CN102649955A - Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker - Google Patents
Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker Download PDFInfo
- Publication number
- CN102649955A CN102649955A CN2012100979126A CN201210097912A CN102649955A CN 102649955 A CN102649955 A CN 102649955A CN 2012100979126 A CN2012100979126 A CN 2012100979126A CN 201210097912 A CN201210097912 A CN 201210097912A CN 102649955 A CN102649955 A CN 102649955A
- Authority
- CN
- China
- Prior art keywords
- microsatellite
- dendrobium
- dna
- gag
- herba dendrobii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 34
- 102000053602 DNA Human genes 0.000 title claims abstract description 8
- 108020004414 DNA Proteins 0.000 title abstract description 18
- 241001678082 Dendrobium huoshanense Species 0.000 title abstract description 8
- 239000003147 molecular marker Substances 0.000 title abstract description 5
- 239000002773 nucleotide Substances 0.000 claims description 25
- 125000003729 nucleotide group Chemical group 0.000 claims description 25
- 239000003550 marker Substances 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 abstract description 7
- 241000026010 Dendrobium candidum Species 0.000 abstract description 4
- 108091081062 Repeated sequence (DNA) Proteins 0.000 abstract description 2
- 241001264163 Dendrobium aduncum Species 0.000 abstract 1
- 244000269590 Dendrobium chrysanthum Species 0.000 abstract 1
- 241001264169 Dendrobium moniliforme Species 0.000 abstract 1
- 238000002591 computed tomography Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 14
- 239000000975 dye Substances 0.000 description 13
- 229910052709 silver Inorganic materials 0.000 description 13
- 239000004332 silver Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 10
- 241001523681 Dendrobium Species 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 238000000137 annealing Methods 0.000 description 8
- 239000012634 fragment Substances 0.000 description 6
- 230000005291 magnetic effect Effects 0.000 description 6
- 230000009182 swimming Effects 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 240000004638 Dendrobium nobile Species 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 241000768494 Polymorphum Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229930003756 Vitamin B7 Natural products 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011735 vitamin B7 Substances 0.000 description 2
- 235000011912 vitamin B7 Nutrition 0.000 description 2
- 241000208140 Acer Species 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- -1 iginates Species 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000005184 irreversible process Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000000627 locus coeruleus Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Images
Abstract
The invention discloses a dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker, which is characterized in that a dendrobium huoshanense microsatellite computer tomography (CT)n enriched library is established, positive clones of a microsatellite sequence are screened and sequenced, 51 clones containing the microsatellite repeated sequence are obtained and are screened to obtain 11 microsatellite molecular markers with high polymorphism Hs39, Hs40, Hs41, Hs42, Hs44, Hs47, Hs48, Hs50, Hs54, Hs56 and Hs57, wherein Hs42 can well discriminate dendrobium huoshanense from dendrobium candidum, dendrobium crispulum Kimura et migo, flat dendrobium chrysanthum Wall and dendrobium aduncum. The microsatellite molecular marker can be used for researching genetic diversity of the dendrobium huoshanense, can identity counterfeit products of the dendrobium huoshanense, has the advantages of accuracy, rapidness and good repeatability, and provides a novel tool for eliminating the false and retaining the true dendrobium huoshanense.
Description
Technical field
The present invention relates to molecular marking technique, be specifically related to a kind of Herba Dendrobii micro-satellite molecule genetic marker.
Background technology
Little satellite (Microsatellite) be called again the repetition of short polyphone (Short Tandem Repeats, STRs), simple repeated sequence (Simple Sequence Repeat, SSRs), SSLP (SSLP).Be meant that is dispersed throughout (being called core sequence, being generally 1～6bps) the simple series connection repetition for repeating unit's composition by several nucleotide pairs in the biological gene group.Its multiplicity is an alterable height between the different genotype of same species; But this section multiple two ends are conservative relatively single-copy sequences; Design primer in view of the above, can analyze the variation of core sequence multiplicity, between different genotype, detect polymorphic through round pcr.According to the formation of repeating unit, the microsatellite DNA sequence is divided into 3 types: single type (pure), compound (compound) and discontinuous form (interrupted).For example:
Single type: ATATATATATATATATATATATAT
Compound: ATATATCACACACACACACAC
Discontinuous form: ATATATCA ATATATCA ATATATA
As a kind of molecule marker, microsatellite DNA has following characteristics: be distributed widely in the Eukaryotic genome, 1 microsatellite locus is just arranged in every according to estimates 6kb length; The multiplicity of core sequence is the height variability between individuality, the polymorphism information capacity is high: be codominance, follow the heredity of broad-mouthed receptacle for holding liquid Dare rule; Select neutrality or the like.
Based on above characteristics, microsatellite DNA is widely used in the research of analysis, assistant breeding, structure finger printing, genetic diversity and aspects such as spore and sibship of structure, the assignment of genes gene mapping, cultivar identification and quality saving, the quantitative character gene of plant genetic collection of illustrative plates.
Herba Dendrobii (Dendrobium huoshanense.C.Z.Tang et S.J.Cheng) has another name called a meter dry measure used in former times, and the orchid family Dendrobium plant originates in the Huoshan, is the superfine product in the medicinal dendrobium, and is described as " the celestial grass of China ".As threatened plant, Herba Dendrobii is existing, and oneself is listed in country-level focused protection plant by China.
