CN102703444B - Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker - Google Patents
Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker Download PDFInfo
- Publication number
- CN102703444B CN102703444B CN 201210165286 CN201210165286A CN102703444B CN 102703444 B CN102703444 B CN 102703444B CN 201210165286 CN201210165286 CN 201210165286 CN 201210165286 A CN201210165286 A CN 201210165286A CN 102703444 B CN102703444 B CN 102703444B
- Authority
- CN
- China
- Prior art keywords
- polygonati officinalis
- radix polygonati
- microsatellite
- dna
- molecular marker
- 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.)
- Expired - Fee Related
Links
- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 30
- 102000053602 DNA Human genes 0.000 title claims description 6
- 239000003147 molecular marker Substances 0.000 title abstract description 5
- 108020004414 DNA Proteins 0.000 title description 14
- 239000002773 nucleotide Substances 0.000 claims description 20
- 125000003729 nucleotide group Chemical group 0.000 claims description 20
- 239000003550 marker Substances 0.000 claims description 9
- 230000002068 genetic effect Effects 0.000 abstract description 7
- 241000756042 Polygonatum Species 0.000 abstract description 6
- 238000012216 screening Methods 0.000 abstract description 5
- 108091081062 Repeated sequence (DNA) Proteins 0.000 abstract description 2
- 238000012163 sequencing technique Methods 0.000 abstract 1
- 230000008859 change Effects 0.000 description 14
- 239000000975 dye Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 229910052709 silver Inorganic materials 0.000 description 9
- 239000004332 silver Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 238000000137 annealing Methods 0.000 description 8
- 238000000246 agarose gel electrophoresis Methods 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
- 239000003814 drug Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 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
- 241000208340 Araliaceae Species 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
- 241000234280 Liliaceae Species 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 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 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 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
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000825107 Hierochloe Species 0.000 description 1
- 235000015466 Hierochloe odorata Nutrition 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 108020004487 Satellite DNA Proteins 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 229930003756 Vitamin B7 Natural products 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
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 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
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 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
- 239000012634 fragment Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation 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
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- LRMQCJCMKQSEJD-UHFFFAOYSA-N oligo b Polymers O1C(N2C3=NC=NC(N)=C3N=C2)C(OC)C(OC(=O)C=2C=C3C4(OC(=O)C3=CC=2)C2=CC=C(O)C=C2OC2=CC(O)=CC=C24)C1COP(O)(=O)OC1C(C(O2)N3C(N=C(N)C(C)=C3)=O)OCC12COP(O)(=O)OC(C1OC)C(COP(O)(=O)OC2C3(COP(O)(=O)OC4C(C(OC4COP(O)(=O)OC4C(C(OC4COP(O)(=O)OC4C(C(OC4COP(O)(=O)OC4C5(COP(O)(=O)OC6C(C(OC6COP(O)(=O)OC6C7(COP(O)(=O)OC8C(C(OC8COP(O)(=O)OC8C9(CO)COC8C(O9)N8C(N=C(N)C(C)=C8)=O)N8C(NC(=O)C=C8)=O)OC)COC6C(O7)N6C(N=C(N)C(C)=C6)=O)N6C(N=C(N)C=C6)=O)OC)COC4C(O5)N4C(N=C(N)C(C)=C4)=O)N4C5=NC=NC(N)=C5N=C4)OC)N4C5=C(C(NC(N)=N5)=O)N=C4)OC)N4C5=C(C(NC(N)=N5)=O)N=C4)OC)COC2C(O3)N2C(N=C(N)C(C)=C2)=O)OC1N1C=CC(=O)NC1=O LRMQCJCMKQSEJD-UHFFFAOYSA-N 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide 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
- 230000008569 process Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a radix polygonati officinalis microsatellite DNA molecular marker, comprising the steps of constructing a radix polygonati officinalis microsatellite (CT) n enrichment library; screening and sequencing microsatellite sequence positive clone; obtaining 55 clones containing the microsatellite repeated sequence; and screening to obtain 9 high polymorphic microsatellite molecular markers Po1, Po2, Po3, Po4, Po5, Po6, Po7, Po8 and Po9. The radix polygonati officinalis microsatellite DNA molecular marker can be used for researching genetic diversity of the radix polygonati officinalis, variation and evolutionary relationship between polygonatum plants and population, has good repeatability and is a reliable and efficient molecular marker.
Description
Technical field
The present invention relates to molecular marking technique, be specifically related to a kind of radix polygonati officinalis micro-satellite molecule genetic marker.
