CN106701960A - Medicago sativa.L ILP (Intron Length Polymorphism) molecular marker primer and application in Medicago sativa.L variety identification - Google Patents
Medicago sativa.L ILP (Intron Length Polymorphism) molecular marker primer and application in Medicago sativa.L variety identification Download PDFInfo
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- CN106701960A CN106701960A CN201710027347.9A CN201710027347A CN106701960A CN 106701960 A CN106701960 A CN 106701960A CN 201710027347 A CN201710027347 A CN 201710027347A CN 106701960 A CN106701960 A CN 106701960A
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- 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
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- 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 invention belongs to the technical field of biologics, and in particular relates to Medicago sativa.L ILP (Intron Length Polymorphism) molecular marker primers and a detection method for application of the Medicago sativa.L ILP molecular marker primers in Medicago sativa.L variety identification. The Medicago sativa.L ILP molecular marker primers comprise 36 pairs of primers. By adopting the Medicago sativa.L ILP molecular marker primers, comparative identification on test e Medicago sativa.L varieties with known e Medicago sativa.L varieties can be completed within 4 hours by using a relatively small number of primer groups, and the Medicago sativa.L ILP molecular marker primers have the advantages of being accurate, efficient, rapid, low in cost, convenient to operate and the like.
Description
Technical field
The invention belongs to biological technical field, and in particular to alfalfa ILP molecular labeling primers and alfalfa variety mirror
Application in fixed.
Background technology
Alfalfa (Medicago sativa L.) is the maximum leguminous forage of grown worldwide area, with extensive raw
State adaptability, the productivity of stabilization and nutritive value higher, the title with " King of Pasture ", in China's agriculture-stock production and
Great function is played in Eco-economic Construction.In recent years, with the fast development of animal husbandry, China alfalfa variety (including
Medicago varia) cultivate and also achieve impressive progress in terms of authorizing work.At present, the authorization of New alfalfa cultivars and variety authentication
Authentication method be mainly by field planting, differentiated according to its morphological feature.But this method wastes time and energy, easily
Influenceed by environment and subjective factor.Further, since parent used in alfalfa variety Breeding Process more concentrates, so as to cause
Traits change is increasingly narrow between improved variety so that carrying out cultivar identification by morphological feature becomes more difficult.Therefore, build
Found a set of quick, accurate, cheap alfalfa variety identification technology, it appears very necessary.
With the development of modern molecular biology, molecular labeling is more and more applied in cultivar identification, such as with PCR
RAPD, SCAR, SSR, ILP based on reaction etc..Compared with other molecular labelings, ILP marks have the advantages that many uniquenesses,
For example, ILP labeled primers are designed at the exon region guarded relatively, with stronger versatility, specificity, codominance, in
The features such as property, reliability and functional dependency.Additionally, ILP mark variations are moderate, can be used for cultivar identification, systematic growth
The aspects such as analysis, analysis of genetic diversity and genetic linkage mapping.
Used as a kind of new molecular labeling, so far, ILP marks are main in paddy rice, corn and soybean isotype plant
In in developed and be mainly used in analysis of genetic diversity and genetic linkage mapses build.Such as Zhao Jianya is marked using ILP
Note have studied 52 parts of genetic diversities of Indica-Japonica hybrid rice (referring to《Shihezi Univ's journal》, the 3rd phase of volume 27 in 2009,
《Using the genetic diversity of ILP molecular marker analysis Indica-Japonica hybrid rices》).But in non-mode plant clover, ILP primers
There is not been reported for exploitation and the application in cultivar identification.Therefore, alfalfa ILP molecule marks are developed in full-length genome level
Remember and study its application in alfalfa variety identification, there is important meaning to alfalfa variety identification and molecular mark
Justice.
The content of the invention
In order to solve the above technical problems, the present invention is adopted the following technical scheme that：
Alfalfa ILP molecular labeling primers, the molecular labeling primer includes 36 pairs as described embodiments.
Application of the alfalfa ILP molecular labeling primers in alfalfa variety identification.
Wherein described alfalfa variety includes table 1 below：
The alfalfa variety information of table 1
A kind of kit for identifying alfalfa variety, comprising described alfalfa ILP molecular labeling primers.
