KR20240029600A - Marker composition for discriminating Panax ginseng 'Cheonryang' cultivar and uses thereof - Google Patents

Abstract

The present invention relates to a marker composition for determining the 'Cheonryang' variety of ginseng and its use. The marker composition of the present invention quickly and accurately distinguishes the 'Cheonryang' variety, which is an excellent variety of ginseng, at the genetic level among various domestic ginseng varieties or collection resources. Therefore, it can be used to solve the problem of seed purity that appears when cultivating ginseng and the problem of unclear varieties that appear during the distribution process. In the cultivation of ginseng, which is greatly influenced by the cultivation environment, it can be used as a basis for selecting varieties suitable for a specific cultivation environment. You will be able to.

Description

Marker composition for discriminating Panax ginseng 'Cheonryang' cultivar and uses thereof}

The present invention relates to a marker composition for determining ginseng 'Cheonryang' variety and its use.

Ginseng ( Panax ginseng ) is a perennial plant belonging to the Araliaceae genus and is a penumbra plant that grows in semi-shade. Ginseng has been known as a mysterious elixir since ancient times, and the root is used medicinally in Oriental medicine. Ginseng contains a large amount of highly functional substances such as saponin, ginsenoside, polyacetylene, acidic polysaccharide, protein, and phenolic substances, so its pharmacological efficacy is superior to that of other crops.

Ginseng is Korea's representative agricultural product, and its production and consumption are steadily increasing every year as the national income level and interest in health increase. However, due to the effects of climate change due to global warming, high temperature and salt damage have occurred recently, causing damage to ginseng. are experiencing many difficulties in stable production. Ginseng varieties cultivated to date have mainly focused on yield and red ginseng processing suitability, and research has been conducted on the development of destructive-resistant varieties to produce ginseng in response to environmental changes such as abnormal climate in the future. Accordingly, the Rural Development Administration developed the ginseng 'Cheonryang' variety, which exhibits high multiplicity and salt resistance.

Therefore, in order to protect and cultivate high-quality ginseng 'Cheonryang' varieties, it is necessary to develop molecular markers that can quickly and accurately analyze and efficiently identify 'Cheonryang' varieties in the field. Molecular markers can reliably search for and identify genetic mutations in all tissues regardless of plant growth by using a small amount of DNA, complementing limitations in morphological and physicochemical discrimination and providing reliability in a short period of time at a relatively low cost. High level analysis is possible.

Meanwhile, Korean Patent No. 1326333 discloses 'SNP primer for distinguishing Korean ginseng Yeonpung varieties and method for distinguishing Yeonpung varieties using the same', and Korean Patent No. 1761290 discloses 'Korean ginseng' based on STS (sequence-tagged sites) markers. A 'primer set for determining ginseng 'Cheonryang' variety and its use' is disclosed, but there is no description of the marker composition for determining ginseng 'Cheonryang' variety of the present invention and its use.

The present invention was derived from the above-mentioned needs, and the present inventors combined the ginseng standard genome sequence (http://ginsengdb.snu.ac.kr/) with sequences produced from 119 ginseng breeding lines, including the Cheonryang variety. In comparison, SNPs that exist in a single region without similar sequences in the ginseng genome were selected, and finally, SNPs that could identify 'Cheonryang' varieties were selected, using one TaqMan probe and primer set (pgTM001) and four fluidimes. (Fluidigm) developed primer sets (pgFD001, pgFD002, pgFD003, pgFD004). In addition, using the above five molecular markers, 14 ginseng varieties (Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonwoon, Seonwon, Cheongseon, Seonhyang, K-1, Cheonryang, Gowon, Geumseon, Geumjin) and 186 ginseng species. The present invention was completed by confirming that the 'Cheonryang' variety could be accurately identified among the collected resources.

