CN103571949A - Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale - Google Patents
Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale Download PDFInfo
- Publication number
- CN103571949A CN103571949A CN201310476400.5A CN201310476400A CN103571949A CN 103571949 A CN103571949 A CN 103571949A CN 201310476400 A CN201310476400 A CN 201310476400A CN 103571949 A CN103571949 A CN 103571949A
- Authority
- CN
- China
- Prior art keywords
- herba dendrobii
- microsatellite
- seq
- hard
- upstream
- 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.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale. The microsatellite molecular markers comprise HZs01, HZs02, HZs03, HZs04, HZs05, HZs06 and HZs07, wherein the HZs01, HZs02, HZs03 and HZs04 are specific sequences of hard-foot dendrobium officinale; the HZs05, HZs06 and HZs07 are specific sequences of soft-foot dendrobium officinale. The obtained specific microsatellite molecular markers of the hard/soft-foot dendrobium officinale can be used for distinguishing the hard-foot dendrobium officinale from the soft-foot dendrobium officinale and are used for cracking down on fake products and identifying medicinal materials such as dried dendrobium officinale. The hard-foot dendrobium officinale and the soft-foot dendrobium officinale can be distinguished by utilizing the microsatellite molecular markers, and the microsatellite molecular markers are high in repeatability and are reliable and effective DNA molecular markers.
Description
Technical field
The invention belongs to plant genome DNA molecular marking technique field, be specifically related to one group of microsatellite molecular marker that can be used for identifying hard pin and weak foot Herba Dendrobii.
Background technology
Herba Dendrobii, as one of most precious traditional rare traditional Chinese medicine of the topmost representative species ，Shi of medicinal dendrobium China, is classified as by honor first of " Chinese nine immortal grass ", have the laudatory title of " gold in medicine ".Just on the books in the < < Shennong Bencaojing > > in the last years of a dynasty or reign of the Eastern Han Dynasty, in the Ancient Times in China multi-section medical science works such as < < Compendium of Materia Medica > > and < < Chinese medicine dictionary > >, all record Herba Dendrobii and there is strengthening immunity, eliminate tumour, suppress the effects such as cancer, to laryngopharyngeal diseases, gastroenteropathy, cataract, cardiovascular disorder, diabetes, tumour all has significant curative effect, particularly Human Lung Cancer cell is had to the inhibiting rate up to 74.7～97.2 ﹪.
Herba Dendrobii is divided into weak foot and the hard large class of pin two conventionally: the most tubbiness of weak foot Herba Dendrobii stem and softness, and dendrobium polysaccharide and dendrobine content are high, and taste is sweet, almost there is no residue after chewing.The stem of noble dendrobium dry product that the processes of turning round through stir-fry limit, limit is concocted helically is exactly first-class " Tiepi Fengdou ".In general, at least 10 kilograms of fresh weak foot Herba Dendrobiis just can be processed into 1 kilogram of Tiepi Fengdou.And authentic wild Herba Dendrobii is conventionally in 1000 yuan of left and right of per kilogram, this causes the price of Tiepi Fengdou to equal to gold.Hard pin Herba Dendrobii stem is long and matter is hard, and stickiness little (dendrobium polysaccharide content is low), can not, by softened by baking, can only to boil waterly take.
On the other hand, wild Herba Dendrobii habitat is unique, breeding difficulty, add excessively and gather, the main range of distribution of Herba Dendrobii such as the Zhejiang of historical document report, Yunnan, Guizhou, Guangxi, Hunan, Anhui, Jiangxi, have been difficult to find large-area wild Herba Dendrobii to distribute in current field investigation.Within 2011, Herba Dendrobii becomes second batch " rare or endangered species of state key first class of protection ", may be for the production of, so over nearly 10 years, the research of Herba Dendrobii artificial culture, large-scale planting technology becomes one of focus.At present, the artificial cultivation technique of Herba Dendrobii is ripe gradually, and industrialized scale constantly expands.Formed the Herba Dendrobii industrial colony that integrates scientific research, plantation, processing, sale.Yet fresh weak foot and hard pin Herba Dendrobii are because of the difference of shape, color and luster aspect, human consumer easily differentiates.But, through curing, the Tiepi Fengdou that forms of the operation such as curling, actually or the hard pin of its raw material weak foot, only according to visual inspection, be just that expert has been difficult to differentiate, let alone ordinary consumer.And pin Herba Dendrobii is because fibre content is high firmly, frangibility, does not more easily make maple bucket, so be often used on market adulterate, pretend to be or mixes in weak foot Tiepi Fengdou.Add existing plant taxonomy or chemical means, be difficult to identify species level, so for many years never to efficient, the science of weak foot and hard pin Herba Dendrobii, general authentication method.
