CN105087804B - For identifying primer sets, kit and the method for identifying Desmodium styracifolium type of Desmodium styracifolium type - Google Patents

For identifying primer sets, kit and the method for identifying Desmodium styracifolium type of Desmodium styracifolium type Download PDF

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CN105087804B
CN105087804B CN201510551808.3A CN201510551808A CN105087804B CN 105087804 B CN105087804 B CN 105087804B CN 201510551808 A CN201510551808 A CN 201510551808A CN 105087804 B CN105087804 B CN 105087804B
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primer
desmodium styracifolium
issr
pcr
dna
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CN105087804A (en
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王学海
许勇
李莉娥
杨仲文
冯芸
尹海龙
杨婷
黄璐
余通
曹儒宾
谢长
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WUHAN KANGLE PHARMACEUTICAL Co.,Ltd.
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Wuhan Guanggu Humanwell Biological Pharmaceutical Co Ltd
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Abstract

The present invention proposes primer sets, the kit for identifying Desmodium styracifolium type and the method for identifying Desmodium styracifolium type for identifying Desmodium styracifolium type.For identifying that the primer sets of Desmodium styracifolium type are made up of following：First primer to the 7th primer, wherein, first primer to the 7th primer has SEQ ID NO respectively：Nucleotide sequence shown in 1~7.The present invention is used to identify that the primer of Desmodium styracifolium type has at least one of following advantages：Accuracy versatile, that Desmodium styracifolium type is identified using the primer is high, objectivity is strong and stability is high.

Description

For identifying primer sets, kit and the identification Desmodium styracifolium of Desmodium styracifolium type The method of type
Technical field
The present invention relates to biological field.In particular it relates to for the primer sets for identifying Desmodium styracifolium type, it is used for The method identified the kit of Desmodium styracifolium type and identify Desmodium styracifolium type.
Background technology
Desmodium styracifolium, Chinese medicine name, for the dry aerial parts of legume Desmodium styracifolium.Its effect is removing dampness through diuresis and removing jaundice, profit Urinate treating stranguria, cure mainly jaundice, heat gonorrhea, urolithiasis, the puckery disease of urine, oedema oliguria.It is distributed in Fujian, Hunan, Guangxi and Guangdong etc. Provinces and regions.
However, the identification for Desmodium styracifolium still has much room for improvement at present.
The content of the invention
It is contemplated that at least solves one of technical problem present in prior art.Therefore, it is an object of the invention to It is proposed a kind of be used to identifying the primer sets of Desmodium styracifolium type, a kind of be used to identify the kit and one kind of Desmodium styracifolium type The method for identifying Desmodium styracifolium type.Using the primer sets for identifying Desmodium styracifolium type, for identifying Desmodium styracifolium type Kit and identify that the method for Desmodium styracifolium type can be with objective, accurate or stably identify the class of Desmodium styracifolium Type.
It should be noted that the present invention is the following discovery based on inventor and completed：
By number of ways such as literature survey, market survey and on-site inspections to Desmodium styracifolium medicinal plant in wild resource Investigated with cultivation resource, it is found that Desmodium styracifolium wild resource is distributed sparse and negligible amounts, cultivation resource turns into market master Circulate commodity.In addition, it is big active constituent content difference to be present between natural crude drugs and cultivation medicinal material, different producing area cultivation medicinal material Etc. mass discrepancy, and the good and the bad of medicinal material has material impact to the quality of product, it must be carried out in process of production strictly Control.Character discriminating is carried out according to features such as its formalness, sections, there is certain subjectivity, to identification person's professional knowledge And skill requirement is very high, and the technology is only capable of differentiating the true and false of Desmodium styracifolium, can not judge its distributed areas, warp can not expire The demand in sufficient market.
The present inventor has found by many experiments, gathers the Desmodium styracifolium of different zones respectively as sample, divides DNA is indescribably taken, is expanded using primer pair DNA fragmentation, the product of amplification is then subjected to gel electrophoresis, and to electrophoresis result Analyzed, identify Desmodium styracifolium type.
In the first aspect of the present invention, the present invention proposes a kind of primer sets for being used to identify Desmodium styracifolium type.According to Embodiments of the invention, this is used to identify that the primer sets of Desmodium styracifolium type to be made up of following：First primer to the 7th primer, its In, first primer to the 7th primer has SEQ ID NO respectively：Nucleotide sequence shown in 1~7.The primer is used to expand Fragment on augmentation desmodium genomic DNA, by analyzing amplified production, further to identify the type of Desmodium styracifolium.By This, what is finally given is used to identify that the primer of Desmodium styracifolium type to have at least one of following advantages：It is versatile, utilize this The accuracy height of primer identification Desmodium styracifolium type, objectivity is strong and stability is high.
According to an embodiment of the invention, the above-mentioned primer sets for being used to identify Desmodium styracifolium type can also have following add At least one technical characteristic：
According to one embodiment of present invention, first primer to the 7th primer has identical molar content.This hair Bright restriction the first primer to the 7th primer need to have identical molar content, it is therefore an objective to which the concentration for eliminating primer is clear to amplified band The influence of the amplification efficiency such as clear degree or specificity.Thus, it is according to embodiments of the present invention to be used to identify Desmodium styracifolium type Primer can further be had stronger versatility, the stronger accuracy of Desmodium styracifolium type is identified using the primer, relatively strong visitor The property seen or higher stability.
In the second aspect of the present invention, the present invention proposes a kind of kit for being used to identify Desmodium styracifolium type.As before It is described, according to an embodiment of the invention, including：It is described previously for identifying the primer sets of Desmodium styracifolium type.Therefore, finally What is obtained is used to identify that the kit of Desmodium styracifolium type to have at least one of following advantages：It is easy to use, versatile, sharp With the accuracy of kit identification Desmodium styracifolium type is high, objectivity is strong and stability is high.
