CN107779521A - A kind of DNA bar code and its purposes in muscat is identified - Google Patents
A kind of DNA bar code and its purposes in muscat is identified Download PDFInfo
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Abstract
The invention discloses a kind of DNA bar code, and its nucleotide sequence is as shown in SEQ ID NO.1.DNA bar code makes public for the first time the ITS sequence of muscat, and effective molecular labeling is provided for the species identification of muscat；Other DNA bar code also includes part conserved sequence, has versatility in the species identification applied to grape.The invention discloses the method for the nucleotide sequence for preparing above-mentioned DNA bar code and primer, can be used in the sequence in acquisition muscat ITS areas.Detection primer and kit the invention also discloses the method for identifying molecules of muscat, for identifying muscat, enter performing PCR amplification using detection primer or kit and obtain grape ITS2 region sequences, then extension increasing sequence is compared with the DNA bar code sequence of muscat or applied to the phylogenetic tree for building grape, realize the accurate identification to grape variety, it is reproducible and qualification process is simple to operate, efficiency high.
Description
Technical field
The invention belongs to species identification technical field, and in particular to a kind of DNA bar code, the purposes of DNA bar code and its
Preparation method, and the detection primer based on its design, the method for identifying molecules of muscat and the examination for identifying muscat
Agent box.
Background technology
Muscat (Vitis rotundifolia) originates in southeastern US, the business metaplasia since 18th century mid-terms
Production plantation, its kind color have green, coppery, red and black etc., and fruit has special fragrance and flavor, can both eaten raw,
Fruit juice, preserved fruit, jam and wine etc. can be processed into again.Muscat is a kind of special grape species, species more other than Vitis
More dyads, primarily now to plant in the U.S., China has to introduce a fine variety on a small quantity, there is the plantation of certain scale in Guangxi in recent years,
But other provinces rarely have cultivation.Studies have found that muscat contains the resveratrol more than vinifera grape decades of times, and
The peculiar natural products such as tannic acid and its derivative that other grape varieties are lacked, 3,5- bioside anthocyanidin.With life bar
The improvement of part, people focus more on for health, therefore muscat has a wide promotion prospect at home, but current Portugal
Grape seedling market is mixed the genuine with the fictitious, and fake and forged event often occurs, and the interests of consumer is incurred loss.Due to the fruit of grape
Outward appearance is influenceed by different cultivation management methods, soil climate condition, and fruit colour is often different, relies solely on traditional shape
Larger limitation be present to distinguish muscat kind in state authentication method.In view of the abundant nutritive value of muscat and
The ever-increasing market demand, it is imperative to establish reliable authentication method for muscat.
With developing rapidly for modern molecular biology particularly DNA sequencing technology, start to develop DNA bar code in the world
Technology (DNA barcoding) differentiates species.DNA bar code technology be 2003 by Canadian zoologist hebert first
It is proposed, be using standard, have enough variations, easily amplification and relatively short DNA fragmentation in species specificity and
Diversity between species and a kind of new biological status identification system created, can be achieved the fast automatic identification to species.Should
Technology has been successfully applied to the fields such as living species classification and identification, and as one of the most rapid Frontier that is in progress.
For the DNA barcoding identification research of plant, due in plant evolution course easily occur hybridization and
Reticulate evolution event, and same gene evolutionary rate in different population is also different, causes the research of plant bar code than dynamic
Thing is more complex, does not find a general DNA barcoding sequence so far, recommend more sequence have matK,
TrnH-psbA, rbcL and ITS etc..
ITS areas are the transcribed spacers in rDNA, by form of tandem repeats of the number in terms of necessarily be present in one or
On multiple chromogene sites, it is between 18S rDNA, 5.8S rDNA and 26S rDNA, comprising by 5.8S rDNA points
Two intervening sequences of ITS1 and ITS2 separated.ITS is amphilepsis in angiosperm, because it has DNA sequence dna length
The features such as consistent variable between genome in conservative, genome (A ' lvarez and Wendel, 2003), in Aconitum, mandarin orange
Tangerine category etc. be used in plant phyletic evolution study infer the origin of species and affiliation (Luo et al., 2005；Li et
al.,2010)。
In recent years, researcher (Shilin Chen et.al.Validation of the ITS2 Region as a
Novel DNA Barcode for Identifying Medicinal Plant Species.PLOSONE.2010,15(1)：
E8613) by being found after contrasting the sample more than 4,000 kinds of plants：The species of ITS2 sequences differentiate that success rate is up to 92.7%,
Therefore, ITS2 sequences are suitable as the standard DNA bar code of plant, to identify wider plant group.ITS2 sequences are present
The second internal transcribed spacer between 5.8S rDNA and 28S rDNA, there is sequence fragment more to guard, mutational site
Easily morphed between more, species, the advantage such as length is shorter, therefore ITS2 sequences are the preferable works that plant identification is classified in genus and species
Tool.
Because ITS sequence analysis can substantially reflect between category, the base pair difference of inter-species, ITS sequence piece in addition
Section is smaller, is easy to analyze, and so far, existing many proof ITS genes are effective in the taxonomic identification of plant.Bar code
Technology provides an effective approach for the species identification of muscat, but only exists a numbering in Genbank at present and be
KF544885 muscat ITS sequence, after Blast compares identification, KF544885 and powdery mildew (Erysiphe
Necator sequence homology) is up to 99%, but with the correlated series of Vitis without homology.Therefore, there is no so far disclosed
Muscat ITS sequence information, due to the missing of ITS sequence information, difficulty is brought for muscat species identification.
The content of the invention
Therefore, the technical problem to be solved in the present invention is solve the ITS sequence letter for lacking muscat in the prior art
Breath, bar codes technique can not be applied to realize the species identification to muscat；So as to provide a kind of DNA bar code, DNA bar code
Include the ITS sequence of muscat, the fast and accurate species identification to muscat can be realized.
The invention provides a kind of DNA bar code, the nucleotide sequence of the DNA bar code is as shown in SEQ ID NO.1.
The invention provides use of the above-mentioned DNA bar code in identification muscat and/or structure grape phylogenetic tree
On the way.
The invention provides the preparation method of the nucleotide sequence of described DNA bar code, comprise the following steps：
A. Vitis 18s rDNA, 5.8s rDNA and 28s rDNA conserved nucleotide sequence design primer are utilized；
B. the genomic DNA of muscat is extracted；
C. using the genomic DNA of the muscat of step b extractions as template, enter performing PCR using the step a primers designed and expand
Increase；
D. pcr amplification product step c obtained is sequenced, and then carries out sequence assembly using sequencing result, obtains DNA bars
The nucleotide sequence of shape code.
