CN107988413A - A kind of method for identifying cucumber variety authenticity and its special SSR primer sets - Google Patents
A kind of method for identifying cucumber variety authenticity and its special SSR primer sets Download PDFInfo
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Abstract
The invention discloses a kind of method for identifying cucumber variety authenticity and its special SSR primer sets.SSR primer sets provided by the present invention are made of 32 primers, and the nucleotide sequences of 32 primers is successively as shown in sequence 1 to sequence 32 in sequence table.SSR primer sets provided by the invention have the advantages that polymorphism is high, reproducible, mark is reliable and stable and easy to count; the present invention has also set up the DNA fingerprint database based on high-flux sequence identification cucumber variety authenticity; available for cucumber variety early stage identification is carried out in seed or Seedling Stage; ensure the authenticity of kind; conscientiously the rights and interests of the protection producer and breeder; and technical support is provided for Cucumber Germplasm and New variety protection, have broad application prospects.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of method of cucumber variety authenticity and its special identified
SSR primer sets.
Background technology
China is that cucumber cultivation area maximum (about 139.6 ten thousand hectares), the highest country of total output (account for the whole world in the world
Yield 70%).Since reform and opening-up, cucumber industry is fast-developing, only by country's authorization, the kind registered just up to more than 300
It is a.Due to breed cucumber, enterprise is small and scattered, and variety managements fail effectively to follow up, and variety certification is mutually uncoordinated between each province and city.
Along with breeding production base managerial confusion, robber educates, kind of illegally buying up phenomenon is rampant, and homonymus or synonymum phenomenon is serious, and seed quality is good
Green bristlegrass is uneven, and seed quality accident happens occasionally.At present, cucumber variety management how is efficiently carried out, protects the producer and breeder
Rights and interests become one of main bugbear for having faced of current cucumber industry development.According to《Non-principal variety of crops registers guide》
Requirement, breediness illustrates with the related character involved in kind DUS test report if any clear and definite associated gene, can be direct
Submit DNA testing results.Therefore, it is badly in need of establishing a set of high throughput DNA fingerprint skill for effectively carrying out cucumber variety authenticity identification
Art system.
SSR (Simple Sequence Repeats) is a kind of single to repeat by several nucleotide (generally 2-6)
The tandem repetitive sequence up to tens nucleotide of position composition, SSR marker have rich polymorphism, reproducible, detection side
The advantages that method is simple.But traditional cucumber SSR marker is without reference to genome mutation group information at present, there are untrue or
Variation situation；In addition being limited to SSR detection modes easily causes untrue, false positive, false negative result, is embodied in following
Problem：1) SSR sources are simple, lack genome and hereditary variation group supports that polymorphism is poor, two bases repeat SSR and easily produce
Draw peak；2) SSR detects amplification region and motif areas there are other SSR, and the interference of Indel/SNP, causes amplified fragments size constant
(false negative) or change；3) traditional SSR detection methods be by gel electrophoresis judge microsatellite marker length, it is necessary to
Reference cultivars are contrasted, so as to add the workload of detection, as a result lack reliable stability and repeatability；4) cannot fit
It should automate, high throughput, the demand of low cost detection.
In recent years, reduced with the continuous development and the continuous of sequencing cost of high throughput sequencing technologies, high-flux sequence with
The amplicon sequencing technologies that multiplex PCR is combined bring new solution for extensive cultivar identification.By to needing to examine
The site design specificity SSR primer sets of survey, different DNA samples is distinguished with different barcode sequences, in single tube
Carry out multiplexed PCR amplification.Deep sequencing is carried out to the amplified production of mixing sample, each site is finally obtained according to sequencing result
The SSR sequence informations of different samples.This method can solve the problems, such as present in above-mentioned tradition SSR detection techniques have very
Wide application prospect.
The content of the invention
The technical problems to be solved by the invention are how to identify cucumber variety.
In order to solve the above technical problems, present invention firstly provides SSR primer sets.The SSR primer sets may include primer pair
M1, primer pair M2, primer pair M3, primer pair M4, primer pair M5, primer pair M6, primer pair M7, primer pair M8, primer pair M9, draw
Thing is to M10, primer pair M11, primer pair M12, primer pair M13, primer pair M14, primer pair M15 and primer pair M16；
The primer pair M1 can be made of primer M1-F and primer M1-R；The primer pair M2 can be by primer M2-F and primer
M2-R is formed；The primer pair M3 can be made of primer M3-F and primer M3-R；The primer pair M4 can be by primer M4-F and drawing
Thing M4-R is formed；The primer pair M5 can be made of primer M5-F and primer M5-R；The primer pair M6 can by primer M6-F and
Primer M6-R is formed；The primer pair M7 can be made of primer M7-F and primer M7-R；The primer pair M8 can be by primer M8-F
Formed with primer M8-R；The primer pair M9 can be made of primer M9-F and primer M9-R；The primer pair M10 can be by primer
M10-F and primer M10-R compositions；The primer pair M11 can be made of primer M11-F and primer M11-R；The primer pair M12
It can be made of primer M12-F and primer M12-R；The primer pair M13 can be made of primer M13-F and primer M13-R；It is described to draw
Thing can be made of M14 primer M14-F and primer M14-R；The primer pair M15 can be by primer M15-F and primer M15-R groups
Into；The primer pair M16 can be made of primer M16-F and primer M16-R.
The primer M1-F can be following A1) or any of A2) single strand dna：
A1) the single strand dna shown in the sequence 1 of sequence table；
A2 single strand dna of the Single-stranded DNA fragments) and A1) limited with more than 85% homogeneity.
The primer M1-R can be following A3) or any of A4) single strand dna：
A3) the single strand dna shown in the sequence 2 of sequence table；
A4 single strand dna of the Single-stranded DNA fragments) and A3) limited with more than 85% homogeneity.
The primer M2-F can be following A5) or any of A6) single strand dna：
A5) the single strand dna shown in the sequence 3 of sequence table；
A6 single strand dna of the Single-stranded DNA fragments) and A5) limited with more than 85% homogeneity.
