KR101752658B1 - SSR molecular markers for discriminating of Codonopsis lanceolata cultivars and uses thereof - Google Patents

SSR molecular markers for discriminating of Codonopsis lanceolata cultivars and uses thereof Download PDF

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KR101752658B1
KR101752658B1 KR1020150153482A KR20150153482A KR101752658B1 KR 101752658 B1 KR101752658 B1 KR 101752658B1 KR 1020150153482 A KR1020150153482 A KR 1020150153482A KR 20150153482 A KR20150153482 A KR 20150153482A KR 101752658 B1 KR101752658 B1 KR 101752658B1
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김호방
김지현
김재준
김세림
길진수
엄유리
이이
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주식회사 바이오메딕
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Abstract

The present invention relates to an SSR primer set for distinguishing a dodec cultivar comprising at least one SSR primer set selected from the group consisting of 10 SSR primer sets, a kit for distinguishing a dodec cultivar including the SSR primer set, The present invention relates to a method for distinguishing dodok cultivars using a mare set, and it is possible not only to efficiently identify dodok cultivars through a specific SSR primer for discriminating dodec cultivars of the present invention, but also to provide information useful for studies on doduk molecular breeding will be.

Description

[0002] SSR molecular markers and their uses for distinguishing dodec cultivars. [0002] SSR molecular markers for discriminating Codonopsis lanceolata cultivars and uses thereof [

The present invention relates to SSR molecular markers and their uses for distinguishing different species of dodec, and more particularly, to a SSR primer set for distinguishing dodec cultivars comprising at least one SSR primer set selected from the group consisting of 10 SSR primer sets, A dodec cultivar discriminating kit comprising an SSR primer set and a method for distinguishing a doduk cultivar using the SSR primer set.

Codonopsis lanceolata is a perennial dicotyledon belonging to the Companulaceae . Doduck is very valuable as a traditional medicinal plant and very popular in East Asia such as China, Japan and Korea.

A number of studies have reported that dodok root contains substances such as saponins, phenylpropanoids, alkaloids, triterpenes and flavonoids. In addition, a number of studies have reported that the components of dodeca affect the immune system or inhibit tumor growth (Sathiyamoorthy et al., 2011, Mol. Biol. Rep . 38: 3541-3549). Ichikawa et al., 2009, J. Nat. Med ., 63: 52-57) reported that there were seven different types of saponins in Doduck. Thus, it was speculated that there would be a considerable diversity in the composition and constituents of the chemicals in the roots grown.

Several DNA markers have been successfully used to analyze genetic distance or genetic relationships in a duck dog. (2002, J. Medicinal., Crop. Sci ., 10: 194-199) studied the genetic relationships of duckweed from Mt. Paektu and several Korean regions using Random Amplified Polymorphic DNA (RAPD). (2001, Korean J. Medicinal Crop Sci . 9: 205-210) We also used the RAPD method to report the identification and genetic relationship between Adenophora triphylla and Doduck. (2006, Plant Cell. Rep . 25: 896-906) reported successful development of inter-simple sequence repeat (ISSR) and RAPD methods, However, the RAPD method is not sufficient for genetic distance studies and further development of additional DNA markers is still needed.

SSR (simple sequence repeat) markers are a very effective tool for the analysis of genetic relationships. In addition, it is a useful tool for studying non-reference plant genomes due to its uniform distribution all over the genome and high inter-individual polymorphism. Thus, a number of studies have been reported that apply SSR markers to crops for phylogenetic analysis or genetic diversity in non-standard plant genomes. These findings confirmed the SSR markers for Codonopsis tangshen and Codonopsis pilosula (2013, Conservation Genet. Resour . 5: 393-395), and found these findings to be used by these two members of the Codonopsis genera Genetic diversity and population structure. However, there have been no studies on the development of SSR markers in Korea, the most important medicinal plant in Korea.

In the present invention, ten new SSR markers were developed based on the genome sequence of Doduck and successfully analyzed the genetic relationships of 53 cultivated doducks collected from 10 regions in Korea using these markers.

