KR20160082292A - Molecular Markers for Selecting radish genetic resources and use thereof - Google Patents

Abstract

The present invention relates to a molecular marker, to a molecular marker amplifying primer set, and a method for identifying the species or the lines of radishes. The method comprises the following steps: separating genome DNA from a radish product sample which are capable of detecting the species or lines of radishes; performing a PCR by using the separated genome DNA as a mold and by using a primer set; and analyzing the amplification and a size of an amplified product by performing dielectrolysis on the amplified product. A molecular marker and a primer set capable of amplifying the same which are defined according to the present invention can easily and rapidly separate various radish genetic sources by lines and species; and can analyze a dielectric structure, thereby being utilized in developing high quality radish modified species, determining import and export places of origin of agricultural and marine products, and ensuring and maintaining radish dielectrics.

Description

Molecular markers for non-genetic analysis and their uses {Molecular Markers for Selecting radish genetic resources and use thereof}

The present invention relates to molecular markers for non-genetic analysis capable of classifying various genomes of radishes and distinguishing different varieties from lines, and uses thereof.

With the development of molecular genetics and the development of mass sequencing techniques, research on specific molecular markers for plants has been accelerated. This has allowed the diversity of plant species to be distinguished at the genomic level, and molecular markers have also been used as important evaluation tools to distinguish or preserve various genetic resources.

On the other hand, the radish belonging to the cabbage division is a root vegetable crop widely cultivated in Asia. It has various roots in morphology and physiologically has a variety of differences in flowering time and nutritional components. Therefore, genes for cultivation and production of radish Research is concentrated on securing the won. In particular, rice is an essential food crop, which is equivalent to major crops such as rice and cabbage, and is therefore an economically important crop. Therefore, various studies on the development of molecular markers have been reported.

However, a method of identifying the species and the variety of the organism developed so far has been used in a morphological or physicochemical method. However, since the chromosomal composition of the crop including the radish belonging to the Chinese cabbage is difficult to establish, In which cultivars and species are distinguished.

Also, in the developed method, most of the molecular markers such as SNP or SSR based on AFLP, RAPD or EST nucleotide sequences were found. However, the molecular markers developed in this way are not only labor-intensive, costly and time-consuming, but also have poor reproducibility, which makes them unsuitable for classifying various genetic resources and for analyzing genomes, There is a problem that it is not suitable for providing sufficient information to be described below.

Thus, the present inventors have developed PCR-based molecular markers based on a non-standard genome, and have been able to analyze various genetic structures of non-genetic resources and find that they can be efficiently used for holding and maintaining diverse genomes.

Korean Patent Publication No. 2013-022328

It is therefore an object of the present invention to provide a molecular marker for the detection of species or lines of radish.

It is a further object of the present invention to provide a primer set capable of detecting and amplifying molecular markers for species or lineage detection of the present invention.

It is another object of the present invention to provide a method or apparatus for identifying species or groups of plants using a primer set according to the present invention.

In order to accomplish the object of the present invention as described above, the present invention provides a method for detecting a species or a system for detecting a non-specific species or system comprising a base sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 1-68 to provide.

The present invention also provides a primer set capable of detecting and amplifying a molecular marker of the present invention.

In one embodiment of the present invention, the primer set has a GC content of 40 to 60%, and PCR can be performed at a temperature of 56 to 60 ° C.

Furthermore, the present invention relates to a method for producing a genetic material comprising the steps of: isolating genomic DNA from a crop sample; Performing PCR using the separated genomic DNA as a template and using the primer set of the present invention; And analyzing the amplified product by electrophoresis to analyze the presence or absence of amplification and the size of the amplification product.

The present invention relates to a method for detecting genomic DNA, comprising the steps of: isolating genomic DNA from a non-crop sample and a molecular marker capable of detecting species or lines of the genome; Performing PCR using the separated genomic DNA as a template and using the primer set of the present invention; And analyzing the amplified product by electrophoresis to analyze the presence or absence of amplification and the size of the amplification product. The molecular markers identified in the present invention and the primer sets capable of amplifying them can easily and quickly distinguish various non-genetic resources by system and species, and can analyze the dielectric structure, thereby developing high-quality unmodified varieties, importing and exporting agricultural and marine products It can be advantageously utilized for discrimination of origin, non-genetic material retention and maintenance, and the like.

FIG. 1 shows the results obtained by applying the molecular markers identified in the present invention to 14 strains and confirming the amplification by electrophoresis through PCR analysis.
Fig. 2 shows the classification of radish species of 14 lines using the molecular markers identified in the present invention.

The inventors are characterized by the development of molecular markers capable of identifying PCR-based species or strains of non-human species.

In general, methods for identifying plant cultivars are divided into two methods: morphological characterization method through cultivation test, assay method using difference of isoenzymes between species or cultivars, and assay method using DNA-based molecular markers. Among them, the method using molecular markers provides information useful for the genetic diversity and phylogeny analysis and the conservation and management of plant genetic resources in crop genetic and breeding studies (Smith et al., 1997. Theor. Appl Genet. 95: 163-173), compared to traditional identification methods based on phenotypes, are not affected by the growing environment and the growth stage of crops, so they are objective and highly reproducible.

On the other hand, systematic taxonomy has not been completed yet, which can be said to be the main crop of food, and there is no molecular marker that can quickly distinguish the genetic resources such as origin and variety.

In this regard, the present inventors have discovered molecular markers capable of distinguishing various kinds of radishes, strains, and origin of plants by a simple and rapid method such as DNA-based PCR.