In the third aspect of the present invention, the present invention proposes a kind of method for identifying Desmodium styracifolium type.As it was previously stated, root According to embodiments of the invention, including：(1) nucleic acid samples are entered with performing PCR amplification, to obtain amplified production, wherein, the nucleic acid Sample contains the genomic DNA from Desmodium styracifolium sample to be identified, the PCR amplifications using foregoing primer sets or Foregoing kit；And the composition of (2) based on the amplified production, determine the class of the Desmodium styracifolium sample to be identified Type.Due to different types of Desmodium styracifolium, the nucleotide sequence of gene is there is otherness between its individual, by Desmodium styracifolium Genomic DNA fragment is expanded, and the composition of amplified production is analyzed, and determines the type of Desmodium styracifolium sample.Thus, most The method of the identification Desmodium styracifolium type obtained eventually has at least one of following advantages：It is easy to operate, primer is versatile, accurate True property is high, objectivity is strong and stability is high.
According to an embodiment of the invention, the method for above-mentioned identification Desmodium styracifolium type can also have following supplementary technology special At least one sign：
According to an embodiment of the invention, the reaction system based on 20 μ L, the PCR amplification conditions are：The μ L of formamide 0.5； 10×Taq buffer 2μL；Mg2+2.0μL；The μ L of template DNA 1.0；dNTP 1.5μL；The μ L of primer 1.0；The μ L of Taq enzyme 1.0；With And the ddH of surplus2O.Inventor optimizes to obtain PCR amplification conditions by many experiments.Addition formamide can strengthen PCR sequences Analysis, prevent phenomena such as background is strong, distinguished sequence analyzes weak ladder, ambiguous signal and too early termination.Work as mould The disperse background accordingly strengthened occurs in plate DNA adding too much, loading wells to swimming lane, otherwise template DNA is too low, amplified production Intensity accordingly weakens, no amplified production.When template DNA concentration is 1.0 μ L, amplified production is basicly stable, band is clear, stably And high resolution.Mg2+For the metal cofactor of polymerase, there is stronger influence to the activity for influenceing polymerase.When primer adds When dosage is 1.0 μ L, amplification is basically identical, but with the gradual increase of primer concentration, starts disperse background enhanced occur Trend, and some amplified bands occur weaken or missing.Thus, the method for identification Desmodium styracifolium according to embodiments of the present invention Can further have easier operation, stronger primer versatility, higher accuracy, stronger objectivity or higher Stability.
According to an embodiment of the invention, step (2) includes：(2-1) carries out gel electrophoresis to the amplified production；And Whether (2-2) there is band based on the gel electrophoresis in predetermined length region, determines the Desmodium styracifolium sample to be identified Type.At least two Desmodium styracifolium sample of nucleic acid are entered with performing PCR amplification, then carries out gel electrophoresis, predetermined length region occurs The sample of band and the sample for not occurring band have otherness, and the type of Desmodium styracifolium is determined according to this difference.Thus, according to The method of the identification Desmodium styracifolium of the embodiment of the present invention can further have easier operation, stronger primer versatility, Higher accuracy, stronger objectivity or higher stability.
According to an embodiment of the invention, in step (2-1), the condition of the gel electrophoresis is：1.5% agarose coagulates Glue, electrophoretic voltage 110V/cm, electrophoresis time 90 minutes.Inventor passes through many experiments, Ago-Gel concentration, electrophoresis electricity Can pressure and electrophoresis time most important to obtain clear band.Inventor passes through conditional filtering, it has unexpectedly been found that, work as agarose The concentration of gel is 1.5%, and electrophoretic voltage 110V/cm, can be effectively to amplified production when electrophoresis time is 90 minutes DNA sample makes a distinction, and inventor has found that, when the concentration of Ago-Gel is more than 1.5%, DNA molecular is in Ago-Gel In translational speed it is very slow, cause all bands all to concentrate in a less region, it is difficult to distinguish, cause electrophoresis Time is grown, and Ago-Gel heating is serious, easily melts.If the concentration of Ago-Gel is less than 1.5%, DNA can be caused Translational speed of the molecule in Ago-Gel is very fast, also results in all bands and all concentrates on a less area In domain, it is difficult to distinguish, or even Ago-Gel can be departed from and entered in electrolyte, cause electrolyte contamination.In addition, invention human hair It is existing, if electrophoresis time more than 90 minutes, can cause testing sample band and Marker to depart from gel, cause electrolyte dirty Dye, if electrophoresis time is less than 90 minutes, testing sample band can be caused can not to be efficiently differentiated with other DNA moleculars and held. If electrophoretic voltage can cause too fast, all DNA of translational speed of the DNA molecular in Ago-Gel more than 110V/cm Molecule is concentrated in less region, and so as to be difficult to differentiate between, while gel can generate heat very fast and cause glue to melt under high voltages Change.If brownout, translational speed of the DNA molecular in Ago-Gel can be caused very slow, cause all bars Band is all concentrated in a less region, it is difficult to is distinguished.Thus, the method for identification Desmodium styracifolium according to embodiments of the present invention Can further have easier operation, stronger primer versatility, higher accuracy, stronger objectivity or higher Stability.
According to an embodiment of the invention, the predetermined length region is that the Desmodium styracifolium sample based on known type determines 's.Thus, the method for identification Desmodium styracifolium according to embodiments of the present invention can further have easier operation, stronger Primer versatility, higher accuracy, stronger objectivity or higher stability.
According to an embodiment of the invention, the Desmodium styracifolium sample in the known source comes from least one following place of production： Warehouse ridge village of golden mean of the Confucian school township of Lingui area of Guilin City；Liuzhou City Rongan County Si Ding towns San Po villages；Wuzhou Cangwu County Xin Di towns Dou Mei villages； Jilin village of Laibin City Xingbin District Qiao Gong townshiies；Lingui county,gui lin Wu Tong towns Dong Ling villages；As if lingui county,gui lin Tian Town；Guilin City faces Golden mean of the Confucian school village of golden mean of the Confucian school township of osmanthus area；Zhanjiang City Suixi County Wu Tang towns；Zhanjiang City Suixi county town moon town Shi Rong villages；Boluo County of Huizhou City Luo Yang Town shaven head village；Shanyi City Haifeng County Mei Long towns silvering solution Jiu Jing villages；Zhanjiang City Suixi County Wu Tang towns；Boluo County of Huizhou City Luo Yang towns lane Mouth village；Zijin County Lan Tangzhen cities Bei Cun；And Huazhou City west of a river causeway road.Desmodium styracifolium is distributed mainly on Fujian, Hunan, Guangxi With the provinces and regions such as Guangdong, so carrying out sample collection in above-mentioned area.Thus, according to embodiments of the present invention identification Desmodium styracifolium Method can further have easier operation, stronger primer versatility, higher accuracy, stronger objectivity or Higher stability.
