CN103937887B - A kind of rapid identification method for grape phylloxera resistance - Google Patents
A kind of rapid identification method for grape phylloxera resistance Download PDFInfo
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- CN103937887B CN103937887B CN201410131800.7A CN201410131800A CN103937887B CN 103937887 B CN103937887 B CN 103937887B CN 201410131800 A CN201410131800 A CN 201410131800A CN 103937887 B CN103937887 B CN 103937887B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention provides a kind of rapid identification method for grape phylloxera resistance, with the DNA of grape rootstock to be detected for template, polymerase chain reaction (PCR) amplification is carried out to the DNA molecular marker mutually chain with grape phylloxera resistance, electrophoresis detection is carried out to the product that amplification obtains, if there is the DNA band corresponding with described DNA molecular marker in electrophoresis result, then illustrate that corresponding grape rootstock has grape phylloxera resistance, if there is not the DNA band corresponding with described DNA molecular marker in electrophoresis result, then illustrate that corresponding grape rootstock does not have grape phylloxera resistance, the present invention can distinguish grape rootstock fast with or without grape phylloxera resistance, solve existing grape phylloxera Resistance Identification and need specific isolation condition, be subject to environmental influence and the problem needing the time limit longer.
Description
Technical field
The invention belongs to grape breeding technical field, relate to the rapid detection to crushing insect grape phylloxera resistance.
Background technology
Grape phylloxera is the crushing insect pest of one of grape, and mainly through taking food root system harm grape, conventional plant protection technology is difficult to control.The most effective prevention method adopts grape phylloxera Resistant rootstock at present, carries out grafting cultivation.But in grape rootstock breeding process, whether the stock of seed selection has Phylloxera resistance, need to carry out the qualification of stock to grape phylloxera resistance, the root nodule numbers that after usual employing grape rootstock root system artificial inoculation grape phylloxera, statistics infection is formed is identified, without nodule formation or nodule formation quantity few for having resistance, perception that what root nodule numbers was many is.But this Resistance Identification needs to carry out avoiding insect pest to spread under special isolation condition, identify the qualification of the needs revision test of more than 2 years in addition, expend time in longer, the envrionment conditions in operating process may affect the accuracy of qualification result.
Summary of the invention
The object of the present invention is to provide a kind of rapid identification method for grape phylloxera resistance.
For achieving the above object, present invention employs following technical scheme.
With the DNA of grape rootstock to be detected for template, polymerase chain reaction (PCR) amplification is carried out to the DNA molecular marker mutually chain with grape phylloxera resistance, electrophoresis detection is carried out to the product that amplification obtains, if there is the DNA band corresponding with described DNA molecular marker in electrophoresis result, then illustrate that corresponding grape rootstock has grape phylloxera resistance, if there is not the DNA band corresponding with described DNA molecular marker in electrophoresis result, then illustrate that corresponding grape rootstock does not have grape phylloxera resistance.
Described DNA molecular marker is microsatellite molecular marker.
Described polymerase chain reaction (PCR) amplification adopts oligodeoxynucleotide sequence Seq01 and Seq02 as primer pair, and Seq01 has the nucleotide sequence as shown in SEQ.ID.NO.1, and Seq02 has the nucleotide sequence as shown in SEQ.ID.NO.2.
The size of described DNA band is about 169bp.
The invention provides one Rapid identification grape rootstock on DNA level and have nonresistant method to grape phylloxera, can distinguish grape rootstock fast with or without grape phylloxera resistance, solving existing grape phylloxera Resistance Identification needs specific isolation condition, is subject to environmental influence and the problem needing the time limit longer.
Accompanying drawing explanation
Fig. 1 has Phylloxera resistance grape rootstock and does not have the result figure that Phylloxera resistance grape rootstock adopts present method to detect; In Fig. 1,1,2,3 swimming lanes are the known grape rootstock result after testing without Phylloxera resistance, 4,5,6 swimming lanes are the grape rootstock result after testing that there will be a known Phylloxera resistance, M swimming lane is molecular weight standard, numeral 250,200 and 150 represents DNA stripe size, unit is base pair (basepair, bp).
Embodiment
Below in conjunction with accompanying drawing, the present invention is elaborated.
