KR20170072484A - Maker for cultivar discrimination in Korean breeding wheat and method for determination using the same - Google Patents

Abstract

The present invention relates to a primer set for identification of domestic wheat varieties comprising a primer set of oligonucleotides comprising primers of SEQ ID NOS: 55, 57, 63, 64, 70 to 75, 93, 94, 118, 119 and 126 to 139, The present invention relates to a method for distinguishing wheat cultivars from domestic wheat varieties, and it is possible to clearly identify all 32 domestic wheat varieties using the primer set.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a marker for discriminating wheat cultivated in Korea,

The present invention relates to a primer set for identification of domestic wheat varieties including 15 primer sets and a method for distinguishing domestic wheat varieties using the primer set.

Since the 1970s, the per capita consumption of grain has been steadily increasing, but more than 99% of wheat demand has been dependent on imported wheat since the mid-1980s. Therefore, efforts to secure domestic self-sufficiency rate and increase production have been continuously carried out, and varieties suitable for use are being developed. The wheat varieties developed by the National Institute of Food Science and Technology (KFDA) have been diversified by use. For example, Kumgang and Dongdong wheat flour were used for bread, and wheat and wheat wheat flours were used for noodles. As the domestic wheat varieties have been developed in various ways, it is necessary to develop molecular markers that can distinguish them. The developed markers can be used not only for the classification of domestic wheat varieties, but also for protecting domestic varieties and protecting genetic resources of wheat.

On the other hand, molecular markers can be usefully used because they can quickly distinguish cultivars without being influenced by cultivation environment or growth period of crops. A microsatellite or simple sequence repeat (SSR) consisting of mainly repeating simple nucleotide sequences, a marker using a nucleotide sequence or an RFLP (Restriction Fragment Length Polymorphism) probe, and a SCAR (Sequence Characterized Amplified Region) . SCAR markers are produced by analyzing band base sequences such as RAPD (Random Amplified Polymorphic DNA) or AFLP (Amplification Fragment Length Polymorphism) markers, and are used in a manner of confirming the presence or absence of bands by using ASAP (Allele-Specific Associated Primer) . Because SCAR markers are relatively insensitive to amplification environments by other molecular markers and can readily read results, they are recognized as efficient marker markers for breed identification with reproducibility, universality and simplicity.

The inventors of the present invention invented a primer set capable of discriminating 13 varieties among 32 domestic wheat varieties in domestic registration patent No. 1546428 and developed an additional primer set to identify 23 kinds of domestic wheat varieties (Plant Breed Biotech 2 (3), 224-230, 2014). However, molecular markers that can identify all domestic wheat varieties have not yet been developed.

Accordingly, the present inventors have made extensive efforts to develop a molecular marker capable of effectively distinguishing all domestic wheat varieties, and as a result, developed a marker set including a primer capable of distinguishing wheat from each variety, and completed the present invention.

The main object of the present invention is to identify domestic wheat cultivars comprising a primer set of oligonucleotides comprising primers of SEQ ID NOS: 55, 57, 63, 64, 70 to 75, 93, 94, 118, 119 and 126 to 139 And the like.

It is another object of the present invention to provide a method for amplifying genomic DNA comprising the steps of amplifying a genomic DNA using the genomic DNA extracted from a domestic wheat as a template and using the primer set; And comparing the amplified product with an amplified product of a domestic wheat variety standard to determine a wheat variety.

It is still another object of the present invention to provide a kit for identifying a domestic wheat variety comprising the primer set.

In one aspect of the present invention, there is provided a primer set for identification of a domestic wheat variety, which comprises an oligonucleotide primer set of SEQ ID NOs: 55 to 57 and 79 to 96.

Specifically, a primer set according to the present invention comprises a KWSM001 primer set consisting of oligonucleotides of SEQ ID NOS: 55 and 57; A KWSM002 primer set consisting of oligonucleotides of SEQ ID NOS: 63 and 64; A KWSM003 primer set consisting of oligonucleotides of SEQ ID NOS: 70 and 72; A KWSM004 primer set consisting of oligonucleotides of SEQ ID NOS: 71 and 72; A KWSM005 primer set consisting of the oligonucleotides of SEQ ID NOs: 73 and 74; A KWSM006 primer set consisting of oligonucleotides of SEQ ID NOS: 73 and 75; A KWSM007 primer set consisting of oligonucleotides of SEQ ID NOS: 93 and 94; A KWSM008 primer set consisting of oligonucleotides of SEQ ID NOs: 118 and 119; A KWSM009 primer set consisting of oligonucleotides of SEQ ID NOS: 126 and 127; A KWSM010 primer set consisting of oligonucleotides of SEQ ID NOS: 128 and 129; A KWSM011 primer set consisting of oligonucleotides of SEQ ID NOS: 130 and 131; A KWSM012 primer set consisting of oligonucleotides of SEQ ID NOS: 132 and 133; A KWSM013 primer set consisting of the oligonucleotides of SEQ ID NOS: 134 and 135; A KWSM014 primer set consisting of oligonucleotides of SEQ ID NOS: 136 and 137; And KWSM015 primer set consisting of the oligonucleotides of SEQ ID NOS: 138 and 139, respectively. The primer set corresponds to a primer set for amplifying SCAR markers, ALFP markers, SSR markers, and SNP markers for discrimination of all 32 domestic wheat varieties.

The Sequence Characterized Amplified Region (SCAR) marker is produced by analyzing the nucleotide sequence of a molecular marker band such as ISSR (Inter Simple Sequence Repeat) or AFLP (Amplification Fragment). The SCAR (Allele-Specific Associated Primer) And the like. Because SCAR markers are relatively insensitive to amplification conditions and can easily read results compared to other molecular markers, they can be used as more accurate and detailed markers for efficient breed discrimination with reproducibility, universality and simplicity .

In one embodiment of the present invention, SCAR markers were developed by extracting DNA from domestic wheat varieties and performing ISSR analysis and AFLP analysis. Specifically, a SCAR marker of KWSM001 to KWSM006 was developed by ISSR analysis, and a primer set containing primers of SEQ ID NOS: 55, 57, 63, 64, and 70 to 75 that specifically amplified the SCAR marker was developed . In addition, SCAR markers of KWSM007 to KWSM009 were developed by AFLP analysis and primer sets of SEQ ID NOS: 93, 94, 118, 119, 126, and 127 were developed that specifically amplify the SCAR markers.

The ISSR (Inter Simple Sequence Repeat) analysis is a method of analyzing DNA by performing PCR analysis using a DNA sequence having a repeating unit of 2 to 4 bp in size as a primer. This technique uses a high binding temperature of a primer and a long base Due to the sequence, the reproducibility and reliability are high, and many polymorphisms are formed per each primer. It also has the great advantage that the analysis is very fast and the development cost is low.

The AFLP (Amplification Fragment) analysis refers to a method of selectively amplifying fragments cleaved by a restriction enzyme. Such assays can be effectively used for mutation analysis by base substitution, inversion, insertion, and deletion as molecular markers. In addition, the above analysis can be used effectively for genetic linkage and gene map analysis, development of SCAR markers, gene cloning, plant evolution, and biodiversity research.

The SSR (Simple Sequence Repeat) marker refers to a marker consisting of a simple repeating base sequence. The markers exist as multi-alleles in most clusters and provide high-level information with simplicity and high reproducibility, and thus can be utilized in genetic diversity analysis of many plant species. In addition, the SNP (Single Nucleotide Polymorphism) marker refers to a marker in which only a single base is different from a polymorphic site in which two or more alleles exist in one locus.

In one embodiment of the present invention, DNA of domestic wheat variety was extracted to develop transcription factors and agricultural trait-specific SSRs and SNP markers. Specifically, markers of KWSM010 to KWSM015 have been developed and primer sets of SEQ ID NOS: 128 to 139 have been developed which specifically amplify the markers.

The marker refers to a nucleotide sequence used as a reference point when identifying a genetically unrelated locus, and the term locus means a position on a genetic map of a molecular marker.

As used herein, the term " primer " refers to a single stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow for the start of the synthesis of the extension product and the specific length and sequence of the primer depends not only on the complexity of the desired DNA or RNA target but also on the conditions of use such as temperature and ionic strength something to do. As a specific example, the specific length and sequence of the primer should be determined in consideration of various conditions such as guanine and cytosine contents (GC contents), GC arrangement, annealing temperature and ionic strength.

Oligonucleotides used as primers in the present invention may comprise a nucleotide analogue, in particular a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid, or an intercalating agent and the like.

The primer set for identification of domestic wheat varieties of the present invention can be used for DNA amplification. Specifically, the primer set for discriminating all 32 domestic wheat varieties includes SCAR markers, SSR markers, Can be used for amplification of SNP markers.

In one embodiment of the present invention, primer set combinations comprising primers of SEQ ID NOS: 55, 57, 63, 64, and 70 to 75 that amplify the KWSM001 to KWSM006 markers were used to identify all, (Example 2), it is possible to distinguish between landscapes, landscapes, Pagoda, Namhae, Olgae, tombs, ornaments, novelties 1, hit, and white wheat. In addition, using primer set combinations of SEQ ID NOS: 93, 94, 118, 119, 126 and 127 for amplifying the primer set and the KWSM007 to KWSM009 markers, , Whitehead, whiteness, lecture, lecture, and zoal were additionally possible (Example 3). Further, it was confirmed that all of 32 domestic wheat varieties could be distinguished using primer set combinations of SEQ ID NOs: 128 to 139, which amplify the primer sets and the KWSM010 to KWSM015 markers (Example 4).

The domestic wheat varieties which can be discriminated by the primer set according to the present invention are all 32 varieties cultivated by the National Institute of Food Science and Technology and 32 wheat varieties which can be discriminated from the above are wheat varieties such as wheat, 1, No. 1, No. 1, No. 1, No. 1, No. 1, No. 1, No. 1, No. 1, No. 1, No. Multiplicity, sue, and zoe.

In a specific embodiment of the present invention, DNA of a domestic wheat variety was extracted, specific bands were detected by ISSR analysis and AFLP analysis, and primers capable of amplifying the specific bands were prepared Examples 2, and 3). In addition, a primer capable of amplifying the transcription factor, the SSR related to the cytoplasmic trait, and the SNP marker was prepared (Example 4). As a result, 15 primer sets for amplifying KWSM001, KWSM002, KWSM003, KWSM004, KWSM005, KWSM006, KWSM007, KWSM008, KWSM009, KWSM010, KWSM011, KWSM012, KWSM013, KWSM014 and KWSM015 were combined, And it was confirmed that the cultivars could be identified.

In another aspect, the present invention provides a method for amplifying genomic DNA comprising the steps of: (a) amplifying a genomic DNA using the genomic DNA extracted from a domestic wheat to be cultivated as a template and using the primer set of claim 1; And (b) comparing the amplified product with an amplified product of a domestic wheat variety standard to determine a wheat variety.

The method of extracting the genomic DNA of domestic wheat in the step (a) can be carried out by a conventional method known in the art. For example, a CRAB method, a phenol / chloroform extraction method, an SDS extraction method, or the like can be used, or a commercially available DNA extraction kit can be used.

In the step (a), the set of primers may be selected from the group consisting of SEQ ID NOs: 55, 57, 63, 64, 70 to 75, 93, 94, 118, 119, Lt; RTI ID = 0.0 > 139 < / RTI > By using the primer set at the same time, it is possible to accurately discriminate all 32 domestic wheat varieties. Specifically, the KWSM001 primer set, KWSM002 primer set, KWSM003 primer set, KWSM004 primer set, KWSM005 primer set, KWSM005 primer set, KWSM005 primer set, KWSM006 primer set, KWSM007 primer set, KWSM008 primer set, KWSM008 primer set, KWSM009 primer set, Set, a KWSM013 primer set, a KWSM014 primer set, and a KWSM015 primer set.

In the step (a), the amplification of the genomic DNA may be carried out by using a polymerase chain reaction (PCR), a ligase chain reaction, a nucleic acid sequence-based amplification, a transcription amplification system -based amplification system, amplification with strand displacement amplification or Qβ replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer set that specifically binds to a target nucleic acid using a polymerase. Such a PCR method is well known in the art, and a commercially available kit may be used. The PCR reaction mixture may include genomic DNA extracted from a domestic wheat variety, a primer set according to the present invention, an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and / or water. The PCR buffer solution may include, but is not limited to, Tris-HCl, MgCl ₂ , KCl, and the like.

The step (b) may include analyzing the amplification product. Preferably, the analysis of the amplified product can be performed by DNA chip, electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement method, but is not limited thereto. As one method of detecting the amplification product, capillary electrophoresis can be performed. Capillary electrophoresis, for example, can use the ABi Sequencer. In addition, gel electrophoresis can be performed, and gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is performed, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter. It can also be visualized using a silver salt kit (Bioneer, Daejeon, Korea).

In the step (b) of the present invention, the analyzed amplified product can be compared with the amplified product of the domestic wheat variety standard to determine the variety.

All 32 domestic wheat varieties which can be distinguished by the molecular markers according to the invention differ in their amplification of the primers against the molecular markers used (Fig. 1). The wheat cultivars in Korea can be easily distinguished by comparing the different amplification statuses.

In another aspect, the present invention provides a domestic wheat variety discriminating kit comprising the primer set.

As a result of analyzing whether the KWSM001 to KWSM015 markers were amplified using the primer set, the kit of the present invention can distinguish wheat varieties of 32 varieties in Korea by confirming whether amplification of each marker is different according to the wheat variety . The kit provided in the present invention may be a kit including essential elements necessary for performing PCR.

For example, in addition to the respective primer pairs specific to the markers, the PCR kit may include a test tube or other appropriate container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq polymer Enzymes such as enzyme and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

As another example, the kit of the present invention may be a DNA chip kit for domestic wheat variety discrimination including elements necessary for carrying out a DNA chip.

The term "DNA chip" of the present invention means one of DNA microarrays capable of confirming each base of hundreds of thousands of DNAs at a time.

The DNA chip kit is formed by attaching nucleic acid species to a glass surface, which is generally not larger than a flat solid support plate, typically a slide for a microscope, in a gridded array. The nucleic acid is uniformly arranged on the chip surface, It is a tool that enables multiple parallel hybridization reactions between the nucleic acid on the chip and the complementary nucleic acid contained in the treated solution on the chip surface.

The use of the primer set according to the present invention makes it possible to clearly identify all 32 domestic wheat varieties, thereby enhancing the protection of breeders, solving the problem of mixing the varieties, It can also be used for research and major morphological characterization.

1 is a photograph showing 16 specific bands amplified from 9 primers by PCR amplification of genomic DNA of 32 wheat varieties in Korea using 54 ISSR primers;
A is UBC807; B is UBC 808; C is UBC812; D is UBC813; E is UBC813; F is UBC815; G is UBC819; H is UBC823; I is UBC836; J is UBC840; K is UBC844; L is UBC846; M is UBC849; N means UBC880; Arrows indicate specific bands.
Fig. 2 is a photograph showing marker information for discrimination of 32 domestic wheat cultivars and marker amplified by the PCR result using the corresponding primers.
FIG. 3A is a photograph showing 19 specific bands amplified as a result of PCR amplification of genomic DNAs of 32 wheat varieties in Korea using the EA-AGG / MA-CTA primer combination.
FIG. 3B is a photograph showing 21 specific bands amplified as a result of PCR amplification of genomic DNA of 32 wheat varieties in Korea using EA-ACG / MA-CAC primer combination.
FIG. 3c is a photograph showing 18 specific bands amplified as a result of PCR amplification of genomic DNA of 32 wheat varieties in Korea using EA-AGC / MA-CTG primer combination.
Fig. 4 is a photograph showing the types of the previously reported transfer factor-related primers.
5 is a photograph showing the types of SSR primers reported in the prior art.
FIG. 6 is a photograph showing the discrimination of all domestic wheat varieties using a primer set of KWSM001 to KWSM015; FIG.
KWSM001 is a primer set consisting of oligonucleotides of SEQ ID NOS: 55 and 57; KWSM002 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 63 and 64; KWSM003 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 70 and 72; KWSM004 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 71 and 72; KWSM005 comprises a primer set consisting of the oligonucleotides of SEQ ID NOS: 73 and 74; KWSM006 comprises a primer set consisting of oligonucleotides of SEQ ID NOs: 73 and 75; KWSM007 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 93 and 94; KWSM008 comprises a primer set consisting of oligonucleotides of SEQ ID NOs: 118 and 119; KWSM009 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 126 and 127; KWSM010 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 128 and 129; KWSM011 is a primer set consisting of oligonucleotides of SEQ ID NOS: 130 and 131; KWSM012 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 132 and 133; KWSM013 comprises a primer set consisting of the oligonucleotides of SEQ ID NOS: 134 and 135; KWSM014 comprises a primer set consisting of oligonucleotides of SEQ ID NOS: 136 and 137; And KWSM015 means a set of primers consisting of the oligonucleotides of SEQ ID NOS: 138 and 139.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: DNA extraction of domestic wheat varieties

Genomic DNA was extracted from the domestic wheat of 32 cultivars listed in Table 1, which was fostered by the National Institute of Food Science and Technology.

number kind number kind One All 17 Joe Eun 2 Tree 18 Invisibility 3 Dahong 19 Little 4 Cheonggye 20 Newcomer 5 Gap 21 Farming 6 Tadong 22 landscape 7 South Sea 23 white lead 8 Us 24 Shinmyul 1 9 All the trees 25 White 10 full 26 hit 11 Tomb 27 sign up 12 Geumgang 28 One hundred 13 Weston 29 Sucess 14 Fresh 30 multiple 15 curio 31 Sue 16 Dense 32 Joa

Specifically, the genomic DNA was extracted from a young leaf and subjected to a mini-prep after grinding. The young leaves of the plants were cut out, quenched with liquid nitrogen, and ground until fine powder was obtained. Immediately after grinding, 30 μl of extraction buffer was added and mixed well with the pulverized powder. Then, it was incubated in a water bath for 30 minutes and centrifuged at 13,000 rpm for 4 to 20 minutes. After centrifugation, the supernatant was transferred to a new tube, 300 μl of isopropyl alcohol was added, and the mixture was slowly invert and pelletized. After centrifugation at 13,000 rpm for 5 minutes, the supernatant was removed except for the pellet, followed by washing with 50 μl of 80% ethanol. After washing, the mixture was centrifuged at 13,000 rpm for 5 minutes. The ethanol was removed except for the pellet. The pellet was dried at room temperature, and sterilized distilled water was added to dissolve the pellet. The extracted DNA was quantified using a nano drop.

Example  2: ISSR  By analysis Molecular marker  Differentiated wheat varieties

Experiments were conducted to develop a set of specific primers for domestic wheat varieties that amplified SCAR markers by ISSR analysis.

Example  2-1: ISSR  Detection of specific bands by analysis

SEQ ID NO: primer The base sequence (5'-3 ') SEQ ID NO: primer The base sequence (5'-3 ') One UBC801 ATATATATATATATT 28 UBC828 TGTGTGTGTGTGTGTGA 2 UBC802 ATATATATATATATATG 29 UBC829 TGTGTGTGTGTGTGTGG 3 UBC803 ATATATATATATATC 30 UBC830 TGTGTGTGTGTGTGTGC 4 UBC804 TATATATATATATATAA 31 UBC831 ATATATATATATATATYA 5 UBC805 TATATATATATATATAC 32 UBC832 ATATATATATATATYC 6 UBC806 TATATATATATATATAG 33 UBC831 ATATATATATATATATYA 7 UBC807 AGAGAGAGAGAGAGAGAGT 34 UBC832 ATATATATATATATYC 8 UBC808 AGAGAGAGAGAGAGAGC 35 UBC833 ATATATATATATATYG 9 UBC809 AGAGAGAGAGAGAGAGAGG 36 UBC834 AGAGAGAGAGAGAGAGAGT 10 UBC810 GAGAGAGAGAGAGAGATAT 37 UBC835 AGAGAGAGAGAGAGAGAGYC 11 UBC811 GAGAGAGAGAGAGAGAC 38 UBC836 AGAGAGAGAGAGAGAGAG 12 UBC812 GAGAGAGAGAGAGAGAGA 39 UBC837 TATATATATATATATART 13 UBC813 CTCTCTCTCTCTCTCTT 40 UBC838 TATATATATATATATARC 14 UBC814 CTCTCTCTCTCTCTA 41 UBC839 TATATATATATATATARG 15 UBC815 CTCTCTCTCTCTCTCTG 42 UBC840 GAGAGAGAGAGAGAGAGAYT 16 UBC816 CACACACACACACACAT 43 UBC841 GAGAGAGAGAGAGAGAGAYC 17 UBC817 CACACACACACACACAA 44 UBC842 GAGAGAGAGAGAGAGAGAYG 18 UBC818 CACACACACACACACAG 45 UBC843 CTCTCTCTCTCTCTCTRA 19 UBC819 GTGTGTGTGTGTGTGTA 46 UBC844 CTCTCTCTCTCTCTCTRC 20 UBC820 GTGTGTGTGTGTGTGTC 47 UBC845 CTCTCTCTCTCTCTCTRG 21 UBC821 GTGTGTGTGTGTGTGTT 48 UBC846 CACACACACACACACART 22 UBC822 TCTCTCTCTCTCTCA 49 UBC847 CACACACACACACACARC 23 UBC823 TCTCTCTCTCTCTCTCC 50 UBC848 CACACACACACACACARG 24 UBC824 TCTCTCTCTCTCTCTCG 51 UBC849 GTGTGTGTGTGTGTGTYA 25 UBC825 ACACACACACACACACT 52 UBC850 GTGTGTGTGTGTGTGTYC 26 UBC826 ACACACACACACACACC 53 UBC876 GATAGATAGACAGACA 27 UBC827 ACACACACACACACACG 54 UBC880 GGAGAGGAGAGGAGA

In order to confirm the bands specifically amplified from the DNA extracted in Example 1, a total of 54 ISSR primers shown in Table 2 were analyzed by PCR reaction, and specific amplified bands were identified 14 primers were selected.