Because the stem of noble dendrobium is a kind of epiphyte, ecotope is required relatively strictness, natural propagation frequency extremely low (＜5%) adds long-term collection, wild plant is endangered.Herba Dendrobii has won fame both at home and abroad because of it has good quality, and Herba Dendrobii is merely nominal on the market for many years, has price but no buyers, and resource is near exhausted.Now on the domestic and international market; All some maple bucket products titled with " Herba Dendrobii ", " Huo Dou " or titles such as " wild gold is dry measures used in former times suddenly "; The real Herba Dendrobii of all non-usefulness processes, and majority is the counterfeit merchandise of Dendrobium Moniliforme, Herba Dendrobii, flat HERBA DENDROBII and hooked dendrobium.
Summary of the invention
The technical problem that the present invention solves: the microsatellite DNA molecule marker of separating clone Herba Dendrobii; Set up Herba Dendrobii microsatellite DNA technical system and carry out the Herba Dendrobii genetic diversity with these molecule markers; The research of Dendrobium plant genetic relation, and carry out the Herba Dendrobii Idioplasm identification.
The technical scheme of technical solution problem of the present invention: Herba Dendrobii microsatellite DNA molecule marker, comprise the structure of the little satellite of Herba Dendrobii (CT) n enriched library, the screening of microsatellite sequence positive colony and order-checking obtain 51 clones that contain little satellite Tumor-necrosis factor glycoproteins.Obtain 11 microsatellite molecular marker Hs39, Hs40, Hs41, Hs42, Hs44, Hs47, Hs48, Hs50, Hs54, Hs56, Hs57 that polymorphum is high; Hs39 is 600 Nucleotide, and Hs40 is 694 Nucleotide, and Hs41 is 503 Nucleotide; Hs42 is 638 Nucleotide, and Hs44 is 437 Nucleotide, and Hs47 is 704 Nucleotide; Hs48 is 558 Nucleotide, and Hs50 is 540 Nucleotide, and Hs54 is 458 Nucleotide; Hs56 is 509 Nucleotide, and Hs57 is 541 Nucleotide.
Description of drawings
Fig. 1: the silver of the DNA of 24 Herba Dendrobii of primer amplified in Hs39 site dyes PAGE figure.
Fig. 2: the silver of the DNA of 20 Herba Dendrobii of primer amplified in Hs40 site dyes PAGE figure.
Fig. 3: the silver of the DNA of 25 Herba Dendrobii of primer amplified in Hs41 site dyes PAGE figure.
Fig. 4: the silver of the DNA of 28 Herba Dendrobii of primer amplified in Hs42 site dyes PAGE figure.
Fig. 5: the silver of the DNA of 24 Herba Dendrobii of primer amplified in Hs44 site dyes PAGE figure.
Fig. 6: the silver of the DNA of 24 Herba Dendrobii of primer amplified in Hs47 site dyes PAGE figure.
Fig. 7: the silver of the DNA of 24 Herba Dendrobii of primer amplified in Hs48 site dyes PAGE figure.
Fig. 8: the silver of the DNA of 31 Herba Dendrobii of primer amplified in Hs50 site dyes PAGE figure.
Fig. 9: the silver of the DNA of 29 Herba Dendrobii of primer amplified in Hs54 site dyes PAGE figure.
Figure 10: the silver of the DNA of 24 Herba Dendrobii of primer amplified in Hs56 site dyes PAGE figure.
Figure 11: the silver of the DNA of 30 Herba Dendrobii of primer amplified in Hs57 site dyes PAGE figure.
Figure 12: 1 Herba Dendrobii (the 1st swimming lane of the primer amplified in Hs42 site; Fragment length is about 331bp), 2 the Henan stem of noble dendrobium (2-3 swimming lanes; Fragment length is about 331bp), 7 Herba Dendrobiis (4-10 swimming lane, fragment length 230-275bp), 7 Dendrobium Moniliformes (the 11-17 swimming lane does not have the product band), 4 flat HERBA DENDROBII (18-21 swimming lanes; Do not have the product band) and the silver of the DNA of 3 hooked dendrobiums (the 22-24 swimming lane does not have the product band) dye PAGE figure.
Embodiment
Below in conjunction with embodiment the present invention is done detailed explanation.
Embodiment 1:
The structure of Herba Dendrobii microsatellite DNA enriched library
1) genomic extraction and enzyme are cut:
CTAB method (Doyle J.DNA protocols for plants-CTAB total DNA isolation [A] .In Hewitt GM with Doyle J introduction; Johnston A (eds.); Molecular Techniques in Taxonomy [M] .Berlin:Springer; 1991,283-293) extract genome.Cut genome with restriction enzyme Sau 3AI (purchasing company) enzyme in TaKaRa.Behind 2% the agarose gel electrophoresis, under UV-light, downcut the dna fragmentation of 400-900bp.Reclaiming test kit (purchasing the company in Axygen) with glue reclaims.
2) add joint:
The product that glue is reclaimed behind the purifying adds joint:
oligo?A(5-GGCCAGAGACCCCAAGCTTCG-3)
oligo?B(5-PO4-GATCCGAAGCTTGGGGTCTCTGGCC-3)，
Under T4 DNA Ligase (purchasing company) effect, spend the night in 16 ℃ of water-baths connections in Fermentas.