In order to solve the above problem, the present invention provides ginseng varieties 'Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonwoon, Seonwon, including oligonucleotide probes of SEQ ID NOs: 1 and 2 and oligonucleotide primer sets of SEQ ID NOs: 3 and 4. Provides a marker composition for identifying ginseng 'Cheonryang' varieties from ', Cheongseon, Seonhyang, K-1, Gowon, Geumseon, and Geumjin'.

In addition, the present invention includes oligonucleotide probes of SEQ ID NOs: 1 and 2 and oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer set of SEQ ID NOs: 5 to 8; Oligonucleotide primer set of SEQ ID NOs: 9 to 12; Oligonucleotide primer set of SEQ ID NOs: 13 to 16; and an oligonucleotide primer set of SEQ ID NOs: 17 and 20. It provides a marker composition for identifying ginseng 'Cheonryang' varieties.

Additionally, the present invention provides a kit for determining ginseng 'Cheonryang' variety, including the marker composition and a reagent for performing an amplification reaction.

Additionally, the present invention includes the steps of isolating genomic DNA from a ginseng sample; Using the isolated genomic DNA as a template and performing an amplification reaction using the marker composition according to the present invention to amplify the target sequence; and determining the genotype of the product of the amplification step. It provides a method for determining the 'Cheonryang' variety of ginseng, including.

The marker composition of the present invention can quickly and accurately distinguish 'Cheonryang' varieties among various domestic ginseng varieties or collection resources at the genetic level, so it can be used to solve the problem of seed purity that appears during ginseng cultivation and the problem of unclear varieties that appear during distribution. It can be used as a basis for selecting varieties suitable for a specific cultivation environment in the cultivation of ginseng, which is greatly influenced by the cultivation environment.

Figure 1 shows 14 ginseng varieties (Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonun, Seonwon, Cheongseon, Seonhyang, K-) using the TaqMan probe and primer set (pgTM001) and Fluidigm primer set (pgFD001~4) of the present invention. 1, Cheonryang, Gowon, Geumseon, Geumjin) and 186 ginseng collection resources (pg001~pg186).
Figure 2 shows 14 ginseng varieties (Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonun, Seonwon, Cheongseon, Seonhyang, K-1 with the TaqMan probe and primer set (pgTM001) and Fluidigm primer set (pgFD001~4) of the present invention. , Cheonryang, Gowon, Geumseon, and Geumjin) are the results of genotyping.
Figure 3 shows 14 ginseng varieties (Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonun, Seonwon, Cheongseon, Seonhyang, K-1 with the TaqMan probe and primer set (pgTM001) and Fluidigm primer set (pgFD001~4) of the present invention. , Cheonryang, Gowon, Geumseon, and Geumjin) and 186 ginseng collection resources (pg001~pg186).

In order to achieve the object of the present invention, the present invention provides ginseng varieties 'Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonun, comprising oligonucleotide probes of SEQ ID NOs: 1 and 2 and oligonucleotide primer sets of SEQ ID NOs: 3 and 4. Provides a marker composition for identifying ginseng 'Cheonryang' varieties from Seonwon, Cheongseon, Seonhyang, K-1, Gowon, Geumseon, and Geumjin'.

The present invention also provides a set of oligonucleotide probes of SEQ ID NOs: 1 and 2 and oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer set of SEQ ID NOs: 5 to 8; Oligonucleotide primer set of SEQ ID NOs: 9 to 12; Oligonucleotide primer set of SEQ ID NOs: 13 to 16; and an oligonucleotide primer set of SEQ ID NOs: 17 and 20. It provides a marker composition for identifying ginseng 'Cheonryang' varieties.

The marker composition of the present invention may contain a molecular marker based on SNP (single nucleotide polymorphism) to distinguish 'Cheonryang' varieties from various ginseng varieties and ginseng cultivation lines. The SNP information is shown in Table 1.

The marker composition of the present invention includes probes of SEQ ID NOs. 1 and 2 containing the bases at the SNP (single nucleotide polymorphism) positions found by analyzing the genomic DNA of the ginseng 'Cheonryang' variety and various breeding lines, and polynucleotides containing the bases at the SNP positions. It contains a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, which can amplify nucleotides (Marker ID_pgTM001 in Table 2).