That is, PCR amplification using the above-mentioned 54 primers was performed by adding 5 μM primer, 10 × buffer, 2.5 mM dNTPs, 5 U / μl i-StarMAX II Taq DNA polymerase (iNtRON, Korea) was added to make a volume of 25 μl. The PCR reaction conditions were 94 cycles of pre-denaturation for 3 min, 95 (30 sec), 55 (30 sec) and 72 (1 min) After repeating 35 cycles, reaction was carried out at 72 for 5 minutes. 5 μl of the amplified product was mixed with 5 μl of electrophoresis 6 × loading buffer (5: 1) and electrophoresed on 1.5% agarose gel.

As a result, 16 specific bands were identified from 9 ISSR primers out of 14 primers (UBC807, UBC808, UBC812, UBC813, UBC815, UBC816, UBC819, UBC823, UBC836, UBC840, UBC844, UBC849, and UBC880 1).

Example  2-2: Selected singularities ISSR Marker Cloning and  Sequencing

Sixteen specific bands identified according to Example 2-1 above were separated from the agarose gel and cloned. DNA purely isolated from agarose gel was inserted into Competent cell (Ecoli, DH5α) in conjunction with T-easy vector (Promega, USA). After that, white colonies on LB medium were again cultured in liquid phase, and only plasmid DNA was extracted to determine the nucleotide sequence. The determined nucleotide sequence was analyzed by NCBI and a primer capable of amplifying the SCAR marker was designed using the nucleotide sequence (Table 3). The primer design used primer3 program.

SEQ ID NO: primer The base sequence (5'-3 ') 55 SCAR812-1F1 AAGAAGAAAAAGAAAGAGAGTTGAG 56 SCAR812-1F2 AAATGATATCCACTCAATCTATCG 57 SCAR812-1R GTTGTGTTTTTATTTGTTTTTGAC 58 SCAR812-2F1 AGTCCACTGGCACTTGCCAAT 59 SCAR812-2R1 ACTCGGCCTACTTGATGTAGT 60 SCAR812-2R2 CTCGTCGAGGCAATCGGAGAGG 61 SCAR812-3F1 GAGAAAGAGGAAGGTGACTGAG 62 SCAR812-3R1 TATAAACCATGTTTCGATATTGTC 63 SCAR812-4F1 GCCGAGATTAAATTTGTTACTACT 64 SCAR812-4R1 AACATTTATGTTTGGTAAATTGAC 65 SCAR813-1F TACTCACGCGGTCACCAATA 66 SCAR813-1R CCAGTCGATGTCATTCGTCA 67 SCAR813-2F GCTATGTGGCGCGGTATTAT 68 SCAR813-2R AAGTAAGTTTGGCCCCCAGT 69 SCAR816-F GTCTCGCATAATGGGCAAGT 70 SCAR816-F1 TGCTGAGGGCATATCTCAACA 71 SCAR816-F2 TGCTGAGGGCATATCTCAACG 72 SCAR816-R GTGTTGCACGACGCTGTACT 73 SCAR808-F GGCTTACTACCTACACTTGACC 74 SCAR808-R1 GCAACAACAACCTTCTCCCAAT 75 SCAR808-R2 AACAACCTTCTCCCAATCCCCT

Example  2-3: Species specific for variety Molecular marker  Development, and Wheat variety  Distinction

A total of 12 pairs of primers were designed according to the above Example 2-2 and PCR was applied to 32 domestic wheat varieties. 5 μM primer, 10 × buffer, 2.5 mM dNTPs and 5 U / μl i-StarMAX II Taq DNA polymerase (Intron, Korea) were added to 3 μl of the genomic DNA quantitatively analyzed in Example 1, The PCR reaction conditions were 94 cycles of 95 cycles for 3 minutes, 95 cycles (30 seconds), 55 to 60 cycles (30 seconds) and 72 cycles (1 minute) In particular, the annealing temperature was varied from 55 to 60 depending on the primer. To confirm the amplified product, 5 를 was mixed with 5 electro of electrophoresis 6X loading buffer (5: 1), electrophoresed on 1.5% agarose gel, and confirmed under UV (Fig. 2).

As a result, a primer set of oligonucleotides comprising the primers of SEQ ID NOS: 55, 57, 63, 64, and 70 to 75 amplifying the SCAR marker of the present invention was developed (Table 4).

Marker name SEQ ID NO: The primer sequence (5'-3 ') Annealing () Product size (bp) Polymorphic type KWSM001 55 F: AAGAAGAAAAAGAAAGAGAGTTGAG 55 359
Presence / absence 57 R: GTTGTGTTTTTATTTGTTTTTGAC KWSM002 63 F: GCCGAGATTAAATTTGTTACTACT 55 312 Presence / absence 64 R: AACATTTATGTTTGGTAAATTGAC KWSM003 70 F: TGCTGAGGGCATATCTCAACA 58 682 Presence / absence 72 R: GTGTTGCACGACGCTGTACT KWSM004 71 F: TGCTGAGGGCATATCTCAACG 58 682 Presence / absence 72 R: GTGTTGCACGACGCTGTACT KWSM005 73 F: GGCTTACTACCTACACTTGACC 58 392 Presence / absence 74 R: GCAACAACAACCTTCTCCCAAT KWSM006 73 F: GGCTTACTACCTACACTTGACC 58 392 Presence / absence 75 R: AACAACCTTCTCCCAATCCCCT

As a result of analyzing the amplification of each SCAR marker using the primer set, it was confirmed that 12 kinds of domestic wheat cultivars can be distinguished (Table 5).

number kind KWSM Distinguished part 001 002 003 004 005 006 One All One One 0 One 0 0 ○ 2 Tree 0 0 0 0 One One × 3 Dahong One 0 0 One 0 0 ○ 4 Cheonggye 0 0 One 0 One One ○ 5 Gap 0 0 0 One One One × 6 Tadong 0 0 0 One 0 0 ○ 7 South Sea One 0 One One 0 0 ○ 8 Us 0 0 0 0 One One × 9 All the trees 0 One 0 0 One One ○ 10 full 0 0 0 0 One One × 11 Tomb 0 One One One One One ○ 12 Geumgang 0 0 0 0 0 One × 13 Weston 0 0 One One One One × 14 Fresh 0 0 0 0 One One × 15 curio One One 0 0 One One × 16 Dense 0 0 One One One One × 17 Joe Eun 0 0 0 One One One × 18 Invisibility One 0 0 0 One One × 19 Little One One 0 One One One ○ 20 Newcomer 0 0 0 0 One One × 21 Farming 0 0 0 0 One One × 22 landscape One One 0 0 0 One ○ 23 white lead 0 0 0 0 0 0 × 24 Shinmyul 1 One One 0 0 0 0 ○ 25 White 0 0 0 0 0 One × 26 hit One 0 0 0 0 One ○ 27 sign up 0 0 0 0 One One × 28 One hundred 0 0 One One 0 0 ○ 29 Sucess 0 0 0 0 One One × 30 multiple One One 0 0 One One × 31 Sue One 0 0 0 One One × 32 Joa One One 0 0 One One ×

As shown in the results of Table 5, KWSM001 was amplified in 12 different cultivars including Oolgae, Daehong, Namhae, Genuine, Anbag, Junior, Landscape, KWSM002 was amplified in 9 varieties including All, Allgreen, Mound, Genuine, Small, Landscape, Shinmi 1, Multi, Zoo. KWSM003 was amplified in 6 cultivars including Cheonggye, Namhae, Tumulus, Seondun, Mungsung and Hanbaek. KWSM004 was amplified in 11 cultivars including Ool, Daehong, Gwangbu, Thadong, Namhae, . KWSM005 was amplified in 21 cultivars such as green, blue sky, silver, we, alley, alcantara, tombstone, westun, bird, genuine, KWSM006 was amplified in 25 cultivars including Grus, Chrysanthemum, Ginkgo, Wu, and Allogue.

When the above 6 primer set combinations were used, olilum was amplified in three primers of KWSM001, KWSM002 and KWSM004. Dalhi wheat was amplified in KWSM001 and KWSM003. Cheonggye wheat was amplified in KWSM003, KWSM005 and KWSM006. Namhae wheat was amplified in three primers of KWSM001, KWSM003 and KWSM004. The allgrill was amplified in KWSM002, KWSM005. The wheat flour was amplified in four primers KWSM002, KWSM003, KWSM004 and KWSM005. The wheat was amplified in four primers KWSM001, KWSM002, KWSM004 and KWSM005. Landscaping wheat was amplified in three primers KWSM001, KWSM002 and KWSM006. Shinmi rice 1 rye was amplified in two primers, KWSM001 and KWSM002. The titration wheat was amplified in two primers, KWSM001 and KWSM006. One hundred wheat is amplified in two primers, KWSM003 and KWSM004, so it can be distinguished from other varieties. That is, it was confirmed that the SCAR markers of KWSM001 to KWSM006 differ in amplification depending on the primer set used for each domestic wheat variety.

Thus, using primer sets including the primers of SEQ ID NOS: 55, 57, 63, 64, and 70 to 75, all of the domestic wheat varieties, oligo, red, blue, It is confirmed that it is possible to distinguish between Yeonbakke, Shinmicam 1, Hit, Hanbyeol.

Example  3: AFLP  By analysis Molecular marker  Differentiated wheat varieties

Experiments were conducted to develop a set of specific primers for domestic wheat varieties that amplify SCAR markers by AFLP analysis.

Example  3-1: AFLP  Detection of specific bands by analysis

SEQ ID NO: primer The base sequence (5'-3 ') 76 EcoRI-adapter EA1 GACTGCGTACC 77 EA2 AATTGGTACGCAGTC 78 MseI-adapter MA1 TACTCAGGACTCATC 79 MA2 GATGAGTCCTGAG 80 Anchored EcoRI-adapter primers CTGCGTACCAATTCA 81 Anchored Msei-adapter primers GATGAGTCCTGAGTAAC 82 EcoRI-selective primers EA-ACG CTGCGTACCAATTCACG 83 EA-AGC CTGCGTACCAATTCAGC 84 EA-AGG CTGCGTACCAATTCAGG 85 EA-ACT CTGCGTACCAATTCACT 86 MseI-selective primers MA-CAC GATGAGTCCTGAGTAACAC 87 MA-CTA GATGAGTCCTGAGTAACTA 88 MA-CTG GATGAGTCCTGAGTAACTG

In order to screen for AFLP primers amplifying specific bands, PCR was performed using the AFLP primers in Table 6 above.

5 μM primer, 10 × buffer, 2.5 mM dNTPs and 5 U / μl i-StarMAX II Taq DNA polymerase (iNtRON, Korea) were added to 3 μl of the genomic DNA quantitatively analyzed in Example 1, The PCR reaction conditions were 94 cycles of 3 cycles of pre-denaturation, 95 cycles (30 sec), 55 cycles (30 sec) and 72 cycles (1 min). The amplified products were diluted to 1/100 of the first reaction and 94 cycles of 3 minutes, 95 (30 seconds), 55 (30 seconds) and 72 (1 minute) For 5 minutes. In order to confirm the product obtained from the second reaction, the product was electrophoresed on a 6% polyacrylamide gel and identified as silver color.

As a result, 18 bands (FIG. 3A) when EA-AGG / MA-CTA primer was combined, 21 bands (FIG. 3B) when EA-ACG / MA- When the CTG primers were combined, 19 bands (Fig. 3C) were isolated and a total of 58 specific bands were identified using the six primer combinations.

Example  3-2: Selected singularities AFLP Marker Cloning and  Sequencing

The specific band identified according to Example 3-1 was ligated with a T-easy vector (Promega, USA) and inserted into competent cells (Ecoli, DH5?). After that, the white colonies on the LB medium were again cultured in liquid phase, and plasmid DNA alone was extracted to determine the base sequence. The determined nucleotide sequence was analyzed by NCBI and primers were designed to amplify the SCAR marker using the nucleotide sequence (Table 7). The primer design used primer3 program.

SEQ ID NO: primer The base sequence (5'-3 ') 89 A13F TGTGTCAAATTCTTCCTTGATG 90 A13R TCAAATTATACCCGTCCAAGAG 91 C1F AACTGTTCGGTTTACAACTTGG 92 C1R GTTTTGGATTTCTCTGAGGATG 93 C2F TAAGGTGGACTCTTCTGGTTGT 94 C2R ACGACATTGTCATATCGTAAAG 95 C4F GCAGGTTGTTTCCTGTAGATTT 96 C4R CATCAAGCTGCTACAACCTAAA 97 C5F AAAGATAGTGTTCCCATTCATG 98 C5R GTAAGCTATGGGGCGCTTCTCT 99 C7F GTGTATTCCAATCCTCCAAAAG 100 C7R AGAGTTCCGTTGTGAGATCAAT 101 C8F CAAAACATTGAGTGAATCCAAA 102 C8R CTGTTGCTTTGATTTTTCCTGT 103 C9F TACTGGTTAGAATCGCACTGAA 104 C9R GTAACCTTCAGACTGGCTTTTG 105 C10F ATGAGTCCTGAGTAACACGACA 106 C10R ACGACTTTGATGTGGCATAGAT 107 C11F CCATCTTCTGCTATATGGAGTC 108 C11R CAACCTACTAGCACCAATCCTG 109 C12F CAAAGTTGCTCCTGTTTAGGAC 110 C12R TACTGACGTTACCCACTTATTG 111 C14F AAGTAGCAAAGTTCCTCCATCC 112 C14R AGGGCATATTTGCTTCAACTAA 113 C15F TTATTCAAATCCTTAGTGATTC 114 C15R TCACTTAGCCATAGGAGTTTTATG 115 C16F AACCATCATCATCTTGTCCTTC 116 C16TR CTTACAATCGCCACTTCACA 117 C16CR CTTACAATCGCCACTTCACG 118 C17F GAATTCAGGCTTCAGTTGATGG 119 C17R AAGGATACAATTGAAGGAACAG 120 G6F GCACCTACAGATTTCCTTTACG 121 G6R GAATTACAAAGCCCTGAAGATG 122 G7F GGGTGAAGAACTCTCGAATATG 123 G7R GTTTGTCGTCCCCTTGAGTAT 124 G8F CTTCAAGAATCTGATTGACACG 125 G8R CTACGTCGATCTTCCTCATCAT 126 G10F CAGAGTATACGAACGAGAACTAAC 127 G10R TGACACGCGATCTCTGATCCTG

Example  3-3: Species specific to variety Molecular marker  Development, distinction of wheat varieties

A total of 5 primers were designed and PCR was applied to 32 domestic wheat varieties. 5 μM primer, 10 × buffer, 2.5 mM dNTPs and 5 U / μl i-StarMAX II Taq DNA polymerase (Intron, Korea) were added to 3 μl of the genomic DNA quantitatively analyzed in Example 1, The PCR reaction conditions were 94 cycles of 95 cycles for 3 minutes, 95 cycles (30 seconds), 55 to 60 cycles (30 seconds) and 72 cycles (1 minute) In particular, the annealing temperature was varied from 55 to 60 depending on the primer. To confirm the amplified product, 5 를 was mixed with 5 electro of electrophoresis 6X loading buffer (5: 1), electrophoresed on 1.5% agarose gel, and confirmed under UV (Fig. 2).

As a result, a primer set of oligonucleotides containing primers of SEQ ID NOS: 93, 94, 118, 119, 126, and 127 amplifying the SCAR marker of the present invention was developed (Table 8).

Marker name SEQ ID NO: The primer sequence (5'-3 ') Annealing () Product size (bp) Polymorphic type KWSM007 93 F: TAAGGTGGACTCTTCTGGTTGT 56 215 Presence / absence 94 R: ACGACATTGTCATATCGTAAAG KWSM008 118 F: GAATTCAGGCTTCAGTTGATGG 58 212 Presence / absence 119 R: AAGGATACAATTGAAGGAACAG KWSM009 126 F: CAGAGTATACGAACGAGAACTAAC 58 468/150 Single or double 127 R: TGACACGCGATCTCTGATCCTG

As a result of combining amplification of each SCAR marker using the primer set and amplification of each SCAR marker analyzed in Example 2, it was confirmed that 10 kinds of domestic wheat varieties could be further distinguished 9).

number kind KWSM Distinguished part 001 002 003 004 005 006 007 008 009 One All One One 0 One 0 0 0 0 0 ○ 2 Tree 0 0 0 0 One One 0 0 0 × 3 Dahong One 0 0 One 0 0 0 0 0 ○ 4 Cheonggye 0 0 One 0 One One 0 0 0 ○ 5 Gap 0 0 0 One One One One 0 0 ○ 6 Tadong 0 0 0 One 0 0 0 0 0 ○ 7 South Sea One 0 One One 0 0 0 0 0 ○ 8 Us 0 0 0 0 One One 0 0 0 × 9 All the trees 0 One 0 0 One One One 0 0 ○ 10 full 0 0 0 0 One One 0 0 0 × 11 Tomb 0 One One One One One One 0 0 ○ 12 Geumgang 0 0 0 0 0 One 0 One 0 ○ 13 Weston 0 0 One One One One 0 0 0 ○ 14 Fresh 0 0 0 0 One One One 0 0 × 15 curio One One 0 0 One One 0 0 0 × 16 Dense 0 0 One One One One One 0 0 ○ 17 Joe Eun 0 0 0 One One One 0 One 0 ○ 18 Invisibility One 0 0 0 One One 0 0 0 ○ 19 Little One One 0 One One One 0 0 0 ○ 20 Newcomer 0 0 0 0 One One One 0 0 × 21 Farming 0 0 0 0 One One 0 0 0 × 22 landscape One One 0 0 0 One One One One ○ 23 white lead 0 0 0 0 0 0 0 0 0 × 24 Shinmyul 1 One One 0 0 0 0 0 0 0 ○ 25 White 0 0 0 0 0 One 0 0 0 ○ 26 hit One 0 0 0 0 One 0 0 0 ○ 27 sign up 0 0 0 0 One One One One 0 ○ 28 One hundred 0 0 One One 0 0 0 One 0 ○ 29 Sucess 0 0 0 0 One One 0 0 0 × 30 multiple One One 0 0 One One 0 0 0 × 31 Sue One 0 0 0 One One One 0 0 ○ 32 Joa One One 0 0 One One One 0 0 ○

Specifically, KWSM007 was amplified in 10 different cultivars including green pine, algae, tomb, sowol, dense, fresh, landscaping, KWSM008 was amplified in five cultivars such as Geumgang, Joeun, Landscape, KWSM009 was amplified only in the landscape.

When the above three primer set combinations were used, the GW, the alum, the tomb, the fresh, the dense, the new, the sue, and the zoo were amplified in one primer of KWSM007. Geumgang, Joeun, and Hanbai were amplified in one primer of KWSM008. Landscapes were amplified on three primers KWSM007, KWSM008, and KWSM009. The courses were amplified in two primers, KWSM007 and KWSM008. That is, the SCAR markers of KWSM007 to KWSM009 were confirmed to be amplified in different domestic wheat cultivars according to the primer set used.

Therefore, the analysis results using the primer set of the primers of SEQ ID NOS: 93, 94, 118, 119, 126 and 127 were combined with the analysis results of Example 2, In addition to the confiscated species, it was confirmed that it is possible to classify Gene, Geumgang, Seondun, Jungsung, Joeun, Jaeun, Baekjung,

Example  4: With metastatic factors Agricultural trait  Related specific SSR, SNP Marker  Differentiated wheat varieties

As a result of the 3B chromosome sequencing analysis of wheat, previously reported SSR primers related to the transfer factor related primer (Fig. 4) and wheat were investigated and applied to the cultivars (Paux et al., 2006; Sadat et al., 2013; Grag et al., 2012). The SSR primers were selected and primers with a PCR product length of 300 bp or more were selected and applied to the cultivars (FIG. 5). The SNP primer was designed to generate a 197 bp PCR product using the nucleotide sequence of the HSP16.9 gene related to the high temperature of the wheat.

5 μM primer, 10 × buffer, 2.5 mM dNTPs and 5 U / μl i-StarMAX II Taq DNA polymerase (Intron, Korea) were added to 3 μl of the genomic DNA quantitatively analyzed in Example 1, The PCR reaction conditions were 94 cycles for 5 minutes, 95 cycles (30 seconds), 55 to 60 cycles (30 seconds) and 72 cycles (1 minute) In particular, the annealing temperature was varied from 55 to 60 depending on the primer. To confirm the amplified product, 5 를 was mixed with 5 electro of electrophoresis 6X loading buffer (5: 1), electrophoresed on 1.5% agarose gel, and confirmed under UV (Fig. 2).

As a result, a primer set of oligonucleotides comprising the primers of SEQ ID NOS: 128 to 139 amplifying the transcription factor, SSR, and SNP marker of the present invention was developed (Table 10)

Marker name SEQ ID NO: The primer sequence (5'-3 ') Annealing () Product size (bp) Polymorphic type KWSM010 128 F: GCATCCCCTCTTTCGTCTC 58 216 Presence / absence 129 R: GCCGTTTCTCCCTTGAGC KWSM011 130 F: CGCAAGGAACGTATGGTTTG 58 129 Presence / absence 131 R: GGTGCTATAGCCCGGTTC KWSM012 132 F: CCAGCCCCTCTACACATTTT 58 295/260 Presence / absence 133 R: GGCCCATTTCCCACTTTCCA KWSM013 134 F: CCAAAAACATGGTTAAAGGGG 58 210/180 Presence / absence 135 R: AACAAAAGTCGGTGCAGTCC KWSM014 136 F: AATGGTATCTATTCCGACCCG 58 200/182/169/152/124 Presence / absence 137 R: CATCCCTAGGCCACTCTGC KWSM015 138 F: GAGGCGGACGAACGTGTTCA 58 197 Presence / absence 139 R: TGACCTCCTCCTTCTTCACG

As a result of combining the amplification of each molecular marker using the primer set and the amplification of each SCAR marker analyzed in Examples 2 and 3, 10 kinds of domestic wheat varieties were further distinguished and all domestic wheat It was confirmed that the cultivars could be distinguished (Fig. 6).

Specifically, KWSM010 was amplified in seven cultivars including Oolgae, Daehong, Namhae, Sawol, Yongsung, Kookjung, and Joa. KWSM011 was amplified in 12 varieties including Oryu, Cheonggye, Gwangpa, Thadong, Namhae, Wolcheon, Tochun, Tombstone, Weston, Genuine, Joeun, and Shinmi. KWSM012 was amplified to 295 bp in six cultivars such as grass, silkworm, white, white, white, and crown, and the other cultivars were amplified to 260 bp. The KWSM0013 amplified the band of 210bp in the case and the other band amplified the band of 180bp. KWSM014 amplified the band of 182 and 200bp in most cultivars and amplified the band of 169bp, 152bp, and 124bp, respectively. KWSM0015 has been amplified only in our landscape.

Therefore, it was confirmed that all 32 domestic wheat cultivars could be identified by combining the analysis results of Examples 2 and 3 with the analysis results using the primer set including the primers of SEQ ID NOS: 128 to 139 (Fig. 6) .

As a result, it was confirmed that all 32 domestic wheat varieties could be discriminated by using the primer sets of Examples 1 to 3 in combination.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> EPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Maker for cultivar discrimination in Korean breeding wheat and          method for determination using the same <130> KPA151131-KR <160> 139 <170> Kopatentin 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC801 primer <400> 1 atatatatat atatatt 17 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC802 primer <400> 2 atatatatat atatatg 17 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC803 primer <400> 3 atatatatat atatatc 17 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC804 primer <400> 4 tatatatata tatataa 17 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC805 primer <400> 5 tatatatata tatatac 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC806 primer <400> 6 tatatatata tatatag 17 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC807 primer <400> 7 agagagagag agagagt 17 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC808 primer <400> 8 agagagagag agagagc 17 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC809 primer <400> 9 agagagagag agagagg 17 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC810 primer <400> 10 gagagagaga gagagat 17 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC811 primer <400> 11 gagagagaga gagagac 17 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC812 primer <400> 12 gagagagaga gagagaa 17 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC813 primer <400> 13 ctctctctct ctctctt 17 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC814 primer <400> 14 ctctctctct ctctcta 17 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC815 primer <400> 15 ctctctctct ctctctg 17 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC816 primer <400> 16 cacacacaca cacacat 17 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC817 primer <400> 17 cacacacaca cacacaa 17 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC818 primer <400> 18 cacacacaca cacacag 17 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC819 primer <400> 19 gtgtgtgtgt gtgtgta 17 <210> 20 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC820 primer <400> 20 gtgtgtgtgt gtgtgtc 17 <210> 21 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC821 primer <400> 21 gtgtgtgtgt gtgtgtt 17 <210> 22 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC822 primer <400> 22 tctctctctc tctctca 17 <210> 23 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC823 primer <400> 23 tctctctctc tctctcc 17 <210> 24 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC824 primer <400> 24 tctctctctc tctctcg 17 <210> 25 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC825 primer <400> 25 acacacacac acacact 17 <210> 26 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC826 primer <400> 26 acacacacac acacacc 17 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC827 primer <400> 27 acacacacac acacacg 17 <210> 28 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC828 primer <400> 28 tgtgtgtgtg tgtgtga 17 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC829 primer <400> 29 tgtgtgtgtg tgtgtgg 17 <210> 30 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> UBC830 primer <400> 30 tgtgtgtgtg tgtgtgc 17 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC831 primer <400> 31 atatatatat atatatya 18 <210> 32 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC832 primer <400> 32 atatatatat atatatyc 18 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC831 primer <400> 33 atatatatat atatatya 18 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC832 primer <400> 34 atatatatat atatatyc 18 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC833 primer <400> 35 atatatatat atatatyg 18 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC834 primer <400> 36 agagagagag agagagyt 18 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC835 primer <400> 37 agagagagag agagagyc 18 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC836 primer <400> 38 agagagagag agagagya 18 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC837 primer <400> 39 tatatatata tatatart 18 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC838 primer <400> 40 tatatatata tatatarc 18 <210> 41 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC839 primer <400> 41 tatatatata tatatarg 18 <210> 42 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC840 primer <400> 42 gagagagaga gagagayt 18 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC841 primer <400> 43 gagagagaga gagagayc 18 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC842 primer <400> 44 gagagagaga gagagayg 18 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC843 primer <400> 45 ctctctctct ctctctra 18 <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC844 primer <400> 46 ctctctctct ctctctrc 18 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC845 primer <400> 47 ctctctctct ctctctrg 18 <210> 48 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC846 primer <400> 48 cacacacaca cacacart 18 <210> 49 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC847 primer <400> 49 cacacacaca cacacarc 18 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC848 primer <400> 50 cacacacaca cacacarg 18 <210> 51 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC849 primer <400> 51 gtgtgtgtgt gtgtgtya 18 <210> 52 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> UBC850 primer <400> 52 gtgtgtgtgt gtgtgtyc 18 <210> 53 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> UBC876 primer <400> 53 gatagataga cagaca 16 <210> 54 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> UBC880 primer <400> 54 ggagaggaga ggaga 15 <210> 55 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-1F1 <400> 55 aagaagaaaa agaaagagag ttgag 25 <210> 56 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-1F2 <400> 56 aaatgatatc cactcaatct atcg 24 <210> 57 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-1R <400> 57 gttgtgtttt tatttgtttt tgac 24 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-2F1 <400> 58 agtccactgg cacttgccaa t 21 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-2R1 <400> 59 actcggccta cttgatgtag t 21 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-2R2 <400> 60 ctcgtcgagg caatcggaga gg 22 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-3F1 <400> 61 gagaaagagg aaggtgactg ag 22 <210> 62 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-3R1 <400> 62 tataaaccat gtttcgatat tgtc 24 <210> 63 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-4F1 <400> 63 gccgagatta aatttgttac tact 24 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SCAR812-4R1 <400> 64 aacatttatg tttggtaaat tgac 24 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCAR813-1F <400> 65 tactcacgcg gtcaccaata 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCAR813-1R <400> 66 ccagtcgatg tcattcgtca 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCAR813-2F <400> 67 gctatgtggc gcggtattat 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCAR813-2R <400> 68 aagtaagttt ggcccccagt 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCAR816-F <400> 69 gtctcgcata atgggcaagt 20 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SCAR816-F1 <400> 70 tgctgagggc atatctcaac a 21 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SCAR816-F2 <400> 71 tgctgagggc atatctcaac g 21 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCAR816-R <400> 72 gtgttgcacg acgctgtact 20 <210> 73 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SCAR808-F <400> 73 ggcttactac ctacacttga cc 22 <210> 74 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SCAR808-R1 <400> 74 gcaacaacaa ccttctccca at 22 <210> 75 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SCAR808-R2 <400> 75 aacaaccttc tcccaatccc ct 22 <210> 76 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-adapter EA1 <400> 76 gactgcgtac c 11 <210> 77 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-adapter EA2 <400> 77 aattggtacg cagtc 15 <210> 78 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> MseI-adapter MA1 <400> 78 tactcaggac tcatc 15 <210> 79 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> MseI-adapter MA2 <400> 79 gatgagtcct gag 13 <210> 80 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Anchored EcoRI-adapter primers <400> 80 ctgcgtacca attca 15 <210> 81 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Anchored MseI-adapter primers <400> 81 gatgagtcct gagtaac 17 <210> 82 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-selective primers EA-ACG <400> 82 ctgcgtacca attcacg 17 <210> 83 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-selective primers EA-AGC <400> 83 ctgcgtacca attcagc 17 <210> 84 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-selective primers EA-AGG <400> 84 ctgcgtacca attcagg 17 <210> 85 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> EcoRI-selective primers EA-ACT <400> 85 ctgcgtacca attcact 17 <210> 86 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MseI-selective primers MA-CAC <400> 86 gatgagtcct gagtaacac 19 <210> 87 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MseI-selective primers MA-CTA <400> 87 gatgagtcct gagtaacta 19 <210> 88 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MseI-selective primers MA-CTG <400> 88 gatgagtcct gagtaactg 19 <210> 89 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> A13F primer <400> 89 tgtgtcaaat tcttccttga tg 22 <210> 90 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> A13R primer <400> 90 tcaaattata cccgtccaag ag 22 <210> 91 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C1F primer <400> 91 aactgttcgg tttacaactt gg 22 <210> 92 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C1R primer <400> 92 gttttggatt tctctgagga tg 22 <210> 93 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C2F primer <400> 93 taaggtggac tcttctggtt gt 22 <210> 94 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C2R primer <400> 94 acgacattgt catatcgtaa ag 22 <210> 95 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C4F primer <400> 95 gcaggttgtt tcctgtagat tt 22 <210> 96 <211> 22 <212> DNA <213> Artificial Sequence <220> <222> The C4R primer <400> 96 catcaagctg ctacaaccta aa 22 <210> 97 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C5F primer <400> 97 aaagatagtg ttcccattca tg 22 <210> 98 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C5R primer <400> 98 gtaagctatg gggcgcttct ct 22 <210> 99 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C7F primer <400> 99 gtgtattcca atcctccaaa ag 22 <210> 100 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C7R primer <400> 100 agagttccgt tgtgagatca at 22 <210> 101 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C8F primer <400> 101 caaaacattg agtgaatcca aa 22 <210> 102 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C8R primer <400> 102 ctgttgcttt gatttttcct gt 22 <210> 103 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C9F primer <400> 103 tactggttag aatcgcactg aa 22 <210> 104 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C9R primer <400> 104 gtaaccttca gactggcttt tg 22 <210> 105 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C10F primer <400> 105 atgagtcctg agtaacacga ca 22 <210> 106 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C10R primer <400> 106 acgactttga tgtggcatag at 22 <210> 107 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C11F primer <400> 107 ccatcttctg ctatatggag tc 22 <210> 108 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C11R primer <400> 108 caacctacta gcaccaatcc tg 22 <210> 109 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C12F primer <400> 109 caaagttgct cctgtttagg ac 22 <210> 110 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C12R primer <400> 110 tactgacgtt acccacttat tg 22 <210> 111 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C14F primer <400> 111 aagtagcaaa gttcctccat cc 22 <210> 112 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C14R primer <400> 112 agggcatatt tgcttcaact aa 22 <210> 113 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C15F primer <400> 113 ttattcaaat ccttagtgat tc 22 <210> 114 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> C15R primer <400> 114 tcacttagcc ataggagttt tatg 24 <210> 115 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C16F primer <400> 115 aaccatcatc atcttgtcct tc 22 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C16TR primer <400> 116 cttacaatcg ccacttcaca 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C16CR primer <400> 117 cttacaatcg ccacttcacg 20 <210> 118 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C17F primer <400> 118 gaattcaggc ttcagttgat gg 22 <210> 119 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C17R primer <400> 119 aaggatacaa ttgaaggaac ag 22 <210> 120 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G6F primer <400> 120 gcacctacag atttccttta cg 22 <210> 121 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G6R primer <400> 121 gaattacaaa gccctgaaga tg 22 <210> 122 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G7F primer <400> 122 gggtgaagaa ctctcgaata tg 22 <210> 123 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> G7R primer <400> 123 gtttgtcgtc cccttgagta t 21 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G8F primer <400> 124 cttcaagaat ctgattgaca cg 22 <210> 125 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G8R primer <400> 125 ctacgtcgat cttcctcatc at 22 <210> 126 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> G10F primer <400> 126 cagagtatac gaacgagaac taac 24 <210> 127 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G10R primer <400> 127 tgacacgcga tctctgatcc tg 22 <210> 128 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KWSM010 F <400> 128 gcatcccctc tttcgtctc 19 <210> 129 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> KWSM010 R <400> 129 gccgtttctc ccttgagc 18 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KWSM011 F <400> 130 cgcaaggaac gtatggtttg 20 <210> 131 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> KWSM011 R <400> 131 ggtgctatag cccggttc 18 <210> 132 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KWSM012 F <400> 132 ccagcccctc tacacatttt 20 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KWSM012 R <400> 133 ggcccatttc ccactttcca 20 <210> 134 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KWSM013 F <400> 134 ccaaaaacat ggttaaaggg g 21 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KWSM013 R <400> 135 aacaaaagtc ggtgcagtcc 20 <210> 136 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KWSM014 F <400> 136 aatggtatct attccgaccc g 21 <210> 137 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KWSM014 R <400> 137 catccctagg ccactctgc 19 <210> 138 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KWSM015 F <400> 138 gaggcggacg aacgtgttca 20 <210> 139 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KWSM015 R <400> 139 tgacctcctc cttcttcacg 20