In the fourth aspect of the present invention, the present invention proposes a kind of method for identifying Desmodium styracifolium type.According to the present invention Embodiment, methods described includes：
The extraction of (3-1) Desmodium styracifolium plant genome DNA
Blade surface is cleaned with absolute ethyl alcohol after the fresh Desmodium styracifolium vanes discoloration silica gel absorbent drying collected, is treated Absolute ethyl alcohol is cut into tiny fragment with scissors after volatilizing, ball milling instrument grind into powder is used rapidly after adding liquid nitrogen.
The blade powder (about 30mg) of milled is quickly adding into 800 μ L and is preheating to 65 DEG C of DNA Extraction buffers (in advance First add RNase 50 μ g) in 65 DEG C of water-baths 25 minutes, every 5 minutes by EP manage it is reverse for several times, prevent blade powder agglomates.Take out EP is managed, and is placed in and on ice, is added isometric Tris- saturated phenols：Chloroform：Isoamyl alcohol (25:24:1) centrifugation is put into after, fully shaking up Machine, 10000rpm centrifugations 10min, takes 600 μ L of supernatant liquid at 20 DEG C.Isometric chloroform is added into supernatant：Isoamyl alcohol (24: 1) it is put into after, shaking up in centrifuge, 10000rpm centrifuges 10min at 20 DEG C, takes the μ L of supernatant 400 in the EP pipes after sterilizing. This supernatant is unpurified DNA solution.
The genomic DNA of extraction is purified from Tiangeng plant genes group extracts kit.To unpurified 700 μ L buffer solution GP2 are added in DNA solution, are fully mixed.The liquid of mixing is transferred in adsorption column CB3,10000rpm centrifugations 30s, discard waste liquid (absorption column volume is 700 μ L, and graded adds centrifugation).500 μ L buffer solution is added into adsorption column CB3 GD (is previously added absolute ethyl alcohol), places 10min, 10000rpm centrifugation 30s, discards waste liquid.600 μ L rinsings are added into CB3 Liquid PW, (being previously added absolute ethyl alcohol) 10000rpm centrifuge 30s, outwell waste liquid.90% ethanol, 10000rpm are added into CB3 30s is centrifuged, outwells waste liquid.CB3 is placed in room temperature and places 15min, volatilizes ethanol.CB3 is put into the EP pipes after sterilizing, added 100 μ L TE buffer solutions (being preheated to 40 DEG C), room temperature are placed 5 minutes, and 10000rpm centrifugation 2min, the solution that centrifugation is obtained is again It is added in adsorption column CB3, room temperature places 3min, 10000rpm centrifugation 2min, obtains Desmodium styracifolium DNA after purification.
The optimization and its stability analysis of (3-2) ISSR-PCR reaction systems
The foundation of (3-2-1) ISSR-PCR amplification programs
Based on the 60 ISSR universal primers announced by Columbia University, with NEW ENGLAND BIOLABS Tm The online calculator of Calculator primer annealing temperatures and the primer-design softwares of Primer Premier 5 are to its annealing temperature Calculated, Tm values are near 50 DEG C.Preliminary program is arranged to：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 45s；50 DEG C of annealing 40s；72 DEG C of extension 60s；Circulation 40 times；72 DEG C of extension 10min；4 DEG C of preservations.
(3-2-2) ISSR-PCR reacts the concentration of working solution
Each reaction substrate concentration is as follows：
Substrate Concentration
10×Taq buffer -
Mg2+ -
dNTPmixture 2.5mM
Primer 10mM
Template DNA ~100ng/uL
Formamide -
ddH2O -
(3-4) ISSR-PCR is expanded and its product analysis
(3-4-1) ISSR-PCR is expanded
Under conditions of the ISSR-PCR reaction systems listed by table 5 and the annealing temperature listed by table 6, to 15 pyrenes acid Sample carries out ISSR-PCR amplified reactions.After amplified reaction, the μ L of PCR reaction products 7 are taken to be mixed with 3 μ L nucleic acid dye working solutions Close, electrophoresis detection, voltage 110V/cm, electrophoresis time 90min, with 100-II are carried out in 1.5% Ago-Gel BpDNA Marker are the length scales of PCR primer fragment.After the completion of electrophoresis, blob of viscose is placed on uv analyzer and takes pictures and remembers Record.According to product carry out gel electrophoresis after result show, the product specificities that two primers of UBC836, UBC890 amplify compared with Difference, it is small to the separating capacity of 15 parts of nucleic acid samples, and UBC808, UBC809, UBC842, UBC844, UBC845, UBC855, Totally 7 primer pair sample template DNA cloning effects are preferable by UBC888, preferably expand the band that preferable 7 primers amplify and make For the object of follow-up cluster analysis.For convenience of description, UBC808 is named as the first primer, UBC809 is named as the second primer, The like, UBC842 to UBC888 is named as the 3rd to the 7th primer.
The ISSR-PCR reaction systems of table 5
Reaction substrate Add volume (μ L)
ddH2O 11.0
Formamide 0.5
10×Taq buffer 2.0
Mg2+ 2.0
Template DNA 1.0
dNTP mixture 1.5
Primer 1.0
Taq enzyme 1.0
The setting of primer annealing temperature in the ISSR-PCR of table 6
(3-4-2) cluster analysis
From electrophoretogram as can be seen that 7 primer coamplifications go out 67 band, wherein polymorphic bandses amount to 51, polymorphic Sex rate up to 76.1%, every primer it is amplifiable go out 5~11 specific bands, average every primer it is amplifiable go out 7.29 rules Band.Amplified production has band to be calculated as 1 in identical migration position, and no band is calculated as 0, builds 0-1 raw data matrixs, is carried out with SPSS Cluster analysis, and clustering tree is established, and cluster result is analyzed.