The primer M2-R can be following A7) or any of A8) single strand dna：
A7) the single strand dna shown in the sequence 4 of sequence table；
A8 single strand dna of the Single-stranded DNA fragments) and A7) limited with more than 85% homogeneity.
The primer M3-F can be following A9) or any of A10) single strand dna：
A9) the single strand dna shown in the sequence 5 of sequence table；
A10 single strand dna of the Single-stranded DNA fragments) and A9) limited with more than 85% homogeneity.
The primer M3-R can be following A11) or any of A12) single strand dna：
A11) the single strand dna shown in the sequence 6 of sequence table；
A12 single strand dna of the Single-stranded DNA fragments) and A11) limited with more than 85% homogeneity.
The primer M4-F can be following A13) or any of A14) single strand dna：
A13) the single strand dna shown in the sequence 7 of sequence table；
A14 single strand dna of the Single-stranded DNA fragments) and A13) limited with more than 85% homogeneity.
The primer M4-R can be following A15) or any of A16) single strand dna：
A15) the single strand dna shown in the sequence 8 of sequence table；
A16 single strand dna of the Single-stranded DNA fragments) and A15) limited with more than 85% homogeneity.
The primer M5-F can be following A17) or any of A18) single strand dna：
A17) the single strand dna shown in the sequence 9 of sequence table；
A18 single strand dna of the Single-stranded DNA fragments) and A17) limited with more than 85% homogeneity.
The primer M5-R can be following A19) or any of A20) single strand dna：
A19) the single strand dna shown in the sequence 10 of sequence table；
A20 single strand dna of the Single-stranded DNA fragments) and A19) limited with more than 85% homogeneity.
The primer M6-F can be following B1) or any of B2) single strand dna：
B1) the single strand dna shown in the sequence 11 of sequence table；
B2 single strand dna of the Single-stranded DNA fragments) and B1) limited with more than 85% homogeneity.
The primer M6-R can be following B3) or any of B4) single strand dna：
B3) the single strand dna shown in the sequence 12 of sequence table；
B4 single strand dna of the Single-stranded DNA fragments) and B3) limited with more than 85% homogeneity.
The primer M7-F can be following B5) or any of B6) single strand dna：
B5) the single strand dna shown in the sequence 13 of sequence table；
B6 single strand dna of the Single-stranded DNA fragments) and B5) limited with more than 85% homogeneity.
The primer M7-R can be following B7) or any of B8) single strand dna：
B7) the single strand dna shown in the sequence 14 of sequence table；
B8 single strand dna of the Single-stranded DNA fragments) and B7) limited with more than 85% homogeneity.
The primer M8-F can be following B9) or any of B10) single strand dna：
B9) the single strand dna shown in the sequence 15 of sequence table；
B10 single strand dna of the Single-stranded DNA fragments) and B9) limited with more than 85% homogeneity.
The primer M8-R can be following B11) or any of B12) single strand dna：
B11) the single strand dna shown in the sequence 16 of sequence table；
B12 single strand dna of the Single-stranded DNA fragments) and B11) limited with more than 85% homogeneity.
The primer M9-F can be following B13) or any of B14) single strand dna：
B13) the single strand dna shown in the sequence 17 of sequence table；
B14 single strand dna of the Single-stranded DNA fragments) and B13) limited with more than 85% homogeneity.
The primer M9-R can be following B15) or any of B16) single strand dna：
B15) the single strand dna shown in the sequence 18 of sequence table；
B16 single strand dna of the Single-stranded DNA fragments) and B15) limited with more than 85% homogeneity.
The primer M10-F can be following B17) or any of B18) single strand dna：
B17) the single strand dna shown in the sequence 19 of sequence table；
B18 single strand dna of the Single-stranded DNA fragments) and B17) limited with more than 85% homogeneity.
The primer M10-R can be following B19) or any of B20) single strand dna：
B19) the single strand dna shown in the sequence 20 of sequence table；
B20 single strand dna of the Single-stranded DNA fragments) and B19) limited with more than 85% homogeneity.
The primer M11-F can be following C1) or any of C2) single strand dna：
C1) the single strand dna shown in the sequence 21 of sequence table；
C2 single strand dna of the Single-stranded DNA fragments) and C1) limited with more than 85% homogeneity.
The primer M11-R can be following C3) or any of C4) single strand dna：
C3) the single strand dna shown in the sequence 22 of sequence table；
C4 single strand dna of the Single-stranded DNA fragments) and C3) limited with more than 85% homogeneity.
The primer M12-F can be following C5) or any of C6) single strand dna：
C5) the single strand dna shown in the sequence 23 of sequence table；
C6 single strand dna of the Single-stranded DNA fragments) and C5) limited with more than 85% homogeneity.
The primer M12-R can be following C7) or any of C8) single strand dna：
C7) the single strand dna shown in the sequence 24 of sequence table；
C8 single strand dna of the Single-stranded DNA fragments) and C7) limited with more than 85% homogeneity.
The primer M13-F can be following C9) or any of C10) single strand dna：
C9) the single strand dna shown in the sequence 25 of sequence table；
C10 single strand dna of the Single-stranded DNA fragments) and C9) limited with more than 85% homogeneity.
The primer M13-R can be following C11) or any of C12) single strand dna：
C11) the single strand dna shown in the sequence 26 of sequence table；
C12 single strand dna of the Single-stranded DNA fragments) and C11) limited with more than 85% homogeneity.
The primer M14-F can be following C13) or any of C14) single strand dna：
C13) the single strand dna shown in the sequence 27 of sequence table；
C14 single strand dna of the Single-stranded DNA fragments) and C13) limited with more than 85% homogeneity.
The primer M14-R can be following C15) or any of C16) single strand dna：
C15) the single strand dna shown in the sequence 28 of sequence table；
C16 single strand dna of the Single-stranded DNA fragments) and C15) limited with more than 85% homogeneity.
The primer M15-F can be following C17) or any of C18) single strand dna：
C17) the single strand dna shown in the sequence 29 of sequence table；
C18 single strand dna of the Single-stranded DNA fragments) and C17) limited with more than 85% homogeneity.
The primer M15-R can be following C19) or any of C20) single strand dna：
C19) the single strand dna shown in the sequence 30 of sequence table；
C20 single strand dna of the Single-stranded DNA fragments) and C19) limited with more than 85% homogeneity.
The primer M16-F can be following D1) or any of D2) single strand dna：
D1) the single strand dna shown in the sequence 31 of sequence table；
D2 single strand dna of the Single-stranded DNA fragments) and D1) limited with more than 85% homogeneity.
The primer M16-R can be following D3) or any of D4) single strand dna：
D3) the single strand dna shown in the sequence 32 of sequence table；
D4 single strand dna of the Single-stranded DNA fragments) and D3) limited with more than 85% homogeneity.
The SSR primer sets specifically can be by the primer pair M1, the primer pair M2, the primer pair M3, the primer
To M4, the primer pair M5, the primer pair M6, the primer pair M7, the primer pair M8, the primer pair M9, described draw
Thing is to M10, the primer pair M11, the primer pair M12, the primer pair M13, the primer pair M14, the primer pair M15
Formed with the primer pair M16.
The application of any of the above-described SSR primer sets falls within protection scope of the present invention.Any of the above-described SSR primers
Any of the application of group can be x1) to x6)：
X1 the kit for being used for identifying cucumber variety) is prepared；
X2 the kit for being used for differentiating cucumber variety true or false) is prepared；
X3 the kit for the genetic relationship for being used to analyze cucumber variety) is prepared；
X4 cucumber variety) is identified；
X5 cucumber variety true or false) is differentiated；
X6 the genetic relationship of cucumber variety) is analyzed.
Kit containing any of the above-described SSR primer sets falls within protection scope of the present invention.
The preparation method of the kit falls within protection scope of the present invention.The preparation method of the kit includes will
The step of each bar primer in any of the above-described SSR primer sets is individually packed.
The application of the kit falls within protection scope of the present invention.The application of the kit can be x4) or x5) or
x6)：
X4 cucumber variety) is identified；
X5 cucumber variety true or false) is differentiated；
X6 the genetic relationship of cucumber variety) is analyzed.
A kind of method identified cucumber to be measured and belong to which kind in 165 cucumber varieties of present invention protection, it may include such as
Lower step：
(1) using the genomic DNA of cucumber to be measured as template, PCR amplification is carried out using any of the above-described SSR primer sets,
Obtain pcr amplification product；Using the genomic DNA of each cucumber variety in standard cucumber variety group as template, using above-mentioned
The one SSR primer sets carry out PCR amplification, obtain pcr amplification product；The standard cucumber variety group is by 165 cucumber varieties
Composition；
(2) pcr amplification product of the pcr amplification product of cucumber to be measured and each standard cucumber variety is subjected to cluster point
Analysis, cucumber to be measured and which standard cucumber variety are same class in cluster analysis, and cucumber to be measured belongs to the standard cucumber variety
In same kind.
The present invention also protects a kind of method for detecting cucumber variety to be measured and whether belonging to the kind in 165 cucumber varieties,
It may include following steps：
(1) using the genomic DNA of cucumber to be measured as template, PCR amplification is carried out using any of the above-described SSR primer sets,
Obtain pcr amplification product；Using the genomic DNA of each cucumber variety in standard cucumber variety group as template, using above-mentioned
The one SSR primer sets carry out PCR amplification, obtain pcr amplification product；The standard cucumber variety group is by 165 cucumber varieties
Composition；
(2) pcr amplification product of cucumber variety to be measured and the pcr amplification product of each standard cucumber variety are compared
Compared with statistical discrepancy number of sites, then makes the following judgment：
If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is more than 2, cucumber variety to be measured
Belong to different cucumber varieties with the standard cucumber variety；Difference number of sites is more, and genetic relationship is more remote；
If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is less than 2, cucumber variety to be measured and
The standard cucumber variety be or it is doubtful be same cucumber variety.
In any of the above-described method, 165 cucumber varieties concretely the northern star of Japan, middle peasant No. 19, strong melon,
Middle peasant No. 15, Zhongnong No.9, CUCUMBER1404, Shen are green 64, Jinyou No.12, Tianjin spring No. 4, Jinyou No.31, Tianjin are No. 2 excellent, Ning Jia 3
Number, No. 6 miscellaneous, miscellaneous No. 2 precious, the peaceful fortune 3 in spring jade, jasper, Shanghai, Jinyou No.36, Tianjin be No. 38 excellent, middle peasant No. 26, MC2065, Tianjin are excellent
20-11, select the beautiful four, winter, No. 11 rich U.S., emerald green, 'Chunhua No.1', Shandong cucumber No. three, C05-016, eastern agriculture 806, the mound an old person having a young heart' a person old in age but young in spirit that pacifies,
Tianjin is No. 48 excellent, what drought, south are No. 1 female, P01, south are No. 2 female, P02, CC3, Ning Feng 09, excellent plus complete female 09, HH1-8-1-2, it is U.S. female 09,
627th, south is No. 3 female, H32, teenage girl, tangshan autumn melon, golden child, capital grind No. 1 mini, capital grind 107, it is green it is emerald green, Tianjin is No. 401 excellent, Tianjin excellent 1
Number, Tianjin is No. 108 excellent, the resistance to H1104 of China, Tianjin are No. 303 excellent, it is blue or green it is beautiful, it is blue or green it is refreshing, successively more, rich U.S. 5032, garden Feng Yuan 6, it is rich U.S. 517,
Rich U.S. No. 28, De Ruite D19, rich No. 10 U.S., longevity dish HG1, longevity dish HG2, DS2121, lucky miscellaneous 9, emerald green jasmine, Tianjin be excellent 35, jasper
