CN106350591B - Construct SSR primer, constructed finger-print and its application of coloured silk Ye Luanshu Variety fingerprinting - Google Patents
Construct SSR primer, constructed finger-print and its application of coloured silk Ye Luanshu Variety fingerprinting Download PDFInfo
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Abstract
The invention discloses SSR primer, constructed finger-print and its applications of building coloured silk Ye Luanshu Variety fingerprinting.The present invention obtains 96 pairs of SSR primers according to Luan Shu transcript profile design data, is further screened out from it the special finger-print that 13 pairs of primers that difference is relatively stable, polymorphism is good are used to construct color Ye Luanshu kind.The present invention further discloses the methods for utilizing screened SSR primer to construct coloured silk Ye Luanshu Variety fingerprinting, comprising: extracts Luan Shu sample DNA as amplification template, carries out PCR amplification with the SSR primer；Amplified production is subjected to Capillary Electrophoresis, primer alpha code is formed according to amplification of the Capillary Electrophoresis result to color Ye Luanshu kind sample DNA, the banding pattern of amplified band length number is added to number, obtain the finger-print of each kind.The screening varieties of SSR primer pair Luan Shu platymiscium of the present invention or identification and the breeding of fine tree species etc. have important application value.
Description
Technical field
The present invention relates to SSR primers, more particularly, to construct the SSR primer of coloured silk Ye Luanshu Variety fingerprinting, this hair
The bright application for further relating to the SSR primer in building coloured silk Ye Luanshu Variety fingerprinting, belongs to color Ye Luanshu varieties systematics figure
The building field of spectrum.
Background technique
Simple sequence repeat marker technology is for molecular labeling skill designed by simple repeated sequence in genomic DNA
Art.Repetitive sequence includes two types in genomic DNA: distributed and tandem type, wherein SSR marker is primarily directed to connect
Microsatellite DNA in repetitive sequence, therefore the microsatellite DNA mark that is otherwise known as.Since microsatellite DNA exists in different plants
Genomic locations are different, number of repetition is different, it is different to repeat different genotype number of repetition in degree difference and same species
Etc. reasons, cause SSR marker polymorphism with higher, while two terminal sequence of SSR conservative with higher again, therefore pass through
The detection of SSR molecular marker polymorphism can quickly be carried out by designing suitable primer.
SSR molecular marker above has a variety of advantages in application, its quantity is abundant, covers whole gene group, it is possible to authenticate
Homozygote and heterozygote out, repeatability is high, is provided simultaneously with the characteristic of multiple alleles, it is more to provide hereditary information amount, and due to two
The conservative of terminal sequence, primer has versatility in genotypical individual of the same race and different；Its polymorphism information amount simultaneously
Also it is much higher than other kinds of molecular labeling.SSR molecular marker technology using very extensive, in genetic diversity, positioning
Gene, map construction and cultivar identification etc. suffer from significant application value.With researchers constantly innovate with it is complete
It is kind, a variety of labelling techniques are derived again by SSR, wherein being most widely used with ISSR and EST-SSR.ISSR labelling technique is
By a kind of New molecular marker technology of the propositions such as Canadian scholar Zietkiewicz, the several bases in SSR sequence both ends are applied
As anchor series, there is the sequence of reversed SSR arrangement to expand its two sides, taken into account a variety of labels such as SSR, RAPD
Advantage, at the same it is easy to operate, it is at low cost, therefore liked deeply by numerous researchers, it is applied to germplasm identification, fingerprint image more
The building of spectrum, the research of kinship and assistant breeding etc..EST-SSR is the SSR marker technology developed in est sequence,
Since est sequence conservative is high compared with non-coding series, EST-SSR is for SSR marker, and repeatability is more preferable, more surely
It is fixed, and the application in medicinal plant, vegetables and fruit is all successfully made practice.Equally in the breeding process of industrial crops
In, EST-SSR label also have high application value, wheat, rice, cotton and millet analysis of genetic diversity and
In cultivar identification screening process, corresponding SSR marker has all played important function.