Described preparation method, the primer include：
Itb-5, its nucleotide sequence is as shown in SEQ ID NO.2；
Itb-3, its nucleotide sequence is as shown in SEQ ID NO.3；
Itc-5, its nucleotide sequence is as shown in SEQ ID NO.4；
Itc-3, its nucleotide sequence is as shown in SEQ ID NO.5.
Described preparation method, the nucleotide sequence of the DNA bar code include：It is part 18s rDNA, whole ITS1, complete
Portion 5.8s rDNA, whole ITS2 and part 28s rDNA nucleotide sequence.
The invention provides a kind of primer sets for being used to identify muscat, the primer sets are included based on above-mentioned DNA
The detection primer of barcode design.
Described primer sets, the primer sets include detection primer as described below：
Itd-5, its nucleotide sequence is as shown in SEQ ID NO.6；
Itd-3, its nucleotide sequence is as shown in SEQ ID NO.7；
Ite-5, its nucleotide sequence is as shown in SEQ ID NO.8；
Ite-3, its nucleotide sequence is as shown in SEQ ID NO.9.
The invention provides a kind of method for identifying molecules of muscat, comprise the following steps：
(1) genomic DNA of grape sample is extracted；
(2) using the genomic DNA of the grape sample of step (1) extraction as template, using as claimed in claims 6 or 7
Primer sets enter performing PCR amplification；
(3) pcr amplification product that step (2) obtains is sequenced, then by shown in sequencing result and SEQ ID NO.1
Nucleotide sequence be compared.
Described method for identifying molecules, the reaction system of the PCR amplifications include：
Master PCR Mix (2X), 12.5 μ L；
Forward Primer, 10 μM, 0.5 μ L；
Reverse Primer, 10 μM, 0.5 μ L；
ddH20,10.5 μ L.
Described method for identifying molecules, the reaction condition of the reaction of the PCR amplifications are：94 DEG C of pre-degeneration 10min；94℃
45s is denatured, 55 DEG C of annealing 45s, 72 DEG C of extension 50s, totally 30 circulate；72 DEG C re-extend 10min.
Be used to identifying the kit of muscat the invention provides a kind of, the kit include above-mentioned primer and/
Or above-mentioned reaction system.
The invention provides the recombinant vector comprising above-mentioned DNA bar code and/or transgenosis recombinant bacterium.
The present invention has the following advantages that compared with the prior art：
(1) DNA bar code provided by the invention, the nucleotide sequence of DNA bar code is as shown in SEQ ID NO.1.This hair
The bright nucleotide sequence for making public for the first time muscat DNA bar code, the wherein nucleotide sequence of DNA bar code both include sequence
The fast ITS1 and ITS2 sequences of variation rate, but comprising sequence evolution than more conservative 5.8S rDNA and part 18s rDNA and
Part 28s rDNA.DNA bar code provided by the invention has filled up the blank of muscat ITS sequence in Genbank, DNA bars
Shape code is on the one hand due to including conservative region sequence, with good versatility during applied to species identification system；On the other hand
There is the variability feature of the conservative and nucleotide sequence in length due to ITS sequence, closed for plant inter-species relationship
Timing accuracy height is sentenced by system.Therefore, the present invention provides effective molecular labeling for the authenticity of muscat.
(2) use of the DNA bar code provided by the invention in identification muscat and/or structure grape phylogenetic tree
On the way.DNA bar code provided by the invention is when for identifying muscat, by the nucleotides of sequence and DNA bar code to be measured
Sequence is compared, using ITS sequence evolutionary rate it is fast the characteristics of, only matched completely with DNA bar code sequence in sequence to be measured
When, the grape variety of sequence to be measured is muscat, DNA bar code degree of accuracy with identification when identifying muscat is high,
Short and reproducible advantage the time required to identification.
On the other hand, DNA bar code provided by the invention can be applied to structure grape phylogenetic tree, and then be used for Portugal
Phylogenetic research between grape inter-species even population.
(3) preparation method of the nucleotide sequence of DNA bar code provided by the invention, can be used in obtaining DNA bar code
Precise sequence, and the simple to operate of preparation method, operating time are short, efficiency high.
The primer designed in preparation method provided by the invention, conserved sequence designs of the primer I tb-5 based on 5.8s rDNA
Obtain, conserved sequences of the Itb-3 based on 28s rDNA designs to obtain, and Itb-5 and Itb-3 expand to obtain 5.8s rDNA, part
Sequence between 28s rDNA and 5.8s rDNA and 28s rDNA；Conserved sequences of the Itc-5 based on 18s rDNA is designed to
Arrive, conserved sequences of the Itc-3 based on 28s rDNA designs to obtain, and Itc-5 and Itc-3 expand to obtain part 18s rDNA, part
Sequence between 28s rDNA and 18s rDNA and 28s rDNA；The DNA bar code of muscat is obtained by sequence assembly
Sequence.Meanwhile primer provided by the invention can also be applied to the ITS region sequences of other kinds in amplification Vitis.
(4) draw provided by the present invention for the primer sets of identification muscat, including the detection designed based on DNA bar code
Thing.When detection primer be based in DNA bar code conserved sequence design when, can expand different grape variety conservative regions it
Between ITS sequence, and by subsequent sequence compare identify whether grape variety is muscat；When detection primer is to be based on DNA
During height series of variation design in bar code, can by directly to the PCR amplifications of grape sample ITS sequence to be measured,
Realize the identification to muscat.
(5) primer sets of identification muscat provided by the invention, wherein primer I td-5 and Ite-5 are based on 5.8s rDNA
Conserved sequence design to obtain, conserved sequences of the primer I td-3 and Ite-3 based on 28s rDNA designs to obtain, Itd-5 and Itd-
3 primer pairs and Ite-5 and Ite-3 primer pairs be able to amplification obtain part 5.8s rDNA, part 28s rDNA and
ITS2 sequences between 5.8s rDNA and 28s rDNA, the cultivar identification realized to muscat is compared by subsequent sequence.By
In ITS2 series jumps site is more, sequence is short, pass through ITS2 sequences carry out species identification the degree of accuracy it is high, inspection provided by the invention
Survey primer can realize the accurate identification to muscat species.
