CN103060438A - Molecular marker fingerprinting for identifying anoectochilus formosanus produced at Huoshan in Anhui and similar species thereof - Google Patents
Molecular marker fingerprinting for identifying anoectochilus formosanus produced at Huoshan in Anhui and similar species thereof Download PDFInfo
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- CN103060438A CN103060438A CN2012104724840A CN201210472484A CN103060438A CN 103060438 A CN103060438 A CN 103060438A CN 2012104724840 A CN2012104724840 A CN 2012104724840A CN 201210472484 A CN201210472484 A CN 201210472484A CN 103060438 A CN103060438 A CN 103060438A
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Abstract
The invention discloses a molecular marker fingerprinting for identifying the anoectochilus formosanus produced at Huoshan in Anhui and the similar species thereof. The molecular marker fingerprinting comprises the steps of preprocessing anoectochilus formosanus samples, extracting DNA of each sample genome, executing ISSR-PCR (inter-simple sequence repeat-polymerase chain reaction), executing agarose gel electrophoresis, building ISSR molecular marker fingerprint of each sample and using the ISSR molecular marker fingerprint for identifying the anoectochilus formosanus germplasm. The method provided by the invention saves time and economic cost, and can obtain the accurate and reliable identification fingerprint just by extracting the DNA of the anoectochilus formosanus plants, executing ISSR-PCR and executing agarose gel electrophoresis; the method identifies the species, which are easy to confuse in appearance, from the essence of inheritance, has the advantages of being simple, convenient and efficient, excellent in repeatability and high in resolution, and so on; and the method can identify the species in seeding stage of plants, and plays an important role in guaranteeing the accuracy and the stability of the Huoshan anoectochilus formosanus crude drug base source.
Description
Technical field
The present invention relates to the finger printing of Herba Anoectochili roxburghii, especially relate to construction process and the application thereof of Herba Anoectochili roxburghii Idioplasm identification finger printing.
Background technology
China's the orchid family (Orchidaceae) is opened lip Cymbidium (Anoectochilus) to be had about 23 kinds, is per nnial herb, and common name Herba Anoectochili roxburghii or Shorthairy Antenoron have the laudatory titles such as " king of medicine ", " gold grass ", " refreshing medicine ", " bird ginseng ".Mainly being distributed in the subtropical zone in China is the provinces and regions such as Fujian, Guangdong, Guangxi, Zhejiang, Jiangxi, Hainan, Yunnan, Sichuan, Guizhou and southern Tibet, wherein take Fujian, Zhejiang, Jiangxi and Taiwan is main product ground.Herba Anoectochili roxburghii is the traditional valuable ingredient of traditional Chinese medicine of China, and the effect of refreshing and detoxicating, nourishing Yin and falling fire, anti-inflammatory analgetic is arranged, and nameless gall, fever, antidiarrheal, snakebite are all had significant curative effect, and uses safely, has no side effect
[3]Measure discovery through relevant department, the content of amino acid composition, composition, content and activity of fighting against senium trace element all is higher than domestic and wild Radix Panacis Quinquefolii in the Leaf of Obtuselobed Snakegourd.Centuries is used as herbal medicine commonly used among the people.Through Fujian platform various big hospital clinical application, the multiple therapeutic efficiency such as Herba Anoectochili roxburghii has liver protection, anti-HBV, anti-oxidant, anti-tumor activity, reducing hyperglycaemia, falls hyperlipidemia, antihypertensive, falls high lithemia, infantile asthma, infantile spasm-obscene words, anti-inflammatory, diuresis, calmness, pain relieving.Simultaneously, the Herba Anoectochili roxburghii plant type is small and exquisite, blade profile is graceful, golden yellow to be netted arrangement be the high indoor sight leaf treasure of ornamental value to vein.Because the fine quality of Herba Anoectochili roxburghii, it is subject to common people's favor day by day, but the classification of Herba Anoectochili roxburghii and not of the same race 's drug effect difference is final conclusion never, among the people, band kind and the middle profile allied species of variegated leaf Cymbidium (Goodyera) yellow or white vein all was used as the Herba Anoectochili roxburghii purchase during the pharmacist belonged to Anoectochilus Roxburghii.Also be with a variety of Herba Anoectochili roxburghiis of all being used as in Anoectochilus Roxburghii genus and the variegated leaf Cymbidium in artificial culture, also have the blood aspidistra also to be Herba Anoectochili roxburghii, cause the Herba Anoectochili roxburghii provenance to mix.This and the requirement of Chinese medicinal materials standardizing standard greatly differ from each other; also can bring risk to large-scale production; so be badly in need of carrying out the research of Herba Anoectochili roxburghii fine provenance screening and identification; to the improved seeds resource of screening and the protection economical character is good, pharmaceutical ingredient content is high, for further large-scale developing and utilizing with scientific research, it provides reference and reference.
