KR101326333B1 - SNP Primers for Yunpoong Distinction of Korean Ginseng (Panax ginseng C.A.Meyer) and Method for Yunpoong Distinciton Using the Same - Google Patents

Abstract

The present invention relates to a SNP primer for distinguishing Korean ginseng lotus cultivars and a method for distinguishing Korean ginseng lotus cultivars using the same.
According to the present invention, the developed SNP primers can be used to quickly and accurately distinguish between varieties of Korean ginseng. Therefore, by developing a system that can easily distinguish foreign ginseng and Korean ginseng lotus cultivars can be useful to protect the rights of domestic farmers.

Description

SNP Primers for Differentiating Korean Ginseng Varieties and Method for Distinguishing Varieties Using the Same {SNP Primers for Yunpoong Distinction of Korean Ginseng (Panax ginseng C.A.Meyer) and Method for Yunpoong Distinciton Using the Same}

The present invention relates to a SNP primer for distinguishing Korean ginseng lotus cultivars and a method for distinguishing Korean ginseng lotus cultivars using the same.

Ginseng (Panax ginseng C.A.Meyer) has been used for a long time as one of the major medicines of the East as a primary treatment for improving fatigue and weakness. This red berry plant, native to Korea and northeastern China, is commonly called Korean ginseng and is now grown worldwide.

The pharmacological component of ginseng was Panax Ginseng quinquefolium ) Scientific research into the active ingredient of ginseng began by separating the amorphous substance, a kind of saponin mixture, from the roots. By emphasizing that it is an active ingredient, intensive research on saponins was conducted, and many studies were actively conducted in the biochemical field by the 1960s. In most ginseng, various active ingredients are found, including ginsenosides, polysaccharides, peptides, polyacetylenic alcohols, and fatty acids.

The pharmacological effects of Korean ginseng include improvement of brain function, pain reduction effect, cancer prevention effect including anti-tumor activity, improvement of immune function, antidiabetic effect, liver function, control of blood pressure, anti-fatigue and anti-stress effect, menopause Modern science has shown that there are improvements in disorders, sexual function, and anti-aging effects including antioxidants. For this reason, Korean ginseng has become the world's most remarkable herbal remedy.

Ginseng is called by various names depending on its origin and cultivation. The genus Panax consists of 17 species, including P. ginseng CAMeyer (Korean ginseng) and P. notoginseng. (Chinese ginseng, Chinese ginseng) and P. quinquefolium (American ginseng, American ginseng) is widely cultivated for its efficacy is known in advance.

Korean ginseng includes three kinds of pure varieties: Jakyung, Chungkyung and Hwangsook. To date, a total of nine varieties have been selected from the three varieties of puree by the pure-line selection method, which include Yunpoong, Gopoong, Sunpoong, Sunwun, Sunweon, Sunhyang, Chunpoong, Chungsun and Gumpoong.

Molecular biological methods used to distinguish ginseng varieties to date include Korean ginseng, including random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), restriction fragment length polymorphism (RFLPs), and simple sequence repeats (SSR) Several approaches have been attempted to elucidate genetic polymorphism between varieties. However, this method has not been put to practical use because of its lack of reproducibility and reliability as a molecular marker for identifying a single variety. In other words, the current cultivation of ginseng varieties are distinguished by external morphological features, there is no molecular marker (molecular marker) to accurately distinguish them. Therefore, there is an urgent need for the development of molecular markers for different varieties to shorten the time period for breeding varieties of ginseng and to clearly distinguish varieties in the distribution process of root ginseng.

The present invention is to provide a molecular marker that can quickly and accurately distinguish between the varieties of Korean ginseng.

In order to solve the above problems, the present invention provides a SNP primer for distinguishing Korean ginseng breeze varieties produced to amplify a polynucleotide characterized in that the 811 th nucleotide B of SEQ ID NO: 1.

The present invention also provides a kit for distinguishing Korean ginseng breeze varieties comprising SNP primers designed to amplify polynucleotides characterized in that 811 nucleotide B of SEQ ID NO: 1 is T.

In another aspect, the present invention comprises the steps of a) extracting DNA from a sample; And b) checking the presence or absence of SNP base "T" corresponding to 811 th nucleotide of SEQ ID NO: 1 in the extracted DNA.

According to the present invention, the developed SNP primers can be used to quickly and accurately distinguish between varieties of Korean ginseng. Therefore, by developing a system that can easily distinguish foreign ginseng and Korean ginseng lotus cultivars can be useful to protect the rights of domestic farmers.