In addition, according to an embodiment of the invention, the present invention is used to identifying primer sets of Desmodium styracifolium type, wide for identifying The kit of desmodium type and the method for identification Desmodium styracifolium type have at least one of advantages below：
1st, according to an embodiment of the invention, the present invention is used to identify that the primer sets of Desmodium styracifolium type have versatility, no With stronger species specificity, design cost is reduced, and accuracy is high, stability is high.
2nd, according to an embodiment of the invention, the method for identification Desmodium styracifolium type is mainly Characters Identification method at present, is had Stronger subjectivity.The present invention identification Desmodium styracifolium type method there is objectivity, can accurately, stably the wide money of determination The type of grass.
3rd, according to an embodiment of the invention, the present invention is used to identify that the kit of Desmodium styracifolium type is easy to use, can Accurately, the type of Desmodium styracifolium stably, is objectively determined.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
Fig. 1 shows 15 parts of template DNA electrophoretograms according to an embodiment of the invention；
Fig. 2 shows ISSR-PCR reactions amplification program according to an embodiment of the invention；
Fig. 3 shows 9 primers according to an embodiment of the invention respectively with the electrophoretogram of two different templates amplifications；
Fig. 4 shows the electrophoretogram of UBC808ISSR-PCR amplifications according to an embodiment of the invention；
Fig. 5 shows the electrophoretogram of UBC809ISSR-PCR amplifications according to an embodiment of the invention；
Fig. 6 shows the electrophoretogram of UBC842ISSR-PCR amplifications according to an embodiment of the invention；
Fig. 7 shows the electrophoretogram of UBC844ISSR-PCR amplifications according to an embodiment of the invention；
Fig. 8 shows the electrophoretogram of UBC845ISSR-PCR amplifications according to an embodiment of the invention；
Fig. 9 shows the electrophoretogram of UBC855ISSR-PCR amplifications according to an embodiment of the invention；
Figure 10 shows the electrophoretogram of UBC888ISSR-PCR amplifications according to an embodiment of the invention；And
Figure 11 shows cluster analysis figure according to an embodiment of the invention.
Embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment Part, carried out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
Embodiment 1
Material, instrument and reagent
1st, Desmodium styracifolium sample collection
15 parts of samples for experiment gather in 2014 7, August in Guangxi, the province of Guangdong two, the fresh blade collected It is put into normal temperature in the valve bag equipped with discoloration silica gel after being wrapped with filter paper in locality to preserve, the holding time is one week.It is 15 parts wide Desmodium crude drug source information is shown in Table 1.
The Desmodium styracifolium source table of table 1
Sample number into spectrum The place of production Cultivation/wild
A Warehouse ridge village of golden mean of the Confucian school township of Lingui area of Guilin City Cultivation
B Liuzhou City Rongan County Si Ding towns San Po villages Cultivation
C Wuzhou Cangwu County Xin Di towns Dou Mei villages Semi-wild
D Jilin village of Laibin City Xingbin District Qiao Gong townshiies Semi-wild
E Lingui county,gui lin Wu Tong towns Dong Ling villages Cultivation
F As if lingui county,gui lin Tian Town Cultivation
G Golden mean of the Confucian school village of golden mean of the Confucian school township of Lingui area of Guilin City Cultivation
H Zhanjiang City Suixi County Wu Tang towns Cultivation
I Zhanjiang City Suixi county town moon town Shi Rong villages Cultivation
J Boluo County of Huizhou City Luo Yang towns shaven head village Semi-wild
K Shanyi City Haifeng County Mei Long towns silvering solution Jiu Jing villages Cultivation
L Zhanjiang City Suixi County Wu Tang towns It is wild
M Boluo County of Huizhou City Luo Yang towns entrance to a lane village It is wild
N Zijin County Lan Tangzhen cities Bei Cun Cultivation
O Huazhou City west of a river causeway road Semi-wild
2nd, instrument and reagent
Thermostat water bath, high speed freezing centrifuge (EPPENDORF CENTRIFUGE 5424R), ball milling instrument (RETECH MM400), ELIASA (SPECTRAMAX M190), micro-wave oven, grads PCR amplification instrument (BIO-RAD), PCR amplification instrument (TECHNE), electrophoresis apparatus (Beijing Jun Yi companies), uv analyzer ZF1-II (praise roc science and technology in Shanghai), pipettor (EPPENDORF)。
Cetyl trimethylammonium bromide (CTAB), PVP-K30, beta -mercaptoethanol, Tris-HCl, EDTA-Na2, NaCl, Chloroform, isoamyl alcohol, Tris alkali, glacial acetic acid, EDTA, NaOH, Tris- saturated phenols, RNase (U.S. SIGMA), Tiangeng novel plant Genome extracts kit (Beijing Tiangeng biochemical technology), 10 × Taq buffer (Mg2+Free) (VAZYME companies), 25mM Mg2+(VAZYME companies), Taq enzyme (VAZYME companies), dNTP MIXTURE (10mM each) (Shanghai life work), PRIMER (on Extra large JaRa bioengineering Co., Ltd synthesis), formamide (Shanghai Jierui Biology Engineering Co., Ltd), 6 × loading Buffer, 50 × TAE, Sybra Green I, Ago-Gel (Shanghai Jierui Biology Engineering Co., Ltd), Wahaha is pure Water, absolute ethyl alcohol.
Embodiment 2
Solution allocation
1st, 50 × TAE solution
Precise 242g Tris alkali, 57.1ml glacial acetic acids, 100ml 0.5mol/L EDTA, pH8.0 is adjusted to NaOH, Water is settled to 1L, stand-by.
Electrophoretic buffer：50 × TAE solution is diluted 50 times with pure water, obtains 1 × TAE working solutions.
2nd, DNA Extraction buffers
2% (W/V) CTAB, 3% (W/V) PVP, 1mol/L Tris-Hcl (pH=8.0) 50mL, 0.5mol/L EDTA- Na2(pH=8.0) 20mL, NaCl 41g, water are settled to 500ml.The μ L of β-thioglycol 1000 are added after sterilizing.
3rd, nucleic acid dye working solution
After Sybra Green I are diluted into 100 times with 1 × TAE working solutions, 6 × loading of 2 times of volumes is added Buffer, fully mix, obtain the working solution of nucleic acid dye.
