CN108034758A

CN108034758A - A kind of method for identifying camellia kind

Info

Publication number: CN108034758A
Application number: CN201810110271.0A
Authority: CN
Inventors: 范瑶飞
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-02-05
Filing date: 2018-02-05
Publication date: 2018-05-15

Abstract

The present invention provides a kind of method for identifying camellia kind, it is related to gardens, technical field of gardening, molecular specificity labeled primers provided by the invention can quickly differentiate camellia flower variety Nujiang camellia, especially, the present invention also provides the detection method for detecting camellia flower variety, the method is simple, fast, accurately, it is that appearance features distinguish the irreplaceable Molecular tools of camellia flower variety, detection method provided by the invention can fundamentally solve camellia in gardens, kind in horticultural applications, between subspecies " homonym ", " synonym " phenomenon, to gardens, the application of gardening plant, popularization and breeding of new variety provide detection method and technical support on molecular level.

Description

A kind of method for identifying camellia kind

Technical field

The present invention relates to the molecular specificity marker of field of garden technology, particularly camellia flower variety " Nujiang camellia " Primer and the method for carrying out Rapid identification to camellia flower variety " Nujiang camellia " using the molecular specificity labeled primers.

Background technology

About 2000 kinds of camellia kind, can be divided into 3 major classes, 12 flower pattern, camellia kind is 306 existing within 2013 More than.《Presbyopic glasses》Recording camellia kind has 19：Agate tea, Heding are red, mound tea, the white mound of any of several broadleaf plants calyx, poplar prince wife tea, palace Powder, pomegranate tea, one twist with the fingers it is red, according to hall is red, late camellia, South Mountain tea etc..Camellia type is more, there is single-lobe, half polyphyll, polyphyll, bent valve, five-pointed star Valve, hexagon, loose shell mould etc..

China's camellia flower plant aboundresources, it is widely distributed, and camellia flower plant phenotype has stronger plasticity, kind Intermolecular hybrid is easy, and kind is large number of, and Traits change is smaller between some kinds, and traditional form classification is difficult quick, accurate and effective Evaluation and differentiation.Academia is all very chaotic to the Name and Description of current camellia flower variety, " synonym " or " homonym " Phenomenon is more serious, lacks the title of unified standard and the taxonomic hierarchies of scientific system, is not easy to cultivar identification, popularization, exchange And the cultivation of new varieties, therefore there is an urgent need for establish a scientific and reasonable camellia assortment identification systems to help camellia to exist There is tremendous development in landscape art field.

The present invention provides a kind of method for identifying camellia kind Nujiang camellia, and the kind is established from molecular level Special DNA fingerprint mark, is camellia in gardens, gardening in order to the cultivation of cultivar identification, popularization, exchange and new varieties Field development provides molecular biology and supports.

The content of the invention

Molecular specificity labeled primers and detection in order to solve the above technical problem, the present invention provides a kind of Nujiang camellia Method.

The present invention is realized with following technical solution：The present invention provides a kind of method for identifying camellia kind, institute Stating method includes extracting the genomic DNA of camellia flower variety tissue to be measured as template, using molecular specificity labeled primers as Amplimer, carries out PCR amplification, electrophoresis detection is carried out to amplified production, if the specific DNA of 600-800bp occurs in electrophoresis result Band, then camellia flower variety to be measured is Nujiang camellia, on the contrary then no.

Further, using the molecular specificity labeled primers as amplimer, PCR amplification is carried out, to amplified production Electrophoresis detection is carried out, if the specific DNA band of 712bp occurs in electrophoresis result, camellia flower variety to be measured is Nujiang camellia.