In addition, the marker composition of the present invention is SEQ ID NO: 5 to SEQ ID NO: 5, which can detect four SNP bases (Marker ID_pgFD001, pgFD002, pgFD003, pgFD004 in Table 2) found by analyzing the genomic DNA of ginseng 'Cheonryang' variety and various breeding lines. Contains 20 primer sets.

The primer set of SEQ ID NOs: 5 to 20 is a primer set that detects SNP base types that are specifically differentiated at the ginseng locus. Specifically, the primer set consists of four oligonucleotides with consecutive sequence numbers forming one primer set, and the four oligonucleotide primers are ASP (allele specific primer) 1, ASP2, LSP (locus specific primer), and STA (specific primer), respectively. target amplification primer). STA and LSP are primer sets used to amplify the target sequence, LSP and ASP are primer sets used to confirm the base at the SNP position, and ASP1 and ASP2 are primer sets used to identify the base at the SNP position. With specific oligonucleotide primers, the final base at the 3' end of ASP represents the SNP position base. Through the fluorescent material FAM attached to the 5' end of the ASP1 primer and HEX attached to the 5' end of the ASP2 primer, the genotype of the ginseng sample can be distinguished and the 'Cheonryang' variety of ginseng can be determined.

In one embodiment of the present invention, SEQ ID NOs: 5, 9, 13, and 17 are ASP1 primers, SEQ ID NOs: 6, 10, 14, and 18 are ASP2 primers, and SEQ ID NOs: 7, 11, 15, and 19 are STA primers. , SEQ ID NOs: 8, 12, 16 and 20 are LSP primers. In addition, the primer may also include sequences added, deleted, or substituted in the base sequences of SEQ ID NOs: 5 to 20 that can bind to the amplification site.

In the present invention, “probe” refers to a single-stranded nucleic acid sequence that hybridizes with a complementary single-stranded target sequence to form a double-stranded molecule (hybrid).

The probes of the present invention were manufactured to include SNP position bases within the sequence of each probe that can distinguish the 'Cheonryang' ginseng variety. Preferably, the probe of SEQ ID NO: 1 or 2 of the present invention can be produced by binding to FAM or VIC fluorescent substance according to the SNP position base shown in Table 2, but is not limited thereto.

In the present invention, “primer” refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be copied, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow for initiation of synthesis of the extension product. The specific length and sequence of the primer will depend on the complexity of the DNA or RNA target desired as well as the conditions under which the primer is used, such as temperature and ionic strength.

The primer includes an oligonucleotide consisting of a fragment of 19 or more, 20 or more, 21 or more, 22 or more, 23 or more consecutive nucleotides in the sequence of SEQ ID NOs: 3 to 20, depending on the sequence length of each primer. can do. For example, the primer (24 oligonucleotides) of SEQ ID NO: 3 may include an oligonucleotide consisting of a segment of 21 or more, 22 or more, or 23 or more consecutive nucleotides in the sequence of SEQ ID NO: 3. In addition, the primer may also include sequences added, deleted, or substituted in the base sequences of SEQ ID NOs: 3 to 20 that can bind to the amplification site.

In the present invention, oligonucleotides used as primers may also include nucleotide analogues, such as phosphorothioates, alkylphosphorothioates or peptide nucleic acids, or May contain an intercalating agent.

The present invention also provides a kit for determining ginseng 'Cheonryang' variety, including the marker composition and a reagent for performing an amplification reaction.

In the kit of the present invention, the marker composition is as described above.

In one embodiment of the present invention, reagents for performing the amplification reaction may include, but are not limited to, DNA polymerase, dNTPs, and buffer.

The kit of the present invention may also further include a user guide describing optimal reaction performance conditions. The guide is a printed material that explains how to use the kit, for example, how to prepare PCR buffer, and the suggested reaction conditions. Instructions include information leaflets in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. Additionally, the guide includes information disclosed or provided through electronic media such as the Internet.