3) the PCR detection tabs connects:
With oligo A is primer; To connect product is that template is carried out pcr amplification; (50 μ l reaction systems are following: 25 μ l GoTaq
Green Master Mix (purchasing the company in Promega); 5 μ l primer oligoA, 5 μ l connect product, add water and mend to 50 μ l.The PCR response procedures: 94 ℃ of preparatory sex change 5 minutes, 94 ℃ of sex change 50 seconds, 56 ℃ of annealing one minute, 72 ℃ were extended 2 minutes, sex change, annealing, three step cycle of extension 25 times, last 72 ℃ were fully extended 10 minutes.After agarose gel electrophoresis 2% detected, the PCR product carried out purifying with purification kit (purchasing the company in Axygen).
4) enrichment with magnetic bead:
Magnetic bead Streptavidin MagneSphere
the Paramagnetic Particles (PMPs) (purchasing the company in Promega) that encapsulates with the affinity element combines the vitamin H on little satellite probe; Since the combination of avidin and vitamin H be one firm; Irreversible process; Therefore simultaneously at the two bonded; Magnetic bead has also adsorbed " little satellite probe and comprise the genomic dna strand of little satellite "; Remove the genomic fragment that does not contain little satellite through wash-out repeatedly at the same time; Open combining of little satellite probe and DNA chain through high-temperature denatured at last, contain the pulsating purpose of little satellite thereby reach enrichment.Hybridization solution is added the magnetic bead of having handled well, and room temperature left standstill 30 minutes.Centrifuge tube is placed on the magnetic force frame, inhales and remove supernatant, use 0.5 * SSC successively, 6 * SSC at room temperature gives a baby a bath on the third day after its birth inferior, uses 6 * SSC again, 2 * SSC, and 1 * SSC washes twice under 60 ℃, use at last under 0.1 * SSC room temperature and wash once.Add the TE damping fluid, 95 ℃ of sex change are 10 minutes on the PCR appearance, collect supernatant.Repeat twice.
5) PCR enrichment:
With the enrichment with magnetic bead product is template; Oligo A is that primer carries out pcr amplification; 25 μ l reaction systems are following; 12.5 μ l GoTaq
Green Master Mix (purchasing company) in Promega; 1.5 μ l primer oligo A, the DNA after the 2.5 μ l enrichments adds water and mends to 25 μ l.The PCR response procedures is following: 94 ℃ of preparatory sex change 3 minutes, and 94 ℃ of sex change 35 seconds, 60 ℃ of annealing property 35 seconds, 72 ℃ were extended 1 minute, sex change, annealing, three step cycle of extension 25 times, last 72 ℃ were fully extended 12 minutes.The PCR product carries out purifying with purification kit (purchasing the company in Axygen) after 2% agarose gel electrophoresis detection.
6) preparation of competent cell:
The single bacterium colony of E.coli DH-5 is inoculated in the LB substratum that does not contain Amp of 5ml on the picking flat board, and 37 ℃ of shaking culture are spent the night.Get the 1ml nutrient solution in the LB of 50ml liquid nutrient medium, 37 ℃ of shaking culture, 2～4h is to logarithmic phase.Survey OD (0.2～0.6).The nutrient solution branch is filled in the EP pipe (every pipe 1.5ml bacterium liquid) water-bath 10～15min.4 ℃ centrifugal, 4000rpm, 10min (earlier cooling after centrifugal again) abandons supernatant.Abandon three pipe and pipes behind the supernatant, and ice bath 30min (first pipe adds 0.1mol/L, and 1ml is the CaCl2 of precooling in the ice in advance, mixing, sucking-off to the second pipe is so with three pipes and a pipe).Take out the back gradient centrifugation, 4 ℃, 4000rpm, 5～8min.Abandon supernatant.Add 200 μ l precooling glycerine (15%)/CaCl2 (0.1M) mixing ,-80 ℃ of preservations.
7) connect conversion:
The purified product of getting after the PCR enrichment connects into TA cloning vector (pMD19-T Simple Vector) (purchasing the company in TaKaRa), and 16 ℃ of water-baths connect 2-3 hour.Take out frozen pipe, on ice recovery 10min; The connection product of 10 μ l is added in the 200 μ l competent cells mixing, ice bath 30min; 42 ℃ of water-bath heat-shocked 90s; Ice bath 2min adds 600 μ l LB substratum, 37 ℃ of shaking culture 1.5h; Get 70 μ l nutrient solutions, splash on the LB/Amp/X-gal/IPTG flat board and coating even (X-Gal needs lucifuge); 37 ℃ of forwards of flat board are placed cultivation 0.5h, in 37 ℃ of baking ovens, be inverted overnight cultures then.Through the cultivation of 12h, treat that the bacterium colony on the flat board is of moderate size, take out flat board and locus coeruleus is fully manifested in 4 ℃ of placements.With white mono-clonal bacterium colony on the toothpick picking flat board of the bacterium of going out, place 96 deep-well plates (deep-well plates has added the LB/Amp substratum that contains the Amp resistance of 400 μ l in advance).After the single bacterium colony picking of white finishes, with non-setting adhesive orifice plate is built the back in 37 ℃, 225rpm shaking culture 4h.
