KR20210061502A - 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 Baek Soo-Oh and Lee Leaf Cattle, and a Discrimination Method Using the Same {InDel Markers for Discrimination of Cynanchum wilfordii and Cynanchum auriculatum and Method for Use thereof}

The present invention relates to a marker for discriminating Baek Soo and two leaf cows, and a method for discrimination using the same, and more specifically, to an InDel marker for discriminating Baek Soo and two leaf cows, and a method for discriminating Baek Soo and two leaf cows using the same.

The Nagoya Protocol, announced in 2010, is an international agreement to share the benefits of utilizing biological resources, and the agreement is in effect in Korea from August 17, 2017. Korea is preparing for the Nagoya Protocol by discovering more than 100,000 species of domestic biological genetic resources and creating a database for the use of resources, centering on the National Institute of Biological Resources and the National Institute of Ecology established in 2012.

As the “Nagoya Protocol” comes into force, protecting and preserving our bio-sovereignty is emerging as a very important issue. Regarding the preservation of our biological resources, the bulbous tree, the representative plant of Mt. Jirisan and Mt. Halla, was exported to the West in 1904 and widely used as a Christmas tree. It was exported to the United States in 1947 and is called'Miss Kim Lilac' and has a high garden tree. The popular clove tree and the Korean native wheat, known as sorona, are all biological resources of Korea that have been exported overseas.

Therefore, various efforts are required to prevent the unauthorized export of Korean endemic organisms and secure biological resources. In particular, in the case of endemic species such as Baeksuo, which prepares for the Nagoya Protocol and has high economic added value, the development of a species-specific marker for securing biological sovereignty is essential.

On the other hand, Cynanchum wilfordii is a vine-like perennial plant in the white rice flower that grows on sunny meadows and beach slopes in the mountains and fields of Korea and China. . Baek Soo-o is characterized as a medicinal herb in many oriental medicine books such as Donguibogam and Donguisusebowon, but it is widely used as a health functional food because it is known to be non-toxic and good for menopausal women. Recently, it has been scientifically proven that Baek Su-Oh does not have side effects due to estrogen activity such as improvement of degenerative arthritis, breast cancer, and endometrial cancer, as well as mechanisms for its efficacy.

On the other hand, Baekshuo and dileaf cattle are both tendril perennial plants belonging to the genus Cynanchum of the Asclepiadaceae family.In Korea, Baekshuo roots and Baekshui cattle roots in China are used for medicinal purposes, respectively, and belong to the same white rice flower genus. They are relatives, but they differ in species. Currently, in the Korean herbal medicine distribution market, sewage is divided into Jeokshou (Jeokshou) and Baekshou (Baekshou) and treated the same.In particular, Baekshou is erroneously known as an improved species of Baekshou imported from China. A completely different plant, which is not listed in the herbal medicine standard, is distributed as Baeksuo. Hasuo, known as Jeoksu-o (Jeok-su-o), has a reddish-brown color and is distinguished from white-colored Baek-su-o (Baek-su-o) because it has a starfish-shaped morphology when sliced. However, in the case of Lee-yeop cattle, the shape and color are the same as those of Baek-su-o. It is very difficult to distinguish with the naked eye. In addition, species identification and classification according to morphological characteristics tends to generate variables according to environmental factors, and species identification through classification according to morphological characteristics requires a lot of time, effort, and cost, and has a lot of experience and expertise. Limited only by researchers. However, recent developments in biotechnology have made it possible to study the diversity of biological species at the DNA level, enabling rapid and accurate species identification through nucleic acid fingerprint analysis.

Patent Registration KR No. 10-1821040, which is a prior art, discloses a marker based on the matK gene of the chloroplast and the InDel region of the trnL-trnF region. In addition, Patent Registration KR No. 10-1866163 has disclosed InDel markers discovered by analyzing the chloroplast genome of Baek Su-Oh and Lee Yeop Soo. In addition, Korean Patent Application Publication No. KR 10-2017-0024388 provides discriminant markers of Ha Su-oh, Baek Su-oh, and Lee Yeop cattle using Single Nucleotide Polymorphism.

