CN106191285A - A kind of method and the application that utilize genome SSR and EST SSR finger printing to differentiate Semen Phaseoli kind - Google Patents
A kind of method and the application that utilize genome SSR and EST SSR finger printing to differentiate Semen Phaseoli kind Download PDFInfo
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Abstract
The present invention provides a kind of method and application utilizing genome SSR and EST SSR finger printing to differentiate Semen Phaseoli kind, belongs to plant variety authentication technique field.The present invention provides the 5 labelling combinations to genome SSR and 5 pairs of EST SSR primer compositions, its nucleotide sequence is respectively as shown in SEQ ID NO.1 20, and then construct the finger printing of 32 kinds of Semen Phaseolis, according to this collection of illustrative plates, the labelling combination using the present invention to provide can complete bean seed cultivar identification within 4 hours and work with Genetic diversity evaluation, had efficient, accurate, low cost, advantage easy and simple to handle.The method of the present invention can carry out effective monitoring to the bean seed true and false; hereditary variation and the sibship of kind is disclosed from DNA level; protect crop varieties; prevent fake and forged kind from coming into the market; also provide technical support for the Appropriate application of excellent germplasm during Semen Phaseoli breed breeding, have a good application prospect.
Description
Technical field
The present invention relates to molecular biology and plant variety authentication technique field, particularly relate to one and utilize genome
SSR and EST-SSR finger printing differentiates method and the application of Semen Phaseoli kind.
Background technology
Semen Phaseoli (Vigna angularis), belongs to pulse family, in the existing bimillennial cultivation history of China.It is that China is important
Cereal crops.Because it has important nutritive value and medical value, develop as important functional food.Closely
Nian Lai, along with adjustment and the change of people's diet structure of crop mix, the demand of Semen Phaseoli is increased by people day by day, plantation
Area expands year by year.Research shows that pest and disease damage has a strong impact on the aspects such as bean seed sprouting and yield.In consideration of it, further investigation is anti-
Worm Semen Phaseoli kind participates in pest-resistant gene and pest-resistant mechanism, the Dominant variety stronger to screening insect resistance capacity and breeding for pest resistance material
The screening tool of material is of great significance.
Owing to introducing a fine variety utilization between region, Semen Phaseoli kind homonymus or synonymum phenomenon is extremely serious, and local varieties genetic similarity is high,
Traditional Identification of morphology and cytology method is difficult to the breed difference accomplishing accurately and really to identify Semen Phaseoli, needs for this
Analysis and Identification is carried out on molecular level.
DNA fingerprinting can identify interracial difference or genetic similarity from DNA level.And microsatellite (
It is referred to as simple repeated sequence, SSR) section of DNA that repeatedly forms of the short sequence tandem sequence repeats that is made up of 1-6 nucleotide.
Owing in microsatellite, the number of repetition of repetitive is different, thus the length of the microsatellite sequence amplified presents polymorphism.Micro-
It is higher that satellite sequence has polymorphism, repeatability and the feature such as good stability, is highly effective molecular marker, is widely used
In the field such as cultivar identification, fingerprint map construction.In order to effectively protect the kind intellectual property of China's Semen Phaseoli, it is necessary in China
Carry out Semen Phaseoli exploitation microsatellite marker exploitation, and be applied to the research that Semen Phaseoli improved seeds " molecular identity card " build.SSR according to
Sequence source is generally divided into genome gSSR (genomic SSR) and EST-SSR (eSSR) two kinds.EST-SSR is based on expression
Sequence label exploitation microsatellite a kind of New molecular marker, compared with genome SSR, EST-SSR have plant species it
Between the advantage of transferability.At present, EST-SSR be widely used in Research of Plant Genomics study such as genetic map construction, compare
The aspects such as mapping, Genetic diversity evaluation, Idioplasm identification, phylogeny and Study on Evolution.
The most conventional cultivation Semen Phaseoli cultivar identification method mainly has: 1, phenotypic character identification method: the method is by difference
Between kind, different cultivars is identified by phenotypic character difference, although the most directly perceived, quick, but the kind close to phenotypic character
The most fubaritic；2, DNA molecular marker method: the method utilizes all kinds of DNA molecular marker to enter different cultivars on gene level
Row is identified, wherein SSR molecular marker is applied because it has the feature such as codominance, high, stable, the fast, economical of amplification of polymorphism
For generally, the most having announced Semen Phaseoli EST-SSR molecular marker number, to have reached 7947 right.How to effectively utilize these molecular markers
Realize the efficient stable to Semen Phaseoli kind and identify it is prior art problem demanding prompt solution.