Embodiment 3
The extraction of Desmodium styracifolium plant genome DNA
Blade surface is cleaned with absolute ethyl alcohol after the fresh Desmodium styracifolium vanes discoloration silica gel absorbent drying collected, is treated Absolute ethyl alcohol is cut into tiny fragment with scissors after volatilizing, ball milling instrument grind into powder is used rapidly after adding liquid nitrogen.
The blade powder (about 30mg) of milled is quickly adding into 800 μ L and is preheating to 65 DEG C of DNA Extraction buffers (in advance Add RNase50ug) in 65 DEG C of water-baths 25 minutes, every 5 minutes by EP manage it is reverse for several times, prevent blade powder agglomates.Take out EP Pipe, is placed in and on ice, adds isometric Tris- saturated phenols：Chloroform：Isoamyl alcohol (25:24:1) centrifugation is put into after, fully shaking up Machine, 10000rpm centrifugations 10min, takes 600 μ L of supernatant liquid at 20 DEG C.Isometric chloroform is added into supernatant：Isoamyl alcohol (24: 1) it is put into after, shaking up in centrifuge, 10000rpm centrifuges 10min at 20 DEG C, takes the μ L of supernatant 400 in the EP pipes after sterilizing. This supernatant is unpurified DNA solution.
Embodiment 4
DNA is purified
The genomic DNA extracted in embodiment 3 is purified from Tiangeng plant genes group extracts kit.To 700 μ L buffer solution GP2 are added in 3 unpurified DNA solution of embodiment, are fully mixed.The liquid of mixing is transferred to adsorption column CB3 In, 10000rpm centrifugation 30s, discard waste liquid (absorption column volume is 700 μ L, and graded adds centrifugation).Add into adsorption column CB3 Enter 500 μ L buffer solution GD (being previously added absolute ethyl alcohol), place 10min, 10000rpm centrifugation 30s, discard waste liquid.To CB3 It is middle to add 600 μ L rinsing liquid PW, (being previously added absolute ethyl alcohol) 10000rpm centrifugation 30s, outwell waste liquid.Added into CB3 90% ethanol, 10000rpm centrifugation 30s, outwells waste liquid.CB3 is placed in room temperature and places 15min, volatilizes ethanol.CB3 is put into and gone out In EP pipes after bacterium, 100 μ L TE buffer solutions (being preheated to 40 DEG C) are added, room temperature is placed 5 minutes, 10000rpm centrifugation 2min, The solution that centrifugation obtains is then added in adsorption column CB3, room temperature places 3min, 10000rpm centrifugation 2min, obtains after purification Desmodium styracifolium DNA.
Embodiment 5
DNA purity detectings
1st, the preparation of 1% (M/V) Ago-Gel
Weigh 0.5g Ago-Gel powder to be put into conical flask, add 50mL 1 × TAE solution, shake up, micro-wave oven adds Heat to agarose powder melts (boiling 2 times), and the Ago-Gel of thawing is cooled into 70 DEG C or so, pours into the punching of glue bed, Comb is inserted, comb is extracted after solidification to be cooled, it is stand-by.
2nd, purity detecting
1% agarose is added to after fully being mixed with 5 μ L DNA solutions with pipettor 3 μ L nucleic acid dyes working solutions of absorption to coagulate In the groove of glue, the electrophoresis 30min under 5V/cm voltage, blob of viscose is put on uv analyzer after the completion of electrophoresis and detects and clap According to record, as a result if Fig. 1, wherein m are the nucleic acid samples numbering in 1000bp DNA Marker, A-O difference corresponding tables 1.By scheming It can be seen that the DNA bands that purifying obtains are clear, disturb in the absence of RNA in 1.
20 μ L DNA mother liquors are drawn with pipettor, after 5 times of dilution, are splined on 96 orifice plates.With ELIASA to its concentration and pure Degree is measured.As a result each DNA sample OD is shown260/OD280Within 1.7-1.9, show DNA purity height, can carry out down The PCR reactions of one step.DNA solution after purification is put in -86 DEG C of refrigerators and frozen above.
Embodiment 6
The optimization and its stability analysis of ISSR-PCR reaction systems
ISSR (inter-simple sequence repeat) is the molecular labeling on the basis of a kind of microsatellite.Its is basic Principle is：Be primer with the microsatellite DNA (SSR sequences) of grappling, i.e., it is random plus 2-4 at the 3' ends of SSR sequences or 5' ends Nucleotides, in PCR reactions, anchor primer can cause specific site to be annealed, and cause the interval complementary with anchor primer less big Repetitive sequence between DNA fragmentation enter performing PCR amplification.Multiple bands of institute's amplification region are differentiated by gel electrophoresis, amplification Bands of a spectrum are mostly dominant performance.
The single-copy sequence for obtaining SSR both sides, development cost drop need to be sequenced in the exploitation of ISSR primers unlike SSR primers It is low.Compared with SSR marker, ISSR primers can be general between different species, has stronger kind special unlike SSR marker The opposite sex；Compared with RAPD and RFLP, the polymorphism that ISSR is disclosed is higher, can obtain the information content times over RAPD, accuracy is several It can be compared favourably with RFLP, detection is very convenient, thus is a kind of very promising molecular labeling.
What term " polymorphism " used in the present invention was known to the skilled person, refer to appropriate frequency at one The phenomenon of two or more variation occurs for the specific genetic locus of some of colony (gene order or non-genomic sequence).
1st, the foundation of ISSR-PCR amplification programs
Based on the 60 ISSR universal primers announced by Columbia University, with NEW ENGLAND BIOLABS Tm The online calculator of Calculator primer annealing temperatures and the primer-design softwares of Primer Premier 5 are to its annealing temperature Calculated, Tm values are near 50 DEG C.Preliminary program, which is set, sees Fig. 2.
2nd, ISSR-PCR reacts the concentration of working solution
Each reaction substrate concentration is shown in Table 2.
The concentration of 2 each reaction substrate of table
Substrate Concentration
10×Taq buffer -
Mg2+ -
dNTPmixture 2.5mM
Primer 10mM
Template DNA ~100ng/uL
Formamide -
ddH2O -
Note："-" represents that without dilution mother liquor is working solution, and "~" represents substantially concentration.