The Huoshan is known orchid---the Herba Anoectochili roxburghii regional distribution is northernmost; the geographical environment that it is special and ecotope make the quality of Huoshan product Herba Anoectochili roxburghii unusual; for protecting and develop this excellent kind, Dabie Mountain orchid child care in imminent danger research establishment associating Anhui Liu Shi Tongji University gives birth to biotechnology limited liability company and has carried out the germ plasm resource identification research.
1994, the Zietkiewicz of Montreal, CAN university etc. has invented the interval segment polymorphism mark of tandem repetitive sequence (ISSR first, Inter simple sequence repeat, ISSR) technology, the ISSR molecular marking technique constantly is promoted and uses in the whole world afterwards.(the Roderetal. because the SSR sequence extensively distributes in genome, 1998), and allelic variation is abundant especially, therefore ISSR compares with RAPD with other polymorphic molecular marker technology RFLP, AFLP, can detect more much higher state property, simultaneously the grappling base makes and only has those sites with the grappling Mismatching could be by target calmly on the genome, avoided the slip of SSR on genome, greatly improved the stability of pcr amplification and repeatable, thus the ISSR molecular marking technique be very suitable for kind between and plant the analysis of lower relation.This paper utilizes the ISSR-PCR technology that 5 provinces, 10 ground such as Anhui, Hubei, Guangdong, Fujian and Taiwan are used for medicinal high-quality Herba Anoectochili roxburghii kind to have carried out differentiating under the kind between population, be intended to set up a comparatively complete reliable Herba Anoectochili roxburghii ISSR-PCR reactive system, for the Population genetic polymorphism of studying from now on the Herba Anoectochili roxburghii geographical provenance and widely fine-variety breeding and hybridization provide reference and reference on the molecular level.
Although carry out the order-checking of PCR product by conserved sequence design primer, identify that according to nucleotide sequence the method for medicinal plant has science and reliability, have that cost is high, program is complicated and the shortcoming such as length consuming time.Research and utilization ISSR molecule marking method of the present invention is identified Herba Anoectochili roxburghii and sibling species thereof, by the ISSR molecular marking fingerprint research to Herba Anoectochili roxburghii and sibling species plant thereof, have rich polymorphism, difference is stable, and operates relative simple, cost economic dispatch advantage.
Summary of the invention
Identify that in order to solve prior art the method cost of medicinal plant is high, program is complicated and the problem of length consuming time, the invention provides a kind of construction process and application of identifying the molecular marking fingerprint of Herba Anoectochili roxburghii that simple and efficient to handle, good reproducibility, resolving power are high and sibling species thereof.
Technical scheme of the present invention is to utilize the ISSR molecular marking technique to make up the molecular marking fingerprint of Herba Anoectochili roxburghii, and utilizes constructed finger printing to realize the evaluation of Huoshan red autumnal leaves Herba Anoectochili roxburghii and sibling species thereof.