The beneficial effects of the invention are as follows：In the case where the time that DNA extraction process is consumed is not calculated, inspection of the invention
Survey method can utilize less primer combination to be completed within 4h to being identified with the comparison of known alfalfa variety for examination alfalfa variety,
With accurate, efficient, quick, low cost and the advantage such as easy to operate, the application has sensitivity higher and accuracy.
Brief description of the drawings
Fig. 1 is specific implementation step flow chart of the present invention；
Fig. 2 is amplification figures of the primer MsILP-07 to alfalfa variety；
Fig. 3 is amplification figures of the primer MsILP-20 to alfalfa variety.
Specific embodiment
It is described in more detail below specific embodiment of the invention.It should be appreciated that may be realized in various forms the present invention
Without that should be limited by embodiments set forth here.Conversely, there is provided these embodiments are able to be best understood from this hair
It is bright, and can by the scope of the present invention it is complete convey to those skilled in the art.
As the "comprising" in specification in the whole text and claim mentioned in or " including " be an open language, therefore should
It is construed to " include but be not limited to ".To implement better embodiment of the invention, so description is specification subsequent descriptions
For the purpose of the rule of specification, the scope of the present invention is not limited to.Protection scope of the present invention is when regarding appended power
Profit requires that the person of defining is defined.
Embodiment：
1. expert evidence DNA is extracted
With reference to SDS methods, concrete operation step is following (see Fig. 1)：
1) 40 full, healthy seeds are chosen, using 20 DEG C of sprouting 72h of double-layer filter paper method；
2) above seed is mixed in mortar and is fully ground, add 600 μ L DNA buffer, continue to be ground.Will
Lapping liquid is transferred to 2.0mL centrifuge tubes；
3) above-mentioned centrifuge tube is transferred to 65 DEG C of water-bath 10min, is shaken up 1-2 times during this；
4) chloroform is added in ventilating kitchen：Isoamyl alcohol volume ratio for 24: 1 the μ L of mixed liquor 600, mixing of turning upside down,
12000 leave heart 10min under room temperature condition, draw 360 μ L of supernatant liquid and are transferred in 1.5ml centrifuge tubes；
5) it is well mixed after the μ L of 3mol/L NaAc (pH=5.2) 36 and the μ L of isopropanol 240 that add precooling, room temperature is placed
After 10min, room temperature 12000 leaves heart 10min, abandons supernatant, and 30s is centrifuged, and is suctioned out residual night with 10 μ L liquid-transfering guns；
6) 75% ethanol of 500 μ L is added, precipitation is upspring, 12000 leave heart 5min, pour out ethanol.Centrifugation 30s, uses
10 μ L liquid-transfering guns suction out residual night, and this step is repeated 2 times, and residual ethanol is dried up in ventilating kitchen；
7) 20 μ L sterile deionized waters are added, fully after dissolving, DNA molecular amount size, profit is detected with 1% agarose electrophoresis
DNA concentration is determined with ultramicron ultraviolet specrophotometer, and is diluted to 25ng/ μ L, 4 DEG C of preservations are stand-by.
2. the ILP-PCR amplifications of expert evidence DNA
The building-up process of primer is the present invention the most difficult, process part the most complicated, most important of which is that early stage
Sequence alignment, and it is conventional method that Primer3.0 carries out design of primers.
From the http of M.sativa gene index 1.2：//plantgrn.noble.org/AGED/ (AGED) database
Download alfalfa high flux transcript profile sequencing sequence totally 112626 conting sequences.From https：//
Www.arabidopsis.org/index.jsp (TAIR 10) downloads arabidopsis whole genome sequence.According to extron between species
The stronger principle of sequence conservation, respectively so that arabidopsis gene group sequence and alfalfa transcript profile sequence are as reference sequences and look into
Sequence is ask, using each 100bp length extron sequence in two ends of perl script screening arabidopsis length of intron≤400bp positions
Row, and determined using local Blast programs pale reddish brown with 200bp arabidopsis exon sequence similarity >=80% of above-mentioned acquisition
Clover transcript profile sequence, finally carries out alfalfa ILP design of primers using the softwares of Primer 3.0.Alfalfa ILP is positive and negative
Primer sequence should be located in each side 100bp sequences respectively, to ensure amplified production as intron sequences.