Korean Patent Laid-Open Publication No. 2014-0106892 discloses a method of identifying a maize gene using an SSR marker. Korean Patent No. 0842434 discloses 'SSR primer derived from ginseng and its use' The SSR molecular markers and the uses thereof for the differentiation of the dodec cultivars of the present invention are different.

The present invention has been made in view of the above-described needs. In the present invention, ten SSR markers are extracted from the partial genomic sequence of Doduck, and genetic diversity among 53 dodocarpy collected using the SSR marker is analyzed, The present inventors completed the present invention by developing an SSR primer capable of effectively distinguishing a dodecan variety.

In order to solve the above problems, the present invention provides an SSR primer set for distinguishing a dodec cultivar comprising at least one SSR primer set selected from the group consisting of 10 SSR primer sets.

The present invention also provides a kit for distinguishing dodoc cultivars comprising the SSR primer set.

The present invention also provides a method for distinguishing dodoc cultivars using the SSR primer set.

According to the present invention, it is possible not only to efficiently discriminate dodok cultivars through SSR primers for distinguishing doduck cultivars, but also to provide information useful for studies on doduk molecular breeding.

FIG. 1 is a diagram showing UPGMA (unweighted pair group method with arithmetic average) tree structure for 53 dodec genomes.

In order to accomplish the object of the present invention, the present invention provides a set of SSR primers for discriminating dodec cultivars comprising at least one SSR primer set selected from the group consisting of ten SSR (simple sequence repeat) primer sets.

The SSR primer set of the present invention specifically comprises SSR primer sets of SEQ ID NOS: 1 and 2; An SSR primer set of SEQ ID NOS: 3 and 4; An SSR primer set of SEQ ID NOS: 5 and 6; An SSR primer set of SEQ ID NOS: 7 and 8; An SSR primer set of SEQ ID NOS: 9 and 10; SSR primer sets of SEQ ID NOS: 11 and 12; An SSR primer set of SEQ ID NOS: 13 and 14; An SSR primer set of SEQ ID NOS: 15 and 16; An SSR primer set of SEQ ID NOS: 17 and 18; And a set of SSR primers of SEQ ID NOs: 19 and 20, respectively.

The SSR primer set of the present invention preferably comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, 8, more than 9 primer sets, most preferably 10 primer sets, i.e. SSR primer sets of SEQ ID NOs: 1 and 2; An SSR primer set of SEQ ID NOS: 3 and 4; An SSR primer set of SEQ ID NOS: 5 and 6; An SSR primer set of SEQ ID NOS: 7 and 8; An SSR primer set of SEQ ID NOS: 9 and 10; SSR primer sets of SEQ ID NOS: 11 and 12; An SSR primer set of SEQ ID NOS: 13 and 14; An SSR primer set of SEQ ID NOS: 15 and 16; An SSR primer set of SEQ ID NOS: 17 and 18; And SSR primer sets of SEQ ID NOS: 19 and 20, respectively.

By using the 10 SSR primer sets at the same time, it is possible to discriminate the dodoc cultivars more efficiently.

The SSR primers may be selected from the group consisting of SEQ ID NOs: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; SEQ ID NOS: 15 and 16; SEQ ID NOS: 17 and 18; And at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 within SEQ ID NOs: 19 and 20 , And oligonucleotides consisting of fragments of 26 or more consecutive nucleotides. For example, the SSR primer (20 oligonucleotides) of SEQ ID NO: 1 is an oligonucleotide comprising 15 or more, 16 or more, 17 or more, 18 or more, or 19 or more consecutive nucleotide fragments in the sequence of SEQ ID NO: . ≪ / RTI > In addition, the SSR primers include SEQ ID NOs: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; SEQ ID NOS: 15 and 16; SEQ ID NOS: 17 and 18; And addition, deletion or substitution of the nucleotide sequences of SEQ ID NOS: 19 and 20.

In the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.

