KR100842434B1 - Ssr primer derived from ginseng and use thereof - Google Patents

Abstract

An SSR(simple sequence repeat) primer pair derived from ginseng is provided to detect DNA polymorphism of the ginseng effectively and be very usefully used for DNA profiling of the ginseng. An SSR primer pair is selected from the group consisting of: a primer pair of SEQ ID : NOs. 1 and 2; a primer pair of SEQ ID : NOs. 3 and 4; a primer pair of SEQ ID : NOs. 5 and 6; a primer pair of SEQ ID : NOs. 7 and 8; a primer pair of SEQ ID : NOs. 9 and 10; a primer pair of SEQ ID : NOs. 11 and 12; a primer pair of SEQ ID : NOs. 13 and 14; a primer pair of SEQ ID : NOs. 15 and 16; a primer pair of SEQ ID : NOs. 17 and 18; a primer pair of SEQ ID : NOs. 19 and 20; a primer pair of SEQ ID : NOs. 21 and 22; a primer pair of SEQ ID : NOs. 23 and 24; a primer pair of SEQ ID : NOs. 25 and 26; a primer pair of SEQ ID : NOs. 27 and 28; a primer pair of SEQ ID : NOs. 29 and 30; a primer pair of SEQ ID : NOs. 31 and 32; a primer pair of SEQ ID : NOs. 33 and 34; a primer pair of SEQ ID : NOs. 35 and 36; a primer pair of SEQ ID : NOs. 37 and 38; a primer pair of SEQ ID : NOs. 39 and 40; a primer pair of SEQ ID : NOs. 41 and 42; and a primer pair of SEQ ID : NOs. 43 and 44 and is derived from ginseng(Panax ginseng C. A. Meyer). A method for detecting DNA polymorphism of the ginseng comprises the steps of: (a) extracting genome DNA from the ginseng; (b) subjecting the extracted genome DNA as a template to PCR using the SSR primer pair; and (c) isolating PCR products obtained from the step(b) by size. A kit for detecting the DNA polymorphism of the ginseng or identifying species of the ginseng comprises the SSR primer pair.

Description

SSR primer derived from ginseng and its use {SSR primer derived from ginseng and use etc}

The present invention relates to an SSR primer isolated from Ginseng (Ginseng; Panax ginseng CA Meyer) and its use, and more particularly, to an SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 44. And it relates to a DNA polymorphism detection method of ginseng comprising performing PCR using the same.

Large quantities of genetic resources are collected annually in most major crops and cultivated crops used for breeding. However, on the other hand, the evaluation of genetic resources has not been sufficiently made. Rapid discrimination between species and subspecies varieties of collected genetic resources is very important for accurate management and protection of genetic resources. In addition to the identification of varieties, the identification and classification of trait traits and genetic relationships of genetic resources are very important for the conservation of genetic resources, the creation of new varieties, and the improvement of crops.

Recent rapid advances in molecular biology have led to the development of nucleic acid fingerprint analysis methods and various DNA markers that enable bio-diversity studies at the nucleic acid (DNA) level. This makes it possible to search useful traits, identify species of species, classify and identify varieties, and analyze flexible relationships of populations in a simple and quick manner with little influence on the external environment. Fingerprinting methods using polymerase chain reaction (PCR) developed so far include randomly amplified polymorphic DNAs (RAPD), amplified fragment length polymorphic DNA (AFLP), and simple sequence repeat (SSR). The RAPD method has a disadvantage of poor reproducibility because the non-specific PCR product is amplified, and the AFLP method is spotlighted by high DNA polymorphism detection, but has a disadvantage of complicated appearance and analysis of a low reproducible band. In contrast, the SSR method is a method of constructing a PCR primer based on nucleotide sequence information of a microsatellite region, which is a DNA repeat sequence. Frequently used. In particular, the SSR method has the advantage that it is not affected by the external environment at all. Due to the superiority of this SSR method, studies are underway to develop SSR markers in several major crops and cultivated crops.

In rice, supersatellite markers have been developed that amplify SSR repeated in the rice genome by PCR technology to identify genotypes of rice varieties (Korean Patent Application No. 2001-34003). Tang's team in the United States developed 879 SSR markers and published a genetic map of sunflowers (Tang et al., Theor. Appl. Genet ., 105: 1124-1136, 2002). In addition, many SSR markers have been developed in barley and soybeans. However, in ginseng, SSR markers have not been developed at all in Korea, and studies related to this have not been made in foreign countries.