More specifically, the present inventors have searched for polymorphic regions in WK10039 and WK10024, which are fixed systems of radish. In the standard genome sequence mapping of these immobilized systems, regions where deletion or insertion (InDel) Only regions with more than 7 NGS sequence readings proving the mutation and the InDel length difference between WK10039 and WK10024 of 10 bp or more were selected.

This is because if the InDel length difference is too short, it is not easy to classify species or phylogeny by PCR method.

Through this search method, the final 68 molecular markers described in the table below were uncovered. These markers were randomly amplified using the Primer3 program for 265 InDel loci, amplicon size of 100 ~ 300 bp, optimal length of 20 bp, Tm of 56 ~ 60 ℃ and GC content of 40-60% The primer was designed. As a specific primer set, as shown in Tables 1 to 2 below, a primer set capable of amplifying the respective molecular markers according to the respective number was designed. A 68-pair primer set consisting of 136 amplicons capable of amplifying these 68 molecular markers can be used to perform PCR once, and it is possible to classify various kinds of radishes by type.

Therefore, the present invention can provide a molecular marker for detecting a species or a system of a non-human species, wherein the molecular marker is preferably a molecule consisting of any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1-68 nucleotides It can be a marker.

In addition, the present invention can provide a primer set capable of detecting and amplifying a molecular marker for detecting species or system of the present invention, wherein the primer set comprises a nucleotide sequence as shown in Tables 1 to 2 ≪ / RTI >

The term "primer set" in the present invention means a short nucleic acid sequence which can form a base pair with a complementary template and functions as a starting point for template strand copying.

In addition, the molecular marker according to the present invention may include any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1-68, or a variant of the nucleotide sequence. Specifically, it may include a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of the nucleotide sequence homology with the nucleotide sequence corresponding to the molecular marker . The "percentage of sequence homology" to a nucleotide sequence (polynucleotide) is confirmed by comparing the comparison region with two optimally aligned sequences, and some of the polynucleotide sequences in the comparison region are identified by reference sequences (I.e., a gap) as compared to a standard (not including additions or deletions).

In addition, the present invention can provide a species or system identification method using a primer set capable of detecting a molecular marker according to the present invention. Preferably, the genomic DNA is isolated from a crop sample. Performing PCR using the separated genomic DNA as a template and using the 68 pairs of primer sets of the present invention; And analyzing the amplified product by electrophoresis to analyze the presence or absence of amplification and size of the amplification product.

In particular, the molecular markers according to the present invention can be amplified using the respective primer sets, that is, when the primer set 1 is used, the molecular markers of SEQ ID NO: 1 can be amplified, And the primer set 3 can amplify the molecular marker of SEQ ID NO: 3. Thus, each primer set can amplify the molecular marker corresponding to each sequence number according to the sequence.

The PCR of the present invention can amplify the target sequence by using 68 pairs of the primer set of the present invention using the non-derived genomic DNA as a template and conducting an amplification reaction. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] 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 can be performed under ordinary conditions. In addition, commercially available kits may be used for such PCR method.

The amplified target sequence of the present invention may be labeled with a detectable labeling substance. In one specific example, the labeling material can be, but is not limited to, a material that emits fluorescence, phosphorescence, or radiation. Preferably, the labeling substance is Ethidium Bromide (EtBr), Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When a radioactive isotope such as ³² P or ³⁵ S is added to the PCT reaction solution during PCR, the amplified product may be synthesized and the radiation may be incorporated into the amplification product and the amplified product may be labeled with radiation .

The detection of the amplified product by the PCR method can be performed by, but not limited to, DNA chip, gel electrophoresis, scatterometry, fluorescence or phosphorescence measurement. As one method of detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can be performed using 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 carried out, 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 the radioactivity is measured by a radiation measuring device such as a Geiger counter or liquid scintillation counter The radiation can be measured using a liquid scintillation counter.

In one embodiment of the invention, the amplified product detection was analyzed by size and amplification of amplified product through gel electrophoresis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Non-fixed system WK10039 Wow WK10024  Search for polymorphism in the liver and search for molecular marker regions that can distinguish the system

In order to search for molecular markers with polymorphism between WK10039 and WK10024, the genomic DNA of WK10024 was extracted using Qiagen DNeasy kit to obtain the nucleotide sequence of the paternal lineage. The extracted gDNA was analyzed by using an Agilent 2100 Bioanalyzer to determine whether it was gDNA that could be used for NGS sequencing. After that, gDNA of high quality gDNA was prepared for NGS sequencing, and gDNA of the prepared library was further subjected to 500 bp paired end shot sequencing using Illumina HiSeq1000 to produce sequences of over 80X. The thus obtained WK10024 The sequence of the system was mapped and aligned using the BWA program to the sequence of WK10039, a non-standard genome. Only the region where the deletion or insertion of the mapped sequence appeared was searched for polymorphism, Was selected to be more than 7, and the difference of InDel length between WK10039 and WK10024 was 10 bp or more.

< Example  2>

Detection of molecular markers capable of classifying radish species

A primer was designed to search 1,039 molecular markers capable of classifying non-specific species by a PCR-based method with the difference of InDel length between WK10039 and WK10024 obtained in Example 1 above 10 bp and depth of 7 or more. PCR was performed under the condition of amplicon size of 100 ~ 300 bp, optimal length of 20 bp, Tm of 58 ℃ and GC content of 40-60% by using Primer3 program for 265 InDel loci arbitrarily selected from 1,039 candidate molecular markers The primer was designed.

