CN107586866A - Thin shell mountain pecan Peach cultivars Moore characteristic sequence, labeled primer and authentication method - Google Patents
Thin shell mountain pecan Peach cultivars Moore characteristic sequence, labeled primer and authentication method Download PDFInfo
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Abstract
The present invention relates to the high thin shell mountain pecan Peach cultivars Moore of a pair of specificity molecular specificity labeled primers, and a kind of method that Rapid identification can be carried out to thin shell mountain pecan Peach cultivars Moore.The primer sequence is as follows：Sense primer：5′‑TGTCAAGGACAAACGGGTGA‑3′；Anti-sense primer：5′‑TGGAGAAGCGAAGTAGTCAACC‑3′.Molecular specificity labeled primers of the present invention can carry out quick Early Identification to thin shell mountain pecan Peach cultivars Moore, and method is simple, quick, accurate, be that appearance features distinguish the irreplaceable Molecular tools of thin shell mountain pecan Peach cultivars.
Description
(1) technical field
The present invention relates to thin shell mountain pecan Peach cultivars Moore characteristic sequence, molecular specificity labeled primers, and utilize and be somebody's turn to do
The method that molecular specificity labeled primers carry out Rapid identification to thin shell mountain pecan Peach cultivars Moore.
(2) background technology
Apocarya (Carya(Wangenh.) K.Koch) it is most to have economy in Juglandaceae hickory
The seeds of value.Apocarya is typical outcrossing plant, and existing production practices are found, the kind configuration of apocarya is
Influence one of its yield principal element.The U.S. is one of original producton location of apocarya, and its center producing region, for many years, its
The cultivar about 1000 of the name of seed selection.China introduces a fine variety existing more than the 100 years history of apocarya, is commonly used at present in production
Kind also have tens, production practices for many years are proved the normal region that the vast subtropical zone in China is apocarya.
But the subject matter that faces of existing China's apocarya producing region is to yield poorly down and unstable, causes this
Phenomenon reason has many aspects, and one of them is exactly that the kind of existing introduction still lacks clear and definite Genetic relationship, is added
The confusion that some filial generations are named, cause to be difficult to effective Juvenile stage and reasonable disposition, be not easy to cultivar identification, push away
Extensively, exchange and the cultivation of new varieties, therefore put forth effort to develop these stable varieties, special DNA fingerprint mark from molecular level
Note is only the Scientific Approaches for realizing the accurate Rapid identification of thin shell mountain pecan Peach cultivars.
Apocarya cultivar identification and Genetic relationship based on SSR molecular marker have been reported both at home and abroad, but have been existed
Detect relatively complicated, the situation of unstable result.
(3) content of the invention
It is an object of the present invention to provide thin shell mountain pecan Peach cultivars Moore characteristic sequence, molecular specificity labeled primers, and
The method for carrying out Rapid identification to primer pair thin shell mountain pecan Peach cultivars Moore using this.
The technical solution adopted by the present invention is：
Thin shell mountain pecan Peach cultivars Moore characteristic sequence, its sequence are as follows：5’-AATTCATTTGAGTTAGATGCTATCT
GGCTTAATTCAACGAAATATAAACATTCCCCGGGAGGAGAATTTCAAACTTGTCAAGGACAAACGGGTGATATTTAA
GTTTGACCAAAAAGCAAGTAAACAAGACTTTCGCCACGTTTTGATGGAAAAATCTTTACTAGTGGAATGACAATTTT
CCTTAGGTTGACTACTTCGCTTCTCCATTGTAGTATAAAACCAAGCCTTAGTTTTAGTCACAGAATTAATTACAATA
AATTATTCA-3’(SEQ ID No.1)
The invention further relates to thin shell mountain pecan Peach cultivars Moore molecular specificity labeled primers, the primer sequence is：
Sense primer：5′-TGTCAAGGACAAACGGGTGA-3′；
Anti-sense primer：5′-TGGAGAAGCGAAGTAGTCAACC-3′.