Accordingly, the present inventors have completed the present invention by developing SSR primer pairs showing a large number of polymorphic variations in various ginseng lines, while continuing to develop SSR markers capable of efficiently evaluating the gene source of ginseng.

Accordingly, it is an object of the present invention to provide an SSR marker and its use capable of efficiently evaluating the gene source of ginseng.

In order to achieve the above object, the present invention provides an SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 44.

In order to achieve another object of the present invention, the present invention provides a method for detecting DNA polymorphism of ginseng and a method for identifying a variety of ginseng, including performing PCR using the SSR primer pair.

In order to achieve another object of the present invention, the present invention provides a kit for detecting DNA polymorphism of ginseng and a kit for identifying a variety of ginseng, including the SSR primer pair, DNA polymerase and PCR reaction buffer.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing an SSR marker capable of specifically analyzing the genetic polymorphism of ginseng.

In the present invention, SSR markers were developed from ginseng by biotin-streptavidin capture (Dixit et al., Mol. Eco.Note, 5: 736-738). The SSR marker isolated from ginseng is the first to be provided by the present invention.

Specifically, the SSR marker of the present invention was developed by the following method. Genomic DNA extracted from ginseng was treated with restriction enzymes to prepare DNA fragments and then ligated with two adapters of AP11 and AP12.

Ligation products were hybridized with biotin-labeled SSR probes and then separated using magnetic beads. PCR was performed using the AP11 primer to amplify and analyze the sequence to obtain 500 DNA fragments containing supersatellite of ginseng.

By analyzing the DNA fragments obtained above, 200 pairs of bidirectional primers were designed to select only fragments containing five or more repeating units and to amplify the supersatellite based on the portion containing the repeating unit. Using the primer pairs, DNA profiling by PCR of various cultivated ginseng strains and varieties was performed to select 22 pairs of SSR primer pairs that exhibited polymorphic variation.

The SSR marker provided by the present invention refers to a pair of PCR primers, and includes a forward primer and a reverse primer. SSR primer pair provided in the present invention has two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 44. Preferably GB-PG-007 (primer pairs represented by SEQ ID NOs: 1 and 2), GB-PG-009 (primer pairs represented by SEQ ID NOs: 3 and 4), GB-PG-014 (SEQ ID NOs: 5 and 6) Primer pairs indicated by (), GB-PG-026 (primary pairs shown by SEQ ID NOs: 7 and 8), GB-PG-033 (primary pairs shown by SEQ ID NOs: 9 and 10), and GB-PG-041 (SEQ ID NO: Primer pairs represented by numbers 11 and 12), GB-PG-043 (primer pairs represented by SEQ ID NOs: 13 and 14), GB-PG-057 (primer pairs represented by SEQ ID NOs: 15 and 16), GB-PG -060 (primary pairs represented by SEQ ID NOs: 17 and 18), GB-PG-065 (primary pairs represented by SEQ ID NOs: 19 and 20), GB-PG-075 (primary pairs represented by SEQ ID NOs: 21 and 22) , GB-PG-077 (primer pairs represented by SEQ ID NOs: 23 and 24), GB-PG-078 (primer pairs represented by SEQ ID NOs: 25 and 26), and GB-PG-114 (SEQ ID NOs 27 and 28) Primer pairs shown), GB-PG-131 (SEQ ID NOs. 29 and 30) Primer pairs), GB-PG-142 (primary pairs represented by SEQ ID NOs: 31 and 32), GB-PG-150 (primary pairs represented by SEQ ID NOs: 33 and 34), GB-PG-152 (SEQ ID NO: 35 Primer pairs represented by and 36), GB-PG-157 (primary pairs represented by SEQ ID NOs: 37 and 38), GB-PG-169 (primer pairs represented by SEQ ID NOs: 39 and 40), GB-PG-177 (Primer pairs represented by SEQ ID NOs: 41 and 42) and GB-PG-186 (primer pairs represented by SEQ ID NOs: 43 and 44). Information on the primer pairs provided in the present invention and the repeat motifs present in the supersatellites amplified by them, the size of the supersatellites to be amplified, T _m (° C.) and the chromosomal alleles in which the supersatellites are present It is listed in Table 1 below.