As a result of the analysis, 68 of the 1,039 candidate molecular markers were found to have at least one polymorphism, and the polymorphism-confirmed primer sets and the molecular markers are shown in Tables 3 and 4 below.

< Example  3>

For molecular label detection Primer  Polymorphism analysis of radish using

The process of confirming whether the genomes of different systems can be actually distinguished by using the 68 molecular markers having the indefinite polymorphism confirmed in the above examples and the primers capable of detecting them were performed. First, gDNA was isolated from the representative fourteen lines shown in Table 5 by the CTAB method, and a total of 20 ng of gDNA was subjected to PCR using the primer sets of Example 2 above. Based on the amplified results, we call it 1 when the band is amplified and 0 when it is not amplified. The results were classified using the NTSYS 2.2 program.

Classification designation Academic name origin Growing System WK10039 Raphanus sativus cv. WK10039 WK10024 R. sativus cv. WK10024 Wild type W1 R. raphanistrum ssp . raphanistrum Vietnam W2 R. raphanistrum ssp . Munra India W3 R. raphanistrum ssp . Mungra India W4 R. sativus there is . raphanistroides Makino Japan W5 R. sativus there is. raphanistroides Makino Korea W6 R. sativus there is. raphanistroides Makino Korea Cultivated type C1 R. sativus there is. longipinnatus LH Bailey cv. Long Scarlet United States of America C2 R. sativus there is. radicula Pers. Korea C3 R. sativus there is. hortensis Becker cv. DB104 Korea C4 R. sativus there is. hortensis Becker cv. DB109 China C5 R. sativus there is. hortensis Becker cv. DB110 China C6 R. sativus there is. hortensis Becker cv. DB113 Japan