Luan Shu platymiscium is deciduous tree, and 4 kinds are shared on taxology, wherein three kinds are Chinese native tree species, respectively
Luan Shu (Koelreuteria paniculata Laxm.), Taiwan Luan Shu (K.formosana Hayta.) and bougainvillea goldraintree root or flower
(K.Bipinnata Franch.).It in addition to this, is koelreuteria integrifoliola tree there are also a mutation in bougainvillea goldraintree root or flower
(K.integrifolia Merr.) has a bud to connect mutation as bright and beautiful leaf goldenrain tree in Luan Shuzhong.Luan Shu is main in Luan Shu platymiscium
Representative plant, compared with other three kinds of Luan Shu platymisciums, it does not have complete two times pinnate compound leaves, and in leaflet top edge
On have crenation, nearly base portion tooth then normal alienation and in deep shape of incising；Capsule is also pushed up with other three kinds of differences in cone
End has the tendency that tapering.Due to itself resistance of Luan Shu, adaptability is good, therefore Luan Shu has extremely in afforestation application
Important role.The bud of bright and beautiful Ye Luanwei Luan Shu connects mutation, and physiologic character is similar with common Luan Shu, its leaf color is special, is
Golden yellow, the stem with unique red, red arteries and veins and red tender tip, the more common Luan Shugeng high of ornamental value have more
Good Landscape Tree Species characteristic.Koelreuteria integrifoliola tree, also known as Mount Huang goldenrain tree are a mutation of bougainvillea goldraintree root or flower, compared with Luan Shu, tool
There are complete two times pinnate compound leaves, fruit is ellipse, wealthy oval or subsphaeroidal, nose-circle or blunt；With bougainvillea goldraintree root or flower phase
Than the leaflet that, difference is mainly reflected in it is usually that the nearly top of full edge or side slightly has sawtooth, and the small limb of bougainvillea goldraintree root or flower
Edge then usually has slightly close, interior curved small sawtooth.In addition to this, have identical physiology with bougainvillea goldraintree root or flower and outside is special
Property, because its rapid, anti-flue dust of growth, can three season views the characteristics of, usually cultivated in flower garden and trade for ornamental, timber
Can furniture processed, seed can be used as the material of oily industry；Its root and flower also have certain medical value simultaneously.
The molecular labeling about Luan Shu platymiscium is developed, the cultivar identification screening and fine tree species to Luan Shu platymiscium
Breeding all has significance, will play extremely important effect for the correlative study of goldenrain tree Pterostyrax plant field.
Summary of the invention
First technical problem to be solved by this invention is to provide the SSR for constructing color Ye Luanshu Variety fingerprinting
Primer；
Another technical problem to be solved by this invention is that the SSR primer is applied to building coloured silk Ye Luanshu kind to refer to
Line map and be applied to cultivar identification.
In order to solve the above technical problems, the technical solution used in the present invention is:
The present invention discloses the SSR primer for constructing color Ye Luanshu Variety fingerprinting first, draws selected from following 13 Duis
Any pair or multipair in object:
Primer pair 1: downstream shown in upstream primer shown in SEQ ID No.1 and SEQ ID No.2 is classified as nucleotides sequence and is drawn
Object composition；Primer pair 2: downstream primer shown in upstream primer shown in SEQ ID No.3 and SEQ ID No.4 is classified as nucleotides sequence
Composition；Primer pair 3: downstream primer group shown in upstream primer shown in SEQ ID No.5 and SEQ ID No.6 is classified as nucleotides sequence
At；Primer pair 4: downstream primer group shown in upstream primer shown in SEQ ID No.7 and SEQ ID No.8 is classified as nucleotides sequence
At；Primer pair 5: downstream primer group shown in upstream primer shown in SEQ ID No.9 and SEQ ID No.10 is classified as nucleotides sequence
At；Primer pair 6: downstream primer group shown in upstream primer shown in SEQ ID No.11 and SEQ ID No.12 is classified as nucleotides sequence
At；Primer pair 7: downstream primer group shown in upstream primer shown in SEQ ID No.13 and SEQ ID No.14 is classified as nucleotides sequence
At；Primer pair 8: downstream primer group shown in upstream primer shown in SEQ ID No.15 and SEQ ID No.16 is classified as nucleotides sequence
At；Primer pair 9: downstream primer group shown in upstream primer shown in SEQ ID No.17 and SEQ ID No.18 is classified as nucleotides sequence
At；Primer pair 10: downstream primer shown in upstream primer shown in SEQ ID No.19 and SEQ ID No.20 is classified as nucleotides sequence
Composition；Primer pair 11: downstream shown in upstream primer shown in SEQ ID No.21 and SEQ ID No.22 is classified as nucleotides sequence and is drawn
Object composition；Primer pair 12: downstream shown in upstream primer shown in SEQ ID No.23 and SEQ ID No.24 is classified as nucleotides sequence
Primer composition；Primer pair 13: under being classified as shown in upstream primer shown in SEQ ID No.25 and SEQ ID No.26 as nucleotides sequence
Swim primer composition.