Background technology
Little satellite (Microsatellite) be called again short polyphone repeat (Short Tandem Repeats, STRs), simple repeated sequence (Simple Sequence Repeat, SSRs), SSLP (SSLP).Refer to that a class is dispersed throughout (being called core sequence, being generally 1～6bps) the simple series connection of forming for repeating unit and repeating by several nucleotide pairs in the biological gene group.Its multiplicity is alterable height between the different genotype of same species, but the two ends of this section repetition are the single-copy sequences of guarding relatively, design primer accordingly, can analyze the variation of core sequence multiplicity by round pcr, between different genotype, detect polymorphic.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, in per 6 kb length 1 microsatellite locus just arranged according to estimates; The multiplicity of core sequence is height variability, polymorphism information capacity height: be codominance, follow the heredity of broad-mouthed receptacle for holding liquid Dare rule between individuality; Select neutrality etc.
Based on above characteristics, microsatellite DNA is widely used in that structure, the assignment of genes gene mapping, cultivar identification and the germplasm of plant genetic collection of illustrative plates are preserved, the research of analysis, assistant breeding, structure finger printing, genetic diversity and the aspects such as spore and sibship of quantitative character gene.
Radix polygonati officinalis has another name called the tail ginseng, beautiful ginseng, and the small bell dish, ground tube and sweet grass are under the jurisdiction of Liliaceae (Liliaceae L.) Polygonatum (Polygonatum Mill.), are per nnial herb, and happiness is cloudy gives birth to, and is distributed widely in area, temperate zone, Eurasia.
The radix polygonati officinalis subterraneous stem can be used as medicine, and its medicinal history has exceeded more than 2,000 year in China.Traditional Chinese medicine thinks that radix polygonati officinalis property is sweet, is slightly cold, attaches to the lung and stomach meridians, can Yin nourishing and lung moistening, reinforcing stomach reg fluid is used for scorching impairment of yin, the disease of thermal burn stomach-Yin.Modern study proof radix polygonati officinalis contains polysaccharide and multiple extract, is delaying senility, and strengthening immunity suppresses tumour, and aspects such as promotion lymphocyte transformation have certain effect.
Since among the people dig excessively serious, in addition the natural crude drugs cycle long, the radix polygonati officinalis wild resource is exhaustion day by day.In fact, radix polygonati officinalis medicine source is comparatively complicated, and little satellite helps to identify the radix polygonati officinalis strain, and distinguishes the pseudo-product of radix polygonati officinalis.Simultaneously, radix polygonati officinalis a series of problems occurred in the cultivation domestication process, and is low as breeding coefficient, even utilize microsatellite marker to the research of radix polygonati officinalis molecular breeding seldom.
Summary of the invention
The technical problem that the present invention solves: the microsatellite DNA molecule marker of separating clone radix polygonati officinalis, set up radix polygonati officinalis satellite DNA technical system and carry out the radix polygonati officinalis genetic diversity with these molecule markers, the research of Polygonatum platymiscium genetic affinity, and carry out the Polygonatum Idioplasm identification.
The technical scheme of technical solution problem of the present invention: radix polygonati officinalis microsatellite DNA molecule marker, comprise the structure of the little satellite of radix polygonati officinalis (CT) n enriched library, the screening of microsatellite sequence positive colony and order-checking obtain 55 clones that contain little satellite tumor-necrosis factor glycoproteins.Obtain 9 microsatellite molecular marker Po1, Po2, Po3, Po4, Po5, Po6, Po7, Po8, Po9 that polymorphism is high; Po1 is 360 Nucleotide, and Po2 is 471 Nucleotide, and Po3 is 447 Nucleotide, and Po4 is 408 Nucleotide, and Po5 is 463 Nucleotide, and Po6 is 497 Nucleotide, and Po7 is 424 Nucleotide, and Po8 is 509 Nucleotide, and Po9 is 342 Nucleotide.
Description of drawings
Fig. 1: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po1 site dyes PAGE figure.
Fig. 2: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po2 site dyes PAGE figure.
Fig. 3: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po3 site dyes PAGE figure.
Fig. 4: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po4 site dyes PAGE figure.
Fig. 5: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po5 site dyes PAGE figure.
Fig. 6: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po6 site dyes PAGE figure.
Fig. 7: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po7 site dyes PAGE figure.
Fig. 8: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po8 site dyes PAGE figure.
Fig. 9: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po9 site dyes PAGE figure.
Embodiment
Do detailed explanation below in conjunction with the present invention of embodiment.
