KR20210110521A - InDel Markers for Discrimination of Cynanchum wilfordii and Cynanchum auriculatum and Method for Use thereof - Google Patents

Abstract

The present invention relates to an InDel marker for discriminating between Cynanchum wilfordii and Cynanchum auriculatum and a discrimination method using the same. In the case of using the InDel marker for species discrimination of Cynanchum wilfordii and Cynanchum auriculatum of the present invention, and a primer to amplify the marker, it is possible to easily distinguish Cynanchum wilfordii from other plants of the genus of Cynanchum such as Cynanchum auriculatum, and classify the species of Cynanchum wilfordii in a rapid, accurate and economical manner.

Description

InDel Markers for Discrimination of Cynanchum wilfordii and Cynanchum auriculatum and Method for Use thereof

The present invention relates to a marker for discriminating between white and bilobed cows, and a discrimination method using the same, and more particularly, to an InDel marker for discriminating between white and bilobed cows, and a method for discriminating between white and bilobed cows using the same.

The Nagoya Protocol, promulgated in 2010, is an international agreement to share the benefits generated by using biological resources. With the National Institute of Biological Resources and the National Institute of Ecology to be established in 2012, Korea is preparing for the Nagoya Protocol by discovering about 100,000 kinds of domestic biological genetic resources and creating a database for resource use.

With the entry into force of the Nagoya Protocol, protecting and preserving our biological sovereignty is emerging as a very important issue. In relation to the preservation of our biological resources, the cypress tree, a representative plant of Mt. Jirisan and Mt. Halla, was exported to the West in 1904 and widely used as a Christmas tree abroad. Cloves, which are popular in Korea, and chrysanthemum wheat, also known as Sorona, are Korean biological resources exported overseas.

Therefore, various efforts are needed to prevent unauthorized export of endemic organisms to Korea and to secure biological resources. In particular, in preparation for the Nagoya Protocol and in the case of an endemic species such as white sagebrush, which has high economic value, it is essential to develop a species-specific marker to ensure biological sovereignty.

On the other hand, Cynanchum wilfordii is a perennial herbaceous perennial in the genus Cynanchum wilfordii, which grows on sunny grasslands and coastal slopes in the mountains and fields of Korea and China. . Baeksuo is described as a herbal medicine in many oriental medicine books such as Donguibogam and Donguisusebowon. Recently, it has been scientifically proven that Baeksuo has no side effects due to estrogen activity such as improvement of degenerative arthritis, breast cancer, and endometrial cancer, as well as the mechanism for its efficacy.

On the other hand, both white succulents and bifoliar papillae are perennial plants belonging to the genus Cynanchum of the family Asclepiadaceae. Although they are related, there is a difference in the species. Currently, in the domestic herbal medicine distribution market, Hasuo is divided into Jeokhasuo (Juksuo) and Baekhasuo (Baeksuo) and treated the same. A completely different plant that is not listed in the Standards of Herbal Medicines, bifoliar Upiso, is being distributed as Baeksuo. Sauerkraut, also known as Jeokhasuo, is reddish-brown in color and has a starfish-shaped morphological feature when sliced, so it is distinguished from white rhododendron. It is very difficult to distinguish visually. In addition, species identification and classification according to morphological characteristics are prone to variables depending on environmental factors, and species identification through classification according to morphological characteristics requires a lot of time, effort, and cost, and requires a lot of experience and expertise. It is only possible, limitedly, by researchers. However, recent developments in biotechnology have made it possible to study biodiversity at the DNA level, enabling rapid and accurate species identification through nucleic acid fingerprint analysis.

Patent registration KR 10-1821040, a prior art, discloses a marker based on the matK gene of chloroplasts and the InDel region of the trnL-trnF region. In addition, patent registration KR 10-1866163 discloses an InDel marker discovered by analyzing the chloroplast genomes of Baeksuo and bilepipizo. In addition, Korean Patent Publication No. KR 10-2017-0024388 provides a discriminative marker for Hausuo, Baeksuo, and Bileaf cows using Single Nucleotide Polymorphism.

However, the markers based on the InDel region derived from the chloroplast gene had a limitation in that they could not distinguish hybrid plants generated by pollen due to the marker characteristics based on the chloroplast gene inherited maternally.