In order to achieve still another object of the present invention,

An SSR primer set according to the present invention; And a reagent for carrying out an amplification reaction.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The manual includes instructions on the surface of the package including a brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

In order to achieve still another object of the present invention,

Isolating the DNA genomic DNA;

Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the SSR primer set according to the present invention; And

And detecting the amplification product. ≪ Desc / Clms Page number 2 >

The method of the present invention comprises separating the genomic DNA from the chrome sample. A method known in the art may be used for separating the genomic DNA from the sample. For example, a CTAB method may be used, or a Wizard prep kit (Promega, USA) may be used. The target sequence can be amplified by performing amplification reaction using the separated genomic DNA as a template and SSR primer set according to an embodiment of the present invention as a primer. 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 pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling material is 6-FAM (6-Carboxyfluorescein), NED, VIC, PET or ROX. When the target sequence is amplified, 6-FAM, NED, VIC, PET or ROX is labeled at the 5'-end of the primer and PCR is carried out. The target sequence can be labeled with a detectable fluorescent labeling substance. Also, the labeling substance may include Cy-5 or Cy-3. When the radioactive isotope such as 32 P or 35 S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The set of SSR primers used to amplify the target sequence is as described above.

The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As a method of detecting the amplification product, gel electrophoresis can be performed, and gel electrophoresis can be performed using acrylamide gel electrophoresis or agarose gel electrophoresis according to the size of the amplification product. In addition, capillary electrophoresis can be performed. Capillary electrophoresis can be performed, for example, using the ABI Genetic Analyzer. In the fluorescence measurement method, when a fluorescent dye is labeled at the 5'-end of the primer and PCR is performed, the target sequence is labeled with a fluorescent label capable of detecting the fluorescence. The fluorescence thus labeled can be measured using a fluorescence analyzer. 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.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Collection and DNA extraction of dodok resources

53 dodok resources were collected in domestic seed companies or farmhouses in the form of fresh roots or seeds. The collected dodok resources are summarized in Table 1 below. The collected roots or seeds were grown in the greenhouse packaging of Chungbuk National University in the spring of 2015. For genomic DNA extraction, leaf tissues were ground with liquid nitrogen and stored at -80 ° C in a cryogenic freezer until DNA extraction. Genomic DNA was extracted according to the method of Doyle and Doyle (Phytochemistry Bulletin, 1987, 19: 11-15).

The 53 dodec resource information number Storage number shape Collection place number Storage number shape Collection place One CL0001 Root Samcheok, Gangwon-do 28 CL0028 Root Yongin-si, Gyeonggi-do (1) 2 CL0002 strain Wonju, Gangwon-do 29 CL0029 strain Yongin-si, Gyeonggi-do (2) 3 CL0003 strain Cheolwon-gun, Gangwon-do 30 CL0030 strain Yongin-si, Gyeonggi-do (3) 4 CL0004 Root Chuncheon, Gangwon-do 31 CL0031 strain Yongin-si, Gyeonggi-do (4) 5 CL0005 Root Pyeongchang-gun, Gangwon-do 32 CL0032 strain Yongin-si, Gyeonggi-do (5) 6 CL0006 Root Baekdu Mountain, Pyeongchang County, Gangwon Province 33 CL0033 Root Jirisan, Sancheong-gun, Gyeongsangnam-do (1) 7 CL0007 Root Mabongsan, Hongcheon-gun, Gangwon-do 34 CL0034 strain Jirisan, Sancheong-gun, Gyeongsangnam-do (2) 8 CL0008 Root Hong-gun County, Gangwon Province 35 CL0035 strain Bonghwa-gun, Gyeongsangbuk-do 9 CL0009 Root Hong-gun, Gangwon-do (1) 36 CL0036 Root Ulleung County, Gyeongsangbuk-do 10 CL0010 Root Hong-gun, Gangwon-do (2) 37 CL0037 Root Youngju City, Gyeongsangbuk-do 11 CL0011 Root Hong-gun, Gangwon-do (3) 38 CL0038 strain Jung-gu, Daejeon city (1) 12 CL0012 Root Hong-gun, Gangwon-do (4) 39 CL0039 strain Jung-gu, Daejeon-si (2) 13 CL0013 Root Hong-gun, Gangwon-do (5) 40 CL0040 strain Songpa-gu Seoul 14 CL0014 Root Hong-gun, Gangwon-do (6) 41 CL0041 strain Jongno-gu, Seoul 15 CL0015 strain Hong-gun, Gangwon-do (7) 42 CL0042 Root Gwangyang City, Jeollanam-do 16 CL0016 strain Hong-gun, Gangwon-do (8) 43 CL0043 Root Jirisan, Gurye-gun, Jeollanam-do 17 CL0017 strain Hong-gun, Gangwon-do (9) 44 CL0044 Root Shinam-gun, Jeollanam-do 18 CL0018 strain Hong-gun, Gangwon-do (10) 45 CL0045 Root Hwasun County, Jeollanam-do 19 CL0019 strain Hong-gun, Gangwon-do (11) 46 CL0046 Root Muju-gun, Jeollabuk-do (1) 20 CL0020 strain Hong-gun, Gangwon-do (12) 47 CL0047 Root Muju-gun, Jeollabuk-do (2) 21 CL0021 strain Hong-gun, Gangwon-do (13) 48 CL0048 Root Seogwipo City, Jeju Island 22 CL0022 strain Hong-gun, Gangwon-do (14) 49 CL0049 Root Jeju City, Jeju Island (1) 23 CL0023 strain Hong-gun, Gangwon-do (15) 50 CL0050 Root Jeju City, Jeju Island (2) 24 CL0024 strain Hong-gun, Gangwon-do (16) 51 CL0051 strain Jeju City, Jeju Island (3) 25 CL0025 strain Namyangju City, Gyeonggi-do 52 CL0052 Root Danyang-gun, Chungcheongbuk-do 26 CL0026 strain Bucheon, Gyeonggi-do 53 CL0053 Root Cheongju-si, Chungcheongbuk-do 27 CL0027 Root Yangpyeong, Gyeonggi