Summary of the invention
It is an object of the invention to provide a kind of efficiently, accurately, low cost utilize genome SSR and EST-SSR fingerprint
Collection of illustrative plates differentiates the method for Semen Phaseoli kind.The present invention also aims to provide the application of the method.
For achieving the above object, the present invention is with 32 parts of representative Semen Phaseoli variety genome DNAs template, by analyzing Semen Phaseoli
Genome sequence and est sequence, synthesis core genome SSR and EST-SSR primer.Through screening, there are 5 pairs of genomes respectively
The molecular marker combination of SSR and 5 pairs of EST-SSR primer compositions can differentiate the difference of 32 parts of Semen Phaseoli kinds completely.
It is little No. 3 that 32 parts of described representative Semen Phaseoli kinds are respectively as follows: capital, and capital is little by 29, and dragon Semen Phaseoli one, little Feng 2, capital is little
38, capital is little by 152, shore Semen Phaseoli two, shore Semen Phaseoli one, Ji Hong tetra-, capital agriculture 2, capital agriculture 5, Ji Semen Ormosiae Hosiei 2, the Liao Dynasty's Semen Phaseoli 1
Number, Baoqing Semen Ormosiae Hosiei, Ji Semen Ormosiae Hosiei 3, protect 9326-16, protect 9327-5, Shanxi Semen Phaseoli 1, protect 947-27, Ji Hong 352, capital agriculture 6
Number, capital agriculture 7, Bai Hong 2, Bai Hong No. 3 numbers, Ji Hong No. 6 numbers, Ji Hong No. 7 numbers, Bai Hong No. 1 number, Bai Hong No. 4 numbers, Bai Hong No. 5 numbers, Bai Hong No. 6 numbers, Ji
Protect Semen Ormosiae Hosiei 2 and the Liao Dynasty's Semen Ormosiae Hosiei 2.
The method screening above-mentioned primer is:
Step one, primary dcreening operation primer: filter out there is between different Semen Phaseoli kinds polymorphism, banding pattern is clear and can stably weigh
Multiple SSR primary dcreening operation primer；
Step 2, sieves primer: utilize the SSR primary dcreening operation that PowerMarker and Popgen computed in software obtains in step one again
The number of alleles (Na) of primer, polymorphism information amount (PIC) and Shannon information index (I), filter out between Semen Phaseoli kind
Polymorphism information content values is the highest, loci number is most, reproducible, banding pattern definition does, chromosome is evenly distributed in amplification
The core primers that builds as Semen Phaseoli Variety fingerprinting storehouse of SSR primer.Based on above-mentioned screening technique, the present invention provides 5 right
The core primers of the labelling combination of genome SSR and 5 pairs of EST-SSR primer compositions, its nucleotide sequence is respectively such as SEQ ID
Shown in NO.1-20.
The most described 5 SSR molecular marker are obtained by following primer pair amplifies: SEQ ID NO.1-2, SEQ ID NO.3-
4, the primer shown in SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10；Described 5 EST-SSR molecular markers
Obtained by following primer pair amplifies: SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID
Primer shown in NO.17-18, SEQ ID NO.19-20.
The test kit combined containing above-mentioned molecular marker falls within protection scope of the present invention.
The invention provides the combination of above-mentioned molecular marker or the test kit containing it application in identifying Semen Phaseoli kind.
The invention provides the combination of above-mentioned molecular marker or the test kit containing it is building Semen Phaseoli varietal characteristic fingerprint image
Application in spectrum.
The invention provides the combination of above-mentioned molecular marker or the test kit containing it is improved or breeding at Germplasm Resource of Vigna Anglaris Ohwi Ohashi
In application.