However, the markers based on the InDel region derived from the chloroplast gene had a limitation in that it was not possible to distinguish hybrid plants caused by pollen due to the marker characteristics based on the maternal chloroplast gene.

In addition, conventional techniques based on the gene of the chloroplast to determine the species of Baeksu-Oh and Epiphytes have been used to determine if there is a mutation in the corresponding region based on the InDel region based on the region containing the high variability of the intron part of the chloroplast. There was a difficulty, and in the case of markers using Single Nucleotide Polymorphism, there was a disadvantage that the analysis was less intuitive than InDel markers.

Accordingly, the present inventors have made extensive research efforts to develop species-specific markers that can distinguish between Baeksuoh and Leeup cattle, and as a result of preparing the standard genome of the plant in the white rice flower, this resulted in individuals such as Baeksuoh, Leeyeopso, and Jeolgwan cattle. After comparing and analyzing the genome information of the single sequence analyzed in, SNP sites present on the autosomal of Baeksu-oh and Leeyeop cattle were secured, and then 9 Indel sites present on the autosomal of Baeksu-oh and Leeyeop cattle were again analyzed. The present invention was completed by discovering that the SNP and Indel sites were cross-checked to reveal that it was possible to easily distinguish between Baek Shou and Iyeop cattle using primers prepared based on the Indel sequence.

Accordingly, the main object of the present invention is to provide a primer set based on Indel markers for species discrimination of Baek Shou and Leeyeop.

Another object of the present invention is to provide a method of discriminating between Baekshou and Leeup cattle by using a primer set based on Indel markers for species identification of Baekshou and Leeupe cattle.

Another object of the present invention is to provide a kit for discriminating Baekshou and Iyeop cattle, including a primer set based on an Indel marker for determining the species of Baek Shou and Iyeop cattle.

Other objects and advantages of the present invention will become more apparent by the following detailed description, 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 sequence of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set represented by the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set represented by the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set represented by the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10; A primer set represented by the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12; A primer set represented by the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14; A primer set represented by the nucleotide sequence 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; Provides a primer set for discriminating Cynanchum wilfordii and Cynanchum auriculatum including one or more primer sets selected from the group consisting of do.

The present inventors prepared a standard genome of a plant in the genus White Rice, and compared and analyzed it with the single sequence genome information analyzed in individuals such as Baek Soo-O, Lee Leaf Cow, and Jeol-Gwan Cow Cow. After securing the SNP site, nine Indel sites present on the autosomes of Baeksu-oh and Iyeoppiso were again discovered, and the SNP and Indel sites were cross-checked and primers prepared based on the same Indel sequence were used. Indel markers that can easily distinguish between Baeksu-oh and Lee-yeop cattle were discovered.

The genetic markers for discriminating the Baek Su-o (Cynanchum wilfordii) and the Cynanchum auriculatum of the present invention are complementary to the corresponding genes present in the autosomal of the Baek Su-O (Cynanchum wilfordii) and the Cynanchum auriculatum. Since it can be combined, it can be used as a marker or a marker composition for discriminating the species of Baekshou and Leeyeoppiso by themselves.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3'terminal hydroxyl group, which can form a complementary template and a base pair, and copy the template. It refers to a short nucleic acid sequence that functions as a starting point for. The length and sequence of the primers should allow the synthesis of the extension product to start, and the specific length and sequence of the primers depend on the conditions of use such as temperature and ionic strength, as well as the complexity of the required DNA or RNA target. 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 content (GC contents), GC sequence, annealing temperature and ionic strength. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates (dNTPs) at an appropriate buffer and temperature.

In the present invention, the "forward primer" and the "reverse primer" are each bound to the 3'-end and the 5'-end of a specific site of a gene amplified by a gene amplification reaction to serve as the starting point of DNA synthesis. It refers to a primer that works.