1 is a diagram showing the position of the SNP specific to the breeze in the nucleotide sequence of the GAPDH gene. A shows the SNP located in the exon 2 region of the GAPDH gene, and B shows the difference in sequence between the mutant and other varieties at the SNP position (1: hurricane, 2: breeze, 3: archaic, 4: kumquat, 5: Whirlwind, 6: American ginseng).
Figure 2 is a photograph showing the results of electrophoresis of the PCR product using the yunF / yunR1 primer pair according to the present invention ( A : 1: heaven, 2: gust, 3: old wind, 4: golden wind, 5: whirlwind, 6: USA 3, M: 1 kb DNA marker; B : 1: hurricane, 2: gust, 3: archaic, 4: golden gust, 5: whirlwind, 6: good luck, 7: sailor, 8: incense, 9: blue wire, 10: Hwang Sook-jong, 11: mimaki-Japanese ginseng variety, 12: American ginseng, M: 1 kb DNA marker).
Figure 3 is a diagram showing the real-time PCR results and melting curve (A: results of real-time PCR amplification using yunF / yunR1 primer pair, B: results of melting curve analysis using yunF / yunR1 primer pair, C: yunF / Real-time PCR amplification using yunR2 primer pair, D: melting curve analysis using yunF / yunR2 primer pair)
Figure 4 is a view showing the melting curve analysis results using a yunF / yunR2 primer pair ( A: SYBR Green I dye, B: Eva Green dye).
Figure 5 shows the nucleotide sequence of the GAPDH gene of the storm varieties.
Figure 6 shows the nucleotide sequence of the GAPDH gene of the yeonyeongi.
Figure 7 shows the nucleotide sequence of the GAPDH gene of the old-fashioned varieties.
Figure 8 shows the nucleotide sequence of the GAPDH gene of Geumpung varieties.
Figure 9 shows the nucleotide sequence of the GAPDH gene of the whirlwind varieties.

The present invention provides SNP primers for discriminating Korean ginseng breeze varieties, which are designed to amplify polynucleotides, wherein 811th nucleotide B of SEQ ID NO: 1 is T.

As can be seen through the following examples, the inventors confirmed that 811th nucleotide in the nucleotide sequence of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was found to be T and other varieties C and G, whereas SEQ ID NO: 1 The 811th nucleotide of the base sequence of GAPDH gene was determined as SNP (single nucleotide polymorphism) position to distinguish the cultivars of Korean ginseng.

Therefore, the present invention uses the SNP position as a molecular marker, thereby providing an SNP primer capable of specifically distinguishing the cultivars of Korean ginseng. By using the SNP primer, Korean ginseng breeze varieties can be quickly and accurately distinguished from other varieties.

According to one embodiment of the present invention, the SNP primer may be composed of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

A primer consisting of the nucleotide sequence of SEQ ID NO: 2 is a forward primer, and a primer consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 is a reverse primer.

The reverse primer consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 is characterized in that the 3 'terminal nucleotide corresponds to the SNP position, and the 3' terminal nucleotide is A so as to specifically detect a cultivar. In addition, the reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3 replaced the base A located second from the 3 'terminal nucleotide by C, and the reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4 was located immediately next to the 3' terminal nucleotide. A was replaced with G. This is to induce an absolute specificity for the brine breed by introducing additional mismatches at the 3 'end.

Therefore, if PCR amplification is performed using the SNP primer pair consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or the SNP primer pair consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4, through the generation of PCR products Korean Ginseng Yeonpung species can be distinguished.

In another aspect, the present invention provides a kit for distinguishing Korean ginseng breeze varieties comprising SNP primers prepared to amplify polynucleotides characterized in that the 811 th nucleotide B of SEQ ID NO: 1.

According to an embodiment of the present invention, the SNP primer consists of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Korean ginseng lotus cultivar discrimination kit according to the present invention, in addition to the SNP primers, including DNA polymerase, dNTPs, etc., including all biological or chemical reagents, instructions for use, etc. Can be. Other configurations of such kits may be appropriately selected by those skilled in the art.

In another aspect, the present invention comprises the steps of a) extracting DNA from a sample; And b) checking the presence or absence of SNP base “T” corresponding to 811 th nucleotide of SEQ ID NO: 1 in the extracted DNA. If the presence of SNP base "T" corresponding to the 811th nucleotide of SEQ ID NO: 1 in the DNA extracted from the sample is confirmed, it can be distinguished as the Korean ginseng yeonpo varieties.