(6) method for identifying molecules of muscat provided by the invention, using above-mentioned detection primer, detection it is accurate
Height, and the step of method for identifying molecules is simple, easy to operate, shortens the qualification time of muscat, identification it is reproducible.This
The reaction system for the PCR amplifications that invention provides and the reaction condition of PCR amplifications can realize the standard to grape ITS2 regional sequences
Really amplification, the identification for muscat.
(7) provided by the present invention for identifying the kit of muscat, including above-mentioned detection primer and/or above-mentioned anti-
System is answered, quick, the accurate identification to muscat can be realized.
(8) recombinant vector and/or transgenosis recombinant bacterium provided by the invention comprising above-mentioned DNA bar code, Neng Gouzuo
For the normative reference of muscat identification.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The required accompanying drawing used is briefly described in embodiment or description of the prior art, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the agarose gel electrophoresis testing result that primer I tb-5 and Itb-3PCR are expanded in the embodiment of the present invention 1,
Band shows DNA Maker and the band of primer I tb-5 and Itb-3 amplification successively from left to right in figure, DNA Maker from upper and
Under show 100bp, 250bp, 500bp, 750bp, 1000bp, the DNA bands of 2000bp sizes successively；
Fig. 2 is the agarose gel electrophoresis testing result that primer I tc-5 and Itc-3PCR are expanded in the embodiment of the present invention 1,
Band shows DNA Maker and the band of primer I tc-5 and Itc-3 amplification successively from left to right in figure, DNA Maker from upper and
Under show 100bp, 250bp, 500bp, 750bp, 1000bp, the DNA bands of 2000bp sizes successively；
Fig. 3 is using muscat Nobel, muscat Ge Weier and this resistance to ratio of muscat in the embodiment of the present invention 2
The phylogenetic tree of special ITS2 sequence constructs；
Fig. 4 is the systematic growth of the ITS2 sequence constructs using No. 1~No. 3 grapes in East China in experimental example 1 of the present invention
Tree.
Embodiment
Illustrate embodiments of the present invention below by way of specific embodiment, unless otherwise indicated, disclosed in this invention
Experimental method use the art routine techniques, all sequencings and primer synthesis are limited by Nanjing Jin Sirui biotechnologies
Company is completed, and used reagent and raw material can be bought by market in embodiment.
Embodiment 1
The present embodiment provides a kind of method for the nucleotide sequence for preparing DNA bar code, specifically includes following steps：
1st, primer is designed
Utilize GenBank (https://www.ncbi.nlm.nih.gov/genbank/) obtaining Vitis, totally 41 kinds not
With the ITS sequence of grape variety, the ITS sequence information of each grape variety is as shown in table 1.With the sub- software of DNAStar softwares
Arrangement is compared in these sequences by SeqMan, so as to obtain 18S, ITS1,5.8S, ITS2 and 28S of each grape variety part
Sequence or the conservative base of sufficient sequence and change base, according to the sequences Design 2 of conservative region to primer I tc-5, Itc-3 and
Itb-5、Itb-3.Wherein conserved sequences of the primer I tb-5 based on 5.8s rDNA designs to obtain, primer I tb-5 nucleotides sequence
Row are as shown in SEQ ID NO.2, and conserved sequences of the Itb-3 based on 28s rDNA designs to obtain, primer I tb-3 nucleotide sequence
As shown in SEQ ID NO.3；Conserved sequences of the Itc-5 based on 18srDNA designs to obtain, and primer I tc-5 nucleotide sequence is such as
Shown in SEQ ID NO.4, conserved sequences of the Itc-3 based on 28s rDNA designs to obtain, and primer I tc-3 nucleotide sequence is such as
Shown in SEQ ID NO.5.
Grape ITS associated sequence informations in the GenBank of table 1
2nd, the genomic DNA of muscat is extracted
Carried with CTAB plant genome DNA rapid extractions kit (being purchased from Beijing Ai Delai bio tech ltd)
Take, concrete operation step is as follows：
(1) taking appropriate muscat, (net purchase is from muscat Nobel of Guangxi China, the muscat lattice of Guangxi China
This resistance to bit of the muscat of Weir or Chinese Guangdong) blade or pulp 100mg or so, it is put into small mortar, adds 60 μ L 65
DEG C preheating lysate PL (added beta -mercaptoethanol to mass concentration be 2%), be fully ground immediately.
(2) transfer is homogenized to a 1.5ml centrifuge tube, vortex oscillation.
(3) 65 DEG C of water-bath 20-60 minutes, during water-bath overturn centrifuge tube with biased sample for several times.
(4) 700 μ L chloroforms/isoamyl alcohol (volume ratio 24 is added:1 mixing), overturn and fully mix a few minutes and (or be vortexed mixed
It is even), 13000rpm is centrifuged 5 minutes.
(5) supernatant is carefully drawn to a new 1.5ml centrifuge tube, is careful not to be drawn onto boundary material.As supernatant compares
Muddiness, then repeat step 4 one times are needed, until obtaining bright supernatant.
(6) relatively accurately estimation supernatant amount, 1.5 times of volume combination liquid PQ of addition (have added anhydrous second in 15ml combination liquid PQ
Alcohol 30ml) after be vortexed at once, fully mix.
(7) mixture (including the precipitation being likely to occur) obtained by previous step is added in an adsorption column AC, (adsorption column is put
Enter in collecting pipe) 13000rpm centrifuge 30 seconds, outwell in collecting pipe waste liquid (first plus 700 μ L centrifugation, abandon waste liquid, add surplus
Remaining solution, centrifuge again).
(8) 500 μ L mortifiers are added and remove liquid IR, 12000rpm centrifugation 30 seconds, abandon waste liquid.
(9) 600 μ L rinsing liquids WB (having added absolute ethyl alcohol 60ml in 15ml rinsing liquids WB), 12000rpm centrifugations 30 are added
Second, discard waste liquid.
(10) 600 μ L rinsing liquids WB, 12000rpm centrifugation 30 seconds is added, discards waste liquid.
(11) adsorption column AC being put back in sky collecting pipe, 13000rpm is centrifuged 2 minutes, removes rinsing liquid as far as possible, in order to avoid drift
Residual ethanol suppresses downstream reaction in washing lotion.
(12) adsorption column AC is taken out, is put into a clean centrifuge tube, adds 100 μ L to elute in the middle part of adsorbed film
Buffer solution EB (elution buffer preheats in 65-70 DEG C of water-bath in advance), room temperature are placed 3-5 minutes, and 12000rpm centrifuges 1 point
Clock.Obtained solution is rejoined in centrifugal adsorbing column, room temperature is placed 2 minutes, and 12000rpm is centrifuged 1 minute.