As a result of analysis, as shown in FIG. 1, when 68 molecular markers identified in the present invention were applied to 14 lines, it was found that each line could be distinguished. Further, the genome group was separated using the electrophoresis result As a result, it was found that the wild type and the cultivar can be separated and sorted, and as shown in FIG. 2, the 68 non-genomic groups can be classified using the primer set for detecting the molecular markers of the present invention.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Molecular Markers for Selecting radish genetic resources and use          the <130> PN1412-362 <160> 68 <170> Kopatentin 2.0 <210> 1 <211> 213 <212> DNA <213> Artificial Sequence <220> <223> RsInD22 <400> 1 tctacaataa cacacgcaca tacaacatat ataacatgtt tagaactcca ataaataaaa 60 actcctacta caagtggcca tttagccttt tcgccgcaag tcagttaccc accttagacg 120 gaaattaata gttgaacaga tatgtagcta aaaaatgttt ttagcttttg tttttttttt 180 caggtgaaga tgacggatag aagtagaatg gag 213 <210> 2 <211> 232 <212> DNA <213> Artificial Sequence <220> <223> RsInD27 <400> 2 tcccaggtta gaatggttta agttgtcggc tgaatcccga aaacaggaca gtagctcaac 60 tagaaacact tgtgagatgt tattcattaa tagaactttg gttctgatcg aaaactcaaa 120 actgtctttt tttttttgaa aaagggcttt aaatcttttt tattgtttac caatgaattt 180 tactaatgga tcctatttta tattctattc tgcataatac aaaatgaacc gt 232 <210> 3 <211> 251 <212> DNA <213> Artificial Sequence <220> <223> RsInD23 <400> 3 atagcgttat acatacacat gcgattcaaa tcatacaatc ttggttccat cataacgata 60 ctcattcgtt gtttattctt tccttggttc tttttcaacc acgacatttg aatgttcata 120 ctaaatttct tctacaagtt gctaatgaaa aatgtttgcg actgtaatat tctaacatat 180 cagctgtatt caaagtttca aacggattat ggattgtaag gtgcttacca taacaccgta 240 gatgaaacga a 251 <210> 4 <211> 201 <212> DNA <213> Artificial Sequence <220> <223> RsInD25 <400> 4 tcttctttcc ttttgtaggc ttgttgttgt tatcgttctt cgctaacgat cccgctgacc 60 cgcaacaatt acccatggct cctttaaaca atgacggatc aaatcaaact ggtcgcagat 120 aggagagatc gaggaatgtt cacgaggtga ttccaagaca atgggggaag atcaagggat 180 ggtggtactt agggatgaaa t 201 <210> 5 <211> 260 <212> DNA <213> Artificial Sequence <220> <223> RsInD24 <400> 5 gtactgtgag acttttcctt gtcataacta aacaacacaa gacacagttg caggcagcaa 60 gacaacatga tatgtataga caacataatc atagagactt gatgagactc aggaactcta 120 tgtagaaaac gtctttaacc aaagttaatt taaaaacatc tttcacaaca taatccatag 180 ctataaaacg aaatcataaa agcatcctta catataatgg tgctaaaaca caaacacatg 240 caaagactac ataaaccgag 260 <210> 6 <211> 194 <212> DNA <213> Artificial Sequence <220> <223> RsInD26 <400> 6 aaaaccagct gaacatccaa ttattgctcc tccaggaaag actggatatg gaatagactg 60 aaattttact agcaccaagt tcaaatatgt cataacatgc caaaacaaaa tctatatcaa 120 cgtacaggtt aatatcttta aggaaaaaag attttaaact cacctgaaaa ccaccttcat 180 ttaaagtacg agct 194 <210> 7 <211> 245 <212> DNA <213> Artificial Sequence <220> <223> RsInD28 <400> 7 acaaagatca ctttagactt cgcctgttcg ccatatgggc tttgttccgg tatctataat 60 aaaagaagaa aaaaatacaa cgaaccattt ttttttgaca tctacaacga accatttata 120 tgtatattat gtgtgttaat taattttact acccatttct ataatatttt aattttgtaa 180 taattcacaa ctatatattc caaagagaaa aagctgtgaa tcgtacttac ctcttgttat 240 tcggc 245 <210> 8 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> RsInD61 <400> 8 attgctgtgt aaatgctagc tagattgtgg atgctattca gaactgtctt ctccctgagt 60 ttgatatttg gttgcagtta gaaatgcagt agattttcac atgattagca gtattatacc 120 tctctgtaac aatggaagaa c 141 <210> 9 <211> 182 <212> DNA <213> Artificial Sequence <220> <223> RsInD62 <400> 9 aggtttgtaa ctgaacttca cacatagtct tcttctgcat cctttttttt atattgttct 60 attgaacctt ggatgggaga agaaaagctt cttcatactt tctcttacaa tcaaaggtga 120 tagattgcag gagaactacg agaaagaggg atctctgctg attattacca cgcagaaatg 180 ga 182 <210> 10 <211> 183 <212> DNA <213> Artificial Sequence <220> <223> RsInD32 <400> 10 agctggtgta tatagtttgg tcaagaaaga aacagacaca aacctggtaa tgcaaaccaa 60 agccgctgtt ttcattgccg gagctcttgt cgcaagaaac aaggcggcca gagacatata 120 cgaaatcatt tggcttcaaa tgcgcaacgc acttccgtgc catcacgtcc caaatactaa 180 gag 183 <210> 11 <211> 221 <212> DNA <213> Artificial Sequence <220> <223> RsInD30 <400> 11 attcaatctg tgctgaaacc tacttacata tgtatatggg gatatgttat tattcgatct 60 gtgccgagac ctagttacat atgaggatgg ccacgtgggc atgcatatat ttagacataa 120 taaataaatg aatattggag gagacaagat agaccgctat aatactgatt gagatgtgga 180 ttgatcctac agacaccatt atcatcattc gccatcatgt c 221 <210> 12 <211> 151 <212> DNA <213> Artificial Sequence <220> <223> RsInD35 <400> 12 agacaaaagg aacagtttcg atttctttga cttttaaaac taaccgtttg ccacaacacg 60 agtgaactaa ctgtaatagc ttaagaagta actctcttat aaacgcaaaa gattttaact 120 ttattatttt cgtgtgtttg gttacattac a 151 <210> 13 <211> 264 <212> DNA <213> Artificial Sequence <220> <223> RsInD40 <400> 13 ttacgttgct ctcgtgagat atacttacgt tgtaaattta tggaaaatcg taggttaaga 60 tcacattcac acatattgac cgatgcaacg acaagtggct tcaacagtac cagtgagatg 120 gcatactact acaatacgta gatatctctc cgtctcataa tcttcaaaat ccaccaccag 180 cttaacatat tacaagatga agtgatgctt cttttttctt tttttcttgg ttaaagaaga 240 cgaaagtgat gtgtggaaag ctat 264 <210> 14 <211> 211 <212> DNA <213> Artificial Sequence <220> <223> RsInD36 <400> 14 gtctcctaat ttgtctgtgt gtttatatca tcttctgctg gaaattaagt acttgtttta 60 gatctggtaa ctttttgcta atcaacttga tgaattgatt gcattaatgt aactccttaa 120 accggatgaa tctcttaaac cgggtttgat tatctcccaa acgaactaca atttgatgaa 180 tttaacacta tacatgtaac tccacacgac a 211 <210> 15 <211> 245 <212> DNA <213> Artificial Sequence <220> <223> RsInD37 <400> 15 tggaaatgaa attctgtctc acctaagttt ttaaagagtt aagaagcaat gtcttgttta 60 agcaaacgag ttgaagcgtg cgtctttcca gcattatcat ttgttggaac gaatctctac 120 ttgtctttcc tcctcctgat tctatatcaa gtttcaatat gttagcttat tgtaatccta 180 ctaataatag acaaatggag ttctttcgga aaaaaaaaat caatttacct ctgtttcctt 240 gatgc 245 <210> 16 <211> 212 <212> DNA <213> Artificial Sequence <220> <223> RsInD38 <400> 16 ttgtcacgat aagtaacgaa cagagttccg gtttggtttt gcatcaataa tcttaaacat 60 aaattaatct tgaataggat tagcaatgat gataaagccg gattaaacaa ggggcctaat 120 aatatttata aaaatgtgtt aatagagtta tcttttaatt gccttctttt tggaacctat 180 ctaatttccc tccatgtggt agatttttcc tc 212 <210> 17 <211> 181 <212> DNA <213> Artificial Sequence <220> <223> RsInD39 <400> 17 tgagcaagtt ttgtacgata acaaaataaa gtaatcttat ataaatcttc tcattggcaa 60 gataagggtg tgatcaagtg ctgtattgct gcaattgtat aaagcaggat tgtactcgag 120 tcaaacttga gcattttctg cattttattc aaacatataa atgcaatcag atgaaaggga 180 a 181 <210> 18 <211> 147 <212> DNA <213> Artificial Sequence <220> <223> RsInD43 <400> 18 ctctctctga agccattaac aaggtatgat agaatcactt tcttttgctc tctggaacat 60 gattagctaa ttctctctct ctctcttgga attcagatcg aggagaagca gagaaacctc 120 tgtctcttgg tcatacagtt catacca 147 <210> 19 <211> 161 <212> DNA <213> Artificial Sequence <220> <223> RsInD57 <400> 19 gtgaaaggaa gggattgaat tcattgagcc gaagcatccg agttgtgttg tatcttagta 60 aggactcctt actaaggagt cagagacggt gatcaattca tgagcaataa taacattctc 120 attttcttta acattactct tcaaaaagag gaggaagcaa a 161 <210> 20 <211> 285 <212> DNA <213> Artificial Sequence <220> <223> RsInD42 <400> 20 actgtaagtc tgcaacatta accaaagagg tgatacttaa agaatgttag gatgagactg 60 gaccataagc gaaatggtca caatatataa tgttgtttat tttttgagat ttgtagggga 120 aaaaaaacaa aaaaggtata cagataattt tggtaaatag ttttggaaga cagcaaaaac 180 acacaatgca ctagcatcgg ttagaaagca aacacaccat tacttttatg cacacaaagt 240 gcagacctaa gagtagcgtg taaaccacta aaagaagagc tttcc 285 <210> 21 <211> 199 <212> DNA <213> Artificial Sequence <220> <223> RsInD44 <400> 21 cgctcttatt gcacttggtt ataaccggtt cagacttctt ccccatgttg ttctcaaaca 60 cctagccatc accacaacat atatatatgc gtcaaaatca gatcatacat agcaaaggcc 120 ttaagatgca atacacatac caaagacaca taaacctaaa atcaatcgag tgtttgtatc 180 gtacctgctt gtacttctt 199 <210> 22 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> RsInD45 <400> 22 cgtaccctaa aaatgatgtc acaatagttt tatcttttca gtgaatctta tttggtgaaa 60 tctgacttac tcagaacaaa gtcaactacc ttttatgtac atcaatgtat acgtcggttt 120 c 121 <210> 23 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> RsInD78 <400> 23 cacacaaggt catgtcagat atcgaattag ccaagtacat tgttaataga caatacaatg 60 aggaacaaat gcatagacgc atgagttctt gactgttatg atccaagcag aaagcaaaat 120 a 121 <210> 24 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> RsInD52 <400> 24 tagtttcaca acctcctctt tctgcttctc ctttgtaaat ctaataaagc aaaatgaggg 60 gactaaaaca ataaactctt ttgtcattgt aatttgtaaa gcaaattttc tcactgacct 120 tgtagtgcaa ggtaaatatt taggctgttt cgtgttacc 159 <210> 25 <211> 178 <212> DNA <213> Artificial Sequence <220> <223> RsInD50 <400> 25 attgaaaagg tgaacaacga gataaaaacg ctgcaactaa attatatatg tgcaacttgg 60 tttacgtatg catattttgt agagtgtatg actatgtatt atgtatagag tcctcaatag 120 tccgtacctg atttgatttt gtactctaga cagcagtgta gctaaacccc tttcattt 178 <210> 26 <211> 185 <212> DNA <213> Artificial Sequence <220> <223> RsInD49 <400> 26 tgatgatagg ctgatagcta aggaacacaa acgtgtttaa aaacacaacg aaatagcact 60 gaactctctc cgagagagtc tctcgtcggt ctataatgat attaacataa taacatcata 120 atatatatat