The present invention provides a kind of Semen Phaseoli varietal characteristic finger printing, with above-mentioned molecular marker combination to different Semen Phaseoli kinds
DNA carry out PCR amplification, PCR primer is carried out native polyacrylamide gel electrophoresis and detects, silver staining develop the color, according to
The mobility record electrophoresis result of stranded DNA molecule amount and primer, the foundation amplified production relative position on running gel,
There is being designated as " 1 " of band on identical migration position, be designated as " 0 " without carry, set up SSR genotype information data, according to primer order
Encoding, the finger-print code obtained is the characteristic fingerprint pattern of Semen Phaseoli kind.
In an embodiment of the present invention, combined after carrying out PCR amplification by the molecular marker of the present invention, according to above-mentioned judgement
Method, it is thus achieved that the characteristic fingerprint pattern of 32 kinds of Semen Phaseoli kinds is as follows:
The invention provides the application in Germplasm Resource of Vigna Anglaris Ohwi Ohashi improvement or breeding of the above-mentioned Semen Phaseoli varietal characteristic finger printing.
Further, the present invention provides a kind of method identifying Semen Phaseoli kind, comprises the steps:
(1) DNA of Semen Phaseoli kind to be checked is extracted, with 10 pairs of primers pair in the molecular marker combination that present invention screening obtains
Respectively the DNA of Semen Phaseoli kind to be checked is carried out PCR amplification；
(2) amplified production is detected by native polyacrylamide gel electrophoresis, and silver staining develops the color, according to amplified production
Relative position on running gel, has being designated as " 1 " of band on identical migration position, without being designated as " 0 " of band, suitable according to primer
Sequence encodes, and is compared by the characteristic fingerprint pattern of the finger-print code obtained with above-mentioned Semen Phaseoli kind, determines to be measured little
Bean kind.
The present invention identifies in the method for Semen Phaseoli kind, the PCR of step (1), its 20 μ L reaction system: DNA profiling 0.8 μ L,
Upstream and downstream primer each 10pmol L-10.2 μ L, 10 × PCR do not contain Mg2+Buffer 2 μ L, 25mmol L-1Mg2+1.2 μ L,
10mmol L-1DNTP 0.5 μ L, 5U μ L-1Taq enzyme 0.2 μ L, ddH2O 14.9μL；
PCR response procedures is: 95 DEG C of denaturations 5min, 95 DEG C of degeneration 30s, 51-60 DEG C of annealing 45s, and 72 DEG C extend 45s,
Carrying out 32-35 circulation, last 72 DEG C extend 5min.
Preferably, 55 DEG C of annealing.Cycle-index is 33.
Wherein, step (2) uses the non-denaturing polyacrylamide gel of 8% to carry out electrophoresis, and electrophoretic buffer is 0.5 ×
TBE, 200V voltage stabilizing electrophoresis 2-2.5h, to sample loading buffer move on to bottom gel time electrophoresis terminate.
The invention provides said method application in Germplasm Resource of Vigna Anglaris Ohwi Ohashi is improved.
The construction method of Semen Phaseoli DNA fingerprinting of the present invention, make use of SSR primer to expand a certain Semen Phaseoli kind DNA
Increase, obtain a specific fingerprint, and one group of SSR primer carries out DNA cloning and will make this kind can this specific kind
Can obtain one group of distinctive DNA fingerprint, primer is carried out again by the method that then the inventive method uses primer combination to differentiate
Analyze and screening, make each kind have found its distinctive DNA fingerprint.The present invention is based on SSR molecular marker identification method
Set up, 10 pairs of SSR primers compositions that utilization is evenly distributed on cultivation Semen Phaseoli chromosome set, polymorphism is good, PCR amplification is stable
The technical system that difference cultivation Semen Phaseoli kind is identified by a set of primer, the amplification of SSR and the EST-SSR primer of the present invention is produced
Between thing banding pattern, difference in size is obvious, it is possible to well distinguish different kinds.To the amplified production between same intravarietal individuality,
Its banding pattern is consistent.The detection method of the present invention can complete bean seed cultivar identification within 4 hours and comment with genetic diversity
Valency works, and has efficient, accurate, low cost, the advantage such as easy and simple to handle.The detection method of the present invention can be effectively to little simultaneously
The bean seed true and false is monitored, and discloses hereditary variation and the sibship of kind from DNA level, protects crop varieties, prevent
Fake and forged kind comes into the market, and also provides Technical Reference for the Appropriate application of excellent germplasm during Semen Phaseoli breed breeding.