Characteristics of SSR Primer Pairs of the Present Invention and Supersatellites Amplified therefrom Primer Name order Sequence number Repeating motif Range of size of supersatellite to be amplified (bp) Tm (℃) Number of alleles GB-PG-007 F: CAGAGCTGGTGGTCGAAG One (GA) ₇ , (GA) ₈ 177-237 52 5 R: ATTCTTTTCTCCAGCGCC 2 GB-PG-009 F: GTCGAATGCAGTGGGGTA 3 (AG) ₅ TG (AG) ₃ 150-334 52 4 R: ACACTACCGGCCACACAC 4 GB-PG-014 F: CCCAGGTGCACAAACAGT 5 (CCG) ₂ CCT (CCG) ₃ 289-301 52 3 R: TTCTCCCCTTTCTCCGTC 6 GB-PG-026 F: CTGGAATCGGAAATGGGT 7 (TGG) ₁₀ , (TGG) ₁₀ 108-177 52 7 R: CAGGCGCCCCTCTAAC 8 GB-PG-033 F: CAGCCCATTATTCCACCA 9 (GCA) ₅ GCC (GCA) ₂ 335-371 52 2 R: ACTCCTCTGCCACGACCT 10 GB-PG-041 F: CAAGTCTATGCGCTCTCCA 11 (TGC) ₃ (TCC) (TGC) ₃ 239-305 52 3 R: GGGCATCTGCAGTCTCTG 12 GB-PG-043 F: AGCCAGGTGCTTGTCTCA 13 (TC) ₃ (CT) (TC) ₅ , (CT) ₉ 90-232 52 5 R: GTCAGACGGATTGCTGCT 14 GB-PG-057 F: GTTCGAGAGTTCGCGATG 15 (GA) ₃ (CA) (GA) ₃ (CA) (GA) ₃ (CA) (GA) ₃ 256-288 52 2 R: CCAGTTTTCCTTCTCGTCC 16 GB-PG-060 F: CGACTGGAATCGGAAATG 17 (TGG) ₄ 163-178 52 3 R: CGCCCCTTCTCAATTCTC 18 GB-PG-065 F: CCGAGCGCTACAAAGAGA 19 (CGC) ₇ 136-157 52 5 R: CCTTCTGCTCAATCGACG 20 GB-PG-075 F: GCGAGCGAGATAACACAAA 21 (GA) ₆ 251-257 52 3 R: TGCTTAAAGCTTGCCTCCT 22 GB-PG-077 F: TAGCAGCACAGCAGCAGA 23 (TA) ₅ 233-235 52 2 R: TCTAGACGCGAGCTCTTCA 24 GB-PG-078 F: GTGACGGGTTAAGAGGGC 25 (GTT) ₂ GAG (GTT) ₃ , (GCG) ₂ CTT (GCG) ₄ 252-264 52 2 R: CTCCTTTCCTTCGCCACT 26 GB-PG-114 F: CCTGGTCGTCATTCTGCT 27 (TGC) ₄ 227-239 52 3 R: AAGGACAAAAAGGGACGC 28 GB-PG-131 F: TCATGATGAGTTGGCGGT 29 (CAG) ₆ (CA) (CAG) ₃ 199-205 52 3 R: TCTAGGCGCTCTTCATGG 30 GB-PG-142 F: CTGGTGATGGAACCGACA 31 (TGG) ₃ (CAG) (TGG) ₃ , (GA) ₉ 162-261 52 6 R: AGTCAGCTCGTCTTCCCC 32 GB-PG-150 F: AATGACACCCGACCATCA 33 (GCA) ₅ 217-250 52 4 R: TGTAACGCATCGTCATCCT 34 GB-PG-152 F: ACCAGGTTGTGTTCGTGG 35 (GT) ₈ 255-259 52 3 R: CCCACAAAATCCCAAATG 36 GB-PG-157 F: GTACAAGGCAGGCGCTC 37 (TGC) ₄ 225-282 52 2 R: AGTCCTGATGAGTTGGCG 38 GB-PG-169 F: TCTAGGCGCTCTTCATGG 39 (TGC) ₂ (TG) (TGC) ₄ 231-276 52 4 R: GGGCATCTGCAGTCTCTG 40 GB-PG-177 F: TTTGATCCGCAACTGTCC 41 (GCA) ₄ 106-286 52 7 R: AATGAGAGGCACCCGAAT 42 GB-PG-186 F: TTGGGAGGTCCAACAGTG 43 (GA) ₄ (AA) (GA) ₃ 174-212 52 2 R: TCTTCTTCATCGCCTCCTC 44

^a F: forward primer ^b R: reverse primer

SSR primer pairs provided in the present invention can be usefully used to detect DNA polymorphism and analyze genetic diversity in ginseng. By using the SSR primers according to the present invention, gene sources of ginseng can be efficiently evaluated and preserved. Accordingly, the present invention provides a method for detecting DNA polymorphism of ginseng comprising performing PCR using the SSR primer pair.