Concrete operations of the present invention may further comprise the steps:
(1) fresh Herba Anoectochili roxburghii sample pretreatment
Fresh Herba Anoectochili roxburghii sample lucifuge is consumed starch more than 6 hours, get fresh Herba Anoectochili roxburghii blade 10 grams, with the above soaked in absolute ethyl alcohol secondary of 50 grams, each 10 minutes, dry;
(2) extraction of sample gene group DNA
Adopt improved CTAB method to extract the Herba Anoectochili roxburghii leaves genomic DNA, operate as follows:
1) pretreated blade is put into frappe ceramic mortar, and add an amount of quartz sand, then repeatedly adding liquid nitrogen fully is ground to Powdered, with spoon the blade powder is transferred to rapidly in the 10ml precooling Eppendorf centrifuge tube, the height of powder reaches 1/3 place of centrifuge tube, stops to add powder;
2) the CTAB Extraction buffer [2% (v/w) CTAB, 1.4M NaCl, 20mM EDTA, 100mM Tris-HCl (PH=8.0), 2% mercaptoethanol] of 65 ℃ of preheatings of adding 4m1 in centrifuge tube;
3) 65 ℃ of temperature are bathed 2h, middle every 10min counter-rotating 1～2 time;
4) the centrifugal 8min of 15000rpm changes supernatant liquor in another centrifuge tube over to;
5) be cooled to room temperature after, softly add isopyknic chloroform and primary isoamyl alcohol solution, wherein the volume ratio of chloroform and primary isoamyl alcohol is 24: 1, the light and slow 40min that puts upside down;
6) the centrifugal 10min of 15000rpm room temperature goes precipitation, and supernatant liquor is changed in another centrifuge tube;
7) add 2/3 volume Virahol at supernatant liquor, the light and slow mixing of putting upside down is placed more than the 1.5h for-20 ℃;
8) the centrifugal 10min of 15000rpm removes supernatant liquor;
9) wash twice to remove salt with 2ml 75% ethanol, wash once with dehydrated alcohol again;
10) remove ethanol, 37 ℃ of dry 20min are dissolved in 600 μ L damping fluids, transfer in the 1.5ml centrifuge tube;
11) adding 5 μ L final concentrations is 50 μ L/mL RnaseA, and 37 ℃ of temperature are bathed 1h;
12) the solution extracting that above-mentioned steps (11) is produced with isopyknic phenol, chloroform and primary isoamyl alcohol solution 2 times, by volume, phenol: chloroform: primary isoamyl alcohol=25:24:1;
13) the centrifugal 10min of 15000rpm gets supernatant liquor;
14) dehydrated alcohol of 2 times of volumes of adding ,-20 ℃ of insulation lh, every 20min upset is once;
15) 15000rpm is centrifugal, uses first 75% ethanolic soln washing precipitation 2-3 time of-20 ℃ of precoolings, again with absolute ethanol washing once.
16) 37 ℃ of temperature are bathed dry DNAs, add the dissolving of 100 μ LTE damping fluids for subsequent use, and 4 ℃ save backup;
(3) ISSR-PCR amplification
Utilize the sample gene group DNA that extracts as template, carry out the ISSR-PCR amplification at German Eppendorf 5331 grads PCR instrument, the PCR reaction system is characterized as: template concentrations 25 ng/L, primer concentration 0.5 μ molL
-1, Mg
2+Concentration 2.0mmolL
-1, dNTP concentration 200 μ molL
-1, Taq polymerase concentration 1U/20 μ l;
The pcr amplification procedure condition is: 94 ℃ of denaturations 8 minutes, and 94 ℃ of sex change 45 seconds, 58-62 ℃ of annealing 45 seconds, 72 ℃ were extended 2 minutes, and 40 circulations of increase are at last in 72 ℃ of eventually extensions 10 minutes;
The amplified production that the ISSR-PCR amplified reaction obtains different Herba Anoectochili roxburghii samples is stored in-20 ℃ of refrigerators;
(4) agarose gel electrophoresis
Take the genomic dna of 4 provinces, 10 ground Herba Anoectochili roxburghiis as template, carry out the ISSR-PCR amplified reaction with two kinds of primers wherein, get amplified production 5 μ L-15 μ L on 1.5% the sepharose under the voltage of 3V/cm electrophoresis 2.5h, develop the color through EB, and record the gel electrophoresis result of amplified production with the gel imaging instrument;
(5) structure of the ISSR-PCR molecular marking fingerprint of each test sample
The ISSR-PCR amplified reaction is obtained the amplified production of different fresh Herba Anoectochili roxburghii samples by gel electrophoresis, set up collection of illustrative plates according to the band that presents: be numbered X-coordinate with the Herba Anoectochili roxburghii strain, the site that exists with band is numbered ordinate zou, at each coordinate interconnecting part in length and breadth, there is amplified band to represent with " ━ ", there is not amplified band not mark, " there are " band or " nothing " to be with total luminance value as selecting foundation, adopt Bandscan software, choose brightness value 〉=0.2 for amplified band is arranged,＜0.2 for there not being amplified band;
(6) the ISSR-PCR molecular marking fingerprint that makes up is preserved, for the identification of Herba Anoectochili roxburghii
The finger printing that unknown sample is set up is under these conditions compared with the Herba Anoectochili roxburghii finger printing that gets, and Herba Anoectochili roxburghii is produced in the identical Huoshan that is of collection of illustrative plates, otherwise is not that Herba Anoectochili roxburghii is produced in the Huoshan.