In addition, prior techniques based on the chloroplast gene for species discrimination of Baeksuo and bilobite are based on the InDel site for a site with high variability in the intron portion of the chloroplast. There were difficulties, and in the case of a marker using a single nucleotide polymorphism, the intuitiveness of analysis was lower than that of the InDel marker.

Accordingly, the present inventors made intensive research efforts to develop a species-specific marker capable of distinguishing between white and bifoliate right ivy, and as a result, created a standard genome of a plant of the genus White rice genus, and used this as an individual After comparing and analyzing the single sequence genomic information analyzed in , the SNP sites present on the autosomes of Baeksuo and bilepidus were obtained, and then the 9 Indel sites present on the autosomes of Baeksuo and bileaf cows were again identified. By cross-checking the SNP and the Indel region, the present invention was completed by revealing that it was possible to easily distinguish between the white succulents and the bilepidus using primers prepared based on the same Indel sequence.

Accordingly, the main object of the present invention is to provide a primer set based on the Indel marker for species discrimination of white succulents and bileaf cows.

Another object of the present invention is to provide a method for discriminating between white and bileaf cows by using a primer set based on the Indel marker for species discrimination of white and bileaf cows.

Another object of the present invention is to provide a kit for discriminating between white and bile pisces, which includes a primer set based on the Indel marker for species discrimination of white rhododendron and bileaf cows.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a primer set represented by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2; a primer set represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4; a primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6; a primer set represented by the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8; a primer set represented by the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10; a primer set represented by the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12; a primer set represented by the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14; a primer set represented by the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16; And SEQ ID NO: 17 and a primer set represented by the nucleotide sequence of SEQ ID NO: 18; Cynanchum wilfordii including one or more primer sets selected from the group consisting of and providing a primer set for discrimination do.

The present inventors prepared a standard genome of a plant of the genus P. genus, and compared and analyzed it with the single sequence genome information analyzed in individuals such as white rhododendron, bifoliate right oxiform, and the like, and analyzed the gene present on the autosomal of baeksuo and bifoliate right genus. After securing the SNP site, nine indel sites existing on the autosomes of Baeksuo and Leeoppizo were again discovered, and the SNP and Indel sites were cross-checked using primers prepared based on the same Indel sequence. Indel markers that can easily distinguish between white and bileaf cows have been discovered.

The gene marker for discriminating Cynanchum wilfordii and Cynanchum auriculatum of the present invention is complementary to the corresponding gene present in the autosomes of Cynanchum wilfordii and Cynanchum auriculatum. Since it is combinable, it can be used as a marker or a marker composition for species discrimination of white succulents and bileaf cows by itself.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and copy the template. It refers to a short nucleic acid sequence that serves as a starting point for The length and sequence of the primer should allow synthesis of the extension product to begin, and the specific length and sequence of the primer will depend on the complexity of the DNA or RNA target required, as well as the conditions of use such as temperature and ionic strength. something to do. As a specific example, the specific length and sequence of the primer should be determined in consideration of various conditions such as guanine and cytosine contents (GC contents), GC alignment, annealing temperature, and ionic strength. Primers are capable of initiating DNA synthesis in the presence of four different nucleoside triphosphates (dNTPs) and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature.

In the present invention, "forward primer" and "reverse primer" bind to the 3'-end and 5'-end of a specific region of a gene to be amplified by a gene amplification reaction, respectively, as a starting point of DNA synthesis. It means a primer that works.

In the present invention, "marker" is a substance that can analyze the species specificity of plants of the genus White rice, and nucleic acids (eg, DNA, mRNA, etc.) related to species identification of Cynanchum wilfordii and Cynanchum auriculatum; organic biomolecules and the like.

In the primer set for discriminating Cynanchum wilfordii and Cynanchum auriculatum of the present invention, the PCR amplification product of Baeksuo species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 is The PCR amplification product of Baeksuo species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 is 350 bp, and the primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 is 321 bp. The PCR amplification product of the amplified Baeksuo species is 333 bp, and the PCR amplified product of the Baeksuo species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8 is 303 bp, the SEQ ID NO: 9 and SEQ ID NO: The PCR amplification product of the Baeksuo species amplified with the primer set represented by the nucleotide sequence of 10 is 339bp, and the PCR amplified product of the Baeksuo species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12 is 305 bp, and the PCR amplification product of Baeksuo species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14 is 301 bp, and the primer set represented by the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16 The PCR amplification product of the amplified Baeksuo species is 357bp, and the PCR amplified product of the Baeksuo species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NO: 18 is 350 bp.