Specifically, the method for detecting DNA polymorphism of ginseng

(a) extracting genomic DNA from ginseng;

(b) performing PCR using the extracted genomic DNA as a template and the SSR primer pair provided by the present invention; And

(c) separating the PCR products by size.

Extraction of genomic DNA in step (a) is performed by phenol / chloroform extraction, SDS extraction (Tai et al ., Plant Mol. Biol. Reporter , 8: 297-303, 1990), CTAB separation method commonly used in the art ( Cetyl Trimethyl Ammonium Bromide; Murray et al. , Nuc. Res. , 4321-4325, 1980) or commercially available DNA extraction kits.

In addition, the PCR in the step (b) may be performed using a PCR reaction mixture containing a variety of components known in the art required for the PCR reaction. The PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water (dH ₂ O) in addition to genomic DNA extracted from ginseng to be analyzed and the SSR primer pair provided in the present invention. The PCR buffer solution includes Tris-HCl, MgCl ₂ , KCl, and the like. At this time, the MgCl ₂ concentration greatly affects the specificity and yield of amplification, and may be preferably used in the range of 1.5-2.5 mM. In general, when Mg ²⁺ is excessive, nonspecific PCR amplification products increase, and when Mg ²⁺ is insufficient, PCR The yield of the product is reduced. The PCR buffer may further include an appropriate amount of Triton X-100. PCR also denatured the template DNA at 94-95 ° C., followed by denaturation; Annealing; And a cycle of extension, followed by general PCR reaction conditions which are finally elongated at 72 ° C. The denaturation and amplification may be performed at 94-95 ° C. and 72 ° C., respectively, and the temperature at the time of binding may vary depending on the type of primer. Preferably it is 52-57 degreeC, More preferably, it is 55 degreeC. The time and number of cycles in each step can be determined according to the conditions generally made in the art. The optimal reaction conditions when performing PCR using the SSR primer pair according to the present invention are as follows: After denature the template DNA for 3 minutes at 95 ℃, 30 seconds at 95 ℃; 30 seconds at 55 ° C .; And repeating 36 cycles of 1 minute 30 seconds at 72 ° C., and finally reacting at 72 ° C. for 5 minutes.

In step (c), DNAs of PCR products may be separated according to sizes according to methods well known in the art. Preferably it can be confirmed by an agarose gel (agarose gel) or polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3100 genetic analyzer-electropherogram). At this time, in order to use the fluorescence analyzer PCR is performed in the step (b) using a primer pair attached to a fluorescent die known in the art. PCR amplification results can be preferably confirmed by polyacrylamide gel electrophoresis, more preferably modified polyacrylamide gel electrophoresis. Electrophoresis results can be analyzed by silver staining after electrophoresis. General PCR performance and resulting analysis methods are well known in the art.

In addition, the present invention provides a method for identifying varieties of ginseng comprising performing PCR using the SSR primer pair.

Specifically, the method of identifying ginseng varieties

(a) extracting genomic DNA from ginseng and control ginseng varieties;

(b) performing PCR using each of the extracted genomic DNA as a template and the SSR primer pair provided by the present invention;

(c) separating each PCR product by size; And

(d) comparing the separation results for each size of step (c) with each other.

Genomic DNA extraction in step (a) to (c), PCR and separation of PCR products by size are as described above, and in step (d) comparison of ginseng and control ginseng varieties is the same combination of SSR primers The size of each PCR product of the ginseng and the control ginseng cultivars used as a sample for the pair is compared with each other. By comparing the size comparison results for each pair of SSR primers, the ginseng serving as a sample can be identified as the same varieties as the control ginseng varieties if the results match both the results of the ginseng and the control ginseng varieties. The identification method of the variety can be useful when the identification of the breed, such as judging whether there is a violation of the labeling of origin through judging whether the overlapping when introducing a new genetic resource and confirm the origin.

Genomic DNA extraction, PCR, and PCR product separation of control ginseng varieties may be performed simultaneously with the sample ginseng, but may be performed prior to the sample ginseng and prepared as a reference for identification. Using such a reference table can be useful because it can be compared with the reference table by performing genomic DNA extraction, PCR and separation of the size of the PCR product for ginseng serving as a sample.

The present invention also provides a kit for detecting DNA polymorphism of ginseng comprising the SSR primer pair according to the present invention. In addition, DNA polymerase and a PCR reaction buffer of the above-described composition may be additionally included in order to easily perform a PCR reaction, and components necessary for performing electrophoresis to confirm amplification of a PCR product are present. It may be further included in the kit of the invention.