8) contain the screening of the positive colony of microsatellite sequence:
To connect the bacterium liquid that transforms gained is template, utilizes primer oligo A and primer (CT) 12 to carry out pcr amplification.15 μ l PCR reaction systems comprise: rTaq enzyme 0.375U (available from TaKaRa company), 10 * Buffer, 1.5 μ l, dNTP 0.2 μ M, MgCl2 1.5 μ M, primer 0.4 μ M (oligo A, (CT) 12), water, template.Response procedures: 94 ℃ of preparatory sex change 3 minutes, 94 ℃ of sex change 50 seconds, 56 ℃ of annealing 35 seconds, 72 ℃ were extended 1 minute, sex change, annealing, three step cycle of extension 30 times.Last 72 ℃ were fully extended 12 minutes.1.5% agarose gel electrophoresis detects.The clone that 2 or 3 bands are arranged in detecting for the PCR product then possibly contain the insertion fragment of little satellite in these clones.
9) positive colony order-checking and design of primers:
Choose the amplification fragment length and insert segmental positive colony bacterium liquid, send the order-checking of the biological (Shanghai) Co., Ltd. of living worker, obtain 51 sequences that contain microsatellite DNA altogether at little satellite that possibly contain of 300-900bp.Utilize software Primer Premier 5.0 sequences to design primer according to the microsatellite DNA two ends.
10) screening has the primer of polymorphum:
Extract the genome of Herba Dendrobii with above-mentioned CTAB method.With primer (table 1) the amplification gene group that designs.Response procedures: 94 ℃ of preparatory sex change 4 minutes, 94 ℃ of sex change 45 seconds, corresponding annealing temperature 30 seconds, 72 ℃ were extended 35 seconds, sex change, annealing, three step cycle of extension 35 times.Last 72 ℃ were fully extended 8 minutes.1.5% agarose gel electrophoresis detects.The PCR product detects with the PAGE electrophoresis.Obtain 11 microsatellite markers that polymorphum is arranged altogether.Hs39 is 600 Nucleotide, and Hs40 is 694 Nucleotide, and Hs41 is 503 Nucleotide; Hs42 is 638 Nucleotide, and Hs44 is 437 Nucleotide, and Hs47 is 704 Nucleotide; Hs48 is 558 Nucleotide, and Hs50 is 540 Nucleotide, and Hs54 is 458 Nucleotide; Hs56 is 509 Nucleotide, and Hs57 is 541 Nucleotide.
11) interpretation of result:
Use Cervus3.0 computed in software expectation heterozygosity and observe heterozygosity.Use the value of Genepop3.4 computed in software Hardy-Weinberg and linkage disequilibrium, describe the characteristic of 11 Herba Dendrobii microsatellite DNA polymorphic sites with this.Can be found out that by accompanying drawing 1-11 the length of 11 microsatellite sequences of the present invention variety all occurred in 20-31 the Herba Dendrobii that supplies examination, band is all in 210-400 Nucleotide scope.This shows that these 11 microsatellite markers of the present invention can be used to study the genetic diversity of Herba Dendrobii, between the Dendrobium plant species and variation between population and evolutionary relationship, have very high repeatability, is a kind of reliable and effective molecule marker.
12) application of Herba Dendrobii microsatellite molecular marker in other several kinds of Dendrobiums
Dye steps such as detection through PCR method with these 11 pairs of micro-satellite primers amplification Dendrobium other 4 kinds of stem of noble dendrobium genomic dnas, electrophoretic separation and silver of Herba Dendrobii; The result finds that wherein the HS-42 primer does not all amplify band at Dendrobium Moniliforme and Herba Dendrobii in the individualities such as flat HERBA DENDROBII and hooked dendrobium in this scope.See accompanying drawing 12.
Table 1 primer property list:
F representes upstream primer in the table 1, and R representes downstream primer.
