KR100842446B1 - Ssr primer derived from ginger and use thereof - Google Patents

Abstract

An SSR(simple sequence repeat) primer pair derived from ginger is provided to detect DNA polymorphism of the ginger effectively and be very usefully used for DNA profiling of the ginger, thereby analyzing whether a newly introduced genetic resource is duplicated with a conventional resource and securing novelty of the newly introduced genetic resource by evaluating the genetic source of the ginger efficiently. 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; and a primer pair of SEQ ID : NOs. 15 and 16 and is derived from ginger(Zingiber officinale Rosc.). A method for detecting DNA polymorphism of the ginger comprises the steps of: (a) extracting genome DNA from the ginger; (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 method for identifying species of the ginger comprises the steps of: (a) extracting genome DNA from the ginger and a control group ginger species, respectively; (b) subjecting each of the extracted genome DNA to PCR using the SSR primer pair; (c) isolating each of PCR products obtained from the step(b) by size; and (d) comparing isolation results by the size of the ginger with that of the control group. A kit for detecting the DNA polymorphism of the ginger or identifying species of the ginger comprises the SSR primer pair.

Description

SSR primer derived from ginger and use thereof

The present invention ginger (Ginger; Zingiber SSR primer isolated from officinale Rosc.) and its use, and more particularly, SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16 and PCR using the same It relates to a DNA polymorphism detection method of ginger.

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 Ginger, the SSR marker has not been developed in Korea at all, and there is little research related to it in foreign countries.

Accordingly, the present inventors have completed the present invention by developing SSR primer pairs that show a large number of polymorphic variations in various ginger strains while researching to develop an SSR marker capable of efficiently evaluating ginger's gene source.

It is therefore an object of the present invention to provide an SSR marker and its use capable of efficiently evaluating ginger's gene source.

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: 16.

In order to achieve another object of the present invention, the present invention provides a method for detecting DNA polymorphism of ginger and a method for identifying a variety of ginger, 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 ginger and a kit for identifying a variety of ginger comprising 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 ginger.

In the present invention, SSR markers were developed from ginger by biotin-streptavidin capture (Dixit et al., Mol. Eco.Note, 5: 736-738). The SSR marker isolated from ginger 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 ginger 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 ginger supernatant.

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. Eight pairs of SSR primer pairs showing polymorphic variation were selected by performing DNA profiling through PCR of various cultivated ginger lines and varieties using the primer pairs.

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: 16. Preferably GB-ZO-033 (primary pairs represented by SEQ ID NOs: 1 and 2), GB-ZO-040 (primary pairs represented by SEQ ID NOs: 3 and 4), GB-ZO-055 (SEQ ID NOs: 5 and 6) Primer pairs represented by a pair), GB-ZO-064 (primer pairs represented by SEQ ID NOs: 7 and 8), GB-ZO-103 (primer pairs represented by SEQ ID NOs: 9 and 10), and GB-ZO-107 (SEQ ID NO: Group consisting of primer pairs represented by numbers 11 and 12), GB-ZO-111 (primer pairs represented by SEQ ID NOs: 13 and 14), and GB-ZO-140 (primer pairs represented by SEQ ID NOs: 15 and 16). Is selected. 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, the T _m (° C.) and the chromosomal alleles in which the supersatellites are present Are 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-ZO-033 F: CAGCAGATTTTTGCTCCG One (GTC) ₆ 86-239 55 6 R: GTCGCGTTCGTGGAAAT 2 GB-ZO-040 F: TCTCCCTCTCGGATCCAT 3 (GGC) ₅ 172-178 60 2 R: ATCCATTGCCTGATGGTG 4 GB-ZO-055 F: GTGAGCAGAAAACAGCCG 5 (CTG) ₅ 270-288 58 4 R: TCGCCAATTGAAGACCAC 6 GB-ZO-064 F: CGTAGGATCTTCCCGACC 7 (GGA) ₅ 252-258 60 3 R: CGAGTGAACCCATGGAGA 8 GB-ZO-103 F: GCTGCGGACTAAATGCTG 9 (CCT) ₂ CTT (CCT) ₄ 117-246 63 3 R: ACGCTAGGGAACAGGGAG 10 GB-ZO-107 F: TCCAAAGGTGCTGTTGCT 11 (GTG) ₂ CTC (GTG) ₄ 181-184 58 2 R: CTGTGTTTTTCTTCGCCG 12 GB-ZO-111 F: TAACCGGGAGAAAACCGT 13 (GA) ₉ , (GAG) ₄ 279-291 52 4 R: ACTCGTCCGATTCCGATT 14 GB-ZO-140 F: AGGGGGCAGTGGAGAG 15 (GGA) ₄ 100-280 55 8 R: ACGTTCCTGCACTTGACG 16

