CN108277295A - Suitable for one group of corn SSR molecular marker of capillary electrophoresis detection technology and its application - Google Patents
Suitable for one group of corn SSR molecular marker of capillary electrophoresis detection technology and its application Download PDFInfo
- Publication number
- CN108277295A CN108277295A CN201810299328.6A CN201810299328A CN108277295A CN 108277295 A CN108277295 A CN 108277295A CN 201810299328 A CN201810299328 A CN 201810299328A CN 108277295 A CN108277295 A CN 108277295A
- Authority
- CN
- China
- Prior art keywords
- corn
- seq
- ssr molecular
- molecular marker
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention provides one group of corn SSR molecular marker suitable for capillary electrophoresis detection technology, it is applied, totally 13 SSR molecular markers, and provides the primer combination of a set of detection SSR molecular marker suitable for Capillary Electrophoresis platform.It can be with using this 13 SSR molecular markers：(1) Maize SSR fingerprint database is built；(2) the maternal Source Tracing of corn sample；(3) corn variety identification and analysis of genetic diversity；(4) corn Kingsoft 6 and 18 kinds of Deng Hai are distinguished.The application of corn SSR molecular marker through the invention can expand the range in corn serviceable indicia site in genomic level；For corn variety, germplasm identification, affiliation evaluation, the researchs such as cytoplasmic inheritance characteristic provide new tool, and application prospect is good.
Description
Technical field
The invention belongs to crops technical field of molecular biology, specifically, being related to being suitable for capillary electrophoresis detection
One group of corn SSR molecular marker of technology and its application.
Background technology
Corn be have grain, warp, fruit, feeding, can polynary purposes one of the important crops of China, the increase pair of total output
National increases in grain production contribution rate occupies first of major cereal crops.With the quickening of breeding process, corn variety quantity is also drastically
Increase.To the structure of the identification of corn variety, analysis of genetic diversity and DNA fingerprint database, can it is objective, fully understand and work as
Preceding corn variety present situation collects variety managements, breed breeding and germ plasm resource and protection is of great significance.
With the development of molecular biotechnology, molecular labeling type and detection means are gradually improved, and various molecular labelings are
Through being widely used in Maize genetic diversity research.In numerous molecular labelings, simple repeated sequence (Simple Sequence
Repeats, SSR) it marks and is answered extensively because having many advantages, such as easy, quick, repeatability height, polymorphism height, codominant marker
With.Traditional SSR operation coordinates some other biotechnology to carry out gene polynorphisms by polyacrylamide gel electrophoresis
Analysis, these method non-automated take, and different allelic variations are difficult to accurately identify, different batches response data is difficult to unification
Processing.
Fluorescent marker capillary electrophoresis detection technology is because having the advantages that efficient, automation, in various plants molecular labeling
Extremely wide application prospect is shown in research.This method uses the fluorochrome label SSR primers of several different colours, then
Different fluorescent markers, the discrepant PCR product of expanding fragment length and standard molecular weight sample are subjected to electricity in same swimming lane
Swimming.By Image Acquisition and analysis, the size of allelic variation amplified fragments can be accurately calculated, realize SSR marker with efficiently,
The combination of automatic technology.In this technical process, the primer of different fluorescent markers how is found out, and is combined and PCR
Product length is variant, effectively can accurately differentiate primer size, is the key technique of SSR.
Invention content
The object of the present invention is to provide one group of corn SSR molecular markers suitable for capillary electrophoresis detection technology.
In order to realize the object of the invention, the present invention by collect derive from a wealth of sources, phenotype and genotype are abundant, representative strong
Corn material is sequenced and is compared to the Maize genome of respective material.The present invention provides one group to be suitable for capillary electricity
Swim detection technique corn SSR molecular marker, the molecular labeling be following 13 SSR molecular markers one or more, 13
A SSR molecular marker is respectively umc1061, umc1663, umc1154, bnlg1209, dupssr21, umc1635, umc1139,
Phi048, umc2294, phi127, phi022, phi128, umc1857.
13 SSR molecular markers pass sequentially through following primer amplification and obtain respectively：SEQ ID NO.1-2, SEQ ID
NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID
NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ
ID NO.23-24, SEQ ID NO.25-26.