(13) obtained DNA will be extracted and be stored in -20 DEG C, it is standby.
3rd, using the genomic DNA that step 2 is extracted as template, using (the purchase of BU-Taq 2 × Master PCR Mix reagents
From biouniquer companies) enter performing PCR amplification, the primer pair Itc-5 and Itc-3 that amplimer is designed using step 1, and
Itb-5 and Itb-3.The reaction system of PCR amplifications is as shown in table 2, and the reaction condition of PCR amplifications is：94 DEG C of pre-degeneration 10min；
94 DEG C of denaturation 45s, 55 DEG C of annealing 45s, 72 DEG C of extension 50s, totally 30 circulate；72 DEG C re-extend 10min.
The reaction system of the PCR of table 2 amplifications
| PCR Mix(2X) | 12.5μL |
| Genomic DNA | 1μL |
| Forward Primer, 10 μM | 0.5μL |
| Reverse Primer, 10 μM | 0.5μL |
| ddH20 | 10.5μL |
4th, the pcr amplification product that step 3 obtains is subjected to electrophoresis, each swimming lane sample-adding on 1% agarose gel electrophoresis
20 μ L, Maker sample-adding 3-5 μ L.Primer I tb-5 and Itb-3 correspondingly expand 5.8s rDNA on rDNA, part 28s rDNA and
With the sequence between 5.8s rDNA and 28s rDNA, the agarose electrophoresis testing result of pcr amplification product as shown in figure 1,
The visible electrophoretic band in position near 600bp；Primer I tc-5 and Itc-3 correspondingly expand rDNA upper parts 18s rDNA, part
Sequence between 28s rDNA and 18s rDNA and 28s rDNA, agarose electrophoresis testing result such as Fig. 2 of pcr amplification product
It is shown, the visible electrophoretic band in position near 1000bp.Pcr amplification product send sequencing after purification, is carried out using sequencing result
Sequence assembly, obtain the nucleotide sequence of muscat DNA bar code.The nucleotide sequence of DNA bar code includes：Part 18s
RDNA, whole ITS1, whole 5.8s rDNA, whole ITS2 and part 28s rDNA nucleotide sequence, the core of DNA bar code
Nucleotide sequence is as shown in SEQ ID NO.1.
5th, the nucleotide sequence of DNA bar code
The nucleotides sequence of the muscat DNA bar code obtained in step 4 is listed in GenBank and is compared, as a result
Display muscat DNA bar code homology highest sequence AM462492.2 is vinifera grape Its correlated serieses, but two sequences
Row homology is only 90%, and the nucleotide sequence of DNA bar code sequence related to an original muscat Its in database
Homology is not present in row.
Embodiment 2
The present embodiment provides a kind of method for identifying molecules of muscat, and the nucleotide sequence based on DNA bar code
Phylogenetic tree construction, specifically include following steps：
1st, grape sample to be measured is used as using muscat Nobel, muscat Ge Weier and this resistance to bit of muscat respectively
This (net purchase from Chinese Guangdong and Guangxi China), according to the extracting method of the genomic DNA shown in embodiment 1, extract three kinds of circles
The genomic DNA of leaf grape.
2nd, using the genomic DNA that step 1 is extracted as template, detection primer Itd-5 and Itd-3 are utilized respectively, and
Two kinds of primer pairs of Ite-5 and Ite-3 enter performing PCR amplification.Detection primer Itd-5 nucleotide sequence as shown in SEQ ID NO.6,
Itd-3 nucleotide sequence as shown in SEQ ID NO.7, Ite-5 nucleotide sequence as shown in SEQ ID NO.8, Ite-3's
Nucleotide sequence is as shown in SEQ ID NO.9.The reaction system of PCR amplifications is as shown in table 2, and the reaction condition of PCR amplifications is：94
DEG C pre-degeneration 10min；94 DEG C of denaturation 45s, 55 DEG C of annealing 45s, 72 DEG C of extension 50s, totally 30 circulate；72 DEG C re-extend 10min.
3rd, the pcr amplification product that step 2 obtains is subjected to electrophoresis, each swimming lane sample-adding on 1% agarose gel electrophoresis
20 μ L, Maker sample-adding 3-5 μ L.Wherein detection primer Itd-5 and Ite-5 is that the conserved sequence based on 5.8s rDNA is designed to
Arrive, conserved sequences of the primer I td-3 and Ite-3 based on 28s rDNA designs to obtain；Itd-5 and Itd-3 primer pairs and Ite-5
Amplification, which is able to, with Ite-3 primer pairs obtains part 5.8s rDNA, part 28s rDNA and 5.8s rDNA and 28s
ITS2 sequences between rDNA.Pcr amplification product send sequencing after purification, as a result as follows：
(1) using muscat Nobel DNA as template, it is 430bp to expand to obtain length using primer I td-5 and Itd-3
DNA fragmentation, the sequencing result of DNA fragmentation is as shown in SEQ ID NO.10.
Expand to obtain the DNA fragmentation that length is 300bp using primer I te-5 and Ite-3, the sequencing result of DNA fragmentation is such as
Shown in SEQ ID NO.11.
(2) using muscat Ge Weier DNA as template, it is 433bp to expand to obtain length using primer I td-5 and Itd-3
DNA fragmentation, the sequencing result of DNA fragmentation is as shown in SEQ ID NO.12.
Expand to obtain the DNA fragmentation that length is 297bp using primer I te-5 and Ite-3, the sequencing result of DNA fragmentation is such as
Shown in SEQ ID NO.13.
(3) using resistance to this bit DNA of muscat as template, expand to obtain length using primer I td-5 and Itd-3 be
423bp DNA fragmentation, the sequencing result of DNA fragmentation is as shown in SEQ ID NO.14.
Expand to obtain the DNA fragmentation that length is 303bp using primer I te-5 and Ite-3, the sequencing result of DNA fragmentation is such as
Shown in SEQ ID NO.15.
The sequencing result of above-mentioned DNA fragmentation is compared with the nucleotide sequence of DNA bar code, finds above-mentioned 6 sequences
Row match completely with DNA bar code, and therefore, the method for identifying molecules for the muscat that the present embodiment provides is applied to roundleaf Portugal
During the species identification of grape, detection it is specific good, testing result is accurate, while also has simple to operate, detection efficiency high and inspection
Survey reproducible advantage.