According to an embodiment of the present invention, step b) may be performed by PCR amplification using an SNP primer prepared to amplify a polynucleotide, wherein 811th nucleotide B of SEQ ID NO: 1 is T. .

In this case, the PCR amplification may be, but is not limited to, general PCR amplification or real-time PCR amplification.

When PCR product is generated as a result of performing general PCR amplification using the SNP primer, it can be distinguished into Korean ginseng cultivar varieties, and real-time PCR amplification using the SNP primer is used to measure the amount of fluorescence of PCR product. When the production of the PCR product is confirmed, it can be distinguished into Korean ginseng yeonryum variety.

At this time, the SNP primer may be made of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

According to the following examples, PCR amplification was performed using the SNP primer pair consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or the SNP primer pair consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4, It was confirmed that only 159bp of PCR product was detected in the sample (FIG. 2).

In addition, as a result of real-time PCR amplification using the SNP primer pair consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or the SNP primer pair consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4, It was confirmed that the cycle curves and melting curves produced distinctive peaks distinguished from other varieties (FIGS. 3 and 4).

Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited by the following examples. In addition, the embodiment of the present invention was carried out after PCR of the tissues of the ginseng growing normally to confirm that the DNA of each part is the same in the individual, DNA results obtained from the root or leaf of the ginseng in the same individual It is based on the same appearance. In addition, all examples of the present invention were applied to the double blind method.

Example  1: experimental materials and DNA  extraction

All ginseng leaves and roots were obtained from Kyung Hee University Ginseng Material Bank. As shown in Table 1, in the present embodiment, Korean ginseng ( P. ginseng ) (heaven, lotus, archaize, geum, breeze, sun cloud, sailor, incense, blue sea), Japanese ginseng (Mimaki) and varieties (hwangsukjong) , And American ginseng ( P. quinquefolius ) was used.

number designation Classification group Total number One Chunpoong Korean cultivar Leaf 9 2 Yunpoong Korean cultivar Leaf 10 3 Gopoong Korean cultivar Leaf 7 4 Gumpoong Korean cultivar Leaf 5 5 Sunpoong Korean cultivar Leaf 5 6 Sunwoon Korean cultivar Leaf 5 7 Sailor Korean cultivar Leaf 5 8 Sunhyang Korean cultivar Leaf 5 9 Chungsun Korean cultivar Leaf 5 10 Hwangsookjong Korean variety Leaf 9 11 Mimaki Japanese cultivar Leaf 5 12 American hemp
(P. quinquefolius ) American ginseng Leaf 2

The ginseng DNA was extracted by two methods.

a. DNA kit method

Plant DNA Isolation Genomic DNA was extracted from plant leaves using SV mini kit (GeneAll, Korea).

b. Modified NaOH-Tris Method

Young leaves (calus or placenta) were removed and placed in a 1.5 ml tube and 50 μl of 4 M NaOH was added. Thereafter, the leaves were ground using Tissue Lyser II (QIAGEN; 5-mm bead, 60 sec, 30 Hz). 5 μl of the grinding material was quickly transferred to a new tube containing 295 μl of 100 mM Tris (pH 8.0), mixed well, and then 1 μl of the mixture was brought to 10 μl of the total volume and used for the real-time PCR reaction.

Example  2: SNP  Location determination

EST  Sequencing

Hairy roots (DC01), 14 year old roots (DC02), 4 year old roots (DC03), flower buds (DC04), leaves (DC05) and ginseng callus (DC06) were used as plant materials for 20,000 EST ( Ginseng Material Bank, Kyung Hee University, Korea). Sequence chromatogram files were transferred to the PHRED program for basecalling. Vector NTI AdvanceTM 10 was then used to clean up the vector sequence, adapter region and poly (A) tail. Sequences with up to 100 bases were removed. EST was combined and processed into a contiguous sequence using CAP3 software. The consensus sequence of each contig and singleton consisted of unique sequences compared to the GenBank database. As a result, the EST was divided into three groups: Known (score 80 or higher, E-value 10-14 or less), unknown (score 40 or more or less than 80, E-value 10-14 or less, 10-2 or more), no hit ( Score below 40, E-value above 10-2).