Described SSR-PCR amplimer be in the following table 1 primer in the primer of itemizing or the combination of the primer more than 1, through the test of 100 many primers relatively, selected primer is as follows:
During more than a primer, every kind of primer add-on is identical, reduces the consumption of corresponding distilled water, keeps cumulative volume constant.
The present invention has following advantage:
(1) the present invention adopts the ISSR molecular marking technique to make up the finger printing of Herba Anoectochili roxburghii, to identifying at the Seedling Stage material, have important effect for the Stability and veracity that guarantees medicinal material Ji Yuan, and utilize chemical chromatographic fingerprinting to identify to compare: sampling less, Proper Sampling Period arbitrarily, is not subjected to the plant growth environment condition influence; With utilize molecule order-checking to identify to compare: link is simple, and by the extraction to the DNA of Herba Anoectochili roxburghii platymiscium, the pcr amplification of ISSR, agarose gel electrophoresis just can be differentiated collection of illustrative plates accurately and reliably, laborsaving, save time, economical;
(2) the invention provides the modification method of more efficiently extraction Herba Anoectochili roxburghii genomic dna;
(3) the present invention is optimized reaction system and the amplification program of pcr amplification, makes the result more stable and reliable;
(4) the amplified production polymorphism of selected primer of the present invention is high, and the gene type ability is very strong, can be used between the Herba Anoectochili roxburghii kind differentiating and plant in discriminating between population.
Description of drawings
Fig. 1 is 4 provinces, 8 ground Herba Anoectochili roxburghii ISSR-PCR experimental result electrophorograms, wherein, swimming lane 1 is Marker:1500bp DNA Ladder for Guangzhou Guangdong, swimming lane 6 Xiamen of Fujian Provinces, swimming lane 7 for Yongan City, Fujian Province, swimming lane M for ShenZhen,GuangDong, swimming lane 5 for Dongguan, Guangdong, swimming lane 4 for the Yingshan Mountains, Hubei, swimming lane 3 for Huoshan, swimming lane 2.
Embodiment
Embodiment
(1) fresh Herba Anoectochili roxburghii sample pretreatment
Fresh Herba Anoectochili roxburghii sample lucifuge is consumed starch more than 6 hours, get fresh Herba Anoectochili roxburghii blade 10 grams, with the above soaked in absolute ethyl alcohol secondary of 50 grams, each 10 minutes, dry;
(2) extraction of sample gene group DNA
Adopt improved CTAB method to extract the Herba Anoectochili roxburghii leaves genomic DNA, operate as follows:
1) pretreated blade is put into frappe ceramic mortar, and add an amount of quartz sand, then repeatedly adding liquid nitrogen fully is ground to Powdered, with spoon the blade powder is transferred to rapidly in the 10ml precooling Eppendorf centrifuge tube, the height of powder reaches 1/3 place of centrifuge tube, stops to add powder;
2) the CTAB Extraction buffer [2% (v/w) CTAB, 1.4M NaCl, 20mM EDTA, 100mM Tris-HCl (PH=8.0), 2% mercaptoethanol] of 65 ℃ of preheatings of adding 4m1 in centrifuge tube;
3) 65 ℃ of temperature are bathed 2h, middle every 10min counter-rotating 1～2 time;
4) the centrifugal 8min of 15000rpm changes supernatant liquor in another centrifuge tube over to;
5) be cooled to room temperature after, softly add isopyknic chloroform and primary isoamyl alcohol solution, wherein the volume ratio of chloroform and primary isoamyl alcohol is 24: 1, the light and slow 40min that puts upside down;
6) the centrifugal 10min of 15000rpm room temperature goes precipitation, and supernatant liquor is changed in another centrifuge tube;
7) add 2/3 volume Virahol at supernatant liquor, the light and slow mixing of putting upside down is placed more than the 1.5h for-20 ℃;
8) the centrifugal 10min of 15000rpm removes supernatant liquor;
9) wash twice to remove salt with 2ml 75% ethanol, wash once with dehydrated alcohol again;
10) remove ethanol, 37 ℃ of dry 20min are dissolved in 600 μ L damping fluids, transfer in the 1.5ml centrifuge tube;
11) adding 5 μ L final concentrations is 50 μ L/mL RnaseA, and 37 ℃ of temperature are bathed 1h;
12) the solution extracting that above-mentioned steps (11) is produced with isopyknic phenol, chloroform and primary isoamyl alcohol solution 2 times, by volume, phenol: chloroform: primary isoamyl alcohol=25:24:1;
13) the centrifugal 10min of 15000rpm gets supernatant liquor;
14) dehydrated alcohol of 2 times of volumes of adding ,-20 ℃ of insulation lh, every 20min upset is once;
15) 15000rpm is centrifugal, uses first 75% ethanolic soln washing precipitation 2-3 time of-20 ℃ of precoolings, again with absolute ethanol washing once.