4th, the structure of phylogenetic tree
It is (specific using the sequencing result in the ITS2 areas of the 3 muscat kinds obtained in step 3 and other grape varieties
Including：Vitis aestivalis；Vitis amurensis；Vitis arizonica；Vitis betulifolia；Vitis
chunganensis；Vitis cinerea；Vitis flexuosa；Vitis heyneana；Vitis labrusca；Vitis
lanata；Vitis latifolia；Vitis longii；Vitis luochengensis；Vitis menghaiensis；
Vitis piasezkii；Vitis pseudoreticulata；Vitis riparia；Vitis tiliifolia；Vitis
vinifera；Vitis vulpina) ITS2 sequences, use adjacent method (N-J, Neighbor with the softwares of Mega 6.0
Joining the systematic evolution tree of ITS2 sequences) is built, Kimure-2-parameter genetic distances are selected with the softwares of Mega 6.0
Model, the bootstrapping confidence level of computing system tree interior joint, phylogenetic tree construction.As a result (Vroits2 as shown in Figure 3：Roundleaf
Grape ITS2 sequences；Vroitdgits2：Using primer I td-5 and Itd-3 the muscat Ge Weier obtained ITS2 sequences；
Vroitdniits2：Using the ITS2 sequences of primer I td-5 and Itd-3 this resistance to bit of muscat obtained；
Vroitdnoits2：Using the ITS2 sequences of primer I td-5 and Itd-3 muscat Nobel obtained；Vroitegits2：
Using primer I te-5 and Ite-3 the muscat Ge Weier obtained ITS2 sequences；Vroiteniits2：Using primer I te-5
With the ITS2 sequences of Ite-3 this resistance to bit of muscat obtained；Vroitenoits2：Obtained using primer I te-5 and Ite-3
Muscat Nobel ITS2 sequences；Vvits2：The ITS2 sequences of vitis vinifera, remaining ITS2 sequence source reference table
The ITS associated sequence informations of grape variety shown in 1), 3 muscat kind Nobels, this resistance to bit, Ge Weier belong to same
One species, that is, belong to muscat.The affiliation of each grape species is substantially divided into 3 major classes, and Vitis longii are independent
For one kind, muscat and Vitis tiliifolia are classified as one kind, and remaining grape species is collected as one kind.Further demonstrate
Validity and feasibility of the DNA bar code provided by the invention in grape variety identification, classification and phylogenetic study.
Embodiment 3
A kind of kit for being used to identify muscat is present embodiments provided, kit includes：
(1) detection primer for being used to expand grape ITS2 region sequences is to Itd-5 and Itd-3, or Ite-5 and Ite-3.Inspection
Primer I td-5 nucleotide sequence is surveyed as shown in SEQ ID NO.6, Itd-3 nucleotide sequence as shown in SEQ ID NO.7,
Ite-5 nucleotide sequence is as shown in SEQ ID NO.8, and Ite-3 nucleotide sequence is as shown in SEQ ID NO.9；
Above-mentioned kit also includes：
(2) reaction system of PCR amplifications, each reaction solution is as shown in table 2 in reaction system；
(3) the DNA bar code standard items of muscat, DNA bar code standard items are to be connected with sequence shown in SEQ ID NO.1
The pMD18-T carriers (pMD18-T is purchased from Takara) of row, or include the genetic engineering bacterium of above-mentioned carrier；
Kit provided by the invention can be applied to the sequence amplification in the ITS2 regions of Vitis different cultivars, enter one
Step, extension increasing sequence is compared with the nucleotide sequence of the DNA bar code shown in muscat SEQ ID NO.1, if comparing
As a result it is 100% matching, then the grape identified is muscat.Or the ITS2 region sequences obtained by expanding and a variety of differences
The ITS2 region sequences (the ITS2 region sequences for including muscat) of grape variety phylogenetic tree construction together, judges detected Portugal
The species of grape sample are sorted out.
Experimental example 1
The method for identifying molecules of muscat shown in Application Example 2, to being purchased from 3 different sellers of East China
Available muscat carries out the identification of the kind true and false：
(1) using the DNA of East China 1 as template, it is 510bp's to expand to obtain length using primer I td-5 and Itd-3
DNA fragmentation, the sequencing result of DNA fragmentation is as shown in SEQ ID NO.16.
Expand to obtain the DNA fragmentation that length is 397bp using primer I te-5 and Ite-3, the sequencing result of DNA fragmentation is such as
Shown in SEQ ID NO.17.
(2) using the DNA of East China 2 as template, it is 476bp's to expand to obtain length using primer I td-5 and Itd-3
DNA fragmentation, the sequencing result of DNA fragmentation is as shown in SEQ ID NO.18.
Expand to obtain the DNA fragmentation that length is 401bp using primer I te-5 and Ite-3, the sequencing result of DNA fragmentation is such as
Shown in SEQ ID NO.19.
(3) using the DNA of East China 3 as template, it is 483bp's to expand to obtain length using primer I td-5 and Itd-3
DNA fragmentation, the sequencing result of DNA fragmentation is as shown in SEQ ID NO.20.
Expand to obtain the DNA fragmentation that length is 404bp using primer I te-5 and Ite-3, the sequencing result of DNA fragmentation is such as
Shown in SEQ ID NO.21.
By the way that above-mentioned sequence is compared with the nucleotide sequence of DNA bar code, it is found that above-mentioned 6 sequences can not be with
The nucleotide sequence of DNA bar code matches completely, is not real muscat purchased from three kinds of grapes of East China therefore.
The construction method for the phylogenetic tree that Application Example 2 provides, utilizes above-mentioned No. 1~No. 3 grapes in East China
Sequencing result phylogenetic tree construction.As a result (Vshuyang1itdits2 as shown in Figure 4：Obtained using primer I td-5 and Itd-3
The ITS2 sequences of the Shuyang 1 obtained；Vshuyang2itdits2：Using primer I td-5 and the Itd-3 Shuyang 2 obtained
ITS2 sequences；Vshuyang3itdits2：Using the ITS2 sequences of primer I td-5 and the Itd-3 Shuyang 3 obtained；
Vshuyang1iteits2：Using the ITS2 sequences of primer I te-5 and the Ite-3 Shuyang 1 obtained；
Vshuyang2iteits2：Using the ITS2 sequences of primer I te-5 and the Ite-3 Shuyang 2 obtained；
Vshuyang3iteits2：Using the ITS2 sequences of primer I te-5 and the Ite-3 Shuyang 3 obtained；Vroits2：Muscat
ITS2 sequences；Vvits2：The ITS2 sequences of vitis vinifera, grape variety shown in remaining ITS2 sequence source references table 1
ITS associated sequence informations), above-mentioned No. 1~No. 3 grapes in East China range vinifera grape, wherein No. 1 plant and No. 2 plant
Belong to same species with vinifera grape, namely No. 1 plant and No. 2 plant belong to vinifera grape on species taxonomy, not seller
The muscat called oneself, No. 3 plant are also close with Vitis vulpina close to vinifera grape.The relationship of each grape species is closed
System is substantially divided into 3 major classes, and Vitis longii are individually for one kind, and muscat and Vitis tiliifolia are classified as one kind, its
Remaining grape species are collected as one kind including the grape from East China.