The present invention provides application of the above-mentioned corn SSR molecular marker in building Maize DNA Fingerprint Database.
The present invention provides application of the above-mentioned corn SSR molecular marker in corn germ plasm resource analysis of genetic diversity.
The present invention provides application of the above-mentioned corn SSR molecular marker in corn identification.
The present invention provides application of the above-mentioned corn SSR molecular marker in corn molecular mark.
The present invention provides application of the above-mentioned corn SSR molecular marker in preparing Maize genome chip.
The present invention provides application of the above-mentioned corn SSR molecular marker in distinguishing corn Kingsoft 6 and 18 kinds of Deng Hai.
Above-described application, includes the following steps：
1) DNA of corn sample to be measured is extracted；
2) using the DNA of step 1) extraction as template, using above-mentioned SSR molecular marker, PCR amplification is carried out；
3) capillary electrophoresis system is used to detect PCR product.
In the step 2) of above application, PCR amplification uses the reaction volume of 20 μ L, contains dNTP0.25mM, forward primer, anti-
Each 0.4 μM to primer, 1.0 unit of Taq archaeal dna polymerases, 1 × PCR buffer solutions (are free of Mg2+), MgCl21.5mM, sample DNA
10-40ng.Response procedures are：94 DEG C of pre-degeneration 4min；94 DEG C denaturation 45s, 60 DEG C annealing 45s, 72 DEG C extension 45s, totally 30
Cycle；72 DEG C of extension 10min, 4 DEG C of preservations.
Further, the present invention provides the kit for distinguishing 18 kind of corn Kingsoft 6 and Deng Hai, contain and be directed to
The specific primer sets of 13 corn SSR molecular markers of the present invention, the nucleotide sequence point of the specific primer sets
Not as shown in SEQ ID NO.1-26.
Above-mentioned 13 pairs of SSR primers provided by the invention can realize genotype data on fluorescent capillary electrophoresis tube platform
Acquisition.Concrete scheme is wherein one ends 5' mark fluorescent group of each pair of primer；PCR reaction systems are prepared to be added
DNA, primer, dNTP, MgCl2, Taq enzyme, Buffer；Run response procedures；Amplified production is in fluorescent capillary electrophoresis tube system
Detection；Initial data is collected using capillary electrophoresis system software kit, quiding gene type software analyzes initial data and obtains piece
The genotype data of segment length format.
Preferably, by the present invention 13 couple specificity SSR primers progress fluorochrome label, selected altogether PET, NED,
Tetra- kinds of fluorescent dyes of VIC, FAM.The PCR product of fluorescent marker ultra-pure water is diluted 30 times；Isometric above-mentioned 4 kinds are taken respectively
Solution is mixed to form mixed liquor after dilution, and 1 μ L 0.5 μ L LZ500 molecular weight internal standards of addition are drawn from mixed liquor and 8.5 μ L are gone
Formamide is added in the special deep-well plates of DNA analysis instrument；Then by it in PCR instrument 95 DEG C denaturation 5min, take out, immediately
It is placed on ice, cooling 10min or more；It is placed to after brief centrifugation 10s on DNA analysis instrument and carries out capillary electrophoresis detection.With
GeneMapper softwares analyze the initial data of collection.Software systems will be according in the position of target peak and same swimming lane
Internal standard LZ500 be compared, directly give the accurate size of target DNA fragments.
In a preferred embodiment of the embodiment of the present invention, using corn Kingsoft 6DNA as template, respectively use FAM,
Four groups of primers of VIC, NED, PET fluorescent marker carry out four Capillary Electrophoresis, obtain four electrophoretogram results；Again with Kingsoft 6
DNA be template, carry out Capillary Electrophoresis with whole primer mixtures of FAM, VIC, NED, PET fluorescent marker and obtain total electrophoresis
Figure result.Total electrophoretogram result is compared with the result of tetra- groups of fluorescent dye primer electrophoresis of FAM, VIC, NED, PET respectively
(Figure 1A-Fig. 1 D), it can be seen that the purpose peak occurred on the independent electrophoretogram of FAM, VIC, NED, PET can be in total electrophoretogram
On distinguish, and the peak of each color is not interfere with each other, that is, illustrates that primer combination (totally 13 pairs of primers) provided by the invention is available
In a Capillary Electrophoresis, purpose band is not interfere with each other, and is easy to judging result.It is identical as template progress that extra large 18DNA is stepped on using corn
Experiment, identical conclusion (Fig. 2A-Fig. 2 D) can be obtained.