3rd, the selection and optimization of ISSR-PCR reaction systems
The determination of ISSR-PCR reaction systems is mainly to the Mg in reaction system2+, dNTP, Taq enzyme, primer, template DNA Concentration optimizes.To Mg2+, dNTP, Taq enzyme, primer, template DNA selected in the experiment condition of Different adding amount, establish Five factors, four horizontal L16(45) orthogonal experiment.The activity of Taq enzyme is 1U/ μ L, and the final volume of reaction system is 20 μ L, wherein 10 × Taq buffer are 2 μ L, and formamide is 0.5 μ L, the DNA for the sample extraction that template DNA sample number into spectrum in through table 1 is A, Primer is selected from universal primer UBC808, and other substrates add reaction system according to table 3, finally use ddH2O complements to 20 μ L, finally 15 μ L paraffin oils are added dropwise and seal liquid level, prevent the volatilization of PCR courses of reaction reclaimed water from causing system unstable.Orthogonal experiment scheme is shown in Table 4。
The L of table 316(45) each factor water product of orthogonal experiment
It is horizontal Mg2+/μL Taq enzyme/μ L dNTP/μL Primer/μ L Template DNA/μ L
1 1.0 0.5 1.0 0.5 1
2 1.5 1.0 1.5 1.0 1.5
3 2.0 1.5 2.0 1.5 2
4 2.5 2.0 2.5 2.0 2.5
The orthogonal experiment scheme of table 4
Mg2+/μL Taq enzyme/μ L dNTP/μL Primer/μ L Template DNA/μ L
1 1.0 0.5 1.0 0.5 1
2 1.5 0.5 1.5 1.0 1.5
3 2.0 0.5 2.0 1.5 2
4 2.5 0.5 2.5 2.0 2.5
5 1.0 1.0 2.0 1.0 2.5
6 1.5 1.0 2.5 0.5 2
7 2.0 1.0 1.0 2.0 1.5
8 2.5 1.0 1.5 1.5 1
9 1.0 1.5 2.5 1.5 1.5
10 1.5 1.5 2.0 2.0 1
11 2.0 1.5 1.5 0.5 2.5
12 2.5 1.5 1.0 1.0 2
13 1.0 2.0 1.5 2.0 2
14 1.5 2.0 1.0 1.5 2.5
15 2.0 2.0 2.5 1.0 1
16 2.5 2.0 2.0 0.5 1.5
After amplified reaction, the μ L of PCR reaction products 7 are taken to be mixed with 3 μ L nucleic acid dye working solutions, in 1.5% agarose Electrophoresis detection is carried out in gel, voltage 110V/cm, electrophoresis time 90min, is produced by PCR of 100-II bpDNA Marker The length scales of thing fragment.Final optimization pass ISSR-PCR reaction systems are shown in Table 5, and electrophoretic band is clear on this condition, become clear, nothing Obvious miscellaneous band.
The ISSR-PCR reaction systems of table 5
Reaction substrate Add volume (μ L)
ddH2O 11.0
Formamide 0.5
10×Taq buffer 2.0
Mg2+ 2.0
Template DNA 1.0
dNTP mixture 1.5
Primer 1.0
Taq enzyme 1.0
Embodiment 7
The screening and its analysis of ISSR-PCR primers
1st, the screening of primer
Under above-mentioned peak optimization reaction system, the 60 ISSR universal primer sequences provided selected from Columbia University are carried out Screening, final screening show that 9 polymorphisms are good, band clearly primer of the primer as this experiment follow-up test after reaction.Connect , the reaction system after optimization is reacted with two different template DNAs, to determine its stability.As a result such as Fig. 3, from electrophoresis It using DNA of the sample number into spectrum as A sample extraction in table 1 is production that template is expanded to obtain that the figure leftmost side 808 to 890, which is respectively, Thing, it is that DNA using sample number into spectrum as K sample extraction is that template is expanded to obtain respectively from the electrophoretogram rightmost side 890 to 808 Product, wherein m is DNA Marker DL1002, and it 100 to 1000bp, 808 is primer UBC808 that length, which is, 809 primers are UBC809, other the like, it will not be repeated here.As can be seen that electrophoretic band is clear in Fig. 3, stability is good.
2nd, the optimization of primer annealing temperature
Above-mentioned 9 primers filtered out are carried out with the investigation of optimum annealing temperature respectively.
The annealing temperature of primer is in optimized selection using grads PCR instrument on the basis of theoretical annealing temperature, set Temperature be 47.9 DEG C, 50 DEG C, 50.3 DEG C, 50.9 DEG C, 51.7 DEG C, 52.8 DEG C, 54.3 DEG C, 56.0 DEG C, 57.4 DEG C, 58.5 DEG C, 59.3℃、59.8℃、60.0℃.Enter performing PCR reaction under the conditions of temperatures above, and be finely adjusted under optimum temperature.Through expanding After increasing reaction, take the μ L of PCR reaction products 7 to be mixed with 3 μ L nucleic acid dye working solutions, electricity is carried out in 1.5% Ago-Gel Swimming detection, voltage 110V/cm, electrophoresis time 90min, the length using 100-II bpDNA Marker as PCR primer fragment Scale.By the readability of electrophoretic band, 9 kinds of primer final annealing temperature are drawn, specific primer annealing temperature is shown in Table 6, its Middle theoretical annealing temperature is the annealing temperature that software obtains according to primer base sequences, and actual annealing temperature is by optimum experimental Obtained optimum annealing temperature.
The setting of primer annealing temperature in the ISSR-PCR of table 6
3rd, annealing temperature stability analysis
The template DNA of 1 sample is randomly selected, under optimal ISSR-PCR reaction systems and annealing temperature, is repeated 3 times PCR reaction experiments, as a result show that ISSR-PCR reaction systems are stable, the actual annealing temperature of primer is good in the system stability inferior It is good.