Obviously, above-described embodiment is only intended to clearly illustrate example, and is not the restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Sequence table
<110>Nanjing Xiaozhuang College
<120>A kind of DNA bar code and its purposes in muscat is identified
<130> NJHA201700057
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 989
<212> DNA
<213>Artificial sequence (DNA barcode)
<400> 1
tcgtaaccaa gggttccgta ggtgaacctg gggaagaatc attgtcgaac ctctgaacga 60
cccgcgaaca cgctacaact ctccgagggg gggcggagcg ggcacgagct cgccacttcg 120
ccgccctttc cccaactcgg gaggccccgg gagccccgcc caccatcgtg gcccgcgcac 180
aggcgggggg tggcccacag ttgccctttt gagtgaacaa cgaaccccgg cgcggaacgc 240
gctaagaaac ctcataatga agcagcgtac tcccgtcgcc ccggtcccga ccgggtgcgt 300
tggcgagtcg tcatcatttc accgaacaca aacgactctc ggcaacggat atctaagctc 360
tcgcatcgat gaagaatgta gcaaaatgcg atacttggtg tgaattgcag aatcccgtga 420
atcatcgagt ctttgaacgc aagttgcgcc cgaagccatt aggccgaggg cacgcctgcc 480
tgggcgtcac gcacccgtcg cccccccacc tcccttccct cgggacgaga ggcacggagg 540
gggcggacat tggcctcccg tgggcgccta agcccgcggt tggccgaaaa tctgtcccgc 600
ggcgacgtac gccacgacga gcggtggatt cgcacgaagc acggctcggc gtcgcgcgcg 660
tcacgtcgcg tcgggacgag aacggccccg gtgaccctcg atcgcgaccc caggtcaggc 720
gggaccaccc gctgagttta agcatatcaa taagcggagg aaaagaaact tacaaggatt 780
cccctagtaa cggcgagcga accgggaaga gcccagcttg agaatcgggc ggccccgccg 840
tccgaattgt agtctggaga agcgtcctca gcggcggacc gggcccaagt cccctggaag 900
ggggcgccgg agagggtgag agccccgtcg tgcccggacc ctgtcgcacc acgaggcgct 960
gtcggcgagt cgggtttttg ggaaatgtg 989
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Itb-5)
<400> 2
cgatgaagaa cgtagcaaaa 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Itb-3)
<400> 3
cattccctaa caacccgact 20
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence (Itc-5)
<400> 4
tcgtaacaag gtttccgtag gtg 23
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Itc-3)
<400> 5
cctcgtggtg cgacagggtc 20
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence (Itd-5)
<400> 6
atcccgtgaa tcatcgagtc tttg 24
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Itd-3)
<400> 7
gcccggtccg ccgctgagga 20
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence (Ite-5)
<400> 8
ttgcagaatc ccgtgaatc 19
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence (Ite-3)
<400> 9
tcgctcgccg ttactagggg aatc 24
<210> 10
<211> 430
<212> DNA
<213>Artificial sequence (Nobel Itd)
<400> 10
ttaggccgag ggcacgcctg cctgggcgtc acgcacccgt cgccccccca cctcccttcc 60
ctcgggacga gaggcacgga gggggcggac attggcctcc cgtgggcgcc taagcccgcg 120
gttggccgaa aatctgtccc gcggcgacgt acgccacgac gagcggtgga ttcgcacgaa 180
gcacggctcg gcgtcgcgcg cgtcacgtcg cgtcgggacg agaacggccc cggtgaccct 240
cgatcgcgac cccaggtcag gcgggaccac ccgctgagtt taagcatatc aataagcgga 300
ggaaaagaaa cttacaagga ttcccctagt aacggcgagc gaaccgggaa gagcccagct 360
tgagaatcgg gcggccccgc cgtccgaatt gtagtctgga gaagcgtcct cagcggcgga 420
ccgggcccaa 430
<210> 11
<211> 300
<212> DNA
<213>Artificial sequence (Nobel Ite)
<400> 11
gccattaggc cgagggcacg cctgcctggg cgtcacgcac ccgtcgcccc cccacctccc 60
ttccctcggg acgagaggca cggagggggc ggacattggc ctcccgtggg cgcctaagcc 120
cgcggttggc cgaaaatctg tcccgcggcg acgtacgcca cgacgagcgg tggattcgca 180
cgaagcacgg ctcggcgtcg cgcgcgtcac gtcgcgtcgg gacgagaacg gccccggtga 240
ccctcgatcg cgaccccagg tcaggcggga ccacccgctg agtttaagca tatcaataag 300
<210> 12
<211> 433
<212> DNA
<213>Artificial sequence (GV Itd)
<400> 12
aagccattag gccgagggca cgcctgcctg ggcgtcacgc acccgtcgcc cccccacctc 60
ccttccctcg ggacgagagg cacggagggg gcggacattg gcctcccgtg ggcgcctaag 120
cccgcggttg gccgaaaatc tgtcccgcgg cgacgtacgc cacgacgagc ggtggattcg 180
cacgaagcac ggctcggcgt cgcgcgcgtc acgtcgcgtc gggacgagaa cggccccggt 240
gaccctcgat cgcgacccca ggtcaggcgg gaccacccgc tgagtttaag catatcaata 300
agcggaggaa aagaaactta caaggattcc cctagtaacg gcgagcgaac cgggaagagc 360
ccagcttgag aatcgggcgg ccccgccgtc cgaattgtag tctggagaag cgtcctcagc 420
ggcggaccgg gcc 433
<210> 13
<211> 297
<212> DNA
<213>Artificial sequence (GV Ite)
<400> 13
attaggccga gggcacgcct gcctgggcgt cacgcacccg tcgccccccc acctcccttc 60
cctcgggacg agaggcacgg agggggcgga cattggcctc ccgtgggcgc ctaagcccgc 120
ggttggccga aaatctgtcc cgcggcgacg tacgccacga cgagcggtgg attcgcacga 180
agcacggctc ggcgtcgcgc gcgtcacgtc gcgtcgggac gagaacggcc ccggtgaccc 240
tcgatcgcga ccccaggtca ggcgggacca cccgctgagt ttaagcatat caataag 297
<210> 14
<211> 423
<212> DNA
<213>Artificial sequence (Nesbitt Itd)
<400> 14
aggccgaggg cacgcctgcc tgggcgtcac gcacccgtcg cccccccacc tcccttccct 60
cgggacgaga ggcacggagg gggcggacat tggcctcccg tgggcgccta agcccgcggt 120