Knowable to foregoing description, alfalfa sequence to be compared is very more (112626), actual design purple out
Russian fenugreek herb ILP primers are also very more.And 36 primers mentioned in the application are by the substantial amounts of creative screening of our early stages
Polymorphism primer high, reproducible is finally obtained, period needs substantial amounts of creative work.
Designed 36 pairs of primers out are as shown in table 2.
2 36 pairs of primer information of table
The 36 pairs of primer pairs material DNA to be identified be given using this method enters performing PCR amplification, and reaction system and program are such as
Under：
1) reaction system：10 μ L systems include expert evidence DNA profiling 25ng, forward and reverse primer each 0.4 μm of ol/L, 5 μ
L2 × power Taq MasterMix (hundred Tykes), residual volume is supplied with ultra-pure water；
2) response procedures：Using touchdown PCR, 94 DEG C first denaturation 30s, 58-61 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 5
Individual circulation；Then 94 DEG C are denatured 30s, 55-58 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30 circulations；72 DEG C extend 7min, 4 DEG C
Preserve.
3. modacrylic acyl ammonia gel electrophoresis is detected with silver staining
As a example by using the double plate sandwich type Vertial electrophorestic tanks of DYCZ-28C, operating procedure is as follows：
1) glass plate is cleaned：Glass plate is cleaned up with running water, with being dried after deionized water rinsing.Use 95% ethanol
Clean twice, blotting paper is dried.Prevent two pieces of glass plates from polluting mutually in operating process.
2) glass plate is assembled：After glass plate is thoroughly dried, electrophoresis offset plate is assembled them into using adhesive tape.
3) offset plate back cover：12.5 μ L TEMED and new 10% over cure prepared are separately added into 10mL 6.0%PAGE glue
The μ L of acid ammonium solution 100 can be used for 2 plate glue back covers.
4) glue：54 μ L TEMED are separately added into 80mL 6.0%PAGE glue and new 10% ammonium persulfate prepared is molten
The μ L of liquid 800 can be used for filling 2 plate glue.Encapsulating after above-mentioned rapid mixing, should at the uniform velocity preventing bubble during encapsulating.Treat that glue fills
Full glass plate interlayer, Great Wall stripping fork is gently inserted on top, and be polymerized at least more than 30min.After glue polymerization, offset plate surface is cleared up
The glue of spilling, gently extracts comb, is cleaned up with water standby.
5) sample preparation：2 μ L6 × sample loading buffer is added in 10 μ L PCR primers, after mixing, in water-bath or PCR
95 DEG C of denaturation 5min, 4 DEG C of cooling more than 10min on instrument.
6) electrophoresis：Wash buffer gel top is drawn with syringe several times, remove bubble and gel pieces, while by glue
Bar sets right.Each well clicks and enters 1 μ L~2 μ L samples.In addition to testing sample, while adding Reference cultivars amplified production.
Electrophoresis under 400V constant pressures, electrophoresis 1.5h~2h (electrophoresis time depends on the magnitude range of amplified fragments).After electrophoresis terminates, carefully
Two pieces of glass plates are separated, gel is removed from glass plate and is prepared silver staining.
7) silver staining：A. the glass plate long for adhering to gel is placed in plastic casing, adds appropriate distilled water, gently rocked and wash down
Film, removes glass plate, and film is rinsed 2 times.B. to the silver nitrate solution that 0.08% is added in the plastic casing equipped with film, make
Solution did not had film, and 60 revs/min are shaken 10min on shaking table.C. silver nitrate is poured into waste liquid barrel, with distilled water rinsed clean glue
Piece, during film is transferred to containing 1.5% NaOH, 0.4% formaldehyde developer solution, 60 revs/min are shaken number on shaking table
Untill there is clear band line in minute.D. film is transferred to lamp box, preservation photo of taking pictures.Wherein, such as primer MsILP-07 pairs
The amplification of alfalfa variety is shown in that Fig. 2, primer MsILP-20 are shown in Fig. 3 to the amplification of alfalfa variety.
8) banding pattern and migration position of the same site amplified fragments of each identification of species are compared, amplification banding pattern is with 0,1
Represent, on identical migration position, there is band to be designated as " 1 ", be designated as " 0 " without band, build " 1 ", " 0 " matrix.Each primer pair 21 is to product
The differentiation information planted is shown in Table 3.
