CN103173546A - Rapid molecule detection method of Pinus massoniana Lamb. and Pinus elliottii, and specific primer pair thereof - Google Patents
Rapid molecule detection method of Pinus massoniana Lamb. and Pinus elliottii, and specific primer pair thereof Download PDFInfo
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- CN103173546A CN103173546A CN2013100760414A CN201310076041A CN103173546A CN 103173546 A CN103173546 A CN 103173546A CN 2013100760414 A CN2013100760414 A CN 2013100760414A CN 201310076041 A CN201310076041 A CN 201310076041A CN 103173546 A CN103173546 A CN 103173546A
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Abstract
The invention discloses a rapid molecule detection method of Pinus massoniana Lamb. and Pinus elliottii, and a specific primer pair thereof. The primer pair is PJ164L/R and has nucleotide sequences which are represented by SEQ. ID. No.1 and SEQ. ID. No.2 respectively in a sequence table, and the primer pair allows a 303bp product to be specifically amplified in Pinus massoniana Lamb. and a 325bp product to be specifically amplified in Pinus elliottii. The rapid molecule detection method of Pinus massoniana Lamb. and Pinus elliottii is established depended on the strong specificity characteristic of the sequences, allows determination to be carried out through the once implementation of a PCR amplification reaction and gel electrophoresis directly according to the fragment of the product, has the advantages of simple operation, accuracy, reliability and high sensitivity, is suitable for the rapid and reliable molecule detection and identification of Pinus massoniana Lamb. and Pinus elliottii, and has great scientific research values to identify the Pinus massoniana Lamb. and Pinus elliottii hybrid seedling in the future.
Description
Technical field
The present invention relates to the evaluation of Pinus massoniana Lamb, slash pine and Hybrid thereof, especially a kind of Pinus massoniana Lamb and slash pine rapid molecular detection method and Auele Specific Primer thereof pair.
Background technology
Pinus massoniana Lamb (Pinus massoniana Lamb.) is the widest coniferous species of China's distribution, be also important use material, afforestation and the industrial raw material seeds in south China area, its timber and rosin are the main raw materials of many forest industry, forest product industry and paper industry.Pinus massoniana Lamb all has distribution in (accounting for territory 1/5) mountain region, Subtropic of China area, and it is the first that the standing forest total area occupies national coniferous species, and accumulation is only second to dragon spruce, fir and tamarack, occupies the 4th of national coniferous species.Slash pine (Pinus elliottii) is one of Introduced Tree Species of successfully promoting of south China artificial forest big area, has that fast-growing, strong adaptability, material are good, the pine resin yield advantages of higher.
Species hybridization is to obtain heterotic main method, is the important content of Forest Tree Genetic Breeding research.Carry out Pinus massoniana Lamb and slash pine species hybridization research, the hybrid generation that how to obtain to have concurrently parents' advantage has far reaching significance to the pine tree improvement of China.Although Pinus massoniana Lamb and slash pine are same subgenus, its country of origin and sibship are all far apart, have no the successfully precedent of hybridization.The pollen of pine tree has air bag, and diffusibility is extremely strong with the wind, is easy to the pine tree hybridization pollination is formed to pollute disturb.Therefore, the evaluation of hybrid generation is to determine whether successful key link of hybridization.
Carrying out hybrid identification in the molecular genetics level is one of the most reliable authentication method at present, yet, there is no at present the molecular biological variety identification method of Pinus massoniana Lamb, slash pine and Hybrid thereof, affect Pinus massoniana Lamb and slash pine species hybridization research and carry out smoothly.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of simple to operate, accurately and reliably, highly sensitive Pinus massoniana Lamb and slash pine rapid molecular detection method and Auele Specific Primer thereof pair, set up Molecular Identification technical system between effective Pinus kind for carrying out Pinus massoniana Lamb and slash pine species hybridization research, the evaluation of in the future wet horse hybridization nursery stock and the tool of concluding the business have been of great significance.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: Pinus massoniana Lamb and slash pine rapid molecular detection method comprise the following steps:
＜1〉DNA extraction
Adopt improved CTAB cracking-silica bead absorption method to extract sample DNA;
＜2〉pcr amplification
With step＜1〉sample DNA carry out pcr amplification,
The PCR reaction system: totally 10 μ L comprise template DNA 1.2 μ L, 10 * Buffer1 μ L, 2mM-dNTP0.2 μ L, 25mu-MgCl
21.1 μ L, r-Taq0.1 μ L, F-primer0.25 μ L, R-primer0.25 μ L, ddH
2O5.9 μ L;
PCR response procedures: 94 ℃ of 4min of denaturation; Gradient cooling amplification 16 circulations, 94 ℃ of 15s for the first time, 60 ℃ of 15s, 72 ℃ of 30s, after this each cycle annealing temperature is all than low 0.5 ℃ of previous circulation; 94 ℃ of 15s of general amplification 15 circulations, 52 ℃ of 15s, 72 ℃ of 30s; Extend 72 ℃ of 20min; 4 ℃ of final preservations;
＜3〉gel electrophoresis
With step＜2〉PCR product electrophoresis on 8% polyacrylamide gel, then silver dyes detection.
In above-mentioned detection method, F-primer is primer PJ164L:CGTCGGTGAATCAGCAGC; R-primer is primer PJ164R:GATAAGCGAGTGGGAAGTA.
The Auele Specific Primer of Pinus massoniana Lamb and slash pine rapid molecular detection method pair, this primer pair is PJ164L/R, has following sequence:
Primer PJ164L:CGTCGGTGAATCAGCAGC(sees SEQ.ID.No.1);
Primer PJ164R:GATAAGCGAGTGGGAAGTA(sees SEQ.ID.No.2).
Key of the present invention is conifer simple repeated sequence (Simple Sequence Repeat, SSR) labeled primer, the EST-SSR primer that the primer is developed from the Pinus est sequence from the applicant.The Pinus est sequence is from the dbEST database, after being downloaded, sequence utilize the online SSR that the GRAMENE website provides to identify that software SSRIT (simple sequence repeat iden-tification tool) carries out the SSR retrieval, the EST-SSRs sequence that retrieves is carried out Primer Premier5 software design of primers, successfully design 248 pairs of EST-SSR primers.Random Pinus massoniana Lamb, each 4 DNA samples of slash pine selected carry out pcr amplification to 248 pairs of primers, have 60 pairs of primer amplifications to go out the purpose product; With each 8 DNA samples of Pinus massoniana Lamb, slash pine, 83 pairs of primers that primary dcreening operation obtains are carried out multiple sieve again, filter out master tape 10 pairs of Auele Specific Primers clearly; At last, adopt 31, the Pinus massoniana Lamb sample of Different Provenances, 27, the slash pine sample of Different Provenances to carry out Screening and Identification for the third time to 10 pairs of Auele Specific Primers, finally selected difference between species obviously to plant the best PJ164 primer pair of interior conservative property as specific molecular detection and identification primer between the kind of Pinus massoniana Lamb and slash pine.The dbEST ID of this primer est sequence is 69147425, totally 815 bases.This primer specific amplified in Pinus massoniana Lamb goes out the product of 303bp, and specific amplified goes out the product of 325bp in slash pine.Based on this, the contriver has set up Pinus massoniana Lamb of the present invention and slash pine rapid molecular detection method, be applicable to fast and reliable Molecular Detection and the evaluation of Pinus massoniana Lamb, slash pine and Interspecific Hybrids thereof, have important practical value for the evaluation of hybridizing result of study.Compare with additive method, the present invention has following technical superiority:
1. the reliable warp of pin-point accuracy detects the Pinus massoniana Lamb of Different Provenances and the DNA sample of slash pine as a result, and detected result 100% is accurate, guarantees the reliability of detected result height.
2. sample is carried out after simple process, pcr amplification and conventional polyacrylamide gel electrophoresis namely can judged result for quick detection method of the present invention easy and simple to handle, need not the product of amplification is carried out digestion with restriction enzyme, whole testing process generally can be completed in 4 hours.
3. the highly sensitive detection method of the present invention of detected result only need provide detected sample template DNA 20ng to get final product its affiliated kind of precise Identification.
4. the draw materials tissue such as convenience, the obvious needle of economic benefit, hibernaculum, globe daisy or organ all can be experiment material, and seedling stage, Juvenile Stage, the equal sampling of large tree of growing up are not subjected to season, site limitation.Can avoid occurring the phenomenon that dragons and fishes jumbled together in nursery stock production and sales process by the pine tree nursery stock being carried out Molecular Identification, also the check for conifer species hybridization result of study provides reliable experimental technique.
Description of drawings
Fig. 1 uses the pcr amplification electrophorogram that the present invention detects 27 slash pine genomic dna samples, and in figure: M is DNA ladder, and 1-27 is the individual DNA samples of 27 Different Provenances slash pine.
Fig. 2 uses the pcr amplification electrophorogram that the present invention detects 31 Pinus massoniana Lamb genomic dna samples, and in figure: M is DNA ladder, and 1-31 is the individual DNA samples of 31 Different Provenances Pinus massoniana Lamb.
Embodiment
＜1〉collection of material
Take the needle of slash pine or Pinus massoniana Lamb, preserve (being mainly to prevent the degraded of leaf tissue and brownization of blade) with ice bag, return with the foam plastic reservoir subband, be placed in-70 ℃ of Refrigerator stores.
＜2〉CTAB-silica bead method is extracted DNA
Add 4ml CTAB extracting solution and 80ul mercaptoethanol, preheating in 65 ℃ of water-baths in 10ml centrifuge tube (eppendorf pipe).Draw materials 1-2g in mortar, add appropriate liquid nitrogen repeatedly to grind to form rapidly finely powdered, go in the CTAB extracting solution of preheating, with sealed membrane, centrifuge tube is sealed to prevent the mercaptoethanol volatilization.Put back in water-bath and be incubated 30 minutes, in insulating process, light rolling several times, keeps shaking up.Take out the Eppenedorf pipe.Be placed in rapid cool to room temperature on ice, add isopyknic 4ml chloroform-primary isoamyl alcohol (24:1), slightly mix and fully, make it be mixed into up hill and dale milk sap, then centrifugal 12000rpm10min under 4 ℃, gently supernatant liquor is drawn 1ml in the 1.5ml centrifuge tube, be put in-20 ℃ of refrigerators standby.
＜3〉pcr amplification
Carry out pcr amplification with above-mentioned sample DNA, reaction system and program are as follows:
The PCR reaction system: totally 10 μ L comprise template DNA 1.2 μ L, 10 * Buffer1 μ L, 2mM-dNTP0.2 μ L, 25mu-MgCl
21.1 μ L, r-Taq0.1 μ L, F-primer0.25 μ L, R-primer0.25 μ L, ddH
2O5.9 μ L;
PCR response procedures: 94 ℃ of 4min of denaturation; Gradient cooling amplification 16 circulations, 94 ℃ of 15s for the first time, 60 ℃ of 15s, 72 ℃ of 30s, after this each cycle annealing temperature is all than low 0.5 ℃ of previous circulation; 94 ℃ of 15s of general amplification 15 circulations, 52 ℃ of 15s, 72 ℃ of 30s; Extend 72 ℃ of 20min; 4 ℃ of final preservations;
＜4〉polyacrylamide gel electrophoresis (PAGE)
With above-mentioned PCR product electrophoresis on 8% polyacrylamide gel, then silver dyes detection, and is specific as follows:
1. gluing will have ear slide (ear down) to steep in anti-silication agent, new liquid 25 minutes, old liquid 30 minutes; Be coated with earless front slide silication agent, evenly put four, light and even is coated with three times along a direction, quiet 10 minutes.
2. sealing to being affixed in rubber frame, is set level two slides, firmly twists extremely, clamps rubber hose; Molten useless agaropectin minute is inhaled a 2500-3000ul sol and is accelerated the two sides slide fully to be shut preventing from cementing leakage along lower of slide edge rapid clockwise; Take horizontal bottom line face level the end of higher than as good, static gel 15 minutes.
3. encapsulating is thoroughly opened with long thin rifle head and is added glue rifle head, adds from the centre, waits for 2 hours, treats that glue fully solidifies.
4. application of sample
5. race glue
6. sled slide
7. silver dyes and takes off band glue sheet glass, uses dd H
2O punching twice, each 2 minutes; Fix 10 minutes with stationary liquid; Use ddH
2O cleans twice, each 2 minutes; Add AgNO
3Solution, silver dyed 6 to 7 minutes; Use ddH
2O cleans twice, each 2 minutes; Add developing solution, wait for peacefully 20 to 30 minutes till show image; Rinse with tap water, stick the order label; The close shot of taking pictures is held level with both hands, keeps label clear.
As illustrated in figs. 1 and 2, use the present invention and detect Pinus massoniana Lamb and slash pine, primer pair PJ164L/R specific amplified in Pinus massoniana Lamb goes out 303bp(and sees SEQ.ID.No.3) product, specific amplified goes out 325bp(and sees SEQ.ID.No.4 in slash pine) product.
Sequence table
Claims (3)
1. a Pinus massoniana Lamb and slash pine rapid molecular detection method is characterized in that comprising the following steps:
＜1〉DNA extraction
Adopt improved CTAB cracking-silica bead absorption method to extract sample DNA;
＜2〉pcr amplification
With step＜1〉sample DNA carry out pcr amplification,
The PCR reaction system: totally 10 μ L comprise template DNA 1.2 μ L, 10 * Buffer1 μ L, 2mM-dNTP0.2 μ L, 25mu-MgCl
21.1 μ L, r-Taq0.1 μ L, F-primer0.25 μ L, R-primer0.25 μ L, ddH
2O5.9 μ L;
PCR response procedures: 94 ℃ of 4min of denaturation; Gradient cooling amplification 16 circulations, 94 ℃ of 15s for the first time, 60 ℃ of 15s, 72 ℃ of 30s, after this each cycle annealing temperature is all than low 0.5 ℃ of previous circulation; 94 ℃ of 15s of general amplification 15 circulations, 52 ℃ of 15s, 72 ℃ of 30s; Extend 72 ℃ of 20min; 4 ℃ of final preservations;
＜3〉gel electrophoresis
With step＜2〉PCR product electrophoresis on 8% polyacrylamide gel, then silver dyes detection.
2. Pinus massoniana Lamb according to claim 1 and slash pine rapid molecular detection method is characterized in that:
Described F-primer is primer PJ164L:CGTCGGTGAATCAGCAGC;
Described R-primer is primer PJ164R:GATAAGCGAGTGGGAAGTA.
3. the Auele Specific Primer of Pinus massoniana Lamb and slash pine rapid molecular detection method pair according to claim 1, is characterized in that
This primer pair is PJ164L/R, has following sequence:
Primer PJ164L:CGTCGGTGAATCAGCAGC;
Primer PJ164R:GATAAGCGAGTGGGAAGTA.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357556A (en) * | 2014-10-20 | 2015-02-18 | 广西壮族自治区林业科学研究院 | Method for detecting relative quantity of massoniana GGPPS gene through real-time quantitative fluorescent RT-PCR |
| CN107557362A (en) * | 2017-10-26 | 2018-01-09 | 南京林业大学 | A kind of authentication method of masson pine cpSSR polymorphism primers and its pine tree sibling species |
| KR101828774B1 (en) | 2014-05-14 | 2018-02-19 | 대한민국 | Method of distinguishing between the red pine and the scot pine using molecular marker |
Citations (1)
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| CN101348830A (en) * | 2008-07-21 | 2009-01-21 | 南京林业大学 | Hybrid Chinese tuliptree rapid molecular detection specific primer and use method thereof |
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2013
- 2013-03-11 CN CN2013100760414A patent/CN103173546A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101348830A (en) * | 2008-07-21 | 2009-01-21 | 南京林业大学 | Hybrid Chinese tuliptree rapid molecular detection specific primer and use method thereof |
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| 《中南林业科技大学学报》 20120430 邵俊培等 马尾松EST-SSR PCR体系的优化 第159-163页 1-3 第32卷, 第4期 * |
| 刘公秉等: "基于松树EST序列的马尾松SSR引物开发", 《分子植物育种》 * |
| 王晓锋等: "基于松属不同树种EST序列的马尾松SSR引物开发", 《湖南林业科技》 * |
| 王润辉等: "湿地松、加勒比松改进的SSR-PCR试验方法", 《广东林业科技》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101828774B1 (en) | 2014-05-14 | 2018-02-19 | 대한민국 | Method of distinguishing between the red pine and the scot pine using molecular marker |
| CN104357556A (en) * | 2014-10-20 | 2015-02-18 | 广西壮族自治区林业科学研究院 | Method for detecting relative quantity of massoniana GGPPS gene through real-time quantitative fluorescent RT-PCR |
| CN104357556B (en) * | 2014-10-20 | 2016-12-07 | 广西壮族自治区林业科学研究院 | A kind of method of real-time quantitative fluorescence RT-PCR detection Pinus massoniana Lamb GGPPS gene relative expression quantity |
| CN107557362A (en) * | 2017-10-26 | 2018-01-09 | 南京林业大学 | A kind of authentication method of masson pine cpSSR polymorphism primers and its pine tree sibling species |
| CN107557362B (en) * | 2017-10-26 | 2019-08-23 | 南京林业大学 | A kind of identification method of masson pine cpSSR polymorphism primer and its pine tree sibling species |
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Application publication date: 20130626 |