According to another aspect of the present invention, the present invention provides a kit for discriminating Cynanchum wilfordii and Cynanchum auriculatum comprising a primer set for discriminating Cynanchum wilfordii and Cynanchum auriculatum. to provide.

The "kit" of the present invention includes genomic DNA derived from a sample to be analyzed, the primer set of the present invention, an appropriate amount of a DNA polymerase (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermisflavus, Thermococcus literalis or thermostable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), dNTP mixture, PCR buffer and water. The PCR buffer may contain KCl, Tris-HCl and MgCl ₂ . In addition, components necessary for performing electrophoresis that can confirm whether the PCR product is amplified may be additionally included in the kit of the present invention. At this time, the MgCl ₂ concentration greatly affects the specificity and quantity of amplification. In general, when the Mg ²⁺ ion is excessive, the non-specific PCR amplification product increases, and when the Mg ²⁺ ion is insufficient, the yield of the PCR product decreases. The PCR buffer may further contain an appropriate amount of BSA.

* According to another aspect of the present invention, the present invention comprises the steps of (a) isolating DNA from a target baeksuo (Cynanchum wilfordii) sample; (b) a primer set using the DNA isolated in step (a) as a template and represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2; a primer set represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4; a primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6; a primer set represented by the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8; a primer set represented by the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10; a primer set represented by the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12; a primer set represented by the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14; a primer set represented by the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16; and a primer set represented by the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18; amplifying a target sequence using one or more primer sets selected from the group consisting of; and (c) the PCR amplification product amplified by the primer set represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 is 321 bp, and PCR amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 The amplification product is 350 bp, the PCR amplification product amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 is 333 bp, and amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 The PCR amplification product obtained is 303 bp, the PCR amplification product amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 is 339 bp, and the primer set represented by the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12 The PCR amplification product amplified with 305 bp, the PCR amplification product amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14 is 301 bp, and the nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 16 When the PCR amplification product amplified with the primer set is 357 bp, and the PCR amplification product amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18 is 350 bp, determining the species as Cynanchum wilfordii; It provides a method for identifying a species of Cynanchum wilfordii, including a.

In the example of the present invention, when the gene sequence was amplified with the nine primer sets of the present invention, it was confirmed that the sequences amplified in the white succulents and the bileaf cows were clearly differentiated, and the bands specific to the beaksuo species were amplified.

Therefore, it is possible to easily discriminate between white and bifoliate right ivy by using the gene marker for species discrimination of white and bifoliate right ivy based on the gene sequence of the Indel region specific to plants of the genus P. Through the difference, it was confirmed that it was possible to easily distinguish between the plants of other genus of white rice and white succulents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a primer set for species discrimination of white-leaved buffalo and bi-leafed buffalo, a kit composition comprising the same, and a method of discriminating between white-leaved buffalo and bileaf cows using the same.

(b) In the case of using the Indel marker for species discrimination of White-leafed right ivy and the primer amplifying the markers of the present invention, it is possible to easily distinguish White-leafed from other plants in the genus of White rice, such as bifoliate rhododendron, and more quickly and accurately. And it is possible to classify the species of Baeksuo in an economical way.