PCR  Amplification

A total of 44 EST sequences were randomly selected from overexpression, stress, color and saponin related genes based on the EST database. Intron-flanking primers were designed to increase the likelihood of length polymorphism at the DNA level. 44 pairs of primer sets were designed using Primer3 (http://frodo.wi.mit.edu/primer3/) and Primer Premier 5.0 software (Premier, Canada). The parameters used at this time are as follows: primer length 20-26 nucleotides (22 nucleotides optimal), PCR product size 150-1200 bp, optimum annealing temperature 58 ° C., GC content 30-50% (45% optimal). The primers were obtained from Genotech Co. Ltd. (Daejeon, Korea).

PCR was performed using the 44 pairs of primer sets for genomic DNA of the gust, gust, arch, gold, gust and P. quinquefolius . PCR amplification was performed using Corbett PCR (Corbett Research, Sydney, Australia, Model: CG1-96). Each PCR was performed in a total volume of 20 μl with 20 ng of template DNA, 0.5 μM of each primer, 200 μM dNTPs, 1.5 mM MgCl ₂ , 1X PCR buffer and 1 U of DNA polymerase (Enzynomics TM, Korea). . Standard PCR amplification was previously denatured at 94 ° C. for 5 minutes, followed by 35 repeated amplification cycles of 30 seconds at 94 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C. It was carried out for 7 minutes at 72 ° C. PCR products were electrophoresed on 1.0% agarose gel, and were observed under UV (DNR MiniBIS Pro Bio-Imagining System, Israel) by ethidium bromide (Et-Br) staining. If the size of the PCR band is greater than expected, the PCR product was sequenced.

DNA  Sequencing

DNA sequences sequenced by direct-sequencing method were analyzed using SeqManII. Sequence alignments were performed using the ClustalW2 and Kalign 2.0 programs (http://www.ebi.ac.uk/Tools/sequence.html). The aligned sequences were analyzed for exon regions, SNPs and InDels sequence positions using BioEdit 7.0 software.

SNP  Location determination

Through sequencing and alignment of the DNA sequence, the breeze had nucleotide T at position 811 in the exon region of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a total length of 2321 bp, but the other varieties and American ginseng were identical. It was confirmed to have nucleotides C and G in. Therefore, this was determined as the SNP position.

Figure 1 shows the SNP position specific to the breeze in the base sequence of the GAPDH gene. A shows the position of the SNP marker primer in the exon 2 region of GAPDH, and B shows the difference in sequence between the mutant and other varieties at the SNP position (1: hurricane, 2: gust, 3: archaic, 4: geum, 5: whirlwind, 6: american ginseng). SNP positions are shown in bold text, and mismatch areas are italicized and underlined.

Example  3: Korean ginseng lotus breeze SNP Primer  design

On the basis of the SNP positions determined in Example 2, allele-specific primers yunR1 and yunR2 were designed to specifically detect Korean ginseng varieties. yunR1 substituted base A, located second from the 3 'terminal nucleotide (SNP position) with C, and yunR2 replaced base A, located next to the 3' terminal nucleotide (SNP position), with G (Table 2, FIG. One). This is to induce specificity for the mutant species by introducing an additional mismatch at the 3 'end of the yunR1 and yunR2 primers.

SEQ ID NO: Name of the primer The sequence (5'-3 ') Location in GAPDH 2 yunF CAA AGG TAT TTT ATT ATC TCT GCT G 676-700 3 yunR1 TAA TTG AGT GTA CAG TAG TCA T C A A 811-835 4 yunR2 TAA TTG AGT GTA CAG TAG TCA TA G A 811-835

Experimental Example 1: SNP for discriminating Korean ginseng breeze varieties Primer Verification

PCR  Amplification

Verification of allelic polymorphism of Yeonpung varieties was confirmed by PCR using SNP primer pair yunF / yunR1 for discriminating Korean Ginseng Yeonpung varieties prepared in Example 3. PCR amplification was performed using Corbett PCR (Corbett Research, Sydney, Australia, Model: CG1-96). Each PCR was performed in a total volume of 20 μl with 20 ng of template DNA, 0.5 μM of each primer, 200 μM dNTPs, 1.5 mM MgCl ₂ , 1X PCR buffer and 1 U of DNA polymerase (Enzynomics TM, Korea). . Standard PCR amplification was previously denatured at 94 ° C. for 5 minutes, followed by 35 repeated amplification cycles of 30 seconds at 94 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C. It was carried out for 7 minutes at 72 ° C. Gradient-PCR reaction conditions are as follows: 35 cycles of 30 seconds at 94 ° C, 30 seconds at 55 ° -64 ° C, 30 seconds at 72 ° C after denaturation at 94 ° C for 5 minutes. Repeated and finally held at 72 ° C. for 7 minutes.

