CN104946641B - Specificity labeled primers and the detection method of the long woods of oil tea breeding No. 3 and No. 21 - Google Patents
Specificity labeled primers and the detection method of the long woods of oil tea breeding No. 3 and No. 21 Download PDFInfo
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Abstract
The present invention relates to the molecular specificity labeled primers of a pair of high long woodss of oil tea breeding of specificity No. 3 and long woods No. 21, and it is a kind of can be to the methods of No. 21 progress Rapid identifications of the long woods of oil tea breeding No. 3 and long woods.The primer sequence is as follows：Sense primer：5 ' AATGAATGGTGCTTTGGATT 3 ', anti-sense primer：5′‑CAAAACCACACCTTGAGTCC‑3′.Molecular specificity labeled primers of the present invention can be to No. 3 and No. 21 quick Early Identifications of progress of the long woods of the long woods of oil tea breeding, and method is simple, quick, accurate, is that appearance features distinguish the irreplaceable Molecular tools of oil tea breeding.
Description
(1) technical field
The present invention relates to the molecular specificity labeled primers and its authentication method of the long woods of oil tea breeding No. 3 and long woods No. 21.
(2) background technology
Oil tea is the woody oil tree species that China's planting area is maximum, distribution is wider.Camellia oleiferaindustry is greatly developed, it is right
Grain and oil safety is ensured, promotes increasing peasant income, Integrated Development of The Mountainous Region and building a New Socialist Countryside are promoted, all with particularly significant
Meaning.China's camellia oleiferaindustry is in the ascendant, and development potentiality is huge.At present, camellia oleifera lam gross area in China's is up to more than 4,500 ten thousand mu, but
Yield is very low, every only 4 kilograms or so of per mu yield tea oil.Its basic reason is that most camellia oleifera lam variety and qualities are poor or seriously move back
Change, and non-popularization and application of improved seeds that substantial amounts of national and provincial careful (recognizing) is fixed.Meanwhile some local seedling quality consciousness
Thin, management is extensive.To ensure the good and fast development of camellia oleiferaindustry, oil tea breeding is fully realized to developing camellia oleiferaindustry
Importance, it is necessary to accelerate oil tea good seed development and strengthen quality control.
China's supervision and management to oil tea seedling quality at present, every system of management layer formulation is essentially relying on, and
Key breakthrough is not yet obtained in technological layer, is especially a lack of the early stage quick identification technology system of oil tea breeding.It is logical at present
The 12 long woods kind series for crossing the authorization of national high quality tree species be respectively long woods No. 3, long woods No. 4, long woods No. 18, long woods No. 21,
Long woods No. 23, long woods No. 26, long woods No. 27, long woods No. 40, long woods No. 53, long woods No. 55, long woods No. 56, long woods No. 166.These
Oil tea breeding has the characteristics that early real high yield, stable yields, seed-producing rate height, oil content height, resistance, wide adaptability.To oil tea seedling
The supervision and management of quality will put forth effort to solve the trouble waters of the upper oil tea seedling of production at present, and to long woods first from technological layer
Oil tea breeding carries out discriminating protection.
Discriminating Main Basiss appearance features to camellia oleifera cultivar at present, but due to the cycle of many morphological characters identification is long,
It is affected by environment big, and kind quantity is on the increase so that and cultivar identification is more and more difficult.Since this century, some bases
In PCR molecular marking technique such as RAPD (Random Amplified Polymorphic DNA, DNArandom amplified polymorphic DNA
DNA), ISSR (Inter-Simple Sequence Repea, Inter-simple sequence repeat) and SRAP
(sequence-related amplified polymorphism, SRAP) has been used for oil tea in succession
Genetic Diversity of Germplasm, genetic distance research, but for point between molecular labeling and the chain of oil tea character, camellia oleifera cultivar
Son differentiates that research is seldom.Moreover it is universal primer used by these molecular marking techniques, its PCR AFLP system is not only multiple
Miscellaneous, poor repeatability, and specificity is not high, therefore be not appropriate for being used for Variety identification, only develop some stable variety, spy
Different DNA fingerprint mark could really be used for the accurate Rapid identification of the kind.
(3) content of the invention
The object of the invention provides the molecular specificity marker of a pair of specific high long woodss of oil tea breeding No. 3 and long woods No. 21
Primer, and it is a kind of can be to the methods of No. 21 progress Rapid identifications of the long woods of the long woods of oil tea breeding No. 3 and long woods.