tggccgaaaa tctgtcccgc ggcgacgtac gccacgacga gcggtggatt cgcacgaagc 180
acggctcggc gtcgcgcgcg tcacgtcgcg tcgggacgag aacggccccg gtgaccctcg 240
atcgcgaccc caggtcaggc gggaccaccc gctgagttta agcatatcaa taagcggagg 300
aaaagaaact tacaaggatt cccctagtaa cggcgagcga accgggaaga gcccagcttg 360
agaatcgggc ggccccgccg tccgaattgt agtctggaga agcgtcctca gcggcggacc 420
ggg 423
<210> 15
<211> 303
<212> DNA
<213>Artificial sequence (Nesbitt Ite)
<400> 15
agccattagg ccgagggcac gcctgcctgg gcgtcacgca cccgtcgccc ccccacctcc 60
cttccctcgg gacgagaggc acggaggggg cggacattgg cctcccgtgg gcgcctaagc 120
ccgcggttgg ccgaaaatct gtcccgcggc gacgtacgcc acgacgagcg gtggattcgc 180
acgaagcacg gctcggcgtc gcgcgcgtca cgtcgcgtcg ggacgagaac ggccccggtg 240
accctcgatc gcgaccccag gtcaggcggg accacccgct gagtttaagc atatcaataa 300
gcg 303
<210> 16
<211> 510
<212> DNA
<213>Artificial sequence (shuyang1 Itd)
<400> 16
atcccgtgaa tcatcgagtc tttgaacgca agttgcgccc gaagccatta ggccgagggc 60
acgcctgcct gggcgtcacg cacccgtcgc ccccccacac ctccctcccc ctcggacggg 120
aggcggagag ggggcggaca ttggcctccc gtgggcgccc cagcccgcgg ttggccgaaa 180
atcggtcccg cggcgacgta cgccacgacg agcggtggat ttcgcacggc tcggcgtcgc 240
gcgcgtcacg tcgcctcagg ggccccccac gagaacggcc cgagaccctc gatcgcgacc 300
ccaggtcagg cgggaccacc cgctgagttt aagcatatca ataagcggag gaaaagaaac 360
ttacaaggat tcccctagta acggcgagcg aaccgggaag agcccagctt gagaatcggg 420
cggccccgcc gtccgaattg tagtctggag aagcgtcctc agcggcggac cgggcccaag 480
tcccctggaa gggggcgccg gagagggtga 510
<210> 17
<211> 397
<212> DNA
<213>Artificial sequence (shuyang1 Ite)
<400> 17
ttgcagaatc ccgtgaatca tcgagtcttt gaacgcaagt tgcgcccgaa gccattaggc 60
cgagggcacg cctgcctggg cgtcacgcac ccgtcgcccc cccacacctc cctccccctc 120
ggacgggagg cggagagggg gcggacattg gcctcccgtg ggcgccccag cccgcggttg 180
gccgaaaatc ggtcccgcgg cgacgtacgc cacgacgagc ggtggatttc gcacggctcg 240
gcgtcgcgcg cgtcacgtcg cctcaggggc cccccacgag aacggcccga gaccctcgat 300
cgcgacccca ggtcaggcgg gaccacccgc tgagtttaag catatcaata agcggaggaa 360
aagaaactta caaggattcc cctagtaacg gcgagcg 397
<210> 18
<211> 476
<212> DNA
<213>Artificial sequence (shuyang2 Itd)
<400> 18
tcccgtgaat cattgagtct ttgaacgcaa gttgcgcccg aagccattag gccgagggca 60
cgcctgcctg ggcgtcacgc acccgtcgcc cccccacacc tccctccccc tcggacggga 120
ggcggagagg gggcggacat tggcctcccg tgggcgcccc agcccgcggt tggccgaaaa 180
tcggtcccgc ggcgacgtac gccacgacga gcggtggatt tcgcacggct cggcgtcgcg 240
cgcgtcacgt cgcctcaggg gccccccacg agaacggccc gagaccctcg atcgcgaccc 300
caggtcaggc gggaccaccc gctgagttta agcatatcaa taagcggagg aaaagaaact 360
tacaaggatt cccctagtaa cggcgagcga accgggaaga gcccagcttg agaatcgggc 420
ggccccgccg tccgaattgt agtctggaga agcgtcctca gcggagaatc gggcaa 476
<210> 19
<211> 401
<212> DNA
<213>Artificial sequence (shuyang2 Ite)
<400> 19
ttttgcagaa tcccgtgaat catcgagtct ttgaacgcaa gttgcgcccg aagccattag 60
gccgagggca cgcctgcctg ggcgtcacgc acccgtcgcc cccccacacc tccctccccc 120
tcggacggga ggcggagagg gggcggacat tggcctcccg tgggcgcccc agcccgcggt 180
tggccgaaaa tcggtcccgc ggcgacgtac gccacgacga gcggtggatt tcgcacggct 240
cggcgtcgcg cgcgtcacgt cgcctcaggg gccccccacg agaacggccc gagaccctcg 300
atcgcgaccc caggtcaggc gggaccaccc gctgagttta agcatatcaa taagcggagg 360
aaaagaaact tacaaggatt cccctagtaa cggcgagcga a 401
<210> 20
<211> 483
<212> DNA
<213>Artificial sequence (shuyang3 Itd)
<400> 20
gaatcccgtg aatcattgag tctttgaacg caagttgcgc ccgaagccat taggccgagg 60
gcacgcctgc ctgggcgtca cgcacccgtc gcccccccac acctccctcc cccccgggga 120
cacgggaggc ggagaggggg cggacattgg cctcccgtgg gcgccccagc ccgcggttgg 180
ccgaaaatcg gtcccgcggc gacgtacgcc acgacgagcg gtggatttcg cacggctcgg 240
cgtcgcgcgc gtcacgtcgc ctcagggacc ccccacgaga acggcccgag accctcgatc 300
gcgaccccag gtcaggcggg accacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ctagtaacgg cgagcgaacc gggaagagcc cagcttgaga 420
atcgggcggc cccgccgtcc gaattgtagt ctggagaagc gtcctcagcg gagaatcggg 480
caa 483
<210> 21
<211> 404
<212> DNA
<213>Artificial sequence (shuyang3 Ite)
<400> 21
tttgcagaat cccgtgaatc attgagtctt tgaacgcaag ttgcgcccga agccattagg 60
ccgagggcac gcctgcctgg gcgtcacgca cccgtcgccc ccccacacct ccctcccccc 120
cggggacacg ggaggcggag agggggcgga cattggcctc ccgtgggcgc cccagcccgc 180
ggttggccga aaatcggtcc cgcggcgacg tacgccacga cgagcggtgg atttcgcacg 240
gctcggcgtc gcgcgcgtca cgtcgcctca gggacccccc acgagaacgg cccgagaccc 300
tcgatcgcga ccccaggtca ggcgggacca cccgctgagt ttaagcatat caataagcgg 360
aggaaaagaa acttacaagg attcccctag taacggcgag cgaa 404
Claims (12)
1. a kind of DNA bar code, it is characterised in that the nucleotide sequence of the DNA bar code is as shown in SEQ ID NO.1.