In the present invention, the term "marker" is a substance capable of analyzing the species specificity of plants in the genus white rice, and nucleic acids (eg, DNA, mRNA, etc.) related to the species identification of Baeksu-o (Cynanchum wilfordii) and Cynanchum auriculatum, Organic biomolecules, such as, and the like.

In the primer set for discrimination of Baekshou (Cynanchum wilfordii) and Cynanchum auriculatum of the present invention, the PCR amplification product of Baekshou species amplified with a primer set represented by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 321 bp, the PCR amplification product of Baek Shou 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 The amplified PCR amplification product of Baekshou species is 333bp, and the PCR amplification product of Baekshou species amplified with a primer set represented by the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 is 303bp, and SEQ ID NO: 9 and SEQ ID NO: The PCR amplification product of Baekshou species amplified with the primer set represented by the nucleotide sequence of 10 is 339 bp, and the PCR amplification product of Baek Shou species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12 is 305bp, the PCR amplification product of Baeksuo species 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 primer set represented by the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16 The PCR amplification product of the amplified Baekshou species is 357bp, and the PCR amplification product of Baekshou species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NO: 18 is 350bp.

According to another aspect of the present invention, the present invention includes a kit for discriminating Cynanchum wilfordii and Cynanchum auriculatum, including a primer set for discriminating Baek Soo Oh (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 DNA polymerase (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermisflavus, Thermostable DNA polymerase obtained from Thermococcus literalis or Pyrococcus furiosus (Pfu)), dNTP mixture, PCR buffer and water. The PCR buffer solution may contain KCl, Tris-HCl and MgCl ₂ . In addition, constituents necessary for electrophoresis that can confirm whether a PCR product is amplified may be additionally included in the kit of the present invention. At this time, the _{concentration of MgCl 2} greatly affects the specificity and quantity of amplification. In general, when Mg ^{2 +} ions are excessive, non-specific PCR amplification products increase, and when Mg ^{2 +} ions are insufficient, the yield of PCR products decreases. An appropriate amount of BSA may be additionally included in the PCR buffer solution.

According to another aspect of the present invention, the present invention comprises the steps of: (a) separating DNA from a sample of Baek Suo (Cynanchum wilfordii); (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 sequence of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set represented by the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set represented by the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set represented by the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10; A primer set represented by the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12; A primer set represented by the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14; A primer set represented by the nucleotide sequence 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 the target sequence using one or more primer sets selected from the group consisting of; And (c) a PCR amplified product amplified with a primer set represented by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 is 321 bp, and a PCR amplified with a primer set represented by the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4 The amplification product is 350 bp, and 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 resulting PCR amplification product is 303 bp, and the amplified PCR amplification product is 339 bp with a primer set represented by the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10, and the primer set represented by the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12 The PCR amplification product amplified by is 305 bp, and the PCR amplification product amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14 is 301 bp, and is represented by the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16. If 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 sequences of SEQ ID NO: 17 and SEQ ID NO: 18 is 350 bp, determining that it is a species of Cynanchum wilfordii; It provides a method for determining the species Baek Shou (Cynanchum wilfordii) containing.

In the embodiment of the present invention, when amplifying the gene sequence with the 9 kinds of primer sets of the present invention, it was confirmed that the sequence amplified in Baekshou and Yiyeop cattle were clearly distinguished, so that Baekshou species-specific bands were amplified.

Therefore, it is possible to easily discriminate between Baekshou and Baekshui cattle, based on the gene sequence of the Indel region specific to the plant in the genus Baekshui, and the length of the PCR amplification product. Through the difference, it was confirmed that it was possible to easily distinguish between the plants of the white rice flower and the white water.

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

(a) The present invention provides a primer set for species identification of Baekshou and Leeyeop cattle, a kit composition including the same, and a method for determining Baekshou and Leeyeop cattle using the same.

(b) In the case of using the Indel marker for species identification of Baekshou and difoliate cattle of the present invention and a primer to amplify the marker, Baekshou may be easily distinguished from other plants in the white rice flower such as Baekshui cattle and more quickly and accurately. It is possible to classify the species of Baekshou in an economical way.