Claims (2)
1. Herba Dendrobii microsatellite DNA molecule marker, it is characterized in that: the label of said microsatellite marker is respectively: Hs39, Hs40, Hs41, Hs42, Hs44, Hs47, Hs48, Hs50, Hs54, Hs56, Hs57;
Its nucleotide sequence is respectively:
Hs39：
gatcagactt?ccttccaact?gccaaccacc?gcctgacttt?cacctgacca?ccactcgata 60
gtcaattgtc?gttagatcac?cgcttgtcca?tcaaacttct?gcccttaagc?tatcggactt 120
ccgcctgact?gctgccagat?ttctactcaa?ctctccaaac?tattataatt?ccacaaccta 180
taagttaaag?gacgggtcaa?cctttgtaat?taactcacat?ttagtgaaat?cttttcatct 240
aggagaggcc?tcatggtttt?tcccactatg?ggttttccac?ataaaaaaat?ctgttgtttt 300
acttctctct?ctctctctct?ctctctctct?ctctctctct?ctctctctct?ctctcaaatt 360
acatgtttgt?atgttggata?tccgtgtatt?gctaagatga?aaaatttaca?cgctcaacca 420
ttgcgaatat?tctaacttgc?aacaatgaca?aaagtcaaac?aaagtcaaac?ataaaatgtg 480
cctttcccta?atagtaatca?tataacaaca?atgactcata?atctttttac?tattatggct 540
ccaagaatat?tgttctaaaa?tattttcata?atcaactaca?tgcaagctgc?acctcaccat 600
Hs40：
gatcagagtt?caacgccagat?tagaaaagtc?cttgtagagg?ccatcacccc?ggcggagggc 60
gcggcatccc?cacaggagaa?ggtcaggttt?gacaagtaat?gtcaaaagat?gatgaaatta 120
ttacgtgcaa?gcacaggacg?tcgtccagag?gcgaattaga?acgcgccacg?ttaccattta 180
ctatataaca?gagtgatgtg?aagcatcttg?attcgcctct?gaactgtccg?gtaactgcac 240
attataattt?ctcccaaaga?tgacatagga?cgaggtgtgg?aggcagagca?gaaaggcatc 300
agatagcatc?atggagataa?tggtggagga?agaagaggat?tagaggcata?gggagagtag 360
aggaggaata?gaggtttctt?gttgggggga?atggatagat?ttgtcattaa?gattgaggcg 420
atgttgtcag?gcaaagactt?tgtacagtca?attatttttc?cacaactcag?cgtctatcat 480
tatacttgta?ccaccttcat?tttgcaacat?taaaaatgta?ttctctctct?ctctctctct 540
ctctctctct?ctctctctct?catacgcatc?tctcaaaatt?cacatatact?tagagagatg 600
ttcaacctga?catatttata?caaatacaag?tgttagaatg?aattgaatac?cacaaattcc 660
agcaagtttg?aagcagccac?taactgattt?tatt 694
Hs41：
gatcaacctc?tggtatcact?tcgagctcta?ttgttcttcc?attaatcttc?ctcttcagat 60
agtatgctac?cagttcttca?tctgtcggat?gaaagcgaaa?gccaggcggc?agagttacag 120
gcgccatttt?tgtactgtta?aatctcttat?caatcctctt?ttgacttttc?tccctctaag 180
ctctgcttca?aaaaaaccat?taaaactatc?tcttgatttg?aacatagtca?gacagaaaat 240
aacagaagga?gagagagaga?gcataccaaa?tctcacataa?gagagagaga?gagagagaga 300
gagagagaga?gagagagaga?gagagagaga?gggcggcggt?ttaagaacct?ctgtagcttt 360
ataaaggcaa?gaaggttgct?ttcctcgcaa?tcgagccttt?actctctctc?tctactttgt 420
tttgctttgg?ctttatatcc?aaatattctg?agattattgt?cctttccttc?atgagccatt 480
ggtccacgtg?gatggacgcc?att 503
Hs42：
gatcgccaca?aaacatcagc?cgcaaataca?tgaagtatgg?aagctcaacg?agattccatc 60
tatccatcaa?ctaagcacac?acagagagag?agagagagag?agagagagag?agagagagag 120
agagagacct?cctgttggaa?ctctcgttgg?ccttgaatac?tatgatgatg?aaacttcttt 180
attgccacac?ttctgttcaa?catttccccc?ttatatacac?atcccagtcc?tccctgtcct 240
atcataaagg?catctgagaa?accaaaggct?gcagtttgca?agtctgatag?tggaaactcc 300
ttgaaatcaa?gagaatcgct?tcctaagtca?ataaacttat?tgcatatttt?ggattttctg 360
cgatggttgc?atctcaacct?ttcaatctgc?tgaacatact?cgtctctctg?atgttggatt 420
ttctgatttt?caagcttaag?cgctgctata?gaagacatgg?ttgaatccaa?ttcaacagtt 480
gccttatctt?tacgatgaaa?catttcacga?acacgaacat?caaggatagc?tacattttcc 540
attgtacttt?gaagttcttt?catgcttttt?tctatttggt?tcactagctc?gttctgcttc 600
aacttggtaa?ctactatgag?ctcttcaagc?tcttctct 638
Hs44：
gatcgggcta?caacattact?tggatttact?acatttttgg?atgtgttgac?tactaactag 60
ccttatcggc?cccacctcga?cgtttctctc?tcaaacgaag?caatggttcg?ctcatataac 120
tcccacttat?ggcataaaac?ctccccgccc?tctctctctc?tctctctctc?tctctctctc 180
tccattcgac?tctcatgtag?gaactgccca?ctagcccctc?ttgcttttct?ccctcattcg 240
catgcgagga?gtgctcgact?ctacctattc?gttcgcatga?ccttgtacat?gagaccctag 300
ccttgtcctc?tctcgtttta?ttgaccgtgc?acccaagtaa?cttagatgcg?attgtccgct 360
cacgtctccg?tctcttcctc?cacgtgtccc?aaactcagct?acgcatgatc?cctaacctga 420
aacccctcgt?tctcttt 437
Hs47：
gatcacaaat?gctgaaacat?cctagcaaat?tatacagttc?atttcaattt?gtgtggacaa 60
gcaaaataac?ttaatttttt?tttccctcca?agagtgggcc?ggttgagaaa?tcaagtagag 120
gcaccaatag?ctgcatccat?ctctgtagag?ttctttatca?agttcttgct?tgaaggcaac 180
ccagcaacga?cattcgcttc?aatgccacat?tcatttgtcc?ctcttaaaat?cttgaaatag 240
ccatcctgaa?gagaaaacat?aaaaaatttt?gatagacaag?tgttaccaaa?actaaaccat 300
acacaaattt?tgccaagtat?tattttgaaa?catacatcac?cccaatttct?attccactgg 360