16) 37 ℃ of temperature are bathed dry DNAs, add the dissolving of 100 μ L TE damping fluids for subsequent use, and 4 ℃ save backup;
(3) ISSR-PCR amplification
Utilize the sample gene group DNA extract as template, carry out the ISSR-PCR amplification at German Eppendorf 5331 grads PCR instrument, take the 40 μ L reaction systems that add two primers as example, add-on is as follows in the PCR pipe of 200 μ L:
The pcr amplification procedure condition is: 94 ℃ of denaturations 8 minutes, and 94 ℃ of sex change 45 seconds, 58-62 ℃ of annealing 45 seconds, 72 ℃ were extended 2 minutes, and 40 circulations of increase are at last in 72 ℃ of eventually extensions 10 minutes;
The amplified production that the ISSR-PCR amplified reaction obtains different Herba Anoectochili roxburghii samples is stored in-20 ℃ of refrigerators;
(4) agarose gel electrophoresis
Take the genomic dna of 4 provinces, 10 ground Herba Anoectochili roxburghiis as template, carry out the ISSR-PCR amplified reaction with two kinds of primers wherein, get amplified production 5 μ L-15 μ L on 1.5% the sepharose under the voltage of 3V/cm electrophoresis 2.5h, develop the color through EB, and record the gel electrophoresis result of amplified production with the gel imaging instrument;
(5) structure of the ISSR-PCR molecular marking fingerprint of each test sample
The ISSR-PCR amplified reaction is obtained the amplified production of different fresh Herba Anoectochili roxburghii samples by gel electrophoresis, set up collection of illustrative plates according to the band that presents: be numbered X-coordinate with the Herba Anoectochili roxburghii strain, the site that exists with band is numbered ordinate zou, at each coordinate interconnecting part in length and breadth, there is amplified band to represent with " ━ ", there is not amplified band not mark, " there are " band or " nothing " to be with total luminance value as selecting foundation, adopt Bandscan software, choose brightness value 〉=0.2 for amplified band is arranged,＜0.2 for there not being amplified band, record result such as following table:
Table 14 provinces 8 ground Herba Anoectochili roxburghii dna fingerprintings
(6) the ISSR-PCR molecular marking fingerprint that makes up is preserved, for the identification of Herba Anoectochili roxburghii
The finger printing that unknown sample is set up is under these conditions compared with the Herba Anoectochili roxburghii finger printing that gets, and Herba Anoectochili roxburghii is produced in the identical Huoshan that is of collection of illustrative plates, otherwise is not that Herba Anoectochili roxburghii is produced in the Huoshan.