Respectively using Kingsoft 6 and Deng Hai 18DNA as template in embodiment, with FAM, VIC, NED, PET fluorescent dye primer group
It closes and carries out disposable Capillary Electrophoresis, respectively obtain the Capillary Electrophoresis figure in Kingsoft 6 and Deng Hai 18 and be compared.From Fig. 3
As can be seen that Kingsoft 6 FAM blues peak occurs in 142bp, steps on sea 18 and occur FAM blues peak at 152bp；Kingsoft 6 is in 132bp
There is NED yellow peak, steps on sea 18 and NED yellow peak occur in 122bp；There is PET red peak in 153bp in Kingsoft 6, steps on sea 18 and exists
There is PET red peak in 143bp.The peak that Kingsoft 6 and each fluorescent marker for stepping on sea 18 occur is not overlapped, can clearly be debated.Therefore,
The fluorescent dye primer combination provided with patent of the present invention can distinguish corn Kingsoft 6 and Deng Hai 18 in a Capillary Electrophoresis.
The amplified production of the different fluorescent markers of the present invention can carry out electrophoresis in same swimming lane, and signal is clear, amplification
Clip size difference is apparent, can accurately calculate clip size, each DNA sample electrophoresis peak type is different, is easy to judge.With sensitive
Degree is high, resolving power is good, as a result accurately and reliably, it is efficiently quick the advantages that.It is combined with primer provided by the invention, it can be quickly and easily
Corn Kingsoft 6 and 18 kinds of Deng Hai are distinguished, realize it is cost-effective, improve efficiency, easy to operate, result is accurately excellent
Gesture.This group of primer provided by the invention can be used for corn fingerprint map construction, cultivar identification and analysis of genetic diversity etc., application
Foreground is very wide.
Description of the drawings
Figure 1A-Fig. 1 D are respectively corn Kingsoft 6SSR fluorescent marker Capillary Electrophoresis figures, wherein Figure 1A passes through for Kingsoft 6
FAM, VIC, NED and PET fluorescent dye primer combine electrophoresis result and the only comparison through FAM labeled primer electrophoresis results.Figure 1B
Be Kingsoft 6 through FAM, VIC, NED and PET fluorescent dye primer combination electrophoresis result with only through VIC labeled primer electrophoresis results
Compare.Fig. 1 C are Kingsoft 6 through FAM, VIC, NED and PET fluorescent dye primer combination electrophoresis result and only through NED labeled primer electricity
The comparison for result of swimming.Fig. 1 D are that Kingsoft 6 is combined electrophoresis result and only marked through PET through FAM, VIC, NED and PET fluorescent dye primer
Remember the comparison of primer electrophoresis result.Each figure comparison result illustrates respectively, the mesh occurred on the electrophoretogram of one-color fluorescence labeled primer
Peak can be combined in four color fluorescent dye primers and distinguish (arrow meaning be shown as occur purpose peak) on electrophoretogram, and it is every
A purpose peak is not interfere with each other, and can clearly read the DNA fingerprint information of kind.Illustrate that primer combination provided by the invention can be used for
Capillary Electrophoresis, purpose band are not interfere with each other, and save time, experiment reagent and consumption while being easy to judging result again
Material.
Fig. 2A-Fig. 2 D are the SSR fluorescent marker capillary electrophoresis detection results that corn steps on sea 18.Wherein, Fig. 2A is to step on sea
18 through FAM, VIC, NED and PET fluorescent dye primer combination electrophoresis result and the only comparison through FAM labeled primer electrophoresis results.