Embodiment 1:
The structure of radix polygonati officinalis microsatellite DNA enriched library
1) genomic extraction and enzyme are cut:
The CTAB method of introducing with Doyle J (Doyle J. DNA protocols for plants-CTAB total DNA isolation [A]. In Hewitt GM, Johnston A (eds.), Molecular Techniques in Taxonomy [M]. Berlin:Springer, 1991,283-293) extract genome.Cut genome with restriction enzyme Sau 3AI enzyme.Behind 2% the agarose gel electrophoresis, under UV-light, downcut the dna fragmentation of 300-900 bp.Reclaiming test kit 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’-PO
4-GATCCGAAGCTTGGGGTCTCTGGCC-3’),
Under T4 DNA Ligase effect, spend the night in 16 ℃ of water-baths connections.
3) the PCR detection tabs connects:
Being primer with oligo A, is that template is carried out pcr amplification to connect product, (50 μ l reaction systems are as follows: 25 μ l GoTaq Green Master Mix, and 5 μ l primer oligo A, 5 μ l connect product, add the water benefit to 50 μ l.The PCR response procedures: 94 ℃ of pre-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.
4) enrichment with magnetic bead:
Dna fragmentation sex change behind the purifying is formed after the strand and the little satellite probe hybridization that contains vitamin H, form " little satellite probe comprises the genomic dna strand of little satellite " complex body.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 6 * SSC successively, 2 * SSC, 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 15 minutes on the PCR instrument, collect supernatant.Repeat twice.
5) PCR enrichment:
Be template with the enrichment with magnetic bead product, oligo A is that primer carries out pcr amplification, and 25 μ l reaction systems are as follows, 12.5 μ l GoTaq Green Master Mix, and 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 as follows: 94 ℃ of pre-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 after 2% agarose gel electrophoresis detection.
6) preparation of competent cell:
The single bacterium colony of E.coli DH-5 α is inoculated in the LB substratum that does not contain Amp of 5 ml on the picking flat board, and 37 ℃ of shaking culture are spent the night.Get 1 ml nutrient solution in the LB of 50 ml liquid nutrient medium, 37 ℃ of shaking culture, 2～4 h are to logarithmic phase.Survey OD(0.2～0.6).The nutrient solution branch is filled to (every pipe 1.5 ml bacterium liquid) in the EP pipe, water-bath 10～15 min.4 ℃ centrifugal, centrifugal again after 4000 rpm, the 10 min(cooling earlier) abandon supernatant.Abandon three pipe and pipes behind the supernatant, ice bath 30 min(first pipe adds 0.1 mol/L, and 1 ml 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 ℃, 4000 rpm, 5～8 min.Abandon supernatant.Add 200 μ l precooling glycerine (15%)/CaCl
2(0.1 M) 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), and 16 ℃ of water-baths connect 2-3 hour.Take out frozen pipe, 10 min on ice recover; The connection product of 10 μ l is added in the 200 μ l competent cells mixing, ice bath 30 min; 42 ℃ of water-bath heat-shocked 90 s; Ice bath 2 min add 600 μ l LB substratum, 37 ℃ of shaking culture 1.5 h; 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 incubation then.Through the cultivation of 12 h, treat that the bacterium colony on the flat board is of moderate size, take out flat board and in 4 ℃ of placements locus coeruleus is fully manifested.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 4 h.
8) screening of positive colony:
The bacterium liquid that transforms gained with connection is template, utilizes primer oligo A and primer (CT)
12Carry out pcr amplification.15 μ l PCR reaction systems comprise: rTaq enzyme 0.375 U, 10 * Buffer, 1.5 μ l, dNTP 0.2 μ M, MgCl
21.5 μ M, and primer 0.4 μ M (oligo A, (CT)
12), water, template.Response procedures: 94 ℃ of pre-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.
9) positive colony order-checking and design of primers:
Choose the amplification fragment length and contain the bacterium liquid of positive colony at 300-900 bp, send the order-checking of the biological (Shanghai) Co., Ltd. of living worker, obtain 51 sequences that contain microsatellite DNA altogether.Utilize software Primer Premier 5.0 sequences according to the microsatellite DNA two ends to design primer.
10) screening has the primer of polymorphism:
Extract the genome of radix polygonati officinalis with above-mentioned CTAB method.With primer (table 1) the amplification gene group that designs.Response procedures: 94 ℃ of pre-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 9 microsatellite markers that polymorphism is arranged altogether.Po1, Po2, Po3, Po4, Po5, Po6, Po7, Po8, Po9; Po1 is 360 Nucleotide, and Po2 is 471 Nucleotide, and Po3 is 447 Nucleotide, and Po4 is 408 Nucleotide, and Po5 is 463 Nucleotide, and Po6 is 497 Nucleotide, and Po7 is 424 Nucleotide, and Po8 is 509 Nucleotide, and Po9 is 342 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 feature of 9 radix polygonati officinalis microsatellite DNA polymorphic sites with this.Can be found out by accompanying drawing 1-12, the diversity that the length of 9 microsatellite sequences of the present invention all occurs in 21 radix polygonati officinalis for examination, this shows, these 9 microsatellite markers of the present invention can be used for the genetic diversity of research radix polygonati officinalis, between the Polygonatum plant species and the variation between population and evolutionary relationship, having very high repeatability, is a kind of reliable and effective molecule marker.