The technical solution adopted by the present invention is：
The long woods of oil tea breeding No. 3 and the molecular specificity labeled primers of long woods No. 21, the primer sequence are as follows：
Sense primer：5 '-AATGAATGGTGCTTTGGATT-3 ',
Anti-sense primer：5′-CAAAACCACACCTTGAGTCC-3′.
The primer pair is to use round pcr, and the long woods of oil tea breeding No. 3 and long woods No. 21 are obtained by a large amount of screening tests
After DNA fragment specific, by the fragment cloning and sequencing, specific primer is designed based on obtained DNA sequence dna, is drawn with this
Thing enters performing PCR amplification to oil tea breeding, and only long woods No. 3 can stablize the specific fragment for obtaining 769bp sizes with long woods No. 21, and
Other camellia oleifera cultivars can not obtain the specific fragment.It should be noted that molecular specificity labeled primers of the present invention only limit
In the discriminating (differentiating whether it is one of long woods No. 3 or long woods No. 21, subsequently can further be identified again) of oil tea breeding, i.e.,
Testing sample is only limitted to oil tea.
The invention further relates to the molecular specificity labeled primers described in a kind of utilization to the long woods of oil tea breeding No. 3 and long woods 21
Number method for carrying out quick discriminating, methods described is：The genomic DNA of camellia oleifera cultivar blade to be measured is extracted as template, with institute
Molecular specificity labeled primers are stated as amplimer, enters performing PCR amplification, electrophoresis detection is carried out to amplified production, if electrophoresis knot
There are the DNA bands of 769bp sizes in fruit, then camellia oleifera cultivar to be measured is the long woods of oil tea breeding No. 3 or long woods No. 21, on the contrary then no；
The molecular specificity labeled primers sequence is：
Sense primer：5 '-AATGAATGGTGCTTTGGATT-3 ',
Anti-sense primer：5′-CAAAACCACACCTTGAGTCC-3′.
The inventive method key is the selection of amplimer, and DNA extractions, PCR reaction systems and reaction condition determine, with
And electrophoresis detection, it can be carried out according to this area conventional method.
Preferably, 20 μ L PCR reaction systems composition of the present invention is as follows：
The PCR amplification conditions are as follows：94 DEG C of pre-degeneration 7min；94 DEG C of denaturation 45min, 61 DEG C of annealing 45s, 72 DEG C of extensions
2min, totally 30 circulations；Most after 72 DEG C of filling-in 7min, final temperature is 4 DEG C.
Specifically, methods described is as follows：
(1) oil tea blade to be measured is taken, liquid feeding nitrogen is ground, and extracts the genomic DNA of oil tea to be measured；The genomic DNA carries
Take using new quick-speed plant extracting genome DNA box (DP3111, BioTeke, the limited public affairs of Tyke biotechnology of Beijing hundred
Department) carry out；
(2) using the genomic DNA of step (1) extraction as template, drawn using the molecular specificity labeled primers as amplification
Thing, enter performing PCR amplification：
The every 20 μ L compositions of PCR reaction systems are as follows：
2×Power Taq PCR Master Mix 10μL
Each 1 μ L of 10 μM of upstream and downstream primers
The μ L of 20ng/ μ L template DNAs 3
ddH2O complements to 20 μ L；
PCR reaction conditions are as follows：
94 DEG C of pre-degeneration 7min；94 DEG C of denaturation 45min, 61 DEG C of annealing 45s, 72 DEG C of extension 2min, totally 30 circulations；Finally
In 72 DEG C of filling-in 7min, final temperature is 4 DEG C.
(3) the μ L of step (2) amplified production 5 are taken, are mixed with the bromjophenol blue buffer solutions of 1 μ L 0.25%, point sample is in 1.5% fine jade
On sepharose, electrophoresis, electrophoresis terminate rear EB dyeing, divided in automatic gel images in 1 × TAE buffer solutions, under 5V/cm voltages
Taken a picture in analyzer, if 769bp DNA bands occurs in electrophoresis result, camellia oleifera cultivar to be measured is long woods No. 3 or long woods No. 21, instead
It is then no.
Beneficial effect of the present invention is mainly reflected in：Molecular specificity labeled primers of the present invention can be No. 3 to the long woods of oil tea breeding
Quick Early Identification is carried out with long woods No. 21, method is simple, quick, accurate, is that appearance features distinguish that oil tea breeding can not
The Molecular tools of replacement.
(4) illustrate
Fig. 1 is the result for entering performing PCR amplification to oil tea breeding；M is DNA molecular amount standard；Numbering 1 and 4 is respectively long woods 3
Number and long woods No. 21, amplified molecular weight be 769bp specific DNA band；Remaining numbering is other long woods oil tea breedings,
The specific DNA band that there are no the more sizes of 700bp produces.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：
(1) oil tea fine breed gene group DNA extraction：
Oil tea breeding young leaflet tablet 0.03g to be measured is taken, liquid feeding nitrogen is thoroughly ground, and the extraction and application of genomic DNA is new fast
Fast plant genome DNA extraction box (DP3111, BioTeke, the Tyke Bioisystech Co., Ltd of Beijing hundred), extraction obtain oil tea
The genomic DNA crude extract of breeding.DNA crude extracts pass through 1.5% agarose gel electrophoresis and DNA/RNA uv-spectrophotometrics
(Nanodrop Technologies, USA) is counted to detect integrality, purity and concentration.OD260/OD280>1.8 DNA sample is used
In subsequent PCR amplification.DNA extracts are standby in -20 DEG C of refrigerator storages.
(2) specific PCR amplification primer is designed, the sequence of primer pair is：
Sense primer：5 '-AATGAATGGTGCTTTGGATT-3 ' and anti-sense primer：5′-CAAAACCACACCTTGAGTCC
- 3 ', by Shanghai, biotechnology Co., Ltd synthesizes.
(3) PCR is expanded：
PCR reaction solutions form：2 × Power Taq PCR Master Mix (PR1700, BioTeke, the life of the Tyke of Beijing hundred
Thing Technology Co., Ltd.) 10 μ L, 10 μM of upstream and downstream special primers are to each 1 μ L, 20ng/ μ L template DNAs 3 μ L, ddH2O 5μL。
Amplified reaction is carried out on Life ECO types amplification instrument (Bioer, Hangzhou BIOER Technology Co., Ltd).
Amplification condition：
94 DEG C of pre-degeneration 7min；
94 DEG C of denaturation 45min, 61 DEG C of annealing 45s, 72 DEG C of extension 2min, totally 30 circulations；
Most after 72 DEG C of filling-in 7min, final temperature is 4 DEG C.
(4) electrophoresis detection：The μ L of step (3) pcr amplification product 5 are taken, are mixed with the bromjophenol blue buffer solutions of 1 μ L 0.25%, point sample
In on 1.5% Ago-Gel, in 1 × TAE buffer solutions, electrophoresis under 5V/cm voltages, after electrophoresis terminates, containing 0.5 μ g/
Dyed in the mL EB aqueous solution 30 minutes, then in the automatic gel image analysis instrument of U.S. Bole (Chemi Doc XRS
Imaging system, Bio-Rad, Hercules, CA, USA) on take a picture.
According to the method described above, to multiple oil tea breedings, (numbering 1~12 represents oil tea breeding and is followed successively by respectively：1：Long woods 3
Number, 2：Long woods No. 4,3：Long woods No. 18,4：Long woods No. 21,5：Long woods No. 23,6：Long woods No. 26,7：Long woods No. 27,8：Long woods 40
Number, 9：Long woods No. 53,10：Long woods No. 55,11：Long woods No. 56,12：Long woods No. 166) specific primer PCR AFLP system carry out
Electrophoresis detection, as a result see Fig. 1.
It is only to be amplified from the long woods of oil tea breeding No. 3 and long woods No. 21 of numbering respectively 1 and 4 that arrow is signified in figure
One clear bright, stable molecular weight is about the specific DNA band of the more sizes of 700bp, and the long woods oil tea of remaining numbering is good
Kind, the special DNA bands that there are no the more sizes of 700bp produce, it is seen that it is good that molecular specificity labeled primers of the present invention are used for oil tea
The long woods of kind No. 3 and the discriminating of long woods No. 21, its stability, specificity are very high.
Claims (3)
1. the long woods of oil tea breeding No. 3 and the molecular specificity labeled primers of long woods No. 21, the primer sequence are as follows：
Sense primer：5 '-AATGAATGGTGCTTTGGATT-3 ',
Anti-sense primer：5′-CAAAACCACACCTTGAGTCC-3′.
2. a kind of molecular specificity labeled primers using described in claim 1 enter to the long woods of oil tea breeding No. 3 and long woods No. 21
The method of row Rapid identification, methods described are：The genomic DNA of camellia oleifera cultivar blade to be measured is extracted as template, with described point
Sub- specificity labeled primers enter performing PCR amplification, electrophoresis detection are carried out to amplified production, if electrophoresis result goes out as amplimer
Existing 769bp specific DNA band, then camellia oleifera cultivar to be measured is the long woods of oil tea breeding No. 3 or long woods No. 21, on the contrary then no；It is described
Molecular specificity labeled primers sequence is：
Sense primer：5 '-AATGAATGGTGCTTTGGATT-3 ',
Anti-sense primer：5′-CAAAACCACACCTTGAGTCC-3′；
The PCR amplification conditions are as follows：94 DEG C of pre-degeneration 7min；94 DEG C of denaturation 45min, 61 DEG C of annealing 45s, 72 DEG C of extensions
2min, totally 30 circulations；Most after 72 DEG C of filling-in 7min, final temperature is 4 DEG C.
3. method as claimed in claim 2, it is characterised in that methods described is as follows：
(1) oil tea blade to be measured is taken, liquid feeding nitrogen is ground, and extracts the genomic DNA of oil tea blade to be measured；
(2) using the genomic DNA of step (1) extraction as template, using the molecular specificity labeled primers as amplimer, enter
Performing PCR expands：
The every 20 μ L compositions of PCR reaction systems are as follows：
2×Power Taq PCR Master Mix 10μL
Each 1 μ L of 10 μM of upstream and downstream primers
The μ L of 20ng/ μ L template DNAs 3
ddH2O complements to 20 μ L；
PCR reaction conditions are as follows：
94 DEG C of pre-degeneration 7min；94 DEG C of denaturation 45min, 61 DEG C of annealing 45s, 72 DEG C of extension 2min, totally 30 circulations；Most after 72
DEG C filling-in 7min, final temperature are 4 DEG C；
(3) the μ L of step (2) amplified production 5 are taken, are mixed with the bromjophenol blue buffer solutions of 1 μ L 0.25%, point sample is in 1.5% agarose
On gel, in 1 × TAE buffer solutions, electrophoresis under 5V/cm voltages, electrophoresis terminates rear EB dyeing, in automatic gel image analysis instrument
Upper photograph, if 769bp DNA bands occurs in electrophoresis result, camellia oleifera cultivar to be measured is long woods No. 3 or long woods No. 21, it is on the contrary then
It is no.
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| CN109694922B (en) * | 2019-02-28 | 2022-03-29 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of pecan variety dependendable |
| CN113604593B (en) * | 2021-07-26 | 2023-08-15 | 中国林业科学研究院亚热带林业研究所 | DNA fragment related to linoleic acid content in oil tea seed oil and application thereof |
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| CN103205424A (en) * | 2013-04-10 | 2013-07-17 | 浙江省林业科学研究院 | Molecule specificity marker primer and identification method of improved variety of camellia oleifera Changlin Number 55 |
| CN103233065A (en) * | 2013-04-10 | 2013-08-07 | 浙江省林业科学研究院 | Molecular specific marker primers for No. 4 and No.32 of an improved variety Changlin of Camellia oleifera and an identification method |
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| CN103205424A (en) * | 2013-04-10 | 2013-07-17 | 浙江省林业科学研究院 | Molecule specificity marker primer and identification method of improved variety of camellia oleifera Changlin Number 55 |
| CN103233065A (en) * | 2013-04-10 | 2013-08-07 | 浙江省林业科学研究院 | Molecular specific marker primers for No. 4 and No.32 of an improved variety Changlin of Camellia oleifera and an identification method |
Non-Patent Citations (2)
| Title |
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| ISSR在油茶品种鉴别和遗传多样性分析中的应用;于小玉等;《南京林业大学学报（自然科学版）》;20130131;第37卷(第1期);第61-66页 * |
| 油茶优良无性系ISSR分子鉴别;张国武等;《林业科学研究》;20071231;第20卷(第2期);第278-282页 * |
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