Fig. 2 B are to step on sea 18 through FAM, VIC, NED and PET fluorescent dye primer combination electrophoresis result and only through VIC labeled primer electrophoresis knots
The comparison of fruit.Fig. 2 C are to step on sea 18 to combine electrophoresis result through FAM, VIC, NED and PET fluorescent dye primer and only mark through NED
The comparison of primer electrophoresis result.Fig. 2 D be step on sea 18 through FAM, VIC, NED and PET fluorescent dye primer combine electrophoresis result with only
Comparison through PET labeled primer electrophoresis results.The comparison result of each figure illustrates respectively, on the electrophoretogram of one-color fluorescence labeled primer
The purpose peak of appearance can four color fluorescent dye primers combine electrophoretogram on distinguish (arrow meaning be shown as occur purpose
Peak), and each purpose peak is not interfere with each other, and can clearly read the DNA fingerprint information of kind.Illustrate primer sets provided by the invention
Conjunction can be used for a Capillary Electrophoresis, and purpose band is not interfere with each other, and save time, experiment examination while being easy to judging result again
Agent and consumptive material.Figure 1A-Fig. 1 D and Fig. 2A-Fig. 2 D are in order to illustrate the same purpose, the reality of two kinds (Kingsoft 6 and Deng Hai 18)
Test result more and can illustrate the practicability and reliability of the primer combination that this patent provides.
Fig. 3 is to carry out capillary electricity to corn Kingsoft 6 and Deng Hai 18 with 13 corn SSR molecular markers provided by the present application
Swimming figure, and to result that the two is compared.As a result illustrate, the peak (blue) for the FAM mesh that 6 (above) of Kingsoft is shown and step on sea 18
The peak (being marked with arrow) of the FAM mesh of (figure below) display is clear and legible, and purpose peak molecular weight is different.Similarly, two kinds
The peak molecular weight of NED, PET mesh of display is different and clear and legible.The primer combination that explanation this patent provides can be to corn
Kingsoft 6 and step on sea 18 carry out kind differentiation.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions.The content not being described in detail in present specification belongs to existing well known to professional and technical personnel in the field
There is technology.
If not specified, biochemical reagents used in the embodiment of the present invention are commercially available, and corn material used is this field
Public corn.
The determination of 1 corn SSR molecular marker of embodiment and primer
36 corn varieties are taken to carry out SSR primer screenings.With denaturing polyacrylamide gel electrophoresis, from 987 pairs of SSR primers
(https://ftp.maizegdb.org/MaizeGDB/FTP/SSRs/) select that site is amplifiable, and banding pattern is clear, polymorphism compared with
High primer is as optional primer.According to optional primer 36 kind allelics molecular weight ranges, PIC values etc., i.e.,
Each combination is made of primer as much as possible, organizes the condition that interior all primer allele ranges are not superimposed, and composition four draws
The merging of object group is respectively synthesized the primer with FAM, VIC, NED, PET fluorophor.It is shown in Table 1.It will be appreciated by those skilled in the art that
Other fluorophors including FAM, VIC, NED, PET also may be selected in the art to modify four groups of primers in table 1, only
It wants every group of inner primer to mark identical fluorophor, is not limited to select any fluorophor.
1 corn SSR Capillary Electrophoresis primer of table and primer combination
Embodiment 2 distinguishes corn variety using SSR molecular marker provided by the invention
(1) DNA rapid extractions
DNA extractions are carried out to corn seed using alkaline-heating method.The method operation is fast and convenient, without poisonous and harmful reagent, fits
Prepared by the DNA for field of plant molecular biology, and to Seed purity assessment, detection GMOs time is greatly shortened,
Detection efficiency is improved, testing cost is reduced and is of great significance.Concrete operation step is：If taking corn seed dry granular, it is placed in
In 1.5mL centrifuge tubes；400 μ L NaOH (1M) are added in centrifuge tube, it is ensured that be completely soaked seed, boiling water bath 5min；From
200 μ L Tris-HCl (1M, pH 8.0), boiling water bath 1min are added in heart pipe；200 μ L TE buffer solutions (pH 8.0) are added, fill
Divide spare after dissolving.
(2) quality and quantity of DNA sample
The OD values that DNA sample 260nm and 280nm are detected on UV detector, select OD260/280Value is 1.8-
1.9 sample is for detecting.
