 pmole), 1㎕ of reverse primer (10 pmole) and 2㎕ of quantified template DNA (5 ng/㎕), and add purified water to adjust the final reaction volume. A total of 20 μl was used.

PCR reaction conditions were predenaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 42-55°C for 30 seconds, and amplification at 72°C for 30 seconds. The amplification process was repeated 30 times, and then final extension was performed at 72°C for 10 minutes to obtain a DNA amplification product. The DNA amplification product was spotted on a 2% agarose gel and electrophoresed for 30 minutes, and when species specificity was confirmed, it was used for species identification.

< Production of species-specific primers for atpF, matK, rbcL , and trnH-psbA barcode sections>

To classify four of the five medicinal plants of Lamiaceae and Chloroplast, four Chloroplast DNA (cpDNA) barcode sections ( atpF, matK, rbcL , and trnH-psbA ) were analyzed. Each nucleotide sequence was aligned using CLC Sequence Viewer to search for nucleotide sequences such as SNPs, insertions, or deletions that are specific to the species, and to design specific primers. To increase specificity when producing specific primers, the SNP was located at the 3'-end and the third base sequence was mismatched from there to the 5'-end to produce a specific primer.

Except for Scutellaria baicalensis , the number of genes registered in the NCBI GenBank was significantly small for the four species of Scutellaria baicalensis, and it was confirmed that there were no overlapping genes in all five scutellaria plant species. Accordingly, a number of species-specific primers were produced by aligning overlapping cpDNA portions and cross-tested, but primers produced using existing registered base sequences were not specifically amplified when PCR was performed. It turns out that it doesn't. The primer preparation method using the GeneBank registered sequence is shown in Figure 2, and the gel electrophoresis image of the PCR amplification performed using the prepared atp primer is shown in Figure 3. In Figure 3, a) is the result using Tsu_F and R primers, which are cotyledon species-specific primers, b) is the result using Bai_F and R primers, which are golden species-specific primers, and in both a) and b), M is 100bp. DNA ladder, samples 1 and 2 are thimble flower samples, 3 and 4 are mountain thimble flower samples, 5 and 6 are cotyledon thimble flower samples, 7 and 8 are toothbrush thimble flower samples, and 9 and 10 are golden samples.

<WGS (Whole Genome De novo Sequencing) analysis and production of species-specific primers of Cotyledon thimbleflower>

In order to produce species-specific primers, there was a lack of literature and prior research related to the genus Thimble, and the base sequences registered in NCBI Genbank were also insufficient. In order to investigate the entire genome of Cotyledon thimbleflower among the four genera of Thimblewort to be identified in the present invention, Whole Genome De novo Sequencing analysis was performed on 10 μg of gDNA and 1 μg of RNA using Illumina HiSeq X-Ten and PacBio RSII platform equipment. .

As a result of examining the entire genes of Cotyledon thimbleflower using Whole Genome De novo Sequencing, 318,741,328 genes (about 31 MB) were obtained, and 14,625 contigs were obtained. This is shown in Table 1 below. The obtained gene was compared for similar regions with the previously registered nucleotide sequence of Thimble genus using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were obtained. This is shown in Figure 4. Additionally, among the annotated DNA, 59 cpDNAs were obtained.

Referring to the results in Figure 4, one DNA from each genus and 9 types of cpDNA barcode genes were selected from the base sequences, and it was determined that there were no similar base sequences to compare, so the size of the PCR product was 100 ~ 100 using NCBI Primer-blast. Primers were prepared to be 1000 bp. Information on the produced primers is shown in Table 2 below. AtpI_0 and rbcL_3 base sequences and primer positions within each base sequence are shown in Figures 5 and 6, respectively.

After performing PCR using the prepared primers, a photograph of the amplification gel is shown in Figure 7. In Figure 7, a is the SL primer (first primer set), b is the SP primer (the fifth primer set), c is the ST primer (the second and third primer sets), and d is the SD primer (the sixth and seventh primers). set), e is the PCR result using SB primers (4th primer set), M is the 100bp ladder, 1 is the thimble species sample, 2 is the mountain thimble sample, 3 is the cotyledon thimble sample, 4 is the toothbrush thimble sample, and 5 is It means golden sample.