Molecular markers for identification is one of current popular important method more scientific and sensitiveer than traditional authentication method.Microsatellite marker (SSR wherein, Simple Sequence Repeat) conduct is with archaeal dna polymerase chain reaction (PCR, Polymerase Chain Reaction) technology and sequencing technologies are basic molecule marker, are considered to one of molecule marker that current genetic variation research intermediate-resolution is the highest, announcement power is the strongest.So far, there is no the relevant report of developing to carry out hard pin and weak foot Herba Dendrobii cultivar identification by Herba Dendrobii microsatellite molecular marker both at home and abroad.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, the microsatellite molecular marker of one group of Herba Dendrobii is provided, it can, for accurately distinguishing hard pin and weak foot Herba Dendrobii, be cracked down on counterfeit goods and identify used with medicinal material for market.
In order to solve the problems of the technologies described above, the invention provides one group of totally 7 Herba Dendrobii microsatellite molecular marker, their primer pair sequence is respectively:
HZs01 primer pair: upstream 5 '-CGAAGGTGGCGAAACAAGAC-3 ';
Downstream 5 '-GAAAACTACAAATTACCAAAGAC-3 ';
HZs02 primer pair: upstream 5 '-AAGTGGATAACGGGGATAAG-3 ';
Downstream 5 '-TTTTGGCAATGAACCTAATG-3 ';
HZs03 primer pair: upstream 5 '-GCAAAAGCTAGGGTAGAGTTC-3 ';
Downstream 5 '-CTCCTTCCCAATGAGCTT-3 ';
HZs04 primer pair: upstream 5 '-GAGAGAAGGGGAGCAATG-3 ';
Downstream 5 '-CTAAGTTCGCACTCATCTCTG-3 ';
HZs05 primer pair: upstream 5 '-GGAGGAAGTAGGGAGTCAAC-3 ';
Downstream 5 '-TAAACCTTATAGAAGCCAAA-3 ';
HZs06 primer pair: upstream 5 '-TGGGCTAACCATTGATTTAC-3 ';
Downstream 5 '-CAGACTTTATTGTTTGCCTAATT-3 ';
HZs07 primer pair: upstream 5 '-CAAATTCTCCGATCTCCGTT-3 ';
Downstream 5 '-AAGTCCCCAAACCTTATCAA-3 '.
As another core content of Herba Dendrobii microsatellite molecular marker of the present invention, the PCR annealing temperature of their primer pair is respectively: HZs01:58 ℃; HZs02:55 ℃; HZs03:55 ℃; HZs04:55 ℃; HZs05:50 ℃; HZs06:53 ℃; HZs07:56 ℃.
Another object of the present invention is that above-mentioned Herba Dendrobii microsatellite molecular marker can be used for hard pin and weak foot Herba Dendrobii to identify, HZs01, HZs02, HZs03, HZs04 are hard pin Herba Dendrobii characteristic sequences, and HZs05, HZs06, HZs07 are weak foot Herba Dendrobii characteristic sequences.
The present invention is on bioinformatics method basis, utilize dendrobium stem est sequence, screening microsatellite molecular marker, and be further used for finding Herba Dendrobii microsatellite molecular marker, select under different time, place and operator's condition, all obtainable 4 stable effectively hard peculiar microsatellite molecular markers of pin Herba Dendrobii and 3 stablize the peculiar microsatellite molecular marker of effective weak foot Herba Dendrobii.