(3) core primers select
By analyzing distribution, polymorphism level, PCR amplification stability and the amplified production banding pattern of primer on chromosome,
The primer that disclosure satisfy that capillary detection technique of fluorescence that selection example 1 determines, is specifically shown in Table 1.
(4) capillary electrophoresis detection
The specific SSR primers filtered out are subjected to fluorochrome label, select tetra- kinds of fluorescence of PET, NED, VIC, FAM altogether
Dyestuff.The PCR product of fluorescent marker ultra-pure water is diluted 30 times；Solution mixes shape after taking isometric above-mentioned 4 kinds of dilutions respectively
At mixed liquor, 1 μ L 0.5 μ L LZ500 molecular weight internal standards of addition are drawn from mixed liquor and 8.5 μ L deionized formamides are added to
In the special deep-well plates of DNA analysis instrument；Then by it, 95 DEG C of denaturation 5min, taking-up are immediately placed on ice in PCR instrument, cooling
10min or more；It is placed to after brief centrifugation 10s on DNA analysis instrument and carries out capillary electrophoresis detection.With GeneMapper softwares pair
The initial data of collection is analyzed.Software systems are compared according to the position of target peak and the internal standard LZ500 in same swimming lane
Compared with directly giving the accurate size of target DNA fragments.The amplification of 18 gained of each group primer amplification corn Kingsoft 6 and Deng Hai in table 1
The purpose band size of product is shown in Table 2.
2 corn Kingsoft 6 of table and the difference peak band and size for stepping on sea 18
| Kingsoft 6 | Step on sea 18 | |
| FAM | 142bp | 152bp |
| NED | 132bp | 122bp |
| PET | 153bp | 143bp |
(5) judgement of primer combination
Using corn Kingsoft 6DNA as template, carried out four times with four groups of primers of FAM, VIC, NED, PET fluorescent marker respectively
Capillary Electrophoresis obtains four electrophoretogram results；Again using Kingsoft 6DNA as template, with FAM, VIC, NED, PET fluorescent marker
Whole primer mixtures carry out Capillary Electrophoresis and obtain total electrophoretogram result.By total electrophoretogram result respectively with FAM, VIC, NED,
The result of tetra- groups of fluorescent dye primer electrophoresis of PET is compared (Figure 1A-Fig. 1 D), it can be seen that mono- in FAM, VIC, NED, PET
The purpose peak occurred on only electrophoretogram can distinguish on total electrophoretogram, and the peak of each color is not interfere with each other, that is, is illustrated
Primer mixture provided by the invention can be used for a Capillary Electrophoresis, and purpose band is not interfere with each other, and is easy to judging result.With jade
Meter Deng Hai 18DNA are that template carries out identical experiment, and identical conclusion (Fig. 2A-Fig. 2 D) can be obtained.
(7) differentiation of 18 kind of Kingsoft 6 and Deng Hai
Respectively using Kingsoft 6 and Deng Hai 18DNA as template in embodiment, with FAM, VIC, NED, PET fluorescent dye primer group
It closes (being shown in Table 1) and carries out disposable Capillary Electrophoresis, respectively obtain the Capillary Electrophoresis figure in Kingsoft 6 and Deng Hai 18 and be compared.