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

The SSR primer pairs provided in the present invention can be usefully used to detect DNA polymorphism and analyze genetic diversity in ginger. Using the SSR primers according to the present invention can efficiently evaluate and preserve the genetic resources of ginger. Accordingly, the present invention provides a method for detecting DNA polymorphism of ginger, including performing PCR using the SSR primer pair.

Specifically, ginger's DNA polymorphism detection method

(a) extracting genomic DNA from ginger;

(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 contains genomic DNA extracted from ginger to be analyzed and an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water (dH ₂ O) in addition to 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 a variety of ginger, including performing PCR using the SSR primer pair.

Specifically, how to identify ginger varieties

(a) extracting genomic DNA from ginger and control ginger 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) the comparison of ginger and control ginger varieties is the same combination of SSR primers The size of each PCR product of the ginger and the control ginger varieties, which are samples for the pair, is compared with each other. By comparing the results of the size comparison for each SSR primer pair, if the sample is consistent with the results of the ginger and the control ginger varieties, the ginger may be identified as the same varieties as the control ginger 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 size separation of PCR products for the control ginger varieties may be performed simultaneously with the sampled ginger, but may be performed prior to the sampled ginger 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 size separation of PCR products for ginger as a sample.

The present invention also provides a kit for detecting DNA polymorphism of ginger 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 ginger comprising an 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.

Ginger that can be applied to the present invention is Zingiber officinale Rosc and variants thereof, but is not limited thereto. Preferably Zingiber It can be officinale Rosc.

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 ginger (Gragen Institute of Agriculture and Biotechnology, Conservation No .: A001) 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 tissue was taken from the young ginger plant 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 ' 17 AP-12 5'-TAGTCCAGATCTAAGCAAGAGCACA-3 ' 18

<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 ginger supernatant order SEQ ID NO: Biotin- (GA) ₂₀ 19 Biotin- (AT) ₂₀ 20 Biotin- (CA) ₂₀ 21 Biotin- (AGC) ₁₅ 22 Biotin- (GGC) ₁₅ 23 Biotin- (AAG) ₁₅ 24 Biotin- (AAC) ₁₅ 25 Biotin- (AGG) ₁₅ 26

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 ginger lines among the primer pairs designed in Example 4, DNA profiling through PCR for 16-point cultivated ginger or ginger varieties shown in Table 4 below ( profiling) analysis was performed.

Ginger used for SSR analysis Deposit number Depositary Institution Collection country (country of origin) Type A001 Rural Development Administration Malaysia Zingiber officinale Rosc. A005 Rural Development Administration Malaysia Zingiber officinale Rosc. A011 Rural Development Administration Malaysia Zingiber officinale Rosc. A012 Rural Development Administration Malaysia Zingiber officinale Rosc. A016 Rural Development Administration Malaysia Zingiber officinale Rosc. A019 Rural Development Administration Malaysia Zingiber officinale Rosc. A022 Rural Development Administration Malaysia Zingiber officinale Rosc. A023 Rural Development Administration Malaysia Zingiber officinale Rosc. A024 Rural Development Administration Malaysia Zingiber officinale Rosc. A033 Rural Development Administration Malaysia Zingiber officinale Rosc. A036 Rural Development Administration Malaysia Zingiber officinale Rosc. A038 Rural Development Administration Malaysia Zingiber officinale Rosc. A043 Rural Development Administration Malaysia Zingiber officinale Rosc. A047 Rural Development Administration Malaysia Zingiber officinale Rosc. A054 Rural Development Administration Malaysia Zingiber officinale Rosc. A057 Rural Development Administration Malaysia Zingiber officinale Rosc.