Claims (2)
1. differentiate that the Huoshan produces the molecular marking fingerprint of Herba Anoectochili roxburghii and sibling species thereof for one kind, its feature is present in and may further comprise the steps:
(1) fresh Herba Anoectochili roxburghii sample pretreatment
Fresh Herba Anoectochili roxburghii sample lucifuge is consumed starch more than 6 hours, get fresh Herba Anoectochili roxburghii blade 10 grams, with the above soaked in absolute ethyl alcohol secondary of 50 grams, each 10 minutes, dry;
(2) extraction of sample gene group DNA
Adopt improved CTAB method to extract the Herba Anoectochili roxburghii leaves genomic DNA, operate as follows:
1) pretreated blade is put into frappe ceramic mortar, and add an amount of quartz sand, then repeatedly adding liquid nitrogen fully is ground to Powdered, with spoon the blade powder is transferred to rapidly in the 10ml precooling Eppendorf centrifuge tube, the height of powder reaches 1/3 place of centrifuge tube, stops to add powder;
2) the CTAB Extraction buffer [2% (v/w) CTAB, 1.4M NaCl, 20mM EDTA, 100mM Tris-HCl (PH=8.0), 2% mercaptoethanol] of 65 ℃ of preheatings of adding 4m1 in centrifuge tube;
3) 65 ℃ of temperature are bathed 2h, middle every 10min counter-rotating 1～2 time;
4) the centrifugal 8min of 15000rpm changes supernatant liquor in another centrifuge tube over to;
5) be cooled to room temperature after, softly add isopyknic chloroform and primary isoamyl alcohol solution, wherein the volume ratio of chloroform and primary isoamyl alcohol is 24: 1, the light and slow 40min that puts upside down;
6) the centrifugal 10min of 15000rpm room temperature goes precipitation, and supernatant liquor is changed in another centrifuge tube;
7) add 2/3 volume Virahol at supernatant liquor, the light and slow mixing of putting upside down is placed more than the 1.5h for-20 ℃;
8) the centrifugal 10min of 15000rpm removes supernatant liquor;
9) wash twice to remove salt with 2ml 75% ethanol, wash once with dehydrated alcohol again;
10) remove ethanol, 37 ℃ of dry 20min are dissolved in 600 μ L damping fluids, transfer in the 1.5ml centrifuge tube;
11) adding 5 μ L final concentrations is 50 μ L/mL RnaseA, and 37 ℃ of temperature are bathed 1h;
12) the solution extracting that above-mentioned steps (11) is produced with isopyknic phenol, chloroform and primary isoamyl alcohol solution 2 times, by volume, phenol: chloroform: primary isoamyl alcohol=25:24:1;
13) the centrifugal 10min of 15000rpm gets supernatant liquor;
14) dehydrated alcohol of 2 times of volumes of adding ,-20 ℃ of insulation lh, every 20min upset is once;
15) 15000rpm is centrifugal, uses first 75% ethanolic soln washing precipitation 2-3 time of-20 ℃ of precoolings, again with absolute ethanol washing once.
16) 37 ℃ of temperature are bathed dry DNAs, add the dissolving of 100 μ LTE damping fluids for subsequent use, and 4 ℃ save backup;
(3) ISSR-PCR amplification
Utilize the sample gene group DNA that extracts as template, carry out the ISSR-PCR amplification at German Eppendorf 5331 grads PCR instrument, the PCR reaction system is characterized as: template concentrations 25 ng/L, primer concentration 0.5 μ molL
-1, Mg
2+Concentration 2.0mmolL
-1, dNTP concentration 200 μ molL
-1, Taq polymerase concentration 1 U/20 μ l;
The pcr amplification procedure condition is: 94 ℃ of denaturations 8 minutes, and 94 ℃ of sex change 45 seconds, 58-62 ℃ of annealing 45 seconds, 72 ℃ were extended 2 minutes, and 40 circulations of increase are at last in 72 ℃ of eventually extensions 10 minutes;
The amplified production that the ISSR-PCR amplified reaction obtains different Herba Anoectochili roxburghii samples is stored in-20 ℃ of refrigerators;
(4) agarose gel electrophoresis
Take the genomic dna of 4 provinces, 10 ground Herba Anoectochili roxburghiis as template, carry out the ISSR-PCR amplified reaction with two kinds of primers wherein, get amplified production 5 μ L-15 μ L on 1.5% the sepharose under the voltage of 3V/cm electrophoresis 2.5h, develop the color through EB, and record the gel electrophoresis result of amplified production with the gel imaging instrument;
(5) structure of the ISSR-PCR molecular marking fingerprint of each test sample
The ISSR-PCR amplified reaction is obtained the amplified production of different fresh Herba Anoectochili roxburghii samples by gel electrophoresis, set up collection of illustrative plates according to the band that presents: be numbered X-coordinate with the Herba Anoectochili roxburghii strain, the site that exists with band is numbered ordinate zou, at each coordinate interconnecting part in length and breadth, there is amplified band to represent with " ━ ", there is not amplified band not mark, " there are " band or " nothing " to be with total luminance value as selecting foundation, adopt Bandscan software, choose brightness value 〉=0.2 for amplified band is arranged,＜0.2 for there not being amplified band;
(6) the ISSR-PCR molecular marking fingerprint that makes up is preserved, for the identification of Herba Anoectochili roxburghii
The finger printing that unknown sample is set up is under these conditions compared with the Herba Anoectochili roxburghii finger printing that gets, and Herba Anoectochili roxburghii is produced in the identical Huoshan that is of collection of illustrative plates, otherwise is not that Herba Anoectochili roxburghii is produced in the Huoshan.