No. 2, river is No. 3 emerald green, bright beautiful, eastern agriculture 804, Chang Chun Mi Ci, Jingyanmini No.2, Tianjin are excellent 308, rich No. 8 U.S., rich U.S. 6913,
13AC230, De Ruite 79, De Ruite Y2,13AC049, De Ruite F16, Zhongnong No. 12, Kant (Condesa) RZ F1, Lvchun County
No. two, Lvchun County's No.1, rich No. 5 large, Tianjin is No. 316 excellent, Tianjin is No. 358 excellent, Dong Fangxiu, middle peasant No. 37, middle peasant No. 50, universe moral 1217,
Universe moral 777, rich No. eight large, field is No. seven proud, field is No. eight proud, emerald green dragon, South Korea cucumber, green emerald, the green excellent cucumber of brothers, grain rains are good
Crisp beautiful fragrant, the grain rains U.S. sharp nova of beautiful, excellent 66 cucumber of monarch, Ying Liang, Peking silicite ultrawhite leaf three, De Ruite 10, De Ruite cucumber L14-
2nd, De Ruite cucumber L14-5, De Ruite cucumber GZ1603, middle peasant No. 27, middle peasant No. 8, middle peasant No. 18, newly grind No. four, glossy 3-
6th, close thorn king cucumber, Ya Meite (Thailand), AMATA765, cucumber, green No. seven rich, green rich ride on Bus No. 11, Man Guan, Tang Chun 100, spring
Light beautiful, difficult to understand, capital are rich 298, capital grinds that pickling treasured, fruit type 101 are ground in No. 3 mini, green elegant, capital, drought treasured 5 is ground in capital, Qiu Mei, capital are ground in capital
Grind 106, capital and grind that spring U.S., Beijing 204 are ground in No. 9 mini, capital, Beijing 403, Xia Mei is ground in capital, smart No. 4 No. 5 mini, green, capital is ground in capital
Grind green exquisite No. 2, capital grinds good elegant, the white highest-ranking imperial concubine cucumber of No. 4 mini, northern agriculture, plucks unbeaten open country king, Kingsoft old cucumber, Beijing white jade
3rd, 7-1,10-1,12-1,13,15-1,17,23,27,38,39,40, A21, A24, A28 and A30.
In any of the above-described method, the reaction system of " carrying out PCR amplification using any of the above-described SSR primer sets "
In, concretely 0.2 μM of the concentration of each primer.The reaction interval of " carrying out PCR amplification using any of the above-described SSR primer sets "
Sequence is concretely：95℃3min；95 DEG C of 30s, 60 DEG C of 4min, 17 circulations；72℃4min.
SSR primer sets provided by the invention have that polymorphism is high, reproducible, mark is reliable and stable and easy to count etc. excellent
Point, genetic relationship of the energy maximum limit accuracy reflection for examination cucumber variety.In addition, the present invention is established based on high pass first
The DNA fingerprint database of sequencing identification cucumber variety authenticity is measured, it is early available for being carried out to cucumber variety in seed or Seedling Stage
Phase is identified, ensures the authenticity of kind, protects the rights and interests of the producer and breeder conscientiously, and be Cucumber Germplasm and new varieties
Protection provides technical support.Method provided by the invention can both identify unknown cucumber variety, can also be to known product
The authenticity of kind is identified.Method provided by the invention have high throughput, it is accurate, inexpensive, easy to operate, save manpower,
Material resources etc. a little, have very wide application prospect.
Brief description of the drawings
Fig. 1 is the dendrogram for establishing 165 cucumber varieties in 16 SSR primer pairs.
Fig. 2 is graph of a relation of the SSR primers logarithm with distinguishing 165 cucumber varieties.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used, is normal unless otherwise specified in following embodiments
Rule biochemical reagents shop is commercially available.Quantitative test in following embodiments, is respectively provided with three repeated experiments, is as a result averaged
Value.
The acquisition of embodiment 1, SSR primer sets for identifying cucumber variety authenticity
The present invention represents the heavy sequencing data of resource based on 49 portions of cucumber, develops suitable high-flux sequence and is used to identify Huang
The SSR primer sets of melon variety authentication.This 49 parts of cucumber resource types are enriched, cover North-China Type (7 parts), indica-type (18 parts),
Japanese type (3 parts), South China type (2 parts), European fruit type (5 parts), America processing type (4 parts), Xishuangbanna type (5 parts) and in
Between type (5 parts), include cucumber main ecotype and economical character substantially, embody as much as possible germplasm representativeness,
With higher genetic diversity.
Specifically, SSR screening criterias are as follows：Without SNP, InDel in motif areas；MAF>0.3 and it is evenly distributed on genome
On；The flanking sequence (200bp) being connected with motif areas makes a variation without poly areas, other SSR, SNP and Indel etc..Pass through analysis 49
The sequencing data of part cucumber resource, obtains SSR primer sequence information.Finally, the present inventor is developed by being uniformly distributed
The SSR primer sets of 16 SSR primer pairs composition in cucumber whole gene group, have higher polymorphism information amount (PIC
Value).The nucleotide sequence of this 16 SSR primer pairs is as shown in table 1.
The title and nucleotide sequence of 1. 16 SSR primer pairs of table
The validity check for the SSR primer sets that embodiment 2, embodiment 1 are developed
165 essential informations for examination cucumber variety in the present embodiment are shown in Table 2.165 are made a living for examination cucumber variety
The external introduced variety of common improved seeds or part in production.
2. 165 cucumber variety information of table
1st, for the acquisition for the genomic DNA for trying cucumber variety
Extract the genome of 165 blades (the mixed cotyledon for taking 5 seeds) for examination cucumber variety respectively using CTAB methods
DNA, obtains the genomic DNA for examination cucumber variety.It must meet for the quality and concentration of the genomic DNA of examination cucumber variety
PCR requirements, Passing Criteria are：Agarose electrophoresis shows that DNA bands are single, without obvious disperse；Ultraviolet specrophotometer
Nanodrop2000 (Thermo) detects A260/A280 ratios and is more than 2.0 in 1.8 or so, A260/A230 ratios；For trying cucumber
The concentration of the genomic DNA of kind>50ng/μL.