2. purposes of the DNA bar code in identification muscat and/or structure grape phylogenetic tree described in claim 1.
A kind of 3. method for the nucleotide sequence for preparing DNA bar code as claimed in claim 1, it is characterised in that including with
Lower step：
A. Vitis 18s rDNA, 5.8s rDNA and 28s rDNA conserved nucleotide sequence design primer are utilized；
B. the genomic DNA of muscat is extracted；
C. using the genomic DNA of the muscat of step b extractions as template, performing PCR amplification is entered using the step a primers designed；
D. pcr amplification product step c obtained is sequenced, and then carries out sequence assembly using sequencing result, obtains DNA bar code
Nucleotide sequence.
4. preparation method according to claim 3, it is characterised in that the primer includes：
Itb-5, its nucleotide sequence is as shown in SEQ ID NO.2；
Itb-3, its nucleotide sequence is as shown in SEQ ID NO.3；
Itc-5, its nucleotide sequence is as shown in SEQ ID NO.4；
Itc-3, its nucleotide sequence is as shown in SEQ ID NO.5.
5. the preparation method according to claim 3 or 4, it is characterised in that the nucleotide sequence bag of the DNA bar code
Include：Part 18s rDNA, whole ITS1, whole 5.8s rDNA, whole ITS2 and part 28s rDNA nucleotide sequence.
6. a kind of primer sets for being used to identify muscat, it is characterised in that the primer sets are included based on described in claim 1
DNA bar code design detection primer.
7. primer sets according to claim 6, it is characterised in that the primer sets include detection primer as described below：
Itd-5, its nucleotide sequence is as shown in SEQ ID NO.6；
Itd-3, its nucleotide sequence is as shown in SEQ ID NO.7；
Ite-5, its nucleotide sequence is as shown in SEQ ID NO.8；
Ite-3, its nucleotide sequence is as shown in SEQ ID NO.9.
8. a kind of method for identifying molecules of muscat, it is characterised in that comprise the following steps：
(1) genomic DNA of grape sample is extracted；
(2) using the genomic DNA of the grape sample of step (1) extraction as template, primer as claimed in claims 6 or 7 is utilized
Group enters performing PCR amplification；
(3) pcr amplification product that step (2) obtains is sequenced, then by sequencing result and the core shown in SEQ ID NO.1
Nucleotide sequence is compared, and/or sequencing result is applied to the phylogenetic tree of structure grape.
9. method for identifying molecules according to claim 8, it is characterised in that the reaction system of the PCR amplifications includes：
PCR Mix (2X), 12.5 μ L；
Forward Primer, 10 μM, 0.5 μ L；
Reverse Primer, 10 μM, 0.5 μ L；
ddH20,10.5 μ L.
10. method for identifying molecules according to claim 8 or claim 9, it is characterised in that the reaction of the reaction of the PCR amplifications
Condition is：94 DEG C of pre-degeneration 10min；94 DEG C of denaturation 45s, 55 DEG C of annealing 45s, 72 DEG C of extension 50s, totally 30 circulate；72 DEG C again
Extend 10min.
11. a kind of kit for being used to identify muscat, it is characterised in that the kit is included described in claim 6 or 7
Primer sets and/or claim 9 described in reaction system.
12. include the recombinant vector and/or transgenosis recombinant bacterium of the DNA bar code described in claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112322634A (en) * | 2020-11-16 | 2021-02-05 | 北京农业生物技术研究中心 | DNA bar code for identifying lily plants and application thereof |
| CN116287372A (en) * | 2022-12-14 | 2023-06-23 | 赣南师范大学 | DNA bar code for identifying Duying of round fruit, identification method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277444A (en) * | 2011-09-19 | 2011-12-14 | 南京农业大学 | Method for quickly distinguishing grape varieties by random amplified polymorphic deoxyribonucleic acid (RAPD) |
| KR20130078111A (en) * | 2011-12-30 | 2013-07-10 | 대한민국(농촌진흥청장) | Scar markers for discrimination of grape cultivars and use thereof |
-
2017
- 2017-10-10 CN CN201710934877.1A patent/CN107779521B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277444A (en) * | 2011-09-19 | 2011-12-14 | 南京农业大学 | Method for quickly distinguishing grape varieties by random amplified polymorphic deoxyribonucleic acid (RAPD) |
| KR20130078111A (en) * | 2011-12-30 | 2013-07-10 | 대한민국(농촌진흥청장) | Scar markers for discrimination of grape cultivars and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| VELASCO.R等: "Accession NO: AM462492.2，Vitis vinifera contig VV78X177628.4, whole genome shotgun sequence", 《GENBANK》 * |
| 刘丽芳等: "《中药分析学》", 31 August 2015 * |
| 宋慧芳等: "分子标记在山葡萄品种鉴定上的应用现状", 《北方园艺》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112322634A (en) * | 2020-11-16 | 2021-02-05 | 北京农业生物技术研究中心 | DNA bar code for identifying lily plants and application thereof |
| CN116287372A (en) * | 2022-12-14 | 2023-06-23 | 赣南师范大学 | DNA bar code for identifying Duying of round fruit, identification method and application |
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