3 21 alfalfa varieties of table are based on " 0 ", " 1 " matrix that 36 ILP molecular labelings build
For the allele in table 3, by taking MsILP-28 as an example, corresponding display is that have 11 allele, but in fact
All it is the corresponding gene of MsILP-28 of amplification, the situation of multiple allele why occurs, because clover is
Allotetraploid, copied along with certain gene may have multiple (i.e. one gene may occur repeatedly in genome, and it
Between introne be possible to variant), it is possible to the situation for many band (allele) occur.
It can be seen that the situation of each primer amplification, 36 pairs of alfalfa ILP molecular labelings are in 21 confessions in table 3
Polymorphic bandses, and stable amplification result can be amplified in examination alfalfa variety, with repeatability.Wherein, 36 pairs of ILP molecules
Coamplification goes out 169 allele bands in being marked at 21 alfalfa varieties, and average each site is 4.7 allele bars
Band.36 pairs of expected heterozygosity (He) scopes of ILP molecular labelings are arrived for 0.16 (MsILP-40, MsILP-53, MsILP-54)
0.88 (MsILP-75), average value is 0.65.Polymorphic sex index (PIC) scope be 0.15 (MsILP-40, MsILP-53,
MsILP-54) to 0.87 (MsILP-75), average value is 0.60.
9) qualification result to each pair primer carries out difference number of sites >=1 item judgement between statistical analysis, different identification of species
Two kinds are different cultivars.Specific cultivar identification the results are shown in Table 4.
4 36 ILP molecular labelings of table distinguish kind information (kind numbering correspondence kind information is shown in Table 1)
By taking MsILP-01 as an example, it enters performing PCR amplification to 21 alfalfa varieties to the monoid that 4 kinds of table, is expanded according to each kind
Increase the band difference for, 21 kinds can be divided into 7 monoids (situation).Wherein, kind 1,18,13 and 14 belongs to monoid
1, because their amplified band is the same；Kind 2 belongs to monoid 2, but their band class as 3 amplified band
The type of strip of type and monoid 1 has difference again.Thereafter similarly.Therefore, if only using if MsILP-01 primers, be only capable of by
21 kind parts distinguish.And if to distinguish 21 kinds completely, it is necessary to using the combination of different primers.Example
Such as, kind 2 and 3 we cannot use MsILP-01, but if the result in conjunction with MsILP-05, it is possible to kind 2
Distinguished with 3, and the above results also provable the application have sensitivity higher and accuracy.
Embodiments of the invention are the foregoing is only, the scope of the claims of the invention is not thereby limited.
SEQUENCE LISTING
<110>Lanzhou University
<120>Alfalfa ILP molecular labeling primers and the application in alfalfa variety identification
<130> 2016
<160> 72
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 1
agacaggttg ttcacattgc 20
<210> 2
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 2
accagcagta tcccaaatct 20
<210> 3
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<212> DNA
<213> Medicago sativa L.
<400> 3
cacaactctg gaatgggaa 19
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<211> 22
<212> DNA
<213> Medicago sativa L.
<400> 4
catctctcgt gttactgaat gc 22
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<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 5
ggactcaact caaagatgcc 20
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<213> Medicago sativa L.
<400> 6
gacccataga caacattcca ag 22
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<213> Medicago sativa L.
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tagacgcttg acccttgag 19
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<212> DNA
<213> Medicago sativa L.
<400> 8
gtggtattcc tctgtcccat 20
<210> 9
<211> 19
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<213> Medicago sativa L.
<400> 9
attgagcgtt acaccaggg 19
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<213> Medicago sativa L.
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tgctgaagta gtggctttga 20
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<400> 11
gctgaaacac acaaggacaa 20
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aacaccgtga gttgaagga 19
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ctcacggtgt ttatgaagaa gg 22
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ccaacctggg cttgtaagt 19
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gttaggaagg aggctgagaa 20
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<212> DNA
<213> Medicago sativa L.
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ggagtggcaa acctgaaa 18
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cacaactctg gaatgggaa 19
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<213> Medicago sativa L.
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catctctcgt gttactgaat gc 22
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<213> Medicago sativa L.
<400> 19
ggactcaact caaagatgcc 20
<210> 20
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<212> DNA
<213> Medicago sativa L.