Preferably, the present invention is used to construct the SSR primer of color Ye Luanshu Variety fingerprinting, including following 7 pairs of primers: drawing
Object is to 2: being classified as downstream primer shown in upstream primer shown in SEQ ID No.3 and SEQ ID No.4 by nucleotides sequence and forms；Primer
To 3: being classified as downstream primer shown in upstream primer shown in SEQ ID No.5 and SEQ ID No.6 by nucleotides sequence and form；Primer pair
4: downstream primer shown in upstream primer shown in SEQ ID No.7 and SEQ ID No.8 being classified as by nucleotides sequence and is formed；Primer pair 6:
Downstream primer shown in upstream primer shown in SEQ ID No.11 and SEQ ID No.12 is classified as by nucleotides sequence to form；Primer pair 8:
Downstream primer shown in upstream primer shown in SEQ ID No.15 and SEQ ID No.16 is classified as by nucleotides sequence to form；Primer pair 9:
Downstream primer shown in upstream primer shown in SEQ ID No.17 and SEQ ID No.18 is classified as by nucleotides sequence to form；And primer pair
12: downstream primer shown in upstream primer shown in SEQ ID No.23 and SEQ ID No.24 being classified as by nucleotides sequence and is formed.
Coloured silk Ye Luanshu kind of the present invention is selected from bright and beautiful leaf goldenrain tree, Shanxi goldenrain tree No. 1, Shanxi goldenrain tree No. 2 or Shanxi goldenrain tree No. 3.
The present invention has carried out the design of SSR primer according to Luan Shu transcript profile data, and according to the stability of design primer, infiltration
The Tm value difference of permeability, the size of product and front and back primer is different to have carried out preliminary screening, obtains 96 pairs of primers.The present invention utilizes four
A sample (Luan Shu sample B 1, B2；Koelreuteria integrifoliola tree sample B 3, B4) annealing temperature of 96 pairs of designed primers and amplification are imitated
Fruit has carried out preliminary screening, as a result has 92 pairs of primer amplifications to go out clear band in 96 pairs of primers, wherein 27 pairs are with difference
The primer of effect occupies 28% ratio in total primer, it was demonstrated that SSR primer of the present invention has certain polymorphism.
The present invention further by six samples (four mutational variety brocade leaf goldenrain trees of Luan Shu, Shanxi goldenrain tree No. 1, Shanxi goldenrain tree No. 2 and
Shanxi goldenrain tree No. 3；2 Luan Shu samples are control) carry out SSR amplified reaction, to the special SSR markers of four mutational varieties of Luan Shu into
Row screening.In results of preliminary screening, 25 pairs of primers are shared in 96 pairs of primers has doubtful inconsistent expansion in six samples
Synergy fruit, the form of expression is mainly by band length is inconsistent and part sample is to embody in the form of two kinds of two close bands etc..According to
Agarose gel electrophoresis as a result, the present invention from it is above-mentioned to Luan Shu four strains have doubtful differential amplification effect primer in select
13 pairs of primers that difference is relatively stable, polymorphism is good are selected, for constructing the special finger-print of color Ye Luanshu four strains.
The present invention further discloses application of the SSR primer in building coloured silk Ye Luanshu Variety fingerprinting.
The invention also discloses application of the SSR primer in color Ye Luanshu cultivar identification.
The construction method of coloured silk Ye Luanshu Variety fingerprinting of the invention, comprising the following steps: (1) extract coloured silk Ye Luanshu sample
DNA；(2) it using the DNA of extraction as template, using the SSR primer as amplimer, establishes PCR reaction system and carries out PCR expansion
Increase；(3) Capillary Electrophoresis is carried out to amplified production, according to Capillary Electrophoresis result to the amplification of color Ye Luanshu sample DNA
Forming primer alpha code adds the banding pattern of amplified band length number to number the finger-print to get each coloured silk Ye Luanshu kind.
If a coloured silk Ye Luanshu sample is passed through a plurality of band occurs with a pair of of primer amplification simultaneously, foundation molecular weight is from small
Connect number with "-" to big sequence (molecular weight unit bp, omits unit herein, only records number).
Wherein, it is 25 μ L that the PCR reaction system, which includes: the total volume of reaction system, wherein 2 × PCR MIX, 12.5 μ
L, 2 μ l of DNA profiling, 2 μ l of upstream primer, downstream primer 0.5 μ l, ddH25 μ l of O3 μ l, M13 albumen.The program of the PCR amplification
It include: 94 DEG C, 3min → 94 DEG C, 30s → (+1 DEG C of the annealing temperature of each pair of primer), 30s → 72 DEG C, 40s → 94 DEG C, 30s →
(+0.5 DEG C of the annealing temperature of each pair of primer), 30s → 72 DEG C, 40s → [94 DEG C, 30s → (annealing temperature of each pair of primer), 30s
→ 72 DEG C, 40s]x35→ 72 DEG C, 7min → 4 DEG C, ∞.Wherein, in upstream primer 5, ' end addition nucleotides sequence is classified as SEQ ID
The sequence of M13 albumen shown in No.27, then carries out PCR amplification again.The annealing temperature of each pair of primer are as follows: the annealing of primer pair 1
Temperature is 53 DEG C；The annealing temperature of primer pair 2 is 52 DEG C；The annealing temperature of primer pair 3 is 55 DEG C；The annealing temperature of primer pair 4
It is 54 DEG C；The annealing temperature of primer pair 5 is 56 DEG C；The annealing temperature of primer pair 6 is 54 DEG C；The annealing temperature of primer pair 7 is 52
℃；The annealing temperature of primer pair 8 is 55 DEG C；The annealing temperature of primer pair 9 is 55 DEG C；The annealing temperature of primer pair 10 is 55 DEG C；
The annealing temperature of primer pair 11 is 53 DEG C；The annealing temperature of primer pair 12 is 53 DEG C；The annealing temperature of primer pair 13 is 52 DEG C.