PCR products were electrophoresed on 1.0% agarose gel, and were observed under UV (DNR MiniBIS Pro Bio-Imagining System, Israel) by ethidium bromide (Et-Br) staining. PCR conditions including annealing temperature (gradient-PCR) were adjusted to reduce non-specific amplification. At annealing temperatures of 58 ° C, breezes were distinguished from other varieties.

2 shows the results of electrophoresis of PCR products. Both A and B were confirmed to produce 160 bp of the product only in lane 2 corresponding to the cultivar varieties ( A : 1: gust, 2: gust, 3: archetype, 4: golden gust, 5: whirlwind, 6: American ginseng, M: 1 kb DNA marker; B : 1: storm, 2: gale, 3: archaic, 4: golden, 5: whirlwind, 6: good fortune, 7: sailor, 8: incense, 9: blue wire, 10: Hwang Suk-jong, 11: Mimaki-Japanese Ginseng Varieties, 12: American Ginseng, M: 1 kb DNA Marker).

Real - time PCR  Amplification

Thermocycling and fluorescence monitoring were performed with Rotor-Gene ™ 6000 (Corbett Life Science, Australia) for real-time PCR.

SYBR Green I conditions: The reaction mix is 9-16 ng DNA, 5 μM of each primer, 100 μM of each dNTPs, 1.5 mM MgCl ₂ , 2X mastermix (SensiMixPlus SYBR, Quantace Ltd, Australia), with TE buffer total volume of 10 μl It consisted of The PCR cycle profile is as follows: after 10 minutes of activation at 95 ° C, denaturation for 10 seconds at 95 ° C, combined annealing for 15 seconds at 60 ° C and 20 seconds at 72 ° C. A total of 40 cycles of a three-stage thermal profile that stretches (green channel setting 'On'). Melting curve analysis was performed at 65 ° C to 85 ° C with 1 ° C in each step.

Eva Green conditions: The reaction mix is 9-16 ng DNA, 5 μM of each primer, 200 μM of each dNTPs, 1.5 mM MgCl ₂ , 2X mastermix (SensiMix HRMTM, Quantace Ltd, Australia), 0.4 ul Eva Green, TE buffer The total volume consisted of 10 μl. The PCR cycle profile is as follows: after 10 minutes of activation at 95 ° C, denaturation for 15 seconds at 95 ° C, combined annealing for 20 seconds at 60 ° C and for 10 seconds at 72 ° C. A total of 40 cycles of a three-stage thermal profile that stretches (green channel setting 'On'). Melting curve analysis was performed at 65 ° C to 85 ° C in 0.1 ° C increments for each step.

Real-time PCR analysis using yunF / yunR1 primers was performed on DNA extracts obtained from the subjects described in Table 1. The fluorescence signal shows that different samples have different amplification concentrations at different cycle counts (FIG. 3A). The melting curve shows the maximum melting point at 79 ° C. (FIG. 3B). To improve the specificity of the marker, base A, located second from the 3 'end, was replaced with G and named yunR2 (Table 2). The modified markers (yunF / yunR2) were found to be more efficient than yunF / yunR1 in the melting curve analysis (Figure 3C-D).

Reproducibility tests on 72 samples using SYBR Green I and Eva Green showed that both dyes produced very distinct peaks, and the breeze was easily found in the melting curve analysis (FIGS. 4A-B).

Attach an electronic file to a sequence list

Claims (7)

SNP primer pair for distinguishing Korean ginseng breeze varieties of SEQ ID NO: 2 and 3 or SEQ ID NO: 2 and 4, which is designed to amplify polynucleotides characterized in that 811 nucleotide B of SEQ ID NO: 1 is T.
delete Kit for distinguishing Korean ginseng breeze varieties comprising SNP primer pairs of SEQ ID NO: 2 and 3 or SEQ ID NO: 2 and 4, designed to amplify polynucleotides characterized in that 811 nucleotide B of SEQ ID NO: 1 is T .
delete a) extracting DNA from a sample; And
b) checking the presence or absence of the SNP base “T” corresponding to 811 th nucleotide of SEQ ID NO: 1 in the extracted DNA;
Step b) is PCR amplification using an SNP primer pair of SEQ ID NO: 2 and 3 or SEQ ID NO: 2 and 4, which is designed to amplify a polynucleotide, characterized in that 811 nucleotide B of SEQ ID NO: 1 is T Korean ginseng breeze varieties method characterized in that performed by.
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