2nd, the preparation of PanelMix primer mixed liquors
Each bar primer shown in artificial synthesized table 1, then each bar primer is mixed, obtain PanelMix primer mixed liquors.
3rd, the preparation of sequencing library
2.0dsDNA is the product of MolecularDevices companies.Bead suspension is morgene companies
Product.3M enzymes are the product of KAPA companies.Magnetic bead is the product of AMPure companies.
Prepare each sequencing library for examination cucumber variety.Prepare the specific step of each sequencing library for examination cucumber variety
It is rapid as follows：
(1) it is quantitative
Using2.0dsDNA for the genomic DNA of examination cucumber variety to carrying out accurate quantitative analysis.
(2) first round PCR amplification
It is template for trying the genomic DNA of cucumber variety, the PanelMix primer mixed liquors prepared using step 2 are carried out
First round PCR amplification, obtains first round pcr amplification product.
The reaction system of first round PCR amplification is 30 μ L, by 8 μ L Panel Mix primer mixed liquors, 50ng for examination cucumber
The genomic DNA of kind, 10 μ L 3M enzymes and water composition.In the reaction system, the concentration of each primer is 0.2 μM.
Response procedures：95℃3min；95 DEG C of 30s, 60 DEG C of 4min, 17 circulations；72℃4min.
(3) purify
A) centrifuge tube is taken, adds first round pcr amplification product and magnetic bead (volume ratio 2:1), gently blown and beaten with pipettor
Uniformly, it is stored at room temperature 2min；Then centrifuge tube is placed on magnetic frame and be stored at room temperature, collect supernatant.
B) it is another to take a new centrifuge tube, add the supernatant magnetic bead (volume ratio 10 that step a) is collected:7) pipettor, is used
Gently piping and druming is uniform, is stored at room temperature 2min；Then centrifuge tube is placed on magnetic frame and be stored at room temperature, abandon supernatant；Into centrifuge tube
The bead suspension of 30 μ L is added, is resuspended, is stored at room temperature 2min；Finally centrifuge tube is placed on magnetic frame and is stored at room temperature, is abandoned
Clearly.
C) after completing step b), the centrifuge tube is taken, 100 μ L 80% (v/v) ethanol waters is added, centrifuge tube is put
In magnetic frame, magnetic bead is adsorbed on different two sides repeatedly with magnetic frame, magnetic bead is sufficiently washed；Then inhaled with magnetic frame
Attached 2min, abandons supernatant, and room temperature is placed clean to ethanol volatilization.
(4) second wheel PCR amplifications
A) reaction system of the second wheel PCR amplification is prepared.
The reaction system of second wheel PCR amplification is 30 μ L, by the magnetic bead after completion step (3), 10 μ L 3M enzymes, 1 μ L
Primer F aqueous solutions (concentration is 10 μM), 1 μ L primer R aqueous solutions (concentration is 10 μM) and 18 μ L water composition.
Primer F：
5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG-3’；
Primer R：
5’-CAAGCAGAAGACGGCATACGAGAT(underscore is GTGACTGGAGTTCCTTGGCACCCGAGA-3 '
Barcode sequences).
B) after completing step a), the reaction system of the second wheel PCR amplification is subjected to PCR amplification, obtains the second wheel PCR amplification
Product.
Response procedures：95℃3min；95 DEG C of 15s, 58 DEG C of 15s, 72 DEG C of 30s, 7 circulations；72℃4min.
Compared with first round pcr amplification product, the second wheel pcr amplification product with the addition of connector and barcode.Each sample
A corresponding specific barcode, purpose are the follow-up high-flux sequence of progress.
(5) purify
A) centrifuge tube is taken, adds the second wheel pcr amplification product and magnetic bead (volume ratio 1:1), gently blown and beaten with pipettor
Uniformly, it is stored at room temperature 2min；Then centrifuge tube is placed on magnetic frame and be stored at room temperature, abandon supernatant.
B) after completing step a), the centrifuge tube is taken, adds the bead suspension of 30 μ L, is resuspended, is stored at room temperature 2min；Most
Centrifuge tube is placed on magnetic frame afterwards and is stored at room temperature, abandons supernatant.
C) after completing step b), the centrifuge tube is taken, 100 μ L 80% (v/v) ethanol waters is added, centrifuge tube is put
In magnetic frame, magnetic bead is adsorbed on different two sides repeatedly with magnetic frame, magnetic bead is sufficiently washed；Then inhaled with magnetic frame
Attached 2min, abandons supernatant, and room temperature is placed clean to ethanol volatilization.
D) after completing step c), the centrifuge tube is taken, is resuspended with 23 μ L pH8.0-8.5,10mM Tris-HCl buffer solutions
Magnetic bead, is then stored at room temperature 2min.
E) after completing step d), the centrifuge tube is taken, is placed in magnetic separtor 5min, supernatant is transferred to centrifuge tube, is obtained
To the sequencing library for examination cucumber variety.
4th, upper machine sequencing
Each sequencing library for examination cucumber variety, upper machine sequencing are taken respectively.
5th, cluster analysis and SSR finger-prints
The sequencing result obtained according to step 4, obtains 165 for examination cucumber varieties in 16 SSR primer pair amplifies
Motif sequence variations information (being shown in Table 3), then using PowerMarker and MEGA7 softwares to 165 for examination cucumber varieties into
Row cluster analysis.
The essential information of 3. 16 pairs of SSR primers of table
165 dendrograms for examination cucumber variety established in 16 SSR primer pairs are as shown in Figure 1.The result shows that 16
A SSR primer pairs can distinguish 165 confession examination cucumber varieties in table 2 completely.It can be seen from the above that the SSR primers that embodiment 1 is developed
Group can be applied to cucumber variety DNA fingerprint database sharing and Varieties identification.
6th, efficiency rating
Varieties identification can reduce workload using sequential analysis mode.The present inventor compares SSR
Primer logarithm is with distinguishing 165 relations for examination cucumber variety discrimination.Test result indicates that (Fig. 2), 16 SSR primer pairs exist
165 reach 100% for the discrimination in examination cucumber variety.