The composition of each PCR reaction mixture (20 μl) was as follows: 40 ng of ginger 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: 27) 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 8 pairs of SSR primer pairs showing polymorphism in the ginger cultivation and ginger varieties collected domestically and internationally. GB-ZO-033 (primer pairs represented by SEQ ID NOs: 1 and 2), GB-ZO- 040 (primary pairs represented by SEQ ID NOs: 3 and 4), GB-ZO-055 (primary pairs represented by SEQ ID NOs: 5 and 6), GB-ZO-064 (primer pairs represented by SEQ ID NOs: 7 and 8), GB-ZO-103 (primary pairs represented by SEQ ID NOs: 9 and 10), GB-ZO-107 (primary pairs represented by SEQ ID NOs: 11 and 12), and GB-ZO-111 (SEQ ID NOs: 13 and 14) Primer pairs), and GB-ZO-140 (primer pairs represented by SEQ ID NOs: 15 and 16) were selected (see Table 1). The size of each PCR fragment amplified by the eight pairs of primers is shown in Table 5 below.

Type and size of amplified supersatellites according to primers (unit: bp) A001 A005 A011 A012 A016 A019 A022 A023 GB-ZO-033 152 152 152 152 86/113 152/239 152 152 GB-ZO-040 178 178 178 178 178 178 178 178 GB-ZO-055 288 288 288 - 288 288 288 288 GB-ZO-064 255/258 255/258 - 255/258 - 255/258 - 255/258 GB-ZO-103 246 246 246 246 246 246 - 246 GB-ZO-107 184 184 184 184 184 184 184 184 GB-ZO-111 279/287 279/287 279/287 279/287 279/287 279/287 - 279/287 GB-ZO-140 268 268 - 100/268 268 268 115 103

A024 A033 A036 A038 A043 A047 A054 A057 GB-ZO-033 - 146 86/152 - 152 - 158 101/152 GB-ZO-040 172/178 - 172/178 - 178 178/181 - - GB-ZO-055 - 282 - 270 288 270 270/279 288 GB-ZO-064 - 252 255/258 252 255/258 252 252 255/258 GB-ZO-103 117 - - - 246 - 243 246 GB-ZO-107 - 181 - - 184 - 181 184 GB-ZO-111 279/287 287/291 - - 279/287 - 284/291 - GB-ZO-140 268 265 268 280 160/268 280 127/265 268

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

Attach sequence list electronic file  

Claims (7)

Primer pairs represented by SEQ ID NOs: 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; A simple sequence repeat (SSR) primer pair having a nucleotide sequence selected from the group consisting of a primer pair represented by SEQ ID NOs: 13 and 14 and a primer pair represented by SEQ ID NOs: 15 and 16. delete The SSR primer pair according to claim 1, which is derived from Ginger ( Zingiber officinale Rosc.). (a) extracting genomic DNA from ginger; (b) using the extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; And (C) method for detecting the DNA polymorphism of ginger comprising the step of separating the PCR product by size. (a) extracting genomic DNA from ginger and control ginger varieties; (b) using each extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; (c) separating each PCR product by size; And (d) a method for identifying a variety of ginger comprising the step of comparing the separation results by size of the ginger and control ginger varieties. Ginger DNA polymorphism detection kit comprising the SSR primer pair of claim 1. Ginger variety identification kit comprising the SSR primer pair of claim 1.