CN107557434A - Thin shell mountain pecan Peach cultivars Van Deman characteristic sequence, labeled primer and authentication method - Google Patents
Thin shell mountain pecan Peach cultivars Van Deman 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 Van Deman 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 Van Deman.The molecular specificity labeled primers sequence is as follows：Sense primer：5′‑ATGCAAGACGCTTGTGGAGA‑3′；Anti-sense primer：5′‑CCGACCCGACCCGATAAAAT‑3′.Molecular specificity labeled primers of the present invention can carry out quick Early Identification to thin shell mountain pecan Peach cultivars Van Deman, 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 Van Deman characteristic sequence, molecular specificity labeled primers and profit
The method that Rapid identification is carried out to thin shell mountain pecan Peach cultivars Van Deman with the molecular specificity labeled primers.
(2) background technology
Apocarya (Carya(Wangenh.) K.Koch) be most have in Juglandaceae hickory through
The seeds for value of helping.Apocarya is typical outcrossing plant, and existing production practices are found, the kind configuration of apocarya
It is to 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,
The cultivar about 1000 of its seed selection name.China introduces a fine variety existing more than the 100 years history of apocarya, and production at present is upper normal
Kind also has tens, and production practices for many years are proved the suitable life that the vast subtropical zone in China is apocarya
Area.
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 Van Deman characteristic sequence, molecular specificity labeled primers
And the method for carrying out Rapid identification to thin shell mountain pecan Peach cultivars Van Deman using the molecular specificity labeled primers.
The technical solution adopted by the present invention is：
Thin shell mountain pecan Peach cultivars Van Deman characteristic sequence, its sequence are as follows：5’-
AATTCGAAATGATGAGTACCTTGTCGTAGCCATGTCCGGATGTAGAAGACAGAATATGCAAGACGCTTGTGGAGAAG
CATTTTTAGTTGTATCCAGAAACAAAAAGAAGAATCGAGAAATCACTTATATCTTGAAACGGGTATCGGATAATTTC
CGATTAAATCAGAAACGGGTACGGTCATTTCTACTTCCGAATAATATTGAGACGGTTCGGGTATTTTATCGGGTCGG
GTCGGTTAAATCGGCTGGGCCGGAATACCGG-3’(SEQ ID No.1)
The invention further relates to thin shell mountain pecan Peach cultivars Van Deman molecular specificity labeled primers, the primer sequence
For：
Sense primer：5′-ATGCAAGACGCTTGTGGAGA-3′；
Anti-sense primer：5′-CCGACCCGACCCGATAAAAT-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 Van Deman specific DNA is obtained by a large amount of screening tests
After fragment (SEQ ID No.1), by the fragment cloning and sequencing, differ larger genetic fragment for sequence and devise more than 1000
To primer, screened and verified in 24 samples, by primary dcreening operation and three times more than repeatability sampling secondary screening, it is final to obtain
Molecular specificity labeled primers, with the specific primer to thin shell mountain pecan Peach cultivars carry out PCR amplifications, only Van Deman can
The specific fragment (SEQ ID No.2) of 181bp sizes is obtained, other thin shell mountain pecan Peach cultivars can not obtain specific piece
Section.It should be 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
It is only limitted to apocarya.
Primer amplified product for features described above Sequence sequences Design is 181bp：5’-
ATGCAAGACGCTTGTGGAGAAGCATTTTTAGTTGTATCCAGAAACAAAAAGAAGAATCGAGAAATCACTTATATCTT
GAAACGGGTATCGGATAATTTCCGATTAAATCAGAAACGGGTACGGTCATTTCTACTTCCGAATAATATTGAGACGG
TTCGGGTATTTTATCGGGTCGGGTCGG-3’(SEQ ID No.2)。
Van Deman are the kinds that Los Angeles,U.S is selected from seedling tree, and tree body is tall and big, and growth potential is vigorous.Female flower is first
Ripe type.The frequent yellowish-brown of exocarp, middle fruit type, mean fruit weight 7.5g or so, nut oblong, top point, bottom is relatively round,
Flatten ellipse in cross section；Nut butter color is to Gold production, and dorsocentral ridge is elongated, and main groove is deep, and base portion crack is obvious.
The invention further relates to the molecular specificity labeled primers described in a kind of utilization to thin shell mountain pecan Peach cultivars Van Deman
The method for carrying out Rapid identification, methods described are：The genomic DNA of thin shell mountain pecan Peach cultivars blade to be measured is extracted as template,
Using the molecular specificity labeled primers as amplimer, enter performing PCR amplification, electrophoresis detection is carried out to amplified production, if electric
There are the DNA bands of 181bp sizes in swimming result, then thin shell mountain pecan Peach cultivars to be measured are Van Deman, on the contrary then no；Described point
Sub- specificity labeled primers sequence is：
Sense primer：5′-ATGCAAGACGCTTGTGGAGA-3′；
Anti-sense primer：5′-CCGACCCGACCCGATAAAAT-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 1 μ L0.25% bromjophenol blues buffer solution, point sample is in 1.5% agar
On sugared gel, in 1 × TAE buffer solutions, electrophoresis under 5V/cm voltages, electrophoresis terminates rear EB dyeing, in automatic gel imaging system
Upper photograph, if 181bp DNA bands occurs in electrophoresis result, thin shell mountain pecan Peach cultivars to be measured are Van Deman；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
Van Deman carry out quick discriminating, and method is simple, quick, accurate, are that appearance features distinguish that thin shell mountain pecan Peach cultivars can not replace
The Molecular tools in generation.