The present invention also includes the steps of isolating genomic DNA from a ginseng sample;

Using the isolated genomic DNA as a template and performing an amplification reaction using the marker composition according to the present invention to amplify the target sequence; and

It provides a method for determining the 'Cheonryang' variety of ginseng, including the step of determining the genotype of the product of the amplification step.

In the method according to one embodiment of the present invention, the marker composition is the same as described above.

The method of the present invention includes the step of isolating genomic DNA from a ginseng sample. Methods for isolating genomic DNA from the ginseng sample can be methods known in the art. For example, the CTAB method can be used, or the Wizard prep kit (Promega) can be used. The target sequence can be amplified by using the isolated genomic DNA as a template and performing an amplification reaction using a primer set according to an embodiment of the present invention. Methods for amplifying target nucleic acids include polymerase chain reaction, ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. ), strand displacement amplification or amplification via Qβ replicase or any other suitable method for amplifying nucleic acid molecules known in the art. Among these, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using polymerase. These PCR methods are well known in the art, and commercially available kits can also be used.

In the method according to one embodiment of the present invention, the ginseng sample may be the root, leaf, stem, or seed of the plant, but is not limited thereto.

The genotype of the product of the amplification step can be determined by various methods known in the art. For example, it may be performed using a DNA chip, capillary electrophoresis, etc., but is not limited thereto. Capillary electrophoresis can be performed using, for example, an ABI Sequencer. In addition, the nucleotide sequence of the polymorphic site can be determined through direct determination of the nucleotide sequence of the nucleic acid by the dideoxy method, or by hybridizing a probe containing the sequence of the SNP site or a probe complementary thereto with the DNA and measuring the degree of hybridization obtained therefrom. A method of determining may be used, but is not limited thereto.

In one embodiment of the present invention, the method for determining the genotype of the product of the amplification step includes detecting the fluorescence of a substance labeled at the 5' end of the primer during amplification of the target sequence using a probe or primer bound to a fluorescent labeling substance. This can be analyzed. Specifically, probes labeled with FAM or VIC depending on the SNP base; Alternatively, the genotype of the ginseng sample can be distinguished through the fluorescent substance FAM attached to the 5' end of the ASP1 primer and HEX attached to the 5' end of the ASP2 primer to determine the 'Cheonryang' variety of ginseng.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Development of SNP markers based on regions that exist as a single copy and do not have homologous genes in the genome

DNA was extracted from 119 ginseng breeding lines, a library was created using the GBS (genotyping-by-sequencing) method (Elshire et al ., PloS one, 2011, 6(5), e19379), and then using Illumina sequencing technology. A large amount of sequence information was obtained. Sequences produced from each genetic resource were mapped to the ginseng standard genome sequence (http://ginsengdb.snu.ac.kr/), compared and analyzed to search for a number of SNPs, among which there were no similar sequences in the ginseng genome. After selecting SNPs that exist in a single region, SNPs that could provide accurate genotype information were finally selected.

Looking at the SNP selection process in detail, first, SNPs with a mapping quality of 20 or less or without polymorphisms in the population were excluded from the raw variants discovered from various ginseng genetic resources, and the minimum read depth was selected to secure as many SNPs as possible and increase accuracy. SNPs with a value of 5 or more and missing data within the group of less than 70% were selected. Among the selected SNPs, those with other SNPs within 50 bp were additionally excluded, and from the GBS analysis results, SNPs with two or more genotypes among the three genotypes (AA, AB, BB) in the population were selected. In order to select SNPs present in a single gene region within the genome, the selected SNP information was used to extract sequences of 150 bp (total 301 bp) on both sides of the SNP from the ginseng standard genome sequence, and then added to the ginseng standard genome sequence using the BLASTN program. SNPs that matched only one place in the genome were selected using .