The present invention realizes by such technical scheme:
Step (1). the acquisition of dendrobium stem est sequence: build the EST library of dendrobium stem flower, complete order-checking and splicing work, obtain dendrobium stem est sequence;
Step (2). the screening that contains micro-satellite est sequence: with the micro-satellite routine analyzer of computer and online microsatellite locus analysis software, checked order dendrobium stem est sequence is screened, obtain the dendrobium stem est sequence that contains microsatellite locus;
Step (3). design of primers: on the dendrobium stem est sequence that contains microsatellite locus, the archaeal dna polymerase chain reaction primer that contains microsatellite locus with primer-design software design amplification, the base numerical control that this primer contains is built in 18～22, two primer annealing temperature head <4 ℃, the DNA fragmentation of amplification is in 350 bases;
Step (4). micro-satellite checking: electrophoretic separation, the silver that utilizes the primer pair Herba Dendrobii genomic dna of above-mentioned steps (3) design to carry out polymerase chain reaction (PCR) amplification, amplified production dyes the increasing again of detection, microsatellite locus, subclone and order-checking confirmation work.
By above-mentioned four steps, the nucleotide sequence (5 '-3 ') of 4 peculiar microsatellite marker primers of hard pin Herba Dendrobii that finishing screen is selected is as follows:
HZs01 primer pair: upstream 5 '-CGAAGGTGGCGAAACAAGAC-3 ';
Downstream 5 '-GAAAACTACAAATTACCAAAGAC-3 ';
HZs02 primer pair: upstream 5 '-AAGTGGATAACGGGGATAAG-3 ';
Downstream 5 '-TTTTGGCAATGAACCTAATG-3 ';
HZs03 primer pair: upstream 5 '-GCAAAAGCTAGGGTAGAGTTC-3 ';
Downstream 5 '-CTCCTTCCCAATGAGCTT-3 ';
HZs04 primer pair: upstream 5 '-GAGAGAAGGGGAGCAATG-3 ';
Downstream 5 '-CTAAGTTCGCACTCATCTCTG-3 ';
The nucleotide sequence (5 '-3 ') of 3 peculiar microsatellite marker primers of weak foot Herba Dendrobii is as follows:
HZs05 primer pair: upstream 5 '-GGAGGAAGTAGGGAGTCAAC-3 ';
Downstream 5 '-TAAACCTTATAGAAGCCAAA-3 ';
HZs06 primer pair: upstream 5 '-TGGGCTAACCATTGATTTAC-3 ';
Downstream 5 '-CAGACTTTATTGTTTGCCTAATT-3 ';
HZs07 primer pair: upstream 5 '-CAAATTCTCCGATCTCCGTT-3 ';
Downstream 5 '-AAGTCCCCAAACCTTATCAA-3 '.
Hard pin Herba Dendrobii and the distinctive microsatellite molecular marker of weak foot Herba Dendrobii are in the application of distinguishing on hard pin and weak foot Herba Dendrobii.
Hard pin Herba Dendrobii and the distinctive microsatellite molecular marker of weak foot Herba Dendrobii that the present invention obtains, can, for the differentiation of hard pin and weak foot Herba Dendrobii, crack down on counterfeit goods and identify with the medicinal material of Tiepi Fengdou for business.The present invention can utilize these microsatellite molecular markers to distinguish hard pin Herba Dendrobii and weak foot Herba Dendrobii, reproducible, is a kind of reliable and effective DNA molecular marker.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of 4 peculiar microsatellite markers of hard pin Herba Dendrobii and 3 peculiar microsatellite markers of weak foot Herba Dendrobii;
Fig. 2 is the analytical results electrophorogram of embodiment 2.
Embodiment
Below that the specific embodiment of the present invention is described in further detail.
Embodiment is not for restriction of the present invention below, and the present invention is not limited only to embodiment below, as long as meet requirement of the present invention, all belongs to protection scope of the present invention.
1. the acquisition of dendrobium stem est sequence
This laboratory builds the EST library of dendrobium stem flower by oneself, completes order-checking and splicing work, obtains a large amount of dendrobium stem est sequences.
2. the screening of dendrobium stem est sequence
Utilize computer microsatellite locus routine analyzer (MISA) and online microsatellite locus analysis software (Microsatellite Repeats Finder), dendrobium stem est sequence is screened, obtain the dendrobium stem est sequence that contains microsatellite locus.
