KR100918018B1 - SSR primer derived from amaranth and use thereof - Google Patents

Abstract

The present invention relates to an SSR primer isolated from Amaranth 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: 24, and performing PCR using the same. It relates to a DNA polymorphism detection method of Amaranth comprising. SSR primer pairs provided in the present invention can effectively detect the DNA polymorphism of the amaranth, and can be very useful for preparing the DNA profile of the amaranth. In addition, by efficiently evaluating Amaranth's genetic resources, it is possible to effectively conduct redundancy analysis and establish novelty with existing resources when introducing new genetic resources.

SSR Primer, Amaranth, DNA Polymorphism

Description

SSR primer derived from amaranth and its use {SSR primer derived from amaranth and use approximately}

The invention amaranth; SSR primers having two sequences selected from relates to the SSR primer, and the use thereof separated in (Amaranth. Amaranthus spp), and more particularly, the group consisting of SEQ ID NO: 1 to SEQ ID NO: 24 pairs, and It relates to a method for detecting DNA polymorphism of Amaranth, including 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 Amaranth, 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 amaranth strains while studying to develop an SSR marker capable of efficiently evaluating the gene source of amaranth.

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

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: 24.

In order to achieve another object of the present invention, the present invention provides a method for detecting DNA polymorphism of Amaranth and a method for identifying varieties of Amaranth, 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 Amaranth and a kit for identifying amaranth varieties 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 the amaranth.

In the present invention, SSR markers were developed from amaranth by biotin-streptavidin capture (Dixit et al., Mol. Eco.Note, 5: 736-738). SSR markers isolated from amaranth are 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 Amaranth 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 carried out using AP11 primers to amplify and analyze the sequence to obtain 500 DNA fragments containing the amaranth supersatellite.

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. The primer pairs were used to select 12 pairs of SSR primer pairs exhibiting polymorphic variation by performing DNA profiling through PCR of various cultivated Amaranth strains and varieties.

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: 24. Preferably GB-AM-013 (primary pairs represented by SEQ ID NOs: 1 and 2), GB-AM-032 (primary pairs represented by SEQ ID NOs: 3 and 4), GB-AM-051 (SEQ ID NOs: 5 and 6) Primer pairs represented by the following formula), GB-AM-071 (primary pairs represented by SEQ ID NOs: 7 and 8), GB-AM-078 (primary pairs represented by SEQ ID NOs: 9 and 10), and GB-AM-099 (SEQ ID NO: Primer pairs represented by numbers 11 and 12), GB-AM-105 (primer pairs represented by SEQ ID NOs: 13 and 14), GB-AM-123 (primer pairs represented by SEQ ID NOs: 15 and 16), GB-AM -129 (primary pairs represented by SEQ ID NOs: 17 and 18), GB-AM-132 (primary pairs represented by SEQ ID NOs: 19 and 20), and GB-AM-136 (primary pairs represented by SEQ ID NOs: 21 and 22) And GB-AM-137 (primary pair represented by SEQ ID NOs: 23 and 24). 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 Standing column Sequence number Repeat motif Range of size of supersatellite to be amplified (bp) Tm (℃) Number of alleles GB-AM-013 ^a F TGAATCCTTATGCGCCAC One (AGG) ₄ 151-160 52 4 ^b R GGAGCCCTGTCCTCATGT 2 GB-AM-032 F CTCCTCGGGAGAAGGTTG 3 (GTG) ₆ 152 ~ 158 55 3 R TGTGTCCCAATCCATCGT 4 GB-AM-051 F GAGGAGACTTGGTGGCCT 5 (AGA) ₅ 232-238 55 3 R TCGGGAGCAATGTAGCAC 6 GB-AM-071 F CCATGAAGAAGACCAAGAGG 7 (AGA) ₅ 160-163 55 2 R CATCCGTCAACGGCTAAA 8 GB-AM-078 F CCCACGAAAACAAACAGG 9 (AG) ₇ , (AG) ₁₁ 97-151 55 6 R CCGTTCTTCGGTGAGATG 10 GB-AM-099 F AAATTGACAATGCGCAGC 11 (TCA) _12, (TCT) ₅ 134-164 50 8 R TTCCTCACCAAAATTGCC 12 GB-AM-105 F GTGATGGTCGTGGTGGAG 13 (GAA) ₄ 133-154 55 4 R GATTCCCTCATCTTCGCC 14 GB-AM-123 F TTGGAGTGGTTTCGGTTG 15 (GA) ₇ 203-231 55 7 R CCACTCTCTCACGTCCCA 16 GB-AM-129 F TTCACGTGGGAAGGAGG 17 (GCT) ₅ 227-248 55 5 R AAAATTAATGGGCCTCGC 18 GB-AM-132 F AACTTTTGCCTCCTGCAA 19 (AAG) ₁₇ 96-147 55 7 R TCAAATGCTGATCCCAGG 20 GB-AM-136 F TCAGCAAAACATGATCAACAA 21 (GAA) ₆ , (CCA) ₆ 192-207 55 5 R GTTGCTGCATTGGTGGTT 22 GB-AM-137 F CGAAGATCATGGGTTTGC 23 (ACA) ₁₀ 200-218 55 7 R TTGAGAATAAGGCGTTGACA 24