Detection of Doduk SSR (simple sequence repeat) marker

Microsatellite enrichment libraries were constructed according to the method of Glenn and Schable (Methods in Enzymology, 2005, 395: 202-222). Briefly, genomic DNA of Dulcite was digested with restriction enzyme Rsa I to obtain DNA fragments corresponding to approximately 300 to 1,000 base pairs, and these fragments were ligated with linkers. The ligated reaction product was used for the double concentration step by hybridization with the biotin labeled microsatellite probe at the 3 'end. The DNA fragments rich in microsatellite sequences were used for the ligation reaction with pGEM-T vector (Promega, USA), and the ligated reaction products were used for transformation of E. coli DH5α strain. M13 forward and reverse primer sets (M13F (-20), 5'-GTAAAACGACGGCCAG-3 ': SEQ ID NO: 21, M13R, 5'-GAAACAGCTATGACCATG-3': SEQ ID NO: 22) were used to identify recombinant clones from the formed colonies. Followed by colony PCR. The amplified product was purified and used for sequencing using an ABI3730 DNA sequencer (Applied Biosystems, USA). After removal of the vector and linker sequences, the nucleotide sequence was assembled using Lasergene SeqMan software (version 7.0.0; DNASTAR, USA) and the duplicated contigs were obtained. Use MISA software (http://www.pgrc.ipk-gastersleben.de/misa) based on a minimum of 3 repetitions for 2 to 6 nucleotides and a spacing of less than 100 bases for complex forms To obtain provisional SSR markers. For PCR primer design, amplification size was 85 ~ 350 bases and primer binding temperature was 57-60 ℃. The primers used in this experiment were synthesized in Biomedic Co., Ltd. The specificity of the primers was verified by a conventional PCR amplification reaction using genomic DNA as a template. The primary selection of the polymorphic markers was carried out by PCR using the same amount of dodeca genomic DNA extracted from 10 different Doduk resources as a template. The PCR amplified product was electrophoresed on 2% agarose gel.