From figure 3, it can be seen that Kingsoft 6 FAM blues peak occurs in 142bp, steps on sea 18 and occur FAM blues peak at 152bp；Kingsoft 6
There is NED yellow peak in 132bp, steps on sea 18 and NED yellow peak occur in 122bp；There is PET red peak in 153bp in Kingsoft 6, steps on
There is PET red peak in 143bp in sea 18.The peak that Kingsoft 6 and each fluorescent marker for stepping on sea 18 occur is not overlapped, clearly may be used
It debates.Therefore, the fluorescent dye primer combination (table 1) provided with patent of the present invention can distinguish corn gold in a Capillary Electrophoresis
Mountain 6 and Deng Hai 18.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Agricultural product quality and safety research institute of Heilongjiang Institute of Agricultural Sciences
<120>Suitable for one group of corn SSR molecular marker of capillary electrophoresis detection technology and its application
<130> KHP181111101.5
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agcaggagta cccatgaaag tcc 23
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tatcacagca cgaagcgata gatg 24
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gcttgcacta gctttagctc catc 24
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cgggatcagt cgttacaaac atag 24
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ccaccacaag acaagacaag aatg 24
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cctgatcgat ctcatcgtcg t 21
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gtcccgggca gaataatacc 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ttcctccttg aagtgctcgt 20
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
gtgcaaacta atccaaagca a 21
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
atgtagggac aaaggaataa atca 24
<210> 11
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gctgagcaga tctttccttg tttc 24
<210> 12
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
aaggagcaga actcggagac g 21
<210> 13
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tttgtaatat ggcgctcgaa aact 24
<210> 14
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
gaagacgcct ccaagatgga tac 23
<210> 15
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
gcaaaccttg catgaacccg attgt 25
<210> 16
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
caagcgtcca gctcgatgat ttc 23
<210> 17
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
attggagtgg ctccattgct t 21
<210> 18
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
cccaccattc tatatattgt tgcca 25
<210> 19
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
atatgcattg cctggaactg gaagga 26
<210> 20
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
aattcaaaca cgcctcccga gtgt 24
<210> 21
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
tgcgcaccag cgactgacc 19
<210> 22
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
gcgggcgacg cttccaaac 19
<210> 23
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
ttgctcggta tgaagaaaat agtctttcc 29
<210> 24
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
atcttgcaac tagactgagg caacca 26
<210> 25
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
ttccttgcca acaaatacaa ggat 24
<210> 26
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
gttcattgct tcatcttgga acct 24
Claims (10)
1. the corn SSR molecular marker suitable for capillary electrophoresis detection technology, which is characterized in that the molecular labeling is following
The one or more of 13 SSR molecular markers, respectively umc1061, umc1663, umc1154, bnlg1209, dupssr21,
Umc1635, umc1139, phi048, umc2294, phi127, phi022, phi128, umc1857.
2. corn SSR molecular marker according to claim 1, which is characterized in that 13 SSR molecular markers respectively according to
It is secondary to be obtained by following primer amplification：SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-
8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID
NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26.
3. application of the corn SSR molecular marker as claimed in claim 1 or 2 in building Maize DNA Fingerprint Database.
4. application of the corn SSR molecular marker as claimed in claim 1 or 2 in corn germ plasm resource analysis of genetic diversity.
5. application of the corn SSR molecular marker described in claims 1 or 2 in corn identification.
6. application of the corn SSR molecular marker in corn molecular mark described in claims 1 or 2.
7. application of the corn SSR molecular marker in preparing Maize genome chip described in claims 1 or 2.
8. application of the corn SSR molecular marker described in claims 1 or 2 in distinguishing corn Kingsoft 6 and 18 kinds of Deng Hai.
9. according to any applications of claim 3-8, which is characterized in that include the following steps：
1) DNA of corn sample to be measured is extracted；
2) using the DNA of step 1) extraction as template, according to the corn SSR molecular marker, PCR amplification is carried out；
3) capillary electrophoresis system is used to detect PCR product.