13 pairs of primers that the present invention is relatively stable by the difference screened, polymorphism is good, use capital letter in a fixed order
Mother number, primer pair 1-13 number be followed successively by A, B, C ... M, by Capillary Electrophoresis as a result, recording every part of coloured silk Ye Luanshu sample
The band that product occur through different SSR primer pair amplifies, according to the length of length each band of journal from small to large, (unit is
Bp omits unit bp herein, only records number)；Occur simultaneously if a coloured silk Ye Luanshu sample is passed through with a pair of of primer amplification
A plurality of band then connects number with "-".The DNA of each coloured silk Ye Luanshu sample will will form after different primers expand by letter
With a series of banding patterns number of Arabic numerals composition, the SSR finger-print of the kind is just formd.
The invention also discloses the color Ye Luanshu Variety fingerprintings that the construction method constructs.
Wherein, the finger-print of the bright and beautiful leaf goldenrain tree are as follows: C243-249D291-293F302H241I232L256；The Shanxi goldenrain tree
No. 1 finger-print are as follows: B128-194C249D291-293F302H247I232L256；The finger-print of the Shanxi goldenrain tree No. 2 are as follows:
B128-134-194-203C243-247D291-293F302H247-256I232L256；The finger-print of the Shanxi goldenrain tree No. 3 are as follows:
B128-134-194-203C249D293F302H241-256I232L256.Wherein, B is the alpha code of primer pair 2, and C is to draw
Object is to 3 alpha code, and D is the alpha code of primer pair 4, and F is the alpha code of primer pair 6, and H is that the letter of primer pair 8 is compiled
Number, I is the alpha code of primer pair 9, and L is the alpha code of primer pair 12；Number is the molecular weight of amplified band.Such as bright and beautiful leaf
C243-249 in the finger-print of goldenrain tree, then it represents that bright and beautiful Ye Luanjing C primer (i.e. primer pair 3) amplification length occurs and is respectively
2 band of 243bp and 249bp.
The invention also discloses a kind of kits for color Ye Luanshu cultivar identification, comprising: PCR amplification primer, 2 ×
PCR MIX and ddH2O, wherein the pcr amplification primer object includes: primer pair 1- primer pair 13；Preferably, including primer pair 2, draw
Object is to 3, primer pair 4, primer pair 6, primer pair 8, primer pair 9 and primer pair 12.Kit of the present invention can be used in color leaf
Luan Shu kind brocade leaf goldenrain tree, Shanxi goldenrain tree No. 1, Shanxi goldenrain tree No. 2 and Shanxi goldenrain tree No. 3 precise Identifications.
Technical solution of the present invention compared with prior art, has the advantages that
The present invention has carried out the exploitation of Luan Shu platymiscium SSR marker using transcript profile data.The present invention is from designed 96
To 13 pairs of primers that difference is relatively stable, polymorphism is good are filtered out in SSR primer, the special of four coloured silk Ye Luanshu kinds is constructed
Finger-print lays a good foundation the popularization for the Luan Shu kind for having important ornamental value for four and planning.The present invention
The SSR primer and have with the finger-print constructed by it to breeding of screening varieties and fine tree species of Luan Shu platymiscium etc.
There is important value.