Whether embodiment 3, detection cucumber variety to be measured belong to 165 methods for the kind in examination cucumber variety
1st, the acquisition of the genomic DNA of cucumber variety to be measured
The blade of cucumber variety to be measured is derived from Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences proving ground.
According to the method for the step 1 in embodiment 2, " cucumber variety to be measured will be replaced with " for the blade of examination cucumber variety "
Blade ", other steps are constant, obtain the genomic DNA of cucumber variety to be measured.
2nd, the preparation of PanelMix primer mixed liquors
With the step 2 in embodiment 2.
3rd, the preparation of sequencing library
According to the method for the step 3 in embodiment 2, " cucumber to be measured will be replaced with " for the genomic DNA of examination cucumber variety "
The genomic DNA of kind ", other steps are constant, obtain the sequencing library of cucumber variety to be measured.
4th, upper machine sequencing
The sequencing library of cucumber variety to be measured is taken, is sequenced.
By the sequencing result of 16 SSR amplified productions of cucumber variety to be measured respectively with 165 for examination cucumber variety (table 2
It is shown) 16 SSR sites contrasted, count two cucumber varieties difference number of sites, then make the following judgment：
If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is more than 2, cucumber variety to be measured
Belong to different cucumber varieties with the standard cucumber variety；Difference number of sites is more, and genetic relationship is more remote；
If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is less than 2, cucumber variety to be measured and
The standard cucumber variety be or it is doubtful be same cucumber variety.
The result shows that cucumber variety to be measured is equal with 165 difference number of sites for examination cucumber variety on 16 SSR sites
For more than 4, therefore, cucumber variety to be measured is not belonging to 165 for any one kind in examination cucumber variety, i.e., cucumber to be measured
Kind is different from 165 for any one kind in examination cucumber variety.
<110>Beijing City Agriculture and Forestry Institute
<120>A kind of method for identifying cucumber variety authenticity and its special SSR primer sets
<160> 32
<170> PatentIn version 3.5
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Claims (9)
1.SSR primer sets, including primer pair M1, primer pair M2, primer pair M3, primer pair M4, primer pair M5, primer pair M6, draw
Thing to M7, primer pair M8, primer pair M9, primer pair M10, primer pair M11, primer pair M12, primer pair M13, primer pair M14, draw
Thing is to M15 and primer pair M16；
The primer pair M1 is made of primer M1-F and primer M1-R；The primer pair M2 is by primer M2-F and primer M2-R groups
Into；The primer pair M3 is made of primer M3-F and primer M3-R；The primer pair M4 is made of primer M4-F and primer M4-R；
The primer pair M5 is made of primer M5-F and primer M5-R；The primer pair M6 is made of primer M6-F and primer M6-R；Institute
Primer pair M7 is stated to be made of primer M7-F and primer M7-R；The primer pair M8 is made of primer M8-F and primer M8-R；It is described
Primer pair M9 is made of primer M9-F and primer M9-R；The primer pair M10 is made of primer M10-F and primer M10-R；It is described
Primer pair M11 is made of primer M11-F and primer M11-R；The primer pair M12 is made of primer M12-F and primer M12-R；
The primer pair M13 is made of primer M13-F and primer M13-R；The primer pair M14 is by primer M14-F and primer M14-R groups
Into；The primer pair M15 is made of primer M15-F and primer M15-R；The primer pair M16 is by primer M16-F and primer M16-
R is formed；
The primer M1-F can be following A1) or any of A2) single strand dna：
A1) the single strand dna shown in the sequence 1 of sequence table；
A2 single strand dna of the Single-stranded DNA fragments) and A1) limited with more than 85% homogeneity；
The primer M1-R can be following A3) or any of A4) single strand dna：
A3) the single strand dna shown in the sequence 2 of sequence table；
A4 single strand dna of the Single-stranded DNA fragments) and A3) limited with more than 85% homogeneity；
The primer M2-F can be following A5) or any of A6) single strand dna：
A5) the single strand dna shown in the sequence 3 of sequence table；
A6 single strand dna of the Single-stranded DNA fragments) and A5) limited with more than 85% homogeneity；
The primer M2-R can be following A7) or any of A8) single strand dna：
A7) the single strand dna shown in the sequence 4 of sequence table；
A8 single strand dna of the Single-stranded DNA fragments) and A7) limited with more than 85% homogeneity；
The primer M3-F can be following A9) or any of A10) single strand dna：
A9) the single strand dna shown in the sequence 5 of sequence table；
A10 single strand dna of the Single-stranded DNA fragments) and A9) limited with more than 85% homogeneity；
The primer M3-R can be following A11) or any of A12) single strand dna：
A11) the single strand dna shown in the sequence 6 of sequence table；
A12 single strand dna of the Single-stranded DNA fragments) and A11) limited with more than 85% homogeneity；
The primer M4-F can be following A13) or any of A14) single strand dna：
A13) the single strand dna shown in the sequence 7 of sequence table；
A14 single strand dna of the Single-stranded DNA fragments) and A13) limited with more than 85% homogeneity；
The primer M4-R can be following A15) or any of A16) single strand dna：
A15) the single strand dna shown in the sequence 8 of sequence table；
A16 single strand dna of the Single-stranded DNA fragments) and A15) limited with more than 85% homogeneity；
The primer M5-F can be following A17) or any of A18) single strand dna：
A17) the single strand dna shown in the sequence 9 of sequence table；
A18 single strand dna of the Single-stranded DNA fragments) and A17) limited with more than 85% homogeneity；
The primer M5-R can be following A19) or any of A20) single strand dna：
A19) the single strand dna shown in the sequence 10 of sequence table；
A20 single strand dna of the Single-stranded DNA fragments) and A19) limited with more than 85% homogeneity；
The primer M6-F can be following B1) or any of B2) single strand dna：
B1) the single strand dna shown in the sequence 11 of sequence table；