Table 1 primer property list:
F represents upstream primer in the table 1, and R represents. downstream primer.
Claims (1)
1. radix polygonati officinalis microsatellite DNA molecule marker, it is characterized in that: the label of described microsatellite marker is: Po1;
Its nucleotides sequence is classified as:
Po1：
gatcacatgc cgatagttcc tcggtcacct agcatattta gtgtcagtct tctagctggc 60
gaataaaaac actaagtcga aatgaactaa ggaaagtgct cttctaactg tgagagagag 120
agagagagag agagagagag agagagagag agatgttggt ccatccctca tggtgtactg 180
tggatttcat tttgcgctct ctagctaatt ggattttagt cagatttttc tagttagttt 240
agcaccactc ataggtagag ctgtaaaatg aactgagttt tggagtgttt gtgttcgttc 300
aataatttac acgagtttaa atttagctga ttctatatga acccaaacaa atactcaatt 360。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210165286 CN102703444B (en) | 2012-05-25 | 2012-05-25 | Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210165286 CN102703444B (en) | 2012-05-25 | 2012-05-25 | Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102703444A CN102703444A (en) | 2012-10-03 |
| CN102703444B true CN102703444B (en) | 2013-08-14 |
Family
ID=46896494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210165286 Expired - Fee Related CN102703444B (en) | 2012-05-25 | 2012-05-25 | Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102703444B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498482B (en) * | 2014-11-28 | 2018-04-13 | 安徽师范大学 | Wingceltis Microsatellite DNA molecular marker and separating screening method |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020033336A (en) * | 2000-10-30 | 2002-05-06 | 박선민 | Food composition comprising extract from Polygonatum odoratum (Mill.) Druce |
| CN1363371A (en) * | 2002-01-11 | 2002-08-14 | 潘文光 | Multifunctional bio-active medicine for preventing and curing AIDS |
| KR20040024008A (en) * | 2002-09-12 | 2004-03-20 | 선만규 | Health food composition for improving constipation |
| KR20040058668A (en) * | 2002-12-27 | 2004-07-05 | 한국화장품주식회사 | Polygonati Rhizoma extract having skin whitening effect and skin whitening cosmetic composition containing the same |
| CN1513968A (en) * | 2002-12-31 | 2004-07-21 | 李玉文 | Special function wine using nucleotide Chinese herbal medicine as main raw material |
| CN1557842A (en) * | 2004-02-02 | 2004-12-29 | 中南大学湘雅二医院 | Process for extracting polygonatum polysaccharides, preparing process for medical preparation and use thereof |
| KR100702901B1 (en) * | 2006-12-24 | 2007-04-09 | 서희동 | A manufacturing method of makgeolli containing medicinal properties which used deep sea water |
| CN1974598A (en) * | 2005-07-26 | 2007-06-06 | 香港中文大学 | Isolated proteins from a traditional Chinese medicine yuzhu and use thereof |
-
2012
- 2012-05-25 CN CN 201210165286 patent/CN102703444B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020033336A (en) * | 2000-10-30 | 2002-05-06 | 박선민 | Food composition comprising extract from Polygonatum odoratum (Mill.) Druce |
| CN1363371A (en) * | 2002-01-11 | 2002-08-14 | 潘文光 | Multifunctional bio-active medicine for preventing and curing AIDS |
| KR20040024008A (en) * | 2002-09-12 | 2004-03-20 | 선만규 | Health food composition for improving constipation |
| KR20040058668A (en) * | 2002-12-27 | 2004-07-05 | 한국화장품주식회사 | Polygonati Rhizoma extract having skin whitening effect and skin whitening cosmetic composition containing the same |
| CN1513968A (en) * | 2002-12-31 | 2004-07-21 | 李玉文 | Special function wine using nucleotide Chinese herbal medicine as main raw material |
| CN1557842A (en) * | 2004-02-02 | 2004-12-29 | 中南大学湘雅二医院 | Process for extracting polygonatum polysaccharides, preparing process for medical preparation and use thereof |
| CN1974598A (en) * | 2005-07-26 | 2007-06-06 | 香港中文大学 | Isolated proteins from a traditional Chinese medicine yuzhu and use thereof |