2. the molecular marking fingerprint of Herba Anoectochili roxburghii and sibling species thereof is produced in a kind of Huoshan of differentiating according to claim 1, its feature is present in, described SSR-PCR amplimer be in the following table 1 primer in the primer of itemizing or the combination of the primer more than 1, test through 100 many primers is compared, and selected primer is as follows:
During more than a primer, every kind of primer add-on is identical, reduces the consumption of corresponding distilled water, keeps cumulative volume constant.
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| CN105018467A (en) * | 2015-07-17 | 2015-11-04 | 福建农林大学 | Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis |
| CN105463088A (en) * | 2015-12-21 | 2016-04-06 | 武汉大学 | Method for authenticating authenticity and/or purity of nelumbo nucifera variety by adopting EST-SSR marker |
| CN106498064A (en) * | 2016-11-08 | 2017-03-15 | 皖西学院 | Differentiate the ISSR fingerprint spectrum methods of three kinds of medicinal dendrobiums and its sibling species |
| CN106508661A (en) * | 2016-11-01 | 2017-03-22 | 玉林师范学院 | Method for constructing anoectochilus formosanus polyploidy resource |
| CN108998565A (en) * | 2018-09-28 | 2018-12-14 | 杭州师范大学 | A kind of molecular specificity labeled primers and its discrimination method of anoectochilus formosanus |
| CN109022610A (en) * | 2018-09-13 | 2018-12-18 | 杭州师范大学 | A kind of molecular specificity labeled primers and its discrimination method of roxburgh anoectochilus terminal bud |
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| CN105018467A (en) * | 2015-07-17 | 2015-11-04 | 福建农林大学 | Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis |
| CN105463088A (en) * | 2015-12-21 | 2016-04-06 | 武汉大学 | Method for authenticating authenticity and/or purity of nelumbo nucifera variety by adopting EST-SSR marker |
| CN105463088B (en) * | 2015-12-21 | 2018-07-03 | 武汉大学 | The method for carrying out the lotus kind true and false and/or Purity is marked using EST-SSR |
| CN106508661A (en) * | 2016-11-01 | 2017-03-22 | 玉林师范学院 | Method for constructing anoectochilus formosanus polyploidy resource |
| CN106508661B (en) * | 2016-11-01 | 2018-07-24 | 玉林师范学院 | A method of creating roxburgh anoectochilus terminal bud polyploid resource |
| CN106498064A (en) * | 2016-11-08 | 2017-03-15 | 皖西学院 | Differentiate the ISSR fingerprint spectrum methods of three kinds of medicinal dendrobiums and its sibling species |
| CN109022610A (en) * | 2018-09-13 | 2018-12-18 | 杭州师范大学 | A kind of molecular specificity labeled primers and its discrimination method of roxburgh anoectochilus terminal bud |
| CN109022610B (en) * | 2018-09-13 | 2021-08-10 | 杭州师范大学 | Molecular specificity marker primer of anoectochilus formosanus and identification method thereof |
| CN108998565A (en) * | 2018-09-28 | 2018-12-14 | 杭州师范大学 | A kind of molecular specificity labeled primers and its discrimination method of anoectochilus formosanus |
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