3. design of primers
On the dendrobium stem est sequence that contains microsatellite locus, utilize online primer-design software Primer Premier3 (http://frodo.wi.mit.edu/primer3/) design dna polymerase chain reaction (PCR) amplification primer, the size of design primer is 20 base left and right, expanding fragment length is in 100～300 bases, and the annealing temperature of two primers differs and is no more than 4 ℃.If contain two microsatellite locus in a DNA sequence dna, and two microsatellite locus are when 100 bases are above, and two microsatellite locus are designed respectively to primer; When two microsatellite locus in 100 bases time, just make two sites within the scope of the amplification of pair of primers during design of primers in same sequence as far as possible; Abandon microsatellite locus at the est sequence at sequence two ends.
4. microsatellite locus sequence verification:
1. archaeal dna polymerase chain reaction amplification
By PCR method the microsatellite marker primer designing increase respectively hard pin and weak foot Herba Dendrobii genomic dna, amplification reaction system and condition are as follows: reaction system is 20 μ l, comprising: 10 * damping fluid, 2 μ l, MgCl
2(25mmol/L) 1.2 μ l, deoxyribonucleotide (10mmol/L) 0.6 μ l, archaeal dna polymerase (5 units/μ l) 0.25 μ l, DNA profiling 50～100ng, primer (25mmol/L) 0.5 μ l, uses dd H
2o is settled to 20 μ l.The reaction conditions of polymerase chain reaction (PCR) amplification is: 94 ℃ of denaturations 5 minutes, then carry out 32 circulations (94 ℃ of sex change 40 seconds, under each micro-satellite primers annealing temperature, renaturation is 40 seconds, 72 ℃ are extended 90 seconds), and last 72 ℃ are extended after 10 minutes eventually in 4 ℃ of preservations.Thereby acquisition amplified production.
The PCR annealing temperature of each micro-satellite primers is respectively: HZs01:58 ℃; HZs02:55 ℃; HZs03:55 ℃; HZs04:55 ℃; HZs05:50 ℃; HZs06:53 ℃; HZs07:56 ℃.
2. electrophoretic separation and silver dye detection
(this sex change damping fluid is 98 ﹪ methane amides in amplified production, to add sex change damping fluid, the EDTA of 10mmol/L pH8.0, 0.25 ﹪ tetrabromophenol sulfonphthalein, the mixed solution of 0.2 ﹪ dimethylbenzene cyanogen), by mass content, be denaturing polyacrylamide gel (0.4 millimeter of thickness) and the 1 * TBE electrophoretic buffer of 4 ﹪, in the Sequi-Gen GT order-checking electrophoresis chamber of producing in U.S. Bio-Rad company, carry out electrophoresis, prerunning after 30 minutes 100 watts of permanent power electrophoresis of loading after approximately 2 hours silver dye detection, silver staining method process is mainly as follows: after electrophoresis, the sheet glass that is stained with gel is inserted to the vinyl disc dying for silver, adding mass content is the acetic acid of 10 ﹪, and on shaking table, slight concussion is fixed for 30 minutes, then adds rinsed with deionized water 3 times, each 5 minutes, gel after acid treatment is inserted in the dyeing dish of cma staining liquid that mass content is 0.1 ﹪, and on shaking table, slight concussion is 30 minutes, then uses 10 seconds of rinsed with deionized water, gel after dyeing is inserted in the colour developing dish of sodium carbonate that mass content is 0.32 ﹪, on shaking table, slightly shake until band is clear and band number no longer increases, on the gel of color development treatment, add stationary liquid (stationary liquid is the acetic acid of 10 ﹪), color development stopping reaction, with distilled water rinsing several minutes, remove the globule on gel and sheet glass, be placed on and on opalescence lamp box, observe and take pictures with digital camera.
3. amplification again and the subclone of microsatellite locus
According to the peculiar electrophoretic band feature preliminary judgement electrophoresis picture of Herba Dendrobii DNA sequence dna microsatellite locus, and check order and further confirm by rubber tapping.Concrete step is: cut off the band that the silver in polyacrylamide gel dyes on the microsatellite locus that signal is the strongest and be placed in pipe, (described TE solution is 10mmol/LTris-HCl to add 10 μ l TE solution, the mixed solution of 1mmol/L EDTA, pH value is 8.0) after, on PCR instrument, 94 ℃ are heated 30 minutes, then centrifuging and taking supernatant liquor is as secondary PCR template, 1. walking under identical PCR reaction system and reaction conditions with above-mentioned, the micro-satellite primers corresponding with each microsatellite locus increases, amplified production carries out separation with the sepharose of 1 ﹪, object band is reclaimed to test kit (QIAquick Gel Extraction Kit with glue, QIAGEN) reclaim, reclaim pMD18-T carrier (TAKARA) for product and carry out 16 ℃ of connections of spending the night, after transforming Top10 competent cell (TAKARA), carry out subclone, last picking positive colony is cultivated, with (the QIAGEN Plasmid Mini Kit of test kit for plasmid extraction, QIAGEN) after extraction plasmid, check order.
