CN104928371B - Specificity labeled primers and the detection method of the long woods of oil tea breeding No. 18 and No. 21 - Google Patents
Specificity labeled primers and the detection method of the long woods of oil tea breeding No. 18 and No. 21 Download PDFInfo
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Abstract
The present invention relates to the molecular specificity labeled primers of the high long woods of oil tea breeding No. 18 of a pair of specificity and long woods No. 21, and a kind of method that can carry out Rapid identification to the long woods of oil tea breeding No. 18 and long woods No. 21.The primer sequence is as follows：Sense primer：5 ' CTCAAGGCTTCACTAACAGCAA 3 ', anti-sense primer：5′‑CTGCCTCTCAATCTTGTTCACC‑3′.Molecular specificity labeled primers of the present invention can carry out quick Early Identification to the long woods of oil tea breeding No. 18 and long woods No. 21, and method is simple, quick, accurate, be 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. 18 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, China's camellia oleifera lam gross area 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 substantial amounts of national and provincial careful (recognizing) fixed non-popularization and application of improved seeds.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 development camellia oleiferaindustry
Importance, it is necessary to accelerate oil tea good seed development and strengthen quality control.
The supervision and management of China at present to oil tea seedling quality, is essentially relying on every system of management layer formulation, and
Key breakthrough is not yet obtained in technological layer, the early stage quick identification technology system of oil tea breeding is especially a lack of.It is logical at present
The 12 long woods kind series for crossing national high quality tree species authorization 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
The features such as oil tea breeding has 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.
At present to the discriminating Main Basiss appearance features of camellia oleifera cultivar, but identified due to many morphological characters cycle length,
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, what these molecular marking techniques were used is universal primer, and 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 mark of a pair of specific high long woodss of oil tea breeding No. 18 and long woods No. 21
Remember primer, and a kind of method that can carry out Rapid identification to the long woods of the long woods of oil tea breeding No. 18 and long woods No. 21.
The technical solution adopted by the present invention is：
The long woods of oil tea breeding No. 18 and the molecular specificity labeled primers of long woods No. 21, the primer sequence are as follows：
Sense primer：5 '-CTCAAGGCTTCACTAACAGCAA-3 ',
Anti-sense primer：5′-CTGCCTCTCAATCTTGTTCACC-3′.
The primer pair is to use round pcr, and the long woods of oil tea breeding No. 18 and long woods No. 21 are obtained by a large amount of screening tests
DNA fragment specific after, by the fragment cloning and sequencing, specific primer is designed based on obtained DNA sequence dna, with this
Primer pair oil tea breeding enters performing PCR amplification, and only long woods No. 18 can stablize the specific piece for obtaining 655bp sizes with long woods No. 21
Section, and other camellia oleifera cultivars can not obtain the specific fragment.It should be noted that molecular specificity labeled primers of the present invention
The discriminating for being only limitted to oil tea breeding (differentiates whether it is one of long woods No. 18 or long woods No. 21, subsequently further reflected again
It is fixed), i.e., testing sample is only limitted to oil tea.
Described molecular specificity labeled primers are utilized to the long woods of oil tea breeding No. 18 and long woods the invention further relates to a kind of
The method of No. 21 progress quick discriminatings, methods described is：The genomic DNA of camellia oleifera cultivar blade to be measured is extracted as template, with
The molecular specificity labeled primers enter performing PCR amplification, electrophoresis detection are carried out to amplified production, if electrophoresis as amplimer
As a result there are the DNA bands of 655bp sizes, then camellia oleifera cultivar to be measured be the long woods of oil tea breeding No. 18 or long woods No. 21, it is on the contrary then
It is no；The molecular specificity labeled primers sequence is：
Sense primer：5 '-CTCAAGGCTTCACTAACAGCAA-3 ',
Anti-sense primer：5′-CTGCCTCTCAATCTTGTTCACC-3′.
The inventive method key is the selection of amplimer, and DNA is extracted, PCR reaction systems and reaction condition are determined, with
And electrophoresis detection, it can be carried out according to this area conventional method.
It is preferred that, 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 denaturation 45min, 60.5 DEG C annealing 45s, 72 DEG C prolong
2min is stretched, 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, extracts the genomic DNA of oil tea to be measured；The genomic DNA is carried
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) genomic DNA using step (1) extraction is drawn as template using the molecular specificity labeled primers as amplification
Thing, enters performing PCR amplification：
The every 20 μ L compositions of PCR reaction systems are as follows：
PCR reaction conditions are as follows：
94 DEG C of pre-degeneration 7min；94 DEG C of denaturation 45min, 60.5 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.
(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, the electrophoresis in 1 × TAE buffer solutions, under 5V/cm voltages, electrophoresis terminates rear EB dyeing, in automatic gel images point
Taken a picture in analyzer, if 655bp DNA bands occurs in electrophoresis result, camellia oleifera cultivar to be measured is long woods No. 18 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. 18 to the long woods of oil tea breeding
Quick Early Identification is carried out with long woods No. 21, method is simple, quick, accurate, be 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 3 and 4 is respectively long woods
No. 18 and long woods No. 21, have amplified the specific DNA band that molecular weight is 655bp；Remaining numbering is that other long woods oil teas are good
Kind, the specific DNA band that there are no many sizes of 600bp is produced.
(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, the extraction and application of genomic DNA is new fast
Fast plant genome DNA extracts box (DP3111, BioTeke, the Tyke Bioisystech Co., Ltd of Beijing hundred), extracts and obtains 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 '-CTCAAGGCTTCACTAACAGCAA-3 ' and anti-sense primer：5′-
CTGCCTCTCAATCTTGTTCACC-3 ', by Shanghai, biotechnology Co., Ltd synthesizes.
(3) PCR is expanded：
PCR reaction solutions are constituted：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, 60.5 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 30 minutes in the mL EB aqueous solution, 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, is as a result shown in Fig. 1.
Arrow meaning is to have amplified one in being only respectively the 3 and 4 long woods of oil tea breeding No. 18 and No. 21 from numbering in figure
Bar clearly becomes clear, stable molecular weight is about the specific DNA band of many sizes of 600bp, and the oil tea breeding of remaining numbering, has no
The special DNA bands for having many sizes of 600bp are produced, and also do not have other non-purpose bands to produce, it is seen that molecular specificity of the present invention
Labeled primer is used for the discriminating of the long woods of oil tea breeding No. 18 and long woods No. 21, and its stability, specificity are very high.
Claims (3)
1. the long woods of oil tea breeding No. 18 and the molecular specificity labeled primers of long woods No. 21, the primer sequence are as follows：
Sense primer：5 '-CTCAAGGCTTCACTAACAGCAA-3 ',
Anti-sense primer：5′-CTGCCTCTCAATCTTGTTCACC-3′.
2. the molecular specificity labeled primers described in a kind of utilization claim 1 enter to the long woods of oil tea breeding No. 18 and long woods No. 21
The method of row Rapid identification, methods described is：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 655bp specific DNA band, then camellia oleifera cultivar to be measured is the long woods of oil tea breeding No. 18 or long woods No. 21,
It is on the contrary then no；The molecular specificity labeled primers sequence is：
Sense primer：5 '-CTCAAGGCTTCACTAACAGCAA-3 ',
Anti-sense primer：5′-CTGCCTCTCAATCTTGTTCACC-3′；
The PCR amplification conditions are as follows：94 DEG C of pre-degeneration 7min；94 DEG C of denaturation 45min, 60.5 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, extracts the genomic DNA of oil tea blade to be measured；
(2) genomic DNA using step (1) extraction, using the molecular specificity labeled primers as amplimer, enters as template
Performing PCR is expanded：
The every 20 μ L compositions of PCR reaction systems are as follows：
PCR reaction conditions are as follows：
94 DEG C of pre-degeneration 7min；94 DEG C of denaturation 45min, 60.5 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；
(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, the electrophoresis in 1 × TAE buffer solutions, under 5V/cm voltages, electrophoresis terminates rear EB dyeing, in automatic gel image analysis instrument
Upper photograph, if 655bp DNA bands occurs in electrophoresis result, camellia oleifera cultivar to be measured is long woods No. 18 or long woods No. 21, it is on the contrary then
It is no.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321768A (en) * | 2011-10-21 | 2012-01-18 | 南京林业大学 | Method for identifying camellia oleifera cultivar and special primer and kit thereof |
| 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 |
| CN103667275A (en) * | 2013-12-13 | 2014-03-26 | 江西省林业科学院 | Oil-tea SSR molecular marker |
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- 2015-06-02 CN CN201510295085.5A patent/CN104928371B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321768A (en) * | 2011-10-21 | 2012-01-18 | 南京林业大学 | Method for identifying camellia oleifera cultivar and special primer and kit thereof |
| 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 |
| CN103667275A (en) * | 2013-12-13 | 2014-03-26 | 江西省林业科学院 | Oil-tea SSR molecular marker |
Non-Patent Citations (3)
| Title |
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| 分子标记技术及其在油茶遗传育种中的应用;张婷等;《湖北畜牧兽医》;20130305;第34卷(第3期);第53-55页 * |
| 利用ISSR和SRAP标记分析油茶遗传多样性;彭方仁等;《南京林业大学学报（自然科学版）》;20120915;第36卷(第5期);第19-25页 * |
| 油茶无性系引种与良种筛选试验;王毅等;《浙江林业科技》;20130315;第33卷(第2期);第39-42页 * |
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