The present invention also provides a kit for identifying a variety of ginseng comprising the SSR primer pair according to the present invention. The breed identification method is as described above. In addition to the DNA polymerase and the PCR reaction buffer of the above-described composition in order to be able to easily perform the PCR reaction may be further included, the components or known to perform electrophoresis to confirm whether the PCR product amplification or not Identification criteria for selected varieties may be further included in the kits of the present invention.

Ginseng that can be applied to the present invention may be Panax ginseng CA Mey and variants thereof, but is not limited thereto. Preferably Panax ginsen g CA Mey.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> Genomic DNA Extraction

Genomic DNA was extracted from cultivated ginseng (Graduate of Agricultural Biotechnology Research Institute, Conservation No .: G03004) according to the method using SDS (Tai et al ., Plant Mol. Biol. Reporter , 8: 297-303, 1990). This will be described in detail as follows. 0.5 g of leaf tissues were taken from young ginseng plants at 1 month of growth and placed in a 1.5 ml tube and quenched with liquid nitrogen. The leaf was then ground finely with a plastic rod immediately. Before the ground leaves were dissolved, 750 μl of extraction buffer (200 mM Tris-HCl, pH 8.0), 200 mM NaCl, 25 mM EDTA, 0.5% SDS) was added and the mixture was stored at 65 ° C. for 30 minutes. An equivalent amount of a phenol (chloro): chloroform: isoamylalchol (25: 24: 1) solution was further added thereto, followed by stirring for about 15 minutes. It was centrifuged for 15 minutes at 13,000 rpm. The supernatant was taken and transferred to a new tube. Equal amount of isopropanol (-20 ° C.) was added to the tube and stored at −20 ° C. for 1 hour. Then, it was centrifuged for 15 minutes at 13,000 rpm. After discarding the supernatant, the precipitated DNA was washed with 70% ethanol. After the DNA was dried, it was sufficiently dissolved by adding TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.4) containing about 300 µl of RNase (50 µg / ml), followed by 1 hour at 37 ° C. Stored.

<Example 2> Ligation with Adapters

500 ng of genomic DNA obtained in Example 1 was digested with Eco RV, Dra I, Sma I, Pvu I, Alu I, Hae III and Rsa I according to the instructions of each restriction enzyme manufacturer. After cleaved DNA was electrophoresed in a 1.2% agarose gel, only DNA fragments of about 300-1500 bp size were obtained by elution from the gel. The DNA fragments obtained were then ligated overnight at 12 ° C. using two adapters (AP-11 and AP-12, 50 ng each) and a T4 DNA ligase (Promega, USA). At this time, in order to increase ligation efficiency, the AP-12 adapter phosphorylated 5 'end with alkaline phosphatase (Promega, USA), and the composition of the reaction solution during ligation was as follows: 1X ligation buffer (Promega). , USA), 50 ng AP11 adapter, 50 ng AP12 adapter, 150 ng DNA fragment, 3 Unit T4 ligation (Promega, USA).

Adapter used in the present invention Adapter name Sequence SEQ ID NO: AP-11 5'-CTCTTGCTTAGATCTGGACTA-3 ' 45 AP-12 5'-TAGTCCAGATCTAAGCAAGAGCACA-3 ' 46

<Example 3> Selection and Isolation of DNA Fragments Containing Supersatellites

<3-1> Magnetic Bead Preparation

The solution was removed after collecting 0.6 ml of magnetic beads (SA-PMPs, Promega, USA; 1 mg / ml) with a magnetic stand (Promega, USA). 0.6 ml of 0.5x Saline-Sodium Citrate (SSC) buffer was added thereto, washed, and collected again using a magnetic separator to remove the solution. Washing with 0.5 × SSC buffer was performed three times. 100 μl of 0.5 × SSC buffer was added to the washed magnetic beads and used within 30 minutes.

<3-2> hybridization

Distilled water was added to the ligation product prepared in Example 2 to 500 μl, and then denatured at 65 ° C. for 10 minutes. Biotin-labeled SSR probe 1.5 μl (20ng) and 20 μl SSC buffer 13 μl were added thereto and left for 10 minutes at room temperature to induce binding. The eight SSR probes used for hybridization are listed in Table 3 below, and the SSR probes were labeled with biotin according to conventional methods known in the art.