PCR amplification and genotyping

The PCR reaction consisted of 20 ng of reaction volume containing 20 ng of genomic DNA, 1 x HS TM Taq DNA polymerase buffer, 1.5 mM MgCl 2 , 0.2 mM dNTP, 0.2 μM of each primer, and 1.25 unit HS TM Taq DNA polymerase (Biometra Thermalcycler, Goettingen, Germany). Conditions for PCR amplification were as follows: Initial heat denaturation (once) -95 ° C for 5 min, DNA amplification (34 total iterations) -94 ° C for 30 sec, 57-60 ° C for 30 sec, Renal reaction (once) -72 캜 for 30 minutes. PCR amplification products were electrophoresed on 2% agarose gel to confirm amplification. Table 2 summarizes the PCR primer set for the Dodec SSR amplification used in this experiment. The 5 'end of the forward primer was labeled with the fluorescent dye 6-FAM, NED, VIC or PET (Applied Biosystems, USA). After PCR amplification, the PCR products were mixed in 0.2 and 9.8 ㎕ ㎕ of Hi-Di formamide and GeneScan ® TM 500 LIZ size standard (Applied Biosystems, USA), 0.2 ㎕. The mixture was denatured at 95 DEG C for 5 minutes and then left in ice. The amplified DNA fragments were separated by capillary electrophoresis on an ABI3730 DNA sequencer equipped with a 50 cm capillary.

Primer set information for amplification of Doduk SSR marker Primer name Elongation temperature (캜) Allele size range (bp) Sequence information (5 '- > 3') (SEQ ID NO: CLSSR-1-F 58 136-166 FAM-GACACAGCATTATCCACGAA (1) CLSSR-1-R GTTTAATTATGCGTTTTGGCTGTC (2) CLSSR-2-F 59 186-215 PET-CACCACTCAATCATGCAAGC (3) CLSSR-2-R GTTTGACGCAGRRGCAGAAAAGAA (4) CLSSR-4-F 61 231-261 FAM-AGGTGGAAACGGTGTCTTTG (5) CLSSR-4-R GTTTGTCCACAGATGCCATTCGTA (6) CLSSR-5-F 59 146-194 FAM-GAGAACAATTATTTAAGAACGGATG (7) CLSSR-5-R GTTTGTCAGGCCCGRAAAAATGAA (8) CLSSR-6-F 59 143-161 PET-TGGACTGTAGATGCCCTGCT (9) CLSSR-6-R GTTTTTAAGCCCAAGGTGTTCGTT (10) CLSSR-7-F 59 128-131 VIC-TAGTTTGGTGGGTAGGTGGA (11) CLSSR-7-R GTTTTGCACTTCCCAAGAAGAAAC (12) CLSSR-9-F 58 161-165 VIC-GCAGAAGTGAGTATGCAAGTAG (13) CLSSR-9-R GTTTGATCATATCTATTGGCATGCA (14) CLSSR-10-F 58 108-126 FAM-CATCCCTCCCTGAAAAATGT (15) CLSSR-10-R GTTTTGAAACTTTATGGGCATCTTG (16) CLSSR-11-F 58 104-112 NED-GTCCTTGCTCACAATTAGCC (17) CLSSR-11-R GTTTTGACAAAATGGTGATGCCTA (18) CLSSR-12-F 58 122-130 PET-GGGACGCATTTTCGTAATC (19) CLSSR-12-R GTTTAGAAATGTTTGTTTATGGGGTG (20)

Data analysis of genetic diversity

DNA peaks were recorded based on presence / absence (1/0) and used to generate data for analysis. The loci and variants of each SSR marker were analyzed. Diversity values for genetic loci were calculated using genetic diversity indices. The values were calculated as follows: h = 1 -? P i 2 , p i The The frequency of the i- th allele. The observed heterozygosity for each locus was calculated for each SSR marker. Polymorphism information content (PIC), a measure of closely related diversity, was estimated using PowerMarker software (version 3.25; http://statgen.ncsu.edu/powermarker). Statistical calculations to analyze the genetic diversity of the collected resources were performed using NTSYS software (version 2.1; http://www.exetersoftware.com). The UPGMA phylogenetic tree was constructed using Jaccard genetic similarity. Bootstrap analysis was performed using WINBOOT software.

Example 1. SSR marker digging

Ten polymorphic SSR markers were identified from the genomic contiguous sequences (226) of Doddock microsomes, each represented by the CLSSR ( Codonopsis lanceolata Simple Sequence Repeat) series (Table 3). Most SSR markers, except for the CLSSR-2 marker, in which two nucleotides were repeated, were four nucleotide repeatable markers. Amplified band sizes ranged from 104 to 261 basepairs and electrophoresis showed single or two bands. From these results, we have obtained a novel SSR marker based on genomic DNA for the analysis of dodec gene.