Detailed description of the invention
Fig. 1 is the effect that part primer amplifies in six samples of Luan Shu；Wherein, (a) is 11,17,27,29 pairs of primer
The amplification of six samples of Luan Shu, wherein No. 17 primers amplify two similar bands in tri- samples of B2, A1, A2,
Single slice is amplified in remaining sample；It (b) is the amplification of 13,16,26,36 pairs of six samples of Luan Shu of primer, wherein drawing
Object 13 amplifies double bands in six samples, but band brightness is not obvious；It (c) is 1,23,24,30 couple of Luan Shu of primer 2
The amplification of six samples, wherein primer 21 amplified band and remaining sample in A2, A4 sample has nuance, primer 30
Amplified band and remaining sample have nuance in sample A3；It (d) is primer 48,67,73,81 expansion pair six samples of Luan Shu
Increase as a result, wherein No. 67 primers amplified band and remaining sample in two samples of A3, A4 has nuance；Be (e) primer 56,
59, the amplification of 60,61 couples of Luan Shu, six samples, wherein primer 60 amplifies two phases in tri- samples of B2, A2, A5
Nearly band, remaining sample amplify single slice；It (f) is the amplification of 70,82,83,87 pairs of six samples of Luan Shu of primer, wherein
Primer 70,87 does not amplify clear band, and primer 82 amplifies double bands in B2 sample, amplified in A1 sample and other
The inconsistent single slice of sample size；(g) be 71,74,75,85 pairs of six samples of Luan Shu of primer amplification, wherein primer
71 amplify the single slice inconsistent with other samples in B2, A1 sample, and primer 74 amplifies two approximations in A5 sample
Band amplifies single slice in remaining sample；
Fig. 2 is semi-capillary electrophoresis result figure (Gel Image)；Wherein, (a), (b), (c) are respectively via software
The Capillary Electrophoresis glue figure of primer 17,48,92 is shown as a result, ordinate is that (1-7 is successively for sample serial number after GeneMarker processing
For sample B 1, B2, A1, A2, A3, A4, A5), abscissa is band length, is the band of amplification difference in box；
Fig. 3 is semi-capillary electrophoresis result figure；Wherein, (a), (b), (c) be respectively via software GeneMarker at
The Capillary Electrophoresis peak figure of primer 17,48,92 shows result after reason；Ordinate represents the expanding effect of primer in the sample,
Abscissa represents the length of amplified band；Sample1-7 is followed successively by sample B 1, B2, A1, A2, A3, A4, A5；
Fig. 4 is semi-capillary electrophoresis result figure；Wherein, (a), (b), (c), (d) are respectively via software
The Capillary Electrophoresis peak figure of primer 13,23,69,71 shows result after GeneMarker processing；Ordinate represents primer in sample
Expanding effect in this, abscissa represent the length of amplified band；Sample 1-7 be followed successively by sample B 1, B2, A1, A2, A3,
A4、A5。
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
Embodiment 1 is used to construct the SSR design of primers and screening of Luan Shu finger-print
1, materials and methods
1.1 experimental material
Experimental material and its source are shown in Table 1, and bright and beautiful Ye Luanwei definite designation divides kind, therefore does not list scientific name.It is acquired for materials equal
Using the young leaflet tablet of current-year branch.
1 experimental material of table and source
The bud of bright and beautiful Ye Luanwei Luan Shu connects mutation；Shanxi goldenrain tree No. 1, No. 2, No. 3 are the seedling from seed breeding product of bright and beautiful leaf goldenrain tree
Kind, each have from Luan Shu and the different phenotypic character of brocade leaf goldenrain tree, be mainly manifested in leaf color, leaf blade size and tender
In terms of stem color, specific phenotypic difference is as shown in table 2.
The comparison of 2 Luan Shu specific breeds of table
1.2 experimental method
1.2.1 the extraction of genomic DNA
Source and physiological property for material: water planting in laboratory, water intaking training branch are taken for the material of Beijing area
New young leaflet tablet is that (note: average indoor temperature is 28 DEG C to experimental material between experiment periods, and day and night temperature is 10 DEG C -12 on item
℃)；For the experimental material of Shanxi area, current year leave piece is taken on the spot, is stored in liquid nitrogen and takes back；Take the fresh blade of Luan Shu
About 100mg takes the CTAB extraction method of improvement to carry out DNA extraction to Luan Shu sample after addition liquid nitrogen is fully ground.
1.2.2SSR-PCR amplification
1.2.2.1 design of primers
The design of SSR primer of the present invention screens the site SSR of species, according to site two sides using building genomic library
Sequence carry out design primer, carried out SSR primer by the Luan Shu transcript profile data that laboratory where the present inventor before obtains
Design, after being handled by QDD software transcript profile data, according to the stability of design primer, permeability, the size of product
And front and back primer Tm value difference it is different carried out preliminary screening, obtain and synthesized 96 pairs of primers, primer concrete condition is shown in Table 3.
Since the source of design primer of the present invention is transcript profile data, therefore SSR marker used belongs to EST-SSR label.
Table 3SSR primer
1.2.2.2 primer screening
For 96 pairs of synthesized primers, the present invention the experiment initial period using four sample DNAs (B1, B2, B3 and
B4 preliminary experiment) has been carried out, three gradients are then taken for the annealing temperature of each pair of primer, with the mean value of its front and back primer Tm
It is each up and down to float 1 DEG C for intermediate degree, it is expected that 96 pairs of more suitable annealing temperatures of primer can be obtained, and it is verified with good
Polymorphism.