<400> 20
gacccataga caacattcca ag 22
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tagacgcttg acccttgag 19
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<213> Medicago sativa L.
<400> 22
gtggtattcc tctgtcccat 20
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attgagcgtt acaccaggg 19
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<212> DNA
<213> Medicago sativa L.
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tgctgaagta gtggctttga 20
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<213> Medicago sativa L.
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gctgaaacac acaaggacaa 20
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aacaccgtga gttgaagga 19
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ctcacggtgt ttatgaagaa gg 22
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<213> Medicago sativa L.
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ccaacctggg cttgtaagt 19
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<212> DNA
<213> Medicago sativa L.
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gaccaacaac accttgacta tc 22
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<212> DNA
<213> Medicago sativa L.
<400> 30
atactcacat cagcaccagc 20
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<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 31
atcctttcaa accacccaag 20
<210> 32
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 32
ggcaaatgct accgttactg 20
<210> 33
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 33
gttaggaagg aggctgagaa 20
<210> 34
<211> 18
<212> DNA
<213> Medicago sativa L.
<400> 34
ggagtggcaa acctgaaa 18
<210> 35
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 35
tgttccctgc tatcaatgtc 20
<210> 36
<211> 18
<212> DNA
<213> Medicago sativa L.
<400> 36
gtgacggcat ccatacaa 18
<210> 37
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 37
aggctgtcct ctccctctat 20
<210> 38
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 38
cggaatccaa cacaatacc 19
<210> 39
<211> 18
<212> DNA
<213> Medicago sativa L.
<400> 39
gggctaaagg gcattaca 18
<210> 40
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 40
agttcctcca ccaagtgaat 20
<210> 41
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 41
acgcagacga atgtatggt 19
<210> 42
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 42
cagggaacct caaacaaca 19
<210> 43
<211> 21
<212> DNA
<213> Medicago sativa L.
<400> 43
cttgtctaca ccgtcttctc c 21
<210> 44
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 44
ccagtgattg ggatagtgg 19
<210> 45
<211> 21
<212> DNA
<213> Medicago sativa L.
<400> 45
gcaatgacag ggatagagtt g 21
<210> 46
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 46
ggcgaacaca agcaatact 19
<210> 47
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 47
tgctgtttct cacggtctt 19
<210> 48
<211> 22
<212> DNA
<213> Medicago sativa L.
<400> 48
ctcacggtca aatgctaagt tc 22
<210> 49
<211> 22
<212> DNA
<213> Medicago sativa L.
<400> 49
gtgttacagc aactgacttg gt 22
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<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 50
atctacaggg aagaaaccca 20
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<211> 19
<212> DNA
<213> Medicago sativa L.
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ggtccactgc tttccgtaa 19
<210> 52
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 52
ccaaccacac tgtcaggat 19
<210> 53
<211> 18
<212> DNA
<213> Medicago sativa L.
<400> 53
tgaagcagag gcagcaat 18
<210> 54
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<212> DNA
<213> Medicago sativa L.
<400> 54
ttgaacccaa caacaccg 18
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<213> Medicago sativa L.
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tttctgtgct ggtcgtgt 18
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<212> DNA
<213> Medicago sativa L.
<400> 56
gaatctctcg tttcctactt gc 22
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<212> DNA
<213> Medicago sativa L.
<400> 57
tgaaacattc gggtcaactg 20
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<212> DNA
<213> Medicago sativa L.
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gcattccagg tcttctcgt 19
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<211> 19
<212> DNA
<213> Medicago sativa L.
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cctattacaa gcagcggct 19
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<211> 19
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<213> Medicago sativa L.
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caatcaggat gaacaaccc 19
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<213> Medicago sativa L.
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ggatggcttt gagcgttt 18
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<212> DNA
<213> Medicago sativa L.
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gaatcagttt aggaaccacc ac 22
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<211> 18
<212> DNA
<213> Medicago sativa L.
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ttgctgcctt tgtcccaa 18
<210> 64
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 64
cgaccttgaa cctgactcca 20
<210> 65
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 65
ggcaaggttg aaaggaaca 19
<210> 66
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 66
acaagggcag cacaagatg 19
<210> 67
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 67
ggcaaggttg aaaggaaca 19
<210> 68
<211> 19
<212> DNA
<213> Medicago sativa L.