Information on the CLSSR marker Marker name SSR motif information CLSSR-1 (CATC) 3 (CATA) 7 CLSSR-2 (CT) 8 (ATCT) 5 CLSSR-4 (CTGT) 4 CLSSR-5 (TATG) 5 CLSSR-6 (AGAA) 7 CLSSR-7 (TATG) 5 CLSSR-9 (CATA) 7 CLSSR-10 (TTGT) 6 CLSSR-11 (AAGA) 7 CLSSR-12 (AAGA) 7

Example 2. Analysis of polymorphism and genetic diversity of SSR markers

We obtained cleanly amplified bands using 10 SSR markers from 53 dodecaggregation sources. All sample polymorphisms were analyzed using the GeneScan TM 500 LIZ ® size standard and the respective band sizes were recorded (Table 4).

Allele size data of SSR markers for 53 dodec gene sources CLSSR-1 CLSSR-2 CLSSR-4 CLSSR-5 CLSSR-6 CLSSR-7 CLSSR-9 CLSSR-10 CLSSR-11 CLSSR-12 CL0001 150 196 231 201/376 148 128 161 114/127 104 124/132 CL0002 150 196/233 235/256 201/390 144/148 128/132 165/169 127 93/104 132/136 CL0003 150 211/219 235 201/376 148/152 128/132 165 118/122 104 128/136 CL0004 150 178 231 201/376 148/156 128/132 161/165 127 104/108 124 CL0005 150 196 231 201/376 148/160 128/132 161/165 124 104/112 132 CL0006 140/154 178 235 201/376 148 128 161 114/120 93/104 124 CL0007 140/154 190/211 235/256 201 128/132 161 127 100/104 124 124 CL0008 150 196 231 183/382 148 128/132 165/173 120 104/112 124/132 CL0009 150/154 178 235/261 201 148 128 161 127 93/108 124 CL0010 150 200/211 256 201/382 148 128/132 161/165 120/124 104 124/128 CL0011 130/154 178/211 235/256 201 140/152 132 161/165 - 93/104 124 CL0012 130 187/207 235 201/382 148/160 128 161/165 - 104 124 CL0013 - 187/239 247 201/376 146/156 128/132 165 - 104 124/136 CL0014 - 178 238/256 201/376 148 128 161/165 131 93/104 132 CL0015 130/154 215/227 238 201/376 148 128/132 165 124 104 124 CL0016 150 178 235 201/376 148 128/159 165 120/124 104 124/132 CL0017 150/154 187 238 201/376 148/156 130/132 161/165 127/131 108/112 124/128 CL0018 130 187 249 201/376 146/148 128/132 161 120 104 132 CL0019 130/150 207 235 201 146/148 130/132 161/165 124/127 104 124/132 CL0020 140/150 178/187 235 190 148/156 128/132 161/169 118 104 124 CL0021 150 178/200 238 201 148 132 165 124/127 104 124 CL0022 140/150 178 235 201/376 148 128 165 124 104 124 CL0023 150 219 247 201/382 148/152 128 161/165 127 104/108 124 CL0024 150/154 200/211 235 201/376 148/160 128/159 161/165 118 104 124 CL0025 130/154 183 235 201/382 148/160 128/130 161 118 93/104 124/128 CL0026 138/140 211/233 235 201 148 128 161 - 104 124 CL0027 150 250 261 201/376 148 132 165 127 104/112 124 CL0028 130/150 205/233 256 201/382 148/156 128/132 165 124 104/108 124/128 CL0029 150 178/196 256 201 148 128/132 161 120 104/108 124/132 CL0030 154/158 215 235 201 148 128/132 161/165 124/127 104/108 124/128 CL0031 150 211/223 235 183/376 146 128/132 161/165 127 100/112 124/128 CL0032 150 178 235/261 201/376 148 128/132 165 127 104/112 124 CL0033 - 190/239 261 201/376 148/160 128/130 173 127 104/108 128/132 CL0034 150 178 256/261 201/376 148/156 128 165 118 108 124/132 CL0035 148/154 190 235 201 146/156 128 165/173 124 104 124 CL0036 150 196 231 201 148/164 128/132 165 120/124 104 124 CL0037 150 196/211 256 201/376 148 128 165 120/122 104 124/128 CL0038 150 178 261 201/376 140/148 128 161 120/124 104 124 CL0039 140/150 205/211 235 201/382 148/160 128/132 161/165 - 104/112 132 CL0040 150/154 178/211 231/256 201 148/156 128 165 120 104/112 132/136 CL0041 130/054 178 256 201/376 148/160 128 165 - 104/112 124/132 CL0042 - 193/223 231 201/376 144/148 128/132 165 124/131 104 124/132 CL0043 - 190/211 235 201/382 148 128 165 124 108/120 124 CL0044 - 178/215 238/256 201/382 136/148 128/132 161/165 - 104/112 124 CL0045 - 178/187 256 201 146/148 128 165 120 104 124 CL0046 130/154 178/207 238/249 201 148/156 128/132 161/169 120/124 104/112 124/128 CL0047 130/140 211 247 201/376 148 128/132 165 124 104/108 124 CL0048 150 196 231 201 148 128 161/165 120/124 104 128/132 CL0049 144 178/233 235 201/382 144/148 128/132 161 - 104/108 124 CL0050 140/150 178/187 235 201/376 148/156 132 165/169 131 104 128/132 CL0051 150/154 211/215 235/261 201/382 148 128/132 161/165 120/124 104/108 124/132 CL0052 - 178 256 201/386 148/156 132 161/169 124 104 124/132 CL0053 130/154 200 235 201/382 148/160 128 161/165 127 104 124/128