1.2.2.3SSR-PCR the optimization of reaction system
One suitable SSR-PCR reaction system is essential for the acquisition of good experimental result.According to this
The existing experiment basis in inventor laboratory, the PCR reaction system taken are selected in 20 μ l and 50 μ l reaction systems,
Specific reaction system is shown in Table 4.
Table 4SSR-PCR reaction system
SSR-PCR response procedures are set as two parts, and first part is on the basis of 1 DEG C of suitable annealing temperature floating, often
A circulation is successively decreased 0.5 DEG C, and 3 circulations are arranged；35 circulations are arranged on the basis of suitable annealing temperature in second part, extend
Time is disposed as 40s, after the reaction was completed, Preliminary detection is carried out in agarose gel electrophoresis.
1.2.3 Capillary Electrophoresis
1.2.3.1 design primer
Several pairs of primers that selection differences are stable from the primer screened, polymorphism is good, recombine primer, all
5 ' the sequences of end addition M13 albumen: TGTAAAACGACGGCCAGT of preceding primer.
1.2.3.2PCR amplification
PCR annealing temperature and response procedures are constant, carry out PCR reaction with the newly synthesized primer with fluorescent protein sequence
Amplification, PCR reaction system are as shown in table 5 below.
(fluorescin is added) in table 5SSR-PCR reaction system
1.2.3.3 detection and data analysis
After the reaction was completed, advanced row agarose gel electrophoresis detection, after band confirmation is errorless, feeding company is examined PCR
It surveys, carries out statistics and list after obtaining peak figure.
1.2.4 fingerprint map construction
With reference to Li Ruifeng etc., (is based on morphology mark to the construction method of finger-print when Li Ruifeng, Gao Peng, Zhu Zicheng, Luan Fei
Note and SSR marker muskmelon main breed taxonomic identification study [J] China's Vegetable, 2014,06:20-27.) finger-print structure
Construction method, selects the primer pair that difference is relatively stable, polymorphism is good from the primer screened, it is used in a fixed order
Capitalization number, by using Capillary Electrophoresis as a result, recording seven Luan Shu sample amplifications of each pair of primer pair as a result, to four
The DNA of a coloured silk Ye Luanshu kind sample forms the banding pattern number of character and digit, as the finger-print of each kind.
2, experimental result
The optimization of 2.1SSR-PCR reaction system
By the preliminary experiment to sample B 1, B2, B3 and B4 progress, the reaction system effect of 50 μ l is significant compared with 20 μ l, can
The error of experimental implementation in operating process is avoided well, therefore is all made of 50 μ l reaction systems in later experiments of the present invention.
Desk study has been carried out to the annealing temperature of 96 pairs of designed primers by sample B 1, B2, B3 and B4, it has been imitated
Fruit has carried out preliminary screening, has 92 pairs of primer amplifications to go out clear band in 96 pairs of primers, wherein 27 pairs are with differential effect
Primer, 28% ratio is occupied in total primer, thus can assert the primer of the present invention have certain polymorphism.
SSR-PCR response procedures: 94 DEG C, 3min → 94 DEG C, 30s → (+1 DEG C of suitable annealing temperature), 30s → 72 DEG C,
40s → 94 DEG C, 30s → (+0.5 DEG C of suitable annealing temperature), 30s → 72 DEG C, 40s → [94 DEG C, 30s → (suitable annealing temperature
Degree), 30s → 72 DEG C, 40s]x35→ 72 DEG C, 7min → 4 DEG C, ∞.The suitable annealing temperature of each pair of primer and amplification situation, then such as
Shown in table 6.
Table 6SSR primer annealing temperature and amplification situation summation
SSR expands difference between 2.2 goldenrain tree Pterostyrax plant varieties
By carrying out SSR amplified reaction to six sample Bs 2, A1, A2, A3, A4 and A5, wherein B2 and A1 is control group, right
The special SSR marker of four mutational varieties (bright and beautiful leaf goldenrain tree, Shanxi goldenrain tree No. 1, Shanxi goldenrain tree No. 2, Shanxi goldenrain tree No. 3) of Luan Shu is screened.?
In results of preliminary screening, 25 pairs of primers are shared in 96 pairs of primers has doubtful inconsistent expanding effect, table in six samples
Existing form is mainly by band length is inconsistent and part sample is to embody (Fig. 1) in the form of two kinds of two close bands etc..