1 is a graph showing the analysis of SNP mutations of plants in the white rice flower (Class Ⅰ: Baek Soo-oh, Class Ⅱ: Baek Soo-oh showing the phenotype of some leaf cows, Class Ⅲ: Bestowed cow, Class Ⅳ: Lee leaf cow, Class V : The genotype is Baeksuoh, Leeyeopwoofiso).
Figure 2 is a schematic diagram showing the result of classification through PCA analysis of the genotype of plants in the genus White rice flower, including Baeksu-oh and Epiphyllum.
3 is a schematic diagram showing the number of SNPs filtered by cause in the process of producing SNP chips for plants in white rice flowers.
FIG. 4 shows a matrix for 113 loci selected as a result of selecting a locus homozygous in a bilobed right cow subject belonging to Group IV and having the same genotype among loci for which indel sites were not found in all of the Baek Shou subjects belonging to Group I. FIG. This is an example.
Figure 5 is a photograph of the results of PCR analysis of the DNA of Baek Shou and Leeyeop cattle with a primer set of SEQ ID NOs: 1 and 2, a primer set of SEQ ID NOs: 3 and 4, and a primer set of SEQ ID NOs: 5 and 6 of the present invention. It's a picture.
Figure 6 is a photograph of the results of PCR analysis of the DNA of Baek Shou and Leeyeop cattle with a primer set of SEQ ID NOs: 7 and 8, a primer set of SEQ ID NOs: 9 and 10, and a primer set of SEQ ID NOs: 11 and 12 of the present invention. It's a picture.
Figure 7 is a photograph of the results of PCR analysis of the DNA of Baek Shou and Leeyeop cattle with a primer set of SEQ ID NOs: 13 and 14, a primer set of SEQ ID NOs: 15 and 16, and a primer set of SEQ ID NOs: 17 and 18 of the present invention. It's a picture.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Determination of the standard genome sequence of Baek Soo-oh and Leeyeop cattle

1-1. PromethION read production

Two kinds of plants in the genus White Rice, Cynanchum wilfordii and Cynanchum auriculatum ) in order to analyze the full-length genome of Oxford Nanopore (ONT) using the promethION platform of Baek Soo-Oh and Lee Yeop Soo. In the decoding process, the promethION platform was used twice to produce a long sequence of more than an average of 5Kb in the genome of Baeksu-oh and Leeyeopso, and the total decoding 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 sequence was 11, which was analyzed to have an accuracy of 90% or more.

[Table 1]

1-2. Production of standard genome assembly of two kinds of plants in the genus White Rice

Base calling of long sequences 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. I did. In addition, the adapter (adaptor) 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 the white rice flower genus Baeksu-oh and bileaflet (Oxford Nanopore Technologies, UK), and the Racon (v.1.4.3) program (Oxford Nanopore Technologies, UK). ), error correction of the long sequence was performed 4 times. In addition, error correction was additionally performed using the r941_prom_high model of Medaka (v.0.8.1) program (Oxford Nanopore Technologies, UK). Finally, polishing was performed using the Pilon program and short-read to create a final assembly.

As a result of the assembly, Baek Soo-o assembled a 229 Mbp-long Baek Soo-Oh full-length genome from a total of 6,365 contigs, and a 250 Mbp-long full-length genome was assembled for a total of 5,524 contigs.

[Table 2]

The results of full-length genomic analysis of the prepared Baek Soo and Lee Yeop cattle were used to develop a marker for distinguishing Baek Soo and Lee Yeop cattle.

Example 2. Selection and sequencing of Baek Shou and Leeyeop cattle samples

In order to produce the SNP chip for differentiating the species of genetic resources in the genus of white rice, including two kinds of plants, Baeksuo and Leeupe cattle, genetic resources of Baeksuo and Leeupe cattle were donated from Chungbuk Agricultural Technology Institute. Baeksuo genome using the “next generation sequencing platform (NGS)” method developed by Illumina for analysis of variants of Baeksuo and dileaf cattle (the sum of nucleotide bases different from the standard genome). It was decoded in 30 to 50 times of the sequence, and the decoding results are summarized in the following [Table 3].