^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 amaranth. The SSR primer according to the present invention can be used to efficiently evaluate and preserve the gene source of amaranth. Accordingly, the present invention provides a method for detecting DNA polymorphism of Amaranth, comprising performing PCR using the SSR primer pair.

Specifically, the method for detecting DNA polymorphism of Amaranth

(a) extracting genomic DNA from amaranth;

(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 and water (dH ₂ O) in addition to genomic DNA extracted from the amaranth 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 Amaranth, including performing PCR using the SSR primer pair.

Specifically, how to identify varieties of Amaranth

(a) extracting genomic DNA from amaranth and control amaranth 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, PCR execution and separation of PCR products in steps (a) through (c) are as described above, and in step (d) comparisons of amaranth and control amaranth varieties are in the same combination of SSR primers. This is done by comparing the size of each PCR product of the amaranth and the control amaranth varieties that are samples for the pair. By comparing the size comparison results for each pair of SSR primers, the amaranth sample can be identified as the same varieties as the control amaranth cultivar if the results are consistent with the results of the sample amaranth and the control amaranth cultivar. 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 amaranth cultivars may be performed simultaneously with the sample amaranth, but may also be performed prior to the sample amaranth and prepared as a reference for identification. Using this 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 the sample of amaranth.

The present invention also provides a kit for detecting DNA polymorphism of Amaranth 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 varieties of amaranth 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.

Amaranth that can be applied to the present invention may be of the genus Amaranthus and variants thereof, but is not limited thereto. Preferably it may be of the genus Amaranthus.

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 the cultivar Amaranth (Dept. of Agricultural Biotechnology, Conservation No .: IT164975) 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 Amaranth 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. Thereafter, the mixture was centrifuged at 13,000 rpm for 15 minutes. 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). To increase ligation efficiency, the AP-12 adapter phosphorylated the 5 'end with alkaline phosphatase (Promega, USA), and the composition of the reaction solution during ligation is 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 ' 25 AP-12 5'-TAGTCCAGATCTAAGCAAGAGCACA-3 ' 26

<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 amaranth supersatellites order SEQ ID NO: Biotin- (GA) ₂₀ 27 Biotin- (AT) ₂₀ 28 Biotin- (CA) ₂₀ 29 Biotin- (AGC) ₁₅ 30 Biotin- (GGC) ₁₅ 31 Biotin- (AAG) ₁₅ 32 Biotin- (AAC) ₁₅ 33 Biotin- (AGG) ₁₅ 34

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

Amaranth Used in SSR Analysis Deposit number Depositary Institution Collection country (country of origin) Type K005397 Rural Development Administration Russia Amaranthus sp. K038161 Rural Development Administration Pakistan Amaranthus caudatus K038169 Rural Development Administration Peru Amaranthus caudatus K038184 Rural Development Administration Ghana Amaranthus cruentus K038200 Rural Development Administration Nepal Amaranthus dubius K038209 Rural Development Administration Zimbabwe Amaranthus hybridus K038221 Rural Development Administration Mexico Amaranthus hypochondriacus K038230 Rural Development Administration India Amaranthus hypochondriacus K038243 Rural Development Administration Portugal Amaranthus powellii K038250 Rural Development Administration France Amaranthus powellii subsp. bouchonii K038258 Rural Development Administration Brazil Amaranthus quitensis K038276 Rural Development Administration United States of America Amaranthus retroflexus IT137653 Rural Development Administration Thailand Amaranthus sp. IT164975 Rural Development Administration Republic of Korea Amaranthus sp. IT197101 Rural Development Administration Mexico Amaranthus crispus 708561 Rural Development Administration Ecuador Amaranthus sp.

The composition of each PCR reaction mixture (20 μl) was as follows: 40 ng of amaranth 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: 35) 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 12 pairs of SSR primer pairs showing polymorphisms in Amaranth cultivars and Amaranth varieties collected domestically and internationally. GB-AM-013 (primary pairs represented by SEQ ID NOs: 1 and 2) 032 (primary pairs represented by SEQ ID NOs: 3 and 4), GB-AM-051 (primary pairs represented by SEQ ID NOs: 5 and 6), GB-AM-071 (primary pairs represented by SEQ ID NOs: 7 and 8), GB-AM-078 (primary pairs represented by SEQ ID NOs: 9 and 10), GB-AM-099 (primary pairs represented by SEQ ID NOs: 11 and 12), and GB-AM-105 (denoted by SEQ ID NOs: 13 and 14) Primer pair), GB-AM-123 (primary pair represented by SEQ ID NOs: 15 and 16), GB-AM-129 (primary pair represented by SEQ ID NOs: 17 and 18), GB-AM-132 (SEQ ID NO: 19 and Primer pairs represented by 20), GB-AM-136 (primary pairs represented by SEQ ID NOs: 21 and 22) and GB-AM-137 (primary pairs represented by SEQ ID NOs: 23 and 24) were selected (see Table 1). ). The size of each PCR fragment amplified by the 12 pairs of primers is shown in Table 5 below.

Type and size of amplified supersatellites according to primers (unit: bp) K005397 K038161 K038169 K038184 K038200 K03209 K038221 K038230 GB-AM-013 154 157 157 154 154 154/157 154 154 GB-AM-032 152 155 152 152 152 152 152/155 152/155 GB-AM-051 238 235 235/238 238 232 235 238 238 GB-AM-071 163 163 160 163 160 163 163 163 GB-AM-078 97/149 97/149 97/149 97/149 97/151 97/149 97/149 97/149 GB-AM-099 164 155 155 158 149/164 161 161 158 GB-AM-105 139/154 151/154 151/154 139/154 154 139/154 139/154 154 GB-AM-123 221 215/221 215/221 221 221 221/223 221/227 221/227 GB-AM-129 239/244 244 244 239 244 239/244 227/242 242 GB-AM-132 96 111 111 96 147 111 114 129 GB-AM-136 201 201 201 201 198 198 201 201 GB-AM-137 209 203 203 209 218 203 215 203

K038243 K038250 K038258 K038276 IT137653 IT164975 IT197101 708561 GB-AM-013 154 154/160 157 151/154 154 154/160 154 154/157 GB-AM-032 158 152/155 152 152 152/155 152/155 152/155 152/152 GB-AM-051 238 238 235/235 238 238 238 238 232 GB-AM-071 160 160 163 160 163 163 163 160 GB-AM-078 97/149 97/147 97/149 97/133 97/149 97/143 97/149 97/151 GB-AM-099 146 134 158 155 158/158 152 164 149 GB-AM-105 154/154 151/154 151/154 133/154 154 154/154 139/154 151/154 GB-AM-123 227/231 203/221 221/221 221/225 221/227 227/231 221/221 221/221 GB-AM-129 242/248 239 244 248 242 227/242 239/239 244 GB-AM-132 132 135 111 132 132 129 96 111 GB-AM-136 207 198/201 201 195 201 201 201 192 GB-AM-137 212 212 203 200 212 206 209 203

As described above, in the present invention, the first SSR marker was developed in Amaranth. SSR primer pairs provided in the present invention can be very useful for effectively detecting the DNA polymorphism of the amaranth to prepare the DNA profile of the amaranth. In this way, by efficiently evaluating the genetic resources of Amaranth, 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 amaranth.

<110> Republic of Korea <120> SSR primer derived from amaranth and use of there <130> NP07-0049 <160> 35 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-013-F <400> 1 tgaatcctta tgcgccac 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-013-R <400> 2 ggagccctgt cctcatgt 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-032-F <400> 3 ctcctcggga gaaggttg 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-032-R <400> 4 tgtgtcccaa tccatcgt 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-051-F <400> 5 gaggagactt ggtggcct 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-051-R <400> 6 tcgggagcaa tgtagcac 18 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-071-F <400> 7 ccatgaagaa gaccaagagg 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-071-R <400> 8 catccgtcaa cggctaaa 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-078-F <400> 9 cccacgaaaa caaacagg 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-078-R <400> 10 ccgttcttcg gtgagatg 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-099-F <400> 11 aaattgacaa tgcgcagc 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-099-R <400> 12 ttcctcacca aaattgcc 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-105-F <400> 13 gtgatggtcg tggtggag 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-105-R <400> 14 gattccctca tcttcgcc 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-123-F <400> 15 ttggagtggt ttcggttg 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-123-R <400> 16 ccactctctc acgtccca 18 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-129-F <400> 17 ttcacgtggg aaggagg 17 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-129-R <400> 18 aaaattaatg ggcctcgc 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-132-F <400> 19 aacttttgcc tcctgcaa 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-132-R <400> 20 tcaaatgctg atcccagg 18 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-136-F <400> 21 tcagcaaaac atgatcaaca a 21 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-136-R <400> 22 gttgctgcat tggtggtt 18 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-137-F <400> 23 cgaagatcat gggtttgc 18 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GB-AM-137-R <400> 24 ttgagaataa ggcgttgaca 20 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AP-11 <400> 25 ctcttgctta gatctggact a 21 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AP-12 <400> 26 tagtccagat ctaagcaaga gcaca 25 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> Biotin- (GA) 20 <400> 27 gagagagaga gagagagaga gagagagaga gagagagaga 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> Biotin- (AT) 20 <400> 28 atatatatat atatatatat atatatatat atatatatat 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> Biotin- (CA) 20 <400> 29 cacacacaca cacacacaca cacacacaca cacacacaca 40 <210> 30 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AGC) 15 <400> 30 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagc 45 <210> 31 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (GGC) 15 <400> 31 ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggc 45 <210> 32 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AAG) 15 <400> 32 aagaagaaga agaagaagaa gaagaagaag aagaagaaga agaag 45 <210> 33 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AAC) 15 <400> 33 aacaacaaca acaacaacaa caacaacaac aacaacaaca acaac 45 <210> 34 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AGG) 15 <400> 34 aggaggagga ggaggaggag gaggaggagg aggaggagga ggagg 45 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> M13 primer <400> 35 tgtaaaacga cggccagt 18  

Claims (7)

delete Primer pairs represented by SEQ ID NOs: 1 and 2; Primer pairs represented by SEQ ID NOs: 3 and 4; A simple sequence repeat (SSR) primer pair selected from the group consisting of a primer pair represented by SEQ ID NOs: 11 and 12 and a primer pair represented by SEQ ID NOs: 17 and 18. 3. The method of claim 2, amaranth (Amaranth;. Amaranthus spp) SSR primer pair, characterized in that derived from. (a) extracting genomic DNA from amaranth; (b) subjecting the extracted genomic DNA to a template and performing PCR using the SSR primer pair of claim 2; And (c) Amaranth DNA polymorphism detection method comprising the step of separating the PCR product by size. (a) extracting genomic DNA from amaranth and control amaranth varieties; (b) performing PCR using each of the extracted genomic DNA as a template and the SSR primer pair of claim 2; (c) separating each PCR product by size; And (d) A method for identifying varieties of amaranth comprising the step of comparing the results of the separation according to the size of the amaranth and the control amaranth varieties. A kit for detecting DNA polymorphism of Amaranth comprising the SSR primer pair of claim 2. A kit for identifying varieties of Amaranth comprising the SSR primer pair of claim 2.