Sequence of probe to isolate supersatellite of ginseng order SEQ ID NO: Biotin- (GA) ₂₀ 47 Biotin- (AT) ₂₀ 48 Biotin- (CA) ₂₀ 49 Biotin- (AGC) ₁₅ 50 Biotin- (GGC) ₁₅ 51 Biotin- (AAG) ₁₅ 52 Biotin- (AAC) ₁₅ 53 Biotin- (AGG) ₁₅ 54

Through hybridization reaction, DNA fragments having the repeat base sequence of genomic DNA fragments will complementarily bind to biotin-labeled SSR probes, which are called SSR probe-template hybrids.

<3-3> Separation of Template

The magnetic bead solution prepared in <3-1> was placed in the SSR probe-template hybrid solution in <3-2> and allowed to stand at room temperature for 10 minutes. Magnetic beads in which the SSR probe-template hybrid was bound were collected by using a magnetic separator in the reaction solution, and the supernatant was removed. The magnetic beads combined with the SSR probe-template hybrid were washed four times with 300 μl of 0.1 × SSC, and then 50 μl of distilled water was added and heated at 90 ° C. for 5 minutes to separate the template.

<3-4> PCR reaction

The PCR reaction was performed using the AP-11 adapter as a primer on the template isolated in <3-3>. The composition of the PCR reaction mixture (20 μl) is as follows: 250 ng adapter-ligated DNA, 10 pmol AP11 primer, 2 mM dNTP, 2 units Taq DNA polymerase, 5 μl 10 × buffer. PCR reactions were heated at 72 ° C. for 2 minutes, 94 ° C. for 3 minutes, and then at 94 ° C. for 30 seconds; 30 seconds at 56 ° C .; And 1 minute at 72 ° C. for 24 cycles, and finally at 72 ° C. for 10 minutes.

In this manner, in the present invention, 500 DNA fragments containing supersatellites were isolated.

<Example 4> Sequencing and Primer Design of Isolated DNA Fragments

DNA fragments containing the supersatellite isolated in Example 3 were cloned into pGEM T-easy vector vector (Promega, USA) to analyze their sequence. Sequence analysis was performed by requesting from Genotech. Only fragments containing 5 or more repeat units were selected based on the analyzed sequences. Based on the part containing the repeating unit, a primer in both directions was designed to amplify the supersatellite. A total of 200 pairs of primer pairs were designed (results not shown).

<Example 5> Selection of SSR Markers

In order to select an SSR primer combination showing a lot of polymorphic variation in various ginseng lines among the primer pairs designed in Example 4, DNA profiling through PCR for 16-point cultivated ginseng or ginseng varieties shown in Table 4 below ( profiling) analysis was performed.

Ginseng Used for SSR Analysis Deposit number Depositary Institution Collection country (country of origin) Type G03004 Rural Development Administration KOR Panax ginseng CA Meyer G03034 Rural Development Administration KOR Panax ginseng CA Meyer G03138 Rural Development Administration KOR Panax ginseng CA Meyer G03146 Rural Development Administration KOR Panax ginseng CA Meyer G03181 Rural Development Administration KOR Panax ginseng CA Meyer G03182 Rural Development Administration JPN Panax ginseng CA Meyer G03184 Rural Development Administration RUS Panax ginseng CA Meyer Gopung Rural Development Administration KOR Panax ginseng CA Meyer Seonpung Rural Development Administration KOR Panax ginseng CA Meyer G04056 Rural Development Administration KOR Panax ginseng CA Meyer G04075 Rural Development Administration KOR Panax ginseng CA Meyer G04085 Rural Development Administration KOR Panax ginseng CA Meyer G04093 Rural Development Administration KOR Panax ginseng CA Meyer G04114 Rural Development Administration CHN Panax ginseng CA Meyer G04116 Rural Development Administration USA Panax ginseng CA Meyer G04121 Rural Development Administration KOR Panax ginseng CA Meyer

The composition of each PCR reaction mixture (20 μl) was as follows: 40 ng of ginseng template DNA isolated by the method of Example 1, 0.2 μM forward primer, 0.6 μM reverse primer, M13 primer labeled with fluorescent material (TGTAAAACGACGGCCAGT, SEQ ID NO: 55) 0.6 μΜ, 1 × PCR reaction solution (10 mM Tris-HCl, pH 8.8), 1.5 mM MgCl ₂ , 50 mM KCl, 0.1% Triton X-100), 1 unit DNA polymerase. PCR reaction was performed after denaturing the template DNA for 3 minutes at 94 ℃, 30 seconds at 94 ℃; 30 seconds at the temperature corresponding to the T _m of each primer (see Table 1); And 35 repetitions under conditions of 45 seconds at 72 ° C., and then 30 seconds at 94 ° C .; 45 sec at 53 ° C .; And 15 repetitions at 45 ° C. for 45 seconds, and finally at 72 ° C. for 15 minutes.