Accompanying drawing explanation
Fig. 1 is 32 parts of representative Semen Phaseoli Variety cluster analysis result figures.
Detailed description of the invention
Following example further illustrate present disclosure, but should not be construed as limitation of the present invention.Without departing substantially from
In the case of present invention spirit and essence, the amendment that the inventive method, step or condition are made or replacement, belong to the present invention
Scope.
If not specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art；
If not specializing, in embodiment, agents useful for same is commercially available.
Embodiment 1 is for identifying the screening of SSR and the EST-SSR primer of Semen Phaseoli kind
32 Semen Phaseoli kinds that the present embodiment is promoted with China's Semen Phaseoli main producing region representativeness (derive from country's farming species matter
Resource conservation center) genomic DNA is template, by analyzing Semen Phaseoli genome sequence and est sequence, synthesizes core genome
SSR and EST-SSR primer.Through screening, the molecular marker group being made up of 5 couples of genome SSR and 5 pairs of EST-SSR primers respectively
Conjunction can differentiate the difference of 32 parts of Semen Phaseoli kinds completely.
32 parts of representative Semen Phaseoli kinds are respectively as follows: light green No. 1, protect 942-40-2, pacify 9910, emerging green No. 1, pacify 05-4, protect
9815-36, Weihe River 8901-32, green No. 8 of Zheng, green No. 1 of Henan, green No. 1 of Hubei Province, green No. 4 of Henan, medium green 3, guarantor 861-10-6, green No. 1 of Ji,
Medium green 5, pacifies black green No. 1 by the greenest No. 6, protects 865-18-9, and lucky green No. 4, Ji green 9346, Jilv 9239, Shanxi Semen Phaseoli 2, the Liao Dynasty is green
No. 6, revive green No. 1, revive green No. 3, Taolv 3, green No. 5 of Henan, green No. 5 of Zheng, Hubei Province green No. 2 and green No. 27 of the Liao Dynasty.
The method screening above-mentioned primer is:
Step one, primary dcreening operation primer: filter out light green No. 1, green No. 8 of Zheng, green No. 1 of Henan, green No. 1 of Hubei Province, green No. 4 of Henan, medium green 3
Number, revive and have between green No. 1 green with Zheng No. 58 different mung bean varieties that polymorphism, banding pattern be clear and at the beginning of the SSR that can stably repeat
Sieve primer；
Step 2, sieves primer: utilize the number of alleles of PowerMarker and Popgen computed in software primer sites again
(Na), polymorphism information amount (PIC) and Shannon information index (I), filter out polymorphism information content values between Semen Phaseoli kind
The highest, loci number is most, reproducible, amplification banding pattern definition does, chromosome is evenly distributed SSR primer is as Semen Phaseoli
The core primers that Variety fingerprinting storehouse builds.
Final screening obtains 5 pairs of genome SSR molecular marker and 5 pairs of EST-SSR molecular markers.It is shown in Table 1.
1 10 pairs of Semen Phaseoli SSR core primers tables (Ag represents genome SSR, and Ae represents EST-SSR) of table
The most corresponding 5 SSR molecular marker of the primer of above-mentioned table 1: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ
Primer shown in ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10；5 EST-SSR molecular markers: SEQ ID
Shown in NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20
Primer.
The structure of embodiment 2 Semen Phaseoli varietal characteristic finger printing and the foundation of Semen Phaseoli cultivar identification method
1, Semen Phaseoli kind total DNA extraction
Semen Phaseoli is planted in soil, incubated at room temperature, take Semen Phaseoli spire after growing two weeks after liquid nitrogen freezing in-80 DEG C of guarantors
Deposit standby.Take each kind Semen Phaseoli tender blade 0.2g of children in 1.5mL centrifuge tube, add liquid nitrogen grinding and become powder.Use CTAB
Method extracts DNA, and operation experiments step is carried out according to test kit description.Detect total with agarose gel electrophoresis and spectrophotometer
The quality of DNA, is then diluted to 100ng/ μ L by STb gene, be stored in-20 DEG C standby.
2, with 32 parts of Semen Phaseoli kinds described in embodiment 1 as material, the 5 couples of genome SSR utilizing embodiment 1 screening to obtain divide
Sub-labelling and the SSR primer Semen Phaseoli kind representative to 32 parts described in embodiment 1 respectively of 5 pairs of EST-SSR molecular marker synthesis
DNA carry out PCR amplification, in PCR reaction test use PCR reaction system be 20 μ L systems: DNA profiling 0.8 μ L, upstream and downstream
Primer each 10pmol L-10.2 μ L, 10 × PCR buffer is (without Mg2+) 2 μ L, Mg2+(25mmol L-1) 1.2 μ L, dNTP
(10mmol L-1) 0.5 μ L, Taq enzyme (5U μ L-1) 0.2 μ L, ddH2O 14.9 μ L, amounts to 20.0 μ L.
PCR response procedures is: 95 DEG C of denaturations 5min, 95 DEG C of degeneration 30s, 51-60 DEG C of annealing 45s, and 72 DEG C extend 45s,
Carrying out 32-35 circulation, last 72 DEG C extend 5min.The present embodiment optimal conditions parameter finds, annealing temperature 55 DEG C, circulates 33
Secondary, optimal comprehensive detection effect can be reached.
Gel electrophoresis and silver staining colour developing: amplified production uses 8% native polyacrylamide gel electrophoresis detection, and silver staining shows
Color, film recording.
3, the structure of Semen Phaseoli kind DNA characteristics finger printing
Mobility record result according to DNA molecular amount standard and primer, according to SSR amplified production on running gel
Position relatively, has being designated as " 1 " of band on identical migration position, without being designated as " 0 " of band, 32 parts of Semen Phaseolis described in constitution and implementation example 1
Variety SSR genotype information data base, encodes according to primer order, and the finger-print code obtained is the fingerprint image of this kind
Spectrum, is shown in Table 2.
Table 2 utilizes 10 pairs of SSR primers to build 32 representative finger print informations promoting Semen Phaseoli kind
Utilize the number of alleles (Na) of PowerMarker and Popgen computed in software primer sites, polymorphism information amount
And Shannon information index (I) (PIC).Table 3 is 10 pairs of SSR primers to 32 parts of Semen Phaseoli kind Genetical Variation results,
In 32 parts of Semen Phaseoli kinds, 10 SSR marker detect 20 allele altogether.Polymorphism information amount (PIC) the change model of primer
Enclosing is 0.3272～0.3711, average out to 0.3566, and Shannon information index (I) excursion is 0.6024～0.6853, flat
It is 0.6577.
3 10 pairs of SSR primers of table are to 32 parts of Semen Phaseoli kind Genetical Variation results
Utilize PowerMarker to calculate 32 interracial genetic similarities of Semen Phaseoli, be shown in Table 4-table 7, between each kind
Genetic similarity is between 0.1～0.9, and the average genetic similarity of 32 Semen Phaseoli kinds is 0.5184, the primer of selection
All there are good polymorphism and distinguishing ability, can effectively distinguish 32 Semen Phaseoli kinds that embodiment 1 is recorded.
The interracial genetic similarity of table 4 Semen Phaseoli (1)
The interracial genetic similarity of table 5 Semen Phaseoli (2)
The interracial genetic similarity of table 6 Semen Phaseoli (3)
The interracial genetic similarity of table 7 Semen Phaseoli (4)
4, the foundation of Semen Phaseoli kind method is identified
(1) DNA of Semen Phaseoli kind to be checked is extracted, with 10 in the molecular marker combination that the embodiment of the present invention 1 screening obtains
To primer to respectively the DNA of Semen Phaseoli kind to be checked being carried out PCR amplification；20 μ L PCR reaction systems: DNA profiling 0.8 μ L, up and down
Trip primer each 10pmol L-10.2 μ L, 10 × PCR do not contain Mg2+Buffer 2 μ L, 25mmol L-1Mg2+1.2 μ L, 10mmol
L-1DNTP 0.5 μ L, 5U μ L-1Taq enzyme 0.2 μ L, ddH2O 14.9μL；
PCR response procedures is: 95 DEG C of denaturations 5min, 95 DEG C of degeneration 30s, and 55 DEG C of annealing 45s, 72 DEG C extend 45s, carry out
33 circulations, last 72 DEG C extend 5min.
(2) using the non-denaturing polyacrylamide gel of 8% to carry out electrophoresis, electrophoretic buffer is that 0.5 × TBE, 200V are steady
Piezoelectricity swimming 2-2.5h, to sample loading buffer move on to bottom gel time electrophoresis terminate.According to amplified production phase on running gel
To position, identical migration position has being designated as " 1 " of band, be designated as " 0 ", according to primer sequential encoding, by obtain without carry
The characteristic fingerprint pattern of finger-print code and above-mentioned Semen Phaseoli kind is compared, and determines Semen Phaseoli kind to be measured.
Embodiment 3 present invention identifies the application of Semen Phaseoli kind method
Extract the DNA of capital agriculture 2 and No. 3 blades of Ji Semen Ormosiae Hosiei.The molecular marker group obtained is screened with the embodiment of the present invention 1
10 pairs of primers in conjunction are to respectively the DNA of Semen Phaseoli kind to be checked being carried out PCR amplification；PCR reaction with reference to the embodiment of the present invention 2
System, PCR response procedures, polyacrylamide gel electrophoresis and the method for bar tape recording, by the finger-print code that obtains with above-mentioned
The characteristic fingerprint pattern of Semen Phaseoli kind is compared, and finds that the finger-print code of capital agriculture 2 is 0110101010011010101, Ji
The finger-print code that Semen Ormosiae Hosiei 3 is 10010110019901100101, with table 2 capital agriculture 2 and the finger-print code of Ji Semen Ormosiae Hosiei 3
Identical, illustrate that the method validation Semen Phaseoli kind accuracy identifying Semen Phaseoli kind applying the embodiment of the present invention 2 to set up is higher, be suitable for
Promote the use of.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. one kind for differentiating the molecular marker combination of Semen Phaseoli kind, it is characterised in that containing 5 SSR molecular marker and 5
EST-SSR molecular marker,
Described 5 SSR molecular marker are obtained by following primer pair amplifies: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ
Primer shown in ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10；
Described 5 EST-SSR molecular markers are obtained by following primer pair amplifies: SEQ ID NO.11-12, SEQ ID
Primer shown in NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20.
2. contain the test kit of molecular marker combination described in claim 1.
3. the molecular marker combination described in claim 1 or test kit the answering in identifying Semen Phaseoli kind described in claim 2
With.
4. the molecular marker combination described in claim 1 or the test kit described in claim 2 refer in structure Semen Phaseoli varietal characteristic
Application in stricture of vagina collection of illustrative plates.
5. a Semen Phaseoli varietal characteristic finger printing, it is characterised in that combine difference with the molecular marker described in claim 1
The DNA of Semen Phaseoli kind carries out PCR amplification, PCR primer is carried out native polyacrylamide gel electrophoresis and detects, and silver staining shows
Color, according to the mobility record electrophoresis result of stranded DNA molecule amount and primer, according to relative on running gel of amplified production
Position, has being designated as " 1 " of band on identical migration position, is designated as " 0 " without carry, sets up SSR genotype information data, according to drawing
Thing order encodes, and the finger-print code obtained is the characteristic fingerprint pattern of Semen Phaseoli kind.
6. Semen Phaseoli varietal characteristic finger printing as claimed in claim 5, it is characterised in that the characteristic fingerprint of 32 kinds of Semen Phaseoli kinds
Collection of illustrative plates is as follows:
7. the method identifying Semen Phaseoli kind, it is characterised in that comprise the steps:
(1) extracting the DNA of Semen Phaseoli kind to be checked, 10 pairs of primers in combining with the molecular marker described in claim 1 are to respectively
The DNA of inspection Semen Phaseoli kind is carried out PCR amplification；
(2) amplified production is detected by native polyacrylamide gel electrophoresis, and silver staining develops the color, according to amplified production at electricity
Relative position on swimming gel, has being designated as " 1 " of band on identical migration position, is designated as " 0 " without carry, enters according to primer order
Row coding, compares the characteristic fingerprint pattern of the finger-print code obtained with the Semen Phaseoli kind described in claim 5 or 6, really
Fixed Semen Phaseoli kind to be measured.
8. method as claimed in claim 7, it is characterised in that the PCR of step (1), its 20 μ L reaction system: DNA profiling 0.8
μ L, upstream and downstream primer each 10pmol L-10.2 μ L, 10 × PCR do not contain Mg2+Buffer 2 μ L, 25mmol L-1Mg2+1.2 μ L,
10mmol L-1DNTP 0.5 μ L, 5U μ L-1Taq enzyme 0.2 μ L, ddH2O 14.9μL；
PCR response procedures is: 95 DEG C of denaturations 5min, 95 DEG C of degeneration 30s, and 51-60 DEG C of annealing 45s, 72 DEG C extend 45s, carry out
32-35 circulation, last 72 DEG C extend 5min.
9. method as claimed in claim 7 or 8, it is characterised in that step (2) uses the non-denaturing polyacrylamide of 8% to coagulate
Glue carries out electrophoresis, and electrophoretic buffer is 0.5 × TBE, 200V voltage stabilizing electrophoresis 2-2.5h, to sample loading buffer move on to bottom gel time
Electrophoresis terminates.
10. described in claim 1 molecular marker combination or claim 2 described in test kit or claim 5 or 6 described in
Semen Phaseoli varietal characteristic finger printing or the application in Germplasm Resource of Vigna Anglaris Ohwi Ohashi is improved of the claim 7-9 arbitrary described method.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610613992.4A CN106191285A (en) | 2016-07-28 | 2016-07-28 | A kind of method and the application that utilize genome SSR and EST SSR finger printing to differentiate Semen Phaseoli kind |
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| CN106480224A (en) * | 2016-12-28 | 2017-03-08 | 中国农业科学院茶叶研究所 | The molecular marker combination of Rapid identification difference albino tea tree breed, method and application |
| CN106636417A (en) * | 2016-12-29 | 2017-05-10 | 中国农业科学院作物科学研究所 | Construction method of pisum sativum SSR (simple sequence repeat) fingerprint |
| CN113795597A (en) * | 2021-02-08 | 2021-12-14 | 中国农业科学院作物科学研究所 | Soybean SNP typing detection chip and application thereof in molecular breeding and basic research |
| CN115011715A (en) * | 2022-04-20 | 2022-09-06 | 黑龙江八一农垦大学 | Fingerprint spectrum for constructing small beans based on SSR molecular markers |
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| HONGLIN CHEN等: ""Development and Validation of EST-SSR Markers from the Transcriptome of Adzuki Bean (Vigna angularis)"", 《PLOS ONE》 * |
| HONGLIN CHEN等: ""Development of SSR markers and assessment of genetic diversity of adzuki bean in the Chinese germplasm collection"", 《MOLECULAR BREEDING》 * |
| 赵波 等: ""基于SSR小豆新品种（系）DNA指纹图谱构建及亲缘分类初探"", 《北京农学院学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106480224A (en) * | 2016-12-28 | 2017-03-08 | 中国农业科学院茶叶研究所 | The molecular marker combination of Rapid identification difference albino tea tree breed, method and application |
| CN106480224B (en) * | 2016-12-28 | 2019-09-24 | 中国农业科学院茶叶研究所 | Molecular labeling combination, method and the application of Rapid identification difference albino tea tree breed |
| CN106636417A (en) * | 2016-12-29 | 2017-05-10 | 中国农业科学院作物科学研究所 | Construction method of pisum sativum SSR (simple sequence repeat) fingerprint |
| CN113795597A (en) * | 2021-02-08 | 2021-12-14 | 中国农业科学院作物科学研究所 | Soybean SNP typing detection chip and application thereof in molecular breeding and basic research |
| WO2022165853A1 (en) * | 2021-02-08 | 2022-08-11 | 中国农业科学院作物科学研究所 | Soybean snp typing detection chip and use thereof in molecular breeding and basic research |
| CN113795597B (en) * | 2021-02-08 | 2023-11-17 | 中国农业科学院作物科学研究所 | Soybean SNP (Single nucleotide polymorphism) typing detection chip and application thereof in molecular breeding and basic research |
| CN115011715A (en) * | 2022-04-20 | 2022-09-06 | 黑龙江八一农垦大学 | Fingerprint spectrum for constructing small beans based on SSR molecular markers |
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