[Table 3]

Example 3. Sequence alignment and SNP analysis of plants of the genus White rice flower

Mutant analysis to identify single nucleotide variation (SNV) mutations was performed as follows by comparing the genome information of a single sequence of an individual in the white rice flower with a standard genome sequence of about 300 Mbp in length.

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

After removing the quality score 30 or less using the mappingQScoreFilter module of the Samtools (ver. 1.9) program, single nucleotide variation (SNV) for each individual was predicted using the Unified Genotyper module of GATK (ver. 3.8). . In the following [Table 4], Group Ⅰ is Baek Soo-oh, Group Ⅱ is Baek Soo-oh, which shows the phenotype of some two-leaf cows, Group Ⅲ is a perfect cow, Group Ⅳ is two-leaf cows, and Group V is a genotype is actually two-leaf cows. It is divided into two types of cows, such as or Baeksu-oh.

As a result of prediction, the ratio of single sequence mapping of plants in the genus White rice flower was found to be 40.58% on average.

[Table 4]

Specifically, the mapping ratio between the six Baek Shou varieties (CBM0134, CBM0134-2, CBM0144, CBM0103, CBM0110, CBM0111, CBM0221) and the three species (CBM0212, CBM0212-2, CBM0212-3) There was no difference in rate). In addition, the aligned reads were found to cover 72.34% of the entire assembled Baekshou standard genome sequence. In addition, the single nucleotide variation (SNV) of Baeksuo individuals identified by the Chungbuk Agricultural Technology Institute was found in about 0.61% of the assembled Baeksuo genome sequence at about 1.4Mbp, and the SNV of the Leeyeop cattle was about 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 cattle compared to the standard genome of Baeksuoh was found to be 2.6 times higher than that of the mutant between the Baeksuo cattle, and the SNV of the Baeksuo cattle was also about 3.6M, which is the genetic difference between the bileaflet and Baeksuoh. It was confirmed that the appearance was similar to (see FIG. 1).

As a result of analyzing the heterozygosity of each individual, Baek Suo CBM0134 used in the reference assembly showed the highest proportion of heterozygous SNV, and CBM0111 was the next highest with 5.47. appear. On the other hand, the ratio of heterozygous SNV in bileaflet cattle was relatively low and similar, 0.88 to 1.69.

Example 4. Development of SNP Marker for Identification of Baek Shou Species

In order to develop a SNP marker for differentiating the species of Baeksu-oh and bileaflete, the SNPs identified in Example 3 were merged for each locus to create a matrix.As a result, SNPs were discovered in a total of 8,508,402 loci. .

Based on the 8.5 Mbp SNP, genotypes of plants in the genus White Rice, including Baekshou and Epiphyllum, were classified through PCA analysis. As a result, in the case of Baek Soo-o (Group II) showing some phenotype of Baek Soo-Oh (Group II) or Baek Soo-Oh (Group V) having a genotype of Baek Soo-Oh, Baek Soo-O (Group I) or Di-Leoped Cow (Group) IV). In particular, it was confirmed that the individual belonging to Group V was located between Baek Su-oh and Lee Yeop Shelter on the PCA (principal components analysis) graph (see FIG. 2).

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

1) If another SNP appears within the bidirectional 200 nucleotide sequence of the target SNP

2) When the target SNP and the bidirectional 200 nucleotide sequence overlap with the repeat sequence of Baek Shou

3) When an insertion/deletion mutation (insertion/deletion, InDel variant) is identified within the target SNP and 200 nucleotide sequences in both directions

In the case of the above three factors, since it may cause an error in primer mapping and genotyping, it was filtered. As a result of filtering, SNP chip with high sensitivity at 7,865 loci, which is 0.92% of the total loci. It was determined that it could be produced (see FIG. 3).

Next, the genetic loci of 6 individuals are the same as the genome sequence assembled with the standard genome, and all the genotypes of the two lobes are identical to the discovered SNP loci, and only the loci that are homozygous. It was further screened. As a result, about 4% of 319 loci were selected.

In order to make the region with high accuracy of SNP prediction into the SNP Chip, 182 (57.10%) SNPs as a result of removing regions with all depths of 10 or less used for SNP analysis in Baek Su-Oh and Lee Yeop Shelter were removed, and as a result, 182 (57.10%) SNPs were SNPs. The locus region was secured, and as a result of removing the region having a read depth of 100 or more, a total of 143 SNP loci regions were secured.

Example 5. Analysis of In-Del Markers of Baek Soo-Oh and Lee Yeop Sopi

GATK (Genome Analysis Tool Kit, ver. 3.8, Broad Institute) to find the indel (insertion and deletion) sites on the autosomal of Baek Soo-Oh and Lee B. cow 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 sites of 16 white rice plants with the standard genome of CBM0134, the number of indel sites discovered for each individual was from 130,213 to 666,406.

[Table 5]

It was found that the average indel variance (274,707) of Baeksu-oh individuals belonging to Group I was less than the average indel variance (504,363) of bileaflet cattle belonging to Group IV.

As a result of creating a matrix by merging the indel loci of the mutant in the genus white rice in [Table 5], 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 clarify the genetic difference between Baek Su-oh and Lee Yeop cattle.

Example 6. Selection of In-Del Markers for Baek Soo-Oh and Lee Yeop Cow

In order to distinguish between Baek Soo-oh and bileaflet, all loci with a read depth of 10 or less were removed from the entire indel variant matrix to secure only regions with a high indel prediction rate. Thereafter, among the loci for which indel sites were not found in all of the Baeksu-Oh individuals belonging to Group I, only 113 loci were obtained by selecting a locus homozygous in the bilobed right cattle individuals belonging to Group IV and having the same genotype (Fig. 4). . Referring to FIG. 4, a blue background is a group I to which Baek Shou individuals belong, and a green background is a group IV to which a cow shelter belongs. The rest of the groups are divided into hybrids of Baek Su-oh and Lee Yeop cattle.

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

[Table 6]

Example 7. In-Del Marker Demonstration of Baek Soo-Oh and Lee Yeop Cow

In order to determine whether Baekshou species can be identified using the Indel marker of the mitochondrial genome selected in Example 6, genomic DNA of Baekshou was isolated and primers based on the Indel marker of [Table 6]. PCR was performed using the set.

Specifically, for separating genomic DNA for genotyping, Baekshou was pulverized and then frozen and pulverized by treatment with liquid nitrogen. 700 µl of microprep buffer was added to the white water powder, followed by incubation 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% volume of isopropanol was added to the separated supernatant and mixed until the DNA was condensed, followed by centrifugation 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 to which 50 mg/L of RNaseA was added was added and left at 37° C. for 120 minutes to remove RNA contained in the DNA pellet to remove the DNA of Baek Shou. Obtained. Thereafter, the concentration of the Baeksuo DNA was measured and diluted to about 100 ng/µl for use as a template in the amplification reaction.

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

For the comparative experiment, dileaf cattle extracted using the same method as the DNA extraction method was used as a control.

For PCR, a PCR reaction product was constructed with the reaction product composition shown in the following [Table 7], and PCR was performed under the conditions of the following [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 subjected to electrophoresis at 100V for 50-60 minutes to confirm the amplification band.

As a result, as shown in Figs. 4 to 6, in the case of amplification using the primer set of [Table 6] prepared to amplify the Indel marker discovered in Example 6, the primer sets of SEQ ID NOs: 1 and 2 In the case of Baeksuo, it was confirmed that a specific band of 321bp in length and 298bp in Baeksuo was amplified, and in the case of the primer sets of SEQ ID NOs: 3 and 4, a specific band of 350bp in Baeksuo, and 312bp in Baeksupiso was amplified. It was confirmed that, in the case of the primer sets of SEQ ID NOs: 5 and 6, a specific band of 333 bp in Baeksuo and 311 bp in Baeksuo was amplified, and in the case of the primer sets of SEQ ID NOs: 7 and 8, Baeksuo was 303bp, and Yeopwoo Lee. It was confirmed that a specific band of length 274 bp was amplified, and in the case of the primer sets of SEQ ID NOs: 9 and 10, it was confirmed that a specific band of length 339 bp and 312 bp in the case of 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 Baeksuo and 274bp in Baeksuo was amplified, and in the case of the primer sets of SEQ ID NOs: 13 and 14 Baeksuo was 301bp, and Baeksuo was 281bp in length. 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 of 357 bp in length and 306 bp in the bileaflet was amplified, and in the case of the primer sets of SEQ ID NOs: 17 and 18 It was confirmed that a specific band of 350 bp and 315 bp in length was amplified.

Therefore, it is possible to easily discriminate between Baekshou and Epiphytes using the genetic marker for discriminating Baekshui species based on the gene sequence of the genome Indel region of the genus Indel plant of the genus Baeksui flower of the present invention. It was confirmed that it was possible to easily distinguish between plants and white water.

In addition, the markers based on the genomic InDel site of the genus white rice flower are hybrid plants generated by pollen unlike markers based on the chloroplast gene inherited from the maternal line It could be confirmed that (Group II) or (corresponding to Group V) having a genotype of Baek Suo] can be distinguished at the source.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is 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 ggtatttaac 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 ttgcctcccg 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 represented by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2; A primer set represented by the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set represented by the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set represented by the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set represented by the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10; A primer set represented by the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12; A primer set represented by the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14; A primer set represented by the nucleotide sequence 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; Baeksuo (Cynanchum wilfordii) and a primer set for discrimination including one or more primer sets selected from the group consisting of. The method of claim 1,
The PCR amplification product of Baek Shou species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 is 321 bp, and the number of white numbers amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 The five kinds of PCR amplification products are 350bp, and the PCR amplification products of Baekshou species amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 are 333 bp, and the bases of SEQ ID NO: 7 and SEQ ID NO: 8 The PCR amplification product of Baek Shou species amplified with the primer set represented by the sequence is 303 bp, and the PCR amplification product of Baek Shou species amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 is 339 bp, The PCR amplification product of Baeksuo species amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12 is 305 bp, and the number of white numbers amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14 The five kinds of PCR amplification products are 301 bp, and the PCR amplification product of Baek Shou species amplified with the primer set represented by the nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 16 is 357 bp, and the bases of SEQ ID NO: 17 and SEQ ID NO: 18 The PCR amplification product of Baekshou species amplified by the primer set represented by the sequence is 350bp, characterized in that Baekshou (Cynanchum wilfordii) and Yiyeopiso (Cynanchum auriculatum) a set of primers for discrimination. Claim 1 or claim 2 Baeksu-o (Cynanchum wilfordii) and yiyeop cow (Cynanchum auriculatum) containing a primer set for identification of Baeksu-o (Cynanchum wilfordii) and yiyeopso (Cynanchum auriculatum) discrimination kit. (a) separating DNA from a sample of the subject Baeksu-o (Cynanchum wilfordii);
(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 sequence of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set represented by the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set represented by the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set represented by the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10; A primer set represented by the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12; A primer set represented by the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14; A primer set represented by the nucleotide sequence 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 the target sequence using one or more primer sets selected from the group consisting of; And
(c) PCR amplification product amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 is 321 bp, and PCR amplification amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 The product is 350bp, and 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 333bp, and amplified with the primer set represented by the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8 The PCR amplification product is 303 bp, and the amplified PCR amplification product is 339 bp with a primer set represented by the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10, and a primer set represented by the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12. The amplified PCR amplification product is 305 bp, the amplified PCR amplification product is 301 bp with the primer set represented by the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14, and the primer represented by the nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 16 If the PCR amplification product amplified by the set is 357 bp, and the PCR amplification product amplified by the primer set represented by the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18 is 350 bp, determining that it is a species of Cynanchum wilfordii; Baeksu-o (Cynanchum wilfordii) species identification method containing.

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