The building of 2.3 Luan Shu four strains finger-prints
According to agarose gel electrophoresis as a result, from the primer for Luan Shu four strains with doubtful differential amplification effect
In have chosen 13 pairs of primers, carried out Capillary Electrophoresis；Obtain each pair of primer each in detail according to Capillary Electrophoresis result
The length of amplified band in kind, and the alpha code of combination primer has obtained the special finger-print of four strains.
The alpha code of fingerprint map construction the primer is specifically as shown in table 7, the finger-print of each kind such as 8 institute of table
Show, semi-capillary electrophoresis result is as shown in Figure 2, Figure 3 and Figure 4.
7 fingerprint map construction the primer alpha code of table
The finger-print of 8 Luan Shu four strains of table
Primer 13 (alpha code B) although amplifying the peak of 128bp and 194bp length in sample A2 (bright and beautiful leaf goldenrain tree),
But its peak value is lower (peak value is at least just valuable in 500bp or more), and carries out repeated authentication with primer 13 to sample A2, finds
Its peak value is continued for being maintained at low-level.Therefore, in order to ensure that the accuracy of experiment, the present invention have given up primer 13 in sample A2
In amplification, two peaks amplified be considered as primer it is not special caused by.
Claims (10)
1. the SSR primer for constructing color Ye Luanshu Variety fingerprinting, which is characterized in that be made of: drawing following 13 pairs of primers
Object is to 1: being classified as downstream primer shown in upstream primer shown in SEQ ID No.1 and SEQ ID No.2 by nucleotides sequence and forms；Primer
To 2: being classified as downstream primer shown in upstream primer shown in SEQ ID No.3 and SEQ ID No.4 by nucleotides sequence and form；Primer pair
3: downstream primer shown in upstream primer shown in SEQ ID No.5 and SEQ ID No.6 being classified as by nucleotides sequence and is formed；Primer pair 4:
Downstream primer shown in upstream primer shown in SEQ ID No.7 and SEQ ID No.8 is classified as by nucleotides sequence to form；Primer pair 5: by
Nucleotides sequence is classified as the composition of downstream primer shown in upstream primer shown in SEQ ID No.9 and SEQ ID No.10；Primer pair 6: by
Nucleotides sequence is classified as the composition of downstream primer shown in upstream primer shown in SEQ ID No.11 and SEQ ID No.12；Primer pair 7: by
Nucleotides sequence is classified as the composition of downstream primer shown in upstream primer shown in SEQ ID No.13 and SEQ ID No.14；Primer pair 8: by
Nucleotides sequence is classified as the composition of downstream primer shown in upstream primer shown in SEQ ID No.15 and SEQ ID No.16；Primer pair 9: by
Nucleotides sequence is classified as the composition of downstream primer shown in upstream primer shown in SEQ ID No.17 and SEQ ID No.18；Primer pair 10:
Downstream primer shown in upstream primer shown in SEQ ID No.19 and SEQ ID No.20 is classified as by nucleotides sequence to form；Primer pair
11: downstream primer shown in upstream primer shown in SEQ ID No.21 and SEQ ID No.22 being classified as by nucleotides sequence and is formed；Primer
To 12: being classified as downstream primer shown in upstream primer shown in SEQ ID No.23 and SEQ ID No.24 by nucleotides sequence and form；Draw
Object is to 13: being classified as downstream primer shown in upstream primer shown in SEQ ID No.25 and SEQ ID No.26 by nucleotides sequence and forms.
2. SSR primer described in accordance with the claim 1, which is characterized in that the coloured silk Ye Luanshu kind is selected from bright and beautiful leaf goldenrain tree, Shanxi goldenrain tree 1
Number, Shanxi goldenrain tree No. 2 or Shanxi goldenrain tree No. 3.
3. application of the SSR primer described in claim 1 in color Ye Luanshu cultivar identification.
4. application of the SSR primer described in claim 1 in building coloured silk Ye Luanshu Variety fingerprinting.
5. according to application described in 3 or 4, which is characterized in that the coloured silk Ye Luanshu kind is selected from bright and beautiful leaf goldenrain tree, Shanxi goldenrain tree No. 1, Shanxi goldenrain tree 2
Number or Shanxi goldenrain tree No. 3.
6. a kind of construction method of coloured silk Ye Luanshu Variety fingerprinting, which comprises the following steps: (1) extract color leaf
The DNA of Luan Shu sample；(2) using the DNA of extraction as template, it is anti-to be established as amplimer using SSR primer described in claim 1 by PCR
It answers system and carries out PCR amplification；(3) amplified production is subjected to Capillary Electrophoresis, according to Capillary Electrophoresis result to color Ye Luanshu
The amplification of the sample DNA of each kind formed primer alpha code add the banding pattern number of amplified band length number to get.