4. order-checking is confirmed
Plasmid by above-mentioned gained, checks order by Sangon Biotech (Shanghai) Co., Ltd., and the microsatellite molecular marker of Herba Dendrobii is confirmed as in the SSR site in analytical sequence.
Two. experimental result
By above-mentioned experimental technique, obtain altogether 4 peculiar microsatellite markers of hard pin Herba Dendrobii and 3 peculiar microsatellite markers of weak foot Herba Dendrobii.And under different time, place and operator's condition, above-mentioned 4 hard pin Herba Dendrobiis and 3 peculiar microsatellite markers of weak foot Herba Dendrobii can be respectively in hard pin and weak foot Herba Dendrobii to specific amplification band.As shown in Figure 1, the hard distinctive microsatellite marker HZs01 of pin Herba Dendrobii, HZs02, HZs03 and HZs04 only can obtain specific amplification band in hard pin Herba Dendrobii, and in weak foot Herba Dendrobii, there is no amplified band; Equally, the hard distinctive microsatellite marker HZs05 of pin Herba Dendrobii, HZs06 and HZs07 only can obtain specific amplification band in weak foot Herba Dendrobii, and there is no amplified band in hard pin Herba Dendrobii.
The nucleotide sequence (5 '-3 ') of 4 peculiar microsatellite marker primers of hard pin Herba Dendrobii is as follows:
HZs01 primer pair: upstream 5 '-CGAAGGTGGCGAAACAAGAC-3 ';
Downstream 5 '-GAAAACTACAAATTACCAAAGAC-3 ';
HZs02 primer pair: upstream 5 '-AAGTGGATAACGGGGATAAG-3 ';
Downstream 5 '-TTTTGGCAATGAACCTAATG-3 ';
HZs03 primer pair: upstream 5 '-GCAAAAGCTAGGGTAGAGTTC-3 ';
Downstream 5 '-CTCCTTCCCAATGAGCTT-3 ';
HZs04 primer pair: upstream 5 '-GAGAGAAGGGGAGCAATG-3 ';
Downstream 5 '-CTAAGTTCGCACTCATCTCTG-3 ';
The nucleotide sequence (5 '-3 ') of 3 peculiar microsatellite marker primers of weak foot Herba Dendrobii is as follows:
HZs05 primer pair: upstream 5 '-GGAGGAAGTAGGGAGTCAAC-3 ';
Downstream 5 '-TAAACCTTATAGAAGCCAAA-3 ';
HZs06 primer pair: upstream 5 '-TGGGCTAACCATTGATTTAC-3 ';
Downstream 5 '-CAGACTTTATTGTTTGCCTAATT-3 ';
HZs07 primer pair: upstream 5 '-CAAATTCTCCGATCTCCGTT-3 ';
Downstream 5 '-AAGTCCCCAAACCTTATCAA-3 '.
In order further to show microsatellite marker that the present invention develops accuracy and the repeatability on the hard pin Herba Dendrobii of difference and weak foot Herba Dendrobii, we have chosen respectively 3 kinds of hard pin Herba Dendrobiis and weak foot Herba Dendrobii in Zhejiang Province, and material list is as follows:
4 peculiar microsatellite marker HZs01 of hard pin Herba Dendrobii that utilize the present invention to develop, HZs02, HZs03 and HZs04,3 peculiar microsatellite marker HZs05 of weak foot Herba Dendrobii, HZs06 and HZs07, respectively to verifying for examination material, result is as shown in Figure 2: 4 peculiar microsatellite marker HZs01 of hard pin Herba Dendrobii, HZs02, HZs03 and HZs04 all obtain specific amplification band in hard pin stem of noble dendrobium material; 3 peculiar microsatellite marker HZs05 of weak foot Herba Dendrobii, HZs06 and HZs07 obtain specific amplification band equally in weak foot stem of noble dendrobium material.Show that hard pin and the accuracy of weak foot Herba Dendrobii microsatellite marker on the hard pin Herba Dendrobii of difference and weak foot Herba Dendrobii that the present invention develops are high, and reproducible.