B2 single strand dna of the Single-stranded DNA fragments) and B1) limited with more than 85% homogeneity；
The primer M6-R can be following B3) or any of B4) single strand dna：
B3) the single strand dna shown in the sequence 12 of sequence table；
B4 single strand dna of the Single-stranded DNA fragments) and B3) limited with more than 85% homogeneity；
The primer M7-F can be following B5) or any of B6) single strand dna：
B5) the single strand dna shown in the sequence 13 of sequence table；
B6 single strand dna of the Single-stranded DNA fragments) and B5) limited with more than 85% homogeneity；
The primer M7-R can be following B7) or any of B8) single strand dna：
B7) the single strand dna shown in the sequence 14 of sequence table；
B8 single strand dna of the Single-stranded DNA fragments) and B7) limited with more than 85% homogeneity；
The primer M8-F can be following B9) or any of B10) single strand dna：
B9) the single strand dna shown in the sequence 15 of sequence table；
B10 single strand dna of the Single-stranded DNA fragments) and B9) limited with more than 85% homogeneity；
The primer M8-R can be following B11) or any of B12) single strand dna：
B11) the single strand dna shown in the sequence 16 of sequence table；
B12 single strand dna of the Single-stranded DNA fragments) and B11) limited with more than 85% homogeneity；
The primer M9-F can be following B13) or any of B14) single strand dna：
B13) the single strand dna shown in the sequence 17 of sequence table；
B14 single strand dna of the Single-stranded DNA fragments) and B13) limited with more than 85% homogeneity；
The primer M9-R can be following B15) or any of B16) single strand dna：
B15) the single strand dna shown in the sequence 18 of sequence table；
B16 single strand dna of the Single-stranded DNA fragments) and B15) limited with more than 85% homogeneity；
The primer M10-F can be following B17) or any of B18) single strand dna：
B17) the single strand dna shown in the sequence 19 of sequence table；
B18 single strand dna of the Single-stranded DNA fragments) and B17) limited with more than 85% homogeneity；
The primer M10-R can be following B19) or any of B20) single strand dna：
B19) the single strand dna shown in the sequence 20 of sequence table；
B20 single strand dna of the Single-stranded DNA fragments) and B19) limited with more than 85% homogeneity；
The primer M11-F can be following C1) or any of C2) single strand dna：
C1) the single strand dna shown in the sequence 21 of sequence table；
C2 single strand dna of the Single-stranded DNA fragments) and C1) limited with more than 85% homogeneity；
The primer M11-R can be following C3) or any of C4) single strand dna：
C3) the single strand dna shown in the sequence 22 of sequence table；
C4 single strand dna of the Single-stranded DNA fragments) and C3) limited with more than 85% homogeneity；
The primer M12-F can be following C5) or any of C6) single strand dna：
C5) the single strand dna shown in the sequence 23 of sequence table；
C6 single strand dna of the Single-stranded DNA fragments) and C5) limited with more than 85% homogeneity；
The primer M12-R can be following C7) or any of C8) single strand dna：
C7) the single strand dna shown in the sequence 24 of sequence table；
C8 single strand dna of the Single-stranded DNA fragments) and C7) limited with more than 85% homogeneity；
The primer M13-F can be following C9) or any of C10) single strand dna：
C9) the single strand dna shown in the sequence 25 of sequence table；
C10 single strand dna of the Single-stranded DNA fragments) and C9) limited with more than 85% homogeneity；
The primer M13-R can be following C11) or any of C12) single strand dna：
C11) the single strand dna shown in the sequence 26 of sequence table；
C12 single strand dna of the Single-stranded DNA fragments) and C11) limited with more than 85% homogeneity；
The primer M14-F can be following C13) or any of C14) single strand dna：
C13) the single strand dna shown in the sequence 27 of sequence table；
C14 single strand dna of the Single-stranded DNA fragments) and C13) limited with more than 85% homogeneity；
The primer M14-R can be following C15) or any of C16) single strand dna：
C15) the single strand dna shown in the sequence 28 of sequence table；
C16 single strand dna of the Single-stranded DNA fragments) and C15) limited with more than 85% homogeneity；
The primer M15-F can be following C17) or any of C18) single strand dna：
C17) the single strand dna shown in the sequence 29 of sequence table；
C18 single strand dna of the Single-stranded DNA fragments) and C17) limited with more than 85% homogeneity；
The primer M15-R can be following C19) or any of C20) single strand dna：
C19) the single strand dna shown in the sequence 30 of sequence table；
C20 single strand dna of the Single-stranded DNA fragments) and C19) limited with more than 85% homogeneity；
The primer M16-F can be following D1) or any of D2) single strand dna：
D1) the single strand dna shown in the sequence 31 of sequence table；
D2 single strand dna of the Single-stranded DNA fragments) and D1) limited with more than 85% homogeneity；
The primer M16-R can be following D3) or any of D4) single strand dna：
D3) the single strand dna shown in the sequence 32 of sequence table；
D4 single strand dna of the Single-stranded DNA fragments) and D3) limited with more than 85% homogeneity.
2. SSR primer sets as claimed in claim 1, it is characterised in that：
The SSR primer sets by the primer pair M1, the primer pair M2, the primer pair M3, the primer pair M4, described draw
Thing is to M5, the primer pair M6, the primer pair M7, the primer pair M8, the primer pair M9, the primer pair M10, described
Primer pair M11, the primer pair M12, the primer pair M13, the primer pair M14, the primer pair M15 and the primer pair
M16 is formed.
Any of 3. the application of the SSR primer sets of claim 1 or 2, is x1) to x6)：
X1 the kit for being used for identifying cucumber variety) is prepared；
X2 the kit for being used for differentiating cucumber variety true or false) is prepared；
X3 the kit for the genetic relationship for being used to analyze cucumber variety) is prepared；
X4 cucumber variety) is identified；
X5 cucumber variety true or false) is differentiated；
X6 the genetic relationship of cucumber variety) is analyzed.
4. the kit containing the SSR primer sets of claim 1 or 2.
5. the preparation method of kit described in claim 4, including each bar in the SSR primer sets of claim 1 or 2 is drawn
The step of thing is individually packed.