1 is a graph showing the analysis of the SNP variation amount of plants of the genus White rice (Class Ⅰ: baeksuo, Class Ⅱ: baeksuo showing some bifoliar right phenotype, Class Ⅲ: intersecting rhododendron, Class IV: bifoliated rhizome, Class V : Two-leaf Upizo whose genotype is Baeksuo).
FIG. 2 is a schematic diagram showing the results of classification through PCA analysis of genotypes of plants of the genus P. genus, including white succulents and bileaf phyllosa.
3 is a schematic diagram showing the number of SNPs filtered by cause in the SNP chip production process for plants of the genus White rice.
4 is a matrix of 113 loci selected as a result of selecting loci having the same genotype and homozygous in a bilobite individual belonging to Group IV among loci for which indel sites have not been discovered in all of the Baeksuo individuals belonging to Group I. This is an example.
5 is a PCR analysis of the DNA of Baeksuo and Leeyeoppiso with the primer sets of SEQ ID NOs: 1 and 2, the primer sets of SEQ ID NOs: 3 and 4, and the primer sets of SEQ ID NOs: 5 and 6 of the present invention. It's a photo.
6 is a PCR analysis of the DNA of Baeksuo and Leeyeoppiso with the primer sets of SEQ ID NOs: 7 and 8, the primer sets of SEQ ID NOs: 9 and 10, and the primer sets of SEQ ID NOs: 11 and 12 of the present invention. It's a photo.
7 is a PCR analysis of the DNA of Baeksuo and Leeyeoppiso with the primer sets of SEQ ID NOs: 13 and 14, the primer sets of SEQ ID NOs: 15 and 16, and the primer sets of SEQ ID NOs: 17 and 18 of the present invention. It's a photo.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

Example 1. Determination of standard genome sequencing of baeksuo and bileaf cows

1-1. PromethION read production

In order to analyze the full-length genome of two types of plants, Cynanchum wilfordii and Cynanchum auriculatum , which are two types of plants in the genus of White rice, the full-length genome sequences of the white and bifoliate Uffizi were obtained using Oxford Nanopore (ONT)'s promethION platform. deciphered. In the decoding process, the promethION platform was used twice to produce an average long sequence of 5Kb or more in the genomes of Baeksuo and Leeoppizo. The total read length, average read length, and longest read length are summarized in [Table 1] below. The average 'quality score (Q=-10log ₁₀ P)' of the analyzed sequences was all 11, which was analyzed to have an accuracy of 90% or more.

[Table 1]

1-2. Manufacture of standard genome assembly of 2 types of plants of the genus White rice

The base calling of the long sequence was performed using the Minknow program and the Filp-Flop program (Oxford Nanopore Technologies, UK), and the quality of the sequences determined by the NanoPlop program (Oxford Nanopore Technologies, UK) was confirmed. did. In addition, adapter sequences were removed with the Porechop program (Oxford Nanopore Technologies, UK).

In addition, the Wtdbg2 (V2.4) program (Oxford Nanopore Technologies, UK) was used to assemble the genomes of white succulents and bileaf genus, which are plants of the genus White Rice, and the Racon (v.1.4.3) program (Oxford Nanopore Technologies, UK). ), error correction of the long sequence was performed 4 times. Then, error correction was additionally performed using the r941_prom_high model of the Medaka (v.0.8.1) program (Oxford Nanopore Technologies, UK). Finally, polishing was performed using the Pilon program and short-read to generate a final assembly.

As a result of the assembly, Suo Baek assembled a 229 Mbp-length full-length genome from a total of 6,365 contigs, and a total of 5,524 contigs, and a 250-Mbp-length full-length genome, assembled the two-leaf Woopiso.

[Table 2]

The results of the full-length genome analysis of the prepared baeksuo and bileaf cows were used to develop markers for distinguishing between the whitefly and bileaf cows.

Example 2. Selection and sequencing of samples from Baeksuo and bileaf cows

In order to produce a SNP chip for species differentiation of white rice genus genetic resources, including two types of plants of white succulents and bifoliar hyacinths, genetic resources of baeksuo and bileaf pisces were donated from Chungbuk Agricultural Research and Extension Services. For the analysis of mutants (the sum of the standard genome and other nucleotide bases) of Baeksuo and bilobite, the Baeksuo genome was used using the “Next generation sequencing (NGS)” method developed by Illumina. The sequence was translated 30 to 50 times, and the translation results are summarized in [Table 3] below.

[Table 3]

Example 3. Single sequence alignment and SNP analysis of plants of the genus White rice

Variant analysis to identify single nucleotide variation (SNV) mutations was performed as follows by comparing the single sequence genome information of individuals in the genus White rice with the Baeksuo standard genome sequence of about 300 Mbp length.