[sequence table]
The SSR sequence (removing carrier) of the hard pin Herba Dendrobii that the present invention obtains and weak foot Herba Dendrobii:
HZs01：
CGAAGGTGGCGAAACAAGACAAGAAGAAGAAGCCTAGGGGGAGGGCCTACAAGCGGATGCAGT
ACAACCGCCGCTTCGTCACCGCCGTCGTTGGATTCGGGAAGAAGAGGGGACCCAACTCGTCCG
AGAAGTAAAGATCTGGATTGCAAAGTTCGTAGGAGTGGAATTCTGATTTTTGTATGTCTTTGG
TAATTTGTAGTTTTC
HZs02：
AAGTGGATAACGGGGATAAGCTTACTTTGGGCTCAGATGTCAATATATTGTGTCTTCACACTC
CCTGCCATACAAAAGGTCATATAAGTTATTATGTCACGAGTAAAGAAGAAGAAGATCCTTCTG
TTTTTACTGGAGACACATTGTTTATTGCTGGTTGTGGAAAGTTCTTTGAAGGAACTGCTGAAG
ATATGTACCAATCATTATCTGTGACATTAGGTTCATTGCCAAAA
HZs03：
GCAAAAGCTAGGGTAGAGTTCGGCTGGACTAGAAGGCGGAAGAGGATGGGATTGAGGTAAGGA
GCTCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTT
TATCTCAGAGATCTGGATGTCGGGGAAAGGGGAAGCTCATTGGGAAGGAG
HZs04：
GAGAGAAGGGGAGCAATGGCGGAGAAGGAGAAGGAGAAGGCTGGTGGCGAGAGGTGGAAGGCA
GCTATTGTGAATCTGTCAGAGATGAGTGCGAACTTAG
HZs05：
GGAGGAAGTAGGGAGTCAACTTCGACTTAATGACAATGAAAATCCATTTGTGACTTCAAAACA
ACTGATGTTCTCTCTCTCTCTCTCTCTCACTTCTCCATCACCCCCCCCCCCCAAAAAAAAGCA
TGTAAAATGTATTATCTCCTTTTTTTGGCTTCTATAAGGTTTA
HZs06：
TGGGCTAACCATTGATTTACCGATCGTCGGGAATTTTACGATTCCTTTGTCCACCAAAGGGGA
GTTGAAGCTTCCTTCCTTCACTGATTTCTTCTCGAGGGAGAAGAGCCCAGAATCCTAATTTTC
ATTACTGTTTCTTTATCTGTGTGTGTGTGGAATAGGAGTTGCACATGTAATTAGGCAAACAAT
AAAGTCTG
HZs07：
CAAATTCTCCGATCTCCGTTAACTAATTAGCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC
TCTCTCTCTCTCTCTCTCTCTCTCTCTCTTAATTTCTCTCCCTTCTGTTTAATAAAAATCTAC
ATAAATATAAATATATGTGCTTTGATGTTATGATCATATCATATGTTATTGATAAGGTTTGGG
GACTT
Claims (5)
1. one group of microsatellite molecular marker for the identification of hard pin and weak foot Herba Dendrobii, it is characterized in that this microsatellite molecular marker comprises HZs01, HZs02, HZs03, HZs04, HZs05, HZs06, HZs07, the nucleotide sequence of these microsatellite marker primers (5 '-3 ') is as follows:
HZs01 primer pair:
Upstream 5 '-CGAAGGTGGCGAAACAAGAC-3 ', as shown in SEQ ID NO.1;
Downstream 5 '-GAAAACTACAAATTACCAAAGAC-3 ', as shown in SEQ ID NO.2;
HZs02 primer pair:
Upstream 5 '-AAGTGGATAACGGGGATAAG-3 ', as shown in SEQ ID NO.3;
Downstream 5 '-TTTTGGCAATGAACCTAATG-3 ', as shown in SEQ ID NO.4;
HZs03 primer pair:
Upstream 5 '-GCAAAAGCTAGGGTAGAGTTC-3 ', as shown in SEQ ID NO.5;
Downstream 5 '-CTCCTTCCCAATGAGCTT-3 ', as shown in SEQ ID NO.6;
HZs04 primer pair:
Upstream 5 '-GAGAGAAGGGGAGCAATG-3 ', as shown in SEQ ID NO.7;
Downstream 5 '-CTAAGTTCGCACTCATCTCTG-3 ', as shown in SEQ ID NO.8;
HZs05 primer pair:
Upstream 5 '-GGAGGAAGTAGGGAGTCAAC-3 ', as shown in SEQ ID NO.9;
Downstream 5 '-TAAACCTTATAGAAGCCAAA-3 ', as shown in SEQ ID NO.10;
HZs06 primer pair:
Upstream 5 '-TGGGCTAACCATTGATTTAC-3 ', as shown in SEQ ID NO.11;
Downstream 5 '-CAGACTTTATTGTTTGCCTAATT-3 ', as shown in SEQ ID NO.12;
HZs07 primer pair:
Upstream 5 '-CAAATTCTCCGATCTCCGTT-3 ', as shown in SEQ ID NO.13;
Downstream 5 '-AAGTCCCCAAACCTTATCAA-3 ', as shown in SEQ ID NO.14.