An automated sequencing device (ABI 3100 Genetic Analyzer, Applied Biosystems, USA) was used to analyze the size of PCR product fragments amplified with different fluorescent materials. For the analysis, 1.5 μl of PCR product, 9.2 μl of Hi-di formamide, and 0.3 μl of internal size standard (Genscan-500 ROX) were mixed and denatured at 94 ° C. for 3 minutes. It was.

As a result, we identified 22 pairs of SSR primer pairs showing polymorphisms in ginseng cultivation and ginseng varieties collected domestically and abroad. GB-PG-007 (primary pairs represented by SEQ ID NOs: 1 and 2), GB-PG- 009 (primary pairs represented by SEQ ID NOs: 3 and 4), GB-PG-014 (primary pairs represented by SEQ ID NOs: 5 and 6), GB-PG-026 (primary pairs represented by SEQ ID NOs: 7 and 8), GB-PG-033 (primary pairs represented by SEQ ID NOs: 9 and 10), GB-PG-041 (primary pairs represented by SEQ ID NOs: 11 and 12), and GB-PG-043 (SEQ ID NOs: 13 and 14) Primer pairs), GB-PG-057 (primary pairs represented by SEQ ID NOs: 15 and 16), GB-PG-060 (primary pairs represented by SEQ ID NOs: 17 and 18), GB-PG-065 (SEQ ID NOs: 19 and Primer pair represented by 20), GB-PG-075 (primary pair represented by SEQ ID NOs: 21 and 22), GB-PG-077 (primer pair represented by SEQ ID NOs: 23 and 24), and GB-PG-078 ( Represented by SEQ ID NOs: 25 and 26 Primer pairs), GB-PG-114 (primary pairs represented by SEQ ID NOs: 27 and 28), GB-PG-131 (primary pairs represented by SEQ ID NOs: 29 and 30), and GB-PG-142 (SEQ ID NO: 31 And primer pairs represented by 32), GB-PG-150 (primer pairs represented by SEQ ID NOs: 33 and 34), GB-PG-152 (primer pairs represented by SEQ ID NOs: 35 and 36), GB-PG-157 (Primary pairs represented by SEQ ID NOs: 37 and 38), GB-PG-169 (primary pairs represented by SEQ ID NOs: 39 and 40), GB-PG-177 (primary pairs represented by SEQ ID NOs: 41 and 42), and GB -PG-186 (primary pairs represented by SEQ ID NOs: 43 and 44) was selected (see Table 1). The size of each PCR fragment amplified by the 22 pairs of primers is shown in Table 5 below.

Type and size of amplified supersatellites according to primers (unit: bp) G03004 G03034 G03138 G03146 G03181 G03182 G03184 Gopung GB-PG-007 177-233 177-231 177-231 177-231 177-231 231 177-231 177-231 GB-PG-009 170-334 334 170-334 150-334 334 334 170-334 170-334 GB-PG-014 301 301 301 301 301 301 301 301 GB-PG-026 126-162 126-168 126 126-168 126-177 126 126 126-156 GB-PG-033 335 335 335 335 335 335 335 335 GB-PG-041 296 296 296 296 296 296 296 296 GB-PG-043 184-206 184-206 90-182 184-206 184-206 184-206 184-232 184-232 GB-PG-057 288 288 288 288 288 288 288 288 GB-PG-060 163-178 163-178 163-178 163-178 163-178 163-178 163-178 163-178 GB-PG-065 136-148 136-157 139-154 139-154 136-148 136-148 139-154 139-148 GB-PG-075 251-253 251-253 251-253 251-253 251-253 251-253 251-253 251-253 GB-PG-077 235 235 235 235 235 235 235 235 GB-PG-078 264 264 264 264 264 264 264 264 GB-PG-114 230-239 230-239 - 230-239 230-239 230-239 230-239 230-239 GB-PG-131 202-205 202-205 202-205 202-205 202-205 202-205 202-205 202-205 GB-PG-142 162-174 162-174 162-174 162-174 166-174 166-174 162-174 162-174 GB-PG-150 217-241 217-241 217-241 217-241 217-241 217-241 217-241 217-241 GB-PG-152 255 255 255 255 255 255 255 255 GB-PG-157 282 282 282 282 282 282 282 282 GB-PG-169 231 231-276 231 231 231 231 231 231-276 GB-PG-177 238-241 106-238 112-286 112-238 238-241 238-241 241 238 GB-PG-186 174 174 174 174 174 174 174 174

