KR20230030838A - 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 species identification method of Scutellaria using the same, and specifically, to a primer set for species identification of Scutellaria and a species identification method of Scutellaria using the same, wherein at least one species selected from a group consisting of Scutellaria indica L., Scutellaria pekinensis var. transitra, Scutellaria indica var tsusimensis, Scutellaria barbata, and Scutellaria baicalensis in Scutellaria can be can identified simply and with high accuracy.

Description

A primer set for species identification of Thimble genus plants and a method for identifying species 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 primer set for species identification of the genus Thimbus and a method for identifying species of the genus Thimble.

In the case of plants, hybrids between various species and genera are frequently reported, and it is difficult to understand the maternal lineage and evolution for the accurate identification of plant taxa due to the complex pattern of polyploidy. Therefore, the development of DNA capable of identifying strains or breeds is desired.

With the recent development of molecular biology, the technology to search for polymorphisms in specific DNA sequences and use them as molecular markers is not affected by the environment and there is little variation between years. 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 that have been used to date include comparing morphological characteristics, qualitative and quantitative analysis of components, and comparing differences at the DNA level. Distinguishing by comparing morphological characteristics can show differences depending on the growth environment of the plant, making it difficult to identify plant species. In the case of classification by component analysis, a taste sensor and a nuclear magnetic resonance spectrometer (NMR spectrometer) were used, but this method has a limitation that components may be affected by the cultivation environment of the production area.

In order to solve the limitations of these conventional methods, molecular biological plant species identification using DNA fragment polymorphisms such as RAPD (Random Amplified Polymorphic DNA), SCAR (Sequence Characterized Amplified Regions), AFLP (Amplified Fragment Length Polymorphism) has been attempted. Also, in recent years, the classification method using DNA barcodes proposed by the CBOL Plant Working Group has been recognized for its usefulness in identifying plant species. In particular, the nrDNA-ITS (internal transcribed spacer) region of nuclear ribosomal DNA (nrDNA) present on the genome not only evolves faster than the coding region of other genes, but also has advantages such as generality, simplicity, and reproducibility. Because of this, it is known as a base sequence that is widely used for phylogeny or search for genetic variation between species. In the case of rRNA genes, there is little difference between species and genera, whereas ITS sections with many mutations can provide characteristics with useful information for phylogenetic analysis because there are thousands of copies per genome.

Currently, a genetic marker capable of accurately identifying the species of plants of the thimble genus ( Scutellaria ), which contains many useful medicinal plants, has not been developed. Thimbleaceae, in the family Lamiaceae, is the second largest genus in the family and includes about 470 taxa worldwide. Many plants of the Thimbleflower genus are mainly distributed in temperate regions and are used as medicinal plants or spices. Thimble flower genus medicinal plants include thimble flower, mountain thimble flower, cotyledon thimble flower, toothbrush thimble flower, and gold. need to develop

The thimble flower ( Scutellaria indica L. ) is a perennial plant, characterized by silky heart-shaped leaves and black spots on the flowers. It is called Hansincho in China, and it is a medicinal plant that has been used in the private sector for pain relief and hemostasis and to make boils go away, or for symptoms of toothache, headache, hematemesis, or hemoptysis.

The mountain radish flower ( Scutellaria pekinensis var. transitra ) is characterized by opposite leaves, hairs on both sides, and sawtooth on the edge. Light purple flowers bloom in May-June, and young shoots and soft leaves are eaten as vegetables in spring.

The cotyledon thimble flower ( Scutellaria indica var. tsusimensis ) is very similar in appearance to the thimble flower, and the leaves are slightly thicker and hairy than the thimble flower. The young leaves are eaten as herbs and used as herbal medicines for detoxification and pain relief.

Toothbrush thimble flower ( Scutellaria barbata ) is also called semi-lily, and is mainly used as a medicine for the aerial part. It has the effect of lowering fever and reducing swelling, and anti-cancer effects have also been studied.

Gold ( Scutellaria baicalensis ) is a naturalized plant native to China and is widely cultivated as a medicinal plant. The root is mainly used, and it is a biological resource with high value as a medicinal and ornamental plant. As for the pharmacological effect, it has the effect of lowering the heat of the heart and lungs, or stopping coughing and blood, and has anti-allergic, antioxidant, anti-inflammatory, and antibacterial effects on the skin, such as atopic dermatitis, bacterial dermatitis such as acne, skin whitening, etc. It is used as an active ingredient in cosmetics.

KR 101804120 B1

The present invention thimble flower ( Scutellaria ) Among plants belonging to the 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 It is an object of the present invention to provide a primer set that can be used to identify or distinguish at least one species selected from the group consisting of golden ( Scutellaria baicalensis ).

In addition, another object 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 identifying species of Thimble genus including the primer set.

According to one aspect of the present invention, including a sixth primer set comprising the nucleotide sequence represented by SEQ ID NO: 11 and the nucleotide sequence represented by SEQ ID NO: 12, thimble flower ( Scutellaria ) to toothbrush thimble flower ( Scutellaria barbata ) A set of primers may be provided for identifying the species of thimblesome plants that can be identified.

In addition, a seventh primer set including the nucleotide sequence represented by SEQ ID NO: 13 and the nucleotide sequence represented by SEQ ID NO: 14; may be further included.

In addition, the sixth primer set may be capable of amplifying a sequence including a species-specific nucleotide sequence within the Scutellaria insignis chloroplast genome (KT750009.1) sequence.

In addition, the seventh primer set may be capable of amplifying a sequence including a species-specific nucleotide sequence within the psbK-psbI intergenic spacer (KX059898.1) sequence.

According to another aspect of the present invention, a first step of isolating nucleic acids from individuals of the genus Thimbus; a second step of performing an amplification reaction using a sixth primer set comprising the nucleotide sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12 using the isolated nucleic acid as a template; And a third step of detecting a product of the amplification reaction; including, a Thimble Flower ( Scutellaria ) in Toothbrush Thimble Flower ( Scutellaria barbata ) A species identification method of a plant of the Thimble Flower genus that can be identified can be provided.

In addition, when the product of the amplification reaction using the sixth primer set is not detected, it can be identified as a toothbrush thimble flower.

In addition, in the second step, a seventh primer set comprising the base sequence represented by SEQ ID NO: 13 and the base sequence represented by SEQ ID NO: 14; is further used, but the product of the amplification reaction using the sixth primer set is detected If not, and a product of the amplification reaction using the seventh primer set is detected, it can be identified as a toothbrush thimble flower.

In addition, a fourth primer set comprising the nucleotide sequence represented by SEQ ID NO: 7 and the nucleotide sequence represented by SEQ ID NO: 8 in the second step; And, a fifth primer set comprising the nucleotide sequence represented by SEQ ID NO: 9 and the nucleotide sequence represented by SEQ ID NO: 10; further using the fourth primer set, the fifth primer set, and the sixth primer set If the product of the amplification reaction used is not detected, it can be identified as toothbrush bone flower.

According to another aspect of the present invention, including a sixth primer set comprising a nucleotide sequence represented by SEQ ID NO: 11 and a nucleotide sequence represented by SEQ ID NO: 12, thimble flower genus ( Scutellaria ) Toothbrush thimble flower ( Scutellaria barbata ) A kit for identifying the species of Thimble genus plants that can identify may be provided.

In addition, a seventh primer set including the nucleotide sequence represented by SEQ ID NO: 13 and the nucleotide sequence represented by SEQ ID NO: 14; may be further included.

Using a primer set or an identification kit including the same according to one aspect of the present invention, PCR is performed to determine specific species of plants of the genus Thimble, specifically thimble flower, mountain thimble flower, cotyledon thimble flower, toothbrush thimble flower, and gold It is possible to discriminate at least one species selected from the group consisting of simple and high accuracy. Through this, it can be usefully used to prevent mixed use between varieties. In addition, it can be applied to a method of providing information for simple but highly accurate identification of plant species of the Thimble genus.

The identification method according to another aspect of the present invention uses a species-specific primer of the Thimble genus plant to identify the plant species of the Thimble genus with more rapid, simple and high accuracy compared to the appearance comparison performed for the conventional identification of Thimble flower species. can do. Through this, it is possible to reduce the time and effort required to identify plant species of the Thimble genus.

Figure 1 is a thimble flower of the 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 golden ( Scutellaria baicalensis ) Comparison of leaf phenotypes of species,
2 shows a method for preparing species-specific primers using atpF registered in GeneBank;
3 is a PCR result of species-specific primers using atpF registered in GeneBank;
Figure 4 shows the results of performing BLAST with the base sequence of all genes of the cotyledonous flower and a number of species of the Thimble genus registered in GeneBank,
Figure 5 shows the position amplified by primers in the AtpI_0 nucleotide sequence and the nucleotide sequence,
Figure 6 shows the rbcL_3 nucleotide sequence and the position amplified by primers in the nucleotide sequence,
7 and 8 are PCR results using the prepared species-specific primers.

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

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

Technical and scientific terms used in this specification may be interpreted as meanings commonly understood by those of ordinary skill in the art to which this invention belongs.

The term DNA marker used in the present invention refers to a specific mark on the DNA of each plant species of the genus Thimbus that can distinguish the species of plants of the genus Thimble. It can be used in combination with species-specific nucleotide sequences.

The term single nucleotide polymorphism (SNP) used in the present invention refers to any position accompanied by a nucleotide sequence having one or more modified bases. A single nucleotide polymorphism is generally defined as a difference from a constructed reference DNA sequence, or as a difference found between subpopulations of individuals taken from the general population.

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

According to one aspect of the present invention, thimble flower genus ( Scutellaria ) Thimble flower among plants ( 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 identifying or distinguishing at least one species selected from the group consisting of may be provided.

The primer set includes a first primer set including a nucleotide sequence represented by SEQ ID NO: 1 and a nucleotide sequence represented by SEQ ID NO: 2; a second primer set comprising the nucleotide sequence represented by SEQ ID NO: 3 and the nucleotide sequence represented by SEQ ID NO: 4; a third primer set comprising the nucleotide sequence represented by SEQ ID NO: 5 and the nucleotide sequence represented by SEQ ID NO: 6; a fourth primer set comprising the nucleotide sequence represented by SEQ ID NO: 7 and the nucleotide sequence represented by SEQ ID NO: 8; A fifth primer set comprising the nucleotide sequence represented by SEQ ID NO: 9 and the nucleotide sequence represented by SEQ ID NO: 10; A sixth primer set comprising the nucleotide sequence represented by SEQ ID NO: 11 and the nucleotide sequence represented by SEQ ID NO: 12; and a seventh primer set comprising the nucleotide sequence represented by SEQ ID NO: 13 and the nucleotide sequence represented by SEQ ID NO: 14; and at least one primer set selected from the group consisting of.

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

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

The species-specific nucleotide sequence includes 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, 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. For the sequence of the aforementioned gene and genome, specific sequence information 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 nucleotide sequence represented by SEQ ID NO: 15. The rbcL_3 gene may include the nucleotide sequence represented by SEQ ID NO: 16.

In a preferred embodiment according to the present invention, a primer set capable of amplifying a sequence including a species-specific nucleotide sequence in the CYC2B gene sequence is used as a first primer set, and a sequence including a species-specific nucleotide sequence in the AtpI gene sequence A primer set capable of amplifying as a second primer set, a primer set capable of amplifying a sequence containing a species-specific nucleotide sequence within the rbcL_3 gene sequence as a third primer set, and the BOP028266 chloroplast genome (MF521633.1 ) A primer set capable of amplifying a sequence including a species-specific nucleotide sequence in the sequence as a fourth primer set, and a primer set capable of amplifying a sequence including a species-specific nucleotide sequence in the trnH-PsbA gene sequence 5 As a primer set, a primer set capable of amplifying a sequence including a species-specific nucleotide sequence in the Scutellaria insignis chloroplast genome (KT750009.1) sequence was used as a sixth primer set, and a psbK-psbI intergenic spacer (KX059898. 1) PCR (polymerase chain reaction) is performed by using a primer set capable of amplifying a sequence containing a species-specific nucleotide sequence in the sequence as the 7th primer set, and using each primer set alone or in combination for cross-validation By doing so, it was used for species identification of the genus Thimble. Specifically, the thimble flower 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 flowers 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 the fifth primer set Using It was confirmed that the mountain thimble flower could be identified, and the toothbrush thimble flower could be identified using the sixth primer set and the seventh primer set.

As described above, at least one primer set selected from the 1st to 7th primer sets can show specific amplification results according to the species of the genus Thimble, so it is easier and more accurate to use them to determine the species within the genus Thimble. There is an advantage that identification can be performed.

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

Specifically, after isolating nucleic acids from individuals of the Thimble genus to be identified, PCR is performed using at least one primer set selected from the group consisting of the first to seventh primer sets using the separated nucleic acids as a template. After performing, species identification can be performed by confirming the PCR reaction product.

Confirmation of the PCR reaction product may be performed by determining the presence or absence of a species-specific amplification reaction by detecting the DNA amplification product or by determining the size of the product. Detection of the product may be performed through 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 the amplification reaction using the first primer set is detected, and amplification using at least one primer set selected from the group consisting of the second primer set and the third primer set If a product of the reaction is not detected, it is identified as a thimble flower, and if 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, as a cotyledon flower It is identified as golden when the product of the amplification reaction using the fourth primer set is detected, and identified as golden radish when the product of the amplification reaction using the fifth primer set is detected, and the seventh primer set If a product of the amplification reaction using the amplification reaction is detected and the product of the amplification reaction using the sixth primer set is not detected, it can be identified as a toothbrush thimble flower.

According to another aspect of the present invention, a composition for species identification of Thimble genus plants comprising the primer set according to one 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 kit for DNA analysis, but is not limited thereto. The PCR kit may be, for example, a kit including essential elements required to perform at least one PCR selected from the group consisting of single PCR, real-time PCR, and multiplex PCR. The essential elements are selected from the group consisting of, for example, test tubes, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase, DNase, RNAse inhibitors, DEPC-water and sterile water It may be at least one, but is not limited thereto. The DNA analysis kit may be, for example, a DNA chip.

The kit can be used to quickly and accurately distinguish a specific species by amplifying and confirming a nucleotide sequence specific to 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 embodiments are only illustrative of the present invention and do not limit the scope of the appended claims, and embodiments within the scope and spirit of the present invention It is obvious to those skilled in the art that various changes and modifications to the are possible, and it is natural that these variations and modifications fall within the scope of the appended claims.

Example

<Nucleic acid extraction from target>

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

About 100 mg of fresh or frozen leaf samples for each plant species were placed in a mortar and pulverized into fine powder using liquid nitrogen. The powdery sample was transferred to a 1.5 ml tube, DNA extraction was performed using Qiagen's DNeasy Plant Mini Kit according to the manual, and RNA extraction was performed 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.

<Set PCR conditions>

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

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

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

In order to classify 4 types of thimble and gold among 5 types of medicinal plants of the genus Lamiaceae, 4 sections of Chloroplast DNA (cpDNA) barcodes ( atpF, Assays were performed for matK, rbcL and trnH-psbA ). Each nucleotide sequence was aligned with CLC Sequence Viewer, and specific primers were designed by searching for nucleotide sequences such as SNPs, insertions, or deletions specific to the species. In order to increase the specificity when preparing a specific primer, a specific primer was prepared by positioning the SNP at the 3'-end and mismatching the third nucleotide sequence in the 5'-side therefrom.

Except for golden ( Scutellaria baicalensis ), the number of genes registered in NCBI GenBank was significantly less for 4 Thimble genus species, and it was confirmed that there were no overlapping gene parts in all 5 scutellaria plant species. Accordingly, a number of species-specific primers were prepared by aligning overlapping cpDNA portions and cross-validated, but primers prepared using previously registered nucleotide sequences were not specifically amplified when PCR was performed. appeared not to A primer preparation method using the GeneBank registration sequence is shown in FIG. 2, and a PCR amplification gel electrophoresis picture performed using the prepared atp primer is shown in FIG. 3. In Figure 3, a) is the result of using the cotyledon thimble flower species-specific primers Tsu_F and R primers, b) is the result of using the golden species-specific primers Bai_F and R primers, a) and b) in both M is 100 bp 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 of Cotyledon Thimble Flower and Production of Species-Specific Primers>

In order to prepare species-specific primers, there is a lack of literature and previous studies related to the genus Thimble, and the nucleotide sequences registered in NCBI Genbank are also insufficient. 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 to investigate the entire gene of the cotyledon flower among the four thimble genus to be identified in the present invention. .

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

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

After PCR was performed using the prepared primers, a photograph of the amplification gel is shown in FIG. 7 . In FIG. 7, a is an SL primer (first primer set), b is an SP primer (fifth primer set), c is an ST primer (second and third primer sets), and d is an SD primer (sixth and seventh primer sets). set), e is the PCR result using the SB primer (fourth primer set), M is a 100bp ladder, 1 is a thimble sample, 2 is a thimble sample, 3 is a cotyledon thimble sample, 4 is a toothbrush thimble sample, 5 is means gold 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) of annotation cpDNA , 1429 bp) , Scutellaria indica var. tsusimensis ) primers (ST1, ST2 primer) and Scutellaria baicalensis isolate BOP028266 chloroplast, complete genome (MF521633.1) Golden ( Scutellaria baicalensis ) primer (SB primer ) can confirm that it is species-specifically amplified. Thimble flower and toothbrush thimble flower were not specifically amplified in the prepared primer.

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

Among plants of the genus Thimble, it was confirmed with reference to the results of FIG. 7 that thimble flower, cotyledon thimble flower, and golden species were specifically amplified and identified when PCR was performed with each of the species-specific primers SP, ST1, ST2, and SB primers produced. . It was found that thimble flower and toothbrush thimble flower species were not specifically amplified, but it was confirmed that they could be identified by cross-validation with other primers. The cross-validation results are shown in FIG. 8 . In FIG. 8, a is a PCR result using SL primer and ST1 primer for discrimination of thimble flower, b is a PCR result using SP primer for discrimination of thimble flower, c is ST1 and 2 for discrimination of cotyledon thimble flower PCR results using primers, d is PCR results using SD 1 and 2 primers for classification of bristles, and e is PCR results using SB primers for gold classification. The meaning of each sample is the same as in FIG. 7 .

Referring to Figure 8, the thimble flower-specific primer (SL primer) prepared from the Scutellaria incana TCP transcription factor (CYC2B) gene, partial cds (KM526800.1) nucleotide sequence was also amplified in thimble flower DNA and cotyledon thimble flower DNA, cotyledon Thimble flower-specific primers, ST1 Primer and ST2 Primer, were used to identify thimble flower upon cross-validation.

In addition, the toothbrush thimble flower-specific primer (SD1 primer) prepared from the Scutellaria insignis chloroplast, complete genome (KT750009.1) nucleotide sequence was amplified from DNA of all other species except toothbrush thimble flower DNA, and Scutellaria indica var. tsusimensis voucher SWUS. Kim 2008-008 psbK-psbI intergenic spacer, partial sequence; Since the primer (SD2 primer) prepared from the chloroplast (KX059898.1) nucleotide sequence is amplified from the DNA of all species including the toothbrush thimble flower, it was shown that the toothbrush thimble flower can be identified during cross-validation when used together.

<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 tccccaaaaa gtggatcccg 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 acctttatt 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 gatttacta 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 ctaaaaagag 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 sixth primer set comprising the nucleotide sequence represented by SEQ ID NO: 11 and the nucleotide sequence represented by SEQ ID NO: 12;
Thimble flower genus ( Scutellaria ) Toothbrush thimble flower ( Scutellaria barbata ) Can be identified, a set of primers for species identification of thimble flower plants.
According to claim 1,
A seventh primer set comprising the nucleotide sequence represented by SEQ ID NO: 13 and the nucleotide sequence represented by SEQ ID NO: 14; further comprising a primer set for species identification of plants of the genus Thimbus.
According to claim 1,
The sixth primer set is capable of amplifying a sequence containing a species-specific nucleotide sequence in the Scutellaria insignis chloroplast genome (KT750009.1) sequence, a primer set for species identification of plants of the genus Thimble.
According to claim 2,
The seventh primer set is capable of amplifying a sequence comprising a species-specific nucleotide sequence in the psbK-psbI intergenic spacer (KX059898.1) sequence.
A first step of isolating nucleic acids from individuals of the genus Thimbus;
a second step of performing an amplification reaction using a sixth primer set comprising the nucleotide sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12 using the isolated nucleic acid as a template; and
A third step of detecting the product of the amplification reaction; including,
Thimble flower genus ( Scutellaria ) Toothbrush thimble flower ( Scutellaria barbata ) Can be identified, species identification method of thimble flower genus plants.
According to claim 5,
A species identification method of a plant of the genus Thimble, which is identified as a toothbrush thimble flower when the product of the amplification reaction using the sixth primer set is not detected.
According to claim 5,
In the second step, a seventh primer set comprising the nucleotide sequence represented by SEQ ID NO: 13 and the nucleotide sequence represented by SEQ ID NO: 14; is further used,
A species identification method of a plant of the genus Thimbus, identified as a toothbrush thimble flower when the product of the amplification reaction using the sixth primer set is not detected and the product of the amplification reaction using the seventh primer set is detected.
According to claim 5,
a fourth primer set comprising the nucleotide sequence represented by SEQ ID NO: 7 and the nucleotide sequence represented by SEQ ID NO: 8 in the second step; and a fifth primer set comprising the nucleotide sequence represented by SEQ ID NO: 9 and the nucleotide sequence represented by SEQ ID NO: 10;
A species identification method of a plant of the genus Thimble, identified as a toothbrush thimble flower when no product of the amplification reaction using the fourth primer set, the fifth primer set, and the sixth primer set is detected.
A 6th primer set comprising the nucleotide sequence represented by SEQ ID NO: 11 and the nucleotide sequence represented by SEQ ID NO: 12, Thimble flower genus ( Scutellaria ) Toothbrush thimble flower ( Scutellaria barbata ) in Thimbleflower plants capable of identifying ( Scutellaria barbata ) Kit for species identification of animals.
According to claim 9,
A seventh primer set comprising the nucleotide sequence represented by SEQ ID NO: 13 and the nucleotide sequence represented by SEQ ID NO: 14; further comprising a kit for species identification of plants of the genus Thimbus.

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