2. one group of microsatellite molecular marker for the identification of hard pin and weak foot Herba Dendrobii as claimed in claim 1, is characterized in that HZs01, HZs02, HZs03, HZs04 are hard pin Herba Dendrobii characteristic sequences.
3. one group of microsatellite molecular marker for the identification of hard pin and weak foot Herba Dendrobii as claimed in claim 1, is characterized in that HZs05, HZs06, HZs07 are weak foot Herba Dendrobii characteristic sequences.
4. one group of microsatellite molecular marker for the identification of hard pin and weak foot Herba Dendrobii as claimed in claim 1, is characterized in that the annealing temperature of primer of the microsatellite molecular marker of hard pin and weak foot Herba Dendrobii is respectively HZs01:58 ℃; HZs02:55 ℃; HZs03:55 ℃; HZs04:55 ℃; HZs05:50 ℃; HZs06:53 ℃; HZs07:56 ℃.
5. one group of microsatellite molecular marker for the identification of hard pin and weak foot Herba Dendrobii as claimed in claim 1 is in the application of distinguishing on hard pin and weak foot Herba Dendrobii.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310476400.5A CN103571949A (en) | 2013-10-12 | 2013-10-12 | Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310476400.5A CN103571949A (en) | 2013-10-12 | 2013-10-12 | Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103571949A true CN103571949A (en) | 2014-02-12 |
Family
ID=50044657
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310476400.5A Pending CN103571949A (en) | 2013-10-12 | 2013-10-12 | Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103571949A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498037A (en) * | 2016-10-09 | 2017-03-15 | 中国中医科学院中药研究所 | Herba Dendrobii authentication method based on double retardance Allele-specific diagnostic PCRs |
| CN114507748A (en) * | 2022-01-25 | 2022-05-17 | 广西壮族自治区药用植物园 | EST-SSR molecular marker, kit and identification method for identifying mesona chinensis benth variety |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1368504A (en) * | 2001-01-31 | 2002-09-11 | 香港中文大学 | DNA sequence of dendrobium and method for discriminating its veriety and judging if it is true or false |
| CN1372005A (en) * | 2001-08-03 | 2002-10-02 | 中国药科大学 | Chinese dendrobe plant and its medicinal materials DNA molecular diagnosis method |
| CN101235377A (en) * | 2007-01-29 | 2008-08-06 | 上海雷允上科技发展有限公司 | Characteristic DNA sequence for dendrobium officinale test-tube plantlet and application thereof |
| CN102649955A (en) * | 2012-04-06 | 2012-08-29 | 安徽师范大学 | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker |
-
2013
- 2013-10-12 CN CN201310476400.5A patent/CN103571949A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1368504A (en) * | 2001-01-31 | 2002-09-11 | 香港中文大学 | DNA sequence of dendrobium and method for discriminating its veriety and judging if it is true or false |
| CN1372005A (en) * | 2001-08-03 | 2002-10-02 | 中国药科大学 | Chinese dendrobe plant and its medicinal materials DNA molecular diagnosis method |
| CN101235377A (en) * | 2007-01-29 | 2008-08-06 | 上海雷允上科技发展有限公司 | Characteristic DNA sequence for dendrobium officinale test-tube plantlet and application thereof |
| CN102649955A (en) * | 2012-04-06 | 2012-08-29 | 安徽师范大学 | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker |
Non-Patent Citations (2)
| Title |
|---|
| 肖冬长 等: "铁皮石斛微卫星SSR设计与应用", 《生物技术通报》 * |
| 胡旭: "金钗石斛EST-SSR标记的开发及其在石斛属植物遗传多样性研究中的应用", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498037A (en) * | 2016-10-09 | 2017-03-15 | 中国中医科学院中药研究所 | Herba Dendrobii authentication method based on double retardance Allele-specific diagnostic PCRs |