ttagcaagta?gctgcaaaac?aaatataaga?gataacaaaa?aaattgtatc?agaattggat 420
ttaatctgta?aaatgtaaaa?gaaatgtgtg?tgtgtgtgtg?tgtgagagag?agagagagag 480
agaactcaaa?cccaataatc?ttctccttgc?tcactagtgc?cccatccaat?taacttaaca 540
gcatgtccac?ctaagacatc?acctgtgata?tatctgtaaa?ctccagattt?gtagtgggca 600
aaatcctgtt?tataagaaaa?taaaaatcag?gtgacctgct?tgattatatt?gtaggattca 660
ttataataaa?gtaaaattaa?acaatgaaaa?tttctatctt?ttgc 704
Hs48：
atcagcaaga?gaagaattat?agatatatag?ccgatataaa?taaagctcat?gttgggatgt 60
tcaggagtag?tccatttgtt?gagcacctaa?accaacttca?atttcctttt?gcactttttt 120
atgaaccctt?tatttggcat?cctcaaggga?aaggccgtaa?tgatgataat?gagaatccag 180
actctgatgg?ttaggagtct?ttacatttct?tcgcttgtat?gtgtatgtgt?gtgtgtgtgt 240
gtgtgtgtgt?gtgtgtgtgt?gtttgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt 300
gtgagagaga?gagagagaga?gagagagaga?gagagagaga?gagagagaga?gagagagaga 360
gagtgtgtgt?gtgtgtgtgt?gtgaagataa?actgtataac?ttctatgagc?cacgccgact 420
taataatcgc?aacgattttg?gctataacta?tctcacgtca?cccagcactg?tagcggagcc 480
aacattacaa?ctctgaggtg?ctagttcata?ttccacagcc?gcaatgccac?cctgtcaagt 540
gccacgatac?tcaagtat 558
Hs50：
gagcccaagc?aaacctcaag?aactcctaaa?tttagcaaat?attcaacata?accctaacca 60
taggataaga?ataactatta?aatgcaaata?atacaaagcc?aaaagttcaa?acagcccttc 120
agattcaaat?ctcctgctca?tctctctctc?tctctctctc?tctctctctc?tctctctctc 180
tctctctctc?tctctctgaa?tgcatggacc?ccaaccaaag?gggggtatct?gattggtttt 240
gcccaggttt?tcgacttttg?tagtattagg?tcggtccttt?cgggatcgac?caaaccagcc 300
cctcctagac?ctgtctaaca?tactcccctc?ccaaagaatc?atcttgtcct?caaggtaaaa 360
ggacgaaaat?tgatgcttca?tcctctcata?tgattcccaa?gtaatatcat?actcaagtag 420
attttttcac?ttaatcaaca?cttcttacgt?ctccctttcg?tactcctacc?aactctagtg 480
gttccaaaat?cacctctaga?ttctctgtca?acatcactga?taactctaca?attgaggtgt 540
Hs54：
gatcaaagtc?tcattggcct?tcccagccaa?cagccactct?tcttcaagct?ccggcctccc 60
gcatgctgat?aatggtggac?gttctctcca?agggcaggga?atcatgctat?aaggtatcag 120
ttcccagctt?ttctctctct?ctctcagtct?ctctctctct?ctctctctct?ctctctctct 180
ctccctctct?ctctctctct?ctctctctct?ctctctctca?tgatttctcg?aatttcgatg 240
gttaggctcg?gaacgccttc?tattcttgct?tggagaagga?ggcggcgacg?aagcattatt 300
aaagtaacaa?tatcaacact?ccgcctgcgg?ccaaacctac?cacaagcgcc?tacgataaac 360
tatttgacgg?cgacagagac?ggcggcgacg?gcgacggtgg?cgacggcggc?gaacggggag 420
aagcatggaa?aggaattctt?tctcccatag?aacccgat 458
Hs56：
gatcagcaga?agcaagaata?aaagtcgaat?gctttcattg?cagccagctt?ttccttgggg 60
aagggggact?taggaagaga?aagagagaga?gagaaagaga?gagagagaga?gagagagaga 120
gagagagaac?tggaaattga?tgcatgagct?caagtccgtt?aactaagaag?cattctgggg 180
ctcatctaat?cttatataac?aatcaggttg?gccatcatac?attcaaactc?agcaatctgc 240
acataaaata?tctgcagaag?aagccacatt?aaagcaacaa?aatttagttt?gaagccattg 300
tcgtctgtgg?ccagggatga?attattaagt?gcggagatca?tacgtagaga?atctggttta 360
tctatgtgaa?ccgtaaaaat?gtagcaaact?gcgttttagc?tgttcaccac?tacgctttta 420
ctattcacaa?agacaaatct?gaatgattct?ttgtatttga?cctctgaacg?taataatctc 480
tcttgaccat?gaccggtaga?agactcaag 509
Hs57：
gatcatagtt?actgtttacc?acttacattc?taagtgtgtc?attctaccat?tcctcttttg 60
tctatttttt?ctcaaaagca?atcaactaat?atcttcccca?atcccatatc?tcatgatgat 120
ttaaagaact?atatattaga?ttcaagtcaa?catcttatgc?ttttccttga?atttgcatgg 180
agttctcata?tttaaatatt?gataagaatt?atcattattc?attgcaaaat?attatctcat 240
tattggggat?atttttttat?tgttctcttc?atatgcctat?atatgcttta?tacaaaacag 300
acaacaaatc?tctctctctc?tctctctctc?tctctctctc?tctctctctc?tcaccttccc 360
atagttgaac?aataaaaaat?tatctttatt?tttttaatag?gcaaacaggg?atgtgacaag 420
gatggagata?ttttgttctc?ttgttcatct?gcttgaacca?actactgcac?aatctatgct 480
taacagcaaa?atgccccaaa?aaagattcaa?cctgccttgt?gttcctgcaa?ccggcgcatc 540
t 541。
2. a kind of Herba Dendrobii microsatellite DNA molecule marker according to claim 1 is characterized in that:
The primer sequence in described Hs39, Hs40, Hs41, Hs42, Hs44, Hs47, Hs48, Hs50, Hs54, Hs56,11 sites of Hs57 is:
Hs39-F CAC?CGC?TTG?TCC?ATC?AAA?CTT?C
Hs39-R CGC?AAT?GGT?TGA?GCG?TGT?AAA?T
Hs40-F AAG?ATT?GAG?GCG?ATG?TTG?T
Hs40-R ACT?TGC?TGG?AAT?TTG?TGG?T
Hs41-F CAG?GCG?GCA?GAG?TTA?CAG
Hs41-R GCC?AAA?GCA?AAA?CAA?AGT?AGA?G
Hs42-F TCC?ATC?TAT?CCA?TCA?ACT?AAG?C
Hs42-R GAC?GAG?TAT?GTT?CAG?CAG?ATT?G
Hs44-F ATG?GCA?TAA?AAC?CTC?CCC?G
Hs44-R GAG?CGG?ACA?ATC?GCA?TCT?AAG?T