The invention provides a kind of by detect with the closely linked DNA molecular marker of grape phylloxera resistance trait gene judge the method whether grape phylloxera resistance exists.The present invention is based on micro-satellite (Simplesequencerepeat, SSR) molecular marking technique, using oligodeoxynucleotide sequence Seq01 and Seq02 provided by the invention as SSR primer pair, with the DNA of grape rootstock to be detected for template, polymerase chain reaction (Polymerasechainreaction is carried out to the specific DNA fragments mutually chain with grape phylloxera resistance and DNA molecular marker, PCR) increase, after electrophoresis detection is carried out to amplification acquisition product, filter out the specific DNA band (between kind length indifference) that size is about 169bp, close linkage relation is there is with grape phylloxera resistance, the grape rootstock to be detected namely with this specific DNA band has grape phylloxera resistance, without the grape rootstock to be detected of this particular bands without grape phylloxera resistance.
Concrete steps of the present invention are as follows:
(1) conventional CTAB method or SDS method is adopted to extract the genomic dna (i.e. sample DNA) at the blade of grape rootstock to be detected, stem section or root system position respectively, the step that CTAB method extracts DNA in sample is as follows: 1. about 0.4g vanes liquid nitrogen freezing is ground into powder fast, proceeds in 2mL centrifuge tube; 2. add CTAB Extraction buffer 700 μ L and the beta-mercaptoethanol 20 μ L of 65 DEG C of preheatings immediately, concuss, to fully mixing, is put in 65 DEG C of water-baths and is incubated 30min, be cooled to room temperature after taking-up; 3. add isopyknic chloroform/primary isoamyl alcohol=24:1, put upside down mixing lightly, the centrifugal 10min of 12000rpm under room temperature, transfer supernatant liquor is in new centrifuge tube; 4. after 3., in supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol=24:1, put upside down mixing lightly, the centrifugal 10min of 12000rpm under room temperature, in transfer supernatant liquor to new centrifuge tube; 5. after 4., in supernatant liquor, the Virahol of equal-volume through-20 DEG C of precoolings is added, after putting upside down mixing to adularescent flocks appearance lightly, being statically placed in-20 DEG C of 30min makes precipitation generate, in 4 DEG C of centrifugal 10min of 12000rpm, outwell supernatant liquor, retain throw out (DNA); 6. add the 70% washing with alcohol throw out (the centrifugal 1min of 13000rpm) of 500 μ L precoolings, outwell supernatant liquor, retain precipitation, lay equal stress on duplicate step once; 7. add absolute ethanol washing precipitation (the centrifugal 1min of 13000rpm) of 500 μ L precoolings, outwell supernatant liquor, after precipitation is standing air-dry, be dissolved in 40 μ LddH
2in O, 4 DEG C are spent the night; 8. get the sample DNA 0.5 μ L agarose gel electrophoresis method after dissolving and detect DNA concentration, purity and palliating degradation degree.The agarose nucleic acid electrophoresis of the OD260/280 of sample DNA between 1.8-2.0, through 0.7% detects DNA sample inclusion-free after testing, without under the condition of obvious degradation, it is that 100ng/uL is stand-by that each grape rootstock sample DNA to be detected is diluted with water to final concentration;
(2) by the strand oligo DNA according to oligodeoxynucleotide sequence seq01 provided by the invention and seq02 synthesis, thin up becomes the 10umol/L aqueous solution respectively, and sequence is respectively:
Seq01:5’-TGTTGGTGTGTGTTTGTACGTG-3’
Seq02:5’-TGTTATTGAGTTAGAATGAAGTGTTGG-3’
(3) add in each 0.2mL thin-walled PCR pipe: the MgCl of the dNTP of 2uL2mmol/L, 1.7uL25mmol/L
2, 2ul10Xtaq damping fluid, each 2.0uL of seq01, seq02 solution that step (2) is prepared, the testing sample DNA solution 2uL that step (1) is prepared, adds Taq polysaccharase 1 unit, adds water to cumulative volume 20ul, slight concussion mixing, brief centrifugation;
(4) above-mentioned PCR pipe is placed in PCR instrument, performs following program: 94 degree, 5 minutes; 94 degree, 60 seconds, 55 degree, 90 seconds, 72 degree, 90 seconds, circulate 35 times; 72 degree, 5 minutes; EP (end of program);
(5) product in PCR pipe is carried out the polyacrylate hydrogel electrophoresis of 6% or the agarose gel electrophoresis of 2.5%, check during different testing sample DNAPCR reacts the DNA band produced;