Referring to Figure 7, Scutellaria pekinensis var. transitra voucher KUS:2006-1082 trnH-PsbA intergenic spacer, partial sequence; and PsbA ( psbA ) gene, partial CDs; Scutellaria pekinensis var. transitra primer (SP primer) prepared from chloroplast (KX060016.1) base sequence, AtpI_0 (scaffold936, location 40496-41245, 750 bp) and rbcL_3 (scaffold2965, location 7668-9095) from annotated cpDNA , Scutellaria indica var. tsusimensis primers (ST1, ST2 primers) produced from Scutellaria baicalensis isolate BOP028266 chloroplast, complete genome (MF521633.1) and golden ( Scutellaria baicalensis ) primers (SB primers) produced from Scutellaria baicalensis isolate BOP028266 chloroplast, complete genome (MF521633.1) ) can be confirmed to be species-specific amplification. Thimble flower and toothbrush thimble flower were not specifically amplified with the primers produced.

< Scutellaria sp. Species identification method using species-specific primers>

Among the plants of the thimble genus, it was confirmed by referring to the results in FIG. 7 that the Snail flower, Cotyledon thimble flower, and Golden bell were specifically amplified and identifiable when PCR was performed with each species-specific primer SP, ST1, ST2, and SB primers produced. . It was found that thimbleflower and toothbrush thimbleflower species were not specifically amplified, but cross-validation with other primers confirmed that they could be identified. The cross-validation results are shown in Figure 8. In Figure 8, a is the PCR result using SL primer and ST1 primer for differentiation of thimble flowers, b is the PCR result using SP primer for differentiation of thimble flower, and c is ST1 and 2 for distinguishing cotyledon thimble flower. This is the result of PCR using primers, d is the result of PCR using SD 1 and 2 primers for identification of Toothbrush thimble flower, and e is the result of PCR using SB primers for identification of Golden Division. The meaning of each sample is the same as in Figure 7.

Referring to Figure 8, the thimble flower-specific primer (SL primer) produced from the Scutellaria incana TCP transcription factor (CYC2B) gene, partial cds (KM526800.1) base sequence was also amplified from thimble flower DNA and cotyledon thimble flower DNA. Thimble flower was able to be identified during cross-validation using ST1 Primer and ST2 Primer, which are specific primers for thimble flower.

In addition, the toothbrush-specific primer (SD1 primer) prepared from the base sequence of Scutellaria insignis chloroplast, complete genome (KT750009.1) was amplified from the DNA of all other species except for the toothbrush-thimbleflower DNA, Scutellaria indica var. tsusimensis voucher SWUS. Kim 2008-008 psbK-psbI intergenic spacer, partial sequence; The primer (SD2 primer) produced from the chloroplast (KX059898.1) base sequence was found to be amplified from the DNA of all species, including Toothbrush thimbleflower, so it could be used together to identify Toothbrush thimbleflower during cross-validation.