6. the application of kit described in claim 4, is x4) or x5) or x6)：
X4 cucumber variety) is identified；
X5 cucumber variety true or false) is differentiated；
X6 the genetic relationship of cucumber variety) is analyzed.
7. a kind of method identified cucumber to be measured and belong to which kind in 165 cucumber varieties, includes the following steps：
(1) using the genomic DNA of cucumber to be measured as template, PCR amplification is carried out using SSR primer sets described in claim 1, is obtained
Pcr amplification product；Using the genomic DNA of each cucumber variety in standard cucumber variety group as template, using claim 1 institute
State SSR primer sets and carry out PCR amplification, obtain pcr amplification product；The standard cucumber variety group is made of 165 cucumber varieties；
(2) pcr amplification product of the pcr amplification product of cucumber to be measured and each standard cucumber variety is subjected to cluster analysis, gathered
Cucumber to be measured and which standard cucumber variety are same class in alanysis, and cucumber to be measured belongs to same with the standard cucumber variety
A kind.
8. a kind of method for detecting cucumber variety to be measured and whether belonging to the kind in 165 cucumber varieties, includes the following steps：
(1) using the genomic DNA of cucumber to be measured as template, PCR amplification is carried out using SSR primer sets described in claim 1, is obtained
Pcr amplification product；Using the genomic DNA of each cucumber variety in standard cucumber variety group as template, using claim 1 institute
State SSR primer sets and carry out PCR amplification, obtain pcr amplification product；The standard cucumber variety group is made of 165 cucumber varieties；
(2) by the pcr amplification product of cucumber variety to be measured compared with the pcr amplification product of each standard cucumber variety, system
Difference number of sites is counted, is then made the following judgment：
If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is more than 2, cucumber variety to be measured and should
Standard cucumber variety belongs to different cucumber varieties；Difference number of sites is more, and genetic relationship is more remote；
If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is less than 2, cucumber variety to be measured and the mark
Quasi- cucumber variety be or it is doubtful be same cucumber variety.
9. method as claimed in claim 7 or 8, it is characterised in that：165 cucumber varieties are the northern star of Japan, middle peasant 19
Number, strong melon, middle peasant No. 15, Zhongnong No.9, CUCUMBER1404, Shen be green 64, Jinyou No.12, Tianjin spring No. 4, Jinyou No.31, Tianjin excellent 2
Number, No. 6 miscellaneous, miscellaneous No. 2 precious, the peaceful fortune 3 in Ning Jia 3, spring jade, jasper, Shanghai, Jinyou No.36, Tianjin be No. 38 excellent, middle peasant No. 26,
MC2065, the excellent 20-11 in Tianjin, select the beautiful four, winter, No. 11 rich U.S., emerald green, 'Chunhua No.1', Shandong cucumber No. three, C05-016, eastern agriculture 806,
Pacify No. 2 female No. 1 female No. 48 excellent mound an old person having a young heart' a person old in age but young in spirit, Tianjin, what drought, south, P01, south, P02, CC3, Ning Feng 09, excellent plus complete female 09, HH1-8-
1-2, it is U.S. female 09,627, south is No. 3 female, H32, teenage girl, tangshan autumn melon, golden child, capital grind No. 1 mini, capital grind 107, it is green it is emerald green, Tianjin is excellent
No. 401, Tianjin is No. 1 excellent, Tianjin is No. 108 excellent, the resistance to H1104 of China, Tianjin are No. 303 excellent, it is blue or green it is beautiful, it is blue or green it is refreshing, successively more, rich U.S. 5032, garden Feng Yuan 6
Number, rich U.S. 517, rich No. 28 U.S., De Ruite D19, rich No. 10 U.S., longevity dish HG1, longevity dish HG2, DS2121, lucky miscellaneous 9, emerald green jasmine,
Tianjin is excellent 35, jasper 2, river are No. 3 emerald green, bright beautiful, eastern agriculture 804, Chang Chun Mi Ci, Jingyanmini No.2, Tianjin are excellent 308, rich U.S. No. 8, Bo Mei
6913rd, 13AC230, De Ruite 79, De Ruite Y2,13AC049, De Ruite F16, Zhongnong No. 12, Kant (Condesa) RZF1,
Lvchun County two, Lvchun County's No.1, rich No. 5 large, Tianjin is No. 316 excellent, Tianjin is No. 358 excellent, Dong Fangxiu, middle peasant No. 37, middle peasant No. 50, universe moral
1217th, universe moral 777, rich No. eight large, field is No. seven proud, field is No. eight proud, emerald green dragon, South Korea cucumber, green emerald, the green excellent cucumber of brothers, paddy
Rain beauty, excellent 66 cucumber of monarch, crisp beautiful fragrant, the grain rains U.S. sharp novas of Ying Liang, Peking silicite ultrawhite leaf three, De Ruite 10, De Ruite cucumber
L14-2, De Ruite cucumber L14-5, De Ruite cucumber GZ1603, middle peasant No. 27, middle peasant No. 8, middle peasant No. 18, new grind No. four, oil
Bright 3-6, close thorn king cucumber, Ya Meite (Thailand), AMATA765, cucumber, green No. seven rich, green rich ride on Bus No. 11, Man Guan, Tang Chun 100,
Spring is beautiful, light difficult to understand, capital are rich 298, capital grind No. 3 mini, green elegant, capital grind pickling treasured, fruit type 101, drought treasured 5 is ground in capital, Qiu Mei is ground in capital,
Capital grind 106, capital grind spring U.S. is ground in No. 9 mini, capital, Xia Mei is ground in Beijing 204, Beijing 403, capital, capital grind it is smart No. 4 No. 5 mini, green,
Capital grinds green exquisite No. 2, good elegant, the white highest-ranking imperial concubine cucumber of No. 4 mini, northern agriculture is ground in capital, to pluck unbeaten open country king, Kingsoft old cucumber, Beijing white
Jade three, 7-1,10-1,12-1,13,15-1,17,23,27,38,39,40, A21, A24, A28 and A30.
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