<400> 68
acaagggcag cacaagatg 19
<210> 69
<211> 22
<212> DNA
<213> Medicago sativa L.
<400> 69
gtgttacagc aactgacttg gt 22
<210> 70
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 70
atctacaggg aagaaaccca 20
<210> 71
<211> 18
<212> DNA
<213> Medicago sativa L.
<400> 71
ccgtgctttc gctttatg 18
<210> 72
<211> 20
<212> DNA
<213> Medicago sativa L.
<400> 72
caccatctcc agaatcaaga 20
Claims (4)
1. alfalfa ILP molecular labeling primers, it is characterised in that the molecular labeling primer includes following 36 pairs, it is positive
The nucleotide sequence of primer and reverse primer is specifically as follows：
(1)MsILP-01：Forward primer sequence is SEQ ID NO.1, and reverse primer sequences are SEQ ID NO.2；
(2)MsILP-05：Forward primer sequence is SEQ ID NO.3, and reverse primer sequences are SEQ ID NO.4；
(3)MsILP-06：Forward primer sequence is SEQ ID NO.5, and reverse primer sequences are SEQ ID NO.6；
(4)MsILP-07：Forward primer sequence is SEQ ID NO.7, and reverse primer sequences are SEQ ID NO.8；
(5)MsILP-08：Forward primer sequence is SEQ ID NO.9, and reverse primer sequences are SEQ ID NO.10；
(6)MsILP-10：Forward primer sequence is SEQ ID NO.11, and reverse primer sequences are SEQ ID NO.12；
(7)MsILP-11：Forward primer sequence is SEQ ID NO.13, and reverse primer sequences are SEQ ID NO.14；
(8)MsILP-20：Forward primer sequence is SEQ ID NO.15, and reverse primer sequences are SEQ ID NO.16；
(9)MsILP-22：Forward primer sequence is SEQ ID NO.17, and reverse primer sequences are SEQ ID NO.18；
(10)MsILP-23：Forward primer sequence is SEQ ID NO.19, and reverse primer sequences are SEQ ID NO.20；
(11)MsILP-24：Forward primer sequence is SEQ ID NO.21, and reverse primer sequences are SEQ ID NO.22；
(12)MsILP-25：Forward primer sequence is SEQ ID NO.23, and reverse primer sequences are SEQ ID NO.24；
(13)MsILP-27：Forward primer sequence is SEQ ID NO.25, and reverse primer sequences are sEQ ID NO.26；
(14)MsILP-28：Forward primer sequence is SEQ ID NO.27, and reverse primer sequences are SEQ ID NO.28；
(15)MsILP-30：Forward primer sequence is SEQ ID NO.29, and reverse primer sequences are SEQ ID NO.30；
(16)MsILP-36：Forward primer sequence is SEQ ID NO.31, and reverse primer sequences are SEQ ID NO.32；
(17)MsILP-37：Forward primer sequence is SEQ ID NO.33, and reverse primer sequences are SEQ ID NO.34；
(18)MsILP-39：Forward primer sequence is sEQ ID NO.35, and reverse primer sequences are SEQ ID NO.36；
(19)MsILP-40：Forward primer sequence is SEQ ID NO.37, and reverse primer sequences are SEQ ID NO.38；
(20)MsILP-41：Forward primer sequence is SEQ ID NO.39, and reverse primer sequences are SEQ ID NO.40；
(21)MsILP-42：Forward primer sequence is SEQ ID NO.41, and reverse primer sequences are SEQ ID NO.42；
(22)MsILP-43：Forward primer sequence is SEQ ID NO.43, and reverse primer sequences are SEQ ID NO.44；
(23)MsILP-45：Forward primer sequence is SEQ ID NO.45, and reverse primer sequences are SEQ ID NO.46；
(24)MsILP-49：Forward primer sequence is SEQ ID NO.47, and reverse primer sequences are SEQ ID NO.48；
(25)MsILP-52：Forward primer sequence is SEQ ID NO.49, and reverse primer sequences are SEQ ID NO.50；
(26)MsILP-53：Forward primer sequence is SEQ ID NO.51, and reverse primer sequences are SEQ ID NO.52；
(27)MsILP-54：Forward primer sequence is SEQ ID NO.53, and reverse primer sequences are SEQ ID NO.54；
(28)MsILP-56：Forward primer sequence is SEQ ID NO.55, and reverse primer sequences are SEQ ID NO.56；
(29)MsILP-66：Forward primer sequence is SEQ ID NO.57, and reverse primer sequences are SEQ ID NO.58；
(30)MsILP-69：Forward primer sequence is SEQ ID NO.59, and reverse primer sequences are SEQ ID NO.60；
(31)MsILP-70：Forward primer sequence is SEQ ID NO.61, and reverse primer sequences are SEQ ID NO.62；
(32)MsILP-72：Forward primer sequence is SEQ ID NO.63, and reverse primer sequences are SEQ ID NO.64；
(33)MsILP-74：Forward primer sequence is SEQ ID NO.65, and reverse primer sequences are SEQ ID NO.66；
(34)MsILP-75：Forward primer sequence is SEQ ID NO.67, and reverse primer sequences are SEQ ID NO.68；
(35)MsILP-86：Forward primer sequence is SEQ ID NO.69, and reverse primer sequences are SEQ ID NO.70；
(36)MsILP-95：Forward primer sequence is SEQ ID NO.71, and reverse primer sequences are SEQ ID NO.72.