7. construction method according to claim 6, which is characterized in that the PCR reaction system includes: the total of reaction system
Volume is 25 μ L, wherein 2 × PCR MIX, 12.5 μ l, 2 μ l of DNA profiling, 2 μ l of upstream primer, downstream primer 0.5 μ l, ddH2O
3 μ l, M13 albumen, 5 μ l；Wherein, in upstream primer 5, ' end addition nucleotides sequence is classified as sequence shown in SEQ ID No.27, then
PCR amplification is carried out again；The program of the PCR amplification includes: 94 DEG C, 3min；94 DEG C, 30s；The annealing temperature+1 of each pair of primer
DEG C, 30s；72 DEG C, 40s；94 DEG C, 30s；+ 0.5 DEG C of the annealing temperature of each pair of primer, 30s；72 DEG C, 40s；94 DEG C, 30s；It is each pair of
The annealing temperature of primer, 30s；72 DEG C, 40s, 35 circulations；72 DEG C, 7min；The annealing temperature of each pair of primer are as follows: primer pair 1
Annealing temperature is 53 DEG C；The annealing temperature of primer pair 2 is 52 DEG C；The annealing temperature of primer pair 3 is 55 DEG C；The annealing of primer pair 4
Temperature is 54 DEG C；The annealing temperature of primer pair 5 is 56 DEG C；The annealing temperature of primer pair 6 is 54 DEG C；The annealing temperature of primer pair 7
It is 52 DEG C；The annealing temperature of primer pair 8 is 55 DEG C；The annealing temperature of primer pair 9 is 55 DEG C；The annealing temperature of primer pair 10 is 55
℃；The annealing temperature of primer pair 11 is 53 DEG C；The annealing temperature of primer pair 12 is 53 DEG C；The annealing temperature of primer pair 13 is 52
℃。
8. the color Ye Luanshu Variety fingerprinting that construction method described in claim 6 or 7 constructs, wherein the coloured silk leaf
Luan Shu kind is bright and beautiful leaf goldenrain tree, Shanxi goldenrain tree No. 1, Shanxi goldenrain tree No. 2 and Shanxi goldenrain tree No. 3.
9. coloured silk Ye Luanshu Variety fingerprinting according to claim 8, which is characterized in that
The finger-print of the brocade leaf goldenrain tree are as follows:
C243-249D291-293F302H241I232L256；
The finger-print of the Shanxi goldenrain tree No. 1 are as follows:
B128-194C249D291-293F302H247I232L256；
The finger-print of the Shanxi goldenrain tree No. 2 are as follows:
B128-134-194-203C243-247D291-293F302H247-256I232L256；
The finger-print of the Shanxi goldenrain tree No. 3 are as follows:
B128-134-194-203C249D293F302H241-256I232L256；
Wherein, B is the alpha code of the primer pair 2 of claim 1, and C is the alpha code of the primer pair 3 of claim 1, and D is
The alpha code of the primer pair 4 of claim 1, F are the alpha code of the primer pair 6 of claim 1, and H is drawing for claim 1
Object is to 8 alpha code, and I is the alpha code of the primer pair 9 of claim 1, and L is the letter of the primer pair 12 of claim 1
Number；Length of the Arabic numerals for the DNA band expanded using the primer after each letter, unit bp.
10. a kind of for identifying the PCR kit of color Ye Luanshu kind, comprising: PCR amplification primer, 2 × PCR MIX and
DdH2O, which is characterized in that the PCR amplification primer is SSR primer described in claim 1.
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| CN103060318A (en) * | 2013-01-11 | 2013-04-24 | 山东省农业科学院作物研究所 | SSR (Simple Sequence Repeat) core primer group developed based on whole genome sequence of foxtail millet and application of SSR core primer group |
| CN104419762A (en) * | 2013-09-09 | 2015-03-18 | 天津市农业质量标准与检测技术研究所 | Method for identifying cauliflower varieties by EST-SSR labels |
| CN105063028A (en) * | 2015-07-31 | 2015-11-18 | 青岛啤酒股份有限公司 | SSR (simple sequence repeat) primer group and method for constructing malt fingerprint maps by aid of primer group |
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| CN103060318A (en) * | 2013-01-11 | 2013-04-24 | 山东省农业科学院作物研究所 | SSR (Simple Sequence Repeat) core primer group developed based on whole genome sequence of foxtail millet and application of SSR core primer group |
| CN104419762A (en) * | 2013-09-09 | 2015-03-18 | 天津市农业质量标准与检测技术研究所 | Method for identifying cauliflower varieties by EST-SSR labels |
| CN105063028A (en) * | 2015-07-31 | 2015-11-18 | 青岛啤酒股份有限公司 | SSR (simple sequence repeat) primer group and method for constructing malt fingerprint maps by aid of primer group |
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