| CN106498037B (en) * | 2016-10-09 | 2019-12-20 | 中国中医科学院中药研究所 | Dendrobium officinale identification method based on double-block site specificity PCR |
| CN114507748A (en) * | 2022-01-25 | 2022-05-17 | 广西壮族自治区药用植物园 | EST-SSR molecular marker, kit and identification method for identifying mesona chinensis benth variety |
| CN114507748B (en) * | 2022-01-25 | 2023-06-27 | 广西壮族自治区药用植物园 | EST-SSR molecular marker for identifying mesona chinensis benth variety, kit and identification method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105624321B (en) | Differentiate the method for yellow stalwart tea tree breed using SSR finger-prints | |
| CN113430300B (en) | SSR molecular marker of mulberry variety Yuehen 123, core primer group and kit thereof, and application of SSR molecular marker | |
| Lin et al. | Genetic identification of edible birds’ nest based on mitochondrial DNA sequences | |
| CN107164471B (en) | Molecular identification method for rapidly identifying truth of beauveria bassiana in traditional Chinese medicine stiff silkworm | |
| CN105002272B (en) | Method for identifying varieties of RAPD (random amplified polymorphic DNA) marked panax japonicus and kindred plants thereof | |
| CN105969872A (en) | Dendrobium officinale EST-SSR primer set, development method and application of primer set in species genetic diversity | |
| CN106636403B (en) | Heat-resistant radish EST-SSR molecular marker, molecular marker primer and application thereof | |
| CN103571949A (en) | Microsatellite molecular markers for identifying hard/soft-foot dendrobium officinale | |
| CN108265122B (en) | PCR method for rapidly identifying authenticity of fritillaria cirrhosa | |
| Li et al. | Molecular identification of traditional medicinal materials | |
| CN104388569B (en) | PCR identification primers and method for identifying B.striata Rchb.f. and B.ochracea Schltr. by using same | |
| Niu et al. | Detection of Panax quinquefolius in Panax ginseng using ‘subtracted diversity array’ | |
| CN107130032A (en) | 6 eel species discrimination methods based on a plurality of DNA bar code | |
| US20200126633A1 (en) | Method for controlling the quality of traditional chinese patent medicines based on metagenomics | |
| CN110527743B (en) | Polymorphic molecular marker for identifying Xinjiang color cotton variety and application | |
| Jiang et al. | Conventional octaplex PCR for the simultaneous identification of eight mainstream closely related Dendrobium species | |
| CN107630014B (en) | A kind of identification method of Aconitum transsectum DNA bar code and Aconitum transsectum based on big data | |
| CN116426680A (en) | Primer, probe and detection method for detecting authenticity of ginseng medicinal material based on second-generation sequencing development | |
| Ren et al. | DNA barcoding of Gaultheria L. in China (Ericaceae: Vaccinioideae) | |
| CN106434976B (en) | Method for identifying true and false of Chinese yam and special primer | |
| CN108588243A (en) | Quickly differentiate specific primer, multiple PCR detection kit and its detection method of adventive Pomacea canaliculata | |
| CN101104868A (en) | Method of herbal authentication based on 5S rRNA spacer sequences | |
| Böhrer et al. | Integrated genome-scale analysis and northern blot detection of retrotransposon siRNAs across plant species | |
| CN116606956B (en) | SNP molecular marker combination for identifying wild buckwheat rhizome produced in south of Yunnan and method and application thereof | |
| CN102618555A (en) | Nucleotide sequence of gamma-alcohol-soluble protein gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140212 |