The present invention have selected known there is Phylloxera resistance grape rootstock kind (comprising Rici, Boerner, V.Riparia181g etc.) and the known grape rootstock kind without Phylloxera resistance (comprising good No. 8 of China, Vitis Amurensis, Vitis davidi etc.) amount to more than 30 and plant and test, wherein (see Fig. 1) in single test, swimming lane 1 ~ 3 is followed successively by good No. 8 of China, Vitis Amurensis, Vitis davidi; Swimming lane 4 ~ 6 is followed successively by Rici, Boerner, V.Riparia181g, and above material is all collected in Xibei Univ. of Agricultural & Forest Science & Technology's Grape Germplasm resource garden;
(6) can determine according to test-results, the band that the DNA fragmentation that the grape rootstock with Phylloxera resistance can produce the specific size (169bp) of position shown in arrow in Fig. 1 is formed, the band that the specific size DNA fragmentation that the grape rootstock without Phylloxera resistance can not produce corresponding shown position is formed; If the swimming lane adding a certain DNA to be detected has the DNA band of this specific size of 169bp to produce, then this testing sample DNA derives from the grape rootstock plant with Phylloxera resistance, if produce without the DNA band of specific size (169bp), then this testing sample DNA derives from the grape rootstock plant without Phylloxera resistance.
Adopt vegetative grape rootstock can extract DNA in any season and detect by blade, root system, stem section, sampling point does not affect the consistence of detected result, and sampling point and sample time do not affect detected result.
The present invention may be used for the DNA molecular marker assisted selection of grape phylloxera resistance trait, for the Seedling selection of the resistant material in grape phylloxera resistance breeding and eliminating of non-resistance material; Meanwhile, with this specific DNA band for road sign, can carry out grape phylloxera resistant gene Fine Mapping or by chromosome walking method close to grape phylloxera resistant gene, thus to lay the first stone for the clone of grape phylloxera resistant gene.
Claims (1)
1. for a rapid identification method for grape phylloxera resistance, it is characterized in that: comprise the following steps:
With the DNA of grape rootstock to be detected for template, polymerase chain reaction (PCR) amplification is carried out to the DNA molecular marker mutually chain with grape phylloxera resistance, electrophoresis detection is carried out to the product that amplification obtains, if there is the DNA band corresponding with described DNA molecular marker in electrophoresis result, then illustrate that corresponding grape rootstock has grape phylloxera resistance, if there is not the DNA band corresponding with described DNA molecular marker in electrophoresis result, then illustrate that corresponding grape rootstock does not have grape phylloxera resistance;
Described DNA molecular marker is microsatellite molecular marker;
Described polymerase chain reaction (PCR) amplification adopts oligodeoxynucleotide sequence Seq01 and Seq02 as primer pair, and the nucleotide sequence of Seq01 is as shown in SEQ.ID.NO.1, and the nucleotide sequence of Seq02 is as shown in SEQ.ID.NO.2;
The size of described DNA band is 169bp.
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| CN201410131800.7A CN103937887B (en) | 2014-04-02 | 2014-04-02 | A kind of rapid identification method for grape phylloxera resistance |
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| CN201410131800.7A CN103937887B (en) | 2014-04-02 | 2014-04-02 | A kind of rapid identification method for grape phylloxera resistance |
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| CN103937887A CN103937887A (en) | 2014-07-23 |
| CN103937887B true CN103937887B (en) | 2015-12-09 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101646769A (en) * | 2007-02-20 | 2010-02-10 | 孟山都技术公司 | Invertebrate micrornas |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101646769A (en) * | 2007-02-20 | 2010-02-10 | 孟山都技术公司 | Invertebrate micrornas |
Non-Patent Citations (2)
| Title |
|---|
| Junke Zhang et al.A framework map from grapevine V3125 (Vitis vinifera ‘Schiava grossa’ × ‘Riesling’) × rootstock cultivar ‘Bö * |
| rner’ (Vitis riparia × Vitis cinerea) to localize genetic determinants of phylloxera root resistance.《Theor Appl Genet》.2009,第119卷第1039－1051页. * |
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