First, the single sequence produced by 16 plants of the genus White rose was aligned with the 229 Mbp-long Baeksuo standard genome sequence using the BWA-MEM (ver. 0.7.17) program, and the Picard (ver. 2.9.2) program By using the MarkDuplicate tool, duplicated reads were masked in the course of the experiment. In order to correct the alignment of the indel region, the realignerTarget Creator (Category Sequence Data Processing Tools) module and the IndelRealigner module of GATK (Genome Analysis Tool Kit, ver. 3.8, Broad Institute) were used.

After removing quality score 30 or lower using the mappingQScoreFilter module of the Samtools (ver. 1.9) program, SNV (Single nucleotide variation) for each individual was predicted using the Unifiedgenotyper module of GATK (ver. 3.8). . In the following [Table 4], Group Ⅰ is Baeksuo, Group Ⅱ is Baeksuoh showing some phenotype of bilobed right genotype, Group Ⅲ is interstitial right phylum, Group Ⅳ is bilobed genotype, and Group V is the actual bilobed genotype. It is divided into two-leaf right wing which appears as white or white swan.

As a result of the prediction, the average sequence mapping ratio of plants of the genus White rice was 40.58%.

[Table 4]

Specifically, the mapping ratio between six Baeksuo cultivars (CBM0134, CBM0134-2, CBM0144, CBM0103, CBM0110, CBM0111, CBM0221) and 3 bileaf cow cultivars (CBM0212, CBM0212-2, CBM0212-3) rate), there was no difference. In addition, the aligned reads were found to cover 72.34% of the total assembled baeksuo standard genome sequence. In addition, the SNV (Single nucleotide variation) of the Baeksoo individual identified at the Chungbuk Agricultural Research and Extension Services was found in about 0.61% of the assembled Baeksuo genome sequence at about 1.4 Mbp, and the SNV of the bileaf cowpea individual was about 1.4 Mbp. It was discovered in about 1.61% of the Baeksuo genome assembled at 3.7Mbp.

Therefore, it was confirmed that the mutant of the bileaf cowhide compared to the Baeksuo standard genome was 2.6 times higher than the mutant between the Baeksuo individuals. It was confirmed that it appeared similar to (see FIG. 1).

As a result of analyzing the heterozygosity of each individual, Suo Baek's CBM0134 individual used for the reference assembly showed the highest percentage of heterozygous SNV, and the CBM0111 individual had the next highest rate of 5.47. appear. On the other hand, the ratio of heterozygous SNV in bilobed cows was 0.88 to 1.69, which was relatively low and similar.

Example 4. Development of SNP markers for identification of Baeksuo species

In order to develop a SNP marker for species differentiation between Baeksuo and bilepipizo, the SNPs identified in Example 3 were merged for each locus and a matrix was prepared. As a result, SNPs were discovered in a total of 8,508,402 loci. .

Based on the SNP of 8.5 Mbp, the genotypes of plants of the genus P. genus, including white succulents and bileaf pifolius, were classified through PCA analysis. As a result, in the case of a bileaf right ivy (Group II) showing a part of the bifoliate right oxiform phenotype or a bileaf winged hyacinth (Group V) having the genotype of the baeksuo genotype, either the baeksuo (Group I) or the bifoliated woopizo (Group I). IV), it was confirmed that they were genetically separated from each other. In particular, it could be confirmed that the individual belonging to Group V was located between Baek Soo-oh and Lee-yeop Upiso on the PCA (principal components analysis) graph (see FIG. 2).

In order to manufacture the SNP Chip for species identification, the following three factors that can hinder the SNP Chip production efficiency were first selected and filtered.

1) When another SNP appears within the 200 nucleotide sequence in both directions of the target SNP

2) When the target SNP and the bidirectional 200 base sequence overlap with Baek Soo-oh's repeat sequence

3) When an insertion/deletion mutation (insertion/deletion, InDel variant) is identified within the target SNP and the bidirectional 200 nucleotide sequence

In the case of the above three factors, it was filtered because it could cause errors in primer mapping and genotyping, and as a result of the filtering, 7,865 loci corresponding to 0.92% of the total locus SNP Chip with high sensitivity was determined to be possible to produce (see FIG. 3).

Next, only those loci that are homozygous with the locus types of the six specimens of Baeksu-oh being identical to the genome sequence assembled into the standard genome, and the genotypes of the three individuals of the bilobed right genus being identical to the discovered SNP locus. further selected. As a result, 319 loci corresponding to about 4% were selected.

In order to make the region with high SNP prediction accuracy into the SNP chip, 182 (57.10%) SNPs were removed as a result of removing the regions where the read depths used for SNP analysis were all 10 or less in Baeksuo and Leeyeopwoopiso. A locus region was secured, and as a result of removing a region having a read depth of 100 or more, a total of 143 SNP locus regions were secured.

Example 5. In-Del marker analysis of Baeksuo and Leeoppizo

GATK (Genome Analysis Tool Kit, ver. 3.8, Broad Institute) to find an indel (insertion and deletion) site on the autosomal of Baeksuo and Leeoppiso from the mapping result of the next-generation nucleotide sequence of Example 2 ) was analyzed using the Unifiedgenotyper module. As a result of comparing the indel regions of a total of 16 plants of the genus White rice with the standard genome of CBM0134, the number of indel sites excavated for each individual was from 130,213 to 666,406.

[Table 5]

The average amount of indel variation (274,707) of Baeksuo individuals belonging to Group I was lower than the average amount of indel variation (504,363) of bileaf cows belonging to Group IV.

As a result of merging the indel loci of the white rice mutant of Table 5 above to create a matrix, insertions/deletions of 1 bp or more were predicted in a total of 3,230,257 regions. The matrix prepared in this way was later used as basic data to elucidate the genetic difference between Baek Su-oh and Lee-yeop Woopiso.

Example 6. Selection of In-Del markers of Suo Baek and Lee Yeop Piso

In order to discriminate between Baeksuo and bilobi, all loci with a read depth of 10 or less were removed from the entire indel mutant matrix to secure only a region with a high indel prediction rate. Afterwards, from among the loci for which indel sites were not discovered in all of the Baeksuo individuals belonging to Group I, only 113 loci were secured by selecting loci having the same genotype and homozygous in the bilobite pisophysis belonging to Group IV (Fig. 4). . Referring to FIG. 4 , the blue background is Group I to which Baeksuo individuals belong, and the green background is Group IV to which the bifoliate right genus belongs. The remaining groups are divided into hybrids of Baeksuo and Leeopyupiso.

Among the 113 loci selected as described above, the portion overlapping with the repeat sequence was removed, and 9 sets of PCR amplification primers were prepared from the 9 Indel regions using the primer 3 program (Table 6).

[Table 6]

Example 7. Demonstration of In-Del Markers of Su-oh Baek and Lee-yeop Piso

In order to find out whether Baeksuo species can be discriminated using the Indel marker of the mitochondrial genome selected in Example 6, the Baeksuo genomic DNA was isolated and the Indel marker-based primers of [Table 6] PCR was performed using the set.

Specifically, for genomic DNA isolation for genotyping, white sorghum was pulverized and then frozen and powdered by treatment with liquid nitrogen. 700 μl of microprep buffer was added to the Baeksuo powder, and incubated at 65° C. for 15 minutes, followed by mixing at intervals of 5 minutes. Then, 700 μl of chloroform:isoamyl alcohol (chloroform:isoamylalcohol = 24:1) was added, mixed for 1 minute, and centrifuged at 11,000 rpm for 15 minutes to obtain a supernatant. About 60% of the volume of isopropanol was added to the separated supernatant and mixed until the DNA was condensed, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was removed to obtain a DNA pellet. After washing and drying the obtained DNA pellet with 70% ethanol, 50 μl of 1xTE buffer containing 50 mg/L of RNaseA was added and placed at 37° C. for 120 minutes to remove the RNA contained in the DNA pellet to obtain Baek Su-oh’s DNA. obtained. Thereafter, the concentration of the Baeksuo DNA was measured and diluted to about 100ng/μl to be used as a template for the amplification reaction.

Then, PCR was performed using the 9 sets of PCR amplification primer sets in Example 6.

For the comparative experiment, bileaf cows extracted using the same method as the DNA extraction method were used as a control.

For PCR, a PCR reaction was constructed with the reaction composition shown in [Table 7] below, and PCR was performed under the conditions of [Table 8] to obtain an amplified marker product.