(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 DL2000marker；Only
Numbering 3 is that thin shell mountain pecan Peach cultivars Van Deman have amplified the specific DNA band that molecular weight is 181bp；Remaining numbering is it
Its thin shell mountain pecan Peach cultivars, the specific DNA band that there are no 181bp 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 0.1g 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′-ATGCAAGACGCTTGTGGAGA-3′；And anti-sense primer：5′-
CCGACCCGACCCGATAAAAT-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 3 thin shell mountain pecan Peach cultivars Van Deman clearly to become clear, surely wherein only from numbering
The specific DNA band that fixed molecular weight is about 181bp, and the thin shell mountain pecan Peach cultivars of remaining numbering, there are no 181bp sizes
Special 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
Early stage for thin shell mountain pecan Peach cultivars Van Deman identifies that its stability, specificity are very high.
Sequence table
<110>Zhejiang Prov. Forest Science Inst
<120>Thin shell mountain pecan Peach cultivars Van Deman characteristic sequence, labeled primer and authentication method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 262
<212> DNA
<213> Carya illinoensis
<400> 1
aattcgaaat gatgagtacc ttgtcgtagc catgtccgga tgtagaagac agaatatgca 60
agacgcttgt ggagaagcat ttttagttgt atccagaaac aaaaagaaga atcgagaaat 120
cacttatatc ttgaaacggg tatcggataa tttccgatta aatcagaaac gggtacggtc 180
atttctactt ccgaataata ttgagacggt tcgggtattt tatcgggtcg ggtcggttaa 240
atcggctggg ccggaatacc gg 262
<210> 2
<211> 181
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 2
atgcaagacg cttgtggaga agcattttta gttgtatcca gaaacaaaaa gaagaatcga 60
gaaatcactt atatcttgaa acgggtatcg gataatttcc gattaaatca gaaacgggta 120
cggtcatttc tacttccgaa taatattgag acggttcggg tattttatcg ggtcgggtcg 180
g 181
<210> 3
<211> 20
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 3
atgcaagacg cttgtggaga 20
<210> 4
<211> 20
<212> DNA
<213> Unknown
<220>
<223>Artificial sequence
<400> 4
ccgacccgac ccgataaaat 20
Claims (5)
1. thin shell mountain pecan Peach cultivars Van Deman characteristic sequence, its sequence are as follows：
5’-AATTCGAAATGATGAGTACCTTGTCGTAGCCATGTCCGGATGTAGAAGACAGAATATGCAAGACGCTTGT
GGAGAAGCATTTTTAGTTGTATCCAGAAACAAAAAGAAGAATCGAGAAATCACTTATATCTTGAAACGGGTATCGGA
TAATTTCCGATTAAATCAGAAACGGGTACGGTCATTTCTACTTCCGAATAATATTGAGACGGTTCGGGTATTTTATC
GGGTCGGGTCGGTTAAATCGGCTGGGCCGGAATACCGG-3’。
2. thin shell mountain pecan Peach cultivars Van Deman molecular specificity labeled primers, the primer sequence are as follows：
Sense primer：5′-ATGCAAGACGCTTGTGGAGA-3′；
Anti-sense primer：5′-CCGACCCGACCCGATAAAAT-3′.
3. a kind of molecular specificity labeled primers using described in claim 1 carry out fast to thin shell mountain pecan Peach cultivars VanDeman
The method of speed identification, methods described are：The genomic DNA of thin shell mountain pecan Peach cultivars blade to be measured is extracted as template, with described
Molecular specificity labeled primers enter performing PCR amplification, electrophoresis detection are carried out to amplified production, if electrophoresis result as amplimer
There is unique 181bp specific DNA band, then thin shell mountain pecan Peach cultivars to be measured are that thin shell mountain pecan Peach cultivars are VanDeman, instead
It is then no；The molecular specificity labeled primers sequence is：
Sense primer：5′-ATGCAAGACGCTTGTGGAGA-3′；
Anti-sense primer：5′-CCGACCCGACCCGATAAAAT-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 1 μ L0.25% bromjophenol blues buffer solution, point sample coagulates in 1.5% agarose
On glue, electrophoresis, electrophoresis terminate rear EB dyeing, can photograph well in automatic gel imaging system in 1 × TAE buffer solutions, under 5V/cm voltages
Phase, if unique 181bp DNA bands occurs in electrophoresis result, thin shell mountain pecan Peach cultivars to be measured are Van Deman, on the contrary then no.
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| CN201710782316.4A CN107557434B (en) | 2017-09-02 | 2017-09-02 | Characteristic sequence, labeled primer and identification method of Carya illinoensis variety Van Deman |
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| CN201710782316.4A CN107557434B (en) | 2017-09-02 | 2017-09-02 | Characteristic sequence, labeled primer and identification method of Carya illinoensis variety Van Deman |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660136A (en) * | 2018-06-26 | 2018-10-16 | 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 Davis |
| CN109652416A (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 Sumner |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
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| CN108660136A (en) * | 2018-06-26 | 2018-10-16 | 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 Davis |
| CN108660136B (en) * | 2018-06-26 | 2020-08-11 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Davis |
| 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 |
| 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 |
| 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 |
| 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 |
| CN109652416A (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 Sumner |
| CN109652416B (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 apocarya variety Sumner |
| 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 |
| 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 |
| 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 |
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