KR101747266B1 - Method and kit for identifying origin of sesame using single nucleotide polymorphism markers - Google Patents

Abstract

The present invention relates to a kit for identifying sesame breeds and a method for identifying sesame varieties using a primer set. More particularly, the present invention relates to kits using a set of single-nucleotide polymorphic primers for identification of sesame varieties, which are polymorphic to known varieties of sesame seeds, and a method for identifying them using the same. By using the primer set according to the present invention as a single base polymorphism marker for sesame breed identification, it can greatly contribute to various fields such as identification of authenticity of sesame breeds, identification of origin and selection of a control varietal for varieties protected varieties.

Description

Methods and kits for identification of sesame varieties using single nucleotide polymorphism (SNP) markers

The present invention relates to a method for identifying a sesame variety using a polymorphic marker. More particularly, the present invention relates to a primer set for identifying sesame cultivars, a kit including the primer set, and a method for identifying sesame cultivars using the primer set.

A DNA marker refers to a specific nucleotide sequence fragment that can be used to determine whether a polymorphism is present on the genome. DNA markers have been used in the field of crop breeding for the development of markers related to traits and marker assisted selection (MAS), gene mapping, quantitative trait locus analysis, purity testing, genetic diversity analysis and breed identification.

In general, methods for identifying plant cultivars are divided into two stages: morphological characterization through cultivation tests, assays based on differences between isoenzymes and DNA analysis methods. DNA analysis method is advantageous in that it is objective and reproducible because it is not influenced by cultivation environment and crop growth stage as compared with phenotype-based identification method. The International Union for the Protection of New Varieties (UPOV) is conducting discussions to utilize DNA markers to investigate the characteristics of new varieties and to grant plant varieties protection rights. Various studies have suggested the possibility of using the nucleotide sequence differences at the gene level as molecular markers for breed identification. Specifically, SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) methods have been proposed in consideration of the polymorphism degree, the reproducibility of analysis, and the distribution of chromosomal patterns among the types of molecular markers.

In Korea, RDA has reported the technology for identification of varieties using SSR molecular markers for rice and soybean and CAPS molecular markers for sesame seeds. Using the SSR molecular marker, the National Seed Source has database of the DNA profile of the crop for the goji, etc. using the DNA profile of the crop and the SNP molecular marker for pepper, Chinese cabbage, watermelon, etc., and as a means to intervene in case of seed dispute, It is a tendency to utilize genetic analysis results in selection of control varieties for varieties of protected varieties using markers.

Another example of the development and utilization of DNA analysis for identification of varieties in Korea is as follows: SNP markers (Patent No. 10-0974821) for identification of commercially available pepper F1 varieties, domestic SNP markers for domestic rice varieties (Patent No. 10-0713669) A variety of assays using genetic markers are being used agriculturally.

The development of molecular genetics and DNA analysis techniques has led to the development of genetic markers including SNPs such as searching genes related to economic traits and discovering disease related genes at molecular level. (genetic marker) has been actively developed.

 As various DNA analysis techniques such as PCR and polymorase chain reaction have been developed, they have been utilized in various industries such as species discrimination and individual identification. In allele-specific PCR (AS-PCR), SNPs are located at the ends of the primers used for gene amplification. An artificial mutation at the ends of the primers is designed so that primer ends (3 'end) And the SNP type of each individual is confirmed by the presence or absence of the gene amplification product by using the characteristic that the single base of the part can not be bound or bound.

On the other hand, human beings have been using sesame seeds since about 3,000 BC, and sesame has been widely cultivated as a health food for reasons of crop function. As of December 2015, the number of registered varieties of registered varieties was 38 varieties, and the number of declarations of domestic production import sales was 72. As a result, the number of varieties of varieties and the number of declarations of production imports are increasing. In addition, it is urgent to protect domestic sesame seeds from imported sesame seeds. In order to test for the protection of sesame varieties, cultivation tests should be conducted. The cultivation environment is affected by the cultivation environment and it takes much time to characterize, so the importance of developing breeding and breeding methods using molecular markers is increasing . Therefore, a technique for quickly and economically discriminating the variety and origin of sesame has been demanded. In the present invention, sesame varieties were identified by using variations such as SNP.

The present invention provides a primer set for identifying sesame varieties.

The present invention provides a kit for identifying sesame varieties comprising the primer set.

The present invention provides a method of identifying a variety of sesame seeds using the primer set.

The present invention establishes a gene analysis system for breed identification of sesame seeds. The SNP markers used in the development of the system are base variants that show variations in individual or varieties of species or interspecies, between sexes, or between nucleotide sequences of chromosomes, and the difference in single base-pare variation Of the polymorphisms of the DNA sequence. Using these characteristics, genetic markers were selected to discriminate the cultivars. Markers were selected from among the selected markers and discriminated by using markers having excellent results in specificity and sensitivity.

The present invention relates to an SSKh2 primer set comprising oligonucleotides of SEQ ID NOS: 1 and 2; An SSKh4 primer set consisting of the oligonucleotides of SEQ ID NOS: 3 and 4; An SSKh5 primer set consisting of the oligonucleotides of SEQ ID NOS: 5 and 6; An SSKh7 primer set consisting of the oligonucleotides of SEQ ID NOS: 7 and 8; An SSKh8 primer set consisting of the oligonucleotides of SEQ ID NOS: 9 and 10; An SSKh10 primer set consisting of the oligonucleotides of SEQ ID NOS: 11 and 12; An SSKh14 primer set consisting of the oligonucleotides of SEQ ID NOS: 13 and 14; An SSKh21 primer set consisting of the oligonucleotides of SEQ ID NOS: 15 and 16; An SSKh24 primer set consisting of the oligonucleotides of SEQ ID NOS: 17 and 18; An SSKh26 primer set consisting of the oligonucleotides of SEQ ID NOS: 19 and 20; An SSKh31 primer set consisting of the oligonucleotides of SEQ ID NOS: 21 and 22; And a set of SSKh33 primers consisting of the oligonucleotides of SEQ ID NOs: 23 and 24.