10. the kit for differentiating 18 kind of corn Kingsoft 6 and Deng Hai, which is characterized in that containing for described in claim 1
The specific primer sets of corn SSR molecular marker, the nucleotide sequence of the specific primer sets is respectively such as SEQ ID
Shown in NO.1-26.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810299328.6A CN108277295B (en) | 2018-04-04 | 2018-04-04 | Corn SSR molecular markers suitable for capillary electrophoresis detection technology and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810299328.6A CN108277295B (en) | 2018-04-04 | 2018-04-04 | Corn SSR molecular markers suitable for capillary electrophoresis detection technology and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108277295A true CN108277295A (en) | 2018-07-13 |
| CN108277295B CN108277295B (en) | 2021-02-09 |
Family
ID=62811031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810299328.6A Active CN108277295B (en) | 2018-04-04 | 2018-04-04 | Corn SSR molecular markers suitable for capillary electrophoresis detection technology and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108277295B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545004A (en) * | 2008-03-28 | 2009-09-30 | 上海市农业科学院 | Waxy maize inbred line Shen-W22 specific molecular marker and application thereof in offspring variety identification |
| CN106326962A (en) * | 2016-08-15 | 2017-01-11 | 安徽农业大学 | Corn two-dimensional code molecule identity card and construction method therefor |
-
2018
- 2018-04-04 CN CN201810299328.6A patent/CN108277295B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545004A (en) * | 2008-03-28 | 2009-09-30 | 上海市农业科学院 | Waxy maize inbred line Shen-W22 specific molecular marker and application thereof in offspring variety identification |
| CN106326962A (en) * | 2016-08-15 | 2017-01-11 | 安徽农业大学 | Corn two-dimensional code molecule identity card and construction method therefor |
Non-Patent Citations (4)
| Title |
|---|
| 张全芳等: "基于荧光SSR标记的玉米自交系遗传结构解析", 《植物遗传资源学报》 * |
| 李丽华等: "20个骨干玉米自交系的SSR指纹图谱构建", 《西南农业学报》 * |
| 王凤格: "玉米通用SSR核心引物筛选及高通量多重PCR复合扩增体系建立", 《科学通报》 * |
| 赵久然等: "中国玉米新品种DNA指纹库建立系列研究*Ⅱ.适于玉米自交系和杂交种指纹图谱绘制的SSR核心引物的确定", 《玉米科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108277295B (en) | 2021-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109706263A (en) | Chain SNP marker and application with wheat stripe rust resisting ospc gene QYr.sicau-1B-1 | |
| CN108374054A (en) | Suitable for one group of rice SSR molecular marker of capillary electrophoresis detection technology and its application | |
| CN103243158B (en) | Method for constructing wheat SSR (single sequence repeat) fingerprint | |
| CN107794304B (en) | Genotyping detection kit for yak individual identification and paternity test | |
| CN110257547B (en) | Corn core SNP marker developed based on KASP technology and application thereof | |
| CN103911372B (en) | Cowpea chloroplast SSR molecule labeled polymorphic primers and screening method thereof, and method for identifying genetic relationship of cowpeas | |
| CN106048042B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits color traits | |
| CN106480228A (en) | The SNP marker of paddy rice low cadmium-accumulation gene OsHMA3 and its application | |
| CN109868328B (en) | SSR molecular marker for identifying paeonia rockii varieties and application | |
| CN109385466A (en) | The KASP Functional marker of resistance gene of rice blast Pi2 a kind of and its application | |
| CN105925721A (en) | Single nucleotide polymorphism marker site, primer and kit for identifying coloring property of peach epidermis and application | |
| CN105506127A (en) | Primers and kit for detecting rice blast resistance genes Pigm and genetic typing method | |
| CN114774570B (en) | Molecular marker closely linked with wheat stem rot resistance QTL and application | |
| CN108642208A (en) | A kind of Cinnamomum and its general SSR molecular marker of relative genus plant and its development approach and application | |
| CN104789648B (en) | Identify molecular labeling and its application of the section haplotypes of rice CMS restoring genes Rf 1 | |
| KR101961653B1 (en) | SNP molecular marker for selecting cultivars of sweet potatoes and uses thereof | |
| CN108384884A (en) | One group of corn SSR molecular marker and its application | |
| CN101928766A (en) | Method for fast appraising purity of cucumber hybrid seed | |
| CN117106967A (en) | Functional KASP molecular marker of rice blast resistance gene and application thereof | |
| CN108220473A (en) | It is marked using chloroplaset InDel and differentiates CMS-S maize material | |
| CN110453003A (en) | Molecular labeling and its application with spinach leaf character main effect QTL compact linkage | |
| CN108531642A (en) | One group of SSR molecular marker and its application for differentiating corn variety | |
| CN114231603B (en) | Primer, reagent, identification method and kit for identifying paeonia boita | |
| CN108660244A (en) | One group of corn SSR molecular marker for being suitable for capillary electrophoresis detection technology and its application | |
| CN108277295A (en) | Suitable for one group of corn SSR molecular marker of capillary electrophoresis detection technology and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |