CN110283932A - A kind of combination of SRAP molecular labeling primer and analysis method for Paris polyphylla resource analysis of genetic diversity - Google Patents
A kind of combination of SRAP molecular labeling primer and analysis method for Paris polyphylla resource analysis of genetic diversity Download PDFInfo
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Abstract
The combination of SRAP molecular labeling primer and analysis method that the present invention provides a kind of for Paris polyphylla resource analysis of genetic diversity, are related to molecular biology DNA marker technology and applied technical field.SRAP molecular labeling primer of the present invention for Paris polyphylla analysis of genetic diversity combines, including following 7 pairs of primers: me2-em10, me3-em5, me3-em7, me3-em9, me3-em10, me4-em1 and me4-em4.It can accelerate the identification speed of Paris polyphylla resource using method provided by the invention, shorten test period, it is as a result reliable and stable, compensate for the biggish deficiency of traditional form identification method experimental error.
Description
Technical field
The present invention relates to molecular biology DNA marker technology and applied technical fields, and in particular to one kind is provided for Paris polyphylla
The SRAP molecular labeling primer of source analysis of genetic diversity combines and analysis method.
Background technique
Paris polyphylla (Paris polyphylla) is Liliaceae (Liliaceae) Paris (Paris L.) perennial herb
Plant is born in 600~3600 meters of height above sea level of the dark and damp place of hayashishita, cheuch side or thick grass more.Its cold nature, bitter, it is slightly poisonous, with
Rhizome is used as medicine, and has swelling and pain relieving, clearing heat and detoxicating, arresting convulsion cool liver and other effects.Its main chemical compositions steroid saponin has suppression
The bioactivity such as bacterium, anti-inflammatory, antitumor, hemostasis, calm, analgesia.In addition, the famous Chinese patent drug Yunnan Baiyao of Paris polyphylla or China,
The primary raw material of the clear, jidesheng sheyao tablets of Gongxuening capsule, total saposins piece, heat toxin etc..Chinese Pharmacopoeia is with paris polyphylla and Hua Chonglou
Rhizome be used as medicine as Paris polyphylla, medical value is higher.Civil reality by the most of paris plants for having sturdy rhizome together
Excavation is sold, and due to long-term artificial excavation, the consumption cumulative year after year of resource, Paris polyphylla wild resource is on the verge of exhaustion.Paris polyphylla growth is special
Property causes that its growth cycle is long, and resource updates are slower.Under this tight market environment, Paris polyphylla resource is by excessive
Picking, wild planting resource are destroyed or even endangered in various degree.Therefore, it is badly in need of to Paris polyphylla resource genetic diversity
It is analyzed, to provide foundation for China especially Poor Mountainous Area Paris bio-diversity conservation and research on utilization.
Molecular labeling is a kind of method with the detection genetic diversity that DNA sequence dna otherness is label, is had not by outer
Influence whether boundary's environment and gene expression, do not influence experimental material character, can the features such as marker number is big and high resolution,
It is widely used in biological heredity research at present.SRAP (related sequence amplified polymorphism, Sequence-related
Amplified Polymorphism) it is to be developed in Brassica Crops by the Li and Quiros of California, USA university in 2001
A kind of molecular labeling of based on PCR out.The label designs ORFs (the open reading to gene by unique decoding for DTMF
Frame) specific region expanded, upstream primer to exon carry out specific amplification, downstream primer is to introne, promoter
Region carries out specific amplified.It is generated because of individual different and introne, promoter and spacer lengths difference of species etc. polymorphic
Property.The label has the characteristics that economical, easy to operate, polymorphism is high, stability is good, at present wheat, rice, arabidopsis,
It is applied in the various plants such as cotton, potato, but is applied to Paris and not yet has been reported that.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of SRAP molecular labelings for Paris polyphylla analysis of genetic diversity
Primer combination and analysis method.It can accelerate the identification speed of Paris polyphylla resource using method provided by the invention, when shortening test
Between, it is as a result reliable and stable, compensate for the biggish deficiency of traditional form identification method experimental error.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of SRAP molecular labeling primers for Paris polyphylla analysis of genetic diversity to combine, including following
7 pairs of primers:
Me2-em10 primer pair, the nucleotide sequence of the forward primer me2 of the me2-em10 primer pair such as SEQ ID
Shown in NO.2, the nucleotide sequence of reverse primer em10 is as shown in SEQ ID NO.18；
Me3-em5 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em5 primer pair such as SEQ ID NO.3
Shown, the nucleotide sequence of reverse primer em5 is as shown in SEQ ID NO.13；
Me3-em7 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em7 primer pair such as SEQ ID
The nucleotide sequence of NO.3, reverse primer em7 are as shown in SEQ ID NO.15；
Me3-em9 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em9 primer pair such as SEQ ID NO.3
Shown, the nucleotide sequence of reverse primer em9 is as shown in SEQ ID NO.17；
Me3-em10 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em10 primer pair such as SEQ ID
Shown in NO.3, the nucleotide sequence of reverse primer em10 is as shown in SEQ ID NO.18；
Me4-em1 primer pair, the nucleotide sequence of the forward primer me4 of the me4-em1 primer pair such as SEQ ID NO.4
Shown, the nucleotide sequence of reverse primer em1 is as shown in SEQ ID NO.9；
Me4-em4 primer pair, the nucleotide sequence of the forward primer me4 of the me4-em4 primer pair such as SEQ ID NO.4
Shown, the nucleotide sequence of reverse primer em4 is as shown in SEQ ID NO.12.
The present invention also provides a kind of methods using SRAP molecular marker analysis Paris polyphylla genetic diversity, including following step
It is rapid:
(1) genome DNA for extracting different Paris polyphylla samples respectively, the SRAP molecular labeling described in above-mentioned technical proposal
Primer combination carries out PCR amplification respectively, respectively obtains pcr amplification product；
(2) it after the pcr amplification product that the step (1) respectively obtains being carried out gel electrophoresis, imaging respectively, respectively obtains
Spectrum information；
(3) spectrum information respectively obtained according to the step (3) carries out cluster and principal coordinate to different Paris polyphylla samples
Analysis, and carry out the statistics of each genetic diversity parameter.
Preferably, the reaction system of PCR amplification is in the step (1), containing without Mg in every 25 μ l reaction system2+'s
10×PCR buffer 3.0μl、Mg2+0.4 μm of 1.5mmol/L, template DNA 40ng, dNTPs 0.25mmol/L, primer ol/
L, Taq enzyme 1U, ddH2O complements to 25 μ l；Above-mentioned reaction mixture is expanded by following procedure: 94 DEG C of initial denaturation 5min,
94 DEG C of denaturation 1min, 35 DEG C of annealing 1min, 72 DEG C of extension 1min, 5 circulations, 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C
Extend 90s, 35 circulations, last 72 DEG C of extensions 7min, 4 DEG C of preservations.
Preferably, the program of the PCR amplification are as follows: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 1min, 35 DEG C of annealing 1min,
72 DEG C of extension 1min, 5 circulations, 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 90s, 35 recycle, and last 72 DEG C
Extend 7min, 4 DEG C of preservations.
Preferably, matrix analysis is carried out to the spectrum information that the step (2) obtains.
Preferably, there is what band occurred to be denoted as 1 same position on electrophorogram, the note that same position does not have band to occur
It is 0, thus generates 0 and 1 matrix.
Preferably, the method for carrying out cluster and principal coordinate analysis to different Paris polyphylla samples in the step (3) includes: benefit
Similarity factor matrix is calculated with SimQual program in NTSYS2.10 software, UPGMA is carried out with SHAN in Clustering program
Cluster；Dendrogram is generated with Three plot module, constructs molecular evolutionary trees；It is carried out according to the similarity factor matrix of calculating
Decenter data conversion, and then carry out principal coordinate analysis.
It preferably, include: to utilize to the method for the statistics for carrying out each genetic diversity parameter in the step (3)
POPGENE1.32 software carries out the calculating of genetic diversity parameter to Paris polyphylla sample；The parameter include: effective number of allele,
Number of alleles, Shannon's information index, gene diversity index genetic diversity parameter, gene diversity Hs in population,
Genetic differentiation coefficient and gene flow genetic variation and genetic differentiation index between the total gene diversity Ht of population, population.
Compared with prior art, the present invention has following technical effect that
(1) SRAP molecular labeling is carried out using PCR reaction of the invention and response procedures, obtained band is clear, more
State property is good, repeated height, is easy to identify the difference of different plants gene level, has established for the research of Paris polyphylla gene level good
Basis.
(2) PCR reaction system of the invention is utilized, the band that the SRAP primer filtered out amplifies in Paris polyphylla has more
State property height, high specificity, stability are strong.
(3) present invention can accelerate the identification speed of Paris polyphylla resource, shorten test period, as a result reliable and stable, compensate for
The deficiency of traditional form identification method.
(4) at low cost, the identification of achievable large quantities of experimental materials in a short time of the invention.
(5) the SRAP molecular labeling that the present invention uses is expanded to the important component ORFs of genome, gene
Diversity can more reflect the diversity of genetic resources；SRAP is evenly distributed in genome, and the information that it is provided is than other points
Son label is more excellent, with a high credibility for Paris polyphylla Genetic diversity evaluation.
(6) genetic diversity between Paris polyphylla population can be disclosed well using method of the invention, differentiate Paris polyphylla germplasm money
Affiliation between source, protection and utilization to Paris polyphylla resource are of great significance.
Detailed description of the invention
Fig. 1 is amplified production electrophoretogram of the me2-em10 primer combination to 33 samples；
Fig. 2 is amplified production electrophoretogram of the me3-em5 primer combination to 33 samples；
Fig. 3 is amplified production electrophoretogram of the me3-em7 primer combination to 33 samples；
Fig. 4 is amplified production electrophoretogram of the me3-em9 primer combination to 33 samples；
Fig. 5 is amplified production electrophoretogram of the me3-em10 primer combination to 33 samples；
Fig. 6 is amplified production electrophoretogram of the me4-em1 primer combination to 33 samples；
Fig. 7 combines the amplified production electrophoretogram to 33 samples for me4-em4 primer, wherein M, 5000bp Marker, 1-
No. 33 corresponding with the Unified number of table 2；
Fig. 8 is that the present invention is based on 33 parts of Paris polyphylla sample clustering figures that SRAP is marked；
Fig. 9 is that the present invention is based on 33 parts of Paris polyphylla sample principal coordinate analysis that SRAP is marked.
Specific embodiment
The present invention provides a kind of SRAP molecular labeling primers for Paris polyphylla analysis of genetic diversity to combine, including following
7 pairs of primers:
Me2-em10 primer pair, the nucleotide sequence of the forward primer me2 of the me2-em10 primer pair such as SEQ ID
Shown in NO.2, the nucleotide sequence of reverse primer em10 is as shown in SEQ ID NO.18；
Me3-em5 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em5 primer pair such as SEQ ID NO.3
Shown, the nucleotide sequence of reverse primer em5 is as shown in SEQ ID NO.13；
Me3-em7 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em7 primer pair such as SEQ ID
The nucleotide sequence of NO.3, reverse primer em7 are as shown in SEQ ID NO.15；
Me3-em9 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em9 primer pair such as SEQ ID NO.3
Shown, the nucleotide sequence of reverse primer em9 is as shown in SEQ ID NO.17；
Me3-em10 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em10 primer pair such as SEQ ID
Shown in NO.3, the nucleotide sequence of reverse primer em10 is as shown in SEQ ID NO.18；
Me4-em1 primer pair, the nucleotide sequence of the forward primer me4 of the me4-em1 primer pair such as SEQ ID NO.4
Shown, the nucleotide sequence of reverse primer em1 is as shown in SEQ ID NO.9；
Me4-em4 primer pair, the nucleotide sequence of the forward primer me4 of the me4-em4 primer pair such as SEQ ID NO.4
Shown, the nucleotide sequence of reverse primer em4 is as shown in SEQ ID NO.12.
In the present invention, the sequence of the SEQ ID NO.2 is as follows:
TGAGTCCAAACCGGAGC；
In the present invention, the sequence of the SEQ ID NO.18 is as follows:
GACTGCGTACGAATTCAG；
In the present invention, the sequence of the SEQ ID NO.3 is as follows:
TGAGTCCAAACCGGAAT；
In the present invention, the sequence of the SEQ ID NO.13 is as follows:
GACTGCGTACGAATTAAC；
In the present invention, the sequence of the SEQ ID NO.15 is as follows:
GACTGCGTACGAATTCAA；
In the present invention, the sequence of the SEQ ID NO.17 is as follows:
GACTGCGTACGAATTCGA；
In the present invention, the sequence of the SEQ ID NO.18 is as follows:
GACTGCGTACGAATTCAG；
In the present invention, the sequence of the SEQ ID NO.4 is as follows:
TGAGTCCAAACCGGACC；
In the present invention, the sequence of the SEQ ID NO.9 is as follows:
GACTGCGTACGAATTATT；
In the present invention, the sequence of the SEQ ID NO.12 is as follows:
GACTGCGTACGAATTTGA。
The present invention also provides a kind of methods using SRAP molecular marker analysis Paris polyphylla genetic diversity, including following step
It is rapid:
(1) genome DNA for extracting different Paris polyphylla samples respectively, the SRAP molecular labeling described in above-mentioned technical proposal
Primer combination carries out PCR amplification respectively, respectively obtains pcr amplification product；
(2) it after the pcr amplification product that the step (1) respectively obtains being carried out gel electrophoresis, imaging respectively, respectively obtains
Spectrum information；
(3) spectrum information respectively obtained according to the step (3) carries out cluster and principal coordinate to different Paris polyphylla samples
Analysis, and carry out the statistics of each genetic diversity parameter.
In the present invention, it is preferred to acquire extraction of the Paris polyphylla tender leaf for complete genome DNA.
In the present invention, it is preferred to extract Paris polyphylla genome DNA using CTAB method, DNA sample is obtained, preferably by extraction
DNA sample be dissolved in TE buffer be placed on -20 DEG C it is spare；DNA sample is preferably diluted to 10ng/ μ with distilled water before PCR amplification
L, the template as pcr amplification reaction.
In the present invention, the PCR reaction system is preferably and contains in every 25 μ l reaction system and be free of Mg2+10 × PCR
buffer 3.0μl、Mg2+1.5mmol/L, template DNA 40ng, dNTPs 0.25mmol/L, primer 0.4 μm of ol/L, Taq enzyme
1U, ddH2O complements to 25 μ l；Above-mentioned reaction mixture is expanded by following procedure: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation
1min, 35 DEG C of annealing 1min, 72 DEG C of extension 1min, 5 recycle, 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 90s,
35 circulations, last 72 DEG C of extensions 7min, 4 DEG C of preservations.
In the present invention, the Mg2+1.5 μ l (being converted into concentration i.e. 1.5mmol/L) of 25mM is added in the 25 μ l system；
The 2.5 μ l of dNTPs (being converted into concentration i.e. 0.25mmol/L) of 2.5mM；The 1.0 μ l of primer of 10 μ Μ (is converted into concentration i.e.
0.40mmol/L)。
In the present invention, the Mg2+Solution is preferably bought in TaKaRa company, for carrying out PCR test.
In the present invention, electrophoresis uses 1% agarose gel electrophoresis, and voltage is preferably 110V, and electrophoresis time is preferably
50min。
In the present invention, it is preferred to carry out matrix analysis to the obtained spectrum information.The method of the matrix analysis is excellent
It is selected as band that is clear on electrophorogram and repeating being denoted as 1, what same position did not had a band is denoted as 0, thus generates 0 He
1 matrix.
In the present invention, the method for carrying out cluster and principal coordinate analysis to different Paris polyphylla samples includes: to utilize
SimQual program calculates similarity factor matrix in NTSYS2.10 software, and it is poly- to carry out UPGMA with SHAN in Clustering program
Class；Dendrogram is generated with Three plot module, constructs molecular evolutionary trees；It is carried out according to the similarity factor matrix of calculating
Decenter data conversion, and then carry out principal coordinate analysis.
In the present invention, the method that the statistics of each genetic diversity parameter is carried out to the Paris polyphylla includes: to utilize
POPGENE1.32 software carries out the calculating of genetic diversity parameter to Paris polyphylla sample；The parameter include: effective number of allele,
Number of alleles, Shannon's information index, gene diversity index genetic diversity parameter, gene diversity Hs in population,
Genetic differentiation coefficient and gene flow genetic variation and genetic differentiation index between the total gene diversity Ht of population, population.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
33 parts of Paris polyphylla samples used in the present invention, wherein 32 parts are picked up from 6 areas of Jinzhai County, Anhui Province, 1 part is picked up from Mount Huang,
Specifically it is shown in Table 1.
1 test material of table number and sampling point information
Embodiment 2
The extraction of Paris polyphylla genomic DNA
The present invention acquires the extraction that 1 Paris polyphylla tender leaf of embodiment is used for complete genome DNA, extracts Paris polyphylla gene using CTAB method
Group total DNA, and by the DNA sample of extraction be dissolved in TE buffer be placed on -20 DEG C it is spare.Preceding distilled water is expanded by DNA sample
It is diluted to 10ng/ μ l, the template as pcr amplification reaction.
The PCR reaction system and primer of Paris polyphylla SRAP label
Through me2-em10, me3-em5, me3-em7, me3-em9, me3-em10, me4-em1 and me4-em4 seven to primer,
10x PCRbuffer (being free of Mg2+) 3.0 μ after each pair of primer does 3 repetition tests, in 25 μ l reaction system of SRAP-PCR
L, Mg2+1.5mmol/L, template DNA 40ng, dNTPs 0.25mmol/L, 0.4 μm of ol/L of primer, Taq enzyme 1U, comprehensive amplification effect
Fruit is best.Pcr amplification reaction program are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 1min, 35 DEG C of annealing 1min, 72 DEG C extend
1min, 5 circulations, 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 90s, 35 recycle, last 72 DEG C of extensions 7min,
4 DEG C of preservations.
SRAP primer sequence is shown in Table 2, using above-mentioned reaction system and response procedures using the SRAP primer sequence of table 2 to 33
The DNA of part Paris polyphylla sample carries out PCR reaction.As a result polymorphism primer combines: me2-em10；me3-em5；me3-em7；me3-
em9；me3-em10；me4-em1；me4-em4；The banding pattern of amplification is stable, clear and reproducible.Fig. 1-7 is respectively above-mentioned draws
Object combines amplification figure, it can be seen that the band that SRAP-PCR reaction system established by the present invention amplifies has polymorphism
The feature that height, high specificity, background understand and stability is strong.
2 SRAP primer sequence of table
Electrophoresis detection: taking 8 μ l pcr amplification product obtained above to carry out electrophoresis, colour developing, and electrophoresis is solidifying using 1% agarose
Gel electrophoresis, voltage 110V, electrophoresis time 50min use DL5000 DNA Marker as standard reference, take pictures and obtain bands of a spectrum letter
It ceases and saves.
SRAP marks the application in Paris polyphylla analysis of genetic diversity
Above-mentioned electrophoresis spectrum information obtained is analyzed, SRAP is dominant marker, in same primer extension product
The consistent band of electrophoretic mobility is considered as the product in same site.Site same on electrophorogram there is into identical amplification item
Band is denoted as " 1 ", without " 0 " is then denoted as, to generate " 0 " and " 1 " matrix diagram.Count total band number and more that each pair of primer amplification goes out
State property band number.Similarity factor matrix is calculated using SimQual program in NTSYS2.10 software, in Clustering program
SHAN carries out UPGMA (unweighted pair-group method with arithmetic means) cluster, uses Three
Plot module generates dendrogram, constructs molecular evolutionary trees.Decenter data conversion is carried out according to the similarity factor matrix of calculating,
And then carry out principal coordinate analysis.
Effective number of allele Ne, number of alleles Na, Shannon's information are calculated separately out using POPGENE1.32
The genetic diversities parameters such as index, Nei ' s gene diversity index, gene diversity Hs, the total gene diversity of population in population
The genetic variation and genetic differentiations index such as genetic differentiation coefficient Gst and gene flow Nm between Ht, population.
Embodiment 3
As a result with analysis
(1) polymorphism analysis
Fig. 1-7 is 7 pairs of primer combinations to the amplified production electrophoretogram of 33 Paris polyphylla samples, has figure can be seen that and is obtained
Band it is clear and abundant.Primer amplification, which the results are shown in Table 3,7 pairs of primer sets and amount to, amplifies 101 band, wherein polymorphism item
97, band, polymorphism percentage (PPB) is 96.04%.Band that different primers are amplified is different, same primers, differently
The band of the Paris polyphylla amplification in area is not also identical, shows 33 relatively rich state properties of sample room and complicated genetic background, for analysis
The affiliation of Paris polyphylla provides may.
3 primer amplification result of table
Note: TNP: total band number is expanded；NPB: polymorphic bands number；PPB: percentage of polymorphisms；PIC: polymorphism information
Content；MI: label index.
4 Paris polyphylla population SRAP analysis of genetic diversity of table
Note: P: polymorphic site percentage；Na: observation number of alleles；Ne: effective number of allele；H:Nei ' s gene
Various degree；I:Shannon ' s information index.
It can be with by number of alleles (Na), effective number of allele (Ne), Nei ' s gene diversity (H) these parameters
Reflect diversity.Ne is bigger, illustrates that allele is more important, and allele effect is bigger.I and H value is bigger, illustrates that the sample is lost
It is higher to pass diversity.Bulk sample species level is it is found that it is that 1.3479, H value is that the Na value of Paris polyphylla, which is 1.9505, Ne value,
0.2192, I value is 0.3511.The total gene diversity of different regions Paris polyphylla population (Ht) is 0.2199, gene diversity in population
It (Hs) is 0.1698, genetic differentiation coefficient (Gst) is 0.2275 between population.Wherein Gst > Hs illustrates genetic variation and genetic differentiation degree between population
Than genetic variation and genetic differentiation Du Genggao in population.Gene flow (Nm) is 1.6973.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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<213>artificial sequence (Artificial Sequence)
<400> 11
gactgcgtac gaattgac 18
<210> 12
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gactgcgtac gaatttga 18
<210> 13
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gactgcgtac gaattaac 18
<210> 14
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gactgcgtac gaattgca 18
<210> 15
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gactgcgtac gaattcaa 18
<210> 16
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gactgcgtac gaattctg 18
<210> 17
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gactgcgtac gaattcga 18
<210> 18
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gactgcgtac gaattcag 18
<210> 19
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gactgcgtac gaattcca 18
Claims (8)
1. a kind of SRAP molecular labeling primer for Paris polyphylla analysis of genetic diversity combines, which is characterized in that including following 7 pairs
Primer:
Me2-em10 primer pair, the nucleotide sequence of the forward primer me2 of the me2-em10 primer pair such as SEQ ID NO.2 institute
Show, the nucleotide sequence of reverse primer em10 is as shown in SEQ ID NO.18；
Me3-em5 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em5 primer pair such as SEQ ID NO.3 institute
Show, the nucleotide sequence of reverse primer em5 is as shown in SEQ ID NO.13；
Me3-em7 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em7 primer pair such as SEQ ID NO.3, instead
To primer em7 nucleotide sequence as shown in SEQ ID NO.15；
Me3-em9 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em9 primer pair such as SEQ ID NO.3 institute
Show, the nucleotide sequence of reverse primer em9 is as shown in SEQ ID NO.17；
Me3-em10 primer pair, the nucleotide sequence of the forward primer me3 of the me3-em10 primer pair such as SEQ ID NO.3 institute
Show, the nucleotide sequence of reverse primer em10 is as shown in SEQ ID NO.18；
Me4-em1 primer pair, the nucleotide sequence of the forward primer me4 of the me4-em1 primer pair such as SEQ ID NO.4 institute
Show, the nucleotide sequence of reverse primer em1 is as shown in SEQ ID NO.9；
Me4-em4 primer pair, the nucleotide sequence of the forward primer me4 of the me4-em4 primer pair such as SEQ ID NO.4 institute
Show, the nucleotide sequence of reverse primer em4 is as shown in SEQ ID NO.12.
2. a kind of method using SRAP molecular labeling primer combinatory analysis Paris polyphylla genetic diversity, which is characterized in that including with
Lower step:
(1) genome DNA for extracting different Paris polyphylla samples respectively, with SRAP molecular labeling primer group described in claim 1
Conjunction carries out PCR amplification respectively, respectively obtains pcr amplification product；
(2) after the pcr amplification product that the step (1) respectively obtains being carried out gel electrophoresis, imaging respectively, bands of a spectrum are respectively obtained
Information；
(3) spectrum information respectively obtained according to the step (3) carries out cluster and principal coordinate analysis to different Paris polyphylla samples,
And carry out the statistics of each genetic diversity parameter.
3. according to the method described in claim 2, it is characterized in that, in the step (1) reaction system of PCR amplification be, often
Containing without Mg in 25 μ l reaction systems2+10 × PCR buffer3.0 μ l, Mg2+1.5mmol/L, template DNA 40ng,
DNTPs 0.25mmol/L, 0.4 μm of ol/L of primer pair, Taq enzyme 1U, ddH2O complements to 25 μ l.
4. according to the method described in claim 3, it is characterized in that, the program of the PCR amplification are as follows: 94 DEG C of initial denaturation 5min,
94 DEG C of denaturation 1min, 35 DEG C of annealing 1min, 72 DEG C of extension 1min, 5 circulations, 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C
Extend 90s, 35 circulations, last 72 DEG C of extensions 7min, 4 DEG C of preservations.
5. according to the method described in claim 2, it is characterized in that, carrying out matrix to the spectrum information that the step (2) obtains
Analysis.
6. according to the method described in claim 5, it is characterized in that, the method for the matrix analysis includes: will be on electrophorogram
Same position has what band occurred to be denoted as 1, and same position does not have what band occurred to be denoted as 0, thus generates 0 and 1 matrix.
7. according to the method described in claim 2, it is characterized in that, gathering in the step (3) to different Paris polyphylla samples
Class and the method for principal coordinate analysis include: to calculate similarity factor matrix using SimQual program in NTSYS2.10 software, with
SHAN carries out UPGMA cluster in Clustering program；Dendrogram is generated with Three plot module, constructs molecular evolutionary trees；
Decenter data conversion is carried out according to the similarity factor matrix of calculating, and then carries out principal coordinate analysis.
8. according to the method described in claim 2, it is characterized in that, to each genetic diversity parameter is carried out in the step (3)
Statistics method include: using POPGENE1.32 software to Paris polyphylla sample carry out the calculating of genetic diversity parameter；The parameter
It include: effective number of allele, number of alleles, Shannon's information index, gene diversity index genetic diversity ginseng
It counts, genetic differentiation coefficient and gene flow genetic variation and genetic differentiation refer between gene diversity Hs, the total gene diversity Ht of population, population in population
Mark.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483282A (en) * | 2016-03-02 | 2016-04-13 | 云南省农业科学院药用植物研究所 | PCR specificity identifying primers and paris polyphylla identifying method adopting same |
| CN107099599A (en) * | 2017-05-26 | 2017-08-29 | 红河学院 | A kind of tsaoko Genetic Diversity of Germplasm analysis method marked based on SRAP |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483282A (en) * | 2016-03-02 | 2016-04-13 | 云南省农业科学院药用植物研究所 | PCR specificity identifying primers and paris polyphylla identifying method adopting same |
| CN107099599A (en) * | 2017-05-26 | 2017-08-29 | 红河学院 | A kind of tsaoko Genetic Diversity of Germplasm analysis method marked based on SRAP |
Non-Patent Citations (2)
| Title |
|---|
| 任李玥等: "胡黄连SRAP反应体系的建立与引物筛选", 《云南中医学院学报》 * |
| 王从彦: "SRAP 标记在植物遗传多样性中的应用进展", 《生物技术》 * |
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