The primer pair is to use round pcr, and the larger kind of shape difference is screened from 24 common kinds and carries out letter
Change the sequencing and comparative analysis of gene, thin shell mountain pecan Peach cultivars Moore DNA fragment specific is obtained by a large amount of screening tests
After (SEQ ID No.1), by the fragment cloning and sequencing, differ larger genetic fragment for sequence and devise 1000 and multipair draw
Thing, screened and verified in 24 samples, by primary dcreening operation and three times more than repeatability sampling secondary screening, point finally obtained
Sub- specificity labeled primers, PCR amplifications are carried out to thin shell mountain pecan Peach cultivars with the specific primer, only Moore can obtain 131bp
The specific fragment (SEQ ID No.2) of size, other thin shell mountain pecan Peach cultivars can not obtain specific fragment.Need to illustrate
, molecular specificity labeled primers of the present invention are only limitted to the identification of thin shell mountain pecan Peach cultivars, i.e. testing sample is only limitted to shell
Hickory nut.
Primer amplified product for features described above Sequence sequences Design is 131bp：5’-TGTCAAGGAC
AAACGGGTGATATTTAAGTTTGACCAAAAAGCAAGTAAACAAGACTTTCGCCACGTTTTGATGGAAAAATCTTTACT
AGTGGAATGACAATTTTCCTTAGGTTGACTACTTCGCTTCTCCA-3’(SEQ ID No.2)
Moore is the kind that Fla. is selected from seedling tree, and tree body is less than normal, and growth potential is medium.Male flower is first
Ripe type, inflorescence is shorter, is good early stage pollinated variety.Small fruit type, mean fruit weight 6.0g or so, nut ellipse, top
It is relatively flat with bottom, it is somewhat flat on cross section；Kernel gold is to light brown, and dorsocentral ridge is narrow, and main groove is close.
Thin shell mountain pecan Peach cultivars Moore is carried out the invention further relates to a kind of molecular specificity labeled primers described in
The method of Rapid identification, methods described are：The genomic DNA of thin shell mountain pecan Peach cultivars 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 131bp sizes in fruit, then thin shell mountain pecan Peach cultivars to be measured are Moore, on the contrary then no；The molecular specificity
Labeled primer sequence is：
Sense primer：5′-TGTCAAGGACAAACGGGTGA-3′；
Anti-sense primer：5′-TGGAGAAGCGAAGTAGTCAACC-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.
The inventive method improves much than the reliability of the SSR marker authentication method of existing thin shell mountain pecan Peach cultivars, and
And the electrophoretic analysis or sequencing up to 1-2bp resolution ratio are also needed to after being expanded unlike SSR marker, this method is only needing regular-PCR
General electrophoresis is carried out afterwards and judges the presence or absence of band, and the inventive method is due to the accuracy and specificity of height in addition, therefore
Requirement to sample is relatively low, and the DNA sample of the tissue such as blade can meet the needs of cultivar identification.
Preferably, PCR amplification system composition of the present invention is as follows：
The PCR amplification conditions are as follows：94 DEG C of pre-degeneration 300s；95 DEG C of denaturation 30s, 56 DEG C of annealing 60s, 72 DEG C extend
50s, totally 30~40 circulations；Most after 72 DEG C of filling-in 300s, final temperature is 4 DEG C.
PCR Buffer final concentration of 1 ×, refer in PCR Buffer concentration of each component in reaction system with 1 ×
PCR Buffer are identical, generally from 10 × PCR Buffer that volume is reaction system volume 1/10.10×PCR Buffer
Composition is：100mM Tris-HCl(pH 8.5)、500mM KCl、25mM MgCl2And 1.0%Triton-X-100, solvent are
ddH2O。
Specifically, methods described is as follows：
(1) apocarya blade to be measured is taken, liquid feeding nitrogen is ground, and apocarya blade to be measured is extracted with SDS-CTAB methods
Genomic DNA；
(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：
Generally, the every 25 μ L compositions of PCR amplification system are as follows：
Or the every 15 μ L compositions of PCR reaction systems are as follows：
PCR reaction conditions are as follows：
94 DEG C of pre-degeneration 300s；95 DEG C of denaturation 30s, 56 DEG C of annealing 60s, 72 DEG C of extension 50s, totally 30~40 circulate；Most
After 72 DEG C of filling-in 300s, 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 131bp DNA bands occurs in electrophoresis result, thin shell mountain pecan Peach cultivars to be measured are Moore；It is on the contrary then no.
Beneficial effect of the present invention is mainly reflected in：Molecular specificity labeled primers of the present invention can be to thin shell mountain pecan Peach cultivars
Moore carries out quick discriminating, and method is simple, quick, accurate, is that appearance features distinguish that thin shell mountain pecan Peach cultivars are irreplaceable
Molecular tools.
(4) illustrate
Fig. 1 is the result that 24 kinds of thin shell mountain pecan Peach cultivars are carried out with PCR amplifications；M is Takara DL2000 marker；Only
Numbering 1 is the specific DNA band that thin shell mountain pecan Peach cultivars Moore has amplified that molecular weight is 131bp；Remaining numbering is other
Thin shell mountain pecan Peach cultivars, the specific DNA band that there are no 131bp sizes produce.
(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) extraction of apocarya variety genome DNA：
Thin shell mountain pecan Peach cultivars young leaflet tablet 0.01g to be measured is taken, liquid feeding nitrogen is thoroughly ground, and the extraction of genomic DNA uses
SDS-CTAB methods, through repeatedly extracting, to extract the genomic DNA crude extract for obtaining thin shell mountain pecan Peach cultivars.DNA crude extracts pass through again
After Magabio Nucleic acid purification kits are purified (rich day Bioer, Hangzhou, China), pass through 1.5% Ago-Gel electricity
Swimming and DNA/RNA ultraviolet specrophotometers (GeneQuant Pro, GE Healthcare) detect integrality, purity and dense
Degree.OD260/OD280>1.8 DNA sample is used for 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′-TGTCAAGGACAAACGGGTGA-3′；And anti-sense primer：5′-TGGAGAAGCGAAGTAGTCA
ACC-3 ', by Shanghai, biotechnology Co., Ltd synthesizes.
(4) PCR is expanded：
PCR reaction solutions form (15 μ L)：
Amplified reaction is carried out on TC-XP type amplification instruments.Amplification condition：94 DEG C of pre-degeneration 300s；95 DEG C denaturation 30s, 56
DEG C annealing 60s, 72 DEG C extension 50s, totally 30 circulation；Most after 72 DEG C of filling-in 300s, final temperature is 4 DEG C.
(4) electrophoresis detection：The μ L of step (3) pcr amplification product 3 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/
Dye in the mL EB aqueous solution 30 minutes, then taken a picture in Bio-rad gel imaging system Gel Doc.
According to the method described above, to 24 thin shell mountain pecan Peach cultivars, (numbering 1~24 represents thin shell mountain pecan Peach cultivars successively respectively
For：1st, Moore, 2, Nacono, 3, Van Deman, 4, Mohawk, 5, Davis, 6, Caddo, 7, Choctaw, 8, Osage, 9,
Mahan, 10, Hirschi, 11, Peruque, 12, Gloria Grande, 13, Sioux, 14, Dependable, 15, Kiowa,
16th, R-2323,17, Stuart, 18, Desirable, 19, Elliott, 20, Pawnee, 21, Pyzner, 22, Schley,
23rd, ShaoXing, 24, Sumner PCR AFLP systems carry out electrophoresis detection, as a result see Fig. 1.
It is respectively that a clear bright, stabilization has been amplified in 1 thin shell mountain pecan Peach cultivars Moore wherein only from numbering
The specific DNA band that molecular weight is about 131bp, and the thin shell mountain pecan Peach cultivars of remaining numbering, there are no the special of 131bp sizes
DNA bands produce, and also do not have other non-purpose bands to produce, it is seen that the molecular specificity labeled primers that the present invention develops are used for
Thin shell mountain pecan Peach cultivars Moore early stage identification, its stability, specificity are very high.
Sequence table
<110>Zhejiang Prov. Forest Science Inst
<120>Thin shell mountain pecan Peach cultivars Moore characteristic sequence, labeled primer and authentication method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 265
<212> DNA
<213> Carya illinoensis
<400> 1
aattcatttg agttagatgc tatctggctt aattcaacga aatataaaca ttccccggga 60
ggagaatttc aaacttgtca aggacaaacg ggtgatattt aagtttgacc aaaaagcaag 120
taaacaagac tttcgccacg ttttgatgga aaaatcttta ctagtggaat gacaattttc 180
cttaggttga ctacttcgct tctccattgt agtataaaac caagccttag ttttagtcac 240
agaattaatt acaataaatt attca 265
<210> 2
<211> 131
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 2
tgtcaaggac aaacgggtga tatttaagtt tgaccaaaaa gcaagtaaac aagactttcg 60
ccacgttttg atggaaaaat ctttactagt ggaatgacaa ttttccttag gttgactact 120
tcgcttctcc a 131
<210> 3
<211> 20
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 3
tgtcaaggac aaacgggtga 20
<210> 4
<211> 22
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 4
tggagaagcg aagtagtcaa cc 22
Claims (5)
1. thin shell mountain pecan Peach cultivars Moore characteristic sequence, its sequence are as follows：
5’-AATTCATTTGAGTTAGATGCTATCTGGCTTAATTCAACGAAATATAAACATTCCCCGGGAGGAGAATTTC
AAACTTGTCAAGGACAAACGGGTGATATTTAAGTTTGACCAAAAAGCAAGTAAACAAGACTTTCGCCACGTTTTGAT
GGAAAAATCTTTACTAGTGGAATGACAATTTTCCTTAGGTTGACTACTTCGCTTCTCCATTGTAGTATAAAACCAAG
CCTTAGTTTTAGTCACAGAATTAATTACAATAAATTATTCA-3’。
2. thin shell mountain pecan Peach cultivars Moore molecular specificity labeled primers, the primer sequence are as follows：
Sense primer：5′-TGTCAAGGACAAACGGGTGA-3′；
Anti-sense primer：5′-TGGAGAAGCGAAGTAGTCAACC-3′.
3. a kind of molecular specificity labeled primers using described in claim 1 carry out quick to thin shell mountain pecan Peach cultivars Moore
The method of identification, methods described are：The genomic DNA of thin shell mountain pecan Peach cultivars 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
Now unique 131bp specific DNA band, then thin shell mountain pecan Peach cultivars to be measured are that thin shell mountain pecan Peach cultivars are Moore, it is on the contrary then
It is no；The molecular specificity labeled primers sequence is：
Sense primer：5′-TGTCAAGGACAAACGGGTGA-3′；
Anti-sense primer：5′-TGGAGAAGCGAAGTAGTCAACC-3′.
4. method as claimed in claim 2, it is characterised in that the PCR amplification conditions are as follows：94 DEG C of pre-degeneration 300s；95℃
30s is denatured, 56 DEG C of annealing 60s, 72 DEG C of extension 50s, totally 30~40 circulate；Most after 72 DEG C of filling-in 300s, final temperature 4
℃。
5. method as claimed in claim 2, it is characterised in that methods described is as follows：
(1) apocarya blade to be measured is taken, liquid feeding nitrogen is ground, and the base of apocarya blade to be measured is extracted with SDS-CTAB methods
Because of a group DNA；
(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 25 μ L compositions of PCR amplification system are as follows：
PCR amplification conditions are as follows：
94 DEG C of pre-degeneration 300s；95 DEG C of denaturation 30s, 56 DEG C of annealing 60s, 72 DEG C of extension 50s, totally 30~40 circulate；Most after
72 DEG C of filling-in 300s, final temperature are 4 DEG C；
(3) the μ L of step (2) amplified production 3 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 unique 131bp DNA bands occurs in electrophoresis result, thin shell mountain pecan Peach cultivars to be measured are Moore, on the contrary then no.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109652589A (en) * | 2019-02-28 | 2019-04-19 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and the identification method of thin shell mountain pecan Peach cultivars Gloria Grande |
| CN109666759A (en) * | 2019-02-28 | 2019-04-23 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and the identification method of thin shell mountain pecan Peach cultivars Wichita |
| CN111719009A (en) * | 2019-03-22 | 2020-09-29 | 云南省林业科学院 | Primer group, kit and method for authenticating apomixis filial generation authenticity of paullo walnuts |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652589A (en) * | 2019-02-28 | 2019-04-19 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and the identification method of thin shell mountain pecan Peach cultivars Gloria Grande |
| CN109666759A (en) * | 2019-02-28 | 2019-04-23 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and the identification method of thin shell mountain pecan Peach cultivars Wichita |
| CN109652589B (en) * | 2019-02-28 | 2022-01-11 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Gloria Grande |
| CN109666759B (en) * | 2019-02-28 | 2022-02-08 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Wichita |
| CN111719009A (en) * | 2019-03-22 | 2020-09-29 | 云南省林业科学院 | Primer group, kit and method for authenticating apomixis filial generation authenticity of paullo walnuts |
| CN111719009B (en) * | 2019-03-22 | 2023-03-14 | 云南省林业科学院 | Primer group, kit and method for authenticating apomixis filial generation authenticity of paullo walnuts |
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