Embodiment 8
ISSR-PCR is expanded and its product analysis
1st, ISSR-PCR is expanded
Under conditions of optimal ISSR-PCR reaction systems and annealing temperature, ISSR-PCR is carried out to 15 points of nucleic acid samples Amplified reaction.After amplified reaction, the μ L of PCR reaction products 7 are taken to be mixed with 3 μ L nucleic acid dye working solutions, in 1.5% agarose Electrophoresis detection is carried out in gel, voltage 110V/cm, electrophoresis time 90min, is produced by PCR of 100-II bpDNA Marker The length scales of thing fragment.After the completion of electrophoresis, blob of viscose is placed on uv analyzer and takes pictures and records.Gel is carried out according to product Result after electrophoresis shows that the product specificities that two primers of UBC836, UBC890 amplify are poor, to 15 parts of nucleic acid samples Separating capacity is small, and UBC808, UBC809, UBC842, UBC844, UBC845, UBC855, UBC888 totally 7 primer pair samples Template DNA expanding effect is preferable, and its electrophoretogram is shown in Fig. 4-10, and wherein m is that Marker DL1002, A-O represent 15 parts of nucleic acid respectively Sample number, specifically it is shown in Table 1.It is preferred that expand the object of band that preferable 7 primers amplify as follow-up cluster analysis.
2nd, cluster analysis
Find out in Fig. 4-10,7 primer coamplifications go out 67 band, and wherein polymorphic bandses amount to 51, polymorphism ratio Rate up to 76.1%, every primer it is amplifiable go out 5~11 specific bands, average every primer it is amplifiable go out 7.29 band. Amplified production has band to be calculated as 1 in identical migration position, and no band is calculated as 0, builds 0-1 raw data matrixs, and physical record is shown in Table 7-13.Cluster analysis is carried out with SPSS, and establishes clustering tree, sees Figure 11.
The UBC808 amplified production bar tape recordings of table 7
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15
0 0 0 0 1 0 0 0 0 0 0 0 0 0 1
1 1 0 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 0 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 0 0 0 0 0 0 1 0 1 0 0 0 0 0
The UBC809 amplified production bar tape recordings of table 8
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15
1 1 1 1 1 1 1 1 1 1 1 0 1 1 1
1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 1 1 0 0 1 0 1 1 0 1 0 1 1 0
0 0 1 1 1 1 1 0 0 0 0 1 0 0 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 0 0 0 0 0 0 0 0
The UBC842 amplified production bar tape recordings of table 9
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15
1 1 0 0 1 1 0 1 1 1 1 1 0 1 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 1 0 0 1 0 0 0 0 0
0 1 0 0 0 0 0 0 0 0 0 1 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0 1 0 0 0 0 1 0
0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 0
0 1 0 1 0 0 1 1 1 0 1 1 1 1 0
The UBC844 amplified production bar tape recordings of table 10
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15
1 1 1 1 1 1 1 1 0 1 0 1 0 1 1
1 1 1 1 1 0 1 0 1 1 1 0 0 1 1
0 1 0 0 1 1 1 1 1 1 1 0 1 1 0
1 1 0 0 0 0 1 1 0 1 0 0 1 0 0
0 0 0 0 1 1 0 1 0 1 1 0 0 0 0
The UBC845 amplified production bar tape recordings of table 11
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15
1 0 1 0 1 1 1 0 1 1 0 1 1 1 0
0 0 1 0 1 1 1 1 1 1 1 1 1 0 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 0 1 1 0 1 0 1 1 1
1 1 1 1 0 0 1 0 0 0 0 0 0 1 0
1 0 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 0 1 1 1 1 1 1 1 1 1 1 1 1
1 1 0 0 0 0 0 0 1 1 0 1 1 0 0
0 1 1 1 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1 0 1 0 0 0 0 0
0 0 0 0 1 0 0 0 0 0 1 0 0 0 0
The UBC855 amplified production bar tape recordings of table 12
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15
1 1 1 1 1 1 0 1 1 1 1 1 0 1 1
0 0 0 0 0 0 0 0 0 0 0 0 1 1 1
1 1 1 1 1 1 0 1 1 1 1 1 1 1 1
1 1 1 0 0 1 1 1 1 1 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 1 1 0
0 1 1 1 0 0 0 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
The UBC888 amplified production bar tape recordings of table 13
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 0 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0 1 0 0 0 0 1 1
0 0 0 1 1 0 1 0 1 1 0 1 0 1 1
0 0 0 0 0 0 0 0 0 1 0 0 0 1 0
3rd, cluster analysis result
The clustering tree set up according to ISSR-PCR molecular marker datas is analyzed, Guangxi province lingui county,gui lin area 4 parts of samples gather for one kind, 3 parts of samples in Zhanjiang, Guangdong Province area individually gather to be gathered for one kind, the two parts of samples in Huizhou City area It is relatively stable for one kind, the Desmodium styracifolium germ plasm resource in this 3 class area.Planted with Laibin City area wide in Liuzhou City Rongan County Desmodium gathers for one kind；The Desmodium styracifolium in Guangzhou Zijin County Lan Tang towns area and the Desmodium styracifolium of Guilin Area gather for one kind； The Desmodium styracifolium in Wuzhou area and the Desmodium styracifolium in Huazhou City area gather for one kind；The Desmodium styracifolium and favour that Shanyi City is planted The Desmodium styracifolium that state city is planted in area gathers for one kind.
From the point of view of regional situation is planted, as if golden mean of the Confucian school township of Guilin City, the Wu Tong towns of Lingui County, the geographical position of Tian Town are nearer, And the Desmodium styracifolium of large area plantation throughout the year, Desmodium styracifolium kind are implanted with certain history, local Desmodium styracifolium resource is sufficiently stable, The annual harvest Desmodium styracifolium time is at the beginning of 11 months, and it is to continue plantation the coming year to use to retain seed, local not introduce a fine variety wide gold from other places Money grass.Similarly Wu Tang counties in Zhanjiang, Guangdong Province are regional with Lingui area plantation situation, after equally also harvesting seed for locality The coming year continues to plant.The two regional cultivated areas are two maximum areas in the area of this resource investigation, wherein extensively Xi Sheng lingui county,gui lin cultivated area is maximum.
With Guilin Area geographical position similar in be Liuzhou City and Laibin City, Liuzhou City Rongan County Si Ding towns are planted wide Desmodium is planted for First Year, and its seed is purchased in Guangdong Province, but from the point of view of cluster analysis, the Desmodium styracifolium kind that locality is planted Matter is still the germplasm of Guangxi province, and the seed that such case may be purchased with seed buyer agent is produced by Guangxi province.Coming The wild Desmodium styracifolium that the Desmodium styracifolium that guest city area is collected is introduced a fine variety for local peasant household, yield very little, in recent years due to purchase Price it is low, most peasant household no longer plants, and replants other crops (such as eucalyptus).
Gather with the Desmodium styracifolium that Guilin Area is planted and also have the medicinal material planted of Guangdong Province Zijin County for one kind, this with During sampling it is inquired ask to introduce a fine variety situation completely the same.Desmodium styracifolium seed is purchased in Guilin used in locality.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this The scope of invention is limited by claim and its equivalent.

Claims (1)

  1. A kind of 1. method for identifying Desmodium styracifolium type, it is characterised in that including：
    The extraction of (3-1) Desmodium styracifolium plant genome DNA：
    Blade surface is cleaned with absolute ethyl alcohol after the fresh Desmodium styracifolium vanes discoloration silica gel absorbent drying collected, is treated anhydrous Ethanol is cut into tiny fragment with scissors after volatilizing, ball milling instrument grind into powder is used rapidly after adding liquid nitrogen；
    The blade powder of milled is quickly adding into 800 μ L and is preheating in 65 DEG C of DNA Extraction buffers 65 DEG C of water-baths 25 minutes, Every 5 minutes by EP manage it is reverse prevent blade powder agglomates for several times, take out EP pipes, be placed in and on ice, add isometric Tris- and satisfy And phenol：Chloroform：Isoamyl alcohol, centrifuge being put into after fully shaking up, 10000rpm centrifuges 10min at 20 DEG C, takes 600 μ L of supernatant liquid, Isometric chloroform is added into supernatant：Isoamyl alcohol, it is put into after shaking up in centrifuge, 10000rpm centrifuges 10min at 20 DEG C, takes For the μ L of supernatant 400 in the EP pipes after sterilizing, this supernatant is unpurified DNA solution；
    The genomic DNA of extraction is purified from Tiangeng plant genes group extracts kit, it is molten to unpurified DNA 700 μ L buffer solution GP2 are added in liquid, fully mixes, the liquid of mixing is transferred in adsorption column CB3,10000rpm centrifugation 30s, Waste liquid is discarded, 500 μ L buffer solution GD is added into adsorption column CB3,10min is placed, 10000rpm centrifugation 30s, discards waste liquid, 600 μ L rinsing liquids PW, 10000rpm centrifugation 30s are added into CB3, waste liquid is outwelled, 90% ethanol is added into CB3, 10000rpm centrifuges 30s, outwells waste liquid, and CB3 is placed in into room temperature places 15min, volatilizes ethanol, and CB3 is put into the EP after sterilizing Guan Zhong, 100 μ L TE buffer solutions are added, room temperature is placed 5 minutes, 10000rpm centrifugation 2min, the solution that centrifugation obtains is added again Enter into adsorption column CB3, room temperature places 3min, 10000rpm centrifugation 2min, obtains Desmodium styracifolium DNA after purification；
    The optimization and its stability analysis of (3-2) ISSR-PCR reaction systems：
    The foundation of (3-2-1) ISSR-PCR amplification programs：
    Based on the 60 ISSR universal primers announced by university of Columbia University, with NEW ENGLAND BIOLABS Tm The online calculator of Calculator primer annealing temperatures and the primer-design softwares of Primer Premier 5 are to its annealing temperature Calculated, near 50 DEG C, preliminary program is arranged to Tm values：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 45s；50 DEG C of annealing 40s；72 DEG C of extension 60s；Circulation 40 times；72 DEG C of extension 10min；4 DEG C of preservations；
    (3-2-2) ISSR-PCR reacts the concentration of working solution：
    Each reaction substrate concentration is as follows：
    Substrate Concentration 10×Taq buffer - Mg2+ - dNTPmixture 2.5mM Primer 10mM Template DNA ~100ng/uL Formamide - ddH2O -
    (3-3) ISSR-PCR is expanded and its product analysis：
    (3-3-1) ISSR-PCR is expanded：
    Under conditions of the ISSR-PCR reaction systems listed by table 5 and the annealing temperature listed by table 6, to 15 points of nucleic acid samples ISSR-PCR amplified reactions are carried out, after amplified reaction, take the μ L of PCR reaction products 7 to be mixed with 3 μ L nucleic acid dye working solutions, Electrophoresis detection, voltage 110V/cm, electrophoresis time 90min, with 100-II bpDNA are carried out in 1.5% Ago-Gel Marker is the length scales of PCR primer fragment, after the completion of electrophoresis, blob of viscose is placed on uv analyzer and takes pictures and records, root The result carried out according to product after gel electrophoresis shows that the product specificities that two primers of UBC890, UBC836 amplify are poor, right The separating capacity of 15 parts of nucleic acid samples is small, and UBC808, UBC809, UBC842, UBC844, UBC845, UBC855, UBC888 are common 7 primer pair sample template DNA cloning effect is preferable, preferably expands the band that preferable 7 primers amplify and gathers as follow-up The object of alanysis,
    The ISSR-PCR reaction systems of table 5
    Reaction substrate Add volume (μ L) ddH2O 11.0 Formamide 0.5 10×Taq buffer 2.0 Mg2+ 2.0 Template DNA 1.0 dNTP mixture 1.5 Primer 1.0 Taq enzyme 1.0
    The setting of primer annealing temperature in the ISSR-PCR of table 6
    (3-3-2) cluster analysis：
    From electrophoretogram as can be seen that 7 primer coamplifications go out 67 band, wherein polymorphic bandses amount to 51, polymorphism ratio Rate up to 76.1%, every primer it is amplifiable go out 5~11 specific bands, average every primer it is amplifiable go out 7.29 band, Amplified production has band to be calculated as 1 in identical migration position, and no band is calculated as 0, builds 0-1 raw data matrixs, is gathered with SPSS Alanysis, and clustering tree is established, and cluster result is analyzed.
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