Hs47-F TGA?AAT?AGC?CAT?CCT?GAA?GAG
Hs47-R GGA?GAA?GAT?TAT?TGG?GTT?TGA?GT
Hs48-F AGG?GAA?AGG?CCG?TAA?TGA?TGA?T
Hs48-R AGA?GTT?GTA?ATG?TTG?GCT?CCG?CTA
Hs50-F CCA?AGC?AAA?CCT?CAA?GAA?CTC
Hs50-R CGA?AAG?GAC?CGA?CCT?AAT?ACT
Hs54-F CAG?CCA?ACA?GCC?ACT?CTT?CTT?C
Hs54-R CGC?CGT?CAA?ATA?GTT?TAT?CGT?AG
Hs56-F GCA?GAA?GCA?AGA?ATA?AAA?GTC?G
Hs56-R ATA?AGA?TTA?GAT?GAG?CCC?CAG?A
Hs57-F CCT?TGA?ATT?TGC?ATG?GAG?TT
Hs57-R TTT?TGG?GGC?ATT?TTG?CTG?TT。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210097912 CN102649955B (en) | 2012-04-06 | 2012-04-06 | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210097912 CN102649955B (en) | 2012-04-06 | 2012-04-06 | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102649955A true CN102649955A (en) | 2012-08-29 |
| CN102649955B CN102649955B (en) | 2013-08-14 |
Family
ID=46692082
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210097912 Expired - Fee Related CN102649955B (en) | 2012-04-06 | 2012-04-06 | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102649955B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468709A (en) * | 2013-07-16 | 2013-12-25 | 南京师范大学 | Chloroplast genome of dendrobium huoshanense and germplasm identification method |
| CN103571949A (en) * | 2013-10-12 | 2014-02-12 | 杭州师范大学 | Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale |
| CN104498482A (en) * | 2014-11-28 | 2015-04-08 | 安徽师范大学 | Pteroceltis tatarinowii microsatellite DNA molecular marker and isolation and screening method thereof |
| CN106119409A (en) * | 2016-09-23 | 2016-11-16 | 皖西学院 | A kind of Chloroplast Simple primer method to identifying Herba Dendrobii |
| CN110484653A (en) * | 2019-09-27 | 2019-11-22 | 广东省生物工程研究所（广州甘蔗糖业研究所） | The identification method and kit of SSR molecular marker primer, mesona kind |
| CN110878376A (en) * | 2019-12-31 | 2020-03-13 | 安徽师范大学 | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof |
| CN113981128A (en) * | 2021-11-22 | 2022-01-28 | 中国热带农业科学院热带作物品种资源研究所 | EST-SSR marker developed based on dendrobium officinale transcriptome sequence and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451163A (en) * | 2008-12-22 | 2009-06-10 | 陈乃富 | Construction and identification method of molecular marking fingerprint of Dendrobium huoshanense and similitude species thereof |
| WO2009102880A2 (en) * | 2008-02-12 | 2009-08-20 | Academia Sinica | Structure and bioactivity of the polysaccharides and oligomers in medicinal plant dendrobium huoshanense |
| CN102168084A (en) * | 2011-04-25 | 2011-08-31 | 安徽师范大学 | Dendrohuoshanense microsatellite deoxyribonucleic acid (DNA) molecule mark |
| CN102370891A (en) * | 2010-08-18 | 2012-03-14 | 浙江中医药大学 | Method for authenticating dendrobium officinale by using HPLC fingerprint |
-
2012
- 2012-04-06 CN CN 201210097912 patent/CN102649955B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009102880A2 (en) * | 2008-02-12 | 2009-08-20 | Academia Sinica | Structure and bioactivity of the polysaccharides and oligomers in medicinal plant dendrobium huoshanense |
| CN101451163A (en) * | 2008-12-22 | 2009-06-10 | 陈乃富 | Construction and identification method of molecular marking fingerprint of Dendrobium huoshanense and similitude species thereof |
| CN102370891A (en) * | 2010-08-18 | 2012-03-14 | 浙江中医药大学 | Method for authenticating dendrobium officinale by using HPLC fingerprint |
| CN102168084A (en) * | 2011-04-25 | 2011-08-31 | 安徽师范大学 | Dendrohuoshanense microsatellite deoxyribonucleic acid (DNA) molecule mark |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468709B (en) * | 2013-07-16 | 2015-03-04 | 南京师范大学 | Chloroplast genome of dendrobium huoshanense and germplasm identification method |
| CN103468709A (en) * | 2013-07-16 | 2013-12-25 | 南京师范大学 | Chloroplast genome of dendrobium huoshanense and germplasm identification method |
| CN103571949A (en) * | 2013-10-12 | 2014-02-12 | 杭州师范大学 | Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale |
| CN104498482B (en) * | 2014-11-28 | 2018-04-13 | 安徽师范大学 | Wingceltis Microsatellite DNA molecular marker and separating screening method |
