CN104962638A - ISSR(Inter-simple Sequence Repeat)-PCR(Polymerase Chain Reaction) molecular marker method for macromitrium gymnostomum - Google Patents
ISSR(Inter-simple Sequence Repeat)-PCR(Polymerase Chain Reaction) molecular marker method for macromitrium gymnostomum Download PDFInfo
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Abstract
The invention relates to an ISSR(Inter-simple Sequence Repeat)-PCR(Polymerase Chain Reaction) molecular marker method for macromitrium gymnostomum. The ISSR-PCR molecular marker method comprises the following steps of firstly, extracting genome DNA (Deoxyribose Nucleic Acid) of the macromitrium gymnostomum; secondly, performing ISSR-PCR amplification, wherein a reaction system for performing the ISSR-PCR amplification in step (2) is as follows: 30mu L reaction system contains 3mu L of 10*LAPCR (Polymerase Chain Reaction ) buffer solution, 50ng of template DNA, 1.5mmol/L of Mg<2+>, 0.25mmol/L of dNTP (Deoxyribonucleoside Triphosphate), 1.5U of Taq enzyme and 0.5mu mol/L of primer. A primer UBC(University of British Columbia) 834 is taken as an example, ISSR amplification comprises conditions of: annealing at the temperature of 94DEG C for 4 minutes, then annealing at the temperature of 94DEG C for 45 seconds, at the temperature of 53DEG C for 45 seconds and at the temperature of 72DEG C for 1 minute for 45 circles, and finally, prolonging the annealing time at the temperature of 72DEG C by 10 minutes. Compared with the prior art, the ISSR-PCR molecular marker method disclosed by the invention has the advantages that a strip amplified by an established ISSR-PCR reaction system for the macromitrium gymnostomum has the characteristics of high stability, high definition and high polymorphism; the shortages in the research on the genetic diversity of the macromitrium gymnostomum are made up; the achievements of the invention can be used for researching the genetic relationship, a molecular marker and biogeography, and has high scientific value and application value.
Description
Technical field
The present invention relates to biology field, especially relate to a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method.
Background technology
Orthotrichaceae (Orthotrichaceae) alpine rush or palm-bark rain cape Rhodobryum (Macromitrium Bridel) is the monoid of general tropical distribution.China alpine rush or palm-bark rain cape Rhodobryum floristics very abundant, hypodontia alpine rush or palm-bark rain cape moss (Macromitrium gymnostomum) is a large kind wherein, because of capsule cap anodontia therefore named be hypodontia alpine rush or palm-bark rain cape moss, this kind of principal feature is that stem crawls to overgrow, close raw reddish-brown rhizoid; Branch is upright, single or have several little brachyplast, reaches 5.0mm.In the middle part of blade, cell is transparent, rectangle, heavy wall, smoothly; Top cell is slightly opaque, circular, the unconspicuous wart of tool.Branches and leaves are curling time dry, linear time moistening, wire lanceolar or ovum shape lanceolar, and sporangium is upright, and ellipticity is cylindrical, has shrinkage, and peristomal teeth lacks.Capsule balaclava shape, long 1.7-2.0mm, once in a while long only 1.5mm, base portion is very wide, and how many division and tool gauffer, without hair.There is the asexual brood cell of the henna strip be made up of 4-10 cell.Sullivant, William Starling delivered the name article of this kind in 1859 first at Proceedings of the American Academy of Arts andSciences.Bryophyte is one of botanic pioneer, in vegitabilia's phylogeny process, have special status.Bryophyte is as the important integral part of the ecosystem; to rock decay, soil organism accumulation, forest swamping etc., there is important Ecological Functions, and it also receives publicity day by day in the fields such as environment protection, medicinal, gardening and the scientific research value in the subjects such as ecology, vegetable chemistry, cytogenetics.Along with the development of DNA molecular marker technology, this technology has been widely used in the aspect such as qualification, diversity analysis of kind by people.
Current DNA molecular marker technology has developed into more than ten and has planted, and conventional means comprises RAPD, ISSR, AFLP, SRAP etc.ISSR (inter-simple sequence repeat, Inter-simple sequence repeat) proposed by Canadian Zietkiewicz etc., its principle utilizes micro-satellite of grappling to be primer, augmental interval is not the sequence between two very large SSR, then by gel electrophoresis strip analyzing samples DNA polymorphism.The advantage of ISSR molecular engineering is without the need to knowing that genomic any information just can mark in advance, can detect in any stage of growth cycle, and design of primers is easy, cost is low, DNA consumption is few, is therefore considered to a kind of more satisfactory molecule marking method.ISSR molecular marking technique has now been widely used in many angiospermous analysis of genetic diversity, but the research for bryophyte is not a lot, rarely seen Skotnicki to cucumber silk moss, Liu Li etc. to mouse tail moss and Li Qianying to the research of the genetic diversity of Haplocladium, use less for ISSR labelling method, domesticly see Wang Chenying etc. to narrow leaf large limit moss, Bryaceae and Wang Yingying to the research of the large hypnum moss in Thousand-Island Lake, Zhejiang, Pogonatum inflexum (Lindb.) Lac. genetic diversity.
Therefore, to research and other the kind gap relatively in addition of hypodontia alpine rush or palm-bark rain cape moss, set up a relatively scientific and reasonable effective ISSR-PCR reaction system, seek best treatment combination, good technical director and theories integration can be provided for follow-up scientific research.
Summary of the invention
Object of the present invention is exactly to provide a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method, to fill up the blank of prior art.
Object of the present invention can be achieved through the following technical solutions:
A kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method, comprises the following steps:
1) genomic dna of hypodontia alpine rush or palm-bark rain cape moss is extracted;
2) ISSR-PCR amplification is carried out.
Step 2) reaction system of carrying out ISSR-PCR amplification is: containing 10 × LA PCR damping fluid 3 μ L in 30 μ L reaction systems, template DNA 50ng, Mg
2+for 1.5mmol/L, dNTP are 0.25mmol/L, Taq enzyme is 1.5U, and primer is 0.5 μm of ol/L.
Described primer is selected from arbitrary in SEQ ID No.1 ~ 10, shown in table specific as follows:
Step 2) condition of carrying out ISSR-PCR amplification is: 94 DEG C of annealing 4min, then 94 DEG C of 45S, 53 DEG C of 45S, 72 DEG C of 1min, 40 circulations, last 72 DEG C of extension 10min.
Step 2) cycle number of carrying out ISSR-PCR amplification is 25-45, is preferably 40.
Compared with prior art, the band stability that the hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR reaction system that the present invention sets up amplifies is high, and sharpness is high, has the feature of very much higher state property.Compensate for the domestic deficiency to hypodontia alpine rush or palm-bark rain cape moss genetic diversity Journal of Sex Research, the research that the achievement of invention can be used for hypodontia alpine rush or palm-bark rain cape moss genetic affinity, molecule marker and biogeography aspect has very large scientific value and using value.
Accompanying drawing explanation
Fig. 1 is the amplification of ISSR-PCR exploration orthogonal experimental design reaction system;
M represents: DL2000Marker; Primer is UBC834; 1-9 is the treatment combination 1-9 of table 3;
Fig. 2 is ISSR-PCR fine tuning orthogonal test figure;
M represents: DL2000Marker; Primer UBC834; 1-9 is the treatment combination 1-9 of table 3
Fig. 3 is the primer amplification result that the applicable hypodontia alpine rush or palm-bark rain cape moss ISSR filtered out increases;
M represents: DL2000Marker; 1,2.UBC807; 3,4.UBC810; 5,6.UBC 811; 7,8.UBC 808; 9,10.UBC 834; 11,12.UBC 835; 13,14.UBC889; 15,16.UBC 880; 17,18.UBC 890; 19,20.UBC 826; 21,22.UBC 842; 23,24.UBC 856.
Fig. 4 is the amplification of different cycle index in ISSR-PCR reaction system;
1-4 be respectively number of cycles 25,30,35,40 and 45; M represents: DL2000; Primer is UBC834.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment 1
Extract hypodontia alpine rush or palm-bark rain cape moss genomic dna.
Embodiment 2
The screening of amplification system and optimization
1, the orthogonal experimental design of exploratory ISSR-PCR reaction system
The peak optimization reaction system (four factor three levels) of Orthogonal Optimum Design screening ISSR, selects L
9(3
4) orthogonal table, the factor-horizontal quadrature contrived experiment table (scheme is in table 2 and table 3) of each composition of design PCR amplification system.Be primer with UBC834, utilize 9 individual system in table 3, ISSR amplification is carried out to hypodontia alpine rush or palm-bark rain cape moss genomic dna.Reaction system is 30 μ L, and except factor listed in table, often pipe is containing 10 × LA PCR buffer 3 μ L, DNA profiling 50ng.Amplification program is: 94 DEG C of annealing 4min, then 94 DEG C of 45S, 53 DEG C of 45S, 72 DEG C 1min40 circulations, and last 72 DEG C extend 10min, 10 DEG C of ° of preservations.
The primer that table 1 ISSR increases and sequence
Orthogonal experimental design [L9 (the 34)] unit of the exploratory ISSR-PCR reaction system of table 2: μ L
Exploration orthogonal experiments is shown in Fig. 1, No. 1, No. 8 do not amplify band, No. 2, No. 3, No. 4, No. 5 process have faint band, and No. 7 bars are with still sharpness is not high, how reproducible the band of No. 6 and No. 9 process is, describe apart from optimum combination deviation little, we are as basic point, and the concentration gradient reducing each factor carries out fine tuning orthogonal test.
2, the foundation of fine tuning ISSR-PCR reaction system
The orthogonal experimental design of fine tuning ISSR-PCR reaction system is in table 3.
The orthogonal experimental design of table 3 fine tuning ISSR-PCR reaction system
Fine tuning orthogonal experiments is shown in Fig. 2, and except No. 5 and No. 6 process, other treatment combination has all run out of obvious band.No. 7, the band that No. 9 process are run out of is few, illustrates that repeatability is not high.No. 1, No. 2, No. 8 have amplified band, but relative No. 3 and No. 4 have been lacked a band, illustrate that repeatability is bad, can show that best combination is No. 4 process thus.Altogether amplified 6 bands, and band being clear, is the treatment combination of the best.
Adopt the system after optimizing, with identical DNA for template, 100 primers that Canadian Columbia University (UBC) provides are screened, finally screening draws UBC807, UBC810, UBC 811, UBC826, UBC834, UBC835, UBC 842, UBC 856, UBC 889, UBC 890 is primer, carries out ISSR amplification, can amplify sharpness high, the band (Fig. 3 represents primer picture) of good stability, illustrates that this reaction system is suitable for hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR and reacts.
3, the determination of optimum cycle number
This test ISSR reacts the primer sequence that adopts Canadian Columbia University (UBC) to provide, according to orthogonal experimental design and the result of annealing temperature that provides, carries out the determination of cycle number.Cycle number is followed successively by: 25, and 30,35,40,45.Eachly be circularly set 2 repetitions.
According to the result of orthogonal test and annealing temperature, carry out the detection of cycle number, as can be seen from Figure 4: when cycle number is 25, when 30, amplification does not almost have band; When cycle number is 35, band is not very clear, and repeatability is not high; When cycle number is 40 and 45, all more clear repeatability of band is all higher, for saving time and experimentation cost, selects cycle number to be 40 proper.
4, conclusion
Utilize orthogonal experimental design, from Mg
2+concentration, dNTP concentration, Taq DNA enzymatic concentration and primer concentration 4 factor 3 level, be optimized analysis to the reaction system of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR, and test the cycle number of PCR on this basis.Result shows: in 30 μ L reaction systems, containing 10 × LA PCR damping fluid 3 μ L in 30 μ L reaction systems, and template DNA 50ng, Mg
2+for 1.5mmol/L, dNTP are 0.25mmol/L, Taq enzyme is 1.5U, and when primer is 0.5 μm of ol/L, and when the suitableeest cycle number of ISSR-PCR is 40, expanding effect is best.For technical foundation and scientific basis have been established in the further research of hypodontia alpine rush or palm-bark rain cape moss genetic diversity.
Above-mentioned is can understand and use invention for ease of those skilled in the art to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (6)
1. a hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method, is characterized in that, comprise the following steps:
1) genomic dna of hypodontia alpine rush or palm-bark rain cape moss is extracted;
2) ISSR-PCR amplification is carried out.
2. a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method according to claim 1, is characterized in that, step 2) reaction system of carrying out ISSR-PCR amplification is: containing 10 × LA PCR damping fluid 3 μ L in 30 μ L reaction systems, template DNA 50ng, Mg
2+for 1.5mmol/L, dNTP are 0.25mmol/L, Taq enzyme is 1.5U, and primer is 0.5 μm of ol/L.
3. a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method according to claim 2, it is characterized in that, described primer is selected from arbitrary in SEQ ID No.1 ~ 10.
4. a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method according to claim 3, it is characterized in that, when described primer is selected from SEQ ID No.5, step 2) condition of carrying out ISSR-PCR amplification is: 94 DEG C of annealing 4min, then 94 DEG C of 45S, 53 DEG C of 45S, 72 DEG C of 1min, 40 circulations, last 72 DEG C extend 10min.
5. a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method according to claim 1, is characterized in that, step 2) cycle number of carrying out ISSR-PCR amplification is 25-45.
6. a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule marking method according to claim 5, is characterized in that, step 2) cycle number of carrying out ISSR-PCR amplification is 40.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048592A (en) * | 2017-12-19 | 2018-05-18 | 广西壮族自治区林业科学研究院 | Hainan wind nanmu ISSR-PCR molecule labelling methods and its primer special |
| CN116445657A (en) * | 2023-06-15 | 2023-07-18 | 西南林业大学 | ISSR-PCR reaction system for garlic fruits, marking method and application |
Citations (1)
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| CN101451163A (en) * | 2008-12-22 | 2009-06-10 | 陈乃富 | Construction and identification method of molecular marking fingerprint of Dendrobium huoshanense and similitude species thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101451163A (en) * | 2008-12-22 | 2009-06-10 | 陈乃富 | Construction and identification method of molecular marking fingerprint of Dendrobium huoshanense and similitude species thereof |
Non-Patent Citations (2)
| Title |
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| 买买提明·苏来曼等: "水藓科植物ISSＲ－ PCＲ反应体系的优化", 《生物技术》 * |
| 汪琛颖等: "真藓科植物ISSR-PCR 反应体系的优化及ISSR 指纹图谱的初步构建", 《安徽农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048592A (en) * | 2017-12-19 | 2018-05-18 | 广西壮族自治区林业科学研究院 | Hainan wind nanmu ISSR-PCR molecule labelling methods and its primer special |
| CN116445657A (en) * | 2023-06-15 | 2023-07-18 | 西南林业大学 | ISSR-PCR reaction system for garlic fruits, marking method and application |
| CN116445657B (en) * | 2023-06-15 | 2023-09-08 | 西南林业大学 | ISSR-PCR reaction system for garlic fruits, marking method and application |
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