Example 2. Distinguishing ginseng ‘Cheonryang’ variety using the SNP marker of the present invention

The effectiveness of molecular markers was tested by performing HRM (High Resolution Melting) analysis on some of the SNPs selected in Example 1, and the information of five SNPs (Table 1) that can identify low-yield cultivars was tested using TaqMan. Probe and primer sets and a Fluidigm-based primer set were designed (Table 2).

Using the developed TaqMan probe and primer set (pgTM001) and Fluidigm primer set (pgFD001~4), 14 ginseng varieties (Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonwoon, Seonwon) provided by the Ginseng Special Crop Department of the Rural Development Administration. , Cheongseon, Seonhyang, K-1, Cheongseon, Gowon, Geumseon, Geumjin) and a total of 200 ginseng genetic resources, including 186 ginseng collection resources (pg001~pg186) collected from ginseng farms across the country and maintained by the Rural Development Administration. Genotype information and polymorphisms were confirmed for the target (Figure 1).

As a result of performing genotyping on 14 ginseng cultivars using the TaqMan probe and primer set (pgTM001), it was confirmed that only the 'Cheonryang' cultivar could be accurately identified. In addition, as a result of performing genotyping on 14 ginseng varieties using the Fluidigm primer set (pgFD001~4), it was confirmed that only the 'Cheonryang' variety could be accurately identified by combining the genotyping results of 4 Fluidigm primer sets. Considering the genotypic diversity of plants, it was found that by combining the results of pgTM001 and pgFD001~4, it was possible to more clearly identify the 'Cheonryang' variety without false positive errors (Figure 2).

In addition, genotyping was performed on a total of 200 ginseng genetic resources using the TaqMan probe and primer set (pgTM001) and Fluidigm primer set (pgFD001~4). As a result, when pgTM001 was used alone, 2 out of 200 individuals were It shows the same genotype as the 'Cheonryang' variety, so there is a possibility of false positives of about 1%, but it was confirmed to have a reliability of 99%. In addition, it was confirmed that when combining the results of four Fluidigm markers (pgFD001~4), 'Cheonryang' varieties can be accurately identified from 200 ginseng resources. Considering the genotypic diversity of plants, it was found that when combining the results of pgTM001 and pgFD001~4, the possibility of false positives was minimized and the 'Cheonryang' variety could be clearly identified (Figure 3).

Through the above results, when the TaqMan probe and primer set (pgTM001) and the Fluidigm primer set (pgFD001~4) of the present invention are applied to ginseng genetic resources such as various ginseng varieties or breeding lines, the genotype can be confirmed and classified in a short time, thereby ginseng' It can be seen that the 'Cheonryang' variety can be efficiently identified.

Claims (5)

Ginseng varieties 'Cheonpung, Yeonpung, Gopung, Seonpung, Geumpung, Seonun, Seonwon, Cheongseon, Seonhyang, K-1, Gowon, comprising oligonucleotide probes of SEQ ID NOs: 1 and 2 and oligonucleotide primer sets of SEQ ID NOs: 3 and 4. A marker composition for distinguishing ginseng 'Cheonryang' varieties from ', Geumseon, and Geumjin'. A set of oligonucleotide probes of SEQ ID NOs: 1 and 2 and oligonucleotide primers of SEQ ID NOs: 3 and 4; Oligonucleotide primer set of SEQ ID NOs: 5 to 8; Oligonucleotide primer set of SEQ ID NOs: 9 to 12; Oligonucleotide primer set of SEQ ID NOs: 13 to 16; and an oligonucleotide primer set of SEQ ID NOs: 17 and 20. A marker composition for determining the 'Cheonryang' variety of ginseng. A kit for determining ginseng 'Cheonryang' variety containing the marker composition of claim 1 or 2 and a reagent for performing an amplification reaction. The kit according to claim 3, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs, and buffer. Isolating genomic DNA from a ginseng sample;
Using the isolated genomic DNA as a template and performing an amplification reaction using the marker composition of claim 1 or 2 to amplify the target sequence; and
A method for determining the 'Cheonryang' variety of ginseng, including the step of determining the genotype of the product of the amplification step.