<110> Bioresources and Technology (B&Tech) Co., Ltd. <120> PRIMER SET FOR IDENTIFYING SPECIES OF SCUTELLARIA SP. AND IDENTIFICATION METHOD USING THE SAME <130> P-2021-0824 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SL primer F <400> 1 tgcttacctg cttccacagg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SL primer R <400> 2 tcggtggcga cgttatatgg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ST1 primer F <400> 3 ttgtggcatc actaaccccc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ST1 primer R <400> 4 aggggtaggc tgaacgtact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ST2 primer F <400> 5 cgcattcctc cagcctatgt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ST2 primer R <400> 6 atcacggcag tagtgtgcaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SB primer F <400> 7 tccccaaaaaa gtggatccccg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SB primer R <400> 8 gggcctcatt ggtaagtgct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SP primer F <400> 9 gaaattactt ttaaattcat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SP primer R <400> 10 gtagtctttc ctagacttta 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SD1 primer F <400> 11 ataacttccc tctagactta 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SD1 primer R <400> 12 tgaatttcaa ttattttttc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SD2 primer F <400> 13 acccttgatt cgcacactga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SD2 primer R <400> 14 tgaggaaacg gacgtaagcc 20 <210> 15 <211> 750 <212> DNA <213> Unknown <220> <223> annotation cpDNA AtpI_0 (ATP synthase subunit a, chloroplastic) <400> 15 atgagtattt attctggtct atttgtggca tcactaaccc ccgctttcca gcttgctagt 60 gtggaagttg gacaacattg gtattggcag cttgggcagt tccaagttca tgcccaagtt 120 ttgattacat cttgggttgt tgctgcgctt ctcttagggt tagcagtttg ggggacacgt 180 agccttaaag ctgtgccaca agggggacaa ggcttggtag aatacgtact tgagtttata 240 cgtgacttga ctcggactca gattggtgaa gaagaatatc gaccttgggt accttttatt 300 ggcactttat ttttgtttat ttttgtatca aattggtcag gggcccttgt accttggaaa 360 gttatgaat tacccaatgg agagcttgcc gctcccacaa acgatattaa tactactgtg 420 gctcttgcat tactaacttc tgtggcttat ttttatgctg gtcttcataa aagaggtcta 480 agctattttg gtaagtacgt tcagcctacc cctgtgttat taccaattaa tattcttgaa 540 gattttacta aacctctgtc gttaagtttc cgactttttg gaaacattct agctgacgaa 600 cttgtcgttg ctgtacttgt ttcattagtc cctcttgtag tacctattcc aatgatgttt 660 ctaggccttt ttactagtgg tattcaagct cttattttcg ccacacttgc tgctgcttat 720 atcggagaat caatggaagg acatcactaa 750 <210> 16 <211> 1428 <212> DNA <213> Unknown <220> <223> annotation cpDNA rbcL_3 (Ribulose bisphosphate carboxylase large chain) <400> 16 atgtcaccac aaactgaaac cagaacaggt actggcttta aagcaggcgt taaggactat 60 cgcctaactt actacactcc tgattatgag accaaagaga ctgatatcct agctgcattt 120 cggatgactc ctcaacctgg agttcctcct gaagaagcag gtgctgcggt tgcagctgaa 180 tcttcaaccg gtacctggac cactgtatgg accgatggac taaccaacct tgatcgttat 240 aagggtcgct gttacgatat tgaacctgtt gctggtgagg aaaaccaata tattgcatat 300 gtggcatatc ctttggacct ttttgaagag ggttcagtca ccaacctgtt tacttctatt 360 gtaggaaacg tattcggctt taaggcactt cgtgctcttc gattggaaga tcttcgcatt 420 cctccagcct atgtgaaaac tttccaaggt cctcctcatg gtatccaggt tgagcgagat 480 aaactaaaca aatacggtcg acctttgctt ggttgtacta ttaagcctaa acttggtctt 540 tctgctaaaa actatggccg ggcagtgtac gaatgcctac gtggtggttt ggactttaca 600 aaagatgatg aaaacgtaaa ctctcagcct ttcatgcgct ggagagatag attccttttt 660 gtagcagagg ctacctttaa agctcaggct gaaactggtg aaatcaaagg acattacctg 720 aatgctacgg ctggtacttc cgaagagatg ctaaaaaagag ctcaatttgc aagagagcta 780 ggagcaccaa tcgtaatgca cgattaccta actggtggct ttactgcaaa cacaagtctt 840 gcacactact gccgtgataa cggtttgctt cttcacattc accgtgctat gcacgcggta 900 attgaccgtc agcgtaacca tggtattcat ttccgtgtct tagcgaaagc acttcgtcta 960 tccggtggtg atcatattca ctctggtaca gtcgtaggta agcttgaagg tgagcgtgaa 1020 gtaacccttg gttttgtaga tcttttgaga gatgactaca ttgaaaaaga ccgaagccga 1080 ggcatttact tcactcaaga ttgggtttct cttcctggtg ttctgcctgt ggcttctggt 1140 ggcattcacg tatggcatat gcctgctctt actgagattt ttggcgatga ctccgtactt 1200 caatttggtg gcggtaccct tggtcaccct tggggcaatg ctcctggtgc tgttgcaaac 1260 cgcgtggcac ttgaagcatg tgttcaagct cgtaatgaag gtcgtgacct agctcgcgaa 1320 ggtaaccaag tgatccgtga agcggctaag tggagcccag agcttgctgc agcctgcgaa 1380 gtttggaaag agatcaagtt tgagtttgaa acaattgata ctctatag 1428

Claims (10)