atatatatat atataacgat cagcttgcaa attcccattc tctttccaaa 180 ctact 185 <210> 27 <211> 272 <212> DNA <213> Artificial Sequence <220> <223> RsInD48 <400> 27 caacaacaga atcaaaggag agtcccaaaa gtcttgatct ttaaggatgg atcaagaaga 60 taaaactaag attgaaacct cacagttctc tagcctctca gcctaactat cgatcattta 120 gtcaacagaa gtcaaggcaa caaaagttaa cttaaaaccc aagaagatac acctagactt 180 agtcaaaaga agtcaatgca acaaaaatta actcaaaccc caattctcta gactccctct 240 gtccaacaac acttagtcaa cagaagtcaa gg 272 <210> 28 <211> 183 <212> DNA <213> Artificial Sequence <220> <223> RsInD51 <400> 28 ctgaggtcga aaacaatgag aatagttaat gcaaaagaat aatactttca tgtaatatct 60 gtaaatgggc aaatacaatg gtggtgatca tgaaacaaag catcggagaa tattctcatt 120 gcagctgtcc gttaaagtcc aatgctatat gtagtttctt cacgcttatt atacccttac 180 acg 183 <210> 29 <211> 227 <212> DNA <213> Artificial Sequence <220> <223> RsInD53 <400> 29 gagggatagg aaccttatcg aatagggatt gggctcgctc ccatggagag gaagcactac 60 tacttttatc catctcaaaa ttatccagga cctgagataa ttaataaata aaaggcaagt 120 caaaaccttg aatagagaaa tcaaagaccc tttacagaga aacattcgat ttttacaagt 180 caataaacaa gagataattg acaaataaaa cacaattccg atttgcg 227 <210> 30 <211> 211 <212> DNA <213> Artificial Sequence <220> <223> RsInD54 <400> 30 ccccctcgtt cttcttttta aaattttgaa gttttaaatc actgaaatac ttaattagaa 60 acttctatag attattttgt aagtcatata tagatactat actctttaaa ttctatctag 120 tattggtgtt atgaacatta aacaatataa acaacgtata tatatatttc ttgttggtga 180 tacattcgca tcgctgattg ttcctatgat t 211 <210> 31 <211> 221 <212> DNA <213> Artificial Sequence <220> <223> RsInD55 <400> 31 aatcgcagac atatagtttc tgccactacc tcgatggggt catcgcagac atatagcttc 60 cctttcaatg aactttgcca tatgtgatct ctttcgtgtt gccactcata tctgtcaccg 120 ggtttgcatc tgctgcacat cctattgttc ttcatagctg tccttcacat cctctatagt 180 tgatgcttgg cagactcaag gtacaacaat cctgaatacc t 221 <210> 32 <211> 233 <212> DNA <213> Artificial Sequence <220> <223> RsInD56 <400> 32 atttcccttt tgtgtgatac tggttgatat ggtttacata tggatataaa accatgttgc 60 agaatcttga tgtttacctc agttacttgt ctagattctt gcataagata ctatggaaac 120 tctcagggaa gataattgat tgagcttcaa caaagtccaa aaaagtatgt ttatatagga 180 actgaagaga gcttaaaatg aagacatttg cttagaaaca acggatttgg tct 233 <210> 33 <211> 190 <212> DNA <213> Artificial Sequence <220> <223> RsInD58 <400> 33 taacatttgc gatcgagaaa agaaaaatat gcataaccta gaaatggaga aacgaagata 60 atcaaaggga atacgagaaa ctgattgcct accatgtata cgaaaatttc agcgaagaat 120 tgggatacag agagacttgg cctttgttct cttgaatcga gaatggggga tatagaggag 180 gaagaaggtg 190 <210> 34 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> RsInD60 <400> 34 aatccaaaac catctgagaa tcgctcaaag aagttactaa gagaaagctg attaaaacgg 60 gtagacacag aatagacttg gcattttaaa atctagtttt aagaatgttg aatggaaaag 120 agagtttgat tacttgacag agatcatctt ctccaa 156 <210> 35 <211> 217 <212> DNA <213> Artificial Sequence <220> <223> RsInD68 <400> 35 ctgcatcaac atccgataca aaacaaaaaa cccttctaga ttataccata tataaaacta 60 tacgtagata aggctgatgc caaacctatt gaaccaacat ctgatacaaa ccacaaaacc 120 ttctagatta taccatttta tctatatgta gataaggcta aagccacact tgtaatgcaa 180 agagtaaccc caagtaagta ccatcatgtc aagtgga 217 <210> 36 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> RsInD65 <400> 36 ccggattcag actactctca atacaaaatt ttattctgaa gtagaatttt caaacgttga 60 tttcacatct gtgaaaaata taaacttaag accctaaatt atatgtctat caaaagaaaa 120 gcacaaccag tcctatta 138 <210> 37 <211> 178 <212> DNA <213> Artificial Sequence <220> <223> RsInD66 <400> 37 atttgagtga aaccaaagta gggaatcaga tcagataaac ttcttattgt cgttacagta 60 tcatgaaaga tacaaaaatg acttttgata ttggcttaaa tcagaaataa atatgaatct 120 ttaatcaata gtatgaacga gatttagatt tagaaaatga gatctggaag agacacat 178 <210> 38 <211> 148 <212> DNA <213> Artificial Sequence <220> <223> RsInD67 <400> 38 agaccaaaat ctcagtgacc ttattttaga tatgaaactc actattgaag ggttaaattt 60 cttggaacaa caatatgaaa gaccttatcc ttgaggaatg agagtgttcg tgcaaactat 120 caatatgatc catgttaact ggaacaac 148 <210> 39 <211> 183 <212> DNA <213> Artificial Sequence <220> <223> RsInD73 <400> 39 acatgctttt ctcttgtttc tcaaaagaga gaaatgtctc ttattaaaca gtatgtaacc 60 aactcacagc tgcttaaaaa tgcagagtgt ttattgatct attgatttcg caatgaattg 120 tttattgatc tatatggcta ctcagtaaat ggttcagaga ttacccagtg cttcttattc 180 tct 183 <210> 40 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> RsInD70 <400> 40 tccctaatga tctcagctta tcctccatgc taaacgtttc ttcataatct gaaatttcaa 60 cialis a 121 <210> 41 <211> 163 <212> DNA <213> Artificial Sequence <220> <223> RsInD71 <400> 41 agctaaagtg