[Table 7]

[Table 8]

Then, the amplified Indel marker product was loaded on a 2.5% agarose gel and electrophoresed at 100V for 50-60 minutes to confirm the amplification band.

As a result, as shown in FIGS. 4 to 6, when amplifying using the primer set of [Table 6] prepared to amplify the Indel marker discovered in Example 6, the primer set of SEQ ID NOs: 1 and 2 In this case, it was confirmed that a specific band of 321 bp in length and 298 bp in bileaf cows was amplified, and in the case of primer sets of SEQ ID NOs: 3 and 4, specific bands of 350 bp in Baeksoo and 312bp in length were amplified. It was confirmed that, in the case of the primer sets of SEQ ID NOs: 5 and 6, it was confirmed that a specific band with a length of 333 bp for Baek Soo Oh and 311 bp for Biyeop Upiso was amplified. It was confirmed that a specific band with a length of 274 bp was amplified for Piso, and in the case of the primer sets of SEQ ID NOs: 9 and 10, it was confirmed that a specific band with a length of 339 bp for Baek Su-o and 312 bp for Bi-yeop Woo Piso was amplified, SEQ ID NO: 11 and In the case of the primer set of 12, it was confirmed that a specific band of 305 bp in length and 274 bp in the bileaf right ivy was amplified. It was confirmed that the band was amplified, and in the case of the primer sets of SEQ ID NOs: 15 and 16, it was confirmed that a specific band with a length of 357 bp for Baeksoo and 306 bp for bileaf cows was amplified, and in the case of primer sets of SEQ ID NOs: 17 and 18, it was confirmed that It was confirmed that a specific band with a length of 350 bp and 315 bp in the bileaf piso was amplified.

Therefore, it is possible to easily discriminate between the genus genus and the genus of the genus by using the gene marker for identification of the species of genus White rhododendron based on the gene sequence of the genomic Indel region of the plant of the genus White rhododendron of the present invention. It was confirmed that it was possible to easily distinguish between plants and white succulents.

In addition, the markers based on the InDel region of the genome of plants of the genus P. genus are different from markers based on chloroplast genes that are inherited maternally, in hybrid plants [in the present invention, Baeksuo showing some phenotype of bifoliar rhizomes generated by pollen]. (Group II) and bileaf cow (Group V) having the genotype of Baeksuo] was confirmed to be fundamentally distinguishable.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Semyung University Division of Industry-Academy Cooperation <120> InDel Markers for Discrimination of Cynanchum wilfordii and Cynanchum auriculatum and Method for Use thereof <130> PN190082AN <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 1 gccggtggct ggttattaac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 2 aggcatcggc atgttctcaa 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 3 tctacgatat caccagcacg g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 4 cagtcacatg ggagcttggt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 5 caccagtgac acctgcagaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 6 aggctgttcc attgctaggc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 7 cggccacacc tttgacaaag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 8 tccttccgcc gcacattatt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 9 tgctgccaca ggagtttgta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 10 ctcagatcca ttgcctccccg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 11 tgcatgaggg agaagttgga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 12 ccagccacag ccagagttaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 13 cccttgagag cacccgaatt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 14 tctcaggcga gggttatgga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 15 tccagggtcc tccttccaat 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 16 aaaaacacca ccggaccagt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer Sequence <400> 17 gggtacgggt ctggcattag 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer Sequence <400> 18 aagaaggggg gtgtgagagt 20

Claims (4)

A primer set composition for discriminating between Cynanchum wilfordii and Cynanchum auriculatum consisting of a primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6. According to claim 1,
The PCR amplification product of the Baeksuo species amplified by the primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 is Cynanchum wilfordii and Cynanchum auriculatum, characterized in that it is a primer for discrimination set. A kit for discriminating between Cynanchum wilfordii and Cynanchum auriculatum comprising a primer set for discriminating between Cynanchum wilfordii and Cynanchum auriculatum of claim 1. (a) isolating the DNA from the target baeksuo (Cynanchum wilfordii) sample;
(b) using the DNA isolated in step (a) as a template and amplifying a target sequence using a primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6; and
(C) when the PCR amplification product amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 is 333 bp, determining the species as Cynanchum wilfordii; Species identification method.

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