| CN104498482A (en) * | 2014-11-28 | 2015-04-08 | 安徽师范大学 | Pteroceltis tatarinowii microsatellite DNA molecular marker and isolation and screening method thereof |
| CN106119409B (en) * | 2016-09-23 | 2019-05-28 | 皖西学院 | A method of Dendrobidium huoshanness is identified with Chloroplast Simple primer pair |
| CN106119409A (en) * | 2016-09-23 | 2016-11-16 | 皖西学院 | A kind of Chloroplast Simple primer method to identifying Herba Dendrobii |
| CN110484653A (en) * | 2019-09-27 | 2019-11-22 | 广东省生物工程研究所（广州甘蔗糖业研究所） | The identification method and kit of SSR molecular marker primer, mesona kind |
| CN110484653B (en) * | 2019-09-27 | 2023-05-19 | 广东省科学院南繁种业研究所 | SSR molecular marker primer, method for identifying mesona chinensis benth variety and kit |
| CN110878376A (en) * | 2019-12-31 | 2020-03-13 | 安徽师范大学 | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof |
| CN110878376B (en) * | 2019-12-31 | 2022-11-22 | 安徽师范大学 | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof |
| CN113981128A (en) * | 2021-11-22 | 2022-01-28 | 中国热带农业科学院热带作物品种资源研究所 | EST-SSR marker developed based on dendrobium officinale transcriptome sequence and application |
| CN113981128B (en) * | 2021-11-22 | 2023-10-20 | 中国热带农业科学院热带作物品种资源研究所 | EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102649955B (en) | 2013-08-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102649955A (en) | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker | |
| Ouziad et al. | Differential gene expressions in arbuscular mycorrhizal-colonized tomato grown under heavy metal stress | |
| KR101331740B1 (en) | SSR primer derived from Paeonia lactiflora and use thereof | |
| CN101988063B (en) | Microsatellite molecular marker of lycoris | |
| Kato | High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures | |
| CN101654707B (en) | Polygonatum cyrtonema microsatellite markers | |
| CN102168084A (en) | Dendrohuoshanense microsatellite deoxyribonucleic acid (DNA) molecule mark | |
| KR101144988B1 (en) | SCAR markers for discrimination of apple cultivars and use thereof | |
| KR101714764B1 (en) | Molecular marker for discrimination of Alexandre melon F1 cultivars and use thereof | |
| CN108396026A (en) | The exploitation and application of the Chang Miho couchgrass indigo plant kernel Characters of decaploid special chemoattractant molecule label and fluorescence in situ hybridization probe | |
| KR101432281B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| Doležalová et al. | Karyotype differentiation in cultivated chickpea revealed by Oligopainting fluorescence in situ hybridization | |
| KR101272530B1 (en) | Chloroplast DNA molecular marker and method of identifying the geographical origin of Korean Iris dichotoma by using thereof | |
| CN101824487A (en) | Method for detecting virus infection in pathological sample by combining PCR (Polymerase Chain Reaction) with nucleic acid probe dot hybridization technology | |
| CN102703444B (en) | Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker | |
| KR101137799B1 (en) | Specific primers for discriminating Suhan strains in Pleurotus ostreatus, and uses thereof | |
| CN102191242B (en) | Paris polyphylla microsatellite DNA molecular markers | |
| KR101432284B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| KR101432287B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| KR102291826B1 (en) | Composition for herbicide-resistant gene modified grass dertermination and grass-breed determination and uses thereof | |
| CN104498482A (en) | Pteroceltis tatarinowii microsatellite DNA molecular marker and isolation and screening method thereof | |
| JP5769377B2 (en) | Specific mold detection system, specific mold detection method, and PCR reaction solution | |
| KR101687581B1 (en) | Primer set for detecting Atractylodes mild mottle pararetrovirus and uses thereof | |
| KR20230109925A (en) | SNP markers and primer sets for high-throughput identification of wheat cultivar and uses thereof | |
| KR101432289B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130814 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |