CN107586867A - Thin shell mountain pecan Peach cultivars Pawnee characteristic sequence, labeled primer and authentication method - Google Patents
Thin shell mountain pecan Peach cultivars Pawnee 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 Pawnee of a pair of specificity characteristic sequence, molecular specificity labeled primers, and a kind of method that Rapid identification can be carried out to thin shell mountain pecan Peach cultivars Pawnee.The molecular specificity labeled primers sequence is as follows：Sense primer：5′‑GCTCTTGTCACTTGGGCCTA‑3′；Anti-sense primer：5′‑TGAGGATGGAGGCTTTTAGGC‑3′.Molecular specificity labeled primers of the present invention can carry out quick Early Identification to thin shell mountain pecan Peach cultivars Pawnee, 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
Should the present invention relates to thin shell mountain pecan Peach cultivars Pawnee characteristic sequence, molecular specificity labeled primers and utilization
The method that molecular specificity labeled primers carry out Rapid identification to thin shell mountain pecan Peach cultivars Pawnee.
(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 Pawnee characteristic sequence, molecular specificity labeled primers and
The method for carrying out Rapid identification to thin shell mountain pecan Peach cultivars Pawnee using the molecular specificity labeled primers.
The technical solution adopted by the present invention is：
Thin shell mountain pecan Peach cultivars Pawnee characteristic sequence, its sequence are as follows：5’-AATTCAATGGCTCTTGTCACTTGG
GCCTATATTGTGGAATTTTGCTTCAGATGGAGATTTCTTATAAAGGTAAAGATTTATGTATTACTACAAAAGTATAA
TGGGGTTTTTGAAGAGCCTAAAAGCCTCCATCCTCATAGGACTCAAGACCATTAGATTGTGTTGAATGATGAGTCAG
TTCCTATGATTTTAAGACCATATAGATACCCTTATTATCAAAAGACAGGGAT-3’(SEQ ID No.1)
The invention further relates to thin shell mountain pecan Peach cultivars Pawnee 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 Pawnee specific DNA piece is obtained by a large amount of screening tests
After section (SEQ ID No.1), by the fragment cloning and sequencing, it is multipair to devise 1000 for the larger genetic fragment of sequence difference
Primer, screened and verified in 24 samples, by primary dcreening operation and three times more than repeatability sampling secondary screening, finally obtain
Molecular specificity labeled primers, PCR amplifications are carried out to thin shell mountain pecan Peach cultivars with the specific primer, only Pawnee can be obtained
The specific fragment (SEQ ID No.2) of 128bp sizes, other thin shell mountain pecan Peach cultivars can not obtain specific fragment.Need
It is noted that 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 only limits
In apocarya.
Primer amplified product for features described above Sequence sequences Design is 128bp：5’-GCTCTTGTCA
CTTGGGCCTATATTGTGGAATTTTGCTTCAGATGGAGATTTCTTATAAAGGTAAAGATTTATGTATTACTACAAAAG
TATAATGGGGTTTTTGAAGAGCCTAAAAGCCTCCATCCTCA-3’(SEQ ID No.2)。
Pawnee was the kind that agri-scientific research office of United States Department of Agriculture (USDA-ARS) is released by crossbreeding, in 1984
Issue, selected by ' Mohawk' × ' Starking Hardy Giant' filial generation in 1963.Tree body is less than normal, growth
Gesture is medium.Male flower first ripe type, but male and female florescence collision probability is higher, male inflorescence is shorter.Middle fruit type, nut is ripe early, average single
Fruit weight 10.0g or so, nut ellipse, top point and bottom are relatively round；Kernel gold, with black splotch, dorsocentral ridge is wide, and master is recessed
Groove width, base portion crack are obvious.
Thin shell mountain pecan Peach cultivars Pawnee is entered the invention further relates to the molecular specificity labeled primers described in a kind of utilization
The method of row Rapid identification, methods described are：The genomic DNA of thin shell mountain pecan Peach cultivars 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 128bp sizes, then thin shell mountain pecan Peach cultivars to be measured are thin shell mountain pecan Peach cultivars Pawnee, 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′.
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 128bp DNA bands occurs in electrophoresis result, thin shell mountain pecan Peach cultivars to be measured are Pawnee；It is on the contrary then
It is 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
Pawnee carries out quick discriminating, and method is simple, quick, accurate, is that appearance features distinguish that thin shell mountain pecan Peach cultivars can not substitute
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 19 is the specific DNA band that thin shell mountain pecan Peach cultivars Pawnee has amplified that molecular weight is 128bp；Remaining numbering is other
Thin shell mountain pecan Peach cultivars, there are no 128bp sizes specific DNA band 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 0.05g 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 on 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, Pawnee, 20, Elliott, 21, Pyzner, 22, Schley,
23rd, ShaoXing, 24, Sumner PCR AFLP systems carry out electrophoresis detection, as a result see Fig. 1.
It is respectively to have amplified one in 19 thin shell mountain pecan Peach cultivars Pawnee clearly to become clear, stably wherein only from numbering
Molecular weight be about 128bp specific DNA band, and remaining numbering thin shell mountain pecan Peach cultivars, there are no the spy of 128bp sizes
Different 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
Identified in thin shell mountain pecan Peach cultivars Pawnee early stage, its stability, specificity are very high.
Sequence table
<110>Zhejiang Prov. Forest Science Inst
<120>Thin shell mountain pecan Peach cultivars Pawnee characteristic sequence, labeled primer and authentication method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 230
<212> DNA
<213> Carya illinoensis
<400> 1
aattcaatgg ctcttgtcac ttgggcctat attgtggaat tttgcttcag atggagattt 60
cttataaagg taaagattta tgtattacta caaaagtata atggggtttt tgaagagcct 120
aaaagcctcc atcctcatag gactcaagac cattagattg tgttgaatga tgagtcagtt 180
cctatgattt taagaccata tagataccct tattatcaaa agacagggat 230
<210> 2
<211> 128
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 2
gctcttgtca cttgggccta tattgtggaa ttttgcttca gatggagatt tcttataaag 60
gtaaagattt atgtattact acaaaagtat aatggggttt ttgaagagcc taaaagcctc 120
catcctca 128
<210> 3
<211> 20
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 3
gctcttgtca cttgggccta 20
<210> 4
<211> 21
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 4
tgaggatgga ggcttttagg c 21
Claims (5)
1. thin shell mountain pecan Peach cultivars Pawnee characteristic sequence, its sequence are as follows：
5’-AATTCAATGGCTCTTGTCACTTGGGCCTATATTGTGGAATTTTGCTTCAGATGGAGATTTCTTATAAAGG
TAAAGATTTATGTATTACTACAAAAGTATAATGGGGTTTTTGAAGAGCCTAAAAGCCTCCATCCTCATAGGACTCAA
GACCATTAGATTGTGTTGAATGATGAGTCAGTTCCTATGATTTTAAGACCATATAGATACCCTTATTATCAAAAGAC
AGGGAT-3’。
2. thin shell mountain pecan Peach cultivars Pawnee molecular specificity labeled primers, the primer sequence are as follows：
Sense primer：5′-GCTCTTGTCACTTGGGCCTA-3′；
Anti-sense primer：5′-TGAGGATGGAGGCTTTTAGGC-3′.
3. a kind of molecular specificity labeled primers using described in claim 1 carry out quick to thin shell mountain pecan Peach cultivars Pawnee
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 128bp specific DNA band, then thin shell mountain pecan Peach cultivars to be measured are that thin shell mountain pecan Peach cultivars are Pawnee, it is on the contrary then
It is no；The molecular specificity labeled primers sequence is：
Sense primer：5′-GCTCTTGTCACTTGGGCCTA-3′；
Anti-sense primer：5′-TGAGGATGGAGGCTTTTAGGC-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 circulate；Most after 72 DEG C of filling-in 300s, final temperature is 4 DEG C.
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, on automatic gel imaging system
Photograph, if unique 128bp DNA bands occurs in electrophoresis result, thin shell mountain pecan Peach cultivars to be measured are Pawnee, on the contrary then no.
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| CN109652428A (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 Sioux |
| CN109652588A (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 Elliott |
| 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 |
| CN109652415A (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 Osage |
| CN109694865A (en) * | 2019-02-28 | 2019-04-30 | 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 Desirable |
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| CN113981125A (en) * | 2021-11-04 | 2022-01-28 | 中国林业科学研究院亚热带林业研究所 | Molecular marker of apocarya variety Creek and application thereof |
| CN114182033A (en) * | 2021-11-04 | 2022-03-15 | 中国林业科学研究院亚热带林业研究所 | SSR molecular markers of carya illinoensis Mahan, Pawnee and Greenriver and application thereof |
| CN114182034A (en) * | 2021-11-04 | 2022-03-15 | 中国林业科学研究院亚热带林业研究所 | SSR molecular marker of apocarya variety McMillian and application thereof |
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| CN109652415B (en) * | 2019-02-28 | 2021-12-17 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of pecan variety Osage |
| 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 |
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| CN109652588B (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 Elliott |
| CN109694865B (en) * | 2019-02-28 | 2022-02-11 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Desirable |
| 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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| CN114182033A (en) * | 2021-11-04 | 2022-03-15 | 中国林业科学研究院亚热带林业研究所 | SSR molecular markers of carya illinoensis Mahan, Pawnee and Greenriver and application thereof |
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