Seonpung G04056 G04075 G04085 G04093 G04114 G04116 G04121 GB-PG-007 177-231 177-233 177-231 177-231 177-231 215-237 215-237 177-231 GB-PG-009 238-334 334 334 170-334 170-334 170 170 334 GB-PG-014 301 301 301 301 301 289-295 289-295 301 GB-PG-026 126 126 126 126 126-168 108 108 126-171 GB-PG-033 335 335 335 335 335 335-371 335-371 335 GB-PG-041 296 296 296 296 296 239-305 239-305 296 GB-PG-043 184-232 184-206 184-206 184-206 184-206 184 182-206 184-232 GB-PG-057 288 288 288 288 288 256-270 256-270 288 GB-PG-060 163-178 163-178 163-178 163-178 163-178 166-178 166-178 163-178 GB-PG-065 136-154 136-154 136-154 139-148 136-154 139 139 139-154 GB-PG-075 251-253 251-253 251-253 251-253 251-253 253-257 253-257 251-253 GB-PG-077 235 235 235 235 235 233-235 233-235 235 GB-PG-078 264 264 264 264 264 252-264 252-264 264 GB-PG-114 230-239 239-245 230-239 239 230-239 227 227 230-239 GB-PG-131 202-205 202-205 202-205 202-205 202-205 199 199 202-205 GB-PG-142 166-174 162-174 166-174 162-174 162-174 213-261 183-261 166-174 GB-PG-150 217-241 217-250 217-241 217-241 217-241 217-223 217-223 217-241 GB-PG-152 255 255 255 255 255 257 259 255 GB-PG-157 282 282 282 282 282 225 225-282 282 GB-PG-169 231 231 231-270 231-270 231-270 231-237 231-237 231-270 GB-PG-177 238-241 238-241 139-235 238-247 238-241 238 139-238 139-238 GB-PG-186 174 174 174 174 174 174-212 174-212 174

As described above, in the present invention, the first SSR marker was developed in ginseng. SSR primer pairs provided in the present invention can be very useful for effectively preparing the DNA profile of ginseng by effectively detecting the DNA polymorphism of ginseng. Through this, by efficiently evaluating the genetic resources of ginseng, it is possible to effectively perform redundancy analysis and establishment of novelty with existing resources when introducing new genetic resources, and to identify varieties of ginseng.

Attach sequence list electronic file  

Claims (7)

Simple sequence repeat (SSR) primer pair having two nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 44. The method of claim 1, wherein the primer pair represented by SEQ ID NO: 1 and 2; Primer pairs represented by SEQ ID NOs: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; Primer pairs represented by SEQ ID NOs: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; Primer pairs represented by SEQ ID NOs: 11 and 12; Primer pairs represented by SEQ ID NOs: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; Primer pairs represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; Primer pairs represented by SEQ ID NOs: 21 and 22; Primer pairs represented by SEQ ID NOs: 23 and 24; Primer pairs represented by SEQ ID NOs: 25 and 26; Primer pairs represented by SEQ ID NOs: 27 and 28; Primer pairs represented by SEQ ID NOs: 29 and 30; Primer pairs represented by SEQ ID NOs: 31 and 32; Primer pairs represented by SEQ ID NOs: 33 and 34; Primer pairs represented by SEQ ID NOs: 35 and 36; Primer pairs represented by SEQ ID NOs: 37 and 38; Primer pairs represented by SEQ ID NOs: 39 and 40; An SSR primer pair selected from the group consisting of a primer pair represented by SEQ ID NOs: 41 and 42 and a primer pair represented by SEQ ID NOs: 43 and 44. The SSR primer pair according to claim 1 or 2, which is derived from Ginseng ( Panax ginseng CA Meyer). (a) extracting genomic DNA from ginseng; (b) using the extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; And (c) DNA polymorphism detection method of ginseng comprising the step of separating the PCR product by size. (a) extracting genomic DNA from ginseng and control ginseng varieties; (b) performing PCR using each of the extracted genomic DNA as a template and the SSR primer pair of claim 1; (c) separating each PCR product by size; And (d) a method of identifying varieties of ginseng comprising the step of comparing the separation results by size of the ginseng and control ginseng varieties. A kit for detecting DNA polymorphism of ginseng comprising the SSR primer pair of claim 1. A kit for identifying a variety of ginseng comprising the SSR primer pair of claim 1.