Further, the camellia DNA extractions include the following steps：Step 1: take camellia flower variety to be detected fresh Young leaflet tablet 2g, is put into rapidly in 2mL Eppendorf pipes, is placed in precooling in liquid nitrogen container；Step 2: by sample from liquid nitrogen container Take out, be put into the mortar of precooling, liquid nitrogen constantly poured into mortar blade is worn into fine powder；Step 3: rapidly will be ground Fine powder be fitted into 2mLEppendorf pipes, add the CTAB Extraction buffers of 65 DEG C of 900 μ L preheating, mix；Step 4: will Eppendorf pipes are placed in 55 DEG C of water-baths, keep the temperature 60min, overturn mix frequently therebetween；Step 5: after room temperature is cooled to, add 500 μ L chloroforms/isoamyl alcohol, reverse mix to solution is in milkiness shape, and 6000rpm centrifugations 5min is layered；Step 6: with cutting off end Upper phase is carefully transferred in another Eppendorf pipes by the 2mL suction nozzles of about 0.5cm；Step 7: 600 μ L isopropanols are added, It is reverse to mix to there is white flock precipitate appearance；Step 8: 4 DEG C, 12000rpm centrifugation 10min precipitation DNA, remove supernatant, successively Precipitation is washed with 800 μ L76% ethanol, 0.2mol/L sodium acetates and 70% ethanol of 1mL；Step 9: it is dissolved in after precipitation is dry added with 3 In the 50 μ L TE of μ L RNaseA, 4 DEG C are stayed overnight or are placed directly within -20 DEG C；It is Step 10: by ultraviolet specrophotometer that DNA is dense Degree is adjusted to 1 μ g/ μ L, spare, if solution colour is too deep after step 9 or obvious muddiness answers repeat step eight, carries out secondary pumping Carry, add 2% PVP, PVP has good effect to the activity for suppressing polyphenol oxidase and cytochrome oxidase.

Further, the reaction solution of the PCR detections includes 10 × PCR Buffer, 2.5 μ L, 10mmol/L dNTPs Molecular specificity labeled primers each 1 described in 2.5 μ L, 25mmol/L MgCl, 22.5 μ L, 5U/ μ L Taq DNA enzymatics 0.2 μ L, 10mM μ L, 20ng/ μ L template DNAs 3 μ L, ddH2O complement to 25 μ L；

The amplified reaction of the PCR detections carries out on TC-XP type amplification instruments, and amplification condition is 95 DEG C of pre-degeneration 3min；95℃ 40s is denatured, 60 DEG C of annealing 40s, 72 DEG C of extension 2min, totally 35 circulate；Most after 72 DEG C of filling-in 7min, final temperature is 4 DEG C；

The molecular specificity labeled primers are：

Primer 4-f：5′- tacacagggctaggctatcg-3′

Primer 4-r：5′- cggctttaagggctagaaaa-3′.

The beneficial effects of the invention are as follows：Camellia is fundamentally solved in gardens, horticultural applications between kind, subspecies " homonym ", " synonym " phenomenon, the inspection on molecular level is provided to Landscape Plants Applied, popularization and breeding of new variety Survey method.

Embodiment

To make the object, technical solutions and advantages of the present invention clearer, the present invention will be made below further detailed Description.

Embodiment 1：The extraction of Nujiang camellia genomic DNA

Material and method

Medicine and reagent：

A. liquid nitrogen；

B.CTAB Extraction buffers:50mmol/L Tris, HCl, pH8.0,0.7mol/LNaCl, 10mmol/L, EDTA PH8.0,1.2%CTAB, 2% the V/V beta -mercaptoethanol used times add；

C. chloroform/isoamyl alcohol 24:1, RNaseA 10mg/mL:With 0.1mol/LNaCl, 0.015mol/L trisodium citrates are matched somebody with somebody System, 15min is handled using preceding with 100 DEG C of boiling water baths, to remove remaining DNase activity；

D. isopropanol；

E.76% ethanol, 0.2mol/L sodium acetates；

F.70% ethanol；

G.TE buffer solutions：10mol/LTris-HCl, 1mmol/LEDTA, pH8.0；Wherein CTAB is purchased from Shanghai life work, RNaseA Magnificent company is purchased from, other medicines are the pure medicine of analysis.

Instrument and equipment：Refrigerated centrifuge, constant water bath box

Operating procedure is as follows：

Step 1: taking the fresh young leaflet tablet 2g of camellia flower variety to be detected, it is put into rapidly in 2mL Eppendorf pipes, is placed in liquid Precooling in nitrogen tank；

Step 2: sample is taken out from liquid nitrogen container, it is put into the mortar of precooling, liquid nitrogen is constantly poured into mortar and grinds blade Into fine powder；

Step 3: ground fine powder is fitted into 2mLEppendorf pipes rapidly, the CTAB for adding 900 μ L, 65 DEG C of preheatings is carried Buffer solution is taken, is mixed；

Step 4: Eppendorf pipes are placed in 55 DEG C of water-baths, 60min is kept the temperature, overturns mix frequently therebetween；

Step 5: after room temperature is cooled to, add 500 μ L chloroforms/isoamyl alcohol, reverse mix to solution is in milkiness shape, 6000rpm from Heart 5min is layered；

Step 6: carefully upper phase is transferred in another Eppendorf pipes with the 2mL suction nozzles for cutting off end about 0.5cm；

It is reverse to mix to there is white flock precipitate appearance Step 7: add 600 μ L isopropanols；

Step 8: 4 DEG C, 12000rpm centrifugation 10min precipitation DNA, remove supernatant, successively with 800 μ L76% ethanol, 0.2mol/L second Sour 70% ethanol of sodium and 1mL washing precipitation；

Step 9: being dissolved in after precipitation is dry in the 50 μ L TE added with 3 μ L RNaseA, 4 DEG C are stayed overnight or are placed directly within -20 DEG C；

Step 10: DNA concentration is adjusted to by 1 μ g/ μ L by ultraviolet specrophotometer, and it is spare, if the solution colour after step 9 Too deep or obvious muddiness answers repeat step eight, carries out secondary extracting, adds 2% PVP, PVP is to suppressing polyphenol oxidase and cell The activity of chromo-oxidase has good effect.

Embodiment 2：Design specific PCR amplification primer

Phylogenetic tree analysis and molecular specificity analysis are carried out for camellia, filters out 4 target genes, source difference For：

1. Genbank Accession No. HM061353.1；

2. Genbank Accession No. HM061351.1；

3. Genbank Accession No. HM061345.1

4. Genbank Accession No. HM061339.1

For above-mentioned target sequence specific primer pair, primer pair is respectively：

Primer 1-f：5′- ccggcgcgaatgccaagg-3′(SEQ ID NO.1)

Primer 1-r：5′- agaagggctgcgggcgga-3′(SEQ ID NO.2)

Primer 2-f：5′- gcatcgatgaagaacgtag-3′(SEQ ID NO.3)

Primer 2-r：5′- gaacgaacgaacgacccg-3′(SEQ ID NO.4)

Primer 3-f：5′- tcgtcaagtcattttaccc-3′(SEQ ID NO.5)

Primer 3-r：5′- atgatgcccccgaacgtacc-3′(SEQ ID NO.6)

Primer 4-f：5′- tacacagggctaggctatcg-3′(SEQ ID NO.7)

Primer 4-r：5′- cggctttaagggctagaaaa-3′(SEQ ID NO.8)

Above-mentioned primer is synthesized by Nanjing Genscript Biotechnology Co., Ltd..

Embodiment 3：PCR amplification:

PCR reaction solution forms：10 × PCR Buffer, 2.5 μ L, 10mmol/L dNTPs, 2.5 μ L, 25mmol/L MgCl22.5 0.2 μ L, 10mM special primers of μ L, 5U/ μ L Taq DNA enzymatics complement to 25 μ to each 1 μ L, 20ng/ μ L template DNAs 3 μ L, ddH2O L。

Amplified reaction carries out on TC-XP type amplification instruments.Amplification condition is 95 DEG C of pre-degeneration 3min；95 DEG C denaturation 40s, 60 DEG C annealing 40s, 72 DEG C extension 2min, totally 35 circulation；Most after 72 DEG C of filling-in 7min, final temperature is 4 DEG C.

Electrophoresis detection：5 μ L of pcr amplification product in Example 3, mix, point with 1 μ L, 0.25% bromjophenol blue buffer solutions Sample is on 1.5% Ago-Gel, in 1 × TAE buffer solutions, electrophoresis under 5V/cm voltages, after electrophoresis, containing 0.5 μ Dyed in the aqueous solution of g/mL EB 30 minutes, then train on the clear automatic gel image analysis instrument of JS-380A and take a picture in Shanghai.

According to the method described above, respectively 5 camellia flower varieties are detected with (numbering 1~5 represents camellia with above-mentioned primer Kind and corresponding swimming lane are followed successively by：Hole 1：Marker；1- Anlongs knurl fruit tea（Hole 2-5, corresponds to primer pair 1-4 respectively）；2- short columns Oil tea (former mutation)（Hole 6-9, corresponds to primer pair 1-4 respectively）；3- Nujiang camellia（10-13, corresponds to primer pair 1-4 respectively）；4- Great Hua Camellia fraternals（14-17, corresponds to primer pair 1-4 respectively）；5- C.yuhsienensis（18-21, corresponds to primer pair 1-4 respectively）.

In four groups of primers, only primer 4 can amplify a clear bright, stable molecular weight from the camellia kind of Nujiang The specific DNA band of about 712bp, and the camellia subspecies kind of remaining numbering, there are no the special of 700bp or so sizes DNA bands produce, and also do not have other non-purpose bands to produce, it is seen that the molecular specificity marker that the present invention develops is used for Nujiang The identification of camellia kind, its stability, specificity are very high.

The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly Enclose, therefore equivalent variations made according to the claims of the present invention, it is still within the scope of the present invention.

Sequence table

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Claims

A kind of 1. method for identifying camellia kind, it is characterised in that the described method includes extract camellia flower variety group to be measured The genomic DNA knitted, using molecular specificity labeled primers as amplimer, carries out PCR amplification, to amplified production as template Electrophoresis detection is carried out, if the specific DNA band of 600-800bp occurs in electrophoresis result, camellia flower variety to be measured is Nujiang camellia, It is on the contrary then no.
2. method according to claim 1, it is characterised in that using the molecular specificity labeled primers as amplimer, PCR amplification is carried out, electrophoresis detection is carried out to amplified production, if the specific DNA band of 712bp, mountain to be measured occurs in electrophoresis result Camellia kind is Nujiang camellia.
3. according to any the method for claim 1 or 2, it is characterised in that the camellia DNA extractions include the following steps： Step 1: taking the fresh young leaflet tablet 2g of camellia flower variety to be detected, it is put into rapidly in 2mL Eppendorf pipes, is placed in liquid nitrogen container Middle precooling；Step 2: sample is taken out from liquid nitrogen container, it is put into the mortar of precooling, liquid nitrogen is constantly poured into mortar by leaf Piece wears into fine powder；Step 3: ground fine powder is fitted into 2mLEppendorf pipes rapidly, 900 μ L, 65 DEG C of preheatings are added CTAB Extraction buffers, mix；Step 4: Eppendorf pipes are placed in 55 DEG C of water-baths, 60min is kept the temperature, is overturned frequently therebetween Mix；Step 5: after room temperature is cooled to, 500 μ L chloroforms/isoamyl alcohol is added, reverse mix to solution is in milkiness shape, 6000rpm Centrifuge 5min layerings；Step 6: carefully upper phase is transferred to the 2mL suction nozzles for cutting off end about 0.5cm another In Eppendorf pipes；It is reverse to mix to there is white flock precipitate appearance Step 7: add 600 μ L isopropanols；Step 8: 4 DEG C, 12000rpm centrifugation 10min precipitation DNA, remove supernatant, successively with 800 μ L76% ethanol, 70% second of 0.2mol/L sodium acetates and 1mL Alcohol washing precipitation；Step 9: be dissolved in after precipitation is dry in the 50 μ L TE added with 3 μ L RNaseA, 4 DEG C overnight or be placed directly within- 20℃；Step 10: DNA concentration is adjusted to by 1 μ g/ μ L by ultraviolet specrophotometer, and it is spare, if the solution colour after step 9 Too deep or obvious muddiness answers repeat step eight, carries out secondary extracting, adds 2% PVP, PVP is to suppressing polyphenol oxidase and cell The activity of chromo-oxidase has good effect.
4. according to any the methods of claim 2-3, it is characterised in that the reaction solution of the PCR detections includes 10 × PCR 2.5 μ L, 10mmol/L dNTPs of Buffer, 2.5 μ L, 25mmol/L MgCl, 22.5 μ L, 5U/ μ L Taq DNA enzymatics, 0.2 μ L, Molecular specificity labeled primers described in 10mM each 1 μ L, 20ng/ μ L template DNAs 3 μ L, ddH2O complement to 25 μ L；

The amplified reaction of the PCR detections carries out on TC-XP type amplification instruments, and amplification condition is 95 DEG C of pre-degeneration 3min；95℃ 40s is denatured, 60 DEG C of annealing 40s, 72 DEG C of extension 2min, totally 35 circulate；Most after 72 DEG C of filling-in 7min, final temperature is 4 DEG C；

The molecular specificity labeled primers are：

Primer 4-f：5′- tacacagggctaggctatcg-3′

Primer 4-r：5′- cggctttaagggctagaaaa-3′.