Claims (5)

A KWSM001 primer set consisting of oligonucleotides of SEQ ID NOS: 55 and 57; A KWSM002 primer set consisting of oligonucleotides of SEQ ID NOS: 63 and 64; A KWSM003 primer set consisting of oligonucleotides of SEQ ID NOS: 70 and 72; A KWSM004 primer set consisting of oligonucleotides of SEQ ID NOS: 71 and 72; A KWSM005 primer set consisting of the oligonucleotides of SEQ ID NOs: 73 and 74; A KWSM006 primer set consisting of oligonucleotides of SEQ ID NOS: 73 and 75; A KWSM007 primer set consisting of oligonucleotides of SEQ ID NOS: 93 and 94; A KWSM008 primer set consisting of oligonucleotides of SEQ ID NOs: 118 and 119; A KWSM009 primer set consisting of oligonucleotides of SEQ ID NOS: 126 and 127; A KWSM010 primer set consisting of oligonucleotides of SEQ ID NOS: 128 and 129; A KWSM011 primer set consisting of oligonucleotides of SEQ ID NOS: 130 and 131; A KWSM012 primer set consisting of oligonucleotides of SEQ ID NOS: 132 and 133; A KWSM013 primer set consisting of the oligonucleotides of SEQ ID NOS: 134 and 135; A KWSM014 primer set consisting of oligonucleotides of SEQ ID NOS: 136 and 137; And a KWSM015 primer set consisting of the oligonucleotides of SEQ ID NOS: 138 and 139.
The method according to claim 1,
The above domestic wheat varieties have been cultivated in a variety of cultivars such as olive, gruel, dahong, cheonggye, Gwangpa, tadong, Namhae, Woori, Alchean, tomb, Geumgang, Seondun, Wherein said primer set is selected from the group consisting of primers, primers, primers, primers, primers, primers, primers, primers, primers, probes, primers, primers and primers.
(a) amplifying genomic DNA using the genomic DNA extracted from a domestic wheat to be cultivated as a template and using the primer set of claim 1; And
(b) comparing the amplified product with an amplified product of a domestic wheat variety standard to determine a variety.
The method of claim 3,
The above domestic wheat varieties are selected from the group consisting of All, Grass, Daehong, Cheonggye, Gwangpa, Tadong, Namhae, Woori, Alchan, Tomb, Geumgang, Seungdun, Seol, Original, Confectionery, Joeun, Wherein the seeds are selected from the group consisting of rice bran, rice paddies, rice paddies, white rice paddies, white rice paddies, white rice paddies, white rice paddies,
A domestic wheat variety discriminating kit comprising the primer set according to claim 1.

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