The 10 confirmed SSR genotypes were polymorphic. Seventy-three unique alleles were detected in 53 resources, with 4 to 17 alleles per locus and an average number of alleles of 7.3. CLSSR-7, CLSSR-9, and CLSSR-12 identified four alleles and CLSSR-11 identified six alleles. CLSSR-2 showed 17 alleles. Of the 73 alleles, 13 alleles appeared in only one resource. Of the 13 alleles present in only one resource, four alleles were detected in CLSSR-1 and CLSSR-2, three in CLSSR-5, and one in CLSSR-6 and CLSSR-11, respectively. The genetic diversity for the genetic loci examined for total dodok resources was 0.63 ( h ), with a minimum of 0.51 (CLSSR-7) and a maximum of 0.86 (CLSSR-2). The mean heterozygosity was 0.49 and was 0.5 or more for the CLSSR-1, CLSSR-2, CLSSR-5, CLSSR-6, CLSSR-7 and CLSSR-11 markers (Table 5).

The mean PIC value was 0.59, and CLSSR-2 showed the highest PIC value of 0.85. Four markers showed a PIC value of less than 0.5. The PIC value reflects the degree of diversity and is a marker that provides enough information if a value of 0.5 or greater is indicated. Even though four of the 10 SSR markers showed a value of less than 0.5, the obtained markers were sufficient to analyze the genetic diversity of the collected dodok resources.

The number of alleles, genetic diversity ( h ), heterozygosity and polymorphism information content (PIC) Marker name Number of alleles Genetic diversity Heterozygosity Polymorphic information content CLSSR-1 8 0.65 0.51 0.61 CLSSR-2 17 0.86 0.51 0.85 CLSSR-4 7 0.76 0.21 0.73 CLSSR-5 7 0.57 0.70 0.52 CLSSR-6 9 0.55 0.60 0.53 CLSSR-7 4 0.52 0.57 0.43 CLSSR-9 4 0.56 0.45 0.48 CLSSR-10 7 0.77 0.36 0.74 CLSSR-11 6 0.51 0.53 0.48 CLSSR-12 4 0.55 0.49 0.50 Average 7.3 0.63 0.49 0.59

Example 3. Analysis of genetic linkage between collected duckuk resources

The genetic distance of the collected resources was analyzed, and UPGMA phylogenetic trees for 53 dodec genomes were generated based on the intercept analysis using SSR markers (FIG. 1). The genetic distance value ranged from 0.74 to 0.95, and there were no genotypes showing remarkable grouping.