The primer set may exhibit high polymorphism for known sesame varieties. Thus, the primer set can be used to identify sesame varieties. The primer set may be used, for example, to identify one or more of the sesame varieties described in Table 2 below.

12 pairs of primer sets containing the oligonucleotides of SEQ ID NOS: 1 to 24 of the present invention are shown in Table 1 below. As used herein, 'marker' may refer to an amplification product obtained through each primer set or each primer set itself.

Marker
name The primer sequence (5 '-> 3') SSKh2 Forward (SEQ ID NO: 1) ATCTAAGGAAAGCTCGGACAGGAC Reverse (SEQ ID NO: 2) AGAAGCACGGCCTTTCAAAAG SSKh4 Forward (SEQ ID NO: 3) ACAATGACGATAAAGAGAAGTAAGAAAGCT Reverse (SEQ ID NO: 4) TGGTTATGTGGAGACTGATGAGTGC SSKh5 Forward (SEQ ID NO: 5) TCTCATGCTTGAAAAGAACAACAGC Reverse (SEQ ID NO: 6) AGCCAGCTTATTTTCGTTTTACTTTTC SSKh7 Forward (SEQ ID NO: 7) TCTTACAAGTATATCAATGAAAGTTTGCGG Reverse (SEQ ID NO: 8) TCAATGCCAGCTGAATTTTCAGAG SSKh8 Forward (SEQ ID NO: 9) ATGCAAGTTGTGCTGTTCTGCTCTAT Reverse (SEQ ID NO: 10) AAGAGACCCCCAAGACAGATACATG SSKh10 (SEQ ID NO: 11) TGTTGAGATTGAGAGGGAGAGAATATCA Reverse (SEQ ID NO: 12) ATCCCTTCACTGTTTTTGTGTTTAACC SSKh14 Forward (SEQ ID NO: 13) CCATGGCATCATAGATAAAGCTTACT Reverse (SEQ ID NO: 14) TCACCAATGTCCATCACCCC SSKh21 Forward (SEQ ID NO: 15) AAAGTGGAACACAACAAGAAAAATAACAT Reverse (SEQ ID NO: 16) CAGCCTTCAAACAAGAAATATAGAATCTG SSKh24 Forward (SEQ ID NO: 17) TCAAAGCTTTTTCAAAAGAATATCAGAG Reverse (SEQ ID NO: 18) TGGAACAGTTCATCACTTTGTGG SSKh26 Forward (SEQ ID NO: 19) AGAGCGGCCGTAAGAGCTG Reverse (SEQ ID NO: 20) TCACTTTTCTCTTCTCCTTCTCCC SSKh31 Forward (SEQ ID NO: 21) CACGAAAAGGACCAAACTATAACC Reverse (SEQ ID NO: 22) TAGAGTCATTAGTTTTCATGCATCTTG SSKh33 Forward (SEQ ID NO: 23) AAATCTTCAATTGGACAAGAAAAATG Reverse (SEQ ID NO: 24) GATTGGAGGAACATAGTGCATTAAC

The oligonucleotide constituting the primer set may include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, a peptide nucleic acid or an intercalating agent. The oligonucleotide may further comprise fluorescent, phosphorescent or radioactive labeling substance. The fluorescent labeling substance may be VIC, NED, FAM, PET, or a combination thereof. The labeling substance may be labeled at the 5 ' end of the oligonucleotide. In addition, the radioactive labeling substance can be incorporated into the amplification product through PCR reaction using a PCR reaction solution in which a radioisotope such as ³² P or ³⁵ S is added.

The primer sets of the present invention can be used to distinguish single nucleotide polymorphic genotypes or to identify insert-deletions (indeles) according to the presence and size of amplification products generated by PCR or AS-PCR.

The SSKh2 primer set consisting of the oligonucleotides of SEQ ID NOS: 1 and 2 distinguishes T and C, which are the 378th nucleotides of SEQ ID NO: 27,

The SSKh4 primer set consisting of the oligonucleotides of SEQ ID NOS: 3 and 4 distinguishes the single base polymorph T, which is the 30 < th > base of SEQ ID NO: 28 from A,

The SSKh5 primer set consisting of the oligonucleotides of SEQ ID NOS: 5 and 6 distinguishes the single base polymorphism G and C, which is the 153rd base of SEQ ID NO: 29, and distinguishes the single base polymorphism A and G,

The SSKh7 primer set consisting of the oligonucleotides of SEQ ID NOS: 7 and 8 distinguishes the single base polymorphism G and C, which is the 30 < th > base of SEQ ID NO:

The SSKh8 primer set consisting of the oligonucleotides of SEQ ID NOS: 9 and 10 distinguishes the single base polymorphism T and C, which is the 26 < th > base of SEQ ID NO:

The SSKh10 primer set consisting of the oligonucleotides of SEQ ID NOS: 11 and 12 distinguishes the single base polymorphism T and G, which is the 26 th base of SEQ ID NO: 32, and distinguishes the single base polymorphism A and G,

The SSKh14 primer set consisting of the oligonucleotides of SEQ ID NOS: 13 and 14 distinguishes the single base polymorphism T, which is the 26 < th > base of SEQ ID NO: 33 from A,

The SSKh21 primer set consisting of the oligonucleotides of SEQ ID NOS: 15 and 16 distinguishes the single base polymorphism T and G, which is the 29 < th > base of SEQ ID NO:

The SSKh24 primer set consisting of the oligonucleotides of SEQ ID NOS: 17 and 18 identifies the insert-deletion indent of the 182nd to 196th bases of SEQ ID NO: 35,

The SSKh26 primer set consisting of the oligonucleotides of SEQ ID NOS: 19 and 20 discriminates single base polymorphism G and C, which is the 264th base of SEQ ID NO: 36,

The SSKh31 primer set consisting of the oligonucleotides of SEQ ID NOS: 21 and 22 identifies the insertion-deletion of the 17th to 27th bases of SEQ ID NO: 37,

And the SSKh33 primer set consisting of the oligonucleotides of SEQ ID NOS: 23 and 24 can identify insertion-deletion of the 144th to 149th bases of SEQ ID NO: 38.

In addition, the present invention relates to a primer set, an SSKh2 primer set, a SSKh4 primer set, A kit for identifying sesame varieties, comprising a set of SSKh33 primers. The kit may further comprise DNA polymerase, dNTP and PCR buffer solution. In addition, components necessary for PCR, specifically AS-PCR or confirmation of amplification products, can be further included in the kit. The PCR buffer may contain KCl, Tris-HCl and MgCl _2. The kit may include a brochure. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

The present invention also relates to a method for screening a genomic DNA of the present invention using genomic DNA derived from sesame as a template and comprising a primer set selected from the group consisting of SSKh2 primer set, SSKh4 primer set, SSKh4 primer set, SSKh5 primer set, SSKh5 primer set, SSKh7 primer set, SSKh8 primer set, SSKh10 primer set, SSKh14 primer set, Amplifying the nucleic acid using primer set, SSKh26 primer set, SSKh31 primer set, and SSKh33 primer set as primers; And determining the varieties of sesame seeds from the resulting amplification products.

The genomic DNA may be derived from the leaves, stems, or combinations thereof. The genomic DNA can be obtained using a conventional method for obtaining DNA from a plant. The genomic DNA can be obtained, for example, using a phenol extraction method.

In this method, amplification of the nucleic acid can be performed using PCR. Specifically, amplification of the nucleic acid can be performed using AS-PCR. The amplification of the nucleic acid was carried out using the SSKh2 primer set, SSKh4 primer set, SSKh5 primer set, SSKh5 primer set, SSKh7 primer set, SSKh8 primer set, SSKh8 primer set, SSKh8 primer set, SSKh10 primer set, SSKh14 primer set, SSKh21 primer set, SSKh21 primer set, May be performed using all of the primer sets.

PCR or AS-PCR methods are well known in the art and commercially available kits can be used. The PCR or AS-PCR reaction can be carried out using a reaction solution containing various components known to be required for PCR or AS-PCR reaction in the art. The reaction solution may be, for example, a genomic DNA derived from sesame to be analyzed, a primer set of the present invention, a DNA polymerase (for example, Taq polymerase), a dNTP mixture, a PCR buffer solution and water .

The method may include detecting the resulting amplification product by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement. When detected by gel electrophoresis, agarose gel electrophoresis or acrylamide gel electrophoresis can be used depending on the amplification product size. In the case of detection through fluorescence measurement, fluorescence can be measured using a fluorescence meter after carrying out PCR using the primer set of the present invention labeled with a fluorescent substance. When detecting by radioactivity, the radioactive material is added to the PCR reaction solution to label the amplified product, and radioactivity is measured using a radioactivity meter such as a Geiger counter or a liquid scintillation counter .

In the above method, the step of determining the sesame breed from the obtained amplification product comprises amplifying the obtained amplification product by amplifying the obtained amplification product with a primer set selected from the group consisting of SSKh2 primer set, SSKh4 primer set, SSKh5 primer set, SSKh7 primer set, SSKh8 primer set, SSKh10 primer set, SSKh14 primer set , The SSKh21 primer set, the SSKh24 primer set, the SSKh26 primer set, the SSKh31 primer set, and the SSKh33 primer set.

Specifically, the step of determining the sesame cultivar from the amplified product may be performed by confirming the size of the amplified product (band) using an automatic nucleotide sequencer by a computer program. For example, the presence and size of products (corresponding to each allele) amplified through the primer set of the present invention for known sesame cultivars are summarized in one table in advance. Specifically, when two allele single nucleotide polymorphisms are present, one allele genotype can be represented by "1" and the other allele genotype can be represented by "0" according to the presence or absence of the amplified product. If there is a single nucleotide polymorphism of A and G, the A genotype may be represented by "1 " and the G genotype may be represented by" 0 ".

That is, genomic DNA was isolated from sesame seeds for breed identification, amplified using the primer set of the present invention, analyzed for the size of the amplified product, and the result of the analysis was amplified from the nucleic acid of the known sesame variety By comparing the results with statistical programs, we can determine the varieties of sesame seeds.

The present invention has established a method and system for identifying sesame varieties using molecular markers in domestic and foreign sesame cultivars. According to the present invention, since it is possible to identify domestic and imported sesame varieties, the present invention can be utilized for agricultural products origin markers. Furthermore, it can contribute to such fields as identification of the authenticity of sesame seeds, arbitration of seed conflicts, and selection of control varieties for varieties.

1 is a schematic diagram of a primer for performing an allele-specific polymerase chain reaction (AS-PCR).
FIGS. 2A, 2B, and 2C show the result of performing a single PCR using 27 pairs of primer sets for discriminating sesame cultivars.
FIG. 3 shows results of multiplex PCR (Mulitiplex PCR) using 12 pairs of primer sets to identify sesame varieties.
FIG. 4 shows the result of confirming amplification products using 12 pairs of primer sets for discriminating sesame cultivars. FIG. 4A shows the amplification product using the SSKh2 primer set, FIG. 4B shows the SSKh4 primer set, FIG. 4C shows the amplification product using the SSKh5 primer set, , FIG. 4F shows the amplification product using the SSKh10 primer set, FIG. 4G shows the amplification product using the SSKh14 primer set, FIG. 4H shows the amplification product using the SSKh21 primer set, FIG. 4I shows the amplification product using the SSKh24 primer set, FIG. 4K shows the amplification products using the SSKh31 primer set, and FIG. 4L shows the amplification products using the SSKh33 primer set.
FIG. 5 is a result of coding allelic genotypes of varieties analyzed by 12 pairs of primer sets to identify sesame varieties as 1 or 0.
FIG. 6a shows the genetic similarity between domestic sesame varieties analyzed by 12 pairs of primer sets to identify sesame varieties. FIG. 6b shows the genetic similarities between domestic and imported sesame varieties analyzed by 12 pairs of primer sets to identify sesame varieties Lt; / RTI >

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Example  1: for sesame breed identification Marker  evaluation

(1) Sesame samples

In the present invention, sesame seeds 123 cultivars described in the following table were used for sesame breed discrimination test. Specifically, samples for the analysis of genome sequence and genetic variation of sesame seeds were classified into 36 varieties of protected varieties, 35 varieties of domestic varieties, 35 varieties of imported varieties, and imported genetic resources of genetic variation centers 52 species were selected.

(2) Sesame genomic DNA isolation

Sesame genomic DNA was isolated using the NucleoSpin ^® Plant Genome Kit (MN Co. Germany). After the sesame sample was pulverized, 225 μl of a lysis buffer was added to the ground sesame sample, and the mixture was reacted at 60 ° C. for 40 minutes. The reacted sample was centrifuged at 8,000 xg for 3 minutes, and 200 μl of the supernatant was taken from the centrifuged sample. 175 μl of the dissolution buffer and 250 μl of ethanol were added to 200 μl of the supernatant, and the mixture was homogenized for 15 seconds and then passed through a column to remove impurities. The column was washed three times with 700 [mu] l of wash buffer and genomic DNA was recovered from the column using 50 [mu] l of sterile distilled water. The extracted genomic DNA was analyzed for concentration and purity using a ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, USA) and used for next-generation sequencing.

(3) Sesame sequence analysis and gene mutation search

Sesame has about 354 Mbp genome with 2n = 26, and genome information already obtained is needed to search SNP and InDel for breed identification of sesame. SNP and InDel information for sesame breed identification were collected from the National Biological Information Center (NCBI) website (www.ncbi.nlm.nih.gov). 136 primers were prepared for sequence analysis in order to search for mutations that could be applied to domestic sesame varieties. Using these primers, we analyzed the nucleotide sequences of seven major domestic sesame seeds (Yumi, Ansan, Super Ansan, 5002, Grammy, Persimmon, Kinta). In order to find the SNP location for breed identification, the results of analysis of the nucleotide sequence by variety were analyzed by ClustalW method using DNASTAR computer program (www. Dnastar.com). A total of 37 SNPs and InDel were identified. Of these, 27 SNPs were selected for easy identification of sesame seeds.

(4) allele specific PCR primer  making

The InDel marker amplified DNA fragments containing InDel with two flanking primers. 2000 Plant Physiol 124, 1483-1492, Kwok, S. et al., 1990 Nucleic Acids Res 18, 999-1005) were used to generate SNP primers (Drenkard, E. et al. (See Fig. 1).

In order to prepare a primer for allele-specific PCR for discriminating sesame cultivars, a modified sequence in which any one or two bases of the bases at positions 2 to 5 from the 3 'end were mutated was prepared. As a result of using a modified sequence in which any one of the bases at positions 2 to 5 from the 3 'end in the base sequence was mutated to A, T, G, or C as a marker for sesame breed identification, It was possible to identify it with other varieties without interfering with other varieties. Thus, in developing a marker to identify sesame varieties, a sense primer and an antisense primer were prepared so that an SNP or an Indel site is 3 'terminus and an artificial inconsistency is induced in the third base. The binding temperature of the SNP marker was 60 ° C and the size was 116 to 490 bp. The genotypes of sesame breeds were determined by amplification of PCR products on agarose gel (see FIG. 2). From this, 27 pairs of primer sets for identification of sesame breed varieties, which are easy to identify 123 varieties, including 71 domestic varieties of sesame and 52 imported varieties, were produced. 27 pairs of primer sets are shown in Table 3 below.

(5) Explored  Sequence re-sequencing of genetic mutations and Gene marker  Selection

Re-sequencing of 27 SNPs was conducted on 71 domestic sesame seed samples. For PCR, the primers were synthesized so that the reaction product was about 700 bp. After the PCR reaction, the reaction products were purified with a multi-well filter plate (Pall Corporation, USA) And using a BigDyeⓡ Terminator (v3.1) Cycle Sequencing Kit (Applied Biosystems, UK).

To 2 μl of the purified PCR product, 1.75 μl of 5 × buffer, 0.5 μl of Big Dye and 5 pmol / μl of a primer were added, followed by addition of sterilized distilled water to a total of 10 μl. The reaction was carried out by heating at 96 占 폚 for 10 seconds, then repeating 34 times at 50 占 폚 for 5 seconds and at 60 占 폚 for 4 minutes. After PCR reaction, the reaction mixture was purified with ethanol and dried. To the dried sample, 8 μl of Hi-Di ™ formamide was added, reacted at 95 ° C. for 2 minutes, and analyzed by ABI 3730xl (Applied Biosystems) The nucleotide sequences of each variety were determined. Sequence analysis was performed using the Lasergene SeqMan program (DNASTAR, Inc., USA) to identify SNPs.

As a result of the base sequence reanalysis, 27 SNP markers which are easy to identify sesame variety were identified. Furthermore, the final 12 SNP markers were selected for efficient breed identification. And multiplex PCR conditions were further established using this. Referring to FIGS. 3A and 3B, DNA of 5002 varieties of Yumi, Gramineae and midworm were amplified using 12 pairs of primer sets, and the amplification products were confirmed by bands. As a result, a product-specific amplification product was formed . In order to improve the accuracy of breed identification, control markers were constructed and used for the RBCL gene located in the chloroplasts as needed.

(6) Allele specific Polymerase chain reaction  (AS- PCR ) To identify sesame seeds

Allele-specific polymerase chain reaction (AS-PCR) was performed on the RBCL gene located in the salt rock as 12 single nucleotide polymorphism (SNP) markers and an internal control marker (RBCL) (SSKh2 primer set, SSKh4 primer set, SSKh5 primer set, SSKh5 primer set, SSKh7 primer set, SSKh7 primer set, SSKh8 primer set, SSKh10 primer set, SSKh10 primer set, SSKh14 primer set, SSKh21 primer set, SSKh24 primer set, SSKh26 Primer set, SSKh31 primer set, SSKh33 primer set and RBCL primer set) were used.

PCR (allele-specific polymerase chain reaction) for the individual analysis of 12 SNP markers was performed by adding 2.5 μl of 10 × buffer, 1 μl of dNTP (2.5 mM each), 50 μl of forward and reverse primer 0.5 μl of each, and 2 U of polymerase (Taq polymerase, Takara, Japan) were added, and the final amount was increased to 25 μl with sterilized distilled water. Gene amplification was carried out using GeneAmp PCR System 9700 (Applied Biosystems, USA). After denaturation at 94 ° C for 15 minutes, the reaction was repeated 30 times at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute and 20 seconds Respectively. PCR amplification products were confirmed by electrophoresis on 3% agarose gel. The results of the single PCR were the same as in Fig.

(7) Marker  Sesame variety identification test

The possibility of identification of the sesame variety of the marker according to the present invention was examined. Polymorphism information content (PIC) values were calculated using the following equation (1). K is the total number of alleles of each allele, and Pi is the frequency of the i-th common band pattern in the marker band (Anderson et al. 1993).

<Formula 1>

The amplification products (bp) and PIC values are shown in Table 4 below.

Serial number Marker name Primer sequence size
(bp) Amplification
Product
size
(bp) transition
Kinds Annealing temperature
(° C) genotype PIC value One SSKh2 F ATCTAAGGAAAGCTCGGACAGGAC 24 398 SNP 60 [C / T] 0.405 2 R AGAAGCACGGCCTTTCAAAAG 21 3 SSKh4 F ACAATGACGATAAAGAGAAGTAAGAAAGCT 30 216 SNP 60 [T / A] 0.299 4 R TGGTTATGTGGAGACTGATGAGTGC 25 5 SSKh5 F TCTCATGCTTGAAAAGAACAACAGC 25 179 SNP 60 [G / C], [A / G] 0.428 6 R AGCCAGCTTATTTTCGTTTTACTTTTC 27 7 SSKh7 F TCTTACAAGTATATCAATGAAAGTTTGCGG 30 313 SNP 60 [G / C] 0.378 8 R TCAATGCCAGCTGAATTTTCAGAG 24 9 SSKh8 F ATGCAAGTTGTGCTGTTCTGCTCTAT 26 399 SNP 60 [T / C] 0.438 10 R AAGAGACCCCCAAGACAGATACATG 25 11 SSKh10 F TGTTGAGATTGAGAGGGAGAGAATATCA 28 269 SNP 60 [T / G], [A / G] 0.405 12 R ATCCCTTCACTGTTTTTGTGTTTAACC 27 13 SSKh14 F CCATGGCATCATAGATAAAGCTTACT 26 200 SNP 60 [T / A] 0.492 14 R TCACCAATGTCCATCACCCC 20 15 SSKh21 F AAAGTGGAACACAACAAGAAAAATAACAT 29 281 SNP 60 [T / G] 0.448 16 R CAGCCTTCAAACAAGAAATATAGAATCTG 29 17 SSKh24 F TCAAAGCTTTTTCAAAAGAATATCAGAG 28 203 INDEL 60 GTTCATCACTTTGTG 0.349 18 R TGGAACAGTTCATCACTTTGTGG 23 19 SSKh26 F AGAGCGGCCGTAAGAGCTG 19 287 SNP 60 [G / C] 0.500 20 R TCACTTTTCTCTTCTCCTTCTCCC 24 21 SSKh31 F CACGAAAAGGACCAAACTATAACC 24 116 INDEL 60 CTTTAACCAAA 0.242 22 R TAGAGTCATTAGTTTTCATGCATCTTG 27 23 SSKh33
F AAATCTTCAATTGGACAAGAAAAATG 26 166
INDEL
60
TAATGC
0.500
24 R GATTGGAGGAACATAGTGCATTAAC 25 25 immanence
Control markers
(rbcL) F TCTTCATATCCACCGTGCAA 20 500
26 R AGCAGCTAATTCAGGACTCC 20

1 and 2 (marker name: SSKh2), SEQ ID NO: 3 and 4 (marker name: SSKh4), SEQ ID NO: 5 and 6 (marker 11 and 12 (Marker Name: SSKh10), SEQ ID NOS: 13 and 14 (Marker Name: SSKh5), SEQ ID NOS: 7 and 8 (Marker Name: SSKh7) 19 and 20 (marker name: SSKh26), SEQ ID NOS: 21 and 22 (marker name: SSKh31), SEQ ID NOS: 15 and 16 (marker name: SSKh21) , And the average PIC value of the 12 markers of SEQ ID NO: 23 and 24 (marker name: SSKh33) was 0.406, confirming that the identification of the sesame variety was sufficiently possible through the marker of the present invention. In order to accurately determine the presence or absence of the alleles (bands) of the marker according to the present invention, the accuracy of identification of sesame varieties was determined by using SEQ ID NOs: 25 and 26 (marker name: RBCL) as the internal control marker.

The gene amplification products of 12 SNP markers can be compared with the database of FIG. 5 through allele-specific polymerase chain reaction (AS-PCR) to identify the breeds and to determine the origin. The presence or absence of alleles (bands) in the marker according to the present invention is determined. When there is a band, the score is coded as "1", and when there is no band, it is coded as "0". FIG. 5 is a result of coding the presence or absence of alleles identified by using the sesame breed identification primer set according to the present invention on 123 varieties of sesame (71 domestic and 52 imported). Genomic DNA is isolated from the sesame seeds for which the breed is to be identified, amplified using the primer set of the present invention, and compared with the result of the amplification product and the result of FIG. 5, thereby identifying the sesame variety and the country of origin. Specifically, the present invention can identify 71 domestic varieties and 52 imported varieties (10 from China, 23 from Uzbekistan, 3 from India and 16 from India) among 123 known varieties using 12 single nucleotide polymorphic markers (SNP markers) It provides a method to distinguish the origin of domestic and imported products, and it is possible to identify 67 domestic varieties.

Genetic similarity values were calculated according to Jaccard's method (Sneath and Sokal, 1973) by inputting the presence of an allele into a computer program of NTSYS pc (version 2.10b) (Rohlf, 2000). Using the calculated genetic similarity, an aggregate analysis was performed by UPGMA (Unweighted Pair-group Method with Arithmetical Average) (Sneath and Sokal, 1973) to prepare a dendrogram, and the results are shown in FIG. FIG. 6 shows the genetic similarity of each sesame variety analyzed by the primer set for discriminating sesame cultivars according to the present invention. As a result, it was confirmed that it is possible to distinguish the origin and the breed using the primer set of the present invention.

<110> Republic of Korea <120> Method and kit for identifying origin of sesame using single          nucleotide polymorphism markers <130> PN114803 <160> 38 <170> KoPatentin 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SSKh2 F-primer <400> 1 atctaaggaa agctcggaca ggac 24 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SSKh2 R-primer <400> 2 agaagcacgg cctttcaaaa g 21 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SSKh4 F-primer <400> 3 acaatgacga taaagagaag taagaaagct 30 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SSKh4 R-primer <400> 4 tggttatgtg gagactgatg agtgc 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SSKh5 F-primer <400> 5 tctcatgctt gaaaagaaca acagc 25 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SSKh5 R-primer <400> 6 agccagctta ttttcgtttt acttttc 27 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SSKh7 F-primer <400> 7 tcttacaagt atatcaatga aagtttgcgg 30 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SSKh7 R-primer <400> 8 tcaatgccag ctgaattttc agag 24 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> SSKh8 F-primer <400> 9 atgcaagttg tgctgttctg ctctat 26 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SSKh8 R-primer <400> 10 aagagacccc caagacagat acatg 25 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> SSKh10 F-primer <400> 11 tgttgagatt gagagggaga gaatatca 28 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SSKh10 R-primer <400> 12 atcccttcac tgtttttgtg tttaacc 27 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> SSKh14 F-primer <400> 13 ccatggcatc atagataaag cttact 26 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSKh14 R-primer <400> 14 tcaccaatgt ccatcacccc 20 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SSKh21 F-primer <400> 15 aaagtggaac acaacaagaa aaataacat 29 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SSKh21 R-primer <400> 16 cagccttcaa acaagaaata tagaatctg 29 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> SSKh24 F-primer <400> 17 tcaaagcttt ttcaaaagaa tatcagag 28 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SSKh24 R-primer <400> 18 tggaacagtt catcactttg tgg 23 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SSKh26 F-primer <400> 19 agagcggccg taagagctg 19 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SSKh26 R-primer <400> 20 tcacttttct cttctccttc tccc 24 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SSKh31 F-primer <400> 21 cacgaaaagg accaaactat aacc 24 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SSKh31 R-primer <400> 22 tagagtcatt agttttcatg catcttg 27 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> SSKh33 F-primer <400> 23 aaatcttcaa ttggacaaga aaaatg 26 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SSKh33 R-primer <400> 24 gattggagga acatagtgca ttaac 25 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RBCL F-primer <400> 25 tcttcatatc caccgtgcaa 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RBCL R-primer <400> 26 agcagctaat tcaggactcc 20 <210> 27 <211> 398 <212> DNA <213> Unknown <220> <223> sesame <400> 27 atctaaggaa agctcggaca ggacgatgtg taatgcttga gatccaatta gctcattcac 60 agcacagggt ctcctggatc tcagcaactg gtttgacctg agattggttt tgctcaaatg 120 catcaacagt tgagttcaga cagcaggacc aaattgccat ggtttcactc aatttcaatg 180 ggaagaatat tgtgttaagc acatatacac ttcttcctgc atgtctgtct tgccatcaaa 240 atatacatag gacaaatcta taaagagcat cataaccaat ctgaagatga ccaccgctcc 300 cataattgac acatttcttg tgtcgctcaa cattggcttc aaaactggtg gtcctgatat 360 ctttcctgtt tttcttcttg ttgaaaggcc gtgcttct 398 <210> 28 <211> 216 <212> DNA <213> Unknown <220> <223> sesame <400> 28 acaatgacga taaagagaag taagaaatct gagtaattac cttcaccagc agcaagattg 60 aagtaaaggt caacaatgtt ggggcaatta tggtagcaag caggcgagca aaggcttgag 120 atgaattcag gtgtcaggaa ggcatcggag gatattccga caaagttacg gtcaacacca 180 catgcattca cgcactcatc agtctccaca taacca 216 <210> 29 <211> 179 <212> DNA <213> Unknown <220> <223> sesame <400> 29 tctcatgctt gaaaagaaca acagcaggaa atctacatga gcaaagacac acagtggttg 60 tacccattag aggtattgca gctctggggc tcactaattg aactttgtgc tgcagcaata 120 tcatgaactt gcagatcgcg aacatgtata tagacaagta aaacgaaaat aagctggct 179 <210> 30 <211> 313 <212> DNA <213> Unknown <220> <223> sesame <400> 30 tcttacaagt atatcaatga aagtttgggg caaaatatga agcatttgga atcaatatac 60 tgatcaactc tgaacatcaa tgcccaatag cctatatcat caatcaagat tcaagaatca 120 atcagaagaa actcgtattc tgagttcaaa aggggatgga aactgaagac tttgaggtga 180 aggcttcaat ttctttgtca tccatccact cggttttcca ttgagaaata atatcagaga 240 cattaatggt agttttgccg tccggcccaa ctgggtaata tggtgaatcc tctgaaaatt 300 cagctggcat tga 313 <210> 31 <211> 399 <212> DNA <213> Unknown <220> <223> sesame <400> 31 atgcaagttg tgctgttctg ctccattctg acccccaaaa cgagcgcgat cagtacrcat 60 cgacgttctt taggcacaag ttggcggaga tgaagtcttt gagaagccag ttgagtgcca 120 agaaacagcg ggatgagggg gaagaagcgc attatagaca gatggagatg gaggcagagt 180 tgcagcgaag gaggcacaac atgcacatgc agcagctgca gcgaatgtcc gattctatcg 240 attttatggg acaaattggg acttcgacca attacactta cagatacttc tgaatatgat 300 gaatattggt tcttgatgtt cagttcggtc cggtctatgt taaatttcga atgttgttgt 360 tggtttgatt tgttcatgta tctgtcttgg gggtctctt 399 <210> 32 <211> 269 <212> DNA <213> Unknown <220> <223> sesame <400> 32 tgttgagatt gagagggaga gaatataact cctcactcct cgtatcggtc ttgtaagacc 60 ggaacttgtc ccaccaatgc atgcctctgt ccttcctcgt tgtgttgtcc ttggggggca 120 gcgttacatc cagaaccagc gccaacactc ccgcaacgaa tggctcagac gagaaaggaa 180 cgttgatcat gtcattgaac tggcaagagc catgaaaacg ttacaaattc agacataaga 240 gtggttaaac acaaaaacag tgaagggat 269 <210> 33 <211> 200 <212> DNA <213> Unknown <220> <223> sesame <400> 33 ccatggcatc atagataaag cttcctaact tgatgtatat ctatccaaac agtccaactt 60 gagccagtct agataaaaac gaaaagaaaa ggtcaatagg accgaacaag tggttgaggt 120 agtcgagcat gggataatgg tgtatggcat gggtgttcta tgctgaggca aaggttttgg 180 ggggtgatgg acattggtga 200 <210> 34 <211> 281 <212> DNA <213> Unknown <220> <223> sesame <400> 34 aaagtggaac acaacaagaa aaataaaatc atagcatacc acagcatgcc aatattacat 60 cactcaccaa ccccatgctt tgggatttat aatgcatgct ctacaatgca aatctgaaaa 120 agctgaaatt ttctctgcta ttctcccact acaaacaatc actattcttg gatccataaa 180 accagagatc ccatctttca tctttcgtat atatcccaat gcttaatctc cagtgctgat 240 actaaggcac cacagattct atatttcttg tttgaaggct g 281 <210> 35 <211> 203 <212> DNA <213> Unknown <220> <223> sesame <400> 35 tcaaagcttt ttcaaaagaa tatcagagta atttaccgaa aaaagaatat cagagtaaaa 60 agatagtgtt agagctaata ttattcattc ttccaagcat acaaaatcaa agaaaatcca 120 agacctggca attcagcctc cctgaaactc cctgggcagc tccaagaaga ggcaggtgaa 180 ccacaaagtg atgaactgtt cca 203 <210> 36 <211> 287 <212> DNA <213> Unknown <220> <223> sesame <400> 36 agagcggccg taagagctga aacacctggc cggacacgtc agcttcttct tgtaacacgg 60 gcccttctca ctgcacacaa aagaagtcct tattatgcca tgtttagagt ggcctccaga 120 cgggccccca taccctgatc cataccctcc accgaaaccc ggcccaaacc ccggtatgtt 180 gaacccgcct ccgggaccca agaatccgcc aggcccagca tcaggttggg gtcgggttgc 240 aagggagagg gtggcggaga ttaggaagaa ggagaagaga aaagtga 287 <210> 37 <211> 116 <212> DNA <213> Unknown <220> <223> sesame <400> 37 cacgaaaagg accaaacttt aaccaaagtt ttggcacaaa cccataccca taccagcagc 60 agaagatcat tagtagaaaa tttgatgcac aagatgcatg aaaactaatg actcta 116 <210> 38 <211> 166 <212> DNA <213> Unknown <220> <223> sesame <400> 38 aaatcttcaa ttggacaaga aaaatgaagg aaaaaacact aaaagggtca aagatattat 60 cattttaagt gaataagtat ttttctcaag aaccaaacag tcacacattg gtggtataat 120 ataaaaaagg ggaagcctca agttaatgca ctatgttcct ccaatc 166