2. application of the alfalfa ILP molecular labeling primers as claimed in claim 1 in alfalfa variety identification.
3. application as claimed in claim 2, wherein described alfalfa variety is including following
4. it is a kind of identify alfalfa variety kit, it is characterised in that comprising the alfalfa ILP molecules described in claim 1
Labeled primer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190096A (en) * | 2017-07-25 | 2017-09-22 | 山东农业大学 | The general molecular labeled primer and its joint development methods and applications of trees |
| CN108441577A (en) * | 2018-05-25 | 2018-08-24 | 兰州大学 | Primer pair, kit and purposes and detection alfalfa product in whether the method containing soybean |
| CN110396555A (en) * | 2019-06-25 | 2019-11-01 | 兰州大学 | Jian Kuo pea ILP molecular labeling primer and the application in cultivar identification |
| CN113817867A (en) * | 2021-11-25 | 2021-12-21 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to alfalfa quality traits and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769492A (en) * | 2005-09-22 | 2006-05-10 | 浙江大学 | General molecular mark development method and designed general Gramineae mark |
| KR20120056520A (en) * | 2010-11-25 | 2012-06-04 | 건국대학교 산학협력단 | Method for developing gene specific molecular markers |
-
2017
- 2017-01-15 CN CN201710027347.9A patent/CN106701960B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769492A (en) * | 2005-09-22 | 2006-05-10 | 浙江大学 | General molecular mark development method and designed general Gramineae mark |
| KR20120056520A (en) * | 2010-11-25 | 2012-06-04 | 건국대학교 산학협력단 | Method for developing gene specific molecular markers |
Non-Patent Citations (2)
| Title |
|---|
| YVES CASTONGUAY 等: "Intron-length polymorphism identifies a Y2K4 dehydrin variant linked to superior freezing tolerance in alfalfa", 《THEORETICAL AND APPLIED GENETICS》 * |
| 杨龙: "利用cDNA/EST序列大规模开发内含子多态性标记的研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190096A (en) * | 2017-07-25 | 2017-09-22 | 山东农业大学 | The general molecular labeled primer and its joint development methods and applications of trees |
| CN108441577A (en) * | 2018-05-25 | 2018-08-24 | 兰州大学 | Primer pair, kit and purposes and detection alfalfa product in whether the method containing soybean |
| CN108441577B (en) * | 2018-05-25 | 2021-05-14 | 兰州大学 | Primer pair, kit and application thereof, and method for detecting whether alfalfa product contains soybeans |
| CN110396555A (en) * | 2019-06-25 | 2019-11-01 | 兰州大学 | Jian Kuo pea ILP molecular labeling primer and the application in cultivar identification |
| CN110396555B (en) * | 2019-06-25 | 2020-07-14 | 兰州大学 | Common vetch I L P molecular marker primer and application thereof in variety identification |
| CN113817867A (en) * | 2021-11-25 | 2021-12-21 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to alfalfa quality traits and application thereof |
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| CN106701960B (en) | 2020-05-22 |
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