KR20060056536A - SSR Primer Isolated from Sesame Plant and Its Use - Google Patents

Abstract

The present invention relates to an SSR primer isolated from sesame plants and its use, and more specifically, SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 38 and PCR using the same The present invention relates to a method for detecting DNA polymorphism and a variety discrimination method of a sesame plant.

SSR primer according to the present invention can be very useful for preparing the DNA profile of sesame plants. Through the efficient evaluation of sesame gene sources, it is possible to effectively conduct redundancy analysis and establish novelty with existing resources when introducing new gene sources. In addition, the SSR primer according to the present invention can effectively detect the DNA polymorphism of the sesame genus plants, through which it is possible to determine the varieties of sesame seeds.

SSR Primer, Sesame, DNA Polymorphism, Variety

Description

SSR primer isolated from sesame plants and its use {SSR primer isolated from Sesamum sp. and use kind}             

1 is a result of PCR amplification of genomic DNA of various sesame lines and varieties with the SSR primer of the present invention to which a fluorescent die is attached, and then the size of the amplified DNA fragments using a fluorescence analyzer.

Figure 2 shows the phylogenetic analysis of the flexible relationship between the strain and varieties from the results of the polymorphism analysis of 29 points of cultivated sesame strains and three points of sesame varieties using 19 pairs of SSR primers according to the present invention. Arabic numerals indicate numbers of the sesame strains and varieties shown in Table 4.

The present invention The present invention relates to SSR primers isolated from sesame plants and their use. More specifically, SSR primer pairs having two nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 38 and performing PCR using the same The present invention relates to a method for detecting DNA polymorphism and varieties of sesame plants.

Most of the important crops and cultivated crops used for breeding are collected in large quantities each year. On the other hand, the evaluation of genetic resources has not been sufficiently carried out. 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 is a very useful tool in the development of genetic engineering, medicine, gene mapping and pharmaceuticals. In particular, the search for useful traits, identification of species of species, classification and identification of species, and analysis of the soft relationships of populations can be performed simply and quickly with little influence on the external environment.

Nucleic acid fingerprinting using polymerase chain reaction (PCR) developed so far includes 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 has been spotlighted by high DNA polymorphism detection, but has the disadvantage of complicated appearance and analysis of a low reproducible band. On the other hand, the SSR method is a method of analyzing a supersatellite in an individual by preparing PCR primers based on nucleotide sequence information of a microsatellite region, a DNA repeat sequence. The supersatellite is a repetitive DNA group that repeats 1 to 5 simple nucleotide sequences and is known to be evenly distributed in the genome of most eukaryotes (Hamada et al., Proc. Natl. Acad. Sci). USA, 79: 6465-6469, 1982; Tautz and Renz, Nucleic Acids Research, 12: 4127-4138, 1984). Since the number of repetitions of the simple base sequence is different between breeds or between individuals, polymorphism occurs when this portion is amplified by a PCR reaction. Such polymorphisms are widely used in genetic studies of animals and plants because they vary widely between breeds and individuals. In particular, the SSR method is frequently used for the identification of species due to the advantages of easy analysis of supersatellites and high reproducibility, and in particular, the SSR method 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.

Since the first method of detecting polymorphisms of supersatellite DNA using PCR has been established, saturation gene maps have been generated with SSR markers only in humans and mammals (Weissenbach et al., Nature 359 (29): 794-801, 1992). Since the development of 13 supersatellite DNA markers in rice (Wu and Tanksley, Mol. Gen. Genet. 241: 225-235, 1993), about 160 markers have been developed in subsequent studies (Panaud et al, Mol). Gen. Genet ., 252: 597-607, 1996; Akagi et al., Korean J. Breeding , 28 (2): 178-183, 1996; Chen et al., Theor.Appl.Genet ., 95: 553 -567, 1997). Korean Patent Application No. 2001-34003 discloses a supersatellite marker for identifying genotypes of rice varieties by amplifying SSR repeated in the rice genome by PCR technique. In addition, Tang's team in the United States has published a genetic map of sunflowers that developed 879 SSR markers (Tang et al., Theor. Appl. Genet ., 105: 1124-1136, 2002). In addition, the Baby Pole (Bell and Ecker, Genomics , 19: 137-1445, 1994), grapes (Thomas and Scott, Theor. Appl. Genet . 86: 985-990, 1993), beans (Akkaya et al., Genetics , 132) : 1131-1139, 1992, Rongwen et al., Theor. Appl. Genet ., 90: 43-48, 1995, Yu et al., Theor.Appl. Genet ., 92: 64-69, 1996), barley ( Plants such as Saghai Maroof et al., Proc. Natl. Acad. Sci . USA, 91: 5466-5470, 1994), wheat (Plaschke et al., Thero. Appl. Genet ., 91: 1001-1007, 1995) Variety classification, genetic resource evaluation, and gene mapping using SSR markers have been carried out.

In sesame, however, no SSR markers have been developed in Korea yet, and there are few reports of related studies in foreign countries.

Accordingly, the present inventors have completed the present invention by developing SSR primers that show many polymorphic variations in various sesame lines and varieties, while continuing to develop SSR markers that can efficiently evaluate sesame gene sources.

Accordingly, it is an object of the present invention to provide an SSR marker and its use capable of efficiently evaluating the genetic resources of sesame plants.

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: 38.

In order to achieve another object of the present invention, the present invention provides a method for detecting DNA polymorphism of sesame genus plants comprising performing PCR using the SSR primer pair.

In order to achieve another object of the present invention, the present invention includes sesame comprising performing PCR using the SSR primer Provides a method for discriminating varieties of genus plants.

In order to achieve another object of the present invention, the present invention provides a kit for detecting DNA polymorphism of sesame genus plants comprising the SSR primer pair, DNA polymerase and PCR reaction buffer.

In order to achieve another object of the present invention, the present invention provides a kit for identifying a variety of sesame genus plants 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 in that it provides an SSR primer that can specifically analyze the genetic diversity of sesame plants. In the present invention, a sesame SSR primer was developed by a novel method combining AFLP and one way PCR. That is, in the present invention, genomic DNA extracted from cultivar sesame seeds ( Sesamum indicum L.) was cut with restriction enzymes to prepare a DNA fragment having a stiky end, and then ligated with four adapters and one-way using an adapter primer. PCR (one way PCR) was performed. The PCR product was then hybridized with a magnetic bead-oligo probe conjugate that could hybridize with the supersatellite present in the sesame genome to amplify the hybridized PCR product, and then sequenced to analyze about 300 containing the supersatellite of sesame. DNA fragments were obtained. By analyzing the sequence of the DNA fragments obtained above, 40 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 primers were used to select 19 pairs of SSR primer combinations that exhibited polymorphic variation by performing DNA profiling through PCR of various cultivated sesame lines and varieties.

The SSR primer provided by the method as described above is the first one disclosed by the present invention.

SSR primer provided by the present invention refers to a PCR primer pair, 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: 38. Preferably GBSSR-5 (SEQ ID NOs 1 and 2), GBSSR-8 (SEQ ID NOs 3 and 4), GBSSR-9 (SEQ ID NOs 5 and 6), GBSSR-10 (SEQ ID NOs 7 and 8), GBSSR-19 (SEQ ID NOs 9 and 10), GBSSR-33 (SEQ ID NOs 11 and 12), GBSSR-34 (SEQ ID NOs 13 and 14), GBSSR-40 (SEQ ID NOs 15 and 16), GBSSR-58 (SEQ ID NOs 17 and 18) ), GBSSR-72 (SEQ ID NOs 19 and 20), GBSSR-83 (SEQ ID NOs 21 and 22), GBSSR-108 (SEQ ID NOs 23 and 24), GBSSR-123 (SEQ ID NOs 25 and 26), GBSSR-135 ( SEQ ID NOs 27 and 28), GBSSR-164 (SEQ ID NOs 29 and 30), GBSSR-173 (SEQ ID NOs 31 and 32), GBSSR-178 (SEQ ID NOs 33 and 34), GBSSR-182 (SEQ ID NOs 35 and 36) , GBSSR-184 (SEQ ID NOs: 37 and 38). For the primer pairs provided in the present invention and the repeat motifs present in the supersatellites amplified by them, the range of the size of the supersatellites to be amplified, Tm (° C.) and the number of alleles in which the supersatellites are present The information is listed in Table 1 below.

Table 1

Characteristics of SSR Primer Pairs of the Present Invention and Supersatellites Amplified therefrom

The SSR primers provided in the present invention can be usefully used for detecting DNA polymorphism and analyzing genetic diversity in sesame plants. Sesame gene source can be efficiently evaluated and preserved using the SSR primer according to the present invention. Therefore, the present invention provides a method for detecting DNA polymorphism of sesame seeds comprising performing PCR using the SSR primer.

Specifically, the method of the present invention

(a) extracting genomic DNA from the sesame plant to be analyzed;

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

(c) electrophoresis of the PCR product.

Extraction of genomic DNA from sesame plants in step (a) can be carried out by any method known in the art. For example, but not limited to, DNA extraction kits using phenol extraction, phenol / chloroform extraction, and SDS extraction (Tai et al ., Plant Mol. Biol. Reporter 8: 297-303, 1990) or otherwise commercially available. Can be used.

In step (b), the PCR may be performed using a PCR reaction mixture containing various components known in the art required for the PCR reaction. The PCR reaction mixture contains an appropriate amount of DNA polymerase, dNTP, PCR buffer and water (dH ₂ O) in addition to genomic DNA extracted from the sesame genus to be analyzed and the SSR primer provided in the present invention. The PCR buffer solution includes Tris-HCl, MgCl ₂ , KCl and the like. At this time, the concentration of MgCl ₂ greatly affects the specificity and quantity of amplification. Preferably 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 TritonX 100. In addition, the PCR is denatured after denatured genomic DNA extracted from the sesame genus plant as a template DNA at 94-95 ℃; Annealing; And after a cycle of extension, and finally under general PCR reaction conditions of elongation 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. Optimal reaction conditions when performing PCR using SSR primers 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.

Step (c) is a step of confirming the band aspect of the PCR amplification product by electrophoresis of the PCR product amplified in the step (b). Electrophoresis can be performed using an agarose gel or a polyacrylamide gel. Preferably polyacrylamide gels, more preferably modified polyacrylamide gels, can be carried out according to methods known in the art. After electrophoresis, the band can be identified by silver staining. The size of the band shown in the electrophoresis can be obtained in units of bp compared to the DNA size marker, and the number of bands of the same size among all the bands amplified by each primer was used as an allele.

Meanwhile, in order to more accurately measure the size of the PCR product amplified by the SSR primer of the present invention, a fluorescent die is attached to both ends of the SSR primer of the present invention to amplify genomic DNA extracted from the plant of sesame, and the amplified DNA fragment The size of these can be analyzed by a fluorescence spectrometer. The fluorescent die is not particularly limited, and examples thereof include FAM, HEX, and TET.

The present invention also provides a kit for detecting DNA polymorphism of sesame seeds containing SSR primer, DNA polymerase and PCR reaction buffer solution according to the present invention. The composition of the PCR reaction buffer is as described above. In addition, the components necessary for performing electrophoresis capable of confirming amplification of PCR products may be further included in the kit of the present invention. For example, it may further include a fluorescent die that can label the SSR primer.

In addition, by detecting the polymorphism of the sesame plant with the method according to the invention it is possible to determine the varieties of the sesame plant. Therefore, the present invention provides a method for identifying a variety of sesame plants and a kit for identifying a variety of sesame plants, including performing PCR using the SSR primer of the present invention.

The cultivation of the sesame plant may be performed in the same manner as the polymorphism detection method of the sesame plant, and the variety may be discriminated from the pattern of the band resulting from the electrophoresis.

A sesame plant in the present invention are filaments stop indicator glutamicum (Sesamum indicum), Cesar car stop fence (S. capense), Cesar stop Lepidocrocite FIG Tomb (S. lepidotum), Cesar stopped tree pilrum (S. triphyllum), Cesar stop S. alatum , sesamum frostatum , sesamum angustifolium , sesamum malloti , sesamum pedarioides , sesamum S. angolense , S. latifolium , S. parviflorum and S. radiatum and the like. Among them, the cultivated sesame seed sesameum indicum is known to be closely related to sesamum prostratum, sesamum latinium and sesamum radidiatum, and is particularly closest to sesamum latinium.

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 synthesized according to a method using SDS (Tai et al ., Plant Mol. Biol. Reporter , 8: 297-303, 1990) from cultivar Sesamum indicum L. (RDA Gene Source, Accession No. 30122) . Each was extracted. 0.5 g of leaf tissues were collected from young sesame 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. 750 μl of extraction buffer (200 mM Tris-HCl pH8.0, 200 mM NaCl, 25 mM EDTA, 0.5% SDS) was added before the leaf sections were dissolved. After adding the same amount of phenol (chloro): chloroform (isoamyl alchol) (25:24: 1), and gently shake by hand for about 5 minutes. The mixed solution was stored at 65 ° C. for 15 minutes, and then centrifuged at 13,000 rpm for 15 minutes. The supernatant was taken and transferred to a new tube. The same amount of isopropanol stored at -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. Precipitated DNA was washed with 70% ethanol. After the DNA was dried, it was sufficiently dissolved by adding TE buffer solution (10 mM Tris-Cl, 1 mM EDTA, pH7.4) containing about 300 µl of RNase (50 µg / ml), followed by 1 hour at 37 ° C. It was stored about.

<Example 2>

Ligation with Adapters

About 500 ng / μl of genomic DNA obtained in Example 1 was double cut into Eco RI and Taq I. After cleaved DNA was electrophoresed in a 1.2% agarose gel, only about 300-1500 bp of DNA was eluated from the gel. Illustrated DNA fragments were sequenced with four adapters ( Eco R I-1, Eco R I-2, Taq I-1, Taq I-2) and T4 DNA ligase (Ligase, Promega) Ligation was performed at 22 ° C. overnight. The sequence of each adapter is shown in Table 2 below.

TABLE 2

Adapter used in the present invention

Adapter name Sequence SEQ ID NO: Eco RⅠ-1 ATC GTA GAC TGC GTA CC 39 Eco RⅠ-2 AAT TGG TAC GCA GTC TAC 40 Taq Ⅰ-1 GAC GAT GAG TCC TGA G 41 Taq Ⅰ-2 CGC TCA GGA CTC AT 42

<Example 3>

One-way PCR

In Example 2, one-way PCR was performed using only one Taq I adapter primer (PRTP-0, SEQ ID NO: 43: gacgatgagtcctgagcga) having a DNA ligated with the adapter as a template and a phosphate attached to the 5 'end. It was. The composition of the PCR reaction mixture (50 μl) is as follows: 250 ng adapter-ligated DNA, 0.5 μM Taq I adapter 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. was repeated for 60 cycles, and finally at 72 ° C. for 5 minutes.

<Example 4>

Selection and Isolation of DNA Fragments Containing Supersatellites

<4-1> Preparation of Magnetic Bead-Oligo Probe Conjugate

Magnetic beads (Roche magnetic particles) l times 5 × SSC

After washing, the resultant was dissolved in 50 μl 5 × SSC again. On the other hand, the fourteen oligo probes described in Table 3 were labeled with biotin according to conventional methods known in the art. Then, 50 μl of magnetic beads dissolved in 5 × SSC, 1.3 μl of each oligo probe, 25 μl of 10 × SSC, and 23.7 μl of distilled water were mixed. The mixture was allowed to stand at room temperature for 15 minutes to induce binding of the magnetic beads and the oligo probe. Magnetic bead-oligoprobe conjugates were washed three times with 100 μl 5 × SSC. The washing was performed by precipitating the conjugate under the tube using the magnetic force of a magnetic separator (Promega). Purity of the magnetic beads-oligoprobe conjugate with increased purity was again dissolved in 50 μl of 10 × SSC and stored at room temperature for a while.                     

TABLE 3

Sequence of probe to isolate sesame supersatellite

order SEQ ID NO: One (AT) ₁₂ 44 2 (AG) ₁₂ 45 3 (CA) ₁₂ 46 4 (ACG) ₈ 47 5 (CTC) ₈ 48 6 (TAA) ₈ 49 7 (AAC) ₈ 50 8 (AAG) ₈ 51 9 (ACG) ₈ 52 10 (AGC) ₈ 53 11 (AGG) ₈ 54 12 (CCG) ₈ 55 13 (CTC) ₈ 56 14 (TAA) ₈ 57

<4-2> hybridization

The PCR product obtained in Example 3 for hybridization was prepared as follows: 10 μl of the PCR product was mixed with 40 μl of distilled water and left for 5 minutes in boiling water for denaturation. It was then transferred directly to ice for DNA fixation. 50 μl of the denatured PCR product and 50 μl of the probe bound to the magnetic beads were mixed and hybridization was induced at 20 ° C. for 20 minutes. Thereafter, the mixture was washed four times for 5 minutes with 100 μl of 2 × SSC, and again for 4 minutes at 30 ° C. with 100 μl of 1 × SSC. 20 μl of 0.15M NaOH was treated for 20 minutes at room temperature. By NaOH treatment, the magnetic beads bound probes are separated separately (chemical denature). The probe combined with the remaining magnetic beads was removed again using a magnetic separator. Chemical denaturation was neutralized by the addition of 2.2 μl 10 × TE buffer and 1.3 μl of 1.24 M acetic acid. Thereafter, the PCR product was desalted using a PCR purification kit or ethanol precipitation.

Desalted PCR products were amplified again for sequencing. At this time, PCR was performed using Eco RI-0 primer (SEQ ID NO: 58: gactgcgtacc aattc) together with the primer used in Example 3. PCR was performed under the same conditions as in Example 3, except that the total number of cycles of the PCR was reduced to 24 cycles. In this way, about 300 DNA fragments containing supersatellites were isolated in the present invention.

Example 5

Sequencing and Primer Design of Isolated DNA Fragments

DNA fragments containing the supersatellite isolated in Example 4 were cloned into pGEM T-easy vector (Promega; USA) to analyze their sequences. Sequence analysis was performed by requesting Macrogen. Only fragments containing at least 5 repeat units were selected based on the isolated sequences. Based on the part containing the repeating unit, a primer in both directions was designed to amplify the supersatellite. A total of 40 pairs of primers were designed.

<Example 6>

Selection of SSR Markers

Among the primer pairs designed in Example 5, in order to select SSR primer combinations exhibiting a large number of polymorphic variation in various cultivars and varieties of various cultivated sesame seeds collected in Korea and abroad, DNA profiling analysis by PCR was performed on the cultivated sesame seeds ( Sesamum indicum L.) and the three-pointed sesame varieties ( Sesamum indicum L.) described in Table 5. Extraction of genomic DNA from the sesame was carried out in the same manner as in Example 1.

Table 4

Cultivated Sesame and Sesame Varieties Used in SSR Analysis

number Deposit number Depositary Institution Collection One 30122 Rural Development Administration Republic of Korea 2 195371 Rural Development Administration Republic of Korea 3 102974 Rural Development Administration Republic of Korea 4 103421 Rural Development Administration Republic of Korea 5 103444 Rural Development Administration Republic of Korea 6 185978 Rural Development Administration Republic of Korea 7 30441 Rural Development Administration Republic of Korea 8 113130 Rural Development Administration Republic of Korea 9 30054 Rural Development Administration Republic of Korea 10 175830 Rural Development Administration Republic of Korea 11 175848 Rural Development Administration Republic of Korea 12 182003 Rural Development Administration Republic of Korea 13 103956 Rural Development Administration Republic of Korea 14 30094 Rural Development Administration   Republic of Korea 15 184433 Rural Development Administration   Afghanistan 16 184440 Rural Development Administration Argentina 17 184438 Rural Development Administration Angola 18 184283 Rural Development Administration Africa 19 184441 Rural Development Administration   Africa 20 167116 Rural Development Administration United States of America 21 167133 Rural Development Administration China 22 184698 Rural Development Administration China 23 169830 Rural Development Administration China 24 184745 Rural Development Administration Africa 25 184536 Rural Development Administration Egypt 26 184340 Rural Development Administration Egypt 27 184458 Rural Development Administration Greece 28 184467 Rural Development Administration India 29 184758 Rural Development Administration Israel

Table 5

Sesame varieties used for SSR analysis

Breed Name Depositary Institution Collection 30 Oil sesame Rural Development Administration Republic of Korea 31 Ansan Sesame Rural Development Administration Republic of Korea 32 Pearl sesame Rural Development Administration Republic of Korea

The composition of the PCR reaction mixture (20 μl) is as follows: 20 ng of sesame genomic DNA, 0.5 μM of each primer, 200 μM of dNTPs, 1 × PCR reaction buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl ₂ , 50 mM KCl, 0.1% Triton X-100), 1 unit DNA polymerase and remaining distilled water. PCR reaction was performed after denaturing the template DNA for 3 minutes at 95 ℃, 30 seconds at 95 ℃; 30 seconds at 55 ° C .; And 1 minute 30 seconds at 72 ° C. was repeated for 36 cycles, and finally at 72 ° C. for 5 minutes.

2 μl of the amplified PCR product was mixed with 4 μl of 6 × loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol FF). The mixed solution was heated at 95 ° C. for 5 minutes, and then 3 μl of the mixed solution was taken and a 6% denaturing polyacrylamide gel (acrylamide: bisacrylamide (19: 1), 7.5 M urea, 1, previously heated to 55 ° C.). X TBE buffer (0.89 M Tris-HCl pH 8.0, 0.89 M boric acid, 0.02 M EDTA pH 8.0). Then, electrophoresis was performed for 1 hour and 30 minutes at 1800V, 60W. The band pattern shown by silver staining was analyzed and polymorphism was analyzed by examining the number of alleles. In addition, the analyzed polymorphism results in NTSYS-pc. V.2.02 (Rohlf 1998) The lineage relationship between sesame lines was investigated by analysis using a statistical program.

In addition, FAM (fluorescent die) at both ends of the primer designed in Example 5 according to Schuelke's PCR method (Schuelke M., Nature Biotechnology, 18: 233-234, 2000) was used to determine the size of the amplified DNA fragments. After amplifying the DNA fragments by attaching 6-carboxy-fluorescience, the size of the amplified DNA fragments was confirmed using a fluorescence analyzer (ABI PriGBSSR 3100 genetic analyzer-electropherogram). PCR amplification was performed after denaturing the template DNA for 3 minutes at 94 ° C., followed by 30 seconds at 94 ° C .; 30 sec at 53 ° C .; 45 cycles at 53 ° C., 1 cycle at 72 ° C. for 10 minutes, and finally at 72 ° C. for 1 minute.

Experimental results, 19 pairs of primers (GBSSR-5, GBSSR-8, GBSSR-9, GBSSR-10, GBSSR-19, GBSSR-33, GBSSR-34, GBSSR) out of a total of 40 pairs of primers designed in Example 5 -40, GBSSR-58, GBSSR-72, GBSSR-83, GBSSR-108, GBSSR-123, GBSSR-135, GBSSR-164, GBSSR-173, GBSSR-178, GBSSR-182, SB-184) It was confirmed that the polymorphism was shown in the cultivated sesame strain of and sesame varieties of three points. The number of alleles amplified by the 19 pairs of primers is shown in Table 1.

In addition, as a result of analyzing the systemic relationship between strains and varieties of domestic and foreign sesame seeds of 32 points from the above-described polymorphism, the strains and varieties of domestic and foreign sesame seeds were identified by their SSR primers. It was clearly identified according to the enemy characteristics.                     

Thus, the 19 pairs of primers were selected as SSR markers of sesame seeds (see Table 1).

As described above, the SSR primer according to the present invention can be very useful for preparing DNA profiles of sesame plants. Through the efficient evaluation of sesame gene sources, it is possible to effectively conduct redundancy analysis and establish novelty with existing resources when introducing new gene sources. In addition, the SSR primer according to the present invention can effectively detect the DNA polymorphism of the sesame genus plants, through which it is possible to determine the varieties of sesame seeds.

<110> Kangwon National University <120> SSR primer isolated from Sesamum sp. and use according <160> 58 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-5 sense primer <400> 1 tcatatatat aaaaggagcc caac 24 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-5 antisense primer <400> 2 gaagaagaga gaagcgatga c 21 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-8 sense primer <400> 3 ggagaaattt tcagagagaa aaa 23 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-8 antisense primer <400> 4 attgctctgc ctacaaataa aa 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-9 sense primer <400> 5 cccaactctt cgtctatctc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-9 antisense primer <400> 6 tagaggtaat tgtggggga 19 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-10 sense primer <400> 7 tagaggtaat tgtggggga 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-10 antisense primer <400> 8 cccaactctt cgtctatctc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-19 sense primer <400> 9 agaaggtgga taggtgaagg 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-19 antisense primer <400> 10 tcacttcttt ctgtgattat gg 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-33 sense primer <400> 11 ttttcctgaa tggcatagtt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-33 antisense primer <400> 12 gcccaatttg tctatctcct 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-34 sense primer <400> 13 caattccacg tcagtgct 18 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-34 antisense primer <400> 14 ggaagccggt cataatcta 19 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-40 sense primer <400> 15 aaagccatgg aaaacggt 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-40 antisense primer <400> 16 gacccgttaa ctccgacc 18 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-58 sense primer <400> 17 ccgtgttcaa ctcgtgtttt 20 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-58 antisense primer <400> 18 atcaggctgc ctctttcg 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-72 sense primer <400> 19 gcagcagttc cgttcttg 18 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-72 antisense primer <400> 20 agtgctgaat ttagtctgca tag 23 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-83 sense primer <400> 21 aagaaacgcc atggacag 18 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-83 antisense primer <400> 22 agcccacttt ccctcctt 18 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-108 sense primer <400> 23 ccactcaaaa ttttcactaa gaa 23 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-108 antisense primer <400> 24 tcgtcttcct ctctcccc 18 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-123 sense primer <400> 25 gcaaacacat gcatccct 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-123 antisense primer <400> 26 gccctgatga taaagcca 18 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-135 sense primer <400> 27 gctgaggagt cttgaagcag 20 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-135 antisense primer <400> 28 cgatatcacc atcacccc 18 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-164 sense primer <400> 29 ggatcccaat cctccattta 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-164 antisense primer <400> 30 tgagatattg gctcccagag 20 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-173 sense primer <400> 31 tttcttcctc gttgctcg 18 <210> 32 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-173 antisense primer <400> 32 cctaaccaac caccctcc 18 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-178 sense primer <400> 33 tccacaaagg accacacc 18 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-178 antisense primer <400> 34 tggccttgaa acctcttct 19 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-182 sense primer <400> 35 ccattgaaaa ctgcacacaa 20 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-182 antisense primer <400> 36 tccacacaca gagagccc 18 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-184 sense primer <400> 37 tcttgcaatg gggatcag 18 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GBSSR-184 antisense primer <400> 38 cgaactatag ataatcactt ggaa 24 <210> 39 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-1 adapter <400> 39 atcgtagact gcgtacc 17 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-2 adapter <400> 40 aattggtacg cagtctac 18 <210> 41 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> TaqI-1 adapter <400> 41 gacgatgagt cctgag 16 <210> 42 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> TaqI-2 adapter <400> 42 cgctcaggac tcat 14 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TaqI adapter primer PRTP-0 <400> 43 gacgatgagt cctgagcga 19 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 44 atatatatat atatatatat atat 24 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 45 agagagagag agagagagag agag 24 <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 46 cacacacaca cacacacaca caca 24 <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 47 acgacgacga cgacgacgac gacg 24 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 48 ctcctcctcc tcctcctcct cctc 24 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 49 taataataat aataataata ataa 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 50 aacaacaaca acaacaacaa caac 24 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 51 aagaagaaga agaagaagaa gaag 24 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 52 acgacgacga cgacgacgac gacg 24 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 53 agcagcagca gcagcagcag cagc 24 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 54 aggaggagga ggaggaggag gagg 24 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 55 ccgccgccgc cgccgccgcc gccg 24 <210> 56 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 56 ctcctcctcc tcctcctcct cctc 24 <210> 57 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe <400> 57 taataataat aataataata ataa 24 <210> 58 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-0 primer <400> 58 gactgcgtac caattc 16  

Claims (7)

SSR primer pair having two nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 38. According to claim 1, SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30, SEQ ID NO: 31 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, and SEQ ID NOs: 37 and 38. The SSR primer pair according to claim 1 or 2, which is derived from sesame seeds ( Sesamum indicum L.). (a) extracting genomic DNA from the sesame plant to be analyzed; (b) using the extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; And (c) A method for detecting DNA polymorphism of a plant in sesame, comprising electrophoresis of a PCR product. (a) extracting genomic DNA from the sesame plant to be analyzed; (b) using the extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; And (c) Varieties of sesame plants varieties comprising the step of electrophoresis of the PCR product. A kit for detecting DNA polymorphism of sesame plants, comprising the SSR primer pair of claim 1 or 2, a DNA polymerase and a PCR reaction buffer. A kit for identifying a variety of sesame plants comprising the SSR primer pair of claim 1 or 2, a DNA polymerase and a PCR reaction buffer.

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