| KR100702901B1 (en) * | 2006-12-24 | 2007-04-09 | 서희동 | A manufacturing method of makgeolli containing medicinal properties which used deep sea water |
Non-Patent Citations (10)
| Title |
|---|
| ISSR法鉴定中药玉竹与小玉竹;周晔等;《中医药学报》;20061031;第34卷(第5期);7-11 * |
| RAPD技术在商品药材玉竹鉴定中的应用;林丽美;《中国优秀硕士论文全文数据库 医药卫生科技辑》;20100515;第二部分试验方法 * |
| 刘婷婷.长梗黄精11个微卫星位点的筛选及跨种扩增.《全国优秀硕士论文全文数据库 农业科技辑》.2012,D047-186. |
| 周晔等.ISSR法鉴定中药玉竹与小玉竹.《中医药学报》.2006,第34卷(第5期),7-11. |
| 李钟.用RAPD技术鉴别湖南道地药材玉竹.《湖南中医药大学2003年度学位论文集》.2003, |
| 林丽美.RAPD技术在商品药材玉竹鉴定中的应用.《中国优秀硕士论文全文数据库 医药卫生科技辑》.2010,E057-1. |
| 潘清平等.玉竹不同品种的ISSR分子鉴定.《中国现代中药》.2008,第10卷(第10期),28-30. |
| 玉竹不同品种的ISSR分子鉴定;潘清平等;《中国现代中药》;20081031;第10卷(第10期);28-30 * |
| 用RAPD技术鉴别湖南道地药材玉竹;李钟;《湖南中医药大学2003年度学位论文集》;20031231;第二部分试验方法 * |
| 长梗黄精11个微卫星位点的筛选及跨种扩增;刘婷婷;《全国优秀硕士论文全文数据库 农业科技辑》;20120215;第二部分试验方法 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102703444A (en) | 2012-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhi et al. | Genomic differentiation among natural populations of orang-utan (Pongo pygmaeus) | |
| CN102649955B (en) | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker | |
| CN101440366B (en) | Specific fragment tightly linked to musk melon powdery mildew resistance gene Pm-2F and obtaining method thereof | |
| CN101654707B (en) | Polygonatum cyrtonema microsatellite markers | |
| CN101886070A (en) | Method for establishing microsatellite molecular marker of scylla paramamosain | |
| CN103361346A (en) | Method for cloning and analyzing populus diversifolia micro RNAs (ribonucleic acids) precursor | |
| CN102703444B (en) | Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker | |
| CN109457023B (en) | Gene combination for identifying sex of gleditsia sinensis, SSR primer combination, identification method and application | |
| CN102191242B (en) | Paris polyphylla microsatellite DNA molecular markers | |
| CN111850138B (en) | Molecular marker, kit and method for distinguishing triangular bream and megalobrama amblycephala in Heilongjiang and hybrid thereof | |
| CN111534630B (en) | SNP molecular marker related to oil content of camellia seed kernels and application thereof | |
| CN114015756A (en) | Paeonia ostii waterlogging-resistant differential expression gene and transcriptome-based sequencing mining method | |
| KR101432281B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| CN102719567A (en) | PCR (Polymerase Chain Reaction) detection primer and method for H120 strain of avian infectious bronchitis virus | |
| CN106148540B (en) | Screen the SNP primer and screening technique of Fugu rubripes seed | |
| CN111394473A (en) | Molecular marker related to chicken antler crowns and typing method and application thereof | |
| CN102168084A (en) | Dendrohuoshanense microsatellite deoxyribonucleic acid (DNA) molecule mark | |
| CN110004232A (en) | The specific gene of east Storehouse midge, the kit and method for identifying east Storehouse midge | |
| WO2015066832A1 (en) | Maize rough dwarf disease resistance-related major quantitative trait locus and use thereof | |
| CN109797235A (en) | With the molecular labeling R112823 of resistance gene of rice blast Pi1 close linkage | |
| NL2030574B1 (en) | Differentially expressed genes (degs) of paeonia ostii waterlogging tolerance and mining method based on rna sequencing (rna-seq) | |
| KR101432287B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| CN103194538B (en) | Rapid identification method for identical twin piglets | |
| CN108531620B (en) | Primer and method for identifying acipenser schrencki, acipenser sibiricus and filial generation thereof | |
| CN104498482A (en) | Pteroceltis tatarinowii microsatellite DNA molecular marker and isolation and screening method thereof |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130814 Termination date: 20200525 |