Claims (8)

An SSKh2 primer set consisting of the oligonucleotides of SEQ ID NOS: 1 and 2;
An SSKh4 primer set consisting of the oligonucleotides of SEQ ID NOS: 3 and 4;
An SSKh5 primer set consisting of the oligonucleotides of SEQ ID NOS: 5 and 6;
An SSKh7 primer set consisting of the oligonucleotides of SEQ ID NOS: 7 and 8;
An SSKh8 primer set consisting of the oligonucleotides of SEQ ID NOS: 9 and 10;
An SSKh10 primer set consisting of the oligonucleotides of SEQ ID NOS: 11 and 12;
An SSKh14 primer set consisting of the oligonucleotides of SEQ ID NOS: 13 and 14;
An SSKh21 primer set consisting of the oligonucleotides of SEQ ID NOS: 15 and 16;
An SSKh24 primer set consisting of the oligonucleotides of SEQ ID NOS: 17 and 18;
An SSKh26 primer set consisting of the oligonucleotides of SEQ ID NOS: 19 and 20;
An SSKh31 primer set consisting of the oligonucleotides of SEQ ID NOS: 21 and 22;
A set of SSKh33 primers consisting of the oligonucleotides of SEQ ID NOS: 23 and 24; The sesame variety identification primer set according to claim 1, wherein the sesame variety includes one or more of the varieties described in the following table
[table]

. The SSKh2 primer set consisting of the oligonucleotides of SEQ ID NOS: 1 and 2 distinguishes between T and C, which is the 378th nucleotide of SEQ ID NO: 27,
The SSKh4 primer set consisting of the oligonucleotides of SEQ ID NOS: 3 and 4 distinguishes the single base polymorph T, which is the 30 &lt; th &gt; base of SEQ ID NO: 28 from A,
The SSKh5 primer set consisting of the oligonucleotides of SEQ ID NOS: 5 and 6 distinguishes the single base polymorphism G and C, which is the 153rd base of SEQ ID NO: 29, and distinguishes the single base polymorphism A and G,
The SSKh7 primer set consisting of the oligonucleotides of SEQ ID NOS: 7 and 8 distinguishes the single base polymorphism G and C, which is the 30 &lt; th &gt; base of SEQ ID NO:
The SSKh8 primer set consisting of the oligonucleotides of SEQ ID NOS: 9 and 10 distinguishes the single base polymorphism T and C, which is the 26 &lt; th &gt; base of SEQ ID NO:
The SSKh10 primer set consisting of the oligonucleotides of SEQ ID NOS: 11 and 12 distinguishes the single base polymorphism T and G, which is the 26 th base of SEQ ID NO: 32, and distinguishes the single base polymorphism A and G,
The SSKh14 primer set consisting of the oligonucleotides of SEQ ID NOS: 13 and 14 distinguishes the single base polymorphism T, which is the 26 &lt; th &gt; base of SEQ ID NO: 33 from A,
The SSKh21 primer set consisting of the oligonucleotides of SEQ ID NOS: 15 and 16 distinguishes the single base polymorphism T and G, which is the 29 &lt; th &gt; base of SEQ ID NO:
The SSKh24 primer set consisting of the oligonucleotides of SEQ ID NOS: 17 and 18 identifies the insert-deletion indent of the 182nd to 196th bases of SEQ ID NO: 35,
The SSKh26 primer set consisting of the oligonucleotides of SEQ ID NOS: 19 and 20 discriminates single base polymorphism G and C, which is the 264th base of SEQ ID NO: 36,
The SSKh31 primer set consisting of the oligonucleotides of SEQ ID NOS: 21 and 22 identifies the insertion-deletion of the 17th to 27th bases of SEQ ID NO: 37, and
Wherein the SSKh33 primer set consisting of the oligonucleotides of SEQ ID NOS: 23 and 24 identifies the insertion-deletion of the 144th to 149th bases of SEQ ID NO: 38. A kit for identifying sesame varieties, comprising a set of primers for identifying the sesame variety of claim 1. Amplifying the nucleic acid using the genomic DNA derived from sesame as a template and the primer set for discriminating sesame variety according to claim 1 as a primer; And
And determining the varieties of sesame from the resulting amplification products. The method according to claim 5, wherein amplifying the nucleic acid is by allele-specific polymerase chain reaction. The method according to claim 5, comprising detecting the obtained amplification product by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement. [Claim 6] The method according to claim 5, wherein the step of determining the varieties of sesame from the obtained amplification product comprises comparing the amplification product obtained with the result of amplifying the nucleic acid of the sesame variety described in the following table by the primer set for discriminating sesame varieties according to claim 1 How to Identify Sesame Varieties That Are Performed
[table]

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