A first primer set comprising a base sequence represented by SEQ ID NO: 1 and a base sequence represented by SEQ ID NO: 2,
A second primer set comprising the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4, and the third primer set containing the base sequence shown in SEQ ID NO: 5 and the base sequence shown in SEQ ID NO: 6 It further includes at least one primer set selected from the group consisting of,
Among Scutellaria plants, thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria barbata ), and gold ( Scutellaria baicalensis ). A set of primers for species identification of plants of the genus Scutellaria, which can identify or distinguish thimble flower ( Scutellaria indica L. ) and cotyledon thimble flower ( Scutellaria indica var. tsusimensis ) from the group consisting of.
According to clause 1,
The first primer set is capable of amplifying a sequence containing a species-specific base sequence in the CYC2B gene sequence,
The second primer set is capable of amplifying a sequence containing a species-specific base sequence in the AtpI gene sequence,
The third primer set is a primer set for species identification of plants of the genus Rhizome, which is capable of amplifying a sequence containing a species-specific base sequence in the rbcL_3 gene sequence.
A second primer set comprising the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4, and the third primer set containing the base sequence shown in SEQ ID NO: 5 and the base sequence shown in SEQ ID NO: 6 Comprising at least one primer set selected from the group consisting of,
Among Scutellaria plants, thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria barbata ), and gold ( Scutellaria baicalensis ). A primer set for species identification of Scutellaria indica var. tsusimensis , which can identify Scutellaria indica var. tsusimensis from the group consisting of.
A first step of isolating nucleic acids from individuals of the thimble flower genus;
A second step of performing an amplification reaction using the isolated nucleic acid as a template and the primer set of claim 1; and
A third step of detecting the product of the amplification reaction,
Among Scutellaria plants, thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria barbata ), and gold ( Scutellaria baicalensis ). A species identification method for plants of the genus Scutellaria that can identify or distinguish thimble flowers ( Scutellaria indica L. ) and cotyledon thimble flowers ( Scutellaria indica var. tsusimensis ) from the group consisting of.
According to clause 4,
When the product of the amplification reaction using the first primer set is detected and the product of the amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is not detected, thimble flower Is identified as
When the product of the amplification reaction using the first primer set is detected and the product of the amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is detected, cotyledon thimble flower A method of identifying species of plants of the genus thimbleflower, identified by .
According to clause 4,
A fourth primer set comprising the base sequence shown in SEQ ID NO: 7 and the base sequence shown in SEQ ID NO: 8 in the second step; And, a fifth primer set comprising the base sequence shown in SEQ ID NO: 9 and the base sequence shown in SEQ ID NO: 10; is further used,
The product of the amplification reaction using the first primer set is detected, the product of the amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is not detected, and the If the product of the amplification reaction using the 4 primer set and the 5th primer set is not detected, it is identified as thimble flower,
A product of an amplification reaction using the first primer set is detected, a product of an amplification reaction using at least one primer set selected from the group consisting of the second primer set and the third primer set is detected, and the fourth primer set is detected. A method for identifying the species of a plant of the genus Thimble, where the product of the amplification reaction using the primer set and the fifth primer set is not detected, and is identified as a cotyledon thimble flower.
According to clause 4,
A sixth primer set comprising the base sequence shown in SEQ ID NO: 11 and the base sequence shown in SEQ ID NO: 12 in the second step; And, a seventh primer set comprising the base sequence shown in SEQ ID NO: 13 and the base sequence shown in SEQ ID NO: 14; is further used,
A product of an amplification reaction using the first primer set, the sixth primer set, and the seventh primer set is detected, and at least one primer set selected from the group consisting of the second primer set and the third primer set If the product of the amplification reaction used is not detected, it is identified as thimble flower,
A product of an amplification reaction using the first primer set, the sixth primer set, and the seventh primer set is detected, and at least one primer set selected from the group consisting of the second primer set and the third primer set A method for species identification of plants of the genus Thimble, which is identified as a cotyledon thimble flower when the product of the amplification reaction used is detected.
A first step of isolating nucleic acids from individuals of the thimble flower genus;
A second step of performing an amplification reaction using the isolated nucleic acid as a template and the primer set of claim 3; and
A third step of detecting the product of the amplification reaction,
Among Scutellaria plants, thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( Scutellaria barbata ), and gold ( Scutellaria baicalensis ). A species identification method of Scutellaria indica var. tsusimensis, which can identify Scutellaria indica var. tsusimensis from the group consisting of.
Containing the primer set of paragraph 1, among Scutellaria plants, thimble flower ( Scutellaria indica L. ), mountain thimble flower ( Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( A kit for species identification of thimble genus plants, which can identify or distinguish thimble flower ( Scutellaria indica L. ) and cotyledon thimble flower ( Scutellaria indica var. tsusimensis ) from the group consisting of Scutellaria barbata ) and gold ( Scutellaria baicalensis ).
Contains the primer set of paragraph 3, and among Scutellaria plants, thimble flower ( Scutellaria indica L. ), Scutellaria pekinensis var. transitra ), cotyledon thimble flower ( Scutellaria indica var. tsusimensis ), toothbrush thimble flower ( A kit for species identification of Scutellaria indica var. tsusimensis, which can identify Scutellaria indica var. tsusimensis from the group consisting of Scutellaria barbata and Scutellaria baicalensis .