ctttttgttt gttttttggt gtcaaataac aatatttaca tctcaaacaa 60 tgaattattt tggaaagaag agcttagaac attgcattag tacttaaaga agagcaatat 120 tcactacatt cctctttttt gtgcacaaaa tgatcgcatt aat 163 <210> 42 <211> 197 <212> DNA <213> Artificial Sequence <220> <223> RsInD72 <400> 42 ctctagtgga agattatccc ctcgtcttcc tgttcctgat cacataccaa ggccttcata 60 tcttgagtct agcgagccac caaagatatc aagtgcgtat cagttttctg attccaaatg 120 cattgtgaat tagactgtgc aggaaattcg gctgtagtaa gtacagatta gtatttcttc 180 tttttctcgt ggttctc 197 <210> 43 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> RsInD81 <400> 43 gaaccaacag tgaagaatct caaggaggcc aaacgtgtac tcagatacgt tcaaggaacg 60 ttggagattg gattgagatg cactcgagtc agtgaaccaa tgctcattgg atactctgat 120 atactgttat attttctaaa ctaaataatt cttacgttac agacttgcac 170 <210> 44 <211> 246 <212> DNA <213> Artificial Sequence <220> <223> RsInD77 <400> 44 caatcttctt ctccatccac aaacatgggg tactgtttgt ttttttagtg tttctattga 60 tttttttttt gttattgtaa tttctagttt atcattgaat tatatagaag attctgcatc 120 agtgcgtgtt tgtttatgga gatgtatgtt gattgcttgt tttcttggtt tgagaaattt 180 taatctaaag atataagagt taataaggag atttagtgtg ttaagtttga tgattggatt 240 gtgtgt 246 <210> 45 <211> 168 <212> DNA <213> Artificial Sequence <220> <223> RsInD75 <400> 45 agtattgacg aactcaaagc aaaatccttg gctaattcca gctattacat aatattagat 60 tgaaaacata aatatagata atacacacat catttgtaca taatttgtta tatgtataaa 120 aaatctctat ccggctctaa gcgaggatta acctttcgca atacctca 168 <210> 46 <211> 166 <212> DNA <213> Artificial Sequence <220> <223> RsInD79 <400> 46 tatccccttg ttcagaactg taagctgtaa atacacaaag aatcgtttaa aacacccaat 60 gattcaccaa acacacacaa aagattagtt tctaatgaat gattccaaga aaaggaggag 120 gcataattac ttggccatga acgtcattag ctaaaccaga ggctaa 166 <210> 47 <211> 173 <212> DNA <213> Artificial Sequence <220> <223> RsInD83 <400> 47 ggtacacaca atcctctgat tttttagtta gtgcaaatag tagtataaat tattttctaa 60 cacaactaaa ttattgagta ccaacaatag attttcctat gatcagaaat tttccacatt 120 attttcactc aaaaatccaa tatgctaaaa tatagaaact ccccagtcac aaa 173 <210> 48 <211> 207 <212> DNA <213> Artificial Sequence <220> <223> RsInD84 <400> 48 catgcaaatg aaccatcaga ttgcttaagc atcacctgcc acggccctgc attattattt 60 ttatatcaga catggataga cagttaaggt gactggtatt gacaatggtt ccgcatattt 120 gatagacaca gagacatgta ccggagccat atacatttat tcccgggaga gaatactata 180 gtatttacca ggatattgcc gaaaaag 207 <210> 49 <211> 184 <212> DNA <213> Artificial Sequence <220> <223> RsInD87 <400> 49 gtcatcgcga tatgattctc tttatatatc actattatta ttactttctt gatggtgtgt 60 aattaaaaat gttgtagcta aaaaaaatgt tatagctatt atacgcaacc cacttctttt 120 attgtttttc attagaagtt tttgacatac aaccgatgct aaaaatgtaa tcccgtgtcc 180 aaat 184 <210> 50 <211> 256 <212> DNA <213> Artificial Sequence <220> <223> RsInD88 <400> 50 acgtgatttt gatttctgaa cctaaagact tatctgatta ataccctccc acattatttt 60 cttgcaccaa aagccatcgt tttagttttc aatcaatctt gtaaaccttt ataacgtttt 120 tatcatgttg gatttggtaa aatcgtgata aagtatcaaa tataaagcag acgtacaaac 180 ctctgataac ttatcacaaa atcttagtat agatcaagat tcacatgttt ttcttgttca 240 gacaacgtat cacaaa 256 <210> 51 <211> 142 <212> DNA <213> Artificial Sequence <220> <223> RsInD90 <400> 51 atacggtttt taggttggtt ctggtaaact gaatggtcat tcctaatgga caatattgca 60 agtgaacggt tcaaaactgt aaagtggtcc aaagagcatc aaaactggga taacgaagtc 120 tcatgagaca gctttattcc tg 142 <210> 52 <211> 186 <212> DNA <213> Artificial Sequence <220> <223> RsInD91 <400> 52 caaagcaaga tagactccag aagctcagta accaatttag acatcaaggt cctcccacct 60 gaaagaaaca agagaacctt ttaatatttc agtagagcag tattcatgta ttttttttta 120 tcagcaaacg aatgaaacag taaaatcaaa atctcacaag cactaagtgc ctaaagtagc 180 atttgg 186 <210> 53 <211> 177 <212> DNA <213> Artificial Sequence <220> <223> RsInD92 <400> 53 ttacgatttc tggttttcat cgtcggttat ggtttcttca gttactttct acaaagaact 60 aaaatggcta tttaatattt actaacacga ggaaaatatt taagatcttc tattttattt 120 atttttttgt tagcccacag tttttccctt catggctttc tcgttctttg tctctaa 177 <210> 54 <211> 139 <212> DNA <213> Artificial Sequence <220> <223> RsInD94 <400> 54 ctgttatcaa cgtattggaa ccatttgatg gccttcttac cgacgctagg catgatcgga 60 caattcaaat agttgaattg tgtttcaagt gcgttgacca gttgaattgt gtgtacaatg 120 cgttgatcag ttttaaagc 139 <210> 55 <211> 188 <212> DNA <213> Artificial Sequence <220> <223> RsInD95 <400> 55 aaagttgaaa gcttgatctc aggaacccaa caaagtatcc gatgaaagct tatgcaatct 60 caattttgca aggatcaagc ttgaagaagc aaatctcaac aactttaaca ttcatcgaca 120 ctgtcatcaa tgatttgatt cattctgcta aaaaaaaaaa aagaaacgaa ctttttgtta 180 cagtttgc 188 <210> 56 <211> 181 <212> DNA <213> Artificial Sequence <220> <223> RsInD96 <400> 56 aacagaaact atccatcgca aaacgggata ccatctatta ataatcacat gggttccaca 60 aacttaccag ccgttcaaca ggctgctgct gcggctgcgg ctgatgaagc aaatgagtct 120 agctcggtgc agaagatact taatgagatc ctgatgaaca accaagctca taatacctca 180 g 181 <210> 57 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> RsInD206 <400> 57 gtagtcggaa agagattcat cagcttcggc ttctcctgct gcccaaacaa aacgttggtg 60 tttctagccg gagatccttt gagaaacctc ccttgttcat ctgcatgatc aatacggtaa 120                                                                          120 <210> 58 <211> 133 <212> DNA <213> Artificial Sequence <220> <223> RsInD209 <400> 58 catcactgaa acttgcattt gttacacaac agtgaaagaa aaaaacaata agaaccaaaa 60 gagggaaaca agttttgtga gaatcatacc gggttccaag tgtgaagatg ccagaatttc 120 tgatgattcc tcc 133 <210> 59 <211> 146 <212> DNA <213> Artificial Sequence <220> <223> RsInD210 <400> 59 cagtagcaag caatgaagta tcaattcgct aatacgttac agtttgtatg cacaatacat 60 aatagcccgt gttcacctaa taactaatgg gcctttaaaa tcaaatcctg taaagttaac 120 tcagcccatt atcacattag ggtttt 146 <210> 60 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> RsInD225 <400> 60 ctacaagcga aaatgaacat gtgtcaaggt atttgttaaa aacaagacga gtcaagattc 60 attatccgcc agaaacatgc aatgatcaaa gctctaacat atgaaacaag aaggaaaagg 120 ccactcacct gacaggatag ataagtccat tttgaagtgc aagaacactg aaacgagtga 180 tcacaaagag tactgaggat accactagtg 210 <210> 61 <211> 174 <212> DNA <213> Artificial Sequence <220> <223> RsInD226 <400> 61 gt; gttttctttt ggtgggtaag ataatgtggg cggaggccat gagcaaactt gaaggcatgg 120 attcatcaga aagagagcgc ctatggcctc agcttgtgca aggttttaaa gatc 174 <210> 62 <211> 146 <212> DNA <213> Artificial Sequence <220> <223> RsInD217 <400> 62 tctcttcttc atatctggtc tcgttgtcaa tctccttcag gtgtggcttt cctcttaaag 60 tcgaaacctt ttttaagatt cctttgcaga tctcctcttc tctttgtaca cttgttctct 120 gtattgattc actgttgtta gggttc 146 <210> 63 <211> 166 <212> DNA <213> Artificial Sequence <220> <223> RsInD233 <400> 63 gcttaatccc tcaaatacga acacaacgcg taatattcat gatactaggg cattaagaca 60 cgtacatgag gacttttcat gttccggagt gtgtggatgg aagatgctta gtgaacgaac 120 gaattcacca aactcgatca caccgtttcg cttcctatca aacata 166 <210> 64 <211> 148 <212> DNA <213> Artificial Sequence <220> <223> RsInD250 <400> 64 tgattttgtt tcgtgattag ggttaatcac tagctcaggg tcagaatagt aatctccgga 60 taataactgc agtaacaaaa acatttagaa acttttgttg ttttaattca aaaactgttt 120 ctgttatata cagttgtttt gagcctcg 148 <210> 65 <211> 136 <212> DNA <213> Artificial Sequence <220> <223> RsInD246 <400> 65 acataatgtc cccactcata actttttttt gtctccataa ctttgtctat gccaccaact 60 atgacaaatt tgaagatata tagtcagtcg catctaaatc tctctttcta atggccatat 120 gtattgatta gcagca 136 <210> 66 <211> 133 <212> DNA <213> Artificial Sequence <220> <223> RsInD247 <400> 66 atctgatggt tggaaagaaa gtgaatctgc tggtgaagat tttaagcttc tgtgggagaa 60 agttcttcag gttgttgagc atcagatgca aacgacgaga taccaattga acttcaatgt 120 gttggtacaa gag 133 <210> 67 <211> 171 <212> DNA <213> Artificial Sequence <220> <223> RsInD64 <400> 67 ttatattccc cttgttttcc gtgttttttt agaaactgaa cttgtaattg gttaaacaac 60 gaaatcgcta aatacactat attttttaaa aagatggaat gaaatgtgag aaataaatct 120 cagtgaattt cttaatgaga gtatgtggcc gcaatgactt atgtagtttt g 171 <210> 68 <211> 224 <212> DNA <213> Artificial Sequence <220> <223> RsInD63 <400> 68 aaagttgctg ccttgttatg tatattagat ttgattctaa gcattaagca cccgttttgg 60 ctcttaagac aagttaagag actctgtctc ttttagacag gtaactcttg tggcaagcag 120 aacaacacgg gaagatggaa gccaagttct ttaggttctt gaagattgtt ggagttggat 180 acaaagccag agctgaggag gctggccgtt tcttatacct taag 224

Claims (4)

A molecular marker for detecting a species or a system of radish comprising any one of the nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 1-68. A primer set capable of detecting and amplifying the molecular markers of claim 1, wherein any primer set selected from the group consisting of primer sets 1 to 68 described in the following table.

3. The method of claim 2,
Wherein the primer set has a GC content of 40 to 60% and is capable of performing PCR at a temperature of 56 to 60 ° C. Isolating the genomic DNA from the non-crop sample;
Performing PCR using the separated genomic DNA as a template and using the primer set of claim 2; And
And analyzing the amplified product by electrophoresis to analyze the amplification product and the size of the amplified product.

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