In the case of CL033 resources collected at Mt. Jiri, it showed a relatively remote genetic distance compared to other resources. CL043, another resource collected from Mt. Jiri, showed a closer relationship with CL001 and CL004 collected in Gangwon Province. In addition, the results of systematic analysis show that CL001 and CL004 collected from Gangwon Province are relatively far away from CL017 and CL031 collected from Gangwon Province and Gyeonggi Province, respectively. These results show that there is no close genetic linkage between collection sites. Between CL027 and CL032 resources collected in Gyeonggi Province and between CL016 and CL022 resources collected in Gangwon Province, the same genetic background was shown for the genetic loci analyzed in this study.

Wild ducks with high saponin content or strong aroma are believed to have spread nationwide during the growing season. Even though consumers demand the origin of crops, this study suggests that it is not easy to identify production sites. It is important to collect and preserve genetic resources that are far from genetically related because of the high chance of obtaining highly resistant varieties or varieties with high functional material content.

In recent years, the development of genetic sequencing technology has made it possible to acquire high-speed large-capacity sequence information. (Gao et al., 2015, Plos ONE. 10: e0117342) reported the transcript analysis results for ginseng ( Codonopsis pilosula ) using a next-generation genome sequence analysis technique and suggested a polysaccharide biosynthetic pathway for ginseng. This report suggests that large quantities of genomic SSR, EST-SSR or single nucleotide polymorphism (SNP) markers can be found in crops without standard genome sequence information. In the future, these molecular markers are expected to be helpful in studying genomics and genetics of wild-type as well as dodok cultivars.

<110> Biomedic Co., LTD <120> SSR molecular markers for discriminating of Codonopsis lanceolata          cultivars and uses thereof <130> PN15332 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gacacagcat tatccacgaa 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gtttaattat gcgttttggc tgtc 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caccactcaa tcatgcaagc 20 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtttgacgca grrgcagaaa agaa 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aggtggaaac ggtgtctttg 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtttgtccac agatgccatt cgta 24 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gagaacaatt atttaagaac ggatg 25 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gtttgtcagg cccgraaaaa tgaa 24 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tggactgtag atgccctgct 20 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gtttttaagc ccaaggtgtt cgtt 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tagtttggtg ggtaggtgga 20 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gttttgcact tcccaagaag aaac 24 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gcagaagtga gtatgcaagt ag 22 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gtttgatcat atctattggc atgca 25 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 catccctccc tgaaaaatgt 20 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gttttgaaac tttatgggca tcttg 25 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gtccttgctc acaattagcc 20 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gtttggacaa aatggtgatg ccta 24 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gggacgcatt ttcgtaatc 19 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gtttagaaat gtttgtttat ggggtg 26 <210> 21 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gtaaaacgac ggccag 16 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaaacagcta tgaccatg 18

Claims (6)

An SSR primer set of SEQ ID NOS: 1 and 2; An SSR primer set of SEQ ID NOS: 3 and 4; An SSR primer set of SEQ ID NOS: 5 and 6; An SSR primer set of SEQ ID NOS: 7 and 8; An SSR primer set of SEQ ID NOS: 9 and 10; SSR primer sets of SEQ ID NOS: 11 and 12; An SSR primer set of SEQ ID NOS: 13 and 14; An SSR primer set of SEQ ID NOS: 15 and 16; An SSR primer set of SEQ ID NOS: 17 and 18; And an SSR primer set of SEQ ID NOs: 19 and 20, respectively. delete An SSR primer set according to claim 1; And a reagent for carrying out an amplification reaction. 4. The kit according to claim 3, wherein the reagent for carrying out the amplification reaction comprises a DNA polymerase, dNTPs and a buffer. Separating the genomic DNA from the chromosome;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the SSR primer set according to claim 1; And
And detecting the amplification product.
6. The method of claim 5, wherein the detection of the amplification product is performed by gel electrophoresis, capillary electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement or phosphorescence measurement.
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KR101912192B1 (en) * 2017-10-13 2018-10-26 충북대학교 산학협력단 Molecular marker and